Functional genomics approaches to neurodegenerative diseases

Many of the neurodegenerative diseases that afflict humans are characterised by the protein aggregation in neurons. These include complex diseases like Alzheimer’s disease and Parkinson’s disease, and Mendelian diseases caused by polyglutamine expansion mutations [like Huntington’s disease (HD) and various spinocerebellar ataxias (SCAs), like SCA3]. A range of functional genomic strategies have been used to try to elucidate pathways involved in these diseases. In this minireview, I focus on how modifier screens in organisms from yeast to mice may be of value in helping to elucidate pathogenic pathways.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Repeat expansion and autosomal dominant neurodegenerative disorders: consensus and controversy

Repeat-expansion mutations cause 13 autosomal dominant neurodegenerative disorders falling into three groups. Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and spinocerebellar ataxias (SCAs) types 1, 2, 3, 7 and 17 are each caused by a CAG repeat expansion that encodes polyglutamine. Convergent lines of evidence demonstrate that neurodegeneration in these diseases is a consequence of the neurotoxic effects of abnormally long stretches of glutamines. How polyglutamine induces neurodegeneration, and why neurodegeneration occurs in only select neuronal populations, remains a matter of intense investigation. SCA6 is caused by a CAG repeat expansion in CACNA1A, a gene that encodes a subunit of the P/Q-type calcium channel. The threshold length at which the repeat causes disease is much shorter than in the other polyglutamine diseases, and neurodegeneration may arise from expansion-induced change of function in the calcium channel. Huntington's disease-like 2 (HDL2) and SCAs 8, 10 and 12 are rare disorders in which the repeats (CAG, CTG or ATTCT) are not in protein-coding regions. Investigation into these diseases is still at an early stage, but it is now reasonable to hypothesise that the net effect of each expansion is to alter gene expression. The different pathogenic mechanisms in these three groups of diseases have important implications for the development of rational therapeutics.     

Modeling Huntington disease in Drosophila

Huntington disease (HD) is an inherited neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in the huntingtin (Htt) gene. Despite years of research, there is no treatment that extends life for patients with the disorder. Similarly, little is known about which cellular pathways that are altered by pathogenic Huntingtin (Htt) protein expression are correlated with neuronal loss. As part of a longstanding effort to gain insights into HD pathology, we have been studying the protein in the context of the fruitfly Drosophila melanogaster. We generated transgenic HD models in Drosophila by engineering flies that carry a 12-exon fragment of the human Htt gene with or without the toxic trinucleotide repeat expansion. We also created variants with a monomeric red fluorescent protein (mRFP) tag fused to Htt that allows in vivo imaging of Htt protein localization and aggregation. While wild-type Htt remains diffuse throughout the cytoplasm of cells, pathogenic Htt forms insoluble aggregates that accumulate in neuronal soma and axons. Aggregates can physically block transport of numerous organelles along the axon. We have also observed that aggregates are formed quickly, within just a few hours of mutant Htt expression. To explore mechanisms of neurodegeneration in our HD model, we performed in vivo and in vitro screens to search for modifiers of viability and pathogenic Htt aggregation. Our results identified several novel candidates for HD therapeutics that can now be tested in mammalian models of HD. Furthermore, these experiments have highlighted the complex relationship between aggregates and toxicity that exists in HD.     

Mouse models of Huntington's disease and methodological considerations for therapeutic trials

Huntington's disease (HD) is an autosomal dominant, progressive, and fatal neurodegenerative disorder caused by an expanded polyglutamine cytosine–adenine–guanine repeat in the gene coding for the protein huntingtin. Despite great progress, a direct causative pathway from the HD gene mutation to neuronal dysfunction and death has not yet been established. One important advance in understanding the pathogenic mechanisms of this disease has been the development of multiple murine models that replicate many of the clinical, neuropathological, and molecular events in HD patients. These models have played an important role in providing accurate and experimentally accessible systems to study multiple aspects of disease pathogenesis and to test potential therapeutic treatment strategies. Understanding how disease processes interrelate has become important in identifying a pharmacotherapy in HD and in the design of clinical trials. A review of the current state of HD mouse models and their successes in elucidating disease pathogenesis are discussed. There is no clinically proven treatment for HD that can halt or ameliorate the inexorable disease progression. As such, a guide to assessing studies in mouse models and salient issues related to translation from mice to humans are included.     

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

Huntington disease (HD) is caused by expansion of a polyglutamine (polyQ) domain in the protein known as huntingtin (htt), and the disease is characterized by selective neurodegeneration. Expansion of the polyQ domain is not exclusive to HD, but occurs in eight other inherited neurodegenerative disorders that show distinct neuropathology. Yet in spite of the clear genetic defects and associated neurodegeneration seen with all the polyQ diseases, their pathogenesis remains elusive. The present review focuses on HD, outlining the effects of mutant htt in the nucleus and neuronal processes as well as the role of cell-cell interactions in HD pathology. The widespread expression and localization of mutant htt and its interactions with a variety of proteins suggest that mutant htt engages multiple pathogenic pathways. Understanding these pathways will help us to elucidate the pathogenesis of HD and to target therapies effectively.     

Genetic correction of Huntington's disease phenotypes in induced pluripotent stem cells

Huntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy.     

Shaping the role of mitochondria in the pathogenesis of Huntington's disease

Intense research on the pathogenesis of Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, revealed multiple potential mechanisms, among which mitochondrial alterations had emerged as key determinants of the natural history of the disease. Pharmacological and genetic animal models of mitochondrial dysfunction in the striatum, which is mostly affected in HD corroborated a key role for these organelles in the pathogenesis of the disease. Here, we will give an account of the recent evidence indicating that the mitochondria-shaping machinery is altered in HD models and patients. Since its correction can counteract HD mitochondrial dysfunction and cellular damage, drugs impacting on mitochondrial shape are emerging as a new possibility of treatment for this devastating condition.     

Protein aggregation and pathogenesis of Huntington's disease: mechanisms and correlations

The formation of insoluble protein aggregates is a hallmark of Huntington's disease (HD) and related neurodegenerative disorders, such as dentatorubral pallidoluysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA) and the spinocerebellar ataxia (SCA) type 1, 2, 3, 6 and 7. These disorders are caused by an expanded polyglutamine (polyQ) tract in otherwise unrelated proteins. They are characterized by late-onset, selective neuropathology, a pathogenic polyQ threshold and a relationship between polyQ length and disease progression. Thus, molecular models of HD and related glutamine-repeat disorders must account for these characteristic features. During the last three years, considerable effort has been invested in the development of in vitro and in vivo model systems to study the mechanisms of protein aggregation in glutamine-repeat disorders and its potential effects on disease progression and neurodegeneration. A selection of these studies is reviewed here. Furthermore, the correlation between aggregate formation and development of HD is discussed.     

Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.     

Destabilization of a non-pathological variant of ataxin-3 results in fibrillogenesis via a partially folded intermediate: a model for misfolding in polyglutamine disease

Ataxin-3 is a member of the polyglutamine family of proteins, which are associated with at least nine different neurodegenerative diseases. In the disease state, expansion of the polyglutamine tract leads to dysfunction and death of neurons, as well as formation of proteinaceous aggregates known as nuclear inclusions. Intriguingly, both expanded and non-expanded forms of ataxin-3 are observed within these nuclear inclusions. Ataxin-3 is the smallest of the polyglutamine disease proteins and in its expanded form causes the neurodegenerative disorder Machado-Joseph disease. Using a non-pathological variant containing 28 residues in its polyglutamine tract, we have probed the folding and misfolding pathways of ataxin-3. We describe here the first equilibrium folding pathway delineated for any polyglutamine protein and show that ataxin-3 folds reversibly via a single intermediate species. We have also explored further the misfolding potential of the protein and found that partial destabilization of ataxin-3 by chemical denaturation leads to the formation of fibrillar aggregates by the non-pathological variant. These results provide an insight into the possible mechanisms by which polyglutamine expansion may affect the stability and conformation of the protein. The implications of this are considered in the wider context of the development and pathogenesis of polyglutamine diseases.     

Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.     

Histone deacetylase activity is retained in primary neurons expressing mutant huntingtin protein

Perturbation of histone acetyl-transferase (HAT) activity is implicated in the pathology of polyglutamine diseases, and suppression of the counteracting histone deacetylase (HDAC) proteins has been proposed as a therapeutic candidate for these intractable disorders. Meanwhile, it is not known whether mutant polyglutamine disease protein affects the HDAC activity in declining neurons, though the answer is essential for application of anti-HDAC drugs for polyglutamine diseases. Here, we show the effect of mutant huntingtin (htt) protein on the expression and activity of HDAC proteins in rat primary cortical neurons as well as in human Huntington's disease (HD) brains. Our findings indicate that expression and activity of HDAC proteins are not repressed by mutant htt protein. Furthermore, expression of normal and mutant htt protein slightly increased HDAC activity although the effects of normal and mutant htt were not remarkably different. In human HD cerebral cortex, HDAC5 immunoreactivity was increased in the nucleus of striatal and cortical neurons, suggesting accelerated nuclear import of this class II HDAC. Meanwhile, western blot and immunohistochemical analyses showed no remarkable change in the expression of class I HDAC proteins such as HDAC1 and HDCA8. Collectively, retained activity in affected neurons supports application of anti-HDAC drugs to the therapy of HD.     

Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.