RESUMO
Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.
Assuntos
Proteína de Replicação A , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Camundongos , DNA/genética , Reparo de Erro de Pareamento de DNA , Doença de Huntington/genética , Proteínas/genética , Ataxias Espinocerebelares/genética , Proteína de Replicação A/metabolismoRESUMO
Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.
Assuntos
Chaperonina com TCP-1 , Macroautofagia , Agregados Proteicos , Animais , Camundongos , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Chaperonina com TCP-1/metabolismo , Proteína Sequestossoma-1/metabolismoRESUMO
Variable, glutamine-encoding, CAA interruptions indicate that a property of the uninterrupted HTT CAG repeat sequence, distinct from the length of huntingtin's polyglutamine segment, dictates the rate at which Huntington's disease (HD) develops. The timing of onset shows no significant association with HTT cis-eQTLs but is influenced, sometimes in a sex-specific manner, by polymorphic variation at multiple DNA maintenance genes, suggesting that the special onset-determining property of the uninterrupted CAG repeat is a propensity for length instability that leads to its somatic expansion. Additional naturally occurring genetic modifier loci, defined by GWAS, may influence HD pathogenesis through other mechanisms. These findings have profound implications for the pathogenesis of HD and other repeat diseases and question the fundamental premise that polyglutamine length determines the rate of pathogenesis in the "polyglutamine disorders."
Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , Peptídeos/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Alelos , Sequência de Bases/genética , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Adulto JovemRESUMO
Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Proteína Fosfatase 1/antagonistas & inibidores , Animais , Peso Corporal , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Guanidinas/química , Células HeLa , Humanos , Doença de Huntington/metabolismo , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Proteostase , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.
Assuntos
Anticorpos Biespecíficos/análise , Transdução de Sinais , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular , Humanos , Mitose , Biossíntese de Proteínas , UbiquitinaçãoRESUMO
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Terapia Genética/métodos , Oligonucleotídeos Antissenso/farmacologia , Animais , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições de Microssatélites , Splicing de RNA , Expansão das Repetições de TrinucleotídeosRESUMO
Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.
Assuntos
Doenças Neurodegenerativas , Sítios de Splice de RNA , Humanos , Sítios de Splice de RNA/genética , Doenças Neurodegenerativas/genética , Códon de Iniciação , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Manifestation of aggregate pathology in Huntington's disease is thought to be facilitated by a preferential vulnerability of affected brain cells to age-dependent proteostatic decline. To understand how specific cellular backgrounds may facilitate pathologic aggregation, we utilized the yeast model in which polyQ-expanded Huntingtin forms aggregates only when the endogenous prion-forming protein Rnq1 is in its amyloid-like prion [PIN+] conformation. We employed optogenetic clustering of polyQ protein as an orthogonal method to induce polyQ aggregation in prion-free [pin-] cells. Optogenetic aggregation circumvented the prion requirement for the formation of detergent-resistant polyQ inclusions but bypassed the formation of toxic polyQ oligomers, which accumulated specifically in [PIN+] cells. Reconstitution of aggregation in vitro suggested that these polyQ oligomers formed through direct templating on Rnq1 prions. These findings shed light on the mechanism of prion-mediated formation of oligomers, which may play a role in triggering polyQ pathology in the patient brain.
Assuntos
Príons , Proteínas de Saccharomyces cerevisiae , Humanos , Príons/genética , Príons/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismoRESUMO
Protein aggregates disrupt cellular homeostasis, causing toxicity linked to neurodegeneration. Selective autophagic elimination of aggregates is critical to protein quality control, but how aggregates are selectively targeted for degradation is unclear. We compared the requirements for autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in clearance of proteotoxic aggregates. Endogenous TAX1BP1 is recruited to and required for the clearance of stress-induced aggregates, whereas ectopic expression of TAX1BP1 increases clearance through autophagy, promoting viability of human induced pluripotent stem cell-derived neurons. In contrast, TAX1BP1 depletion sensitizes cells to several forms of aggregate-induced proteotoxicity. Furthermore, TAX1BP1 is more specifically expressed in the brain compared to other autophagy receptor proteins. In vivo, loss of TAX1BP1 results in accumulation of high molecular weight ubiquitin conjugates and premature lipofuscin accumulation in brains of young TAX1BP1 knockout mice. TAX1BP1 mediates clearance of a broad range of cytotoxic proteins indicating therapeutic potential in neurodegenerative diseases.
Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Autofagia , Encéfalo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas de Neoplasias/deficiência , Doenças Neurodegenerativas/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/patologia , Feminino , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipofuscina/genética , Lipofuscina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Ratos , Ratos Sprague-Dawley , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.
Assuntos
Reparo de Erro de Pareamento de DNA , Proteína Huntingtina , Doença de Huntington , Células-Tronco Pluripotentes Induzidas , Expansão das Repetições de Trinucleotídeos , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Genes Modificadores , Proteína 3 Homóloga a MutS/genética , Proteína 3 Homóloga a MutS/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Sistemas CRISPR-Cas , Estudo de Associação Genômica AmplaRESUMO
Antisense oligonucleotides represent a novel therapeutic platform for the discovery of medicines that have the potential to treat most neurodegenerative diseases. Antisense drugs are currently in development for the treatment of amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease, and multiple research programs are underway for additional neurodegenerative diseases. One antisense drug, nusinersen, has been approved for the treatment of spinal muscular atrophy. Importantly, nusinersen improves disease symptoms when administered to symptomatic patients rather than just slowing the progression of the disease. In addition to the benefit to spinal muscular atrophy patients, there are discoveries from nusinersen that can be applied to other neurological diseases, including method of delivery, doses, tolerability of intrathecally delivered antisense drugs, and the biodistribution of intrathecal dosed antisense drugs. Based in part on the early success of nusinersen, antisense drugs hold great promise as a therapeutic platform for the treatment of neurological diseases.
Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos/farmacologia , Distribuição Tecidual/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Doenças Neurodegenerativas/genéticaRESUMO
Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.
Assuntos
Núcleo Celular/efeitos dos fármacos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , RNA Polimerase II/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fatores de Elongação da Transcrição/antagonistas & inibidores , Regiões 3' não Traduzidas , Animais , Núcleo Celular/enzimologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Descoberta de Drogas/métodos , Metabolismo Energético/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Huntingtina/biossíntese , Proteína Huntingtina/genética , Células Jurkat , Células MCF-7 , Camundongos Transgênicos , Mutação , NF-kappa B/biossíntese , NF-kappa B/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.
Assuntos
Doença de Huntington , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteínas Mutantes/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Nucleotidiltransferases/genética , DNA , Apoptose/genética , Proteína 1 Homóloga a MutL/genéticaRESUMO
Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Dobramento de Proteína , Dicroísmo Circular , Estrutura Secundária de Proteína , Humanos , Transferência Ressonante de Energia de Fluorescência , Temperatura , Conformação ProteicaRESUMO
The striato-nigral (Str-SN) circuit is composed of medium spiny neuronal projections that are mainly sent from the striatum to the midbrain substantial nigra (SN), which is essential for regulating motor behaviors. Dysfunction of the Str-SN circuitry may cause a series of motor disabilities that are associated with neurodegenerative disorders, such as Huntington's disease (HD). Although the etiology of HD is known as abnormally expanded CAG repeats of the huntingtin gene, treatment of HD remains tremendously challenging. One possible reason is the lack of effective HD model that resembles Str-SN circuitry deficits for pharmacological studies. Here, we first differentiated striatum-like organoids from human pluripotent stem cells (hPSCs), containing functional medium spiny neurons (MSNs). We then generated 3D Str-SN assembloids by assembling striatum-like organoids with midbrain SN-like organoids. With AAV-hSYN-GFP-mediated viral tracing, extensive MSN projections from the striatum to the SN are established, which formed synaptic connection with GABAergic neurons in SN organoids and showed the optically evoked inhibitory postsynaptic currents and electronic field potentials by labeling the striatum-like organoids with optogenetic virus. Furthermore, these Str-SN assembloids exhibited enhanced calcium activity compared to that of individual striatal organoids. Importantly, we further demonstrated the reciprocal projection defects in HD iPSC-derived assembloids, which could be ameliorated by treatment of brain-derived neurotrophic factor. Taken together, these findings suggest that Str-SN assembloids could be used for identifying MSN projection defects and could be applied as potential drug test platforms for HD.
Assuntos
Doença de Huntington , Organoides , Humanos , Doença de Huntington/patologia , Doença de Huntington/metabolismo , Organoides/patologia , Organoides/metabolismo , Substância Negra/patologia , Substância Negra/metabolismo , Corpo Estriado/patologia , Corpo Estriado/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Diferenciação Celular , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Células-Tronco Pluripotentes/metabolismo , OptogenéticaRESUMO
Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.
Assuntos
Doença de Huntington , MicroRNAs , Humanos , Regiões 3' não Traduzidas/genética , Endodesoxirribonucleases , Exodesoxirribonucleases/genética , Estudo de Associação Genômica Ampla , Doença de Huntington/genética , MicroRNAs/genética , Enzimas MultifuncionaisRESUMO
The neurodegenerative disorder, Huntington disease (HD), manifests as disorders of movement, cognition and mood. Although studies report abnormal corticostriatal synaptic function early in HD mouse models, less is known about cortical-cortical activity across brain regions and disease stages. Recently, we reported enhanced mesoscale spread of cortical responses to sensory stimulation in vivo at early-manifest stages of two HD mouse models. Here, we investigated cortical excitability of zQ175 HD-model mice compared to their wild-type littermates across different cell types, ages and/or cortical regions using ex vivo electrophysiology. Cortical pyramidal neurons (CPNs) in somatosensory cortex of zQ175 mice showed intrinsic hyper-excitability at 3-4 months, but hypo-excitability at early-manifest stage (8-9 months); reduced frequency of spontaneous excitatory postsynaptic currents (sEPSCs) was seen at both ages. In contrast, motor cortex CPNs in early-manifest zQ175 mice showed increased intrinsic excitability and sEPSC frequency. Large-amplitude excitatory discharges recorded from CPNs in early-manifest zQ175 mice showed increased frequency only in somatosensory cortex, suggesting the intrinsic hypo-excitability of these CPNs may be compensatory against cortical network hyper-excitability. Similarly, in early-manifest zQ175 mice, region-dependent differences were seen in fast-spiking interneurons (FSIs): somatosensory but not motor FSIs from early-manifest zQ175 mice had reduced intrinsic excitability. Moreover, CPNs showed decreased frequency of spontaneous inhibitory postsynaptic currents and increased excitatory-inhibitory (E-I) balance of evoked synaptic currents in somatosensory cortex. Aberrant large-amplitude discharges and reduced inhibitory drive may therefore underlie E-I imbalances that result in circuit changes and synaptic dysfunction in early-manifest HD.
Assuntos
Excitabilidade Cortical , Doença de Huntington , Camundongos , Animais , Doença de Huntington/metabolismo , Células Piramidais/metabolismo , Interneurônios/metabolismo , Fenômenos EletrofisiológicosRESUMO
Huntington's disease (HD) is a neurodegenerative genetic disorder caused by an expansion in the CAG repeat tract of the huntingtin (HTT) gene resulting in behavioural, cognitive, and motor defects. Current knowledge of disease pathogenesis remains incomplete, and no disease course-modifying interventions are in clinical use. We have previously reported the development and characterisation of the OVT73 transgenic sheep model of HD. The 73 polyglutamine repeat is somatically stable and therefore likely captures a prodromal phase of the disease with an absence of motor symptomatology even at 5-years of age and no detectable striatal cell loss. To better understand the disease-initiating events we have undertaken a single nuclei transcriptome study of the striatum of an extensively studied cohort of 5-year-old OVT73 HD sheep and age matched wild-type controls. We have identified transcriptional upregulation of genes encoding N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in medium spiny neurons, the cell type preferentially lost early in HD. Further, we observed an upregulation of astrocytic glutamate uptake transporters and medium spiny neuron GABAA receptors, which may maintain glutamate homeostasis. Taken together, these observations support the glutamate excitotoxicity hypothesis as an early neurodegeneration cascade-initiating process but the threshold of toxicity may be regulated by several protective mechanisms. Addressing this biochemical defect early may prevent neuronal loss and avoid the more complex secondary consequences precipitated by cell death.
Assuntos
Modelos Animais de Doenças , Ácido Glutâmico , Doença de Huntington , Neurônios , Receptores de N-Metil-D-Aspartato , Animais , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Ovinos , Neurônios/metabolismo , Neurônios/patologia , Ácido Glutâmico/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , RNA-Seq , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Morte Celular/genética , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Animais Geneticamente Modificados , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Humanos , Transcriptoma/genética , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Neurônios Espinhosos MédiosRESUMO
The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1's C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation.
Assuntos
Chaperonas Moleculares/metabolismo , Peptídeos/metabolismo , Linhagem Celular , Dictyostelium/metabolismo , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Serina/metabolismo , Ubiquitina/metabolismoRESUMO
Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo.