Death by polyglutamine: expanding our knowledge

Huntingtons disease treatments In 1872, George Huntington, an American physician who at the time was only 22 years old, published a short communication in the "Medical and Surgical Reporter" (Philadelphia) describing a hereditary form of chorea (uncontrollable dance-like movements similar to St. Vitus dance). As a young boy, George had accompanied his father in his visits to families in East Hampton, New York, who were affected by the dreaded disease and noticed that if the parents presented with the disease, their progeny had a great chance of acquiring it. These observations led him to write the above-noted first description of the disease, which ultimately was named after him. In 1993, the gene mutation causing Huntington's disease (HD) was discovered thanks to the impetus of Dr. Nancy Wexler (Columbia University) and collaborators.1 It consists of an elongation of CAG (coding for glutamine) repeats in a previously unknown gene located in the short arm of chromosome 4. When the number of CAG repeats exceeds 39, the disease will inexorably occur at some point in life. HD is a devastating, incurable neurodegenerative disease. Its prevalence is relatively high in Western European nations, Venezuela, the United States, Canada, and Oceania (5-10/100 000) but very low in Asian and most African countries (0.1-0.5/100 000). Although victims of the disease are thus relatively rare, the number of people at risk is significantly higher, emphasizing the need for finding new treatments to alleviate symptoms and improve the quality of life for patients suffering from HD. The hallmark histopathology of HD is cell degeneration in striatum and cerebral cortex, which explains the emergence of motor (chorea), cognitive, and psychiatric symptoms. The probable causes of cell death are still unknown, but significant strides have been made in the past few years to understand disease mechanisms. Such advances have been made possible in part by the generation of genetic animal models of HD. Before the advent of such models, excitotoxic models (eg, intrastriatal injection of quinolinic acid) were the only tools for mechanistic studies and were based on the idea that excessive glutamate release from corticostriatal terminals or heightened sensitivity of glutamate receptors in striatal neurons was the principal cause of cell death.2 The excitotoxic models, although certainly possessing some heuristic value, proved to be simplistic and to have important shortcomings, notably in that they did not allow examination of disease progression. Although also imperfect, genetic animal models have provided a vast number of mechanistic insights and have emphasized the fact that functional alterations, more than cell death, can explain a variety of HD symptoms.3 In particular, synaptic disconnection and miscommunication between basal ganglia structures and the cerebral cortex appear to play a crucial role. The R6/2 transgenic mouse model, created in the laboratory of Dr. Gillian Bates (then at King's College London) just 3 years after the discovery of the gene mutation,4 has been instrumental in the exploration of new therapies for HD and the gold standard for drug testing. Since then, numerous animal models have been created that allowed further insights into disease mechanisms and progression. Although no model can reproduce yet the whole gamut of human HD symptoms, animal models represent an invaluable source of knowledge and probably the only way to develop a real cure. The present Special Issue (SI) on HD is a small compendium of recent advances arising from the utilization of animal and cellular models for better understanding of disease mechanisms and for discovering new potential treatments. The authors are well-renowned leaders in their respective fields, and each one has provided either original data or detailed reviews. The 9 articles included in this issue can be considered a snapshot of the current status of HD research using those models. In no way does it aim to be a comprehensive overview of the vast amount of research presently being conducted in the HD field. However, we hope that this SI will attract the attention of investigators working on HD and other neurodegenerative diseases that may share common mechanisms. Although the functions of the normal huntingtin protein are still unclear, there is substantial evidence that, among others, it plays a key role in proper brain development, transcriptional regulation, vesicle transport, and synaptic transmission.5, 6 From a systems perspective, it is of great interest that communication along the cortico-basal ganglia-thalamocortical loop is disrupted, lending support to the idea that HD is, above all, a synaptopathy.7 Disrupted connectivity is best exemplified in Anton Reiner's (University of Tennessee) review examining striatal neuron inputs and outputs in HD8 (pp. 250–280). Professor Reiner is a world-renowned authority in the field of HD and one of the discoverers of the increased vulnerability of enkephalin-containing striatal projection neurons (SPNs) constituting the indirect pathway. In particular, he is interested in anatomical changes occurring in patients and in animal models during HD progression. In his detailed review, Professor Reiner dissects the topography and chronology of connectivity changes occurring in HD. From early loss of cortical and thalamic inputs to striatum to late disruption of direct pathway SPNs, he is able to explain the changing patterns of motor dysfunction in HD patients. Abnormalities in glutamate release and/or glutamate receptor sensitivity have long been invoked to explain striatal cell death in HD. Although probably not exclusive, anatomical and physiological data support glutamate involvement. George Rebec (Indiana University) has been studying corticostriatal communication and how glutamate transport in the corticostriatal pathway is altered in HD animal models9 (pp. 281–291). Using a wide array of techniques including in vivo electrophysiological recordings, measurement of glutamate release, and computational modeling Professor Rebec and his team have demonstrated important changes in striatal and cortical neuron firing patterns, as well as dysregulation of dynamic changes in extracellular glutamate and ascorbate. By demonstrating deficits in astrocyte-mediated glutamate transport in the striatum, he has paved the way for new ideas to treat HD. He has shown that increased expression of glutamate transport proteins restores glutamate and ascorbate homeostasis in striatum while normalizing striatal neuronal activity and improving behavioral signs. However, in HD not only glutamate transmission goes awry. Dysregulation of glutamate release along the corticostriatal pathway triggers compensatory mechanisms to prevent cell damage. The function of GABA, the main inhibitory neurotransmitter in the brain, also is altered in HD. Upregulation of phasic GABA release in striatum, as well as deficits in tonic GABA, has been demonstrated in mouse models. Maurice Garret (Université de Bordeaux) is an expert on GABA receptor structure and function in the brain. Recently, he has focused his attention on uncovering alterations of GABA receptors in animal models. He demonstrated, beyond doubt, that glutamate and dopamine are not the only neurotransmitters affected by the malady, as the GABA system also is deeply altered. In addition, GABA is the main neurotransmitter used by SPNs of both direct and indirect pathways. Thus, any changes in GABA function occurring in striatum will have an effect on key output regions including the external globus pallidus and the substantia nigra pars reticulata. In his insightful review10 (pp. 292–300), Dr. Garret examines specific changes in GABAA receptor subunits in different basal ganglia regions and highlights potential therapeutic avenues by targeting selective GABAA receptor subunits to treat nonmotor symptoms such as sleep disturbances commonly observed in HD. The laboratory of Lynn Raymond (University of British Columbia, Vancouver) is well known for its contributions to our understanding of the role that glutamate extrasynaptic NMDA receptors play in excitotoxicity. Deleterious effects are likely mediated by dysregulation of calcium influx and handling in different cell compartments. Dr. Raymond and her collaborators James P. Mackay and Wissam B. Nassrallah discuss how abnormal calcium handling can lead to neuronal degeneration11 (pp. 301–310). As many alterations observed in HD animal models occur very early in development, they wonder whether changes are a primary cause or the result of compensatory mechanisms. This is a provocative idea emphasizing the capabilities of the brain to repair itself. Their review also highlights possible therapies focused on restoring calcium homeostasis. Calcium influx, either via NMDA receptors or voltage-gated and store-operated channels, is not the only process affected in HD. Potassium channels also play an important role as they are involved in myriad of cellular processes, most of them designed to prevent neuronal hyperexcitability. Potassium channel dysfunction was first suspected by studies showing that SPNs are more depolarized in HD mouse models, pointing to decreased inward rectification due to loss of Kir channel expression. Working in Baljit Khakh's laboratory (University of California Los Angeles), Xiaoping Tong (presently a Professor at Shanghai Jiao Tong University School of Medicine) discovered that astrocytic Kir4.1 channels were deficient in HD mouse models. This deficit was accompanied by increased extracellular potassium, which in turn could explain depolarized resting membrane potentials of affected SPNs. Further, upregulation of Kir4.1 channels ameliorated the HD phenotype. In their contribution to the SI12 (pp. 311–318), Professor Tong and collaborators review evidence summarizing recent progress on the function of neuronal and astrocytic potassium channels and their involvement in HD pathology using animal models. They also illustrate how targeting potassium channels can be used for the treatment of HD. The altered interplay between neurotransmitters and channels explains many of the molecular cascades leading to neurodegeneration in HD. But this is not, by any stretch of the imagination, the whole story. Cellular energy metabolism is deeply disturbed in HD. In particular, inefficient use of lactate by HD neurons contributes to metabolic failure. Maité A. Castro (Universidad Austral de Chile), an expert in cellular energy metabolism, and colleagues demonstrate that HD cells exhibit decreased expression of the neuronal glucose transporter GLUT3, which could be the cause of insufficient energy supply13 (pp. 343–352). Further, stimulation of lactate uptake by ascorbic acid could be restored by overexpressing GLUT3. Again this opens new avenues for HD treatment. Another potential avenue targets transcriptional dysregulation, a major cause of HD pathology. Francesca Fusco (Fondazione Santa Lucia, Rome) and Antonella Cardinale review recent neuroprotective strategies via phosphodiesterase (PDE) inhibition and consequent upregulation of cyclic nucleotide signaling in HD14 (pp. 319–328). They argue that, since mutant huntingtin alters cAMP response element-binding protein (CREB), which in turn promotes transcription of brain-derived neurotrophic factor (BDNF), strategies aimed at increasing CREB transcription could have neuroprotective effects in HD animals. As PDEs are enzymes that catabolize cAMP and/or cGMP in the cell, their inhibition could be beneficial. Also highlighting the beneficial effects of neurotrophic factors, Elizabeth Hernández-Echeagaray (Universidad Nacional Autónoma de México) and colleagues examine the role of neurotrophin-3 (NT-3) in neuroprotection and synaptic plasticity in the striatum of an animal model of HD15 (pp. 353–363). They convincingly demonstrate that normal synaptic plasticity in the corticostriatal pathway is aberrant in HD model mice but it can be restored by administration of NT-3. One of the most challenging aspects of HD research relating to therapies is whether cell loss can be prevented or cells can be replaced. In human HD and, to a lesser degree, in genetic animal models, striatal and cortical cell loss is massive. Fortunately, regenerative medicine is becoming a reality and specific cell types may be able to be replaced using stem cells that can be induced to adopt particular phenotypes. Recent studies demonstrate that cells implanted in the brain of HD model mice can survive and differentiate. Notably, most studies demonstrate clear benefits on HD symptoms with some showing a possible mechanism of action that includes the release of neurotrophic factors such as BDNF. Sandra M. Holley (University of California Los Angeles, UCLA), working in the Cepeda-Levine laboratory, and associates at the University of California Davis (UCD) and at the University of California Irvine (UCI) review the use of stem cells in HD animal models16 (pp. 329–342). In their California Institute for Regenerative Medicine (CIRM)-funded studies, they showed that human-derived neural stem cells implanted in HD mice can protect or repair damaged tissue, delay disease progression, decrease pathologies, and increase production of neurotrophic molecules. In this SI, they also provide a more detailed electrophysiological characterization of human-derived neural stem cells implanted in HD mice. We would like to thank all the contributors who took time from their busy schedules to write comprehensive reviews or high-quality original papers that will certainly help the neuroscience community in general and the HD field in particular. Finally, we would like to thank CNSNT and its Editor-in-Chief, Ding-Feng Su, as well as its Managing Editor, Buddy Zhou, for encouraging the compilation of this SI on HD. Without their input and guidance, this SI would not have been possible.

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disease. HD has no cure, and patients pass away 10-20 years after the onset of symptoms. The causal mutation for HD is a trinucleotide repeat expansion in exon 1 of the huntingtin gene that leads to a polyglutamine (polyQ) repeat expansion in the N-terminal region of the huntingtin protein. Interestingly, there is a threshold of 37 polyQ repeats under which little or no disease exists; and above which, patients invariably show symptoms of HD. The huntingtin protein is a 350 kDa protein with unclear function. As the polyQ stretch expands, its propensity to aggregate increases with polyQ length. Models for polyQ toxicity include formation of aggregates that recruit and sequester essential cellular proteins, or altered function producing improper interactions between mutant huntingtin and other proteins. In both models, soluble expanded polyQ may be an intermediate state that can be targeted by potential therapeutics. In the first study described herein, the conformation of soluble, expanded polyQ was determined to be linear and extended using equilibrium gel filtration and small-angle X-ray scattering. While attempts to purify and crystallize domains of the huntingtin protein were unsuccessful, the aggregation of huntingtin exon 1 was investigated using other biochemical techniques including dynamic light scattering, turbidity analysis, Congo red staining, and thioflavin T fluorescence. Chapter 4 describes crystallization experiments sent to the International Space Station and determination of the X-ray crystal structure of the anti-polyQ Fab MW1. In the final study, multimeric fibronectin type III (FN3) domain proteins were engineered to bind with high avidity to expanded polyQ tracts in mutant huntingtin exon 1. Surface plasmon resonance was used to observe binding of monomeric and multimeric FN3 proteins with huntingtin.

Huntington’s disease (HD) is a devastating, genetic, neurodegenerative disease for which there is currently no effective therapy. The polyglutamine (polyQ) expansion that causes HD is in the first exon (HDx1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids (AAs) and two proline (polyP) repeat domains, modulate the toxicity of mutant Htt (mHtt). The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies; iAbs), we tested various epitopes for their roles in mHDx1 toxicity, aggregation, localization and turnover. Three domains in the P-rich region (PRR) of HDx1 are defined by iAbs: MW7 binds the two polyP domains, and Happs 1 and 3, two new iAbs, bind the unique, P-rich epitope located between the two polyP epitopes. In cultured cells, we find that the three PRR-binding iAbs, as well as VL12.3, which binds an epitope in the N-terminal 17 AA segment, decrease the toxicity and aggregation of mHDx-1, but they do so by different mechanisms. The PRR-binding iAbs have no effect on Htt localization, but they cause a significant increase in the turnover rate of mHtt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. These results suggest that the PRR domain regulates mHtt stability and toxicity. Thus, compromising this pathogenic epitope by iAb binding represents a novel therapeutic strategy for treating HD. We have tested this hypothesis by delivering both VL12.3 and Happ1 to the brains of HD model mice using an AAV2/1 viral vector with a modified CBA promoter. VL12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in assays of motor and cognitive deficits as well as in the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128 and BACH models of HD. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment possibility for HD.

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD

Polyglutamine (polyQ) diseases are hereditary neurodegenerative disorders caused by an abnormal expansion of a trinucleotide CAG repeat in the coding region of their respective associated genes. PolyQ diseases mainly display progressive degeneration of the brain and spinal cord. Nine polyQ diseases are known, including Huntington’s disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA), and six forms of spinocerebellar ataxia (SCA). HD is the best characterized polyQ disease. Many studies have reported that transcriptional dysregulation and post-translational disruptions, which may interact with each other, are central features of polyQ diseases. Post-translational modifications, such as the acetylation of histones, are closely associated with the regulation of the transcriptional activity. A number of groups have studied the interactions between the polyQ proteins and transcription factors. Pharmacological drugs or genetic manipulations aimed at correcting the dysregulation have been confirmed to be effective in the treatment of polyQ diseases in many animal and cellular models. For example, histone deaceylase inhibitors have been demonstrated to have beneficial effects in cases of HD, SBMA, DRPLA, and SCA3. In this review, we describe the transcriptional and post-translational dysregulation in polyQ diseases with special focus on HD, and we summarize and comment on potential treatment approaches targeting disruption of transcription and post-translation processes in these diseases.

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins

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Investigating neurodegenerative diseases with small molecule modulators Reka Rebecca Letso Elucidation of the mechanisms underlying cell death in neurodegenerative diseases has proven difficult, due to the complex and interconnected architecture of the nervous system as well as the often pleiotropic nature of these diseases. Cell culture models of neurodegenerative diseases, although seldom recapitulating all aspects of the disease phenotype, enable investigation of specific aspects of these disease states. Small molecule screening in these cell culture models is a powerful method for identifying novel small molecule modulators of these disease phenotypes. Mechanistic studies of these modulators can reveal vital insights into the cellular pathways altered in these disease states, identifying new mechanisms leading to cellular dysfunction, as well as novel therapeutic targets to combat these destructive diseases. Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are quite rare. Small molecule protein upregulators with targeted activities would be of great value in the study of the mechanisms underlying many loss of function diseases. We developed a highthroughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease Spinal Muscular Atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analog designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via translation, an effect which may be associated with the translational regulator mammalian target of rapamycin (mTOR). These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA. Small molecule modulators of a disease phenotype, such as cell death, have the potential to reveal novel mechanisms regulating disease processes. This was exemplified by a screen for small molecule inhibitors of cell death caused by a pathogenic, misofolded mutant huntingtin protein in a cell culture model of Huntington’s Disease (HD). These cell death inhibitors were found to target protein disulfide isomerase (PDI), an oxidoreductase known to be important in endoplasmic reticulum quality control of protein folding. However, our studies utilizing the small molecule PDI inhibitors determined that the cell death observed in this system was due to a pro-apoptotic function of PDI involving proteins of the mitochondrial outer membrane. We have begun studies aimed at identifying the binding mode of these novel small molecule inhibitors of PDI, in efforts to develop more potent and efficacious analogs for testing in animal models of HD. These studies have helped defined a novel mechanism linking protein misfolding to cell death, and may prove to be relevant to a broader range of protein misfolding diseases. Table of contents List of Figures and Tables v Acknowledgements vii Chapter 1: Spinal Muscular Atrophy 1 I. Treating inherited neurodegenerative diseases 1 a. Gain of function neurodegenerative diseases 2 b. Loss of function neurodegenerative diseases 5 II. Spinal Muscular Atrophy Disease 7 a. SMA disease pathogenesis 8 b. SMA disease phenotypes 9 c. Genetics of SMA 10 d. Animal models of SMA 15 III. Survival of Motor Neuron protein: Structure and Function 23 a. SMN expression and localization 23 b. SMN domain structure and functions 25 c. SMN interacting partners 28 i. The SMN Complex 29 ii. SMN interacts with hnRNP Q/R and β-actin mRNA 30 iii. SMN and profilin 32 iv. The possible role of actin dynamics in Spinal Muscular Atrophy 33 d. Neuronal function(s) of SMN 34 IV. Current state of small molecule therapeutics for SMA 46

Huntington’s disease (HD) is a neurodegenerative disease that affects four to seven individuals per 100,000. The onset of symptoms usually begins in middle age, although approximately 5% become symptomatic as juveniles. Death occurs approximately 15 years following the onset of symptoms, which include choreic movements, cognitive decline and psychiatric changes. HD is an autosomal dominant inherited disease that is associated with an expansion of a trinucleotide (CAG) repeat located on chromosome 4. Physicians rely on a positive family history, and diagnostic and genetic tests to detect the expansion in the number of CAG trinucleotide repeats in the HD gene to confirm the diagnosis. More than 99% of HD patients have 40 or more CAG triplet repeats and, therefore, targeted mutational analysis is greater than 99% sensitive. Individuals with 26 triplet repeats or less are normal, and while those with 27–35 repeats may not demonstrate symptoms themselves, their offspring may have the disease. Individuals with 36–39 repeats may or may not exhibit symptoms. The College of American Pathology/American College of Medical Genetics Biochemical and Molecular Genetics Resource Committee has emphasized the need to standardize the methodology for the determination of the accurate number of CAG repeats. This will prevent false-positive or -negative results when conducting predictive or prenatal testing of at-risk individuals. The National Institute of Standards and Technology is developing a standard reference material to provide these positive and negative controls needed by clinical testing laboratories. The use of a HD standard reference material will provide the quality control and assurance that data from different laboratories are both comparable and accurate.

A novel class of inherited human neurodegenerations is now known to be caused by expanded CAG repeats encoding polyglutamines. Polyglutamine-containing protein fragments have been shown to accumulate as aggregates in the nucleus and in the cytoplasm, and to induce cell death when expressed in cultured cells, leading to the proposal that polyglutamine aggregation is an important step in the pathogenesis. Supporting this, nuclear inclusions containing expanded polyglutamines have been identified in neurones from the brains of patients and in neurones from transgenic mouse models of this class of neural disorders. We analysed the consequences of polyglutamine expression in PC12 neuronal cells. Activated SEK1 accumulated with nuclear but not cytoplasmic polyglutamine aggregations, which consequently triggers cell death. Cell death induced by polyglutamine expression was inhibited by a dominant-negative SEK1 (DN-SEK1), but not by DN-SEK1 tagged with a nuclear export signal. Steady state SEK1 expression itself was enhanced two to three-fold. Nuclearly aggregated polyglutamines, which were identified in PML bodies, co-localized with not only activated SEK1 but also activated c-Jun. We also observed that nuclear inclusion-positive neurones from brains with Huntington's disease expressed SEK1. This study provides molecular links between the neurodegeneration observed in polyglutamine diseases, cell death signalling kinase cascades and nuclear subdomains related to cell death. We propose that the nuclear PML bodies containing polyglutamine aggregates activate the SEK1-JNK kinase cascade, resulting in the transduction of a death signal.

Silmitasertib, an FDA-designated orphan CK2 inhibitor, ameliorates neuropathology and motor dysfunction in a Huntington's disease mouse model.

With the gradual advancement of research methods and technologies, various biological processes have been identified as playing roles in the pathogenesis of neurodegenerative diseases. However, current descriptions of these biological processes do not fully explain the onset, progression, and development of these conditions. Therefore, exploration of the pathogenesis of neurodegenerative diseases remains a valuable area of research. This review summarizes the potential common pathogeneses of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, frontotemporal lobar dementia, and Lewy body disease. Research findings have indicated that several common biological processes, including aging, genetic factors, progressive neuronal dysfunction, neuronal death and apoptosis, protein misfolding and aggregation, neuroinflammation, mitochondrial dysfunction, axonal transport defects, and gut microbiota dysbiosis, are involved in the pathogenesis of these six neurodegenerative diseases. Based on current information derived from diverse areas of research, these biological processes may form complex pathogenic networks that lead to distinctive types of neuronal death in neurodegenerative diseases. Furthermore, promoting the regeneration of damaged neurons may be achievable through the repair of affected neural cells if the underlying pathogenesis can be prevented or reversed. Hence, these potential common biological processes may represent only very small, limited elements within numerous intricate pathogenic networks associated with neurodegenerative diseases. In clinical treatment, interfering with any single biological process has proven insufficient to completely halt the progression of neurodegenerative diseases. Therefore, future research on the pathogenesis of neurodegenerative diseases should focus on uncovering the complex pathogenic networks, rather than isolating individual biological processes. Based on this, therapies that aim to block or reverse various targets involved in the potential pathogenic mechanisms of neurodegenerative diseases may be promising directions, as current treatment methods that focus on halting a single pathogenic factor have not achieved satisfactory efficacy.

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington’s disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10 nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, only FAN1, OGG1, and PCNA showed significant suppression. When the methylation profile of HD cells was analyzed, FAN1 and OGG1 showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

Genomic clones encompassing the Huntington disease (HD) mutation were used to isolate a probe that detects size changes in the restriction fragments that contain the HD trinucleotide repeat (TNR). This probe also detects a frequent Sau3A polymorphism (allele sizes 1.8-kb and 2.7kb), which maps approximately 950bp from the TNR. Examination of a number of HD families established that the frequency of the Sau3A alleles did not differ significantly between control and HD populations; however, the HD expansion was always present on a chromosome that contained the 1.8-kb Sau3A allele. This association between a specific allele and the HD TNR expansion was significant and could provide a clue to the chromosomal elements that produce the trinucleotide expansion on the Huntington disease chromosome.

Protein misfolding and aggregation have been recognized as a common molecular pathogenesis of various neurodegenerative diseases including Alzheimer's, Parkinson's, and the polyglutamine (polyQ) diseases. The polyQ diseases, including Huntington's disease and various spinocerebellar ataxias, are caused by abnormal expansions of the polyQ stretch (>35-40) within disease-causative proteins. Recently, defects in protein degradation in the brain have been shown to cause neurodegeneration in genetically-engineered mice, highlighting two important roles of protein degradation systems in neurodegenerative diseases; 1) their dysfunction in the pathogenesis and 2) their activation as a therapy. However, it is indispensable to eliminate only the pathogenic proteins to avoid deleterious side effects. Aiming to selectively degrade the expanded polyQ proteins, we employed QBP1, a peptide which specifically binds to the expanded polyQ stretch. We designed a chimeric protein with the Hsc70 binding motif, a signal sequence for chaperone-mediated autophagy, fused to QBP1 (Hsc70BM-QBP1), and found that Hsc70BM-QBP1 accelerates the selective degradation of expanded polyQ proteins in cell culture. Gene therapy using a viral vector expressing Hsc70BM-QBP1 effectively ameliorates the motor dysfunction and premature death in polyQ disease model mice. We propose that our therapeutic strategy to selectively degrade the pathogenic proteins can also be applied to other neurodegenerative diseases.