Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction.
Ontology highlight
ABSTRACT:
SUBMITTER: Ghosh R
PROVIDER: S-EPMC8432155 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full-length HTT or an N-terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP-positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50-61.
| S-EPMC5129569 | biostudies-literature
Project description:Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)(n) repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.
| S-EPMC3245261 | biostudies-literature
Project description:The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates -100?nm in diameter and up to -1-2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.
| S-EPMC3508451 | biostudies-literature
Project description:PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)-inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103QExon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose- and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-?B activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.
| S-EPMC6177601 | biostudies-literature
Project description:Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several ?m) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.
| S-EPMC4785097 | biostudies-other
Project description:The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.
| S-EPMC34592 | biostudies-literature
Project description:Many neurodegenerative diseases, such as Huntington's disease, are hallmarked by the formation of intracellular inclusion bodies (IBs) that are decorated with ubiquitin, proteasomes and chaperones. The apparent enrichment of ubiquitin and components involved in protein quality control at IBs suggests local ubiquitin-dependent enzymatic activity. In this study, we examine recruitment of ubiquitin to IBs of polyglutamine-expanded huntingtin fragments (mHtt) by using synthesized TAMRA-labeled ubiquitin moieties. We show that intracellular TAMRA-ubiquitin is dynamic at mHtt IBs and is incorporated into poly-ubiquitin chains of intracellular substrates, such as mHtt, in a conjugation-dependent manner. Furthermore, we report that mHtt IBs recruit catalytically active enzymes involved in (de)-ubiquitination processes based on novel activity-based probes. However, we also find that the overexpression of the GFP-ubiquitin reporter, unlike the endogenous ubiquitin and TAMRA-ubiquitin, becomes irreversibly sequestered as a ring-like structure around the mHtt IBs, suggesting a methodical disadvantage of GFP-tagged ubiquitin. Our data provide supportive evidence for dynamic recruitment of ubiquitin and ubiquitin (de)-conjugating activity at mHtt initiated IBs.
| S-EPMC5780509 | biostudies-literature
Project description:BackgroundThe potential benefit of cysteamine for Huntington's disease has been demonstrated in HD animal models. Cysteamine and its derivate cystamine were shown to reduce neuropathology and prolong lifespan. Human studies have demonstrated safety, and suggestive results regarding efficacy. Despite all the studies available in vivo, there are only few in vitro studies, and the mechanism of action of cysteamine remains unclear.ObjectiveThe objective of this study is to assess the capacity of cysteamine for neuroprotection against mutant Huntingtin in vitro using cellular models of HD, and to provide initial data regarding mechanism of action.MethodsWe tested the neuroprotective properties of cysteamine in vitro in our primary neuron and iPSC models of HD.ResultsCysteamine showed a strong neuroprotective effect (EC50 = 7.1 nM) against mutant Htt-(aa-1-586 82Q) toxicity, in a nuclear condensation cell toxicity assay. Cysteamine also rescued mitochondrial changes induced by mutant Htt. Modulation of the levels of cysteine or glutathione failed to protect neurons, suggesting that cysteamine neuroprotection is not mediated through cysteine metabolism. Taurine and Hypotaurine, which are metabolites of cysteamine can protect neurons against Htt toxicity, but the inhibition of the enzyme converting cysteamine to hypotaurine does not block either protective activity, suggesting independent protective pathways. Cysteamine has been suggested to activate BDNF secretion; however, cysteamine protection was not blocked by BDNF pathway antagonists.ConclusionsCysteamine was strongly neuroprotective with relatively high potency. We demonstrated that the main neuroprotective pathways that have been proposed to be the mechanism of protection by cysteamine can all be blocked and still not prevent the neuroprotective effect. The results suggest the involvement of other yet-to-be-determined neuroprotective pathways.
| S-EPMC7039181 | biostudies-literature
Project description:There exists strong correlation between the extended polyglutamines (polyQ) within exon-1 of Huntingtin protein (Htt) and age onset of Huntington's disease (HD); however, the underlying molecular mechanism is still poorly understood. Here we apply extensive molecular dynamics simulations to study the folding of Htt-exon-1 across five different polyQ-lengths. We find an increase in secondary structure motifs at longer Q-lengths, including ?-sheet content that seems to contribute to the formation of increasingly compact structures. More strikingly, these longer Q-lengths adopt supercompact structures as evidenced by a surprisingly small power-law scaling exponent (0.22) between the radius-of-gyration and Q-length that is substantially below expected values for compact globule structures (?0.33) and unstructured proteins (?0.50). Hydrogen bond analyses further revealed that the supercompact behavior of polyQ is mainly due to the "glue-like" behavior of glutamine's side chains with significantly more side chain-side chain H-bonds than regular proteins in the Protein Data Bank (PDB). The orientation of the glutamine side chains also tend to be "buried" inside, explaining why polyQ domains are insoluble on their own.
| S-EPMC5835228 | biostudies-literature
Project description:Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.
| S-EPMC8170701 | biostudies-literature