摘要

亨廷頓氏病（HD）是一個(gè)中年發(fā)病的神經(jīng)退行性疾病，特征為不自主運(yùn)動(dòng)，性格改變和癡呆。它發(fā)病在10-20年后發(fā)展到死亡。目前還沒有治愈的方法，治療這種致命的疾病。在血液透析患者中，蛋白的杭丁頓蛋白含有多聚束的異常擴(kuò)張，從而導(dǎo)致紋狀體神經(jīng)元選擇性死亡。杭丁頓蛋白的功能，以及由突變導(dǎo)致功能失調(diào)仍知之甚少。

本dissertation的第一章介紹了亨廷頓氏病的研究現(xiàn)狀：亨廷頓氏病（HTT），引起疾病的蛋白質(zhì)；有毒的通路誘導(dǎo)變異的亨廷頓氏病和建議的治療策略，建議用他們來干預(yù)治療；可用的細(xì)胞和動(dòng)物模型，最后用基因治療神經(jīng)退行性變疾病，如亨廷頓氏病。

第二章介紹了利用慢病毒載體表達(dá)突變亨廷頓氏病（HTT）片段感染原代培養(yǎng)的細(xì)胞模型的新發(fā)展。該模型的特點(diǎn)是廣義的轉(zhuǎn)基因表達(dá)，神經(jīng)元的慢性病理，神經(jīng)功能障礙和最終細(xì)胞死亡。

ABSTRACT

Huntington’s disease (HD) is a mid-life-onset neurodegenerative disorder characterized by involuntary movements, personality changes and dementia. It progresses to death within 10-20 years after onset. There is currently no cure to treat this fatal disease. In HD patients, the protein huntingtin contains an abnormal expansion of a polyglutamine tract, which leads to the selective death of striatal neurons. The functions of huntingtin, as well as the dysfunctions induced by the mutation are still poorly understood.

The first chapter of this thesis describes the state of the art in the study of Huntington’s disease: huntingtin (htt), the protein which induces the disease; the hypothesis of toxic pathways induced by the mutant htt and the proposed therapeutic strategies to interfere with them; the available cellular and animal models and finally; the promise of gene therapy for neurodegenerative diseases such as Huntington’s disease.

The second chapter presents a new cellular model of the developed by infecting primary cultures with lentiviral vectors expressing a mutant htt fragment. This model is characterized by generalized neuronal transgene chronic pathology, expression, neuronal dysfunction and finally cell death. The slow progression in this model allows the study of the cascade of events leading to cell death. These unique characteristics allow the investigation of the pathological events induced by htt expression with analytical techniques on the entire neuronal population. The chapter ends with the description of an experiment showing the neuroprotective effects of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic actor (BDNF) on thiese neurons.

The third chapter describes the delivery of CNTF in transgenic HD mice which express the human full-length htt (YAC72). CNTF was delivered directly to the affected neurons by striatal lentiviral injection. The sustained one-year delivery was not associated with side effects; it was correlated to reduced hyperactivity and a reduction in degenerating neurons. However, a neuroprotective effect was difficult to establish, due to the very mild and subtle pathology occurring in these animals.#p#分頁(yè)標(biāo)題#e#

The fourth chapter considers delivery of BDNF in two genetic rodent HD models: in the mild phenotypic HD mice (YAC128) and a more severe rat model based on the lentiviral delivery of mutant htt in the striatum. No neuroprotective effect of BDNF could be detected in either animal model.

The conclusions of this thesis discuss the potential of lentiviral vectors in modeling neurodegenerative diseases in vitro and in vivo, as well as their potential role in the treatmentof such diseases. The perspective gained by the developed cellular model toward the understanding of the molecular events induced by mutant huntingtin is discussed, as well as the difficulties in testing neuroprotective approaches on animal models of HD.

1. INTRODUCTION介紹
The central nervous system (CNS) is the most complex, amazing and still mysterious structure of our body, controlling functions such as voluntary movements and perception, but also learning, creativity, abstract thinking and emotions. Multiple pathways are orchestrating these functions in networks of highly specialized neurons. Evolution has provided a robust skull and backbone to protect the CNS from outside mechanical injuries, and a molecular obstacle, the blood-brain barrier, to protect it from endogenous chemical agents. However, the brain is still vulnerable, as revealed by the emergence of neurodegenerative diseases associated with dramatic increases in human live expectancy. Dysfunctions in this complex system can lead to a wide range of different severe pathologies such as Alzheimer’s, Parkinson’s, Amyotrophic Lateral Sclerosis or Huntington’s disease. These disorders are characterized by a specific degeneration of neurons in well-defined areas of the CNS. The molecular bases of these specific neuronal losses are not yet fully understood, but major medical research efforts are concentrated on finding therapies to slow down or stop the course of these disorders.

Cellular and animal models have been developed to mimic these complex pathologies.Models faithfully reproducing a human disease are indispensable tools to analyze, understand, and finally, test interventions against the disorder. This helps explain the great research interest in Huntington’s disease (HD). Although it is rare compared to Parkinson’s and Alzheimer’s, it is the paradigm of a dominant monogenetic disease. The genetic factor, underlying HD was identified in 1993 as a mutation in a unknown protein: a polyglutamine expansion in huntingtin. This is the primary cause of neuronal dysfunction, accumulation of insoluble proteins and neuronal death, which are events common to most of chronic neurodegenerative diseases. With the technological advances in molecular biology and gene transfer techniques, mutant huntingtin can be delivered to living cells to model HD in cell cultures but also in animals. These genetic models are important tools to understand the toxic mechanisms leading to neuronal death, as well as to test therapeutic approaches.#p#分頁(yè)標(biāo)題#e#

The first goal of this thesis was to develop a cellular genetic model for Huntington’s disease, closely mimicking the typical features of the pathology which can be used for fundamental research as well as a platform to develop potential therapies. This goal was strongly inspiredby the seminal paper of Saudou et. al. in 1998, which described a new cellular model by transfecting a mutant huntingtin fragment in primary striatal neurons. In that model, neurons experience similar chronic molecular events and death as is observed in the human pathology. The fast kinetics and the scarce transfection yield limit the analysis of pathological events to single cell analysis. To overcome this disadvantage, we used a lentiviral vector, which can efficiently infect non-dividing cells such as neurons and therefore increase the neuronal population expressing mutant htt. The elegant, slow kinetics of the pathological events leading to neuronal death, combined with a generalized neuronal transgene expression are unique features of this cellular paradigm which allow the analysis of population dysfunctions at different time points and open new avenues to understanding the impact of mutant htt.

The second goal of this thesis was the evaluation of therapeutic strategies, first in the new cellular model, then in genetic animal models. Neurotrophic factors have been proposed to slow down, or arrest disease progression or even promote regeneration in neurodegenerative diseases. CNTF and BDNF have shown a specially strong protection spectrum in different paradigms of striatal neuron injuries, both in vitro and in vivo. We have therefore tested if the lentiviral mediated delivery of these factors could represent an efficient treatment for HD. Complete rescue from neuronal death in vitro and long-term delivery of trophic factors in vivo without any side effects are encouraging with regard to both gene therapy and the possible beneficial effects of these trophic factors.

1.1.HUNTINGTON’S DISEASE 亨廷頓氏病
1.1.1. Eponym Huntington: introduction to the disease
Huntington’s disease is a fatal neurodegenerative disease named after George Summer
Huntington (Figure 1) who first described the disorder in 1872 (Huntington, 2003). His
original paper published under the title “ On Chorea” is brief, complete and still up-to-date in
major aspects. Going through Huntington’s publication represents an excellent introduction to
the disorder.

Inheritance家族遺傳
‘When either or both the parents have shown manifestations of the disease, and more especially when these manifestations have been of a serious nature, one or more of the offspring almost invariably suffer from the disease if they live to adult age. But if by any chance these children go through life without it, the thread is broken and the grandchildren and the great-grandchildren of the original shakers may rest assured that they are free from the disease.’#p#分頁(yè)標(biāo)題#e#

This description of the autosomal dominant trait of the mutation is remarkable, because the knowledge of genetics was poorly integrated by the scientific community at that time and Mendel’s Laws describing the principle of hereditary transmission were still largely ignored,although they had been published seven years before in 1865 in a book under the title Versuche über Pflanzen-hybride (Treaties on Plant Hybrids). These theories were rediscovered in the early 20th century, when the cell and chromosome structures were better understood, and significant interest for HD, especially its hereditary aspect, was stimulated by Huntington’s paper.

Dementia癡呆
‘The tendency to insanity, and sometimes that form of insanity which leads to suicide, is marked. I know of several instances of suicide of people suffering from this form of chorea, or who belonged to families in which the disease existed. As the disease progresses the mind becomes more or less impaired, in
many amounting to insanity, while in others both mind and body gradually fail until death relieves them of their sufferings. At present I know of two married men, whose wives are living, and who are constantly making love to some young lady, not seeming to be aware that there is any impropriety in it. They are suffering from chorea to such an extent that they can hardly walk, and would be thought, by a stranger, to be intoxicated. They are men of about fifty years of age, but never let an opportunity to flirt with a girl go past unimproved. The effect is ridiculous in the extreme’.


A second aspect of the hereditary chorea he observed is dementia and depression. Mood and behavioral disturbance, memory impairment and personality changes are typical clinical features of the disorder, occurring in general before the onset of chorea. Indeed, suicide attempts are more frequent in persons carrying the HD mutation. These secondary treats of the disorder are often ignored as they are less impressive than the abrupt motor dysfunctions and can only be perceived by relatives living close to HD patients. Antidepressant and antipsychotic drugs are often being prescribed to HD patients.

Adult onset成年型
‘Its third peculiarity is its coming on, at least as a grave disease, only in adult life. I do not know of a single case that has shown any marked signs of chorea before the age of thirty or forty years, while those who pass the fortieth year without symptoms of the disease are seldom attacked.’

Huntington’s disease is predominantly an adult disorder, with the average onset between 35 and 42 years (Quinn and Schrag, 1998). However, the disease can start at any age and 6% are juvenile forms, with an onset before the age of 20 (Figure 2). These early onsets have quite a 。different clinical description with the absence of abrupt movements and chorea. As it will be discussed in chapter 1.2.2 the age of onset is closely correlated with the extent of the mutation in the huntingtin gene.#p#分頁(yè)標(biāo)題#e#

Movement disorders運(yùn)動(dòng)障礙
It begins as an ordinary chorea might begin, by the irregular and spasmodic action of certain muscles, as of the face, arms, etc. These movements gradually increase, when muscles hitherto unaffected take on the spasmodic action, until every muscle in the body becomes affected (excepting the involuntary ones), and the poor patient presents a spectacle, which is anything but pleasing to witness. I have never known a recovery or even an amelioration of symptoms in this form of chorea; when once it begins it clings to the bitter end. No treatment seems to be of any avail, and indeed ，nowadays its end is so well known to the sufferer and his friends, that medical advice is seldom sought. It seems at least to be one of the ，incurables’.

These uncontrolled and involuntary movements are the most peculiar trait of the disorder. They were probably the cause for the famous witch trials in Salem, Massachusetts in 1692, where several girls were judged and executed as witches possessed by the devil (Trask, 1997). George Huntington concluded his essay with the sad statement that no cure is available and that the death of the affected persons is inevitable. More than a century after Huntington’s paper, and more than ten years after the discovery of the HD gene, still no effective treatment is available and the disease progresses uninterrupted to the death of the patient.

1. Epidemiology of HD and the discovery of huntingtin流行病學(xué)的HD和杭丁頓蛋白的發(fā)現(xiàn)
Huntington’s disease is the most prevalent disorder in a family of nine neurodegenerative diseases that are caused by a polyglutamine expansion in the associated proteins. HD affects both sexes with the same frequency. The highest prevalence is in Europe and North America, with 4-8 cases per 100’000 (Squitieri et al., 1994). In Switzerland about 400 persons are affected by the disorder (Laccone et al., 1999). HD is notably rare in Finland and in Japan (Harper, 1992). An extremely high occurrence was found within the 15’000 members of a large group of inter-related families living in fishing villages along the borders of Lake Maracaibo in Venezuela (Negrette, 1955). The common ancestor of these families was a woman with el mal de San Vito, as the locals named Huntington’s disease, who had 10 children about 200 years ago. This sad peculiarity led to the foundation of the Venezuela Huntington’s disease project with Nancy Wexler as principal investigator. The aim of that project was to identify the chromosomal region, and ultimately the gene, responsible for HD. These big families were therefore genetically screened, and in 1983 a transcript called IT15 ，(interesting transcript 15) located on the 4th chromosome was shown to be associated with the ，disease (Gusella et al., 1983). This was the first gene linked to a disease to be mapped. Ten years later, in 1993, the gene was finally isolated and called the HD gene or huntingtin (htt) (The Huntington's Disease Collaborative Research Group, 1993). The mutation leading to HD was identified as a polyglutamine expansion in the N-terminal region of the protein. The discovery of htt was a major breakthrough for HD research allowing the development of genetic models both in vitro and in vivo, and also opened the possibility to perform genetic screening for the mutation on individuals at risk, including human fetuses or even for in vitro fertilized eggs prior to implantation.#p#分頁(yè)標(biāo)題#e#

Huntington’s disease is a progressive neurodegenerative disorder characterized by chorea (which means dance in Greek) rigidity and dementia; moreover its juvenile form is frequently associated with seizures. This chapter describes the typical decline of an HD patient from the onset until death. Psychiatric symptoms are believed to occur before the development of neurological symptoms, but a study performed by Shiwach and Norbury concluded that asymptomatic HD gene carriers do not have a greater incidence of psychiatric disorders than non-gene carriers born from a HD parent (Shiwach and Norbury, 1994). However, what the study revealed is that at-risk persons have often psychiatric episodes, probably associated with the uncertainty of having inherited the mutation. The authors conclude that neither depression nor psychiatric disorders are significant pre-neurological indicators of the disease. In contrast to this, attention, learning and planning are affected even before the onset of clinical symptoms (Rosenberg et al., 1995). The definition of onset is often unclear and imprecise. A common definition is the time of the first appearance of persistent motor abnormalities, which can be of different nature. Ocular motor abnormalities, especially the saccadic movement, are observed in early HD and are often tested to assess the onset of the disease (Berardelli et al., 1999).

The initial symptoms vary from person to person. In general, involuntary movements of face, fingers, feet or thorax manifest the onset. Symptoms develop gradually and are initially very subtle and hard to detect, as they only weakly influence the normal life of the affected person, who keeps his independence. In concomitance, abrupt mood changes, unusual apathy, ，irritability, anger and depression are commonly observed. Minor motor abnormalities usually precede the obvious signs of dysfunction, which follow at least 3 years later (Folstein et al., 1986; Penney et al., 1990).

As HD progresses, the physical, intellectual and emotional symptoms become more marked. In many cases, the affected person develops overt choreiform movements of the head, neck, arms and legs. These characteristic movements are present in 90 percent of patients. In juvenile forms, the patient will often experience muscle rigidity and bradykinesia without chorea. This form of the disease is also called ‘Westphal variant’ of HD and affects 6% of HD patients (Hayden et al., 1981; Gervais et al., 2002; Zackowski et al., 2002). Weight loss is one ，of the features of advanced HD and is not related to a poor dietary intake (Morales et al., 1989).

Bradykinesia, rigidity and dystonia gradually appear and often dominate the final stage of the disease, in which the patient will become severely rigid, grossly akinetic and dysphagic. About 20 percent are incontinent. A global decline in cognitive capabilities is present at the final stage. Due to severe dementia and progressive motor dysfunction, patients with advanced HD may become unable to walk, have poor dietary intake, eventually cease to talk, and become unable to care for themselves, potentially requiring long-term institutional care. Fatal complications may result from injuries related to serious falls, poor nutrition, infection, choking, inflammation of the lungs and heart failure .#p#分頁(yè)標(biāo)題#e#

2. The neuropathology of HD   HD神經(jīng)病理學(xué)
The neurodegenerative process starts in the striatum, which is so called because of its striped structure (Figure 3). More than 95% of striatal neurons are medium spiny projecting neurons (MSN), which use gamma-aminobutyric acid (GABA) as their primary inhibitory neurotransmitter (Chesselet and Delfs, 1996). These neurons are most severely affected, resulting in atrophy of the striatum, first in the caudate nucleus, then in the putamen. Postmortem brains of advanced state HD patients are recognized by the dilatation of the lateral ventricles due to striatal shrinkage. Glial proliferation is observed in concomitance with neuronal cell loss (Robitaille et al., 1997).

The first degenerating subpopulation of striatal neurons belongs to the so-called ‘indirect’ pathway (see chapter 1.1.5). These neurons express enkephalin and are enriched in dopamine receptor D2 (Reiner et al., 1988; Albin et al., 1991; Richfield et al., 1995; Sapp et al., 1995). In contrast, striatal interneurons, including large spiny cholinergic cells, medium aspiny neurons as well as somatostatin-, neuropeptide Y-, nitric oxide synthase (NOD)- and parvalbumin positive neurons are spared (Ferrante et al., 1985; Graveland et al., 1985; Ferrante et al., 1987a; Ferrante et al., 1987b; Sieradzan and Mann, 2001). The neuronal loss in the cortex is less severe. Large neurons in layer V and VI, which project to the striatum, are mostly affected (Hedreen et al., 1991).

Vonsattel et al. have proposed a system for grading the severity of neuropathology by macroscopic and microscopic criteria (Vonsattel et al., 1985). The grades correlate closely with the extent of clinical disability and are ranking from grade 0 (no discernible neuropathology) to grade 4 (95% of neuronal loss in caudate nuclei).

Characteristic insoluble protein aggregates are found in HD but also in other polyglutamine diseases as well as other neurological disorders, such as the Lewy bodies in Parkinson’s disease, the plaques and tangles in Alzheimer’s disease or the Bunina bodies in ALS. Theseaggregates, which are ubiquitinated, are called neuronal nuclear inclusions (NIIs) or dystrophic neuritic inclusions (DNIs), depending on their sub-cellular localization (DiFiglia et al., 1997). Inclusions are mainly found in the striatum and cortex and partially overlap with the neuronal pathology. Their direct impact on the disease is still under debate, as both protective as well as toxic functions have been described (Klement et al., 1998; Saudou et al., 1998; Sisodia, 1998; Kim et al., 1999; Rubinsztein et al., 1999; Yang et al., 2002).

3. Link between chorea and striatal degeneration舞蹈病和紋狀體變性之間的聯(lián)系
The primary motor dysfunctions in HD are due to the degeneration of a subset of striatal neurons. The striatum belongs to a system called basal ganglia (Figure 3), which are a collection of subcortical nuclei that are involved in the control of movement. The basal ganglia do not receive direct sensory input and send little output to the spinal cord. The major flow of information arrives from the cortex into the striatum and returns to the cortex through the thalamus, which is the major output structure of the basal ganglia. A classical model developed by Albin et. al. in 1989 described two major neuronal motor pathways, which act in opposition, and correctly predicts the motor impairment in Huntington’s disease. The ‘direct pathway’ promotes movements by a relay of two inhibitory synapses, first in the globus pallidum internal segment and the substantia nigra pars reticulata (Gpi/SNr) and second in the thalamus; the ‘indirect pathway’ inhibits movement by three inhibitory connections in the globus pallidum external segment (Gpe), the substantia nigra pars compacta (SNc) and the thalamus with a relay of GABAergic inhibition between the SNc and SNr. A more recent view of the basal ganglia functions indicates that in addition from the striatum, the subthalamic nucleus also receives input from the cortex (Mink and Thach, 1993; Nambu et al., 2000) and sends output to the Gpi/SNr. This new cortico-subthalamo-pallidal loop is called ‘hyperdirect pathway’ (Nambu et al., 1996) and acts by inhibiting the thalamus and cortex. As this loop is not modified in HD, the model proposed by Albin correctly predicts the motor defects present in Huntingon’s disease (Figure 3) as well as in Parkinson’s disease and ballism.#p#分頁(yè)標(biāo)題#e#