44: Genetic Mechanisms in Degenerative Diseases of the Nervous System

• Expanded Trinucleotide Repeats Characterize Several Neurodegenerative Diseases

  • Huntington Disease Involves Degeneration of the Striatum

  • Spinobulbar Muscular Atrophy Is Due to Abnormal Function of the Androgen Receptor

  • Hereditary Spinocerebellar Ataxias Include Several Diseases with Similar Symptoms but Distinct Etiologies

• Parkinson Disease Is a Common Degenerative Disorder of the Elderly

• Selective Neuronal Loss Occurs After Damage to Ubiquitously Expressed Genes

• Animal Models Are Powerful Tools for Studying Neurodegenerative Diseases

  • Mouse Models Reproduce Many Features of Neurodegenerative Diseases

  • Invertebrate Models Manifest Progressive Neurodegeneration

• Several Pathways Underlie the Pathogenesis of Neurodegenerative Diseases

  • Protein Misfolding and Degradation Contribute to Parkinson Disease

  • Protein Misfolding Triggers Pathological Alterations in Gene Expression

  • Mitochondrial Dysfunction Exacerbates Neurodegenerative Disease

  • Apoptosis and Caspase Modify the Severity of Neurodegeneration

• Advances in Understanding the Molecular Basis of Neurodegenerative Diseases Are Opening Possibilities for Approaches to Therapeutic Intervention

• An Overall View

The major degenerative diseases of the nervous system—Alzheimer, Parkinson, and the triplet-repeat diseases (Huntington and the spinocerebellar ataxias)—afflict nearly 5 million people in the United States alone, and more than 25 million people throughout the world. Although this is a relatively small percentage of the population, these diseases bring a disproportionate amount of suffering and economic loss, not only to their victims but also to the families and friends of the afflicted.

Most of these disorders strike in mid-life or later; aging itself may in fact contribute to susceptibility. With the exception of Alzheimer disease, the first symptoms to appear usually involve loss of control of fine motor movements, although Huntington disease can first manifest itself in cognitive deficits. Nevertheless, the end result is the same. After a lengthy period of progressive deterioration, usually 10 to 20 years, the affected individual dies a terrible, helpless death.

The late-onset neurodegenerative diseases can be grouped conceptually into two categories: sporadic (unknown etiology) and inherited. Alzheimer disease and Parkinson disease are predominantly sporadic; inherited forms afflict a small number of patients. The triplet-repeat diseases, however, are notable for their dominant pattern of inheritance and the dynamic nature of the pathological mutation, an elongation of a CAG repeat tract that is subject to further expansion. Among the triplet-repeat neurodegenerative diseases are Huntington disease, the spinocerebellar ataxias, dentatorubropallidoluysian atrophy, and spinobulbar muscular atrophy. Identification of the molecular basis of some of these disorders has facilitated diagnosis and classification and provides hope for eventual treatment.

Huntington Disease Involves Degeneration of the Striatum

Huntington disease usually strikes in early or middle adulthood and affects 5 to 10 people per 100,000. The clinical presentation includes loss of motor control, cognitive impairment, and affective disturbance. Motor-control problems most commonly manifest themselves early as chorea, involuntary jerky movement that involves the small joints at first but then gradually creates instability of gait as the trunk and legs are affected. Fast, fluid movements are replaced by rigidity and bradykinesia (difficulty initiating action and unusually slow movements).

Cognitive impairment—such as difficulty in planning and executing complex functions—typically appears along with the involuntary movements but may be detected by formal neuropsychological testing even prior to motor dysfunction. Affective disturbances (psychiatric and behavioral features) include depression, irritability, social withdrawal, and disordered sleep. Hypomania and increased energy occur in 10% of the patients, whereas frank psychosis with delusions occurs in a smaller subset.

In adult patients the disease progresses inexorably to death some 17 to 20 years after onset. Juvenile-onset cases suffer a more rapid course of the disease and within only a few years typically develop bradykinesia, dystonia (spasm of the neck, shoulders, and trunk), rigidity (resistance to the passive motion of a limb), seizures, and severe dementia.

The pathological hallmark of Huntington disease is degeneration of the striatum, with the caudate nucleus being more affected than the putamen. Loss of a class of inhibitory interneurons in the striatum, the medium spiny neurons, reduces inhibition of neurons in the external pallidum (see Chapter 43). The resulting excessive activity of the pallidal neurons inhibits the subthalamic nucleus, which could account for the choreiform movements. As the disease progresses and striatal neurons projecting to the internal pallidum degenerate, rigidity replaces chorea. Abnormalities in corticostriatal projections are thought to contribute to pathogenesis. Juvenile cases suffer a more severe and generalized pathology that often includes cerebellar Purkinje cells.

Huntington disease is an autosomal dominant disorder and one of the first human diseases to have its gene mapped using polymorphic DNA markers. It is caused by expansion of a translated CAG repeat that encodes a glutamine tract in the huntingtin protein. Normal or wild-type alleles have 6 to 34 repeats, whereas disease-associated alleles typically have 36 or more repeats and are quite unstable when transmitted from one generation to the next, especially through paternal germ cells.

The dynamic nature of the mutation, expanding in successive generations, accounts for the greater severity of the disease in juvenile-onset cases, a phenomenon known as anticipation. The length of the repeat correlates inversely with the age of onset, one of the many common features of neurodegenerative diseases caused by CAG-repeat expansions (Figure 44–1). Huntington disease-like 2 (HDL2), a rare neurodegenerative disorder that is clinically similar to Huntington disease, is caused by CTG expansion in junctophilin 3.

Huntington disease appears to be a true dominant disease in that patients homozygous for the condition do not differ significantly from their heterozygous siblings. The expanded glutamine tract causes the huntingtin protein to gain toxic function in addition to its normal function. Huntingtin is expressed throughout the brain in the cytoplasm, where it associates with microtubules, with a minor fraction present in cell nuclei. Although its precise functions are unknown, huntingtin is an essential protein in normal embryonic development as shown by mouse knock-out studies; it is also essential for neuronal integrity in the postnatal brain.

Spinobulbar Muscular Atrophy Is Due to Abnormal Function of the Androgen Receptor

Spinobulbar muscular atrophy (Kennedy disease), the only X-linked disorder among the neurodegenerative diseases discussed in this chapter, is caused by expansion of a translated CAG repeat in the androgen receptor protein, a member of the steroid hormone receptor family. Only males manifest symptoms; the mutant androgen receptor is toxic when in the nucleus, and such localization requires the male hormone androgen. Proximal muscle weakness is usually the presenting symptom; eventually the distal and facial muscles weaken as well. Muscle wasting is prominent, secondary to degeneration of motor neurons.

Bulbar dysfunction results from loss of brain stem motor neurons. Many patients also develop gynecomastia, late hypogonadism, and sterility, indicating the loss of androgen receptor function. Individuals lacking androgen receptor function without expansion of CAG repeats do not, however, develop motor neuron degeneration. It thus appears that the glutamine expansion causes a partial loss of function that accounts for the secondary sexual characteristics and a partial gain of function that affects neurons and produces the neurological phenotype.

Hereditary Spinocerebellar Ataxias Include Several Diseases with Similar Symptoms but Distinct Etiologies

The spinocerebellar ataxias and dentatorubropallidoluysian atrophy are dominantly inherited neurodegenerative diseases that, for all their heterogeneity, are characterized predominantly by dysfunction of the cerebellum, spinal tracts, and various brain stem nuclei. The basal ganglia, cerebral cortex, and peripheral nervous system are also affected in some subtypes or in isolated cases (Table 44–1).

The two clinical features common to all the spinocerebellar ataxias are ataxia and dysarthria. These typically appear in mid-adulthood and gradually worsen, making walking impossible and speech incomprehensible. The brain stem dysfunction manifests itself through difficulties in swallowing and breathing and eventually causes death.

Features such as chorea or dementia are associated more strongly with one spinocerebellar ataxia than others, but these symptoms are so variable that they cannot be reliably used to refine the diagnosis. Even individuals within the same family can present a quite different clinical picture. Thus, although the spinocerebellar ataxias are single-gene Mendelian disorders, individual genetic makeup and environmental influences clearly affect the clinical-pathological situation.

For example, Machado-Joseph disease and spinocerebellar ataxia type 3 (SCA3) had been regarded clinically as distinct diseases before it was discovered that they are caused by mutations in the same gene. The clinical confusion arose by historical accident. The most prominent features of the Portuguese families first studied by Machado and Joseph were bulging eyes, faciolingual fasciculations, parkinsonism, and dystonia, whereas the first SCA3 patients had features more reminiscent of SCA1 (hypermetric saccades and brisk reflexes in addition to the characteristic ataxia and dysarthria). We now know that these apparent clinical differences are at least partially attributable to differences in length of the CAG repeats. Nonetheless, differences in the activity of other proteins caused by genetic variations are probably also at play.

Although the age of onset within each type of ataxia depends on the number of CAG repeats in the gene (Figure 44–1), the toxicity of the abnormally long glutamine tract in the protein product depends on the protein: Expanded glutamine tracts have different effects in different proteins. For example, very short repeat lengths that are detrimental to Purkinje cells in SCA6 are nonpathogenic in other SCAs. In fact, the CAG expansion in SCA6 is the shortest of all the spinocerebellar ataxias: 21 to 33 repeats in mutants compared to fewer than 18 in normal alleles. In contrast, the gene responsible for SCA7 normally tolerates a few dozen CAG repeats, and in the disease state undergoes some of the largest expansions seen in any spinocerebellar ataxia (hundreds of CAGs). (Table 44–2.)

Besides tolerating different CAG repeat lengths, the gene products of mutated genes in polyglutamine diseases vary widely in function. The affected gene product in SCA1, ataxin-1, seems to be important for learning and memory; it is predominantly a nuclear protein that shuttles to the cytoplasm and can bind RNA in vitro, which suggests that it might play a role in RNA transport and processing. The affected gene product in SCA6, CACNA1A, is the α_1A subunit of the voltage-gated Ca²⁺ channel; interestingly enough, loss-of-function mutations in the gene (not caused by CAG repeats) have been reported in patients with episodic ataxia and familial hemiplegic migraine. In SCA17 the affected gene product is the TATA box-binding protein, an essential transcription factor. The affected product in dentatorubropallidoluysian atrophy, atrophin-1, is thought to be a corepressor based on functional studies of its probable ortholog in Drosophila. Despite these differences, some pathogenetic mechanisms may be common to the polyglutamine diseases, as discussed later in this chapter.

A few spinocerebellar ataxias are caused by unstable trinucleotide repeats other than CAG (Table 44–2). Spinocerebellar ataxia type 8 is caused by an expansion of a CTG repeat in the 3′ untranslated region of a transcribed RNA with no open reading frames. The mutation responsible for SCA12 is a CAG repeat, but it occurs in a noncoding region upstream of a brain-specific regulatory subunit of the protein phosphatase 2A. Spinocerebellar ataxia type 10 is unique in that it is caused by massive expansion of a pentanucleotide (ATTCT) repeat in the intron of a novel gene. The pathogenic mechanisms accounting for the dominant phenotypes in spinocerebellar ataxia types 8, 10, and 12 are not yet known.

Parkinson disease, one of the more common neurodegenerative disorders, affects 1% to 2% of the population older than 65 years of age. Patients with Parkinson disease suffer from a resting tremor, bradykinesia, rigidity, and impairment in their ability to initiate and sustain movements. Affected individuals walk with a distinctive shuffling gait, and their balance is often precarious. Spontaneous facial movements are greatly diminished, such that the face has a mask-like, expressionless appearance. The pathological hallmark of Parkinson disease is progressive loss of dopaminergic neurons, mainly in the substantia nigra (see Chapter 43).

Although the majority of parkinsonian cases are sporadic, studies of rare familial cases have provided insight into the genetic factors that predispose individuals to this disorder. Here we focus on how the genetic bases of some forms of Parkinson disease provides insights into sporadic Parkinson disease and link the pathogenic mechanism of parkinsonism to that seen in the polyglutamine disorders.

Both autosomal dominant and recessive inheritance patterns have been documented in familial parkinsonism. To date, several genetic loci have been mapped (designated PARK1–PARK8, PARK10, and PARK11), and the genes for all but three of these loci (PARK3, PARK10, and PARK11) have been identified (Table 44–3).

Parkinson disease type 1 (PARK1) is the locus for the dominantly inherited Parkinson disease caused by mutations in the gene α-synuclein. Two mutations in α-synuclein have been identified: A53T has been described in several Greek families, whereas A30P has been identified in one German family. Mutations in α-synuclein have not been identified in sporadic Parkinson disease. However, because the α-synuclein protein is a primary component of the Lewy bodies in the substantia nigra of patients with sporadic disease as well as those with PARK1, α-synuclein mutations could play an important role in the pathogenesis of the sporadic disease.

The function of the α-synuclein protein is not yet known, but its abundance in presynaptic terminals suggests a role in presynaptic function and perhaps synaptic plasticity. Patients with α-synuclein mutations differ from those with sporadic Parkinson disease in that the age of onset is earlier (a mean of 45 years), and they exhibit fewer tremors and more rigidity, cognitive decline, myoclonus, central hypoventilation, orthostatic hypotension, and urinary incontinence.

Parkinson disease type 2 (PARK2) is an autosomal recessive disease characterized by early onset (as young as three years of age), dystonia, brisk deep-tendon reflexes, and cerebellar signs in addition to the classic Parkinson disease phenotype. More than 60 different mutations have been identified in the gene PARK2, and most are clearly inactivating, demonstrating that this form of the disease is caused by loss of function of the gene product, parkin. Whereas α-synuclein mutations have not been detected in the sporadic disease, mutations in the parkin gene have been found in isolated cases of early-onset Parkinson disease and in one patient with onset at 65 years of age. Loss of dopaminergic neurons in the substantia nigra is typical of this form of the disease, but Lewy bodies are not as common as in sporadic or PARK1 cases.

The parkin gene encodes an E3 ubiquitin ligase of the RING-finger family that transfers activated ubiquitin to lysine residues in proteins destined for degradation by proteasomes. The ligase is quite specific and transfers ubiquitin to only a few substrates. Some substrates for parkin have been identified, including a putative transmembrane G protein-coupled receptor named parkin-associated endothelin receptor-like receptor (Pael-R), the synaptic vesicle protein CDCrel-1, the O-glycosylated form of α-synuclein, the α-synuclein interactor synphilin-1, and parkin itself.

A missense mutation, I93M, in the gene encoding ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been identified in a family with an apparently autosomal dominant Parkinson disease, PARK5. UCH-L1 is an abundant protein in the brain and is thought to cleave polyubiquitin chains as the ubiquitinated proteins are being degraded by the proteasome. This activity is decreased in individuals with the 193M mutant. A homologous protein is necessary for the formation of memory in the marine mollusk Aplysia.

Parkinson disease type 6 (PARK6) is caused by mutations in a gene that encodes a PTEN-induced putative kinase 1 (PINK1), a mitochondrial protein kinase, whereas Parkinson disease type 7 (PARK7) is caused by mutations in a gene that encodes DJ-1, a protein that may function as a sensor of oxidative stress. Mutations in the gene encoding the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson disease type 8 (PARK8) as well as a small percentage of sporadic Parkinson disease cases.

A perplexing aspect of these neurodegenerative diseases is that the altered gene products are widely and abundantly expressed not only in the nervous system but also in other tissues, yet the phenotypes are predominantly neurological. Moreover, the phenotypes reflect dysfunction in only specific groups of neurons (Figure 44–2), a phenomenon referred to as neuronal selectivity.

Why are striatal neurons the most vulnerable in Huntington disease, whereas the Purkinje cells are targeted in the spinocerebellar ataxias (SCA)? Why are the dopaminergic neurons in the substantia nigra primarily affected in Parkinson disease even though α-synuclein, parkin, DJ-1, PINK1, LRRK2, and UCH-L1 are abundant in many other neuronal groups? Although definitive answers are not yet available, there are some clues.

In the polyglutamine diseases the selectivity of the cellular pathology diminishes as the length of the glutamine tract increases: the more severe the mutation, the greater the number of neuronal groups affected. This is especially evident in the early-onset forms characterized by extremely long repeats. Juvenile SCA1 patients suffer from dystonia, rigidity, and cognitive impairment, features that overlap with Huntington disease and dentatorubropallidoluysian atrophy. Juvenile SCA7 patients can suffer seizures, delusions, and auditory hallucinations, and infantile cases also develop somatic features, including short stature and congestive heart failure. Spinocerebellar ataxia type 7 also causes progressive blindness owing to dystrophy of both rods and cones; interestingly, cases of infants with SCA2 who also suffer retinal degeneration have recently been reported.

These and similar observations suggest that various cell types have different thresholds of vulnerability to toxic proteins with expanded glutamine tracts. Retinal cells, for example, seem more resistant to polyglutamine toxicity than cerebellar neurons, but more vulnerable than cardiac myocytes. Once the number of glutamines in the tract expands beyond a certain length—which varies from one protein to the next—no cell is safe.

Studies using mouse models suggest that protein misfolding is responsible for polyglutamine disorders. The longer the glutamine tract, the more severe the misfolding and protein accumulation. As the tracts become very long, even cells with lower concentrations of disordered gene product become vulnerable. Indeed, studies of animal models show that even a doubling in concentration can be the difference between phenotypic manifestation and apparent normality. It is therefore conceivable that the neurons affected in each disease have more mutant protein than do the less vulnerable neurons. Although not detectable by current immunolabeling techniques, this increment would nevertheless be sufficient to interfere with cellular function if the neuron was exposed to the toxic mutant protein over decades.

Other major contributors to selective vulnerability might be variations in the levels of proteins that interact with or help dispose of the mutant proteins. Variations in the genes encoding such proteins could contribute to the clinical variability that is so prominent among ataxia families.

Why are neurons affected before other cells? As the organism ages, slight insults that have small detrimental effects could be exacerbated by the extra challenge the mutant protein presents to the protein-folding machinery. Because neurons are post-mitotic, they might be especially sensitive to perturbations in the balance of intracellular factors. If the organism could survive the neurological assault long enough, other tissues might also eventually show signs of distress.

Animal models have proven extremely valuable for probing the pathogenesis of various neurodegenerative diseases and investigating therapies. Because of the ease in making mutations, the mouse has been the favored animal for modeling neurological disorders, but Drosophila models also have proven useful in the delineation of genetic pathways.

Mouse Models Reproduce Many Features of Neurodegenerative Diseases

With the exception of PARK2, the neurodegenerative diseases discussed here are caused mostly by a gain of function rather than loss of function. Thus most of the genetically engineered mice that model these diseases are created using two techniques. In transgene experiments an allele harboring the mutant gene is overexpressed, whereas in knock-in experiments a human mutation, such as an expanded CAG tract, is inserted into an endogenous mouse locus to control expression of the gene product temporally and spatially (see Box 3–2). In some mutants, such as spinocerebellar ataxia types 1, 2, 3, and 7, dentatorubropallidoluysian atrophy, and some Huntington disease models, a full-length cDNA with either wild-type or expanded alleles is overexpressed either in a particular class of neurons or in a larger population of cells. In other mutants, such as SCA3, Huntington disease, and spinobulbar muscular atrophy, truncated versions of the coding regions are expressed.

Knock-in mice have been generated for Huntington disease and spinocerebellar ataxia types 1 and 7. These models confirm that features other than the length of the expanded glutamine tract affect the toxicity of the tract. As noted above, the same expansion in two different host proteins affects cells differently. For example, in human patients 37 repeats cause SCA2 but not SCA3 (see Figure 44–1 and Table 44–2). In mouse models, however, the relationship of the length of the tract to the rest of a given host protein is a good predictor of toxicity.

Severe, widespread, nonselective neuronal dysfunction occurs in both transgenic mice in which the glutamine tract constitutes a major portion of the protein and mice bearing a truncated protein with a relatively large glutamine tract. In contrast, mice that express full-length proteins containing a CAG repeat develop a neurological syndrome that progresses more slowly. Similarly, weakly expressing promoters generally produce more selective neuronal dysfunction (Figure 44–3). In some cases expression of the full-length protein with even a moderately large expansion does not cause neurological dysfunction, but a truncated version bearing a similar repeat size produces the disease phenotype. In sum, a glutamine tract of a certain length is more toxic when in isolation or flanked by short peptide sequences, that is, when it occupies a larger proportion of the protein.

In knock-in mice with 80 to 111 glutamine repeats, neurological dysfunction is barely detectable; only when the repeat length is expanded to approximately 150 glutamines does a neurological phenotype become apparent. Because polyglutamine toxicity takes time to exert its effects, longer repeats are necessary to see a phenotype during the short life span of a mouse. However, massive overproduction of the mutant protein, as in transgenic mice, can compensate for a moderate repeat length and brevity of exposure to the toxic repeat. Indeed, overproduction of wild-type ataxin 1 or α-synuclein in mice results in mild neurological dysfunction.

Analysis of brain tissue from patients and experimental mice reveals that misfolded proteins tend to accumulate in various neurons, sometimes forming visible aggregates (Figure 44–4). Lewy bodies and abnormal accumulation of α-synuclein, as observed for years in patients, develop in mouse models of Parkinson disease. Although protein accumulation is common to all these neurodegenerative disorders in humans and their respective mouse models, the localization of the accumulated protein in the cell varies, and location within the cell is a factor in the protein's pathogenicity. For example, mutant ataxin 1 that remains in the cytoplasm because of a mutant nuclear-localization signal exerts no detectable toxic effects.

The fact that mutated proteins accumulate in mouse models that do not overproduce the proteins as well as in human patients who carry a single mutant allele suggests that neurons have difficulty clearing the proteins. Support for this hypothesis comes from the finding that ubiquitin and proteasome components, the machinery of protein degradation, localize to the site of protein aggregates in both human and mouse tissues.

Invertebrate Models Manifest Progressive Neurodegeneration

Several invertebrate models have been used to study polyglutamine proteins and α-synuclein. The similarities in the pathogenic effects of these proteins across species are remarkable.

Flies with high levels of human α-synuclein develop age-dependent, progressive degeneration of dopaminergic neurons and have α-synuclein–immunoreactive cytoplasmic aggregates reminiscent of Lewy bodies. As in the mouse model, high levels of either wild-type or mutant α-synuclein in flies induce this phenotype, and the toxic effect of the expanded glutamine tract is reduced as more amino- or carboxyl-terminal amino acids are added to the polypeptide. Overproduction of wild-type or mutant ataxin-1 in flies induces progressive neuronal degeneration that is dependent on protein levels but is more severe for the mutant when protein levels are similar.

Polyglutamine toxicity has also been evaluated in the nematode Caenorhabditis elegans by expressing an amino terminal fragment of huntingtin containing glutamine tracts of different length. Neuronal dysfunction and cell death occur in worms expressing expanded alleles.

Protein Misfolding and Degradation Contribute to Parkinson Disease

Proteins with expanded glutamine tracts and mutant α-synuclein accumulate in the neurons of patients and various animal models, indicating that these abnormal proteins are not degraded efficiently. Molecular chaperones, which facilitate cell protein refolding and degradation (see Chapter 4), are redistributed to the site of these protein aggregates.

The gradual accumulation of proteins with expanded glutamine tracts along with chaperones and components of the ubiquitin-proteasome degradation pathway, suggests that the glutamine tract expansion alters the folding state of the native protein, which in turn recruits the activity of the protein-folding and degradation machinery. When that machinery cannot process the protein, the protein molecules accumulate, eventually forming aggregates. Wild-type α-synuclein and ataxin-1 may have some tendency to misfold even in the absence of a mutation; when they are produced at sufficiently high levels the number of misfolded molecules increases, and their toxicity becomes apparent.

Evidence in support of this idea first came from studies in cell culture in which overproduction of chaperones both reduces protein aggregation and mitigates the toxicity of expanded glutamine tracts in proteins. In contrast, blocking the proteasome inhibits protein degradation and thus enhances aggregation and toxicity. Genetic studies in flies and mice provide even more compelling evidence. Overproduction of at least one chaperone, such as Hsp70, Hsp40, or tetratricopeptide protein 2, suppresses polyglutamine toxicity in Drosophila and mouse models of Parkinson disease and several different ataxias, whereas loss of chaperone function aggravates neurodegeneration (Figure 44–5).

The importance of the ubiquitin-proteasome pathway and protein degradation in the spinocerebellar ataxias is further supported by genetic modification in animal models. In a Drosophila model of SCA1, haploinsufficiency for ubiquitin, ubiquitin carrier enzymes, or a ubiquitin carboxyl-terminal hydrolase aggravates neurodegeneration. Loss of function of the E3 ubiquitin ligase Ube3a in mice exacerbates ataxin-1–induced neurodegeneration, even though the Purkinje cells develop no nuclear inclusions. It appears that inclusions are part of the cell's attempt to sequester the mutant protein and thereby limit its toxic effects. Cells that are unable to form aggregates of the mutated proteins suffer the worst damage from polyglutamine toxicity. Indeed, the knock-in mouse models of spinocerebellar ataxia types 1 and 7 show conclusively that cells that form aggregates survive longer; cerebellar Purkinje cells, the prime targets in this disease, are the last to form nuclear aggregates.

Studies of Parkinson disease further underscore the importance of the ubiquitin-proteasome pathway and reveal additional parallels with the polyglutamine diseases. The parkin gene, mutated in Parkinson disease type 2, encodes an E3 ubiquitin ligase that has among its targets the O-glycosylated form of α-synuclein. Levels of this glycosylated form of α-synuclein are increased in brains of patients with deformed parkin despite the absence of Lewy bodies. This suggests that Lewy-body formation requires ubiquitination of α-synuclein, and again indicates that the misfolded protein is sequestered when it is not completely degraded.

How do misfolded α-synuclein or expanded glutamine tracts disrupt neuronal function? A protein that resists degradation or has an aberrant conformation might have its normal function in the cell enhanced, as happens with glutamine-expanded ataxin-1. Part of the gain of toxic function involves alterations in gene expression.

Protein Misfolding Triggers Pathological Alterations in Gene Expression

One of the key consequences of misfolding as a result of expanded glutamine tracts is alteration in gene expression. This was first suspected when it was realized not only that most of the mutant proteins accumulate in the cell nucleus, but also that they interact with or affect the function of key transcriptional regulators. For example, CREB-binding protein interacts with huntingtin exon 1. Moreover, overproduction of polyglutamine proteins reduces levels of histone acetylation in cells, an effect that can be reversed by overproduction of CREB-binding protein.

Alterations in gene expression are among the earliest events in pathogenesis, occurring within days of expression of the mutant transgene in mouse models of SCA1 and Huntington disease. Many of the genes whose expression is altered are involved in Ca²⁺ homeostasis, synaptic transmission, and transduction of sensory events into neural signals. In fly models of SCA1 several modifiers of the neurodegenerative phenotype are transcriptional cofactors. Alterations in gene expression may also occur because of altered RNA processing or stability. Ataxin-1 binds RNAs in vitro, and several genetic modifiers in fly models of SCA1 encode proteins involved in RNA binding or processing.

Mitochondrial Dysfunction Exacerbates Neurodegenerative Disease

Evidence for mitochondrial dysfunction in polyglutamine disorders and Parkinson disease comes from morphological and functional studies. Lymphoblast mitochondria from patients with Huntington disease as well as brain mitochondria from a transgenic mouse model for Huntington disease have a lower membrane potential and depolarize at lower Ca²⁺ loads than do control mitochondria.

Several proteins implicated in Parkinson disease affect mitochondrial function and integrity. For example, in Drosophila the loss of PINK1 leads to mitochondrial dysfunction that can be rescued by parkin. LRRK2, which interacts with parkin, is localized in part to the outer mitochondrial membrane. Thus, given the functions and interactions of these proteins, mitochondrial dysfunction is likely to be a key contributor to the Parkinson disease phenotype.

Apoptosis and Caspase Modify the Severity of Neurodegeneration

Although animal studies of most neurodegenerative diseases have demonstrated that symptoms appear long before detectable cell death, loss of neurons is a hallmark of the end stage of all these disorders. Although many factors are implicated in the death of neurons, such as altered Ca²⁺ homeostasis and decreased induction of neuronal survival factors (eg, brain-derived neurotrophic factor in Huntington disease), there is specific evidence that the caspase activity critical for apoptosis is a contributing factor in neurodegenerative diseases. Some of the polyglutamine proteins, such as huntingtin, AR, ataxin-3, and atrophin-1, are substrates for caspases in vitro. This raises the possibility that the proteases liberate the fragments of these proteins with expanded glutamine tracts. As discussed earlier, such fragments are even more damaging than the full-length protein.

Intranuclear huntingtin increases production of caspase-1 in cells; this could lead to apoptosis and caspase-3 activation. Hip-1, a protein that interacts with huntingtin, forms a complex that activates caspase-8. This process might be enhanced by the glutamine expansion in huntingtin, because Hip-1 binds less avidly to mutant huntingtin than to the wild-type protein. In Drosophila production of the anti-apoptotic protein p35 results in partial rescue of the pigment loss induced by mutant ataxin-3.

In summary, expansions of polyglutamine tracts as well as several missense mutations in proteins implicated in neurodegenerative diseases alter the host protein, leading to its accumulation or abnormal interactions. The neuronal dysfunction results from the downstream effects of such abnormal interactions (Figure 44–6).

The discovery of the genetic bases and pathogenic mechanisms of various neurodegenerative diseases gives us hope that therapies for these diseases will soon emerge.

Dopamine replacement therapy has so far been the only option for Parkinson disease and other conditions with parkinsonian features, but it is not ideal. Patients tend to develop tolerance and require higher and higher doses of the drugs. Side effects therefore become as troubling as the symptoms being treated. Patients with Huntington disease and spinocerebellar ataxia are in a worse state: There are no treatments that slow the progressive loss of motor coordination.

The identification of protein misfolding as a key step in pathogenesis has led investigators to search for drugs that safely induce chaperone activity and that can be tested in the various animal models. Geldanamycin, a drug known to activate heat-shock chaperones, suppresses polyglutamine-induced protein aggregation and toxicity in cell culture. Because some glutamine-expanded proteins interact with or sequester CREB-binding protein, a protein with histone acetylase activity, inhibitors of histone deacetylase such as sodium butyrate and suberoylanilide hydroxamic acid have been used in mammalian cells, yeast cells, and Drosophila models expressing a polypeptide with an expanded glutamine tract. These treatments suppress cell death and improve the viability of cells of fruit flies—a very promising finding.

Cystamine, an inhibitor of the transglutaminase that is thought to promote protein aggregation, improves neuronal function. Moreover, caspase inhibitors enhance the survival of mice expressing an expanded glutamine tract in huntingtin exon 1. These results suggest that several pathways can be targeted for therapeutic intervention. Ideally, therapies would be targeted at some of the earliest pathogenic stages, at which neuronal dysfunction rather than cell death causes the neurological phenotype. Intervention at an early stage could prove beneficial clinically, for it might halt the disease and even allow recovery of function. Indeed, in mouse models of Huntington disease and SCA1 in which expression of the mutant gene can be turned on and off, neuronal dysfunction is reversible. When expression of the transgene is turned off, the neurons have a chance to clear the mutant polyglutamine protein and regain normal activity.

Because most neurodegenerative diseases progress over a period of decades, pharmacological interventions that even slightly modulate one or more of the pathways described earlier could delay disease progression or modestly improve function, which would greatly enhance the quality of life for patients suffering from these devastating disorders.

The identification of genes causing several forms of Parkinson disease and the various polyglutamine neurodegenerative diseases has allowed the accurate diagnosis and classification of these clinically heterogeneous disorders. More importantly, studies in cell culture and model organisms have revealed a pathogenic mechanism common to all of these diseases: protein misfolding. Mutations that cause the respective proteins to adopt an altered conformation gradually induce neuronal dysfunction either because of abnormal protein interactions or because of intracellular protein accumulation and altered activity. The identification of pathways that mediate some of the pathogenic effects is likely to lead to the discovery of drugs that can first be tested in model animals and then applied in humans.