Huntingtons Disease

1. Introduction

Huntington's disease (HD) is a fully penetrant genetic neurodegenerative disorder that is inherited as an autosomal dominant mutation of the HD gene (136). Affected individuals begin to exhibit symptoms in the third to fifth decade of life with some cases of juvenile (under 20 years old) and late onset (over 65 years old) (137,138). Clinical manifestations of the disease include progressive chorea, emotional disturbances, and dementia. Neuropathological features of HD include an extensive loss of neurons and astrogliosis in the striatum, primarily the caudate nucleus. Histologically, intracellular inclusions consisting of ubiquitinated polyglutamine aggregates have been found in neurons of the striatum and other less affected areas such as neocortex.

The HD gene encoding the huntingtin protein was first localized to chromosome 4p16.3 in 1983 (139). The normal function of huntingtin remains an enigma; however, cloning of the normal and diseased gene revealed the mutant form of the gene contains an increased number of glutamine encoding CAG repeats in the amino terminus (139). Normal individuals contain 11 to 34 repeats, whereas affected individuals contain 40 or greater repeats (140,141). A study by Ashizawa et al. (141) provided evidence that repeat numbers correlated with age of onset and clinical symptom severity; however, the mechanism by which the expression of this mutant protein leads to cell death remains elusive.

2. Possible Mechanisms of Disease

Understanding the normal function of huntingtin may prove pivotal in understanding how the mutant form leads to the selective loss of medium spiny neurons in the striatum. In the absence of this knowledge, studies have suggestedpossible mechanisms by which the mutant HD gene product causes cell death and provided a basis for developing therapeutic strategies.

One proposed mechanism involves a pathway of cellular protein degradation. The ubiquitin/proteasome pathway is the major protein degradation pathway of the cell. The protea-some, a cylindrical peptidase-containing complex that cleaves ubiquitinated proteins into their amino acid constituents, is believed to be involved in the degradation of huntingtin (142,143). In Huntington's disease, ubiquitinated polyglutam-ine aggregates have been identified, suggesting the digested

Figure 6 P-Glucuronidase expression following FIV-mediated gene transfer into the striata of MPS VIII mice and its effect on lysosomal storage. Eight-week-old MPS VII (gusmps) mice were injected unilaterally into the striatum with 1 X 106 transduction units of a feline immunodeficiency virus (FIV) vector expressing p-glucuronidase (FIVPgluc). Six weeks later the animals were sacrificed and analyzed for transgene expression, p-glucuronidase enzyme activity, and lysosomal storage profiles. Transgene-positive cells were detected near the injection site as revealed by in situ RNA analyses (A). p-Glucuronidase activity in the brain of a MPS VII mouse injected with FIVPgluc was found to encompass a wide volume of the brain as determined by histological staining (red staining; B). Representative examples of lysosomal storage in the striatum (C), cortex (E), and hippocampus (G) in nontreated 8 to 12-week-old MPS VII mice are shown. Noticeable lysosomal storage is evident at this age. Analysis of age-matched, FIVPglu-treated MPS VII mice shows significant correction of the storage deficit in the contralateral striatum (D), cortex (F), and hippocampus (H). In data not shown, treated animals exhibited improved learning and memory behavior, indicating that lentivirus-based delivery of P-glucuronidase to the CNS of an animal with preestablished lysosomal storage disease can reverse the neurological deficits caused by the disease. (From Ref. 132, © 2002 National Academy of Sciences.) See the color insert for a color version of this figure.

Figure 6 P-Glucuronidase expression following FIV-mediated gene transfer into the striata of MPS VIII mice and its effect on lysosomal storage. Eight-week-old MPS VII (gusmps) mice were injected unilaterally into the striatum with 1 X 106 transduction units of a feline immunodeficiency virus (FIV) vector expressing p-glucuronidase (FIVPgluc). Six weeks later the animals were sacrificed and analyzed for transgene expression, p-glucuronidase enzyme activity, and lysosomal storage profiles. Transgene-positive cells were detected near the injection site as revealed by in situ RNA analyses (A). p-Glucuronidase activity in the brain of a MPS VII mouse injected with FIVPgluc was found to encompass a wide volume of the brain as determined by histological staining (red staining; B). Representative examples of lysosomal storage in the striatum (C), cortex (E), and hippocampus (G) in nontreated 8 to 12-week-old MPS VII mice are shown. Noticeable lysosomal storage is evident at this age. Analysis of age-matched, FIVPglu-treated MPS VII mice shows significant correction of the storage deficit in the contralateral striatum (D), cortex (F), and hippocampus (H). In data not shown, treated animals exhibited improved learning and memory behavior, indicating that lentivirus-based delivery of P-glucuronidase to the CNS of an animal with preestablished lysosomal storage disease can reverse the neurological deficits caused by the disease. (From Ref. 132, © 2002 National Academy of Sciences.) See the color insert for a color version of this figure.

mutant form of huntingtin creates aggregates leading to an apparent apoptotic cell death (144,145).

Huntingtin has been shown to interact with huntingtin-asso-ciated protein 1 (HAP-1) (146), huntingtin-interacting protein land 2 (HIP-1,-2) (147,148) glyceraldehydes-3-phosphate dehydrogenase (GAPDH) (149), and calmodulin (150). HAP-1 binding to huntingtin is enhanced by increased glutamine repeat length (146). It is believed to be involved in vesicular membrane trafficking, but this has not been proven (151). Unlike HAP-1, HIP-1 binding to huntingtin is inversely related to the polyglutamine residue number. The loss of this interaction in a HD neuron may affect the cytoskeletal architecture given HIP-1's similarity to cytoskeletal proteins (148). HIP-2 is an ubiqui-tin-conjugating enzyme that may be involved in the proteaso-mal degradation or aggregation of mutant huntingtin (152). The glycolytic enzyme, GAPDH, binds to cleaved huntingtin fragments in vitro (149). The increased binding of GAPDH to cleaved huntingtin fragments may alter cellular energy production, leading to membrane depolarization, increased intracellu-lar calcium, and cell death. Huntingtin can from a complex with calmodulin in a Ca2+-dependent manner, but the mutant form binds independent of calcium (150). Subsequent activation of downstream targets of calmodulin, such as those activated in excitotoxic cell death, may lead to cell death. Further understanding of the interactions of huntingtin with the aforementioned proteins or other proteins will allow for targeted gene therapy. Based on our current understanding, therapies designed to block apoptosis, relieve oxidative stress and prevent the expression and accumulation of degraded mutant huntingtin may help to relieve or prevent symptoms in affected patients.

3. Potential Gene-based Therapies for HD

Yamamoto et al. (153) demonstrated that continuous expression of mutant protein is necessary to maintain intracellular inclusions and symptoms. A therapy designed to decrease mutant protein levels by antisense therapy may prove useful. In this strategy, a viral vector expressing an antisense RNA or ribozyme (154) to huntingtin could be delivered to the striatum. Alternatively, if a technology were available to reduce the glutamine repeats and/or replace the mutant allele with a normal allele, a permanent reversal of the disease might be accomplished. The use of chimeroplasts, target-specific RNA/ DNA oligonucleotides, could possibly allow correction of the genetic deficit, but this may not be technically feasible for such a large mutation (155-157).

A second approach takes advantage of the well-characterized neurotrophic factors, endogenous soluble proteins that regulate survival, growth, morphological plasticity, or synthesis of proteins for differentiated functions of neurons (157). The delivery of the neurotrophic factors GDNF, BDNF, or CNTF have shown neuroprotective effects when given prior to a quinolinic acid challenge in rodent and primate models of HD (158-160). The neuroprotective effect of lentiviral vector-mediated delivery of CNTF in the quinolinic acid model for HD is illustrated in Fig. 7. Further analysis is needed to evaluate the full potential of neurotrophic factor treatment for HD.

A third approach would be to target mechanisms of apop-totic cell death and oxidative stress. Similar to neurotrophic factors, this approach may delay the progression of the disease, but does permanently correct the genetic deficit. The antiapoptotic gene, bcl-2, has been shown to inhibit neuronal apoptosis both in vitro and in vivo (161-171). Delivery of the bcl-2 gene to affected neurons may prevent or delay cell death that occurs in HD. If oxidative free radicals contribute to cell death, the expression of gene products capable of directly or indirectly lowering or removing free radicals [e.g., superoxide dismutase (SOD), catalase, glutathione reductase

(GR), glutathione peroxidase (GPO), and glutathione (GSH)] might prove useful in combating neuronal loss.

HD is one of a family of trinucleotide repeat disorders, which includes spinal and bulbar muscular atrophy, spinocerebellar ataxia (types 1, 2,3,5, and 7), and dentatorubropallido-lusian atrophy. Elucidating the mechanisms by which the mutant form of huntingtin leads to disease may aid in the development of successful gene therapies for HD and possibly other related trinucleotide repeats disorders.

E. Stroke

1. Introduction

Hypoxic injuries, such as stroke, are the genesis of substantial morbidity and mortality in neonates and older individuals. Oxygen deprivation can lead to profound effects on motor function and cognition (172), leading to severe disability and diminished quality of life. Stroke is most commonly the result of embolic obstruction that results in either reduced or complete loss of blood flow to downstream fields. Two types of cell death appear to occur following a stroke. At the ischemic core, necrosis is readily apparent and has been linked to elevated extracellular glutamate and intracellular calcium levels. Necrotic cell death involves nonspecific DNA degradation, nuclear pyknosis, diminished membrane integrity, and mito-chondrial swelling. Neuronal degeneration within this area following blood vessel obstruction occurs very rapidly (minutes) following the ischemic insult, and due to this time limitation, does not represent a viable target for gene-based therapeutics. In brain areas more distal to the ischemic core, termed the prenumbra, neurons undergo a more delayed form of cell death (hours to days) that exhibits dependence6 de novo gene expression (173-175). This delayed neuronal death, or

GFP CNTF "off1 CNTF "on"

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