CHAPTER 30: BIOLOGY 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. Each mature brain is composed of 100 billion neurons, several million miles of axons and dendrites, and >10¹⁵ synapses. Neurons exist within a dense parenchyma of multifunctional glial cells that synthesize myelin, preserve homeostasis, and regulate immune responses. Measured against this background of complexity, the achievements of molecular neuroscience have been extraordinary. This chapter reviews selected themes in neuroscience that provide a context for understanding fundamental mechanisms underlying neurologic disorders.

The landscape of neurology has been transformed by modern molecular genetics. Several hundred neurologic and psychiatric disorders can now be diagnosed through genetic testing (http://www.ncbi.nlm.nih.gov/sites/GeneTests/?db=GeneTests). The vast majority of these represent highly penetrant mutations that cause rare neurologic disorders; alternatively, they represent rare monogenic causes of common phenotypes. Examples of the latter include mutations of the amyloid precursor protein in familial Alzheimer’s disease, the microtubule-associated protein tau (MAPT) in frontotemporal dementia, and α-synuclein in Parkinson’s disease. These discoveries have been profoundly important because the mutated gene in the familial disorder often encodes a protein that is also pathogenetically involved (although not mutated) in the typical, sporadic form. The common mechanism involves disordered processing and, ultimately, aggregation of the protein leading to cell death (see “Protein Aggregation and Neurodegeneration,” below).

There is optimism that complex genetic disorders, caused by combinations of both genetic and environmental factors, have now become tractable problems. Genome-wide association studies (GWAS) have been carried out in many complex neurologic disorders, with many hundreds of variants identified, nearly all of which confer only a small increment in disease risk (1.15- to 1.5-fold). GWAS studies are rooted in the “common disease, common variant” hypothesis, as they examine potential risk alleles that are relatively frequent (e.g. >5%) in the general population. More than 1500 GWAS studies have been carried out, with notable successes such as the identification of 110 risk alleles for multiple sclerosis (Chap. 45). Furthermore, using bioinformatics tools, risk variants can be aligned in functional biologic pathways to identify novel pathogenic mechanisms as well as to reveal heterogeneity (e.g., different pathways in different individuals). Despite these successes, many experienced geneticists question the real value of common disease-associated variants, particularly whether they are actually causative or merely mark the approximate locations of more important—truly causative—rare mutations.

This debate has set the stage for the next revolution in human genetics, made possible by the development of increasingly efficient and cost-effective high-throughput sequencing methodologies. It is already possible to sequence an entire human genome in approximately an hour, at a cost of only $1300 for the entire coding sequence (“whole-exome”) or $3000 for the entire genome; it is certain that these costs will continue to decline. This makes it feasible to look for disease-causing sequence variations in individual patients with the possibility of identifying rare variants that cause disease. The utility of this approach was demonstrated by whole-genome sequencing in a patient with Charcot-Marie-Tooth (CMT) neuropathy, in which compound heterozygous mutations were identified in the SH3TC2 gene, which then were shown to co-segregate with the disease in other members of the family.

It is increasingly recognized that not all genetic diseases or predispositions are caused by simple changes in the linear nucleotide sequence of genes. Disease-causative mutations also occur commonly in noncoding sequences of DNA. For example, large intronic GGGGCC repeat expansions in a gene of unknown function C9orf 72 (chromosome 9 open reading frame 72) were recently identified as a common cause of both frontotemporal dementia and amyotrophic lateral sclerosis (ALS). This mutation is the most common cause of both familial and sporadic ALS identified thus far. It was shown to be associated with TDP-43 (tar DNA binding protein-43) inclusions in both hippocampal and cerebral neurons. Interestingly, despite the absence of a start codon, the three alternate dipeptide sequences consisting of two amino acids are translated and found in postmortem brain tissue of affected patients. Three potential pathogenic mechanisms have been proposed, including (1) haploinsufficiency, (2) repeat RNA-mediated toxicity, and (3) dipeptide protein toxicity. The possibility of RNA toxicity is supported by the finding of intranuclear RNA foci containing C9orf 72 hexanucleotide repeats and specific RNA-binding proteins. The C9orf 72 mRNA forms quadruplexes of DNA and RNA, which then can halt transcription and also bind transcription factors. Mutations in both TARDP (transactive region DNA binding protein) and FUS (fused in sarcoma) also encode DNA/RNA-processing polypeptides and are a cause of familial and sporadic ALS.

As the complex architecture of the human genome becomes better defined, many disorders that result from alterations in copy numbers of genes (“gene-dosage” effects) resulting from unequal crossing-over are also likely to be identified. As much as 5–10% of the human genome consists of nonhomologous duplications and deletions, and these appear to occur with a much higher mutational rate than is the case for single base pair mutations. The first copy-number disorders to be recognized were Charcot-Marie-Tooth disease type 1A (CMT1A), caused by a duplication in the gene encoding the myelin protein PMP22, and the reciprocal deletion of the gene causing hereditary liability to pressure palsies (Chap. 53). Gene-dosage effects are causative in some cases of Parkinson’s disease (α-synuclein), Alzheimer’s disease (amyloid precursor protein), spinal muscular atrophy (survival motor neuron 2), the dysmyelinating disorder Pelizaeus-Merzbacher syndrome (proteolipid protein 1), late-onset leukodystrophy (lamin B1), and a variety of developmental neurologic disorders. It is likely that copy-number variations contribute substantially to normal human genomic variation for numerous genes involved in neurologic function, regulation of cell growth, and regulation of metabolism. It is also already clear that gene-dosage effects will influence many behavioral phenotypes, learning disorders, and autism spectrum disorders. Deletions at ch30q and ch15q have been associated with schizophrenia, and deletions at 15q and 16p with autism. Interestingly, the 16p deletion is also associated with epilepsy. Duplications of the X-linked MeCP2 gene cause autism in males and psychiatric disorders with anxiety in females, whereas point mutations in this gene produce the neurodevelopmental disorder Rett’s syndrome. The understanding of the role of copy number variation in human disease is still in its infancy.

The role of splicing variation as a contributor to neurologic disease is another area of active investigation. Alternative splicing refers to the inclusion of different combinations of exons in mature mRNA, resulting in the potential for many different protein products encoded by a single gene. Alternative splicing represents a powerful mechanism for generation of complexity and variation, and this mechanism appears to be highly prevalent in the nervous system, affecting key processes such as neurotransmitter receptors and ion channels. Numerous diseases are already known to result from abnormalities in alternative splicing. Increased inclusion of exon 10–containing transcripts of MAPT can cause frontotemporal dementia. Aberrant splicing also contributes to the pathogenesis of Duchenne’s, myotonic, and facioscapulohumeral muscular dystrophies; ataxia telangiectasia; neurofibromatosis; some inherited ataxias; and fragile X syndrome, among other disorders. It is also likely that subtle variations of splicing will influence many genetically complex disorders. A splicing variant of the interleukin 7 receptor α chain, resulting in production of more soluble and less membrane-bound receptor, is associated with susceptibility to multiple sclerosis (MS) in multiple different populations.

Epigenetics refers to the mechanisms by which levels of gene expression can be exquisitely modulated, not by variations in the primary genetic sequence of DNA but rather by postgenomic alterations in DNA and chromatin structure, which influence how, when, and where genes are expressed. DNA methylation and methylation and acetylation of histone proteins that interact with nuclear DNA to form chromatin are key mediators of these events. Epigenetic processes appear to be dynamically active even in postmitotic neurons. Imprinting refers to an epigenetic feature, present for a subset of genes, in which the predominant expression of one allele is determined by its parent of origin. The distinctive neurodevelopmental disorders Prader-Willi syndrome (mild mental retardation and endocrine abnormalities) and Angelman’s syndrome (cortical atrophy, cerebellar dysmyelination, Purkinje cell loss) are classic examples of imprinting disorders whose distinctive features are determined by whether the paternal or maternal copy of the chromosome of the critical genetic region 15q11-13 is responsible. In a study of discordant monozygotic twins for MS in which the entire DNA sequence, transcriptome (e.g., mRNA levels), and methylome were assessed genome-wide, tantalizing allelic differences in the use of the paternal, compared to maternal, copy for a group of genes were identified. Preferential allelic expression, whether due to imprinting, resistance to X-inactivation, or other mechanisms, is likely to play a major role in determining complex behaviors and susceptibility to many neurologic and psychiatric disorders.

Another advance is the development of transgenic mouse models of neurologic diseases, which has been particularly fruitful in producing models relevant to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and ALS. These models are useful in both studying disease pathogenesis and developing and testing new therapies. New transgenic mouse models with conditional expression have fostered investigations in which late gene expression avoids developmental compensation or in which the reversibility of a disease phenotype can be examined by turning a gene off after the disease phenotype has manifested. One can also examine the effects of gene expression in specific subsets of neurons, such as entorhinal cortex, or selectively in neurons, astrocytes, or microglia. Models in both Caenorhabditis elegans and Drosophila have also been extremely useful, particularly in studying genetic modifiers and therapeutic interventions.

The resting potential of neurons and the action potentials responsible for impulse conduction are generated by ion currents and ion channels. Most ion channels are gated, meaning that they can transition between conformations that are open or closed to ion conductance. Individual ion channels are distinguished by the specific ions they conduct; by their kinetics; and by whether they directly sense voltage, are linked to receptors for neurotransmitters or other ligands such as neurotrophins, or are activated by second messengers. The diverse characteristics of different ion channels provide a means by which neuronal excitability can be exquisitely modulated at both the cellular and the subcellular levels. Disorders of ion channels—channelopathies—are responsible for a growing list of human neurologic diseases (Table 30-1). Most are caused by mutations in ion channel genes or by autoantibodies against ion channel proteins. One example is epilepsy, a syndrome of diverse causes characterized by repetitive, synchronous firing of neuronal action potentials. Action potentials are normally generated by the opening of sodium channels and the inward movement of sodium ions down the intracellular concentration gradient. Depolarization of the neuronal membrane opens potassium channels, resulting in outward movement of potassium ions, repolarization, closure of the sodium channel, and hyperpolarization. Sodium or potassium channel subunit genes have long been considered candidate disease genes in inherited epilepsy syndromes, and recently such mutations have been identified. These mutations appear to alter the normal gating function of these channels, increasing the inherent excitability of neuronal membranes in regions where the abnormal channels are expressed.

Whereas the specific clinical manifestations of channelopathies are quite variable, one common feature is that manifestations tend to be intermittent or paroxysmal, such as occurs in epilepsy, migraine, ataxia, myotonia, or periodic paralysis. Exceptions are clinically progressive channel disorders such as autosomal dominant hearing impairment. The genetic channelopathies identified to date are all uncommon disorders caused by obvious mutations in channel genes. As the full repertoire of human ion channels and related proteins is identified, it is likely that additional channelopathies will be discovered. In addition to rare disorders that result from obvious mutations, it is also likely that less penetrant allelic variations in channel genes or in their pattern of expression might underlie susceptibility to some apparently sporadic forms of epilepsy, migraine, or other disorders. For example, mutations in the potassium channel gene Kir2.6 have been found in many individuals with thyrotoxic hypokalemic periodic paralysis, a disorder similar to hypokalemic periodic paralysis but precipitated by stress from thyrotoxicosis or carbohydrate loading.

Synaptic neurotransmission is the predominant means by which neurons communicate with each other. Classic neurotransmitters are synthesized in the presynaptic region of the nerve terminal; stored in vesicles; and released into the synaptic cleft, where they bind to receptors on the postsynaptic cell. Secreted neurotransmitters are eliminated by reuptake into the presynaptic neuron (or glia), by diffusion away from the synaptic cleft, and/or by specific inactivation. In addition to the classic neurotransmitters, many neuropeptides have been identified as definite or probable neurotransmitters; these include substance P, neurotensin, enkephalins, β-endorphin, histamine, vasoactive intestinal polypeptide, cholecystokinin, neuropeptide Y, and somatostatin. Peptide neurotransmitters are synthesized in the cell body rather than the nerve terminal and may colocalize with classic neurotransmitters in single neurons. A number of neuropeptides are important in pain modulation including substance P and calcitonin gene-related peptide (CGRP), which causes migraine-like headaches in patients. As a consequence, CGRP receptor antagonists have been developed and shown to be effective in treating migraine headaches. Nitric oxide and carbon monoxide are gases that appear also to function as neurotransmitters, in part by signaling in a retrograde fashion from the postsynaptic to the presynaptic cell.

Neurotransmitters modulate the function of postsynaptic cells by binding to specific neurotransmitter receptors, of which there are two major types. Ionotropic receptors are direct ion channels that open after engagement by the neurotransmitter. Metabotropic receptors interact with G proteins, stimulating production of second messengers and activating protein kinases, which modulate a variety of cellular events. Ionotropic receptors are multiple subunit structures, whereas metabotropic receptors are composed of single subunits only. One important difference between ionotropic and metabotropic receptors is that the kinetics of ionotropic receptor effects are fast (generally <1 ms) because neurotransmitter binding directly alters the electrical properties of the postsynaptic cell, whereas metabotropic receptors function over longer time periods. These different properties contribute to the potential for selective and finely modulated signaling by neurotransmitters.

Neurotransmitter systems are perturbed in a large number of clinical disorders, several of which are highlighted in Table 30-2. One example is the involvement of dopaminergic neurons originating in the substantia nigra of the midbrain and projecting to the striatum (nigrostriatal pathway) in Parkinson’s disease and in heroin addicts after the ingestion of the toxin MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine).

A second important dopaminergic system arising in the midbrain is the mediocorticolimbic pathway, which is implicated in the pathogenesis of addictive behaviors including drug reward. Its key components include the midbrain ventral tegmental area (VTA), median forebrain bundle, and nucleus accumbens (see Fig. 60-2). The cholinergic pathway originating in the nucleus basalis of Meynert plays a role in memory function in Alzheimer’s disease.

Addictive drugs share the property of increasing dopamine release in the nucleus accumbens (Chap. 60). Amphetamine increases intracellular release of dopamine from vesicles and reverses transport of dopamine through the dopamine transporters. Patients prone to addiction show increased activation of the nucleus accumbens following administration of amphetamine. Cocaine binds to dopamine transporters and inhibits dopamine reuptake. Ethanol inhibits inhibitory neurons in the VTA, leading to increased dopamine release in the nucleus accumbens. Opioids also disinhibit these dopaminergic neurons by binding to μ receptors expressed by γ-aminobutyric acid (GABA)–containing interneurons in the VTA. Nicotine increases dopamine release by activating nicotinic acetylcholine receptors on cell bodies and nerve terminals of dopaminergic VTA neurons. Tetrahydrocannabinol, the active ingredient of cannabis, also increases dopamine levels in the nucleus accumbens. Blockade of dopamine in the nucleus accumbens can terminate the rewarding effects of addictive drugs.

Not all cell-to-cell communication in the nervous system occurs via neurotransmission. Gap junctions provide for direct neuron-neuron electrical conduction and also create openings for the diffusion of ions and metabolites between cells. In addition to neurons, gap junctions are also widespread in glia, creating a syncytium that protects neurons by removing glutamate and potassium from the extracellular environment. Gap junctions consist of membrane-spanning proteins, termed connexins, that pair across adjacent cells. Mechanisms that involve gap junctions have been related to a variety of neurologic disorders. Mutations in connexin 32, a gap junction protein expressed by Schwann cells, are responsible for the X-linked form of CMT disease (Chap. 53). Mutations in either of two gap junction proteins expressed in the inner ear—connexin 26 and connexin 31—result in autosomal dominant progressive hearing loss (Chap. 29). Glial calcium waves mediated through gap junctions also appear to explain the phenomenon of spreading depression associated with migraine auras and the march of epileptic discharges. Spreading depression is a neural response that follows a variety of different stimuli and is characterized by a circumferentially expanding negative potential that propagates at a characteristic speed of 20 m/s and is associated with an increase in extracellular potassium.

The fundamental issue of how memory, learning, and thinking are encoded in the nervous system is likely to be clarified by identifying the signaling pathways involved in neuronal differentiation, axon guidance, and synapse formation, and by understanding how these pathways are modulated by experience. Many families of transcription factors, each comprising multiple individual components, are expressed in the nervous system. Elucidation of these signaling pathways has already begun to provide insights into the cause of a variety of neurologic disorders, including inherited disorders of cognition such as X-linked mental retardation. This problem affects ~1 in 500 males, and linkage studies in different families suggest that as many as 60 different X-chromosome-encoded genes may be responsible. The formation of RNA-DNA duplexes that block transcription has also been observed with the CGG repeat expansions that occur in fragile X gene-associated mental retardation. Rett’s syndrome, a common cause of (dominant) X-linked progressive mental retardation in females, is due to a mutation in a gene (MECP2) encoding a DNA-binding protein involved in transcriptional repression. Because the X chromosome comprises only ~3% of germline DNA, then by extrapolation, the number of genes that potentially contribute to clinical disorders affecting intelligence in humans must be potentially very large. As discussed below, there is increasing evidence that abnormal gene transcription may play a role in neurodegenerative diseases, such as Huntington’s disease, in which proteins with polyglutamine expansions bind to and sequester transcription factors. A critical transcription factor for neuronal survival is CREB (cyclic adenosine monophosphate responsive element-binding) protein, which also plays an important role in memory in the hippocampus. The regulatory gene repressor element 1-silencing transcription factor (REST) coordinates the expression of neuroprotective stress genes during normal aging. It turns off genes involved in cell death and pathology and boosts protective factors. High levels of REST are associated with normal cognition even in the presence of both amyloid plaques and neurofibrillary tangles. Although REST increases with normal aging, it fails to increase in the nucleus in patients with Alzheimer’s disease and is found clumped with amyloid in autophagosomes.

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. Molecular interactions between the myelin membrane and axon are required to maintain the stability, function, and normal life span of both structures. A single oligodendrocyte usually ensheaths multiple axons in the central nervous system (CNS), whereas in the peripheral nervous system (PNS), each Schwann cell typically myelinates a single axon. Myelin is a lipid-rich material formed by a spiraling process of the membrane of the myelinating cell around the axon, creating multiple membrane bilayers that are tightly apposed (compact myelin) by charged protein interactions. Several inhibitors of axon growth are expressed on the innermost (periaxonal) lamellae of the myelin membrane (see “Stem Cells and Transplantation,” below). A number of clinically important neurologic disorders are caused by inherited mutations in myelin proteins of the CNS or PNS (Fig. 30-1). Constituents of myelin also have a propensity to be targeted as autoantigens in autoimmune demyelinating disorders (Fig. 30-2).

Specification to oligodendrocyte precursor cells (OPCs) is transcriptionally regulated by the Olig 2 and Yin Yang 1 genes, whereas myelination mediated by postmitotic oligodendrocytes depends on a different transcription factor, myelin gene regulatory factor (MRF). It is noteworthy that in the normal adult brain, large numbers of OPCs (expressing PDGFR-α and NG2) are widely distributed but do not myelinate axons, even in demyelinating environments such as in lesions of MS. Several families of molecules have now been identified that regulate oligodendrocyte differentiation and myelination, including LINGO-1, PSA-NCAM, hyaluronan, Nogo-A, the Wnt pathway, notch signaling (and its receptor Jagged), and the retinoic acid receptor RXRγ; all are inhibitory, with the exception of RXRγ, which is excitatory. All are also potential targets for myelin repair therapies, and a monoclonal antibody against LINGO-1 is in clinical testing for remyelination in MS. Very recently, a series of observations has called into question the traditional concept that axon-derived cues are always required for myelination to occur. Fixed (i.e., dead) axons could be efficiently myelinated by oligodendrocytes in vitro, as could artificial polystyrene nanowires of a similar diameter. This led to development of new high-throughput screening assays based on myelination of polystyrene nanowires to identify compounds that could promote myelination.

Macrophages and microglia represent the major cell types in the nervous system responsible for antigen presentation and innate immunity. Brain macrophages are derived from either hematopoietic stem cell–derived bone marrow monocytes or from brain microglia that migrate from the yolk sac early in embryogenesis before the blood-brain barrier is formed. In a murine model of autoimmune demyelination, experimental allergic encephalomyelitis (EAE) (Fig. 30-3), macrophages derived from bone marrow monocytes, but not microglia, were found to represent the critical population that initiated inflammatory demyelination at paraxonal regions near nodes of Ranvier. An additional, unexpected role for brain microglia was also identified in the regulation of neural circuits through pruning of excitatory synapses and control of dendritic spine densities; mice depleted of microglia during development exhibited a variety of cognitive learning and behavioral deficits, including abnormal social behaviors. Remarkably, depletion of microglia in adult mice by administration of a selective inhibitor of colony-stimulating factor receptor 1 (CSFR1) was followed by their rapid repopulation, suggesting that a pool of resident microglial precursor cells may exist throughout the CNS.

The human microbiome represents the collective set of genes from the 10¹⁴ organisms living in our gut, skin, mucosa, and other sites. Different microbial communities are associated with different 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.

There is compelling evidence that 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 interleukin (IL) 10 and IL-13 and 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.

In addition to nonspecific effects on immune homeostasis mediated by cytokines and regulatory cells, some microbial proteins can trigger, in susceptible individuals, 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. 45); the neural ganglioside Gm1 and similar sialic acid–containing structures from Campylobacter jejuni in Guillain-Barré syndrome (Chap. 54); and the sleep-promoting protein hypocretin and hemagglutinin from H1N1 influenza virus in narcolepsy (Chap. 24), among others.

Recently, a number of tantalizing observations have incriminated the microbial environment in the pathogenesis of a much wider spectrum of neurologic conditions and behaviors, extending well beyond the traditional boundaries of immune-mediated pathologies. This is perhaps not surprising, as it has long been known in neurology 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 developed in a completely germ-free environment displayed less anxiety, lower responses to stressful situations, more exploratory locomotive behaviors, and impaired memory formation compared with non-germ-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.

The enteric autonomic nervous system in humans provides a bidirectional neural connection between the brain and gut. The vagus nerve, which innervates the upper gut and proximal colon, has been implicated in anxiety- and depression-like 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.

Another area of emerging interest is in a possible contribution of the gut microbiome to autism and related disorders. Children with autistic spectrum disorders have long been known to have gastrointestinal disturbances, and it has been claimed that the severity of dysbiosis correlates with the severity of autism. A murine model of autism was recently 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 gut permeability.

Neurotrophic factors (Table 30-3) are secreted proteins that modulate neuronal growth, differentiation, repair, and survival; some have additional functions, including roles in neurotransmission and in the synaptic reorganization involved in learning and memory. The neurotrophin (NT) family contains nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, and NT4/5. The neurotrophins act at TrK and p75 receptors to promote survival of neurons. BDNF is linked to synaptogenesis. Certain polymorphisms are linked to increased risk for Alzheimer’s disease (AD), and BDNF is depleted in Huntington’s disease. Because of their survival-promoting and antiapoptotic effects, neurotrophic factors are in theory outstanding candidates for therapy of disorders characterized by premature death of neurons such as occurs in ALS and other degenerative motor neuron disorders. Knockout mice lacking receptors for ciliary neurotrophic factor (CNTF) or BDNF show loss of motor neurons, and experimental motor neuron death can be rescued by treatment with various neurotrophic factors including CNTF, BDNF, and vascular endothelial growth factor (VEGF). However, in phase 3 clinical trials, growth factors were ineffective in human ALS. The growth factor glial-derived neurotrophic factor (GDNF) is important for survival of dopaminergic neurons. Direct infusions of GDNF showed initial promise in Parkinson’s disease, but the benefits were not replicated in a larger clinical trial.

The nervous system is traditionally considered to be a nonmitotic organ, in particular with respect to neurons. These concepts have been challenged by the finding that neural progenitor or stem cells exist in the adult CNS that are capable of differentiation, migration over long distances, and extensive axonal arborization and synapse formation with appropriate targets. These capabilities also indicate that the repertoire of factors required for growth, survival, differentiation, and migration of these cells exists in the mature nervous system. In rodents, neural stem cells, defined as progenitor cells capable of differentiating into mature cells of neural or glial lineage, have been experimentally propagated from fetal CNS and neuroectodermal tissues and also from adult germinal matrix and ependyma regions. Human fetal CNS tissue is also capable of differentiation into cells with neuronal, astrocyte, and oligodendrocyte morphology when cultured in the presence of growth factors.

Once the repertoire of signals required for cell type specification is better understood, differentiation into specific neural or glial subpopulations can be directed in vitro; such cells could also be engineered to express therapeutic molecules. Another promising approach is to use growth factors, such as BDNF, to stimulate endogenous stem cells to proliferate and migrate to areas of neuronal damage.

A major advance has been the development of induced pluripotent stem cells. Using this technique, adult somatic cells such as skin fibroblasts are treated with four pluripotency factors (SOX2, KLF4, cMYC, and Oct4), and this generates induced pluripotent stem cells (iPSCs). These adult-derived stem cells sidestep the ethical issues of using stem cells derived from human embryos. The development of these cells has tremendous promise for both studying disease mechanisms and testing therapeutics. As yet there is no consensus on the best way to generate and differentiate the iPSCs; however, techniques to avoid using viral vectors and use of Cre-lox systems to remove reprogramming factors result in a better match of gene expression profiles with those of embryonic stem cells. Over the years, the field of directed differentiation has used three main strategies to specify neural lineages from human pluripotent stem cells. These strategies are embryoid body formation, coculture on neural-inducing feeders, and direct neural induction. Thus far, iPSCs have been made from patients with all of the major human neurodegenerative diseases, and studies using them are under way.

Although stem cells hold tremendous promise for the treatment of debilitating neurologic diseases, such as Parkinson’s disease and spinal cord injury, it should be emphasized that medical application is in its infancy. Major obstacles are the generation of position- and neurotransmitter-defined subtypes of neurons and their isolation as pure populations of the desired cells. This is crucial to avoid persistence of undifferentiated embryonic stem (ES) cells, which can generate tumors. The establishment of appropriate neural connections and afferent control is also critical. For instance, human ES motor neurons will need to be introduced at multiple segments in the neuraxis, and then their axons will need to regenerate from the spinal cord to distal musculature.

Experimental transplantation of human fetal dopaminergic neurons in patients with Parkinson’s disease has shown that these transplanted cells can survive within the host striatum; however, some patients developed disabling dyskinesias, and this approach is no longer in clinical development. The possibility that iPSCs will be used in Parkinson’s disease was strengthened by studies showing that they can be differentiated into dopaminergic neurons. The dopaminergic neurons were then shown to rescue the parkinsonian phenotype in a MPTP-induced primate model with excellent dopaminergic neuron survival function and lack of neural overgrowth. The correction of tau mutations in iPSC-derived neurons has been shown to reverse the toxic phenotype in dendrite retraction and cell death.

Another new use for iPSCs is to screen drugs as potential treatments for neurodegenerative and other diseases. The feasibility of this has been shown using iPSC-induced macrophages from patients with Gaucher’s disease, and verifying the efficacy of protein chaperones in these cells as a means of stabilizing the mutant glucocerebrosidase and increasing its activity and the duration of its effects. Other approaches are to attempt to reduce expression of proteins, such as amyloid, tau, and α-synuclein, implicated in the pathogenesis of neurodegenerative diseases. One difficulty has been that reprogramming cells to iPSCs resets their identity back to an embryonic age, which is a hurdle in modeling of late-onset diseases. One approach to this has been to express a fragment of the mutated gene, such as a portion of lamin A, which causes premature aging in progeria. This approach showed that dendrite degeneration and progressive loss of tyrosine hydroxylase expression, as well as enlarged mitochondria and Lewy body precursor inclusions, were induced in iPSC-derived dopaminergic neurons with progerin-induced aging.

Studies of transplantation for patients with Huntington’s disease have also reported encouraging, although very preliminary, results. OPCs transplanted into mice with a dysmyelinating disorder effectively migrated in the new environment, interacted with axons, and mediated myelination; such experiments raise hope that similar transplantation strategies may be feasible in human disorders of myelin such as MS. The promise of stem cells for treatment of both neurodegenerative diseases and neural injury is great, but development has been slowed by unresolved concerns over safety (including the theoretical risk of malignant transformation of transplanted cells), ethics (particularly with respect to use of fetal tissue), and efficacy.

In developing brain, the extracellular matrix provides stimulatory and inhibitory signals that promote neuronal migration, neurite outgrowth, and axonal extension. After neuronal damage, reexpression of inhibitory molecules such as chondroitin sulfate proteoglycans may prevent tissue regeneration. Chondroitinase degraded these inhibitory molecules and enhanced axonal regeneration and motor recovery in a rat model of spinal cord injury. Several myelin proteins, specifically Nogo, oligodendrocyte myelin glycoprotein (OMGP), and myelin-associated glycoprotein (MAG), may also interfere with axon regeneration. Sialidase, which cleaves one class of receptors for MAG, enhances axonal outgrowth. Antibodies against Nogo promote regeneration after experimental focal ischemia or spinal cord injury. Nogo, OMGP, and MAG all bind to the same neural receptor, the Nogo receptor, which mediates its inhibitory function via the p75 neurotrophin receptor signaling.

Excitotoxicity refers to neuronal cell death caused by activation of excitatory amino acid receptors (Fig. 30-4). Compelling evidence for a role of excitotoxicity, especially in ischemic neuronal injury, is derived from experiments in animal models. Experimental models of stroke are associated with increased extracellular concentrations of the excitatory amino acid neurotransmitter glutamate, and neuronal damage is attenuated by denervation of glutamate-containing neurons or the administration of glutamate receptor antagonists. The distribution of cells sensitive to ischemia corresponds closely with that of N-methyl-D-aspartate (NMDA) receptors (except for cerebellar Purkinje cells, which are vulnerable to hypoxia-ischemia but lack NMDA receptors); and competitive and noncompetitive NMDA antagonists are effective in preventing focal ischemia. In global cerebral ischemia, non-NMDA receptors (kainic acid and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate [AMPA]) are activated, and antagonists to these receptors are protective. Experimental brain damage induced by hypoglycemia is also attenuated by NMDA antagonists.

Excitotoxicity is not a single event but rather a cascade of cell injury. Excitotoxicity causes influx of calcium into cells, and much of the calcium is sequestered in mitochondria rather than in the cytoplasm. Increased cytoplasmic calcium causes metabolic dysfunction and free radical generation; activates protein kinases, phospholipases, nitric oxide synthase, proteases, and endonucleases; and inhibits protein synthesis. Activation of nitric oxide synthase generates nitric oxide (NO•), which can react with superoxide (O•₂) to generate peroxynitrite (ONOO⁻), which may play a direct role in neuronal injury. Another critical pathway is activation of poly-ADP-ribose polymerase, which occurs in response to free radical–mediated DNA damage. Experimentally, mice with knockout mutations of neuronal nitric oxide synthase or poly-ADP-ribose polymerase, or those that overexpress superoxide dismutase, are resistant to focal ischemia.

Another aspect of excitotoxicity is that it has been demonstrated that stimulation of extrasynaptic NMDA receptors mediates cell death, where as stimulation of synaptic receptors is protective. This has been shown to play a role in excitotoxicity in transgenic mouse models of Huntington’s disease, in which using low-dose memantine to selectively block the extrasynaptic receptors is beneficial.

Although excitotoxicity is clearly implicated in the pathogenesis of cell death in stroke, to date treatment with NMDA antagonists has not proven to be clinically useful. One approach has been to use an inhibitor of the postsynaptic density-95 protein that uncouples NMDA receptors from neurotoxic pathways, including the generation of nitric oxide. This approach was effective in a primate stroke model and in a phase 2 clinical trial of stroke associated with endovascular repair of cerebral aneurysms. Transient receptor potentials (TRPs) are calcium channels that are activated by oxidative stress in parallel with excitotoxic signal pathways. In addition, glutamate-independent pathways of calcium influx via acid-sensing ion channels have been identified. These channels transport calcium in the setting of acidosis and substrate depletion, and pharmacologic blockade of these channels markedly attenuates stroke injury. These channels offer a potential new therapeutic target for stroke.

Apoptosis, or programmed cell death, plays an important role in both physiologic and pathologic conditions. During embryogenesis, apoptotic pathways operate to destroy neurons that fail to differentiate appropriately or reach their intended targets. There is mounting evidence for an increased rate of apoptotic cell death in a variety of acute and chronic neurologic diseases. Apoptosis is characterized by neuronal shrinkage, chromatin condensation, and DNA fragmentation, whereas necrotic cell death is associated with cytoplasmic and mitochondrial swelling followed by dissolution of the cell membrane. Apoptotic and necrotic cell death can coexist or be sequential events, depending on the severity of the initiating insult. Cellular energy reserves appear to have an important role in these two forms of cell death, with apoptosis favored under conditions in which ATP levels are preserved. Evidence of DNA fragmentation has been found in a number of degenerative neurologic disorders, including AD, Huntington’s disease, and ALS. The best characterized genetic neurologic disorder related to apoptosis is infantile spinal muscular atrophy (Werdnig-Hoffmann disease), in which two genes thought to be involved in the apoptosis pathways are causative.

Mitochondria are essential in controlling specific apoptosis pathways. The redistribution of cytochrome c, as well as apoptosis-inducing factor (AIF), from mitochondria during apoptosis leads to the activation of a cascade of intracellular proteases known as caspases. Caspase-independent apoptosis occurs after DNA damage, activation of poly-ADP-ribose polymerase, and translocation of AIF into the nucleus. Redistribution of cytochrome c is prevented by overproduction of the apoptotic protein BCL2 and is promoted by the proapoptotic protein BAX. These pathways may be triggered by activation of a large pore in the mitochondrial inner membrane known as the permeability transition pore, although in other circumstances, they occur independently. The permeability transition pore is made up of dimers of ATP synthase and is activated by cyclophilin D, leading to large calcium fluxes across the inner mitochondrial membrane. Certain forms of congenital muscular dystrophies are caused by mutations in collagen VI, which leads to increased activation of the permeability transition pore. Recent studies suggest that blocking the mitochondrial pore reduces both hypoglycemic and ischemic cell death. Mice deficient in cyclophilin D, a key protein involved in opening the permeability transition pore, are resistant to necrosis produced by focal cerebral ischemia.

The possibility that protein aggregation plays a role in the pathogenesis of neurodegenerative diseases is a major focus of current research. Protein aggregation is a major histopathologic hallmark of neurodegenerative diseases. Deposition of β-amyloid is strongly implicated in the pathogenesis of AD. Genetic mutations in familial AD cause increased production of β-amyloid with 42 amino acids, which has an increased propensity to aggregate, as compared to β-amyloid with 40 amino acids. Furthermore, mutations in the amyloid precursor protein (APP), which reduce the production of β-amyloid, protect against the development of AD and are associated with preserved cognition in the elderly. Mutations in genes encoding the MAPT lead to altered splicing of tau and the production of neurofibrillary tangles in frontotemporal dementia and progressive supranuclear palsy. Familial Parkinson’s disease is associated with mutations in leucine-rich repeat kinase 2 (LRRK2), α-synuclein, 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 Parkinson’s disease, is a ubiquitin ligase. The characteristic histopathologic feature of Parkinson’s disease is the Lewy body, an eosinophilic cytoplasmic inclusion that contains both neurofilaments and α-synuclein. Huntington’s disease 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 mutations and cytoplasmic inclusions containing superoxide dismutase. An important finding was the discovery that ubiquinated 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. In autosomal dominant neurohypophyseal diabetes insipidus, mutations in vasopressin result in abnormal protein processing, accumulation in the endoplasmic reticulum, and cell death.

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 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 mitofuscins 1 and 2 (MF 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 Parkinson’s disease, parkin and PINK1, cause abnormal mitochondrial morphology and result in impairment of the ability of the cell to remove damaged mitochondria by autophagy.

One major scientific question is whether protein aggregates directly contribute to neuronal death or whether they are merely secondary bystanders. A current focus in all the neurodegenerative diseases is on small protein aggregates termed oligomers. These may be the toxic species of β-amyloid, α-synuclein, and proteins with expanded polyglutamines such as are associated with Huntington’s disease. Protein aggregates are usually ubiquinated, 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, Parkinson’s disease, and Huntington’s disease. Autophagy is particularly important to the health of neurons, and failure of autophagy contributes to cell death. In Huntington’s disease, a failure of cargo recognition occurs, contributing to protein aggregates and cell death. Rapamycin, which induces autophagy, exerts beneficial therapeutic effects in transgenic mouse models of AD, Parkinson’s disease, and Huntington’s disease.

There is other evidence for lysosomal dysfunction and impaired autophagy in Parkinson’s disease. Mutations in glucocerebrosidase are associated with 5% of all Parkinson’s disease cases as well as 8–9% of patients with dementia with Lewy bodies. Therefore, this is the most important genetic cause of both disorders thus far identified. There appear to be reciprocal interactions between glucocerebrosidase and α-synuclein. It has been shown that glucocerebrosidase concentrations and enzymatic activity are reduced in the substantia nigra of sporadic Parkinson’s disease patients. Furthermore, 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 as well as in mice in which the enzyme has been inhibited. Furthermore, it is known that α-synuclein inhibits the activity of glucocerebrosidase. Therefore, there is bidirectional feedback between α-synuclein and glucocerebrosidase. An attractive therapeutic intervention could be to use protein chaperones to increase the activity and duration of action of glucocerebrosidase. This would also reduce α-synuclein levels and block the degeneration of dopaminergic neurons.

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. Recently, mutations in VPS35 were shown to be a cause of late-onset autosomal dominant Parkinson’s disease. 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 new 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.

The LRRK2 mutations were shown to have effects on clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. LRRK2 mutations also are linked to elevated protein synthesis mediated by ribosomal protein s15 phosphorylation. Blocking this phosphorylation reduces LRRK2-mediated neurite loss and cell death in human dopamine and cortical neurons.

Interestingly, in experimental models of Huntington’s disease and cerebellar degeneration, protein aggregates are not well correlated with neuronal death and may be protective. A substantial body of evidence suggests that the mutant proteins with polyglutamine expansions in these diseases bind to transcription factors and that this contributes to disease pathogenesis. In Huntington’s disease, there is dysfunction of the transcriptional co-regulator, PGC-1α, a key regulator of mitochondrial biogenesis. There is evidence that impaired function of PGC-1α is also important in both Parkinson’s disease and AD, making it an attractive target for treatments. Agents that upregulate gene transcription are neuroprotective in animal models of these diseases. A number of compounds have been developed to block β-amyloid production and/or aggregation, and these agents are being studied in early clinical trials in humans. Another approach under investigation is immunotherapy with antibodies that bind β-amyloid, tau, or α-synuclein. These studies have shown efficacy in preventing the spread of amyloid, tau, and α-synuclein in animal studies, raising hopes that this could lead to effective therapies by blocking neuron-to-neuron propagation. Two large clinical trials of β-amyloid immunotherapy, however, did not show efficacy, although this therapeutic strategy is still being studied.

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 Creutzfeldt-Jakob disease (CJD) are filled with the PrP^Sc 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 (Chap. 40). Prions are self-propagating by imposing their conformations on the normal, precursor protein; 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 are 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 (Fig. 30-5).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 30-4). Over the past decade, an expanding body of experimental data has accumulated implicating 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 PrP^Sc prions. In animals, PrP^Sc 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 more recently in transgenic (Tg) mice inoculated with a synthetic Aβ peptide folded into a prion. Studies with the tau protein have shown that it not only features in the pathogenesis of AD, but also causes such illnesses as 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 has demonstrated that tau can become a prion and multiply in the brain. In contrast to the Aβ and tau prions, a strain of α-synuclein prions found in the brains of patients who died of multiple system atrophy (MSA) killed the Tg mouse host ~90 days after intracerebral inoculation, whereas α-synuclein prions formed spontaneously in Tg mouse brains killed recipient mice in ~200 days.

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 only be encoded within a nucleic acid. Subsequently, many studies demonstrated that strain-specified variation is enciphered in the conformation of PrP^Sc, but the molecular mechanisms responsible for the storage of 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.

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).

All mammalian prion proteins adopt a β-sheet-rich conformation and appear to readily oligomerize as this process becomes self-propagating. Control of the self-propagating state of benign mammalian prions is not well understood but is critical for the well-being of the host. In contrast, pathogenic mammalian prions appear to multiply exponentially, but the mechanisms by which they cause disease are poorly defined. We do not know if prions multiply as monomers or as oligomers; notably, the ionizing radiation target size of PrP^Sc prions seems to suggest it is a trimer. The oligomeric states of pathogenic mammalian prions are thought to be the toxic forms, and assembly into larger polymers, such as amyloid fibrils, seems to be a mechanism for minimizing toxicity.

To date, there is no medication that halts or even slows a 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 several drugs that cross the blood-brain barrier have been identified that prolong the lives of mice infected with scrapie prions, none have been identified that extend the lives of Tg mice that replicate human CJD prions. Despite doubling 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.

Systems neuroscience refers to study of the functions of neural circuits and how they relate to brain function, behavior, motor activity, and cognition. Brain imaging techniques, primarily functional magnetic resonance imaging (fMRI) and position emission tomography (PET), have made it possible to investigate, noninvasively and in awake individuals, cognitive processes such as perception, making judgments, paying attention, and thinking. This has allowed insights into how networks of neurons operate to produce behavior. Many of these studies at present are based on determining the connectivity of neural circuits and how they operate, and how this can be then modeled to produce improved understanding of physiologic processes. fMRI uses contrast mechanisms related to physiologic changes in tissue, and brain perfusion can be studied by observing the time course of changes in brain water signal as a bolus of injected paramagnetic gadolinium contrast moves through the brain. More recently, to study intrinsic contrast-related local changes in blood oxygenation with brain activity, blood-oxygen-level-dependent (BOLD) contrast has been used to provide a rapid noninvasive approach for functional assessment. These techniques have been reliably used in the field of both behavior and cognitive sciences. One example is the use of fMRI to demonstrate mirror neuron systems, imitative pathways activated when observing actions of others. Mirror neurons are thought to be important for social conditioning and for many forms of learning, and abnormalities in mirror neurons may underlie some autism disorders.

Both structural and functional connectivity methods show large-scale network dysfunction in AD and frontotemporal dementia. The networks targeted have been defined as the default network in AD and the salience network in frontotemporal dementia. The default network is characterized by an area of reduced glucose metabolism in the temporoparietal cortex, which precedes the onset of dementia and which is an area preferentially affected by amyloid deposition. These networks are now thought to be pathways accounting for the spread of abnormally templated proteins (prions; see above), including β-amyloid, tau, and α-synuclein.

Other examples of the use of fMRI include the study of memory, revealing that not only is hippocampal activity correlated with declarative memory consolidation, but it also involves activation in the ventral medial prefrontal cortex. Consolidation of memory over time results in decreased activity of the hippocampus and progressively stronger activation in the ventral medial prefrontal region associated with retrieval of consolidated memories. fMRI has also been used to identify sequences of brain activation involved in normal movements and alterations in their activation associated with both injury and recovery, to plan neurosurgical operations, and remarkably, to reconstruct actual visual images from the occipital cortex. Noninvasive brain-computer interfaces have extraordinary potential to advance the development of robotics and exoskeleton devices guided by brain activity for patients with a variety of nervous system afflictions. Diffusion tensor imaging is a recently developed MRI technique that can measure macroscopic axonal organization in nervous system tissues; it appears to be useful in assessing myelin and axonal injuries as well as brain development. Advances in understanding neural processing have led to the development of the ability to demonstrate that humans have online voluntary control of human temporal lobe neurons.

Multitasking capabilities, including attention to tasks when faced with distractions, decline as we age due to a decline in medial prefrontal cognitive control system. When faced with a multitasking challenge, video game training can improve cognitive control capabilities by augmenting prefrontal suppression of the default network and, as measured by electroencephalography and fMRI, result in improved performance that is sustained and, importantly, transfers to other cognitive tasks not associated with the training paradigm.

A significant recent advance in neuropathology has discovered that sodium dodecyl sulfate (SDS) detergent treatment can render the brain transparent (CLARITY), removing lipids while preserving most protein and structural elements and providing opportunities to identify brain structures and neural networks with unprecedented detail.

A therapeutic technology that has long-reaching implications for the development of novel interventions for neurologic, including behavioral, conditions has been the development of deep-brain stimulation as a highly effective therapeutic intervention for treating excessively firing neurons in the subthalamic nucleus of patients with Parkinson’s disease and the precingulate cortex in patients with depression.

The BRAIN initiative, grand in scope, was launched in 2013 to speed development of advances to understand, treat, repair, and prevent common neurologic disorders that, in aggregate, affect more than 1 billion people worldwide. The initial goal of BRAIN is to bring together experts in neurobiology (including optogenetics), engineering, information technology, and other fields to develop novel visualization and electrophysiologic methods to better define and understand neural circuits and all the connections among individual neurons. The announcement of the BRAIN initiative followed just weeks after a similarly ambitious program, the Human Brain Project (HBP), was unveiled by the European Union. The HBP seeks to model individual neurons, neural circuits, and ultimately the entire brain using computer technologies. Its architects also envision layering clinical and biomarker data from large health care databases to identify biosignatures associated with human phenotypes, possibly leading to a fundamental reclassification of disease, a concept also proposed by others, including in a 2011 report of the National Academy of Sciences (Toward Precision Medicine: Building a Knowledge Network for Biomedical Research and a New Taxonomy of Disease). These two ambitious projects are expected to be complementary and over time will hopefully become increasingly integrated. Emerging discoveries are also certain to stimulate a range of ethical questions, many of which are not unique to the neurosciences but do come into sharpest focus in this area. These include possible risks to the privacy of personal information about our health, cognitive capabilities, or behavioral attributes; the sanctity of our private thoughts; as well as concerns regarding neuroenhancement technologies, including their potential military use. Both the BRAIN and HBP initiatives have put into place strong ethical components to ensure that these programs are carried out, from the outset and to the fullest extent possible, consistent with guiding ethical principles that include respect for individuals, public beneficence, justice and fairness, democratic deliberation, and transparency.