3294 PART 13 Neurologic Disorders

Rolling

Activated

lymphocyte

Flow

LFA-1 α4 Integrin

VCAM ICAM

Triggering Strong adhesion

Gelatinases

Antibody

complement

B cell

B cell

B cell

T cell

Antigen

presentation TNFα, LT, and

GM-CSF

Basal lamina

Microglia/macrophages

T cell

activation

Chemokines

and cytokines

TNF, IFN, free radicals,

vasoactive amines,

complement, proteases,

cytokines, eicosanoids

Chemokines

IL-1, IL-12

IFN-γ

IL-2

Heat shock

proteins? Activated

Microglia/

macrophages

Astrocytes

Blood-brain

barrier

endothelium

Fc receptor

Extravasation

CD 31

Myelin damage

Brain tissue

FIGURE 424-1 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.

Promote learning and memory

IL-1α | TNF | C1q

BDNF

Pruning or elimination of synapses

Uptake of aggregated proteins

Phagocytosis of debris

Proinflammatory (A1) astrocyte

FIGURE 424-2 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 secreting cytokines (such as IL-1α, TNF, 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. 417-2). (Reproduced with permission

from J Herz et al: Myeloid cells in the central nervous system. Immunity 46:943, 2017.)

3295Pathobiology of Neurologic Diseases CHAPTER 424

classical complement pathway molecules, including C1q, complement

receptor 3 (CR3), and CR5.

Microglia are located throughout the brain parenchyma, whereas

brain macrophages occur primarily in perivascular regions, including

the meninges and choroid plexus. Brain macrophages are believed to

be 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, large numbers of hematogenously derived monocytes enter

the brain parenchyma. In the EAE model (Fig. 424-1), macrophages

derived from bone marrow monocytes, but not microglia, are the

critical population that initiates inflammatory demyelination at paraxonal regions near nodes of Ranvier. Brain macrophages have multiple

proinflammatory functions, including promoting adhesion, attraction,

and activation of B and T lymphocytes; providing antigen-specific

activation of T cells via antigen presentation of specific 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

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 can

be revealed by single-cell sequencing methods, depending on the specific context and environmental cues.

Evidence also supports a primary role for microglia and brain

macrophages in neurodegenerative diseases, in contrast to earlier

views in which their role was seen as largely secondary and involving

phagocytosis of cell debris. Approximately half of all genes implicated

in genome-wide association studies in AD implicate innate immune

processes and microglia. Under different experimental conditions,

these cells can be either protective or pathogenic. As examples of the

former, macrophages could promote spatial memory in mice when

activated by interleukin (IL)-4 produced by invading lymphocytes.

Also, secretion of BDNF by microglia promoted synaptic plasticity and

improved learning and memory. Microglia and brain macrophages also

promoted clearance of pathogenic β-amyloid aggregates in AD-prone

mice; also, disruption of brain macrophages by knockout of CCR2, a

chemokine required for entry of bloodstream monocytes into the CNS,

exacerbated AD pathology.

On the other hand, disease-worsening effects of microglia and macrophages are likely to predominate in other situations. A direct role for

microglia in human AD was suggested by genetic evidence implicating

the phagocytosis-associated gene TREM2 in AD susceptibility. TREM2

is a microglial receptor that can bind amyloid, induce proliferation

and migration of microglia, and possibly limit the spread of diseaseassociated AD aggregates. Loss-of-function mutations in TREM2

increase AD risk threefold. In a mouse model of AD, overexpression

of TREM2 blocked AD pathology and rescued performance on tests

of learning and memory. A clinical trial testing the value of an agonist

monoclonal antibody against TREM2 is underway.

Other immune system genes implicated in susceptibility to AD and

other late-life dementias also represent promising targets for future

therapy. Activation of the classical complement cascade, noted above,

is assuming an increased role in concepts of pathogenesis, as follows;

synapses targeted for elimination express the complement proteins

C1q and C3, the levels of which increase in the presence of excess

β-amyloid; C3-bearing synapses are then targeted for elimination by

microglia that express the complement 3 receptor (CR3); and knockout

of C3 can rescue the clinical and pathologic abnormalities associated

with neurodegeneration in AD-prone mice.

In familial frontotemporal degeneration (FTD) due to mutations

of progranulin (Chap. 432), a prominent immune pathology has also

been identified, with activated microglia expressing high levels of

proinflammatory cytokines. When progranulin is deleted in mice, an

age-dependent microglial activation phenotype results, associated with

upregulation of complement and other genes associated with innate

immunity, enhanced pruning of inhibitory synapses, and behavioral

manifestations reminiscent of human FTD. Moreover, inhibition of

complement activation can rescue all of these deficits. These data

indicate a primary role for microglial activation in FTD caused by

mutations in progranulin, likely mediated through enhanced lysosomal

trafficking, increased production of C3 complement, and excessive

synaptic pruning in brain regions affected in FTD. Although it is likely

that the specific mechanisms of complement-dependent neurodegeneration will 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.

ASTROCYTES

Astrocytes represent half or more of all cells in the CNS. Traditionally

thought to function as simple interstitial supporting cells that provide scaffolds for neuronal migration and contribute to homeostasis,

emerging data indicate far more pleiotropic functions for this cell type.

Astrocytes, 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

communication 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 maintenance

of neuron health.

One characteristic of the response to many types of brain injury is

reactive astrocytosis, or the formation of a glial scar. Recent work has

highlighted the transcriptional and functional heterogeneity of reactive

astrocytes that, depending on the context, could promote neurotoxicity

or aid in protection and repair. In a model of brain ischemia, reactive

astrocytes promoted tissue repair after injury. By contrast, in other

inflammatory and degenerative states, reactive astrocytes appear to

actively contribute to the injury process. Secreted products of activated

microglia, specifically IL-1a, TNF, and C1q, can induce astrocytes

to transform to a disease-promoting phenotype. Such cells lose the

capacity to phagocytose synapses and myelin debris, and become

toxic in vitro to neurons and mature oligodendrocytes, possibly via

complement-mediated damage. Interestingly, OPCs, abundant in

active lesions of MS (Chap. 444) despite the inflammatory milieu,

are resistant to astrocyte-mediated killing. Reactive astrocytes could

promote damage in disorders as varied as AD (Chap. 431), Parkinson’s

disease (PD) (Chap. 435), and ALS (Chap. 437), despite the distinct

etiologies and pathologies of these conditions.

LYMPHATICS OF THE CENTRAL

NERVOUS SYSTEM

Two recently identified lymphatic structures of the CNS are the glymphatic and deep dural lymphoid systems, responsible for clearance of

debris in the CNS, and likely also serving roles in immune surveillance. 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 concept

termed immune privilege. However, the immune privilege status of the

brain is only relative and not absolute. Also, given the high metabolic

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 granulations, 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

architecture involving lymphoid-like structures and astroglial cells.

3296 PART 13 Neurologic Disorders

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

penetrating 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 perivenous 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 neurodegenerative disease, such as β-amyloid associated with AD

(Chap. 431), 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 antigenspecific stimulation of B cells in MS (Chap. 444).

A second recently identified pathway consists of a plexus of small

lymphatic-like vessels 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 are

comprised of cells that appear to represent a lymphoid drainage system

distinct from vascular endothelium. These sinus-associated lymphoid

structures may be most important in clearing 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 interrelationships are only beginning to be

understood.

MICROBIOTA AND NEUROLOGIC DISEASE

The human microbiome (Chap. 471) represents the collective set of

genes from the 1014 organisms living in our gut, skin, mucosa, and

other sites. For each gene encoded in the human genome, 1000 microbial genes exist within our bodies, and these can encode a wide variety

of molecules that directly or indirectly affect nervous system development, maintenance, and function. Different microbial communities are

associated with different genetic backgrounds, ethnicities, diets, and

environments. In any individual, the predominant gut microbiota can

be remarkably stable over decades, but also can be altered by exposure

to certain microbial species, for example by ingestion of probiotics.

Gut microbes can shape immune responses through the interaction

of their metabolism with that of humans. These gut–brain interactions

are likely to be important in understanding the pathogenesis of many

autoimmune neurologic diseases. For example, mice treated with

broad-spectrum antibiotics are resistant to EAE, an effect associated

with decreases in 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.

Oral administration of polysaccharide A (PSA) from Bacillus fragilis

also protects mice from EAE, via increases in IL-10. 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 promote inflammation (such as Akkermansia muciniphila

and Acinetobacter calcoaceticus) and those that induce regulatory

immune responses (such as Parabacteroides distasonis).

In addition to nonspecific effects on immune homeostasis mediated

by cytokines and regulatory lymphocytes, some microbial proteins

might trigger a cross-reactive immune response against a homologous 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. 445); HLA molecules with A. muciniphila peptides in MS (Chap. 444); the neural

ganglioside Gm1 and similar sialic acid–containing structures from

Campylobacter jejuni in Guillain-Barré syndrome (Chap. 447); and the

sleep-promoting protein hypocretin and hemagglutinin from an H1N1

influenza virus in narcolepsy (Chap. 31).

Microbial genes also encode molecules that can affect development of neurons and glia, and influence myelination and plasticity.

Bacterial-derived short-chain fatty acids, for example, regulate production of brain-derived neurotrophic factor (BDNF). Bacteria also

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 also influence development and activity of the enteric nervous system, which

communicates bidirectionally with the CNS via the vagus nerve that

innervates the upper gut and proximal colon. As these gut–brain relationships become better 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 classic studies demonstrating that products of gut microbes

can worsen hepatic encephalopathy, forming the basis of treatment

with antibiotics 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 nongerm-free 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. Intestinal microbiota were also found to be

required for the normal development and function of brain microglia,

potentially linking these behavioral effects to specific cellular targets in

the CNS. Remarkably, the actions of gut microbial species on microglia

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, associated 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

gastrointestinal disturbances, and the severity of dysbiosis appears

to correlate with the severity of autism. In several murine models of

autism, manipulation of the gut microbiome ameliorated the behavioral abnormalities. 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); remarkably, oral treatment of

offspring with B. fragilis corrected a range of autistic behaviors in these

mice and also improved GI 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 neurodegenerative

diseases, possibly mediated in part 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

3297Pathobiology of Neurologic Diseases CHAPTER 424

(vitamin B3) metabolite; a small preliminary clinical trial of nicotinamide 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 is further strengthened by epidemiologic data

indicating that vagotomy may be protective against PD. In related

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.

PATHOLOGIC PROTEINS, PRIONS, AND

NEURODEGENERATION (FIG. 424-3)

■ PROTEIN AGGREGATION AND CELL DEATH

The term protein aggregation has become widely used to describe easily

recognizable hallmarks of neurodegeneration. While such neuropathologic 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

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

causing 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 is

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 majority 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 thereafter, 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

atrophy (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 recessive early-onset PD-like illness, is a ubiquitin ligase.

The characteristic histopathologic feature of PD is the Lewy body, an

eosinophilic cytoplasmic inclusion that contains both neurofilaments

and α-synuclein. Huntington’s disease (HD) and cerebellar degenerations are associated with expansions of polyglutamine repeats in

Wt prion

form

Mutant

prion

form

Mutant

precursor

Wt

precursor

Age-dependent

mutant prion formation

Inherited NDs

Sporadic NDs

A

A

B

1 2

i ii

3

Prions causing neurodegradation

Aβ plaque

α-Synuclein

Lewy body

Tau tangle

+

+

Amyloid

fibrils

FIGURE 424-3 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 AD, neurofibrillary tangles in AD and other tauopathies, or Lewy bodies in 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. (Reproduced with permission from SB Prusiner: Biology and

genetics of prions causing neurodegeneration. Annu Rev Genet 47:601, 2013.)

3298 PART 13 Neurologic Disorders

proteins, which aggregate to produce neuronal intranuclear inclusions.

Familial ALS is associated with superoxide dismutase (SOD1) mutations and cytoplasmic inclusions 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.

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 proteins (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 fragmentation 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 component 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 contributes to cell death. In HD, a failure of cargo recognition occurs, contributing to protein aggregates and cell death.

There is other evidence for lysosomal dysfunction and impaired

autophagy in PD. Mutations in glucocerebrosidase (GBA) are associated 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 degradation 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 the endosome-to-Golgi complex. 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 therapeutic 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 (Chap. 430).

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 neuropathologic 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 neurodegenerative diseases have been offered, but none is supported by substantial

experimental evidence. The late onset might be due to a second event

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 mechanisms 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 neuropathology, but also the prion etiology (Table 424-1). Over the past

decade, an expanding body of experimental data has accumulated connecting prions in each of these illnesses. In addition to kuru and CJD,

Gerstmann-Sträussler-Scheinker 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 disease (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 synaptically connected neurons. Moreover, each of these

prions causes a distinct constellation of neurodegenerative diseases.

Evidence for a prion etiology of AD comes from a series of transmission experiments initially performed in marmosets and subsequently

in transgenic mice expressing the mutant APP from which the Aβ

peptide is derived (Table 424-1). 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

3299Pathobiology of Neurologic Diseases CHAPTER 424

found together in the brains of AD patients. These findings indicated

that AD is a double-prion disease (Table 424-1); 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 prions in

their brains with the frequent diagnosis of AD. The third double-prion

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 different 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 424-1).

For many years, the most frequently cited argument against prions

was the existence of strains that produced distinct clinical presentations

and different patterns of neuropathologic lesions. Some investigators

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 enciphered in

the conformation of PrPSc, but the molecular mechanisms responsible

for the storage of this biologic information remains enigmatic. 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.

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 functions 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).

Many but not all prion proteins adopt a β-sheet-rich conformation and appear to readily oligomerize as this process becomes selfpropagating. Control of the self-propagating state of benign mammalian

TABLE 424-1 Prion-Based Classification of

Neurodegenerative Diseases

NEURODEGENERATIVE DISEASE

CAUSATIVE PRION

PROTEINS

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-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

prions is less well understood than that of pathogenic mammalian 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 oligomeric states

of pathogenic mammalian prions are thought to be toxic; larger polymers, such as amyloid fibrils, seem to be a mechanism for minimizing

toxicity.

To date, there is no medication that halts or even slows one human

neurodegenerative disease. 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 resistance 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 invasive 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 lentiviruses 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 progenitor 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

3300 PART 13 Neurologic Disorders

spinal cord as well as retinal cells, glial cells including astrocytes and

oligodendrocytes, immune cells, and peripheral nervous system cells.

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 patients. 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

would be possible to explore how disease phenotypes may vary according 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 roadblocks remain. There are no standard reprogramming or derivation

protocols, and the different methods can result in considerable variability 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 disease 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 autism spectrum disorder,

schizophrenia, PD, AD, and ALS are complex disorders, resulting

from an unknown combination 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 cellular 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 the human forebrain 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 variability in such “wholebrain organoids” can be a liability for controlled studies, and can be

reduced by the use of more 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. Nonetheless, while still only reflecting

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 neurodevelopmental disorder producing severe microcephaly with simplified 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-associated

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

differentiation compared to wild-type controls. Introducing functional

CDK5RAP2 by electroporation partially rescued the disease phenotype, 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 microcephaly that had not already been described in mouse models.

In a study using cortical organoids to model Miller-Dieker syndrome

(MDS), a severe congenital form of lissencephaly or “smooth-brain,”

features of the human disease were observed that had not been noted in

murine models. Classical lissencephaly is a genetic neurologic disorder

associated with mental retardation and intractable epilepsy, and MDS

is a severe form of the disorder. Cortical folding in humans begins

toward the end of the second trimester, a stage of development that has

not yet been modeled in organoids, but gyrencephaly depends upon

earlier events such as neural progenitor cell proliferation and neuronal

migration, 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

spindle 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 highlight how organoid technology can provide new

and valuable perspectives on the pathophysiology of disorders of in

utero development.

■ ACQUIRED NEURODEVELOPMENTAL

DISORDERS: ZIKA

The recent outbreak of Zika virus (ZIKV) and associated microcephaly

cases in the Americas provided a test case for the utility of brain

3301Pathobiology of Neurologic Diseases CHAPTER 424

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. Neural progenitor cells (radial glia) were readily infected

in vitro with subsequent progenitor cell death and involution of

organoid size. Forebrain 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 alteration in the cleavage plane of mitotic radial

glia associated with premature 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 libraries 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

Autism spectrum disorders (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 iPSCderived disease models. iPSC-derived neurons have been used to study

the pathophysiology of disorders associated with ASD that are caused

by monogenic mutations, including Fragile X, Rett, and Timothy

syndromes.

Fragile X is the most common heritable cause of intellectual

disability, affecting 1 in 4000 males and 1 in 8000 females, and is a

leading genetic cause of ASD. Patients also have speech delay, growth

and motor abnormalities, hyperactivity, and anxiety. The causative

mutation lies in the FMR1 gene and produces a CGG triplet repeat

expansion 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 following differentiation. Fragile X stem cell lines can therefore be used to study the mechanism of FMR1 silencing, an effort that

is ongoing. Neurons 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 strategies that could be able to remove the repressive

epigenetic marks induced by the mutation and rescue the phenotype.

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 defect gene usually die

in infancy, most patients are girls. Random inactivation of the X

chromosome 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 dysfunction, 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 regulation 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 inactivation 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 synapses, 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 potassium/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

expression 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 coding for a voltage-gated calcium channel, and neuron defects in Timothy

syndrome organoids were rescued by selectively altering calcium channel 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 interneuron 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

polygenic 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 exuberant

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 underscores

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 factors.

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.

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 phenotypes 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

3302 PART 13 Neurologic Disorders

abnormal microRNA expression, disordered cyclic AMP and Wnt signaling, abnormal stress responses, diminished neuronal connectivity,

fewer neuronal processes, problems with neuronal differentiation, and

mitochondrial 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 protocols, 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

clearance 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. Recent failures of anti-β-amyloid therapies, which were highly

effective in mouse models, have led to a search for alternative models

that might be more predictive of therapeutic effectiveness in humans.

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 carrying mutations in AD-causative genes as well as from sporadic AD

cases. The disease neurons developed hallmarks of AD including intracellular 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 systems, 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 diagnostic 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 derivation 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 numbers 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 potential 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 programs 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 ventralizing factor. Generating human motor neurons proved more complex, 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 pathophysiology. 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 ESC-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 noncell autonomous toxic mechanism 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 additional cell types such

as microglia. A variety of approaches including co-culture of specific

cell types, three-dimensional spinal cord organoids, 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 huntingtin 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-offunction, 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 neurons where they misfold and form aggregates that are

visible as cellular inclusions. Affected cells eventually die, possibly

as a result of metabolic 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 metabolic capacity, increased oxidative stress