Chapter 3 The Genetic Basis and Molecular Pathogenesis of Huntington's Disease

Chapter 3

The Genetic Basis and Molecular Pathogenesis of Huntington’s Disease NEIL W. KOWALL, STEPHAN KUEMMERLE, and ROBERT J. FERRANTE

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81 Genetic Basis of Huntington’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . 82 Neuronal Degeneration in Huntington’s Disease . . . . . . . . . . . . . . . . . . . 83 Normal Distribution and Function of Huntington in the Brain . . . . . . . . . . . 84 TrinucleotideRepeat Expansion and the Molecular Pathogenesis of Huntington’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Distribution of Huntingtin in Huntington’s Disease: Nuclear Inclusions . . . . . . 86

INTRODUCTION Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder characterized clinically by progressive cognitive decline and chorea. Onset is typically in adulthood but a more fulminant juvenile form is well recognized. Initial clinical manifestations may be behavioral so the diagnosis may be missed for years until the classical movement disorder develops. Psychosis, obsessive thought disorder, and dementia are common. Mood disorders, personality changes, irritable and explosive behavior, schizophrenia-like behavior, suicidal behavior, sexuality changes, and specific cognitive deficits can occur. The choreiform movement disorder is progressive, affects the extremities and face, and is associated with Advances in Cell Aging and Gerontology Volume 3, pages 81-92 Copyright 0 1999 by JAI Press Inc. A11 rights of reproductionin any form reserved. ISBN: 0-7623-0405-7

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slowness and clumsiness of fine movements and postural instability (Thompson et al., 1988). Reflexes are typically unaffected and tone is reduced in early stages but is increased later (Homberg and Huttunen, 1994). There are no sensory abnormalities. In late stages patients are cachectic, dystonic, rigid, and bedridden. The disease typically runs its course over several years. Studies show that the likelihood of HD in a patient with the typical clinical features of this disorder but no history of affected relatives is at least 75%. The most plausible explanations for seemingly sporadic patients with HD are nonpaternity and mild, late-onset disease that is overlooked by other family members (Bateman et a]., 1992), but new mutations may also occur (Andrew and Hayden, 1995; Alford et al., 1996).

GENETIC BASIS OF HUNTINGTON’S DISEASE The discovery of the HD gene on chromosome 4 in 1993 was a major step forward (Huntington’s Disease Collaborative Research Group, 1993; Gusella and MacDonald, 1995). The gene was initially called IT 15 (interesting transcript 15) or the huntingtin gene. The genetic abnormality was unexpected; rather than a simple missense mutation or deletion, the HD gene is expanded with an excess number of CAG trinucleotide repeats that result in a long stretch of polyglutamine in the expressed protein. Expansion of trinucleotide repeats is now recognized as a major cause of neurological disease (Plassart and Fontaine, 1994). At least eight disorders result from trinucleotide repeat expansion: X-linked spinal and bulbar muscular atrophy (Kennedy’s syndrome, SBMA), two fragile X syndromes of mental retardation (FRAXA and FRAXE), myotonic dystrophy, HD, spinocerebellar ataxia type 1 (SCA 1, 6p), spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD, 14q) and dentatorubral-pallidoluysian atrophy (DRPLA, 12p). The expanded trinucleotide repeats are unstable, and the phenomenon of anticipation (i.e., worsening of disease phenotype over successive generations) correlates with increasing expansion size. These disorders may be subdivided into two classes: Fragile X and myotonic dystrophy are multisystem disorders usually associated with large expansions of untranslated repeats, whereas the neurodegenerative disorders-SBMA, Huntington’s disease, SCAl , SCA3, and DRPLA-are caused by smaller expansions of CAG repeats within the protein coding portion of the gene. Polyglutamine expansion appears to be a common element that may lead to a toxic gain of function effect (La Spada et al., 1994). SBMA, or Kennedy’s syndrome, is an X-linked, adult-onset motor neuronopathy caused by expansion of a trinucleotide (CAG) repeat in the androgen receptor gene. The length of this repeat varies as it is passed down through SBMA families, and correlates inversely with the age of onset of the disease. The motor neuron degeneration that occurs in this disease is probably caused by a toxic gain of function in the androgen receptor protein (Brooks and Fischbeck, 1995).

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NEURONAL DEGENERATION IN HUNTINGTON’S DISEASE Even though the genetic abnormality in HD has been defined, the underlying mechanism of neuronal degeneration has not been discovered. Neuropathological studies of human brain identified selective patterns of neuronal degeneration in HD and have driven the creation of animal models (Kowall et al., 1987; Beal, 1992). Initially it was hypothesized that excitotoxic injury caused neuronal degeneration in HD (Coyle and Schwarcz, 1976; McGeer and McGeer, 1976). Animal studies showed that NMDA-type excitotoxins reproduce the patterns of neuronal loss found in HD (Beal et al., 1991; Ferrante et al., 1993). An abnormality affecting the NMDA receptor, reduced levels of an endogenous NMDA antagonist or increased production of an endogenous excitotoxin were thought to be potential causes of HD but no abnormality related to glutamate receptors was identified (Beal et al., 1990, 1992; Bruyn and Stoof, 1990; Pearson and Reynolds, 1992). More recently it has been thought that HD may result from impaired mitochondrial oxidative phosphorylation because an identical pattern of differential neuronal loss can be produced in rodents and primates by mitochondrial electron transport chain inhibitors (Beal, 1992; Brouillet et al., 1994,1995). These inhibitors cause partial energy failure that triggers NMDA-receptor mediated excitotoxic injury, decreased free radical scavenging and increased production of free radicals (Beal, 1994). Other indirect lines of evidence suggest a possible role for mitochondrial abnormalities in the pathogenesis of HD as well (Blass et al., 1988; Parker et al., 1990; Gu et al., 1996). There is direct evidence of abnormal electron transport chain activity in HD. Decreased activity of complex IYIII of the electron transport chain has been found in the caudate nucleus, but not in other brain areas in HD (Mann et al., 1990; Gu et al., 1996). Cytochrome oxidase (complex IV) abnormalities have also been reported in the caudate nucleus (Brennan et al., 1985; Gu et al., 1996). Studies of platelets from HD patients suggest that complex I activity may be selectively decreased in HD patients although it is normal in at-risk family members (Parker et al., 1990). Complex I is composed of over 30 subunits, the majority of which are encoded by nuclear DNA (Hatefi, 1985). Other electron transport chain complexes, including complex 11, complex I11 (ubiquinol cytochrome c reductase), and complex IV (cytochrome oxidase) are normal in blood platelets (Parker et al., 1990). Depletion of complex JV activity parallels the pattern of neuronal loss in HD striatum (Ferrante et al., 1988). Other indirect lines of evidence suggest a possible role for mitochondria in the pathogenesis of HD. Tellez-Nagel et al. (1973) performed ultrastructural studies on brain biopsies of four HD patients and found evidence of mitochondrial abnormalities and increased lipofuscin, a pigment that accumulates as a consequence of free radical-mediated membrane damage. The delayed onset of HD may be a result of the contribution of age-related mutations of mitochondrial DNA that cause decreased mitochondrial function, as shown by Trounce and co-workers in skeletal muscle (Trounce et al., 1989). Even though the neuron is postmitotic, the mitochon-

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dria in neurons continue to proliferate (Linnane et al., 1989). Focal putaminal degeneration has been reported in a family with Leber’s disease caused by a point mutation in NADH-dehydrogenase (Larsson et al., 1991). Encephalopathy also occurs in patients with MELAS, another disease characterized by a deficiency in complex I (Wallace, 1991). Jenkins and co-workers have found increased anaerobic metabolism in the cerebral cortex of patients with HD using magnetic resonance spectroscopy, further suggesting that an abnormality of oxidative phosphorylation occurs in this disease (Jenkins et al., 1993). Systemic administration of electron transport chain inhibitors to animals causes focal basal ganglia pathology. Intravascular administration of rotenone, an electron transport chain complex 1 inhibitor, causes degeneration of the striatum and pallidum in rats (Ferrante et al., 1997b). Oral or intraperitoneal administration of 3-nitropropionic acid (3NP), an electron transport chain complex I1 inhibitor, produces striking focal pathology that resembles excitotoxin lesions (Borlongan et al., 1995; Brouillet et al., 1995). Lesions can be blocked with theNMDA antagonist MK8Ol or free radical spin trapping agents (Schulz et al., 1995). Interestingly, chronic food restriction, a manipulation that extends life span in rodents and monkeys (Finch and Morgan, 1997), greatly reduces the vulnerability of striatal neurons to 3NP toxicity and improves behavioral outcome (Bruce-Keller et al., 1998). The latter findings support a role for age-related increases in free radical production or neuronal susceptibility to free radical damage, or both, in HD. How do these experimental findings relate to the underlying genetic cause of HD? The answer lies with a better understanding of the physiological role and pathological effects of the huntingtin gene product.

NORMAL DISTRIBUTION AND FUNCTION OF HUNTINGTON IN THE BRAIN Sharp et al. (1995) found widespread expression of huntingtin, most prominently in neurons with no enrichment in the striatum. They found that it was localized to the cytoplasm, especially in nerve terminals, and was loosely associated with membranes and the cytoskeleton. Hersh and colleagues used immunocytochemical methods with antipeptide antibodies and found that huntingtin is present throughout the brain enriched in large neurons and striatal patches associated with microtubules, and to a lesser degree synaptic vesicles (Gutekunst et al., 1995). Trottier et al. (1995) used a series of monoclonal antibodies raised against different parts of the protein and found huntingtin in perikarya of some neurons, neuropil, varicosities, and as punctate staining likely to be synapses. DiFiglia et al. (1995) found huntingtin associated with synaptic vesicles, especially in the somatodendritic compartment. Hoogeveen et al. (1993) found a cytoplasmic localization in various cell types including neurons. In most neurons, huntingtin was present in the nucleus. No difference in intracellular localization was found between normal and mutant cells (Hoogeveen et al., 1993). Most recently, Ferrante et al. (1997a) have identified

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that huntingtin is heterogeneously distributed and enriched in neurons that degenerate in HD. Double staining studies show that it is co-localized with calbindin and is not present in NADPH diaphorase neurons. The preferential distribution of huntingtin in neurons that are vulnerable to degeneration in HD is consistent with the hypothesis that mutant huntingtin manifests a toxin “gain-of-function’’ that directly causes preferential degeneration of neurons expressing high levels of the protein. This observation has been confirmed by others (Kosinski et al., 1997), but some investigators dispute this finding (Gourfinkel-An et aI., 1997). The physiological role of huntingtin remains a mystery. Targeted disruption of the HD gene leads to fetal death, possibly owing to increased apoptosis (Duyao et al., 1995;Zeitlin et al., 1995).Recent work suggests that huntingtin may be involved in the intracellular trafficking of nutrients in early embryonic stages (Dragatsis and Zeitlin, 1998). The localization of huntingtin with microtubules suggests it could play a role in intracellular or axonal transport (Tukamoto et al., 1997). Excitotoxic lesions of the striatum lead to increased expression of neuronal huntingtin (Tatter et a]., 1995). It is likely that huntingtin plays a role in cellular survival or response to injury, or both, but little more can be stated at the present time. Huntingtin-associatedproteins have been described.Huntingtin-associatedprotein-1 (HAP-I) is enriched in brain and binds with increased affinity to mutant huntingtin (Li et al., 1995). However, HAP-1 is not enriched in areas of HD pathology (Bertaux et al., 1998). A second protein called huntingtin-interacting protein (HIP-1) interacts with huntingtin in a similar manner (Kalchman et al., 1997).HIP-1 is associated with the cytoskeleton and abnormal binding may contribute to a cytoskeletal disruption and cell death in HD.

TRINUCLEOTIDE REPEAT EXPANSION A N D THE MOLECULAR PATHOGENESIS OF HUNTINGTON’S DISEASE Nakayabu et al. (1998) recently found that mismatching of nucleotide pairs makes double-stranded DNA unstable and triggers the slippage of DNA polymerase, thereby leading to expansion of trinucleotide repeats. Kang et al. (1995) found that DNA triplets beyond a certain length interfere with the progression of DNA polymerase, giving rise to expanded sequences. Surprisingly, CTG repeats are expanded at least eight times more frequently than other triplet permutations in Escherichia coli. The structure of CTG repeats, or unique aspects of their interaction with DNA polymerase, may explain this effect. The relationship between expanded polyglutamine tracts in huntingtin and the well-establisheddefects in cellular energetics in HD has not been explained. Burke and associates (1996) reported that huntingtin binds to the glycolytic enzyme glyceraldehyde-3-phosphatedehydrogenase. There is a suggestion, which remains to be reconfirmed, that long stretches of polyglutamine may inhibit activity of this enzyme. Because neurons are highly dependent on glucose as a source of energy, impaired glycolysis could lead to neuronal cell death. Recently, huntingtin has been

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shown to specifically bind to cystathionine p-synthase (Boutell et al., 1998). Inhibition of this enzyme would lead to increased homocysteine levels that could potentially lead to excitotoxicity. In either case, the pattern of degeneration in HD may reflect differential sensitivity to energy impairment and the distribution pattern of huntingtin in specific neuronal populations.

DISTRIBUTION OF HUNTINCTIN IN HUNTINGTON’S DISEASE: NUCLEAR INCLUSIONS When the distribution of huntingtin was studied in HD brain, an unexpected finding was made. Intense huntingtin immunoreactivity was found in neuronal intranuclear inclusions and in dystrophic neurites in the striatum and cerebral cortex (DiFiglia et al., 1997). These aggregates also contain ubiquitin immunoreactivity. Tissue transglutaminase may catalyze cross-linking reactions between cellular proteins and the expanded polyglutamine domain in huntingtin, leading to the deposition of high molecular weight protein aggregates (Gentile et al., 1998). Transglutaminase has also been localized to the nucleus and may contribute to nuclear inclusion formation (Lesort et al., 1998). It is possible that these inclusions are neurotoxic and lead to the neuronal degeneration in HD. Bates and colleagues (Davies et al., 1997) found that transgenic mice expressing mutant huntingtin develop similar neuronal intranuclear inclusions prior to developing neurological signs. Transfection of neuroblastoma cells with mutant huntingtin constructs results in cytoplasmic and cellular aggregates (Cooper et al., 1998). Truncated huntingtin forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli (Hackam et al., 1998; Martindale et al., 1998). Caspase-3 appears to be the enzyme responsible for proteolytic cleavage of huntingtin and other mutant proteins with expanded polyglutamine tracts that accumulate in trinucleotide repeat disorders (Wellington et al., 1998). Indeed, all of the neurological diseases associated with expanded trinucleotide repeats may be characterized by neuronal nuclear inclusions (Ross, 1997; Davies et al., 1998). Huntingtin has a propensity to associate with other intracellular inclusions and been reported to be present in both neurofibrillary tangles and Pick bodies (Singhrao et al., 1998). The hypothesis that intracellular aggregates are toxic and directly lead to neuronal cell death has been challenged. Intraneuronal inclusions are prominent in spared subsets of neurons in both HD brain (Figure 1) and in transgenic animal models of HD (Figure 2). Indeed, in spinocerebellar ataxia type 7, inclusion distribution is very widespread and does not correspond to the restricted distribution of cell loss (Holmberg et al., 1998).

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Figure 1. Double immuno- and enzyme histochemical staining for N-terminal huntingtin (EM-48, courtesy of Dr. Xiao Li, Emory University) immunostaining in striatal neuronal populations from Huntington’s disease (HD) tissue specimens and those containing calbindin (A), acetycholinesterase (B), and NADPH-diaphorase (C and D). lntranuclear neuronal aggregates can be observed in neurons (arrowheads) in vulnerable calbindin neurons (A). These inclusions are also present in unstained neurons (arrows) in this tissue preparation. Cytosolic aggregation of EM-48 i s present in many NADPH-diaphorase neurons (C). Of great interest i s the observation that nuclear aggregates are also seen in spared acetycholinesterase and NADPH-diaphorase striatal neurons in H D (B and D), suggesting a dissociation between huntingtin aggregation and neuronal death in this disease.

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Figure 2. Double immunostaining for N-terminal huntingtin (EM-48) and NADPHdiaphorase enzyme histochemical staining in a transgenic mouse model for Huntington’s disease (Gill Bates; R6/2). Large numbers of intranuclear neuronal inclusion bodies are present within the striatum of these animals (A, arrowhead). lntranuclear neuronal inclusions are also observed within NADPH-diaphorase striatal neurons as in the human HD.

ACKNOWLEDGMENTS This work was supported by NIH grants AG 13846 (NWK), NS 35255 (RJF), and the Department of Veterans Affairs.

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