04 - 435 Pathobiology of Neurologic Diseases

435 Pathobiology of Neurologic Diseases

used successfully in this setting, as has iodinated contrast CTA, which has promise for replacing diagnostic spinal angiography for some indications.

INTERVENTIONAL NEURORADIOLOGY This rapidly developing field is providing new therapeutic options for patients with challenging neurovascular problems. Available proce­ dures include detachable coil therapy for aneurysms, particulate or liquid adhesive embolization of arteriovenous malformations, stent retrieval systems for embolectomy in acute stroke, balloon angioplasty and stenting of arterial stenosis or vasospasm, transarterial or trans­ venous embolization of dural arteriovenous fistulas and CSF-venous fistulas of the spine, balloon occlusion of carotid-cavernous and verte­ bral fistulas, endovascular treatment of vein-of-Galen malformations, preoperative embolization of tumors, and thrombolysis of acute arte­ rial or venous thrombosis. Many of these disorders place the patient at high risk of cerebral hemorrhage, stroke, or death. The highest complication rates are found with the therapies designed to treat the highest risk diseases. The advent of electrolytically detach­ able coils ushered in a new era in the treatment of cerebral aneurysms (Chap. 440). Two randomized trials found reductions of morbidity and mortality at 1 year among those treated for aneurysm with detachable coils compared with neurosurgical clipping. In many centers, coil­ ing has become standard therapy for many proximal circle of Willis aneurysms. PART 13 Neurologic Disorders Finally, recent studies of stent retrieval systems used to withdraw emboli have shown improved clinical outcomes in patients presenting with large-vessel occlusions and signs of acute stroke (Chap. 438). ■ ■FURTHER READING Bambach S et al: Arterial spin labeling applications in pediatric and adult neurologic disorders. J Magn Reson Imaging 55:698, 2020. Choi JW, Moon WJ: Gadolinium deposition in the brain: Current updates. Korean J Radiol 20:134, 2019. Mandell DM et al: Intracranial vessel wall MRI: Principles and expert consensus recommendations of the American Society of Neuroradi­ ology. AJNR Am J Neuroradiol 38:218, 2017. Nadjir R et al: Neuroradiology: The Core Requisites, 5th ed. Philadelphia, Elsevier, 2024. Pelz DM et al: Interventional neuroradiology: A review. Can J Neurol Sci 16:1, 2020. Schönmann C, Brockow K: Adverse reactions during procedures: Hypersensitivity to contrast agents and dyes. Ann Allergy Asthma Immunol 124:156, 2020. Tournier JD: Diffusion MRI in the brain—theory and concepts. Prog Nucl Magn Reson Spectrosc 112-113:1, 2019. Watson RE et al: MR imaging safety events: Analysis and improve­ ment. Magn Reson Imaging Clin N Am 28:593, 2020. Stephen L. Hauser, Arnold R. Kriegstein,

Stanley B. Prusiner

Pathobiology of

Neurologic Diseases The human nervous system is the organ of consciousness, cognition, ethics, and behavior; as such, it is the most intricate structure known to exist. More than one-third of the 23,000 genes encoded in the human genome are expressed in the nervous system, and for many, different isoforms are expressed that further increase the result­ ing specificities and potential functionality. Each mature brain is

composed of 100 billion neurons, several million miles of axons and dendrites, and >1015  synapses. Neurons exist within a dense paren­ chyma of multifunctional glial cells that synthesize myelin, preserve homeostasis, and regulate immune responses. Measured against this background of complexity, the achievements of molecular neurosci­ ence have been extraordinary. Advances have occurred in parallel with the development of new enabling technologies—in bioengineering and computational sciences; imaging; and cell, molecular, and chemical biology—and moving forward it is likely that the pace of new discover­ ies will only increase. This chapter reviews a few of the most dynamic areas in neuroscience, specifically highlighting advances in immunol­ ogy and inflammation, neurodegeneration, and stem cell biology. In each of these areas, recent discoveries are providing context for an understanding of the triggers and mechanisms of disease and offering new hope for prevention, treatment, and repair of nervous system inju­ ries. Discussions of the neurogenetics of behavior, advances in addic­ tion science, and diseases caused by network dysfunction can be found in Chap. 462 (Biology of Psychiatric Disorders); and new approaches to rehabilitation via harnessing of neuroplasticity, neurostimulation, and computer–brain interfaces are presented in Chap. 500 (Emerging Neurotherapeutic Technologies). NEUROIMMUNOLOGY AND NEUROINFLAMMATION Neuroimmunology traditionally comprises the science of immunemediated diseases of the nervous system, especially autoimmune diseases such as multiple sclerosis, myasthenia gravis, and GuillainBarré syndrome, as well as disorders in which immune-mediated neurologic damage occurs in the context of infection or neoplasia. More recently, recognition that neuroinflammation and the innate immune system play key roles in an expanded category of neurologic disorders, and neurodegenerative disorders in particular, has focused renewed attention on the intrinsic cellular components in the central nervous system (CNS) that mediate tissue damage, especially microg­ lia/macrophages and astrocytes, and also on oligodendrocytes, which are now recognized as central players across a wide range of brain disorders. It is also increasingly recognized that extensive networks of communication exist between each of these cell types and that result­ ing non-cell-autonomous pathologies are likely to underlie many human CNS disorders. ■ ■MICROGLIA AND MACROPHAGES These represent the most abundant cell types in the nervous system responsible for antigen presentation and innate immunity. Brain microglia (“small glue”) are derived from a primitive macrophage population in the yolk sac that migrates to the nervous system early in embryogenesis before the blood-brain barrier is formed. Once in the CNS, a variety of cell signals mediate microglial proliferation, migra­ tion, and differentiation. Microglia maintain their cell numbers pri­ marily through self-renewal and not repopulation from the circulation; however, it is now also clear that under some conditions peripherally derived macrophages that become microglia-like can replace damaged or defective microglia throughout the life span. Most microglia receive survival signals through colony-stimulating factor 1 receptor (Csf1r), via its natural ligands Csf1 produced by astrocytes and oligoden­ drocytes, and interleukin (IL) 34 produced by neurons. Depletion of microglia by administration of a selective inhibitor of Csf1r (PLX5622) was followed by rapid repopulation, which led to identification of a second population of ramified microglial precursor cells that do not require Csf1r signaling. Microglia have traditionally been divided into “resting” and “activated” states, the former characterized by an exten­ sively ramified appearance and the latter by a globular amoeboid phe­ notype. A variety of factors and cues, including type 1 interferons, can prime resting microglia toward an activated state, and these “primed” microglia are then hyperresponsive to a variety of secondary immune challenges including infection. Recently, single-cell transcriptome sequencing of microglia has also revealed a high degree of heterogene­ ity that was previously unappreciated, the functional consequences of which are largely unknown.

Promote learning and memory BDNF Phagocytosis of debris Proinflammatory (A1) astrocyte Uptake of aggregated proteins FIGURE 435-1  The multifunctional microglial cell. Microglia have diverse functions that can support healthy development and maintain homeostasis or contribute to tissue damage in pathologic conditions. Homeostatic functions include promotion of learning and memory through secretion of soluble proteins such as brain-derived neurotrophic factor (BDNF); participation in normal synaptic pruning; and clearing cellular debris and protein aggregates via phagocytosis. However, in pathologic states, activated microglia also contribute to tissue damage by targeting normal healthy neurons and synapses; by promoting formation of β-amyloid or other misfolded proteins deposited in neurodegenerative diseases; and by secreting cytokines (such as interleukin 1α, tumor necrosis factor, and the complement component C1q) incriminated in induction of neurotoxic A1 astrocytes. In addition, microglia have diverse functions in adaptive immunity, including roles in antigen presentation and immune regulation (Fig. 435-2). (Reproduced with permission from J Herz et al: Myeloid cells in the central nervous system. Immunity 46:943, 2017.) Microglia play critical roles in sculpting neuronal populations dur­ ing development and across the life span, through secretion of brainderived neurotrophic factor (BDNF) and other trophic factors that promote neuronal survival, and also via production of reactive oxygen species (ROS) and other molecules that mediate cell death. Microglia regulate development and maintenance of neural circuits through pruning of excitatory synapses and control of dendritic spine densities (Fig. 435-1). Mice depleted of microglia during development exhibit a variety of cognitive, learning, and behavioral deficits, including abnor­ mal social behaviors. These processes are dependent on classical com­ plement pathway molecules, including secretion of C1q and expression of complement receptor 3 (CR3) and CR5. Abnormalities in these microglial-dependent networks are now recognized as critical to the pathogenesis of most neurodegenerative and age-related pathologies. Activation of the complement cascade has assumed a major place in current concepts of pathogenesis of Alzheimer’s disease (AD) and other dementias, as follows. Synapses targeted for elimination are tagged by the complement proteins C1q and C3, the levels of which increase in the presence of excess β-amyloid. C3-bearing synapses are targeted for elimination by microglia that express CR3, and knockout of C1q or C3 can rescue the clinical and pathologic abnormalities asso­ ciated with neurodegeneration. Modification of aberrant complement activity represents an attractive approach for treatment of neurodegen­ eration from multiple causes, not only AD, but also Parkinson’s and Huntington’s disease, among others, in which microglia are responsible for clearing pathologic protein aggregates. An ideal therapeutic would target the deleterious microglial functions while preserving ones that are central to homeostasis such as synaptic pruning. Another consid­ eration, especially given the diverse roles of the complement system in all organs of the body, is potential off-target toxicities. Clinical trials are now underway with an inhibitory monoclonal antibody against C1q for Huntington’s disease and amyotrophic lateral sclerosis (ALS). While the specific mechanisms of complement-dependent neurode­ generation will likely differ in distinct neurodegenerative conditions, these data provide hope that complement-pathway interventions could represent an approach to control of neurodegenerative pathologies mediated at least in part through the innate immune system. Genetic evidence also supports a primary role for microglia in numerous neurodegenerative conditions and disease states, in con­ trast to earlier views in which their role was seen as largely secondary

Pruning or elimination of synapses IL-1α | TNF | C1q CHAPTER 435 Pathobiology of Neurologic Diseases and involving phagocytosis of cell debris. More than half of all genes implicated in genome-wide association studies in AD implicate innate immune processes and microglia. One direct genetic link is the phagocytosis-associated gene TREM2 (triggering receptor expressed on myeloid cells 2). TREM2 is a microglial receptor that can bind amyloid, induce proliferation and migration of microglia, and pos­ sibly limit the spread of disease-associated AD aggregates. Moreover, a soluble TREM2 cleavage product promotes neuroinflammation and inhibits aggregation of β-amyloid. Loss-of-function mutations in TREM2 increase AD risk up to fourfold. In one mouse model of AD, overexpression of TREM2 blocked AD pathology and rescued perfor­ mance on tests of learning and memory; however, in other models, including tau models, the effects of TREM2 targeting were found to be stage specific or inconsistent. A clinical trial of an agonist monoclonal antibody against TREM2 is underway. Immune system genes implicated in susceptibility to other late-life dementias also represent promising targets for therapy. For example, ~10% of all cases of frontotemporal degeneration (FTD) are due to heterozygous mutations of the gene granulin (GRN), which encodes the protein progranulin expressed in neurons and microglial cells (Chap. 443). Progranulin is a neurotrophic factor essential for lyso­ somal function and microglial homeostasis. GRN mutations with resulting haploinsufficiency of progranulin are highly penetrant for FTD. When progranulin is deleted in mice, an age-dependent microg­ lial activation phenotype results, associated with upregulation of proinflammatory neurotoxic cytokines, complement components, and other genes associated with innate immunity, along with enhanced pruning of inhibitory synapses and behavioral manifestations remi­ niscent of human FTD. Remarkably, inhibition of complement activa­ tion can rescue all of these deficits. These data indicate a primary role for microglial activation in FTD caused by mutations in GRN, likely mediated through increased production of C3 complement, enhanced lysosomal trafficking, and excessive synaptic pruning in affected brain regions affected. Clinical trials underway for neurodegeneration due to progranulin deficiency include a monoclonal antibody to block lysosomal degradation of progranulin, as well as gene and protein replacement therapies. Microglia are generally considered the most important source of antigen-presenting cells in the brain, as they express class II major histocompatibility complex molecules enabling antigen presentation

to CD4 T cells. In Parkinson’s disease (PD), neurotoxic T cells reactive against epitopes of α-synuclein are commonly found, and it has been postulated that antigen presentation by microglia may have initiated this autoimmune response. Beyond a traditional role as antigenpresenting cells, microglia are now understood to also have a range of other interactions with T and B lymphocytes. For example, secre­ tion of CCL3 by microglia, which is enhanced with aging, promotes the recruitment of memory CD8 T cells, which might account for the presence of CD8 T cells in a wide range of neuropathologic conditions including multiple sclerosis (MS).

Microglia are located throughout the brain parenchyma, while the closely related brain macrophages occur primarily in perivascular regions, including the meninges and choroid plexus. Like microglia, brain macrophages are derived from yolk sac precursors that appear to enter the brain at an early developmental stage and propagate locally, although some choroid plexus macrophages may also be replenished at low levels from the bloodstream on a continuing basis. Under inflammatory conditions, however, large numbers of hematog­ enously derived monocytes readily enter the brain parenchyma. In the disease model, experimental autoimmune encephalomyelitis (EAE), macrophages derived from bone marrow monocytes are the critical population that initiates inflammatory demyelination at paraxonal regions near nodes of Ranvier (Fig. 435-2). Brain macrophages have multiple proinflammatory functions, including promoting adhesion, attraction, and activation of B and T lymphocytes; providing antigenspecific activation of T cells via antigen presentation of immunogenic peptides, including autoantigens, complexed to surface class II major histocompatibility complex (MHC II) molecules; and contributing to cell injury through generation of oxidative stress and cytotoxicity. By contrast, microglia have been traditionally thought to downregulate PART 13 Neurologic Disorders Triggering Strong adhesion Rolling Flow Activated lymphocyte CD 31 Blood-brain barrier endothelium Chemokines and cytokines Astrocytes Heat shock proteins? Activated Microglia/ macrophages IFN-γ IL-2 Fc receptor Chemokines IL-1, IL-12 Brain tissue TNF, IFN, free radicals, vasoactive amines, complement, proteases, cytokines, eicosanoids Myelin damage FIGURE 435-2  A model for experimental allergic encephalomyelitis (EAE). Crucial steps for disease initiation and progression include peripheral activation of preexisting autoreactive T cells; homing to the central nervous system (CNS) and extravasation across the blood-brain barrier; reactivation of T cells by exposed autoantigens; secretion of cytokines; activation of microglia and astrocytes and recruitment of a secondary inflammatory wave; and immune-mediated myelin destruction. ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; LFA-1, leukocyte function-associated antigen-1; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

inflammatory responses and promote tissue repair in EAE. This model of the relative roles of macrophage and microglial cells is certainly an oversimplification, and more nuanced functions of these cell types have been revealed by single-cell sequencing methods, demonstrating previ­ ously unappreciated heterogeneity influenced by location, context, and environmental cues. ASTROCYTES Astrocytes represent half or more of all cells in the CNS. Thought ear­ lier to function as simple interstitial supporting cells that provide scaf­ folds for neuronal migration and contribute to homeostasis, emerging data indicate far more pleiotropic functions for this cell type. Astro­ cytes, like microglia, play profound roles in the life of synapses by secreting factors (such as apolipoprotein E, thrombospondins, and glypicans) that regulate development, maintenance, and pruning of presynaptic and postsynaptic structures. Influenced by local neuronal activity, astrocytes actively phagocytose synapses. Pruning of synapses and clearance of apoptotic cells by astrocytes are mediated through the scavenger receptor multiple EGF-like domains 10 (Megf10), a high-affinity receptor for C1q. Astrocytes also participate in dynamic regulation of vascular tone, in part through astrocyte-astrocyte com­ munication mediated through gap junctions and calcium waves modulated by neuronal activity; support blood-brain barrier and glymphatic (see below) integrity through extension of foot processes to vascular structures and expression of aquaporin-4 water channels; and carry out additional metabolic functions essential for mainte­ nance of neuron health. Recent work has highlighted the transcriptional and functional heterogeneity of reactive astrocytes that, depending on the con­ text, could regulate inflammation, promote neurotoxicity, or aid in Extravasation B cell Gelatinases LFA-1 α4 Integrin VCAM ICAM Basal lamina Microglia/macrophages T cell activation B cell T cell Antigen presentation TNFα, LT, and GM-CSF B cell Antibody complement

protection and repair. As examples, in response to cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) pro­ duced by infiltrating lymphocytes, astrocytes can augment neuroin­ flammation via secretion of chemokines such as CCL2 and CXCL10 that attract lymphocytes and monocytes to the CNS. In MS and other neuroimmune disorders, production of B-cell activation factor (BAFF) by astrocytes within lesions promotes the survival and activation of pathogenic autoreactive B cells. Under other conditions, astrocytes appear to limit inflammation, through expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced by interferon γ produced by natural killer cells, or by secretion of the regulatory cytokine IL-27. And in still other settings, astrocytes can secrete factors that promote neuronal survival and axon regeneration, for example, by secretion of wingless-type MMTV integration site 1 (Wnt1), a neuroprotective factor for dopaminergic neurons. It is also now clear that bidirectional crosstalk between immune system cells and astrocytes can profoundly influence the brain’s response to injury. In the best-studied example, activated microglia, through secretion of IL-1a, TNF, and C1q, can induce astrocytes to transform to a reactive disease-promoting phenotype. Such cells lose the capacity to phagocytose synapses and myelin debris and become toxic in vitro to neurons and mature oligodendrocytes, via different mechanisms including complement-mediated damage and production of nitric oxide (NO) and free radicals. Interestingly, oligodendrocyte progenitor cells (OPCs), abundant in active lesions of MS (Chap. 455) within the inflammatory milieu, are resistant to astrocyte-mediated killing. In ALS models modeled on human ALScausing mutations in SOD1 and C9orf72 genes, astrocyte expression of the mutant genes promoted neurotoxicity, indicating a noncell-autonomous disease mechanism mediated through astrocytes. Similarly, in AD, astrocytes have been implicated in several genetic forms of AD, including amyloid accumulation mediated through apolipoprotein E (ApoE), which in the nervous system is expressed primarily in astrocytes. Thus, reactive astrocytes could promote dam­ age in disorders as varied as MS (Chap. 455), AD (Chap. 442), PD (Chap. 446), and ALS (Chap. 448), despite the distinct etiologies and pathologies of these conditions. ■ ■OLIGODENDROCYTES AND MYELIN Myelin is the multilayered insulating substance that surrounds axons and speeds impulse conduction by permitting action potentials to jump between naked regions of axons (nodes of Ranvier) and across myelinated segments. Oligodendrocytes contact axons at paranodes, where sodium and potassium channels essential for saltatory con­ duction are clustered. Molecular interactions between the myelin membrane and axon are required to maintain the stability, function, and normal life span of both structures. The process of myelination is directed both by axon-derived cues as well as the physical proper­ ties of the axon-membrane curvature. Importantly, ongoing neuronal activity influences both the differentiation of oligodendrocytes as well as the extent of myelination, a process referred to as adaptive myelina­ tion. A single oligodendrocyte usually ensheaths multiple axons in the CNS, whereas in the peripheral nervous system (PNS), each Schwann cell typically myelinates a single axon. Oligodendrocytes can increase their surface length by as much as 2000-fold, the maintenance of which places significant metabolic demands on the cell. Myelin is a lipid-rich material formed by a spiraling process of the membrane of the myelinating cell around the axon, creating mul­ tiple membrane bilayers that are tightly apposed (compact myelin) by charged protein interactions. From an evolutionary perspective, an increase in the efficiency of nerve conduction enabled by CNS myelin has permitted expanded neuronal connectivity and brain complexity without the need to dramatically increase brain size. CNS myelin has also evolved to provide critical neuroprotective support to axons. Not surprisingly, myelin pathologies cause or contribute to many neurologic conditions. Several clinically important neuro­ logic disorders result from inherited mutations in myelin proteins (Chap. 457). Constituents of myelin have a propensity to be targeted as autoantigens in autoimmune demyelinating disorders, in part due

to molecular similarities between myelin and microbial cell wall con­ stituents (Chaps. 455 and 456). Alterations in myelin are increasingly recognized as a contributor to cognitive changes associated with aging, including dementia. For example, in AD models, oligodendrocyte damage can promote the deposition of disease-associated amyloid plaques. Oligodendrocytes can also be the site of disease-associated protein deposition in some neurodegenerative disorders, for example, with toxic accumulation of α-synuclein aggregates in multiple system atrophy (Chap. 451). Evidence also supports a role for oligodendro­ cyte and myelin pathology in disorders as varied as ALS (Chap. 448), traumatic brain injury (Chap. 454), and stroke (Chap. 437), among other conditions.

Premyelinating OPCs are highly motile cells that migrate exten­ sively during development and in the adult brain following injuries to the myelin sheath. OPCs migrate along the inner (or abluminal) surface of endothelial cells, a process regulated by Wnt pathway sig­ naling and upregulation of the chemokine receptor Cxcr4 that drives their attachment and retention to the vasculature. In the normal adult brain, large numbers of OPCs are widely distributed. Follow­ ing demyelination, remyelination is largely dependent on OPCs that differentiate into myelin-producing oligodendrocytes and produce characteristic thinly remyelinated fibers. In some situations, a second population of regenerating oligodendrocytes derived from neural stem cells can mediate more effective remyelination, with thicker lamellae and greater functional preservation of axons. A C14 labeling study from human MS lesions indicated that a third population of nonmitotic preexisting oligodendrocytes may represent an additional source of remyelinating cells. CHAPTER 435 Pathobiology of Neurologic Diseases Both acquired demyelinating disorders, such as MS, and inherited ones, such as Pelizaeus-Merzbacher disease (duplication or deletion of the CNS proteolipid protein gene) and adrenoleukodystrophy (muta­ tions in the ABCD1 gene responsible for transport of very-long-chain fatty acids into the peroxisome for degradation), are associated with progressive axonal loss. Loss of oligodendrocyte support can produce axonal damage through a variety of mechanisms, including reductions in the supply of glucose and other essential nutrients; an increased axonal workload; impaired glutamate and calcium buffering; mito­ chondrial damage; loss of neurotrophins; enhanced susceptibility to reactive oxygen species including nitric oxide; and failure to maintain normal synapses. A number of molecules have been identified that regulate oligo­ dendrocyte differentiation and myelination, including LINGO-1, hyaluronan, chondroitin sulfate proteoglycan, the Wnt pathway, Notch (and its receptor Jagged), fibrinogen, and the M1 muscarinic receptor Chrm1, all of which are inhibitory. Other targets are the retinoic acid receptor RXRγ, vitamin D, and thyroid hormone, all of which promote oligodendrocyte maturation. All are also potential targets for myelin repair therapies. In the EAE model of autoimmune demyelination (Fig. 435-2), oligodendrocyte-specific knockout of Chrm1 improved remyelination, protected axons, and restored function, directly dem­ onstrating that remyelination can be neuroprotective following injury. A pivotal trial of a monoclonal antibody against LINGO-1 in patients with acute optic neuritis failed to improve clinical outcomes, a disap­ pointing result given that the antibody appeared to have promising clinical effects in an earlier phase 2 trial. In a preliminary trial of chronic optic neuritis, a promising result was reported with clemastine, an antihistamine and M1 muscarinic receptor antagonist, raising hope that clinically effective remyelination might be achievable even in a chronic demyelinating condition. LYMPHATICS OF THE CENTRAL

NERVOUS SYSTEM Two recently identified lymphatic structures of the CNS are the glym­ phatic and deep dural lymphoid systems, responsible for clearance of debris in the CNS, and likely also serving roles in immune surveil­ lance. The brain has traditionally been considered to lack a classical lymphatic system, and immune responses against antigens are less effectively generated in the CNS than in other organ systems, a con­ cept termed immune privilege. However, the immune privilege status

of the brain is relative and not absolute. Also, given the high meta­ bolic demands of the brain, some mechanism for efficient removal of solute and debris must be present. One well-established pathway involves the passive flow of solutes from the brain parenchyma into the cerebrospinal fluid (CSF), and their exit via the arachnoid granu­ lations, as well as along cranial and spinal nerve roots to a series of lymphoid structures located in the cribriform plate, nasal mucosa, and elsewhere.

The glymphatic system derives its name from a distinctive archi­ tecture involving lymphoid-like structures and astroglial cells. CSF synthesized in the arachnoid villi circulates through the ventricles and subarachnoid space surrounding the convexities of the brain and spinal cord and exits through conduits surrounding arterioles penetrat­ ing into the brain parenchyma. These spaces are lined by endothelial cells internally and by astrocyte foot processes that form the external walls. Aided by arterial propulsion, CSF moves out of these specialized conduits and into astrocytes via foot processes rich in aquaporin-4 water channels, and then in the interstitium of brain parenchyma, picks up solutes and particulate debris that are then carried to peri­ venous spaces where they passage to exit the brain and drain into the lymphatic system. In mice, knockout of aquaporin-4 markedly reduced the flow of interstitial fluids in the brain, underscoring the critical role of astrocyte uptake of CSF in this process. Interstitial flow in the CNS is also impaired with aging, possibly related to changes in astrocytic aquaporin-4 expression. A fascinating aspect of the glymphatic system is that the transport of fluids and solutes accelerates with sleep, arguing for a critical role for sleep in promoting clearance of debris needed to meet the high metabolic demands of the nervous system. Furthermore, in disease models, aggregated proteins associated with neurodegenera­ tive disease, such as β-amyloid associated with AD (Chap. 442), were also more efficiently cleared during sleep. Indeed, in mice genetically engineered to produce excess β-amyloid and develop AD-like cognitive decline, sleep deprivation increased accumulation of amyloid plaques. Glymphatic pathways are also likely to represent an important egress pathway for lymphocytes in the CNS and a route for lymphocyte encounters with CNS antigens in cervical lymph nodes. In this regard, deep cervical lymph nodes may be a site for antigen-specific stimula­ tion of B cells in MS (Chap. 455). PART 13 Neurologic Disorders A second pathway consists of a plexus of small lymphatic-like ves­ sels located on the external surface of meningeal arteries and deep dural sinuses (including the sagittal and transverse sinuses), structures that exit the brain along the surface of veins and arteries and drain to the deep cervical lymph nodes. These conduits appear to represent a lymphoid drainage system distinct from vascular endothelium. These sinus-associated lymphoid structures may be most important in clear­ ing solutes from the CSF, in contrast to the glymphatic system that likely functions to remove waste products from the brain interstitium; however, the exact functions of these two systems and their interrela­ tionships are only beginning to be understood. MICROBIOTA AND NEUROLOGIC DISEASE The human microbiome (Chap. 484) represents the collective set of genes from the 1014 organisms living in our gut, skin, mucosa, and other sites. The aggregate number of genes encoded by bacteria living in and around us (i.e., the microbiome) outnumber our own genome by a factor of 100, and these can encode a wide variety of molecules that directly or indirectly affect nervous system development, main­ tenance, and function. Distinctive microbial communities have been found in individuals with different genetic backgrounds, ethnicities, diets, and environments. While progressively evolving with age, in any individual, the predominant gut microbiota can be remarkably stable over decades, but also can be altered by exposure to certain microbial species (e.g., by ingestion of probiotics) or the frequent use of antibiotics. Gut microbes can shape immune responses through the interaction of their metabolism with that of humans. These gut–brain interac­ tions are likely to be important in understanding the pathogenesis of many autoimmune neurologic diseases. For example, mice raised in a germ-free environment (or treated with broad-spectrum antibiotics)

are resistant to EAE, an effect associated with decreased production of proinflammatory cytokines and conversely more production of the immunosuppressive cytokines IL-10 and IL-13 as well as an increase in regulatory T and B lymphocytes. Intestinal microbiota from patients with MS were found to promote EAE when transferred to germ-free mice, possibly due to imbalances between bacterial species that pro­ mote inflammation (e.g., Akkermansia muciniphila and Acinetobacter calcoaceticus) and those that induce regulatory immune responses (e.g., Parabacteroides distasonis, Prevotella copri, and certain species of Clostridium). Recent studies have shown that the transcription fac­ tor aryl hydrocarbon receptor (AHR) is an important regulator of the differentiation of murine and human regulatory T cells and microglia. AHR is activated by endogenous physiologic ligands, but also by meta­ bolic products of the commensal flora. In addition to nonspecific effects on immune homeostasis mediated by cytokines and regulatory lymphocytes, some microbial proteins could trigger a cross-reactive immune response against a homolo­ gous protein in the nervous system, a mechanism termed molecular mimicry. Examples include cross-reactivity between the astrocyte water channel aquaporin-4 and an ABC transporter permease from Clostridia perfringens in neuromyelitis optica (Chap. 456); human leukocyte antigen (HLA) molecules with A. muciniphila peptides of A. muciniphila and Epstein-Barr virus in MS (Chap. 455); the neural ganglioside Gm1 and similar sialic acid–containing structures from Campylobacter jejuni in Guillain-Barré syndrome (Chap. 458); and the sleep-promoting protein hypocretin and hemagglutinin from an H1N1 influenza virus in narcolepsy (Chap. 33). Microbial genes also encode molecules that can affect development of neurons and glia, and influence myelination and plasticity. Bacterialderived short-chain fatty acids, for example, regulate production of brain-derived neurotrophic factor (BDNF). Bacteria produce a variety of neurotransmitters including γ-aminobutyric acid (GABA) and serotonin and other neuroactive peptides that can modulate the hypothalamic-pituitary axis. Gut microbiota influence development and activity of the enteric nervous system, which communicates bidi­ rectionally with the CNS via the vagus nerve that innervates the upper gut and proximal colon. As these gut–brain relationships become bet­ ter defined, a role for the microbial environment in the pathogenesis of a much wider spectrum of neurologic conditions and behaviors seems likely, extending well beyond the traditional boundaries of immune-mediated pathologies. In this regard, it has long been known that gut bacteria can influence brain function, based mostly on clas­ sic studies demonstrating that products of gut microbes can worsen hepatic encephalopathy, forming the basis of treatment with antibiot­ ics for this condition. Mice that develop in a germ-free environment display less anxiety, lower responses to stressful situations, more exploratory locomotive behaviors, and impaired memory formation compared with non-germfree counterparts. These behaviors were related to changes in gene expression in pathways related to neural signaling, synaptic function, and modulation of neurotransmitters. Moreover, this behavior could be reversed when the germ-free mice were co-housed with non-germ-free mice. As noted above, intestinal microbiota were found to be required for the normal development and function of brain microglia, poten­ tially linking these behavioral effects to specific cellular targets in the CNS. Some actions of gut microbial species on microglia also appear to be sex- and age-specific. The vagus nerve has been implicated in anxiety- and depressionlike behaviors in mice. Ingestion of Lactobacillus rhamnosus induced changes in expression of the inhibitory neurotransmitter GABA1b in neurons of the limbic cortex, hippocampus, and amygdala, associ­ ated with reduced levels of corticosteroids and reduced anxiety- and depression-like behaviors. Remarkably, these changes could be blocked by vagotomy. A related area of emerging interest is in a possible contribution of the gut microbiome to autism and related disorders. Children with autistic spectrum disorders (ASD) have long been known to have gas­ trointestinal disturbances, and the severity of dysbiosis appears to cor­ relate with the severity of autism. In several murine models of autism,

manipulation of the gut microbiome ameliorated the behavioral abnor­ malities. A role for the proinflammatory cytokine IL-17 was implicated as a possible mediator in producing the ASD-like changes. In mice, an ASD-like disorder could be induced in offspring after injecting the pregnant mother with the viral RNA mimic, polyinosinic:polycytidylic acid (poly I:C); oral treatment of offspring with Bacteroides fragilis cor­ rected a range of autistic behaviors in these mice and also improved gastrointestinal dysfunction. These preclinical data led to a small uncontrolled study of fecal gut transplantation in children with ASD that reported encouraging results but will need to be confirmed in rigorous controlled trials. There has been considerable interest in the possible role of the microbiome in a variety of vascular, traumatic, and neurodegenera­ tive diseases, possibly mediated through actions on innate immunity and microglia. In SOD1 transgenic ALS-prone mice, a germ-free environment exacerbated disease progression, and symptoms could be ameliorated by increasing levels of A. muciniphila or its nicotinamide (vitamin B3) metabolite; a small preliminary clinical trial of nicotin­ amide supplementation subsequently reported encouraging results in ALS patients. In a PD model, injection of misfolded α-synuclein into the gut triggered deposition of α-synuclein in the brain, an effect that was blocked when the vagus nerve was severed. This supported a prion mechanism (see below) for PD pathogenesis, in which vagal transport of aggregated α-synuclein might seed the CNS via the vagus nerve. The concept of a gut origin of PD is also consistent with clinical and pathologic studies, and by some epidemiologic data suggesting that vagotomy may be protective against PD. In transgenic mice that over­ express human α-synuclein, transplantation of intestinal microbiota from PD patients worsened motor deficits, α-synuclein deposition, and neuroinflammation, and conversely, pathology could be ameliorated with germ-free housing conditions or antibiotic treatment. In other Sporadic NDs Prions causing neurodegradation

Wt precursor + A A Age-dependent mutant prion formation Inherited NDs i ii Mutant prion form Mutant precursor + B FIGURE 435-3  Neurodegeneration caused by prions. A. In sporadic neurodegenerative diseases (NDs), wild-type (Wt) prions multiply through self-propagating cycles of posttranslational modification, during which the precursor protein (green circle) is converted into the prion form (red square), which generally is high in β-sheet content. Pathogenic prions are most toxic as oligomers and less toxic after polymerization into amyloid fibrils. The small polygons (blue) represent proteolytic cleavage products of the prion. Depending on the protein, the fibrils coalesce into Aβ amyloid plaques in Alzheimer’s disease (AD), neurofibrillary tangles in AD and other tauopathies, or Lewy bodies in Parkinson’s disease (PD) and dementia with Lewy bodies. Drug targets for the development of therapeutics include: (1) lowering the precursor protein, (2) inhibiting prion formation, and (3) enhancing prion clearance. B. Late-onset heritable neurodegeneration argues for two discrete events: The (i) first event is the synthesis of mutant precursor protein (green circle), and the (ii) second event is the age-dependent formation of mutant prions (red square). The highlighted yellow bar in the DNA structure represents mutation of a base pair within an exon, and the small yellow circles signify the corresponding mutant amino acid substitution. Green arrows represent a normal process; red arrows, a pathogenic process; and blue arrows, a process that is known to occur but unknown whether it is normal or pathogenic. (Used with permission of Annual Reviews, from [Biology and genetics of prions causing neurodegeneration, SB Prusiner, 47:601, 2013 permission conveyed through Copyright Clearance Center, Inc.)

work, a protein of Escherichia coli, named Curli, has been shown to misfold and potentially serve as a template for subsequent propagation of misfolded α-synuclein. The possibility that a bacterial protein could initiate the cascade of events leading to PD is an extraordinary, but still unproven, hypothesis.

Thus, there are a variety of mechanisms whereby the microbiome— in gut, skin, and other bodily surfaces—can modulate healthy brain function or influence susceptibility to and expression of a variety of brain diseases. The specific mechanisms are likely to vary with each condition but are likely to include promoting autoimmunity through molecular mimicry; disrupting immune homeostasis; modulating brain function via bacterial metabolites that travel in the circula­ tion and cross the blood-brain barrier; influencing neural activity through signals generated in the enteric nervous system; or even the possibility that prion-like aggregates are formed in the gut and passage to the CNS via the vagus nerve. Although no proven benefit of modulating the microbiome exists for any brain disease today, this situation is likely to change as information about specific disease-associated microbial communities rapidly increases, along with improved methods to fine-tune their proportions and overall diversity in humans. CHAPTER 435 Pathobiology of Neurologic Diseases PATHOLOGIC PROTEINS, PRIONS, AND NEURODEGENERATION (FIG. 435-3) ■ ■PROTEIN AGGREGATION AND CELL DEATH The term protein aggregation has become widely used to describe easily recognizable hallmarks of neurodegeneration. While such neuropatho­ logic hallmarks including plaques, neurofibrillary tangles (NFTs), and inclusion bodies are often thought to cause neurologic dysfunction, numerous new discoveries over the past several decades have rendered this view increasingly unlikely. Instead, protein aggregates represent

Wt prion form Aβ plaque Amyloid fibrils Tau tangle α-Synuclein Lewy body

accumulations of toxic proteins that may become less harmful when they are sequestered into plaques, NFTs, and inclusion bodies.

Most mutations in the amyloid precursor protein (APP) gene caus­ ing familial AD are concentrated within the Aβ peptide. Many of these mutations increase production of the Aβ42 peptide composed of β-amyloid with 42 amino acids, which has an increased propensity to adopt a prion conformation, as compared to β-amyloid with 40 amino acids. In contrast, mutations in the APP that reduce the production of β-amyloid protect against the development of AD and are associated with preserved cognition in the elderly. The most common cause of NFTs is AD, but the precise molecular events that produce tangles are unknown. Mutations in the MAPT gene encoding tau stimulate NFT formation in familial frontotemporal dementia, inherited progressive supranuclear palsy, and other familial tauopathies. Like AD, the major­ ity of most tauopathies as well as PD are sporadic. The second most common neurodegenerative disease is PD. The saga of α-synuclein and PD begins in 1996 with the identification of a mutation in a family of Greek descent. With this family and others, there were sufficient patients to establish genetic linkage. Soon there­ after, immunostaining showed that α-synuclein was present in Lewy bodies, and the following year staining of glial cytoplasmic inclusions (GCIs) was identified in the brains of deceased multiple-system atro­ phy (MSA) patients. Subsequently, brains from deceased MSA patients transmitted the disease to transgenic mice, establishing that the α-synucleinopathies are prion diseases. Before the α-synuclein (SCNA) gene was found to cause familial PD, other genes such as the leucine-rich repeat kinase 2 (LRRK2) were found to modify the onset of PD; other similar PD modifier genes include parkin, PINK1, and DJ-1. PINK1 is a mitochondrial kinase (see below), and DJ-1 is a protein involved in protection from oxidative stress. Parkin, which causes autosomal reces­ sive early-onset PD-like illness, is a ubiquitin ligase. The characteristic histopathologic feature of PD is the Lewy body, an eosinophilic cyto­ plasmic inclusion that contains both neurofilaments and α-synuclein. Huntington’s disease (HD) and cerebellar degenerations are associated with expansions of polyglutamine repeats in proteins, which aggregate to produce neuronal intranuclear inclusions. Familial ALS is associated with superoxide dismutase (SOD1) mutations and cytoplasmic inclu­ sions containing superoxide dismutase. An important finding was the discovery that ubiquitinated inclusions observed in most cases of ALS and the most common form of frontotemporal dementia are composed of TAR DNA-binding protein 43 (TDP-43). Subsequently, mutations in the TDP-43 gene, and in the fused in sarcoma gene (FUS), were found in familial ALS. Both of these proteins are involved in transcription regulation as well as RNA metabolism. PART 13 Neurologic Disorders Another key mechanism linked to cell death is mitochondrial dynamics, which refers to the processes involved in movement of mitochondria, as well as in mitochondrial fission and fusion, which play a critical role in mitochondrial turnover and in replenishment of damaged mitochondria. Mitochondrial dysfunction is strongly linked to the pathogenesis of a number of neurodegenerative diseases such as Friedreich’s ataxia, which is caused by mutations in an iron-binding protein that plays an important role in transferring iron to iron-sulfur clusters in aconitase and complex I and II of the electron transport chain. Mitochondrial fission is dependent on the dynamin-related pro­ teins (Drp1), which bind to its receptor Fis, whereas mitofusins 1 and 2 (MFN 1/2) and optic atrophy protein 1 (OPA1) are responsible for fusion of the outer and inner mitochondrial membrane, respectively. Mutations in MFN2 cause Charcot-Marie-Tooth neuropathy type 2A, and mutations in OPA1 cause autosomal dominant optic atrophy. Both β-amyloid and mutant huntingtin protein induce mitochondrial frag­ mentation and neuronal cell death associated with increased activity of Drp1. In addition, mutations in genes causing autosomal recessive PD, parkin and PINK1, cause abnormal mitochondrial morphology and result in impairment of the ability of the cell to remove damaged mitochondria by autophagy. As noted above, one major scientific question is whether protein aggregates directly contribute to neuronal death or whether they are merely secondary bystanders. A focus in all the neurodegenerative diseases is on small-protein aggregates termed oligomers. How many

monomers polymerize into a particular disease-specific oligomer has been elusive. Whether oligomers are the toxic species of β-amyloid, α-synuclein, or proteins with expanded polyglutamines such as the one causing HD remains to be established. Protein aggregates are usually ubiquitinated, which targets them for degradation by the 26S com­ ponent of the proteasome. An inability to degrade protein aggregates could lead to cellular dysfunction, impaired axonal transport, and cell death by apoptotic mechanisms. Autophagy is the degradation of cystolic components in lysosomes. There is increasing evidence that autophagy plays an important role in degradation of protein aggregates in the neurodegenerative diseases, and it is impaired in AD, PD, FTD, and HD. Autophagy is particularly important to the health of neurons, and failure of autophagy contrib­ utes to cell death. In HD, a failure of cargo recognition occurs, contrib­ uting to protein aggregates and cell death. There is other evidence for lysosomal dysfunction and impaired autophagy in PD. Mutations in glucocerebrosidase (GBA) are associ­ ated with 5% of all PD cases as well as 8–9% of patients with dementia with Lewy bodies. Notably, glucocerebrosidase and enzymatic activity are reduced in the substantia nigra of sporadic PD patients. α-Synuclein is degraded by chaperone-mediated and macro autophagy. The degra­ dation of α-synuclein has been shown to be impaired in transgenic mice deficient in glucocerebrosidase, and α-synuclein inhibits the activity of glucocerebrosidase; thus, there appears to be bidirectional feedback between α-synuclein and glucocerebrosidase. The retromer complex is a conserved membrane-associated protein complex that functions in endosome to Golgi transport. The retromer complex contains a cargo selective complex consisting of VPS35, VPS26, and VPS29, along with a sorting nexin dimer. Mutations in VPS35 were shown to be a cause of late-onset autosomal dominant PD. The retromer also traffics APP away from endosomes, where it is cleaved to generate β-amyloid. Deficiencies of VPS35 and VPS26 were also identified in hippocampal brain tissue from AD. A potential thera­ peutic approach to these diseases might therefore be to use chaperones to stabilize the retromer and reduce the generation of β-amyloid and α-synuclein. PRIONS AND NEURODEGENERATIVE DISEASES As we have learned more about the etiology and pathogenesis of the neurodegenerative diseases, it has become clear that the histologic abnormalities that were once curiosities, in fact, are likely to reflect the etiologies. For example, the amyloid plaques in kuru and CreutzfeldtJakob disease (CJD) are filled with the PrPSc prions that have assembled into fibrils. The past three decades have witnessed an explosion of new knowledge about prions. For many years, kuru, CJD, and scrapie of sheep were thought to be caused by slow-acting viruses, but a large body of experimental evidence argues that the infectious pathogens causing these diseases are devoid of nucleic acid. Such pathogens are called prions, which are composed of host-encoded proteins that adopt alternative conformations that undergo self-propagation. Prions impose their conformations on the normal, precursor proteins, which in turn become self-templating, resulting in faithful copies; most prions are enriched for β-sheet and can assemble into amyloid fibrils. Similar to the plaques in kuru and CJD that are composed of PrP prions, the amyloid plaques in AD are filled with Aβ prions that have polymerized into fibrils. This relationship between the neuropatho­ logic findings and the etiologic prion was strengthened by the genetic linkage between familial CJD and mutations in the PrP gene, as well as (as noted above) between familial AD and mutations in the APP gene. Moreover, a mutation in the APP gene that prevents Aβ peptide formation was correlated with a decreased incidence of AD in Iceland. The heritable neurodegenerative diseases offer an important insight into the pathogenesis of the more common sporadic ones. Although the mutant proteins that cause these disorders are expressed in the brains of people early in life, the diseases do not occur for many decades. Many explanations for the late onset of familial neurodegen­ erative diseases have been offered, but none is supported by substantial experimental evidence. The late onset might be due to a second event

TABLE 435-1  Prion-Based Classification of Neurodegenerative Diseases CAUSATIVE PRION PROTEINS NEURODEGENERATIVE DISEASE Creutzfeldt-Jakob disease (CJD) Kuru Gerstmann-Sträussler-Scheinker disease (GSS) Fatal insomnia Bovine spongiform encephalopathy (BSE) Scrapie Chronic wasting disease (CWD) Feline spongiform encephalopathy Transmissible mink encephalopathy PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc PrPSc Alzheimer’s disease (AD) Down syndrome ALS-parkinsonism dementia complex (PDC) of Guam Aβ → tau Aβ → tau Aβ → tau Parkinson’s disease (PD) Dementia with Lewy bodies Multiple-system atrophy α-Synuclein α-Synuclein α-Synuclein Frontotemporal dementias (FTDs) Posttraumatic FTD Chronic traumatic encephalopathy (CTE) Tau, TDP43, FUS (C9orf72, progranulin) Tau Amyotrophic lateral sclerosis (ALS) SOD1, TDP43, FUS (C9orf72) Huntington’s disease (HD) Huntingtin in which a mutant protein, after its conversion into a prion, begins to accumulate at some rather advanced age. Such a formulation is also consistent with data showing that the protein quality-control mecha­ nisms diminish in efficiency with age. Thus, the prion forms of both wild-type and mutant proteins are likely to be efficiently degraded in younger people but are less well handled in older individuals. This explanation is consistent with the view that neurodegenerative diseases are disorders of the aging nervous system. A new classification for neurodegenerative diseases can be proposed based on not only the traditional phenotypic presentation and neuro­ pathology but also the prion etiology (Table 435-1). An expanding body of experimental data has accumulated connecting prions in each of these illnesses. In addition to kuru and CJD, Gerstmann-SträusslerScheinker disease (GSS) and fatal insomnia in humans are caused by PrPSc prions. In animals, PrPSc prions cause scrapie of sheep and goats, bovine spongiform encephalopathy (BSE), chronic wasting dis­ ease (CWD) of deer and elk, feline spongiform encephalopathy, and transmissible mink encephalopathy (TME). Similar to PrP, Aβ, tau, α-synuclein, superoxide dismutase 1 (SOD1), and possibly huntingtin all adopt alternative conformations that become self-propagating, and thus, each protein can become a prion and be transferred to synapti­ cally connected neurons. Moreover, each of these prions causes a dis­ tinct constellation of neurodegenerative diseases. Evidence for a prion etiology of AD comes from a series of transmis­ sion experiments initially performed in marmosets and subsequently in transgenic mice expressing the mutant APP from which the Aβ peptide is derived (Table 435-1). Synthetic mutant Aβ peptides folded into a β-sheet-rich conformation exhibited prion infectivity in cultured cells. Studies of the tau protein have shown that it not only features in the pathogenesis of AD but also causes the frontotemporal dementias including chronic traumatic encephalopathy, which has been reported in both contact sport athletes and military personnel who have suffered traumatic brain injuries. A series of incisive studies using cultured cells and Tg mice have demonstrated that both tau and Aβ prions are found together in the brains of AD patients. These findings indicated that AD is a double-prion disease (Table 435-1); unexpectedly, two more double-prion diseases have been identified recently. Patients with Down syndrome, from 6–72 years of age, all had both Aβ and tau pri­ ons in their brains with the frequent diagnosis of AD. The third doubleprion disease has been found in the Chamorro people on Guam as well as Japanese living on the Kii peninsula: both groups of people develop ALS with dementia and both have Aβ and tau prions in their brains.

In contrast to Aβ and tau prions, α-synuclein prions cause very dif­ ferent illnesses, i.e., PD, dementia with Lewy bodies (DLB), and MSA. Brains from MSA patients inoculated into Tg(SCNA∗A53T) mice died ~90 days after intracerebral inoculation, whereas mutant α-synuclein (A53T) prions formed spontaneously in Tg mouse brains that killed recipient Tg mice in ~200 days (Table 435-1).

For many years, the most frequently cited argument against prions was the existence of strains that produced distinct clinical presenta­ tions and different patterns of neuropathologic lesions. Some investi­ gators argued that the biologic information carried in different prion strains could be encoded only within a nucleic acid. Subsequently, many studies demonstrated that strain-specified variation is enci­ phered in the conformation of PrPSc, but the molecular mechanisms responsible for the storage of this biologic information remains enig­ matic. The neuroanatomical patterns of prion deposition have been shown to be dependent on the particular strain of prion. Convincing evidence in support of this proposition has been accumulated for PrP, Aβ, tau, and α-synuclein prions. The most persuasive information on prion strains comes from studies in yeast where the tools of yeast genetics allowed inciteful investigations to be performed in ways that could not be accomplished in mammals. CHAPTER 435 Although the number of prions identified in mammals and in fungi continues to expand, the existence of prions in other phylogeny remains undetermined. Some mammalian prions perform vital func­ tions and do not cause disease; such nonpathogenic prions include the cytoplasmic polyadenylation element-binding (CPEB) protein, the mitochondrial antiviral-signaling (MAVS) protein, and T cell– restricted intracellular antigen 1 (TIA-1). Pathobiology of Neurologic Diseases Many but not all prion proteins adopt a β-sheet-rich conforma­ tion and appear to readily oligomerize as this process becomes self-propagating. Control of the self-propagating state of benign mam­ malian prions is less well understood than that of pathogenic mamma­ lian prions, which appear to multiply exponentially. We do not know if prions multiply as monomers or as oligomers; notably, the ionizing radiation target size of PrPSc prions suggests it is a trimer. The oligo­ meric states of pathogenic mammalian prions are thought to be toxic; larger polymers, such as amyloid fibrils, seem to be a mechanism for minimizing toxicity. The development of drugs designed to inhibit the conversion of the normal precursor proteins into prions or to enhance the degradation of prions focuses on the initial step in prion accumulation. Although a dozen drugs that cross the blood-brain barrier have been identified that prolong the lives of mice infected with scrapie prions, none has been identified that extends the lives of Tg mice that replicate human CJD prions. Despite doubling or tripling the length of incubation times in mice inoculated with scrapie prions, all of the mice eventually succumb to illness. Because all of the treated mice develop neurologic dysfunction at the same time, the mutation rate as judged by drug resis­ tance is likely to approach 100%, which is much higher than mutation rates recorded for bacteria and viruses. Mutations in prions seem likely to represent conformational variants that are selected for in mammals where survival becomes limited by the fastest-replicating prions. The results of these studies make it likely that cocktails of drugs that attack a variety of prion conformers will be required for the development of effective therapeutics. NEURAL STEM CELL BIOLOGY Normal and genetically modified (“transgenic”) mice are the most widely used model systems to study features of human nervous system diseases. However, modeling genetic diseases in rodents is limited to the relatively small number of monogenic human diseases where the specific gene mutations are known and is further limited by species differences. The latter can be particularly important in brain regions such as the cerebral cortex that have undergone significant evolutionary expansion in humans. These shortcomings, which likely contribute to the low probability that therapeutic efficacy translates from animal models to humans, can potentially be overcome through stem cell models that enable the use of human cells and tissues to model human diseases. The advent of new stem cell technologies

is transforming our understanding of the pathobiology of human neurologic diseases. Stem cell platforms are being used to screen for therapeutic agents, to uncover adverse drug effects, and to discover novel therapeutic targets.

Among the most exciting recent advances in stem cell technology is the ability to convert somatic cells, either skin fibroblasts or blood cells, into pluripotent stem cells known as induced pluripotent stem cells (iPSCs). This technology has introduced an entirely new and powerful approach to study the pathobiology of heritable diseases. Pluripotent stem cells can be easily obtained through minimally inva­ sive procedures such as a skin biopsy or blood sample and converted to pluripotency through application of a cocktail of reprogramming factors to create iPSCs. Initially, a set of four programming factors, Oct3/4, Klf-4, Sox2, and c-Myc, was delivered to cells using lenti­ viruses that stably integrated the reprogramming factor genes into the iPSC genome, potentially altering disease phenotypes and also abrogating expression of native genes at the DNA sites where the factors integrated. Newer techniques have been developed that use nonintegrating approaches such as through the use of Sendai virus, messenger RNA (mRNA), or episomal vectors that circumvent these problems. Once created, iPSC lines can be expanded indefinitely to produce a limitless supply of stem cells. These cells are the starting material for the derivation of specific cell types based on protocols that use small molecules, proteins, or direct gene induction to recapitulate developmental programs. Most current protocols derive neuronal progenitors through dual-SMAD inhibition, a step that involves the use of small-molecule inhibitors to block endoderm and mesodermal cell fates, thereby creating neural cells by default. Multiple protocols have been developed over the last decade for creating large numbers of human neuron progenitor cell types and directing them toward specific nervous system cell fates, including neuron subtypes from multiple regions of brain and spinal cord as well as retinal cells, glial cells including astrocytes and oligodendrocytes, immune cells, and peripheral nervous system cells. PART 13 Neurologic Disorders The primary medical benefit of iPSC technology is that it enables the creation of patient-specific cells or tissues that are genetically matched to individual subjects. This approach enables the study of not only monogenetic disorders but also sporadic forms of disease and complex polygenic disorders including those with unidentified risk loci. Furthermore, by deriving iPSC cell lines from multiple patients, it is possible to explore how disease phenotypes may vary accord­ ing to genetic background. Another approach that has been used to generate specific neuron and glial cell types from somatic cells such as fibroblasts is through direct reprogramming. This approach relies on a cocktail of specific transcription factors to directly convert somatic cells into the alternate desired cell type. This approach bypasses the epigenetic reset that accompanies cells as they are reprogrammed to a pluripotent state. The advantage of this approach is that age-related epigenetic signatures are not erased, so that derived neurons may more readily reflect diseases that manifest in older cells. Despite the advantages of using in vitro models of nervous system diseases derived from patient-specific iPSCs, several potential road­ blocks remain. There are no standard reprogramming or derivation protocols, and the different methods can result in considerable vari­ ability in the disease phenotypes reported by different laboratories. Confidence in the specificity of a particular phenotype is therefore increased if it has been validated across multiple laboratories. There is also the problem of inherent variability between patient lines that may result from their different genetic backgrounds. One solution, available only in the case of monogenic disorders, is to use isogenic controls generated using gene editing, such as with CRISPR-Cas9 technology, to create disease and control lines on an identical genetic background. However, because differences in genetic background can influence the penetrance of a particular trait, it will still be necessary to compare dis­ ease lines from multiple patients to discern a true disease phenotype. For polygenic disorders where the causative mutations are unknown, it will not be possible to create isogenic controls, and in these situations, the best strategy for improving reliability and sensitivity is to compare lines from multiple patients.

ORGANOIDS Most nervous system disorders, including ASD, schizophrenia, PD, AD, and ALS, are complex disorders, resulting from an unknown combi­ nation of gene mutations, and manifest not only in specific cell types but also in alterations of the local tissue environment. These disorders are difficult to model in animals, but they are approachable using three-dimensional human iPSC stem cell models, often referred to as “organoids.” Organoids are derived from pluripotent stem cells that are directed along a tissue-specific lineage through the timed application of growth factors, genes, or small-molecule activators or inhibitors, and allowed to aggregate into three-dimensional structures. With time, cell intrinsic programs are spontaneously engaged and the cel­ lular aggregates begin to self-organize and develop into structures that recapitulate the complex topographical and cellular diversity of normal organ development. In this way, it has been possible to create, at least in part, in vitro brainlike organoids that resemble parts of the human brain at early stages of development. When allowed to develop from an anterior neural tube stage, these structures can become heterogeneous, containing regions with forebrain, midbrain, and/or hindbrain identity and can often include retina-like structures. The high degree of vari­ ability in such “whole-brain organoids” can be a liability for controlled studies and can be reduced by the use of directed protocols that restrict outcomes to more defined brain regions, such as forebrain, cortex, or ganglionic eminence. A variety of protocols have now been developed to generate organoids with specific regional identity, and fusing organoids of different regional identity with each other has been used to reproduce cellular interactions such as neuronal migration across regions. Many protocols are focused on modeling cortical development, and they can reproduce developmental features including a diversity of progenitor and neuronal cell types topographically distributed within ventricular and subventricular progenitor regions and rudimentary cortical layers. However, the organoids follow a human developmental timetable and still remain at stages roughly comparable to late fetal development even after 6–9 months of culture. Moreover, they lack key cell types such as endothelial cells, pericytes, and microglia, and have few if any astrocytes or oligodendrocytes. Recently, it has become possible to derive these cell types independently from stem cells and then combine them with brain organoids to create tissue-diverse brain organoids that also contain, for example, vascular or immune cells. Nonetheless, while still only reflect­ ing rudimentary organizational and compositional features, organoids have become attractive models to study human brain development and the pathophysiology of human nervous system diseases in the context of a partially organized brainlike structure. ■ ■BRAIN DEVELOPMENT AND DEVELOPMENTAL DISORDERS: MICROCEPHALY AND LISSENCEPHALY Transcriptional analysis has suggested that the neurons produced by most stem cell protocols resemble early- to mid-gestational stages of human brain development. The immaturity of stem cell–derived human neurons may limit their utility for modeling adult diseases, but it does make them ideally suited for the study of brain development and the pathophysiology of neurodevelopmental disorders. Primary autosomal recessive microcephaly (MCPH) is a rare neu­ rodevelopmental disorder producing severe microcephaly with simpli­ fied cortical gyration and intellectual disability. MCPH was one of the first disorders to be studied using cerebral organoids. Mutations in genes encoding microtubule spindle components and spindle-associ­ ated proteins are the most frequent causes of congenital microcephaly. Among them is cyclin-dependent kinase 5 related activator protein 2 (CDK5RAP2). Skin fibroblasts derived from a single microcephalic patient carrying a mutation in CDK5RAP2 were used to generate four iPSC lines. Cerebral organoids grown from these cell lines contained fewer proliferating progenitor cells and showed premature neural dif­ ferentiation compared to wild-type controls. Introducing functional CDK5RAP2 by electroporation partially rescued the disease pheno­ type, supporting the notion that failure of the founder population of neural progenitors to properly expand underlies the smaller brain. This study demonstrated that brain organoids derived from patients with microcephaly can be used to reproduce features of the disease but

did not reveal new insights or disease features of CDK5RAP2 micro­ cephaly that had not already been described in mouse models. Organoids, however, are well-suited to model human microcephaly because the phenotype manifests at early prenatal ages and can be assessed by organoid size, which can serve as a proxy for a micro­ cephaly phenotype. For example, a loss-of-function screen of candi­ date microcephaly human genes was carried out in organoids using CRISPR-lineage tracing; 173 potential microcephaly genes were tested, and novel mechanisms involved in brain size control were discovered including a pathway involved in endoplasmic reticulum function and extracellular matrix production. Cortical organoids have also been used to model lissencephaly or “smooth brain.” Miller-Dieker syndrome (MDS), a severe congenital form of lissencephaly, was modeled in organoids and features of the human disease were observed that had not been noted in murine models. Classical lissencephaly is a genetic neurologic disorder associ­ ated with intellectual disability and intractable epilepsy, and MDS is a severe form of the disorder. Cortical folding in humans begins toward the end of the second trimester, but gyrencephaly depends upon earlier events such as neural progenitor cell proliferation and neuronal migra­ tion, which can be modeled in organoids. The human organoid model of MDS exhibited several neural progenitor cell phenotypes that had already been reported in mouse models, including altered mitotic spin­ dle orientation and neuronal migration defects. But the organoids also displayed a mitotic defect in a specific neural stem cell subtype, the outer radial glia cell (oRG), that had not been observed in mice. oRG cells are enriched in the outer subventricular zone, a proliferative region that is large in primates and not present in rodents. These cells are particularly numerous in the developing human cortex and are thought to underlie the developmental and evolutionary expansion of the human cortex. oRG cells from MDS patients behaved abnormally and had arrested or delayed mitoses. MDS organoids also identified noncell autonomous defects in Wnt signaling as an underlying mechanism. These insights into mechanistic and cell-type-specific features of human disease high­ light how organoid technology can provide new and valuable perspec­ tives on the pathophysiology of disorders of in utero development. ■ ■ACQUIRED NEURODEVELOPMENTAL DISORDERS: ZIKA The outbreak of Zika virus (ZIKV) and associated microcephaly cases in the Americas provided a test case for the utility of brain organoids to model acquired human microcephaly. Despite a correlation between Zika infection rates and the incidence of congenital microcephaly, compelling evidence that ZIKV caused microcephaly was lacking in the early phases of the epidemic. The causal link between ZIKV and congenital microcephaly was buttressed by two studies in 2016 that used human iPSC-derived neural progenitor cells and organoids to demonstrate ZIKV tropism for human neural progenitor cells. Neu­ ral  progenitor cells (radial glia) were readily infected in vitro with subsequent progenitor cell death and involution of organoid size. Fore­ brain organoids were further used to highlight the role of the flavivirus entry factor, AXL, in determining viral tropism, and were also used to explore the disease mechanism by demonstrating upregulation of the innate immune receptor toll-like receptor 3 (TLR) in response to ZIKV infection. Stem cell–derived models of human brain development have also demonstrated centrosomal abnormalities in radial glia and altera­ tion in the cleavage plane of mitotic radial glia associated with prema­ ture neural differentiation. Mouse models are also being used to study the pathophysiology of congenital ZIKV syndrome, but the availability of unlimited numbers of human neural cells produced using stem cell technology has enabled high-throughput screening assays to test librar­ ies of clinically approved compounds for potential therapeutic agents. This strategy has already highlighted several compounds that could potentially help protect against ZIKV microcephaly. ■ ■NEURODEVELOPMENTAL DISORDERS:

AUTISM AND SCHIZOPHRENIA ASDs are complex and heterogeneous neurodevelopmental disorders usually manifesting in childhood with difficulties in social interaction, verbal and nonverbal communication, and repetitive behaviors. The

cellular and molecular mechanisms underlying ASD are thought to arise at stages of fetal brain development, making them well-suited for exploration using human iPSC-derived disease models. iPSC-derived neurons have been used to study the pathophysiology of disorders associated with ASD that are caused by monogenic mutations, includ­ ing fragile X, Rett, and Timothy syndromes.

Fragile X is the most common heritable cause of intellectual dis­ ability, affecting 1 in 4000 males and 1 in 8000 females, and is a lead­ ing genetic cause of ASD. Patients also have speech delay, growth and motor abnormalities, hyperactivity, and anxiety. The causative muta­ tion lies in the FMR1 gene and produces a CGG triplet repeat expan­ sion from a normal number of 5–20 to >200, leading to epigenetic silencing of the FMR1 gene and loss of the fragile X mental retardation protein. The epigenetic mechanism means that unlike a simple gene deletion that would lead to ubiquitous loss of expression, the FMR1 locus becomes hypermethylated and epigenetically silenced during differentiation; thus, FMR1 protein is expressed by the early embryo and becomes absent only around the beginning of the second trimester. Interestingly, this expression pattern is recapitulated during cellular differentiation in stem cell models. Pluripotent fragile X stem cell lines have been derived from embryos identified through preimplantation genetic diagnosis and by reprogramming skin fibroblasts from fragile X patients to create iPSC lines. In both cases, FMR1 was expressed by the pluripotent stem cells but underwent transcriptional silencing fol­ lowing differentiation. Fragile X stem cell lines can therefore be used to study the mechanism of FMR1 silencing, an effort that is ongoing. Neu­ rons generated from fragile X iPSC cells reproduce features observed in neurons from transgenic FMR1 mouse models and patients, including stunted neurites with decreased branching, increasing confidence in the iPSC model. In addition to providing a model that can be used to study disease pathogenesis, fragile X iPSC-derived neurons could be used to screen for potential therapeutic agents or gene-editing strate­ gies to remove the repressive epigenetic marks induced by the mutation and rescue the phenotype. CHAPTER 435 Pathobiology of Neurologic Diseases Rett syndrome is an X-linked neurodevelopmental disorder with dominant inheritance caused by a mutation in the MECP2 gene. Because males carrying one copy of the defective gene usually die in infancy, most patients are girls. Random inactivation of the X chromo­ some in girls results in mosaic cellular expression of the mutation that circumvents fatality and produces a variable phenotype. The symptoms are present in early childhood and include microcephaly associated with developmental delay, autistic-like behaviors and cognitive dys­ function, seizures, and repetitive motor actions; these then progress to include difficulties with gait, swallowing, and breathing before usually stabilizing with patients surviving to adulthood. The pathophysiology of Rett syndrome is presumed to involve abnormal epigenetic regula­ tion leading to decreased transcriptional repression of genes whose overexpression produces the disease phenotype, although this concept has been contested. In one of the first studies to use iPSC modeling to study Rett syndrome, it was discovered that when fibroblasts from patients were reprogrammed to pluripotent stem cells, X inactiva­ tion was erased. In apparent recapitulation of endogenous events, X chromosome inactivation re-occurred during neuronal differentiation, producing a mosaic of cells carrying the mutant gene intermingled with normal cells. Rett neurons had fewer dendritic spines and syn­ apses, smaller cell bodies, and reduced network activity. Another iPSC model of Rett syndrome highlighted the potential role of altered inhibitory function. Rett neurons were found to have a deficit of potas­ sium/chloride cotransporter (KCC2) that is developmentally regulated and normally leads to a switch in GABA signaling from excitatory at embryonic ages to inhibitory by birth. In Rett neurons, KCC2 expres­ sion level was low, and the functional switch in GABA effects was delayed, contributing to some of the disease features and possibly accounting for the developmental onset of the disease. One curious feature of some iPSC Rett lines was that despite the mosaic expression of the mutation, disease phenotypes were observed in all cells. Possibly, this could reflect a noncell autonomous effect, but as in all iPSC disease models, confidence in disease-specific features will be increased when similar phenotypes are seen across multiple independent studies.

Timothy syndrome, another severe neurodevelopmental disease associated with ASD, has been modeled using iPSC-derived organoids. Timothy syndrome is caused by a mutation in the CACNA1C gene cod­ ing for a voltage-gated calcium channel, and neuron defects in Timothy syndrome organoids were rescued by selectively altering calcium chan­ nel activity. In one study, two separate organoids were produced with different regional identity, one represented neocortex and one a more ventral structure known as the medial ganglionic eminence, which is the source of most cortical interneurons. The two organoids were then fused together to allow the interneurons to migrate into the cortex, mimicking their endogenous behavior. The ability to model interneu­ ron migration led to the discovery of a cell-autonomous migration defect in the disease-carrying neurons.

The majority of nervous system diseases, including ASD, are poly­ genic and cannot be modeled in animals but can be modeled using patient-derived iPSCs. For example, a subset of patients with ASD have large head size, and a cohort of patients with this phenotype was used to generate iPSCs that were converted to neural progenitor cells and forebrain neurons. The progenitors had an accelerated cell cycle and produced an excess of inhibitory interneurons and had exuber­ ant cellular overgrowth of neurites and synapses. This last feature is in contrast to the decrease in spines and synapses observed in other iPSC models of ASD such as fragile X and Rett syndrome and under­ scores the need for replication and validation of purported disease phenotypes given the high variability based on differences between stem cell lines, protocols, patient genetic background, and other fac­ tors. Moreover, the clinical features of most neuropsychiatric diseases reflect disorders in processes such as circuit formation and refinement that occur after birth and may be difficult to capture at the fetal stage of development reflected in stem cell models. PART 13 Neurologic Disorders Patient stem cells have also been used by multiple groups to study the pathophysiology of schizophrenia, producing a variety of diverse and sometimes contradictory results. Reports claim obvious pheno­ types such as disruptions in the adherens junctions of forebrain radial glia or aberrant neuronal migration, although such gross abnormalities observed at the equivalent of in utero stages of development seem very unlikely to underlie a disease that usually manifests at adolescence or young adulthood. Other studies report abnormalities related to abnor­ mal microRNA expression, disordered cyclic AMP and Wnt signaling, abnormal stress responses, diminished neuronal connectivity, fewer neuronal processes, problems with neuronal differentiation, and mito­ chondrial abnormalities, among others. While the pathophysiology of as complex a neurodevelopmental disorder as schizophrenia may be multidimensional, it is unclear which, if any, of the reported findings in iPSC models reflect the true pathology of schizophrenia. Progress will likely depend on the adoption of more standard and reproducible pro­ tocols, more rigorous identification of cell types, markers of regional identity, and indicators of maturity. ■ ■ALZHEIMER’S DISEASE As noted above, the leading concept of AD pathogenesis, the amyloid hypothesis, suggests that an imbalance between production and clear­ ance of β-amyloid leads to excessive accumulation of β-amyloid peptide and the formation of NFTs within neurons, composed of aggregated hyperphosphorylated tau proteins. Additionally, aggregates of amyloid fibrils are deposited outside neurons in the form of neuritic plaques. Among the causes of familial AD are mutations in genes involved in β-amyloid production, including amyloid precursor protein (APP) and presenilin 1 and 2. Shortly after the introduction of iPSC technology, human stem cell–derived neurons were generated from patients carry­ ing mutations in AD-causative genes as well as from sporadic AD cases. The disease neurons developed hallmarks of AD including intracel­ lular accumulation of β-amyloid and phosphorylated tau, as well as secretion of APP cleavage products, features that could be reduced by adding β- or γ-secretase inhibitors or β-amyloid-specific antibodies. The neurons also demonstrated other disease features observed in postmortem AD tissues. However, extracellular β-amyloid aggregation and NFTs were not robustly modeled in these two-dimensional sys­ tems, presumably because secreted factors were able to readily diffuse

away. The use of three-dimensional organoids to model AD overcame this limitation, presumably by recreating a more faithful extracellular matrix. Organoid models promoted the aggregation of β-amyloid, and more readily recapitulated the pathologic features of AD, including the formation of NFTs and neuritic plaques. It is hoped that the new stem cell models, particularly organoid models, will accelerate our understanding of AD by enabling the study of human disease-carrying cells in a quasi in situ setting. These new models may lead to discovery of novel druggable targets and new diag­ nostic and prognostic biomarkers. One concern is that the pathogenic features of AD usually appear in the sixth or seventh decade of life and progress slowly over years, while most protocols for the deriva­ tion of human cortical neurons generate cells over weeks or months and most remain comparable to immature neurons at fetal stages of development. Nonetheless, these young cells have been used to model neurodegenerative diseases such as AD and HD that strike patients in middle to late adulthood. Possibly the onset of disease phenotype is accelerated in stem cell models due to increased cellular stress, which appears to be a feature of stem cell culture, or disease features may actually have a subtle onset at earlier stages than generally suspected. Indeed, 3-year-old children at genetic risk of developing early-onset AD appear to have smaller hippocampal size and lower scores on memory tests than children in a nonrisk group. The phenotypes of adult neurodegenerative diseases that are visible at fetal stages may or may not correspond to those manifest at later, adult stages, but they may offer the possibility of devising preventative strategies effective at very early stages of the disease. ■ ■CELL TYPE DISORDERS: ALS AND

HUNTINGTON’S DISEASE In diseases such as ALS, PD, and HD, that mostly target specific neuron subtypes, stem cells provide an ideal means to study the vulnerable human cell populations. By enabling the production of unlimited num­ bers of normal and diseased human midbrain dopaminergic neurons for the study of PD, medium spiny striatal neurons for HD, and spinal and cortical motor neurons for ALS, iPSC approaches have the poten­ tial to transform our understanding and management of these diseases. Stem cell–derived neurons serve as platforms to explore mechanisms of cell vulnerability, to screen drugs for neural protection, and potentially to derive neurons for replacement therapy. ■ ■AMYOTROPHIC LATERAL SCLEROSIS One of the first protocols for producing neurons of a specific subtype from embryonic stem cells recapitulated normal developmental pro­ grams to generate mouse spinal motor neurons. Pluripotent mouse stem cells underwent neural induction and adopted a caudal identity through the application of retinoic acid, and subsequently adopted motor neuron fate through the action of Sonic hedgehog (Shh), a ven­ tralizing factor. Generating human motor neurons proved more com­ plex, requiring additional steps, such as early exposure to the growth factor, FGF2. The first application of stem cell–derived motor neurons to study ALS involved the use of mouse motor neurons generated from transgenic mice expressing a mutation in the superoxide dismutase 1 (SOD1) gene, the most common mutation responsible for familial ALS. Only 5–10% of ALS cases are familial, but the known mutations provide a useful entry point to tease apart the causative pathophysiol­ ogy. Mutations in SOD1 produce ALS through a toxic gain of function for which the mechanism remains unclear, despite the use of multiple transgenic animal and iPSC models. The use of mouse embryonic stem cell–derived motor neurons, however, demonstrated that toxic factors secreted by SOD1 astrocytes contribute to the death of motor neurons. Interestingly, stem cell–derived interneurons were spared, indicating a specific vulnerability of motor neurons. These findings helped establish the notion that a non-cell-autonomous toxic mecha­ nism contributes to ALS pathogenesis and may ultimately lead to novel treatment strategies. These findings also highlight that modeling the full pathophysiology of ALS may require the reproduction of a complex environment including motor neurons, astrocytes, and possibly addi­ tional cell types such as microglia. A variety of approaches including

co-culture of specific cell types, three-dimensional spinal cord organ­ oids, and microfluidic organ-on-chip models are being explored to achieve a more complete facsimile of spinal cord organization. Similar to other neurologic disorders where a clearly defined phenotype has been observed in human stem cell–derived models, there is hope that drug screening using human disease-expressing cells will identify a potential therapeutic compound. ■ ■HUNTINGTON’S DISEASE HD is caused by an expansion in CAG triplet repeats in the huntingtin gene, which leads to an expanded polyglutamine tract in the hunting­ tin protein. HD is dominantly inherited, with symptoms of cognitive decline and uncontrollable gait and limb motions beginning in the third to fifth decade of life with progression to dementia and death approximately 20 years later. Mutant huntingtin causes a toxic gain of function, with the degree of effect related to the CAG repeat length. For example, a CAG length of 40–60 repeats produces adult-onset HD, whereas repeats of 60 or more produce juvenile-onset disease. Although it has been 25 years since the discovery of this causative mutation, the disease mechanism remains poorly understood. Excess huntingtin protein and protein fragments accumulate in specific subtypes of neu­ rons where they misfold and form aggregates that are visible as cellular inclusions. Affected cells eventually die, possibly as a result of meta­ bolic toxicity. The medium spiny neurons of the striatum are the most vulnerable neurons, spurring ongoing attempts to produce replacement cells derived from stem cells, but neuron loss is widespread including in the cortex, complicating a cell replacement approach for this disease. HD iPSCs have been generated from patients with various CAG repeat lengths, but those from juvenile-onset disease with the longest repeat lengths have been favored as being most likely to express robust disease phenotypes at an early stage. This is particularly important given the immature stage of maturation of stem cell–derived human neurons. This approach has been able to produce disease phenotypes observed in patients including huntingtin protein aggregation, decreased meta­ bolic capacity, increased oxidative stress with mitochondrial fragmen­ tation, and apoptosis enhanced by withdrawal of growth factor support. However, many of these phenotypes were observed in pluripotent cells prior to neural differentiation and in neural progenitors and a broad array of CNS neurons in contrast to the cell type–specific features of the disease. Nonetheless, neurons that assumed striatal fate appear to be more vulnerable to stress and apoptosis than other cell types. As with other iPSC models of nervous system diseases, there have so far been few efforts to validate results in multiple iPSC lines having different genetic backgrounds but with similar CAG repeat lengths. An HD con­ sortium has been formed to address this problem by generating a series of iPSC lines from multiple patients. An alternative strategy to validate disease phenotypes has been to use gene editing to create isogenic iPSC lines that are corrected to produce wild-type control and HD iPSC lines against the same genetic background. FUTURE PERSPECTIVES Despite early successes, it may prove difficult to reconstitute neuro­ degenerative disease conditions in human cells in vitro over a short course of time because the pathogenic changes of degenerative dis­ eases progress slowly and commence in the later stages of life. The differentiation and maturation of human neurons from stem cell lines occur over a span of months, which may not be long enough to establish the aged-brain conditions under which patients develop robust neurodegenerative pathology. Possible manipulation through gene editing or by application of aging-associated stresses, such as DNA-damaging agents or proteasome inhibitors, may accelerate the expression of degenerative phenotypes in human iPSC-derived cellular models. Stem cell–derived organoid models are also ideal platforms to apply methods for cellular-level visualization such as CLARITY and multielectrode recording techniques to better evaluate threedimensional organoid structures and explore early-forming circuits. These applications are only just beginning. Two-dimensional cell cultures are ideal for production and evalua­ tion of large numbers of specific cells of a particular identity but may

not provide the complex extracellular environment necessary to model certain disease processes, such as extracellular protein aggregation. These features can be best modeled using three-dimensional organ­ oids, but current methods do not reproduce all the relevant features of brain tissue. Optimization will be needed to better reproduce the cellular composition of brain, including endothelial cells, astrocytes, microglia, and oligodendrocytes. It may also be necessary to combine different brain regions generated separately, possibly by fusion of tis­ sues such as dorsal cortex, subpallium, thalamus, retina, and others in so-called “assembloids.” One roadblock has been the limited ability to recreate tissues or neurons with regional brain identity, such as hip­ pocampus, thalamus, or cerebellum. However, recently regionalized organoids have been created using innovative microfluidic chambers and the imposition of morphogen gradients such as bone morpho­ genetic proteins (BMPs) and WNTs. More faithful organoid models could also emerge through the application of bioengineered scaffolds, matrices, or perfusion systems that might allow the growth of larger structures, a feature currently limited due to the emergence of a necrotic core when nonperfused organoids exceed a certain size. Of course, not all aspects of mature brain architecture and function will be modeled by these tissue structures, particularly as they generally repre­ sent prenatal stages of development, but perhaps the most precocious events in disease etiology can be captured and investigated, and these may share mechanistic pathways with disease features that manifest at later stages.

CHAPTER 435 Pathobiology of Neurologic Diseases The current excitement surrounding human stem cells has more to do with their promise to improve on animal models of disease, for which their potential appears unlimited, rather than on their use as a source for cell-based therapies, where the potential has thus far been relatively limited. Even without new insights into disease pathogen­ esis, it is likely that iPSC models such as brain organoids will serve as drug-screening platforms for discovery of novel therapeutics and for detection of off-target and toxic effects. The failure of many neu­ rotherapeutic approaches to translate from animal models to clinical practice underscores the need for better predictive models, and stem cell models and brain organoids based on human cells may be ideally suited to bridge this divide. A CURRENT PERSPECTIVE ON NEURAL STEM CELLS IN THE CLINIC The prospect of stem cell therapies to treat diseases or injuries of the nervous system has captured the attention of researchers, clinicians, and the public. The pace of research is usually slow and deliberate, but in the stem cell arena, there has been enormous pressure to accelerate the pace of progress in order to bring cell-based therapies to the clinic. Expectations have been raised, and clinics have already begun offering unproven or dangerous treatments to a public that can be, in some situ­ ations, ill-informed and vulnerable to exploitation. Nonetheless, there has been remarkable progress over the past few years toward legitimate stem cell–based therapies for a number of nervous system disorders. There are multiple clinical trials underway fueling cautious optimism that stem cells will eventually realize the promise of regenerative therapy for at least some currently untreatable or incurable nervous system diseases. ■ ■PARKINSON’S DISEASE Pursuit of a cell-based therapy for PD using fetal-derived midbrain cells has been ongoing for many decades but with disappointing results. Following anecdotal success in a handful of patients who appeared to improve following striatal grafts of fetal midbrain dopaminergic cells, two National Institutes of Health (NIH)-sponsored double-blind con­ trol studies were launched in the 1990s. However, only a small number of younger patients showed some benefit, and several patients devel­ oped spontaneous dyskinetic movements related to the therapy. These efforts constituted a failed trial as the treated patients who did not experience side effects failed to improve significantly. However, tech­ niques to extract dopaminergic cells from fetal tissue have improved, and on the basis of encouraging results in individual transplanted patients, some of whom have managed to go off their Parkinson’s

medication, a new trial of fetal cell transplantation for PD was con­ ducted in Europe. Unfortunately, enrollment was unexpectedly slow and the trial was terminated after several years without reaching the anticipated number of patients. There was no clear clinical improve­ ment among the treated patients, possibly due to difficulty harvesting sufficient fetal cells to reach the targeted number per patient. Despite the disappointing clinical outcome, enthusiasm remains high for a bet­ ter outcome with stem cell–derived dopamine neurons where, among other differences, cell number is not a limitation.

The dyskinesias that curtailed the NIH trials in the 1990s were eventually ascribed to an abundance of serotonergic neurons that were inadvertently included in some of the cell grafts. Protocols for deriving dopaminergic neurons from stem cells would presumably avoid this complication by providing a more purified cell population, and sev­ eral groups around the world have been aggressively pursuing a stem cell–based approach to PD. In 2018, researchers from Kyoto University in Japan started a phase 1/2 clinical trial to treat PD using stem cells. The investigators chose to use iPSCs derived from a healthy person who had the most common HLA haplotype in Japan. The iPSCs were used to make dopamine-secreting neurons. Seven patients have had the reprogrammed stem cells surgically delivered into the brain and have been followed for two years postinjection to assess safety and pos­ sible efficacy. The U.S. Food and Drug Administration (FDA) recently approved the first clinical trial of a stem cell–derived dopamine neuron for the treatment of PD in the United States. These cells, derived from an embryonic stem cell line, have been delivered to 10 patients in a phase 1 clinical trial to assess safety, tolerability, and preliminary effi­ cacy. A European trial of embryonic stem cell–derived dopaminergic neurons led by scientists in Sweden and the United Kingdom began in 2023. Four patients given a low dose experienced no acute adverse effects, and four more will be treated with a higher dose, and all will be followed for 36 months to evaluate safety and tolerability. PART 13 Neurologic Disorders A clinical trial using iPSC-generated dopaminergic neurons to treat patients with PD was begun in 2018 in Japan. The cell line was derived from a healthy individual with the most common HLA hap­ lotype in the Japanese population. The clinical product consisted of 80% dopamine neuron progenitor cells and a population of glial cells. Because the cell line was genetically altered, the karyotype, plasmid survival, and genomic and epigenomic abnormalities were evaluated and known carcinogenic gene mutations were screened. A single-arm, nonrandomized and open-phase 1/2 study was begun in 2018 with ~5 million cells transplanted stereotactically to the putamen bilaterally. Seven patients have been treated thus far. ■ ■BATTEN’S DISEASE AND PELIZAEUSMERZBACHER DISEASE One of the first cell-based clinical trials for a neurologic disease tar­ geted patients suffering from an untreatable childhood disorder, Batten disease. Batten disease is an autosomal recessive metabolic disorder resulting from an inability to synthesize a lysosomal enzyme critical to brain function. The phase 1 trial involved six patients with infantile and late-infantile forms of the disease who received neural stem cells rather than any specific postmitotic cell type. Neural stem cells derived from donated fetal tissue were expanded in vitro prior to surgical graft­ ing into the brain. This approach was not without risk, as the neural stem cells were proliferating and could potentially form an abnormal growth. The rationale was that the cells would be capable of synthesiz­ ing and secreting the missing lysosomal enzyme and would therefore serve as a delivery device. Animal studies using a transgenic mouse model of Batten disease demonstrated rescue, and this promising result led to a small phase 1 trial. The phase 1 study was considered a success as no adverse events were reported and the cells appeared to be safe, though there was no clinical improvement and no clear evidence of whether the cells had dispersed, transformed into neurons or glia, or indeed survived at all. Despite clearing the phase 1 trial, the company did not pursue further trials for Batten disease, but instead initiated clinical trials using the same cell product for several other indica­ tions, including an inherited fatal dysmyelination syndrome known as Pelizaeus-Merzbacher disease (PMD). The human neural stem cells

have both neurogenic and gliogenic potential, and when delivered to white matter regions in experimental animals, most persisting cells had become oligodendrocytes. This supported use of the cells to promote myelin formation in conditions such as PMD. The company also initi­ ated trials in spinal cord injury. However, the spinal cord trial failed to achieve sufficient benefit in phase 2 and the company ceased its work on stem cell therapies. ■ ■SPINAL CORD INJURY There is a pressing need for novel therapies for spinal cord injury, with

1 million patients worldwide suffering from spinal cord injuries and no effective treatment options. Not surprisingly, there has been intense interest in achieving a stem cell treatment for this condition and dozens of early-stage clinical trials, and anecdotal treatment results have been reported by investigators around the globe. The vast majority have not been blinded controlled trials, but rather individual reports treating a handful of patients, and somewhat surprisingly, most are using mes­ enchymal stem cells (MSCs) or hematopoietic stem cells that normally generate either bone, cartilage, fat, or blood cells. As described below, the rationale for the use of MSCs for neurologic conditions is based on vague and poorly understood mechanisms of action. A series of stem cell trials designed to treat subacute spinal cord injury is underway in the United States and Europe using neural stem cells or their derivatives as potential therapeutic agents. The first to enter clinical trials in the United States was based on a protocol designed to generate oligodendrocytes from pluripotent embryonic stem cells. Evidence of efficacy was obtained in animal models fol­ lowing surgical grafting of cells to sites of spinal cord injury. However, evidence of myelination of host axons was minimal, and other mecha­ nisms were invoked for improvement in gait, including trophic support and immune modulation. Regulatory permission for a phase 1 trial for subacute midthoracic injury was initially stalled by concern over abnormal growths at sites of cell deposit in some animals, but this was satisfactorily addressed and patient trials commenced. However, fol­ lowing a change in leadership, the stem cell program was terminated. The program was acquired by another company that has resumed the spinal cord injury trial and received regulatory approval to advance to include cervical-level injuries. The current phase 1/2a multicenter clinical trial is an open-label, single-arm trial testing three sequential escalating doses administered 21–42 days after injury in 25 patients with subacute severe cervical spinal cord injuries. No adverse events have been reported for 21 patients at 2 years posttreatment. A laterstage comparative clinical trial is now planned to probe for possible efficacy. In 2020, an open-label, single-arm clinical trial for subacute spinal cord injury patients was begun in Japan using iPSC-derived neural stem and progenitor cells injected directly into the damaged spinal cord. The rationale for injecting neuronal precursor cells into the injured spinal cord is not clear as no specific mechanism of action has been demonstrated. Trophic support and neuroimmune modulation have been proposed, and while studies in mice show that newborn neu­ rons can form synapses onto host spinal neurons, how this could lead to improved locomotor or sensory function remains unclear. Results of the trial have yet to be reported. Initial clinical trials for stem cell therapies that address grave medi­ cal conditions such as spinal cord injury often involve small numbers of patients, and larger pivotal trials that can confirm clinical benefit can take a long time to conduct. In a controversial move designed to accelerate the regulatory approval process, in 2014 the Ministry of Health of Japan authorized the use of conditional and time-limited (CTL) approval for regenerative medical products. This designation can be applied when safety has been demonstrated but efficacy has not yet been fully established and enables the manufacturer to market and sell the product. CTL approval converts to full approval if postmar­ keting clinical data demonstrate clinical benefit, but if no convincing clinical benefit can be demonstrated within 7 years, the product must be withdrawn. The effort to bring MSC treatments to patients with currently untreatable neurologic conditions has consequently had a recent boost

in Japan. Autologous bone marrow–derived stem cells were evaluated in an open-label trial involving 13 patients who had experienced severe spinal cord injury 1 month prior to treatment. Mesenchymal stem cells were taken from the patient’s own bone marrow, expanded in number, and delivered back to the patient by intravenous infusion. Six months after treatment, 12 of the patients improved by at least one level on the American Spinal Injury Association impairment scale that ranks muscle contraction and touch, although patients with the same or even greater degree of injury can show spontaneous improvement. As is the case for most MSC therapies, the proposed mechanism of action was quite vague, including reducing inflammation, protecting existing neu­ rons, or replacing damaged neurons. It is also unclear how intravenous delivery could accomplish any of the proposed actions. In 2018, on the basis of the results, unpublished at the time, Japan’s health ministry gave conditional (CTL) approval for the treatment, called Stemirac. This became the first stem cell therapy for spinal cord injury to receive government approval for sale to patients. But the approval of a therapy that may carry risk following a small, unblinded, and uncontrolled study without actual proof of efficacy raised considerable concern among scientists in the stem cell community. Charging patients for such an unproven therapy raises even more ethical concerns. Patients in Japan can now be charged for their treatment while trials to test efficacy are still proceeding. ■ ■AMYOTROPHIC LATERAL SCLEROSIS The possibility of treating ALS by replacing dying motor neurons with stem cell–derived substitutes has excited interest, but this pros­ pect seems very remote. Even if new neurons are able to integrate into spinal cord circuits and become properly innervated, they would have to grow long axons that would take many months to years to project to appropriate targets and attract myelinating Schwann cells. Furthermore, cells would need to be grafted at multiple spinal cord and brainstem levels, and the upper motor neuron deficit would need to be treated by replacing projecting neurons in the motor cortex. An additional complication is the recent finding that spinal motor neurons have unique segmental identity, and replacement cells might need to be generated with a range of molecular identities in order to integrate at multiple spinal levels. This would still leave unaddressed the toxic effects recently shown to be produced in ALS by diseased astrocytes and microglia that could attack the replacement cells. A more tractable near-term solution would be to graft support cells that could rescue or protect endogenous motor neurons from damage. This approach was tried in a mouse model of ALS. Human fetal stem cell–derived neural progenitor cells engineered to express glial cell line–derived neurotrophic factor (GDNF), a growth factor known to provide tro­ phic support for neurons, were grafted to the spinal cord of young ALS mice. The cells dispersed and were able to rescue motor neurons, a very promising result, but disappointingly, the animals became weak and died at the same rate as untreated control animals. ALS is a deadly disease with no known treatment; thus in the hope that patients will respond differently from mice, a phase 1/2a clinical trial based on this approach was approved by the FDA in 2016 and completed in 2019. Patients had the GDNF-producing progenitor cells surgically grafted unilaterally into the lumbar spinal cord. Although disease progression appeared to slow in some treated patients, this was not statistically significant. Of note, several patients developed painful schwannomas, a result of off-targeting deposit of the GDNF-secreting cells close to the dorsal horn root entry zone. Autopsy study of 13 of the 18 treated patients who died within 2 years of treatment showed that the grafted cells had survived. Based on these results, a new trial has begun. With the notion that rescuing lower motor neurons alone may not be suffi­ cient to arrest progression of a disease that affects both upper and lower motor neurons, this trial aims to enroll 16 patients who will have the GDNF-secreting progenitor cells grafted to the motor cortex in an area controlling hand movement. The goal is to try to slow the progression of the upper motor deficit associated with ALS. Among the many MSC-based clinical trials for ALS, two are par­ ticularly notable. Corestem, a stem cell company in South Korea, launched a phase 1 open-label study demonstrating the safety and

feasibility of intrathecal injections of autologous bone marrow–derived MSCs in seven patients with ALS. This was followed by a phase 2 trial that demonstrated safety and efficacy for slowing disease progression. On the basis of these results, Corestem received conditional approval in South Korea in 2014 to market the first stem cell therapy for ALS. By 2021, >300 patients had received this cell treatment. However, full approval is contingent on the results of a randomized, double-blind, placebo-controlled, multicenter phase 3 study, which began in 2021. The trial aims to enroll 115 patients who will receive repeated intrathe­ cal injections of the autologous cells. The goal is to slow or prevent ALS progression and delay death for up to 3 years.

The importance of conducting proper phase 3 clinical trials to deter­ mine therapeutic efficacy in ALS is underscored by the recent experi­ ence of BrainStorm Cell Therapeutics. In 2016, the company reported preliminary positive results for its bone marrow MSC cell therapy in an uncontrolled study of nearly 50 ALS patients. Based on those results, the company launched a phase 3, multicenter, placebo-controlled, ran­ domized, double-blind trial of 189 ALS patients. In 2020, the company reported that there was no significant clinical improvement in the treatment group. Interestingly, despite the failed clinical trial, a public campaign led by ALS patients and advocates called on the FDA to approve the stem cell treatment. The social media response prompted the FDA to take the unusual step of releasing a public statement under­ scoring the lack of efficacy, and in 2023, an FDA advisory committee voted against approval. The company responded by withdrawing its Biologics License Application and announcing a phase 3b trial that will focus on early-stage patients. CHAPTER 435 Pathobiology of Neurologic Diseases ■ ■EPILEPSY A phase 1/2 clinical trial was launched in 2022 using stem cell– derived inhibitory interneurons to treat medically intractable temporal lobe epilepsy. Somatostatin (SST)-expressing inhibitory interneurons, matching the predominant cortical interneuron subtype, were derived from an embryonic stem cell line. Following demonstration of efficacy in a mouse model of temporal lobe epilepsy, 10 patients who had been determined to have a seizure focus in the hippocampus had stem cell– derived interneurons surgically delivered to the epileptic hippocampus. The first five patients received a low dose followed by five patients who received a higher dose. All 10 patients will be followed for 2 years after treatment. As in the PD clinical trials, all patients received antirejec­ tion therapy for the first 12 months. The current standard of care for patients with intractable temporal lobe epilepsy is surgical ablation or resection of the epileptic hippocampus. Surgically treated patients can have seizure remission but often have cognitive decline, particularly if the focus is in the dominant hemisphere. Early indications are that the cell-based therapy can not only reduce seizures but also spare or improve cognitive function. ■ ■MACULAR DEGENERATION Because iPSC technology enables the generation of pluripotent stem cells from adult somatic cells, it has enabled the production of patientspecific autologous cells for cell replacement therapy. Following Shinya Yamanaka’s discovery of iPSCs, the Japanese government has invested in bringing iPSC-derived cell therapy to the clinic. Banks of iPSC lines selected to capture the diversity of HLA haplotypes found in the Japanese population have been produced in the hope that these will allow cell therapies to be matched to individual patient haplotypes in order to avoid immune rejection. While these stem cell banks were still being produced, the first Japanese study to use stem cells was approved in 2013 and involved patients who were to receive customized therapy using cells derived from their own skin fibroblasts. The targeted disease was age-related macular degeneration, a common cause of blindness in the elderly that results from loss of retinal pigment epithelial (RPE) cells. RPE cells are relatively easy to generate from pluripotent stem cells, making replacement therapy an attractive target in this condition. A challenge is to coax the replacement cells to recreate an epithelium in the subretinal space. The Japanese approach involves surgical inser­ tion of a biofilm seeded with RPE cells into the retina. One patient was treated with their own stem cell–derived RPE cells, but prior to treating