Polyglutamine Expansion Research Articles

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths.

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27 , Q27 P11 , Q34 , Q35 , Q36 , Q40 , Q50 , and Q50 P11 . In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.

Impaired Nuclear Export of Polyglutamine-Expanded Androgen Receptor in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR). Prior studies have highlighted the importance of AR nuclear localization in SBMA pathogenesis; therefore, in this study, we sought to determine the role of AR nuclear export in the pathological manifestations of SBMA. We demonstrate here that the nuclear export of polyQ-expanded AR is impaired, even prior to the formation of intranuclear inclusions of aggregated AR. Additionally, we find that promoting AR export with an exogenous nuclear export signal substantially reduces its aggregation and blocks hormone-induced toxicity. Moreover, we show that these protective effects are conferred by destabilization of the mutant protein due to an increase in proteasomal degradation of the cytoplasmic AR. Despite a growing body of evidence that global disruption of nucleo/cytoplasmic transport occurs in ALS and HD, our data suggest that no such global disruption occurs in models of SBMA; rather, AR-specific mechanisms, including reduced phosphorylation at Serine 650, are likely responsible for the impaired nuclear export of polyQ-expanded AR.

Ironing out the Brain.

Our understanding of the broad principles of cellular and systemic iron homeostasis in man are well established with the exception of the brain. Most of the proteins involved in mammalian iron metabolism are present in the brain, although their distribution and precise roles in iron uptake, intracellular metabolism and export are still uncertain, as is the way in which systemic iron is transferred across the blood-brain barrier. We briefly review current concepts concerning the uptake and distribution of iron in the brain, before turning to the ways in which brain iron homeostasis might be regulated. The distribution of iron between different brain regions is then discussed as is the increase in brain iron with normal aging, and the different forms in which iron is present. The increased levels of iron found in specific brain regions and their potential contribution to neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease and other polyglutamine expansion diseases, amyotrophic lateral sclerosis, Friedreich's ataxia, as well as a number of neurodegenerative diseases with iron accumulation, are discussed. The interactions between neuroinflammation and iron are presented, and the chapter concludes with a review of current clinical studies and discussion of the potential and efficacy of iron chelation therapy in the treatment of neurodegenerative diseases.

Cell Death Mechanisms in a Mouse Model of Retinal Degeneration in Spinocerebellar Ataxia 7

Spino-cerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by neurodegeneration of the brain, cerebellum, and retina caused by a polyglutamine expansion in ataxin7. The presence of an expanded polyQ tract in a mutant protein is known to induce protein aggregation, cellular stress, toxicity, and finally cell death. However, the consequences of the presence of mutant ataxin7 in the retina and the mechanisms underlying photoreceptor degeneration remain poorly understood. In this study, we show that in a retinal SCA7 mouse model, polyQ ataxin7 induces stress within the retina and activates Muller cells. Moreover, unfolded protein response and autophagy are activated in SCA7 photoreceptors. We have also shown that the photoreceptor death does not involve a caspase-dependent apoptosis but instead involves apoptosis inducing factor (AIF) and Leukocyte Elastase Inhibitor (LEI/L-DNase II). When these two cell death effectors are downregulated by their siRNA, a significant reduction in photoreceptor death is observed. These results highlight the consequences of polyQ protein expression in the retina and the role of caspase-independent pathways involved in photoreceptor cell death.

HNRNP Q suppresses polyglutamine huntingtin aggregation by post‐transcriptional regulation of vaccinia‐related kinase 2

Misfolded proteins with abnormal polyglutamine (polyQ) expansion cause neurodegenerative disorders, including Huntington's disease. Recently, it was found that polyQ aggregates accumulate as a result of vaccinia-related kinase 2 (VRK2)-mediated degradation of TCP-1 ring complex (TRiC)/chaperonin-containing TCP-1 (CCT), which has an essential role in the prevention of polyQ protein aggregation and cytotoxicity. The levels of VRK2 are known to be much higher in actively proliferating cells but are maintained at a low level in the brain via an unknown mechanism. Here, we found that basal levels of neuronal cell-specific VRK2 mRNA are maintained by post-transcriptional, rather than transcriptional, regulation. Moreover, heterogeneous nuclear ribonucleoprotein Q (HNRNP Q) specifically binds to the 3'untranslated region of VRK2 mRNA in neuronal cells to reduce the mRNA stability. As a result, we found a dramatic decrease in CCT4 protein levels in response to a reduction in HNRNP Q levels, which was followed by an increase in polyQ aggregation in human neuroblastoma cells and mouse cortical neurons. Taken together, these results provide new insights into how neuronal HNRNP Q decreases VRK2 mRNA stability and contributes to the prevention of Huntington's disease, while also identifying new prognostic markers of HD.

Новые подходы в исследовании молекулярного патогенеза спиноцеребеллярной атаксии 2-го типа

Spinocerebellar ataxia type 2 (SCA2) represents a progressive hereditary disease, genetically caused by a polyglutamine expansion in the ataxin-2 protein. The effective ways of the therapeutic treatment as well as the disease-modifiyng therapy are not available yet for SCA2 patients. At present, patients can get only the symptomatic treatment or palliative care. Many research groups study the physiological, molecular, and biochemical changes in the cerebellar neurons from SCA2 patients and different model systems to find new therapeutic targets for SCA2 treatment. Comprehensive approaches in the research of SCA2 pathogenesis allowed to get the new data about the molecular mechanism of the disease and to suggest the possible strategies for the potential therapy of the disease. In this article the relevant data about the genetic basis of SCA2 are summarized, the known to date properties and functions of ataxin-2 protein are described, the mechanisms of the cerebellar cells degeneration are discussed together with the dicturbances of the cellular physilological functions and the related impairments of the cerebellar curcuits, the information about the modern model systems that allow to study the SCA2 bases is presented, and also the data about new approaches in molecular mechanism research are presented, describing the SCA2 pathology bases such as aggregation, oxidative stress, the dysfunction of the cell and calcium signaling. The role of the autophagy and microglia in the molecular pathogenesis of SCA2 is also discussed.

TiO2 Nanoparticles Catalyze Oxidation of Huntingtin Exon 1-Derived Peptides Impeding Aggregation: A Quantitative NMR Study of Binding and Kinetics.

Polyglutamine expansion within the N-terminal region of the huntingtin protein results in the formation of intracellular aggregates responsible for Huntington's disease, a fatal neurodegenerative condition. The interaction between TiO2 nanoparticles and huntingtin peptides comprising the N-terminal amphiphilic domain without (httNT) or with (httNTQ10) a ten-residue C-terminal polyglutamine tract, is investigated by NMR spectroscopy. TiO2 nanoparticles decrease aggregation of httNTQ10 by catalyzing the oxidation of Met7 to a sulfoxide, resulting in an aggregation-incompetent peptide. The oxidation agent is hydrogen peroxide generated on the surface of the TiO2 nanoparticles either by UV irradiation or at low steady-state levels in the dark. The binding kinetics of nonaggregating httNT to TiO2 nanoparticles is characterized by quantitative analysis of 15N dark state exchange saturation transfer and lifetime line broadening NMR data. Binding involves a sparsely populated intermediate that experiences hindered rotational diffusion relative to the free state. Catalysis of methionine oxidation within the N-terminal domain of the huntingtin protein may potentially provide a strategy for delaying the onset of Huntington's disease.

Upregulation of miR-25 and miR-181 Family Members Correlates with Reduced Expression of ATXN3 in Lymphocytes from SCA3 Patients.

Spinocerebellar ataxia type 3 (SCA3), the most common spinocerebellar ataxia, is caused by a polyglutamine (polyQ) expansion in the protein ataxin-3 (ATXN3). Silencing the expression of polyQ-expanded ATXN3 rescues the cellular disease phenotype. This study investigated the differential expression of microRNAs (miRNAs), small noncoding RNAs targeting gene expression, in lymphoblastoid cells (LCs) from SCA3 patients and the capability of identified deregulated miRNAs to target and alter ATXN3 expression. MiRNA profiling was performed by microarray hybridization of total RNA from control and SCA3-LCs. The capability of the identified miRNAs and their target sites to suppress ATXN3 expression was analyzed using mutagenesis, reverse transcription PCR, immunoblotting, luciferase reporter assays, mimics and precursors of the identified miRNAs. SCA3-LCs showed significantly decreased expression levels of ATXN3 and a significant upregulation of the ATXN3-3'UTR targeting miRNAs, miR-32 and miR-181c and closely related members of the miR-25 and miR-181 family, respectively. MiR-32 and miR-181c effectively targeted the 3'UTR of ATXN3 and suppressed the expression of ATXN3. The simultaneous upregulation of closely related miRNAs targeting the 3'UTR of ATXN3 and the significantly reduced ATXN3 expression levels in SCA3-LCs suggests that miR-25 and miR-181 family members cooperatively bind to the 3'UTR to suppress the expression of ATXN3. The findings further suggest that the upregulation of miR-25 and miR-181 family members in SCA3- LCs reflects a cell type-specific, protective mechanism to diminish polyQ-mediated cytotoxic effects. Thus, miRNA mimics of miR-25 and miR-181 family members may prove useful for the treatment of SCA3.

Biodegradable Nanoparticles Containing Mechanism Based Peptide Inhibitors Reduce Polyglutamine Aggregation in Cell Models and Alleviate Motor Symptoms in a Drosophila Model of Huntington's Disease.

Detailed study of the molecular mechanism behind the pathogenesis of Huntington's disease (HD) suggests that polyglutamine aggregation is one of the fundamental reasons for HD. Despite the discovery of many potential molecules, HD therapy is still limited to symptomatic relief. Among these molecules, few mechanism based peptide inhibitors of polyglutamine aggregation (QBP1, NT17 and PGQ9P2) have shown promising activity; however, poor blood-brain barrier (BBB) penetration, low bioavailability, and low half-life may hinder their therapeutic potential. Hence, to deliver them to the brain for assessing their efficacy, we have designed and synthesized peptide loaded poly-d,l-lactide- co-glycolide (PLGA) nanoparticles of less than 200 nm in size by carbodiimide chemistry and nanoprecipitation protocols. For brain delivery, PLGA nanoparticles were coated with polysorbate 80 which aids receptor mediated internalization. Using the in vitro BBB model of Madin-Darby canine kidney cells and healthy mice, the translocation of polysorbate 80 coated fluorescent nanoparticles was confirmed. Moreover, QBP1, NT17, and PGQ9P2 loaded PLGA nanoparticles showed dose dependent inhibition of polyglutamine aggregation in cell models of HD (Neuro 2A and PC12 cells) and improved motor performance in Drosophila model of HD. Additionally, no toxicity in cells and animals confirmed biocompatibility of the nanoparticulate formulations. Based on this work, future studies can be designed in higher animal models to test peptide loaded nanoparticles in HD and other polyglutamine expansion related diseases.

Loss of zebrafish Ataxin-7, a SAGA subunit responsible for SCA7 retinopathy, causes ocular coloboma and malformation of photoreceptors.

Polyglutamine (polyQ) expansion in Ataxin-7 (ATXN7) results in spinocerebellar ataxia type 7 (SCA7) and causes visual impairment. SCA7 photoreceptors progressively lose their outer segments (OSs), a structure essential for their visual function. ATXN7 is a subunit of the transcriptional coactivator Spt-Ada-Gcn5 Acetyltransferase complex, implicated in the development of the visual system in flies. To determine the function of ATXN7 in the vertebrate eye, we have inactivated ATXN7 in zebrafish. While ATXN7 depletion in flies led to gross retinal degeneration, in zebrafish, it primarily results in ocular coloboma, a structural malformation responsible for pediatric visual impairment in humans. ATXN7 inactivation leads to elevated Hedgehog signaling in the forebrain, causing an alteration of proximo-distal patterning of the optic vesicle during early eye development and coloboma. At later developmental stages, malformations of photoreceptors due to incomplete formation of their OSs are observed and correlate with altered expression of crx, a key transcription factor involved in the formation of photoreceptor OS. Therefore, we propose that a primary toxic effect of polyQ expansion is the alteration of ATXN7 function in the daily renewal of OS in SCA7. Together, our data indicate that ATXN7 plays an essential role in vertebrate eye morphogenesis and photoreceptor differentiation, and its loss of function may contribute to the development of human coloboma.

Recent advances in understanding dominant spinocerebellar ataxias from clinical and genetic points of view.

Spinocerebellar ataxias (SCAs) are rare types of cerebellar ataxia with a dominant mode of inheritance. To date, 47 SCA subtypes have been identified, and the number of genes implicated in SCAs is continually increasing. Polyglutamine (polyQ) expansion diseases ( ATXN1/SCA1, ATXN2/SCA2, ATXN3/SCA3, CACNA1A/SCA6, ATXN7/SCA7, TBP/SCA17, and ATN1/DRPLA) are the most common group of SCAs. No preventive or curative treatments are currently available, but various therapeutic approaches, including RNA-targeting treatments, such as antisense oligonucleotides (ASOs), are being developed. Clinical trials of ASOs in SCA patients are already planned. There is, therefore, a need to identify valid outcome measures for such studies. In this review, we describe recent advances towards identifying appropriate biomarkers, which are essential for monitoring disease progression and treatment efficacy. Neuroimaging biomarkers are the most powerful markers identified to date, making it possible to reduce sample sizes for clinical trials. Changes on brain MRI are already evident at the premanifest stage in SCA1 and SCA2 carriers and are correlated with CAG repeat size. Other potential biomarkers have also been developed, based on neurological examination, oculomotor study, cognitive assessment, and blood and cerebrospinal fluid analysis. Longitudinal studies based on multimodal approaches are required to establish the relationships between parameters and to validate the biomarkers identified.

Conserved Pbp1/Ataxin-2 regulates retrotransposon activity and connects polyglutamine expansion-driven protein aggregation to lifespan-controlling rDNA repeats

Ribosomal DNA (rDNA) repeat instability and protein aggregation are thought to be two major and independent drivers of cellular aging. Pbp1, the yeast ortholog of human ATXN2, maintains rDNA repeat stability and lifespan via suppression of RNA–DNA hybrids. ATXN2 polyglutamine expansion drives neurodegeneration causing spinocerebellar ataxia type 2 and promoting amyotrophic lateral sclerosis. Here, molecular characterization of Pbp1 revealed that its knockout or subjection to disease-modeling polyQ expansion represses Ty1 (Transposons of Yeast) retrotransposons by respectively promoting Trf4-depedendent RNA turnover and Ty1 Gag protein aggregation. This aggregation, but not its impact on retrotransposition, compromises rDNA repeat stability and shortens lifespan by hyper-activating Trf4-dependent turnover of intergenic ncRNA within the repeats. We uncover a function for the conserved Pbp1/ATXN2 proteins in the promotion of retrotransposition, create and describe powerful yeast genetic models of ATXN2-linked neurodegenerative diseases, and connect the major aging mechanisms of rDNA instability and protein aggregation.

Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington's Disease.

Huntington’s disease (HD) is a hereditary neurodegenerative disease that is caused by polyglutamine expansion within the huntingtin (HTT) gene. One of the cellular activities that is dysregulated in HD is store-operated calcium entry (SOCE), a process by which Ca2+ release from the endoplasmic reticulum (ER) induces Ca2+ influx from the extracellular space. HTT-associated protein-1 (HAP1) is a binding partner of HTT. The aim of the present study was to examine the role of HAP1A protein in regulating SOCE in YAC128 mice, a transgenic model of HD. After Ca2+ depletion from the ER by the activation of inositol-(1,4,5)triphosphate receptor type 1 (IP3R1), we detected an increase in the activity of SOC channels when HAP1 protein isoform HAP1A was overexpressed in medium spiny neurons (MSNs) from YAC128 mice. A decrease in the activity of SOC channels in YAC128 MSNs was observed when HAP1 protein was silenced. In YAC128 MSNs that overexpressed HAP1A, an increase in activity of IP3R1 was detected while the ionomycin-sensitive ER Ca2+ pool decreased. 6-Bromo-N-(2-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (C20H22BrClN2), identified in our previous studies as a SOCE inhibitor, restored the elevation of SOCE in YAC128 MSN cultures that overexpressed HAP1A. The IP3 sponge also restored the elevation of SOCE and increased the release of Ca2+ from the ER in YAC128 MSN cultures that overexpressed HAP1A. The overexpression of HAP1A in the human neuroblastoma cell line SK-N-SH (i.e., a cellular model of HD (SK-N-SH HTT138Q)) led to the appearance of a pool of constitutively active SOC channels and an increase in the expression of STIM2 protein. Our results showed that HAP1A causes the activation of SOC channels in HD models by affecting IP3R1 activity.

CYP46A1 protects against NMDA-mediated excitotoxicity in Huntington's disease: Analysis of lipid raft content

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (mHtt) protein leading to degeneration of striatal neurons. Excitotoxicity, consecutive to overstimulation of N-methyl d-aspartate receptors (NMDARs) has a pivotal role in many neurological disorders including HD. Mutant Htt causes enhanced NMDA sensitivity, alteration of NMDAR expression and localization in neurons. Excitotoxic events initiate neuronal death in numerous ways, including activation of apoptotic cascades. Among the NMDAR subunits involved in glutamatergic-mediated excitotoxicity, GluN2B has been extensively reported. In addition to excitotoxicity, alteration of cholesterol metabolism has been observed in HD, with a decrease of cholesterol precursor synthesis along with an increase of cholesterol accumulation, which is deleterious for neurons. Expression of Cholesterol Hydroxylase enzyme, CYP46A1, which converts cholesterol into 24 S-hydroxycholesterol is down-regulated in HD. We found that CYP46A1 overexpression is beneficial in HD neurons and mouse model, but the mechanisms involved still remain unclear. In this study we addressed the effect of CYP46A1 on NMDAR-mediated excitotoxicity in HD primary neurons and its role in modulating cholesterol and localization of GLUN2B in lipid rafts. We showed that CYP46A1 is protective against NMDAR-mediated excitotoxicity in two different HD neuronal cell models. Cholesterol as well as GluN2B level in lipid raft, are significantly increased by mHtt. Despite a clear effect of CYP46A1 in reducing cholesterol content in lipid raft extracts from wild type neurons, CYP46A1 overexpression in HD neurons could not normalize the increased cholesterol levels in lipid rafts. This study highlights the beneficial role of CYP46A1 against NMDAR-mediated excitotoxicity and gives further insights into the cellular mechanisms underlying CYP46A1-mediated neuroprotection.

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-sequencing (RNA-Seq) to identify pathways that are disrupted in diseased muscle using AR113Q knockin mice. This analysis unexpectedly identified substantially diminished expression of numerous ubiquitin/proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age, hormone, and glutamine length dependent and arose due to a toxic gain of function conferred by the mutation. Moreover, altered gene expression was associated with decreased levels of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.

Calmidazolium Chloride and Its Complex with Serum Albumin Prevent Huntingtin Exon1 Aggregation

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing an expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules for HD, which is currently untreatable. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Because it is hydrophobic, CLC was adsorbed to the vial surface and could not sustain an inhibition effect for a longer duration. The use of bovine serum albumin (BSA) prevented CLC adsorption by forming a BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, the NT17 part of Httex1. Furthermore, biocompatible CLC-loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.

HTT Gene Premutation Allele Frequencies in the Russian Federation

Huntington disease (Huntington chorea, HD) is a severe neurodegenerative disease determined by polyglutamine. Polyglutamine expansion in exon 1 of the HTT gene causes Huntington disease. To date, less than 35 CAG triplets are suggested to be present in normal alleles. Alleles bearing from 27 to 35 CAG repeats are generally considered as intermediate or premutation. Alleles with the number of CAG repeats varying from 36 to 39 demonstrate reduced penetrance and result in late manifestation of the disease. To date, no studies based on representative samples of Russian residents estimating the frequencies of premutation and alleles with reduced penetrance have been published. Meanwhile, this is extremely important for genetic counseling of patients bearing such alleles and for the calculation of risks for their offspring. In the present study, the analysis of samples of Russian patients with incoming diagnoses of Huntington chorea (N = 1092), Wilson–Konovalov disease (N = 333), Hallervorden–Spatz disease (N = 33) and a control group consisting of 230 unexamined individuals from the Russian Federation under 45 years of age was carried out. A spectrum of CAG repeat length in the HTT gene in patients with HD was obtained. Patients with HD were detected in the samples of patients with incoming diagnoses of Wilson–Konovalov disease and Hallervorden–Spatz disease. This observation indicates the complexity of differential diagnosis of these diseases. An assumption was made that an allele with 36 CAG repeats should be classified as premutation. We confirmed the previously observed trend for the increased number of CAG repeats in the case of paternal transmission of the mutant allele. The frequency of premutation allele of 2.6% in Russian residents was established.

Glial expression of disease-associated poly-glutamine proteins impairs the blood-brain barrier in Drosophila.

Expansion of poly-glutamine (polyQ) stretches in several proteins has been linked to neurodegenerative diseases. The effects of polyQ-expanded proteins on neurons have been extensively studied, but their effects on glia remain unclear. We found that expression of distinct polyQ proteins exclusively in all glia or specifically in the blood-brain barrier (BBB) and blood-retina barrier (BRB) glia caused cell-autonomous impairment of BBB/BRB integrity, suggesting that BBB/BRB glia are most vulnerable to polyQ-expanded proteins. Furthermore, we also found that BBB/BRB leakage in Drosophila is reflected in reversed waveform polarity on the basis of electroretinography (ERG), making ERG a sensitive method to detect BBB/BRB leakage. The polyQ-expanded protein Atxn3-84Q forms aggregates, induces BBB/BRB leakage, restricts Drosophila lifespan and reduces the level of Repo (a pan-glial transcriptional factor required for glial differentiation). Expression of Repo in BBB/BRB glia can rescue BBB/BRB leakage, suggesting that the reduced expression of Repo is important for the effect of polyQ on BBB/BRB impairment. Coexpression of the chaperon HSP40 and HSP70 effectively rescues the effects of Atxn3-84Q, indicating that polyQ protein aggregation in glia is deleterious. Intriguingly, coexpression of wild-type Atxn3-27Q can also rescue BBB/BRB impairment, suggesting that normal polyQ protein may have a protective function.