Intranuclear Protein Aggregates Research Articles

VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models.

Pathogenic variants in valosin-containing protein (VCP) cause multisystem proteinopathy (MSP), a disease characterized by multiple clinical phenotypes including inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia (FTD). How such diverse phenotypes are driven by pathogenic VCP variants is not known. We found that these diseases exhibit a common pathologic feature: ubiquitinated intranuclear inclusions affecting myocytes, osteoclasts, and neurons. Moreover, knock-in cell lines harboring MSP variants show a reduction in nuclear VCP. Given that MSP is associated with neuronal intranuclear inclusions comprised of TDP-43 protein, we developed a cellular model whereby proteostatic stress results in the formation of insoluble intranuclear TDP-43 aggregates. Consistent with a loss of nuclear VCP function, cells harboring MSP variants or cells treated with VCP inhibitor exhibited decreased clearance of insoluble intranuclear TDP-43 aggregates. Moreover, we identified 4 compounds that activate VCP primarily by increasing D2 ATPase activity, where pharmacologic VCP activation appears to enhance clearance of insoluble intranuclear TDP-43 aggregate. Our findings suggest that VCP function is important for nuclear protein homeostasis, that impaired nuclear proteostasis may contribute to MSP, and that VCP activation may be a potential therapeutic by virtue of enhancing the clearance of intranuclear protein aggregates.

P.352 - Intranuclear protein aggregation in myofibrillar myopathies

P.352 - Intranuclear protein aggregation in myofibrillar myopathies

Polyglutamine aggregation in Huntington's disease and spinocerebellar ataxia type 3: similar mechanisms in aggregate formation.

Polyglutamine (polyQ) diseases are characterized by the expansion of a polymorphic glutamine sequence in disease-specific proteins and exhibit aggregation of these proteins. This is combated by the cellular protein quality control (PQC) system, consisting of chaperone-mediated refolding as well as proteasomal and lysosomal degradation pathways. Our recent study in the polyQ disease spinocerebellar ataxia type 3 (SCA3) suggested a distinct pattern of protein aggregation and PQC dysregulation. To corroborate these findings we have investigated immunohistochemically stained 5 μm sections from different brain areas of Huntington's disease (HD) and SCA3 patients. Irrespective of disease and brain region, we observed peri- and intranuclear polyQ protein aggregates. A subset of neurones with intranuclear inclusions bodies exhibited signs of proteasomal dysfunction, up-regulation of HSPA1A and re-distribution of DNAJB1. The extent of the observed effects varied depending on brain area and disease protein. Our results suggest a common sequence, in which formation of cytoplasmic and nuclear inclusions precede proteasomal impairment and induction of the cellular stress response. Clearly, impairment of the PQC is not the primary cause for inclusion formation, but rather a consequence that might contribute to neuronal dysfunction and death. Notably, the inclusion pathology is not directly correlated to the severity of the degeneration in different areas, implying that different populations of neurones respond to polyQ aggregation with varying efficacy and that protein aggregation outside the neuronal perikaryon (e.g. axonal aggregates) or other effects of polyQ aggregation, which are more difficult to visualize, may contribute to neurodegeneration.

Regulation of BACE1 by miR-29a/b in a cellular model of Spinocerebellar ataxia 17

Polyglutamine diseases are a class of neurodegenerative disorders characterized by expansion of polyglutamine repeats, protein aggregation and neuronal cell death in specific regions of the brain. The expansion of a polyglutamine repeat in the TATA binding protein (TBP) causes a neurodegenerative disease, Spinocerebellar Ataxia 17 (SCA17). This disease is characterized by intranuclear protein aggregates and selective loss of cerebellar neurons, including Purkinje cells. MicroRNAs are small, endogenous, regulatory non-coding RNA molecules that bind to messenger RNAs with partial complementarity and interfere in their expression. Here, we used a cellular model of SCA17 where we expressed TBP with 16 (normal) or 59 (pathogenic) polyglutamines and found differential expression of several microRNAs. Specifically, we found two microRNAs, miR-29a/b, were down-regulated. With miR-29a/b down regulation, we found an increased expression of targets of miR-29a/b -beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), p53 upregulated modulator of apoptosis (PUMA) and BAK, increased cytochrome c release and apoptosis. Restoration of miR-29a/b in the pathogenic polyglutamine background reduced the BACE1expression. While, antagomiRs against miR-29a/b resulted in an increase in BACE1 levels and neuronal apoptosis. In spite of the elevation of BACE1 in Alzhemiers disease, its role in neuronal cell death has not been established. Here, we show that increased BACE1 expression is not sufficient to cause apoptosis. However restoring level of BACE1 to normal in polyglutamine cells partially reduced neuronal apoptosis. We show a role for the miR-29a/b-BACE1 regulatory interaction in SCA17, suggesting that this microRNA could be part of a common molecular mechanism leading to neuronal cell death in multiple neurodegenerative disorders. The identification of a common mechanism of microRNA mediated neurodegeneration not only improves our understanding of the process, but also provides promising and novel therapeutic targets.

Abnormalities of the nucleus and nuclear inclusions in neurodegenerative disease: a work in progress

Neurodegenerative diseases are characterized pathologically by the abnormal accumulation of pathogenic protein species within the cell. Several neurodegenerative diseases feature intranuclear protein aggregation in the form of intranuclear inclusion bodies. Studies of these intranuclear inclusions are providing important clues regarding the cellular pathophysiology of these diseases, as exemplified by recent progress in defining the genetic basis of a subset of frontotemporal dementia cases. The precise role of intranuclear inclusion bodies in disease pathogenesis is currently a focus of debate. The present review provides an overview of the diverse family of neurodegenerative diseases in which nuclear inclusions form part of the neuropathological spectrum. In addition, current pathogenetic concepts relevant to these diseases will be reviewed and arguments for and against a protective role for intranuclear inclusions will be presented. The relationship of pathological intranuclear inclusions to functional intranuclear bodies will also be discussed. Finally, by analogy with pathological intranuclear inclusions, I will speculate on the possibility that intranuclear protein aggregation may represent a constitutive cellular protective mechanism occurring in neurons under physiological conditions.

Aging and neurodegeneration: Molecular mechanisms of neuronal loss in Huntington's disease

Huntington's disease (HD) is a fatal, genetically based late-onset neurodegenerative disorder in which a loss of neostriatal neurons is a main characteristic. The CAG trinucleotide repeat expansion encoding polyglutamine tract induces progressive deficits in intra- and inter-cellular signalling, and subsequent clinical signs developed with aging process. CAG-induced neurodegeneration and disease-onset shows aging-dependent pattern. Proposed mechanism of neurodegeneration includes intranuclear or intracellular protein aggregates, proteolytic cleavage of huntingtin (cf. caspase, calpain), altered transcription or other neurotransmitter signalling deficits. Recently, stem cell transplantation is of benefit to protect neurons against neurodegeneration and recover the functional deficit in the experimental HD model. This review focuses on current knowledge of molecular mechanisms in neurodegeneration and potential therapeutic targets in HD.

Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO 2 nanoparticles

Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO 2) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO 2 nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology.

An expanded glutamine repeat destabilizes native ataxin-3 structure and mediates formation of parallel beta -fibrils.

The protein ataxin-3 contains a polyglutamine region; increasing the number of glutamines beyond 55 in this region gives rise to the neurodegenerative disease spinocerebellar ataxia type 3. This disease and other polyglutamine expansion diseases are characterized by large intranuclear protein aggregates (nuclear inclusions). By using full-length human ataxin-3, we have investigated the changes in secondary structure, aggregation behavior, and fibril formation associated with an increase from the normal length of 27 glutamines (Q27 ataxin-3) to a pathogenic length of 78 glutamines (Q78 ataxin-3). Q78 ataxin-3 aggregates strongly and could be purified only when expressed with a solubility-enhancing fusion-protein partner. A marked decrease in alpha-helical secondary structure accompanies expansion of the polyglutamine tract, suggesting destabilization of the native protein. Proteolytic removal of the fusion partner in the Q78 protein, but not in the Q27 protein, leads to the formation of SDS-resistant aggregates and Congo-red reactive fibrils. Infrared spectroscopy of fibrils reveals a high beta-sheet content and suggests a parallel, rather than an antiparallel, sheet conformation. We present a model for a polar zipper composed of parallel polyglutamine beta-sheets. Our data show that intact ataxin-3 is fully competent to form aggregates, and posttranslational cleavage or other processing is not necessary to generate a misfolding event. The data also suggest that the protein aggregation phenotype associated with glutamine expansion may derive from two effects: destabilization of the native protein structure and an inherent propensity for beta-fibril formation on the part of glutamine homopolymers.

The selective vulnerability of nerve cells in Huntington's disease.

It is now more than 7 years since the genetic mutation causing Huntington's disease (HD) was first identified. Unstable CAG expansion in the IT15 gene, responsible for disease, is translated into an abnormally long polyglutamine (polyQ) tract near the N-terminus of the huntingtin protein. The presence of expanded polyQ in the mutant protein leads to its abnormal proteolytic cleavage with liberation of toxic N-terminal fragments that tend to aggregate, probably first in the cytoplasm. Subsequent nuclear translocation of the cleaved mutant huntingtin is associated with formation of intranuclear protein aggregates and neurotoxicity, probably involving apoptotic cascades. These processes, which can be experimentally modelled in cultured neuronal and non-neuronal cells, seem to underlie neurodegeneration in HD, and also other polyQ disorders, such as dentatorubro-pallidoluysian degeneration, spinal and bulbar muscular atrophy and the spinocerebellar ataxias. They do not, however, explain why within the corpus striatum and cerebral cortex certain nerve cells are susceptible to disease and others are not. In the human HD brain, vulnerable pyramidal neurones within the deeper layers of the cerebral cortex frequently contain large intranuclear inclusions composed of N-terminal fragments of huntingtin. Such inclusions are, however, rare within neurones of the striatum, even in the medium spiny neurones preferentially lost from this region. While inclusions per se do not seem to be neurotoxic, they may provide a surrogate marker of molecular pathology. Recent studies indicate that the nuclear accumulation of mutant huntingtin interferes with transcriptional events. Of particular importance may be the effect on the genes encoding neurotransmitter receptor proteins, especially those involved with glutamatergic neurotransmission. Such changes may trigger or facilitate a low-grade, chronic excitotoxicity of the glutamatergic cortical projection neurones on their target cells in the striatum, already partly compromised by the toxic effects of the HD mutation. This combination of insults, for anatomical reasons experienced predominantly by striatal projection neurones, would eventually cause their selective demise.

An isoform of ataxin-3 accumulates in the nucleus of neuronal cells in affected brain regions of SCA3 patients.

Autosomal dominant spinocerebellar ataxias (SCA) form a group of clinically and genetically heterogeneous neurodegenerative disorders. The defect responsible for SCA3/Machado-Joseph disease (MJD) has been identified as an unstable and expanded (CAG)n trinucleotide repeat in the coding region of a novel gene of unknown function. The MJD1 gene product, ataxin-3, exists in several isoforms. We generated polyclonal antisera against an alternate carboxy terminus of ataxin-3. This isoform, ataxin-3c, is expressed as a protein of approximately 42 kDa in normal individuals but is significantly enlarged in affected patients confirming that the CAG repeat is part of the ataxin-3c isoform and is translated into a polyglutamine stretch, a feature common to all known CAG repeat disorders. Ataxin-3 like immunoreactivity was observed in all human brain regions and peripheral organs studied. In neuronal cells of control individuals, ataxin-3c was expressed cytoplasmatically and had a somatodendritic and axonal distribution. In SCA3 patients, however, C-terminal ataxin-3c antibodies as well as anti-ataxin-3 monoclonal antibodies (1 H9) and anti-ubiquitin antibodies detected intranuclear inclusions (NIs) in neuronal cells of affected brain regions. A monoclonal antibody, 2B6, directed against an internal part of the protein, barely detected these NIs implying proteolytic cleavage of ataxin-3 prior to its transport into the nucleus. These findings provide evidence that the alternate isoform of ataxin-3 is involved in the pathogenesis of SCA3/MJD. Intranuclear protein aggregates appear as a common feature of neurodegenerative polyglutamine disorders.

Heat-induced intranuclear protein aggregation and thermal radiosensitization.

In the current study, the hypothesis that thermal radiosensitization is (indirectly) caused by heat-induced denaturation and aggregation of nuclear proteins is further investigated. Thermotolerant rodent cells showed a reduced intranuclear protein aggregation as compared with non-tolerant cells immediately after a heat treatment. This was reflected in the extent of radiosensitization when the cells were X-irradiated immediately after a heat treatment. When heat and radiation were separated in time, a faster disaggregation was found in thermotolerant cells, which was paralleled by a more rapid decline of radiosensitization. Cells transfected with hsp72 showed protection against heat-induced nuclear protein aggregation and reduced thermal radiosensitization. Transfection with hsp27 resulted in an accelerated nuclear protein disaggregation and accelerated decline of thermal radiosensitization. Despite a significant overall correlation between TER and nuclear protein aggregation, the slopes of the correlation curves for the individual cell lines deviated significantly. Yet, the experiments support the hypothesis that radiosensitization is primarily caused by inhibition of DNA repair as a result of the presence of denatured and aggregated proteins in the cell nucleus. Expression of hsps (e.g. in thermotolerant cells), by affecting nuclear protein aggregation, can have an impact on thermal radiosensitization.

Importance of the ATP-Binding Domain and Nucleolar Localization Domain of HSP72 in the Protection of Nuclear Proteins against Heat-Induced Aggregation

Heat treatment of cells results in an increased protein content of nuclei when isolated after the heat treatment (intranuclear protein aggregation). In a previous study, the role of HSP72 was investigated using Rat-1 fibroblasts stably transfected with the human HSP72 gene. It was observed that the expression of human HSP72 in Rat-1 cells (HR cells) confers heat resistance. The initial heat-induced increase in the nuclear protein content was lower in HR cells as compared to the parent Rat-1 cells. In the present communication, the effects of overexpression of intact or mutant human HSP72 in Rat-1 cells on heat-induced increase in intranuclear protein aggregation and their relationship to cells' thermal sensitivity were examined. Four closely related cell lines were used for this study: Rat-1 cells which constitutively expressed the intact human HSP72, or mutant human HSP72 either missing its ATP-binding domain or nucleolar localization domain, and wild type Rat-1 cells. Our results show that expression of the intact human HSP72 or mutant human HSP72 missing its ATP-binding domain confers heat resistance and protects cells against heat-induced intranuclear protein aggregation. On the other hand, cells expressing mutant human HSP72 missing its nucleolar localization domain demonstrated heat shock responses similar to control Rat-1 cells.

On the role of hsp72 in heat-induced intranuclear protein aggregation.

Heat treatment of cells results in an increased protein content of nuclei and nuclear matrices when isolated after the heat treatment. This increase of TX-100 insoluble protein is interpreted as being the result of protein denaturation and subsequent aggregation. After the heat treatment cells can (partly) recover from these aggregates. Recent data suggest that heat shock proteins (hsps) might be involved in the recovery (disaggregation) from these heat-induced insoluble protein complexes. In this report, the role of hsp72 in the process of aggregation and disaggregation was investigated using: non-tolerant rat-1 cells, thermotolerant rat-1 cells (rat-1 TT), and transfected rat-1 cells constitutively expressing the human inducible hsp72 gene (HR-24 cells). After heating the various cells, it was observed that the expression of the human hsp72 confers heat resistance (43-45 degrees C). Heat-induced intranuclear protein aggregation was less in HR and rat-1 TT cells as compared to nontolerant rat-1 cells. After heat treatments leading to the same initial intranuclear protein aggregation, rat-1 TT cells recovered more rapidly from these aggregates, while HR cells recovered at the same rate as nontolerant rat-1 cells. Our data suggest that increased levels of hsp72 can confer heat resistance at the level of initial (nuclear) heat damage. Elevated levels of hsp72 alone, however, do not enable cells to recover more rapidly from heat-induced intranuclear protein aggregates.

Association of HSP72 with the nuclear (TX-100-insoluble) fraction upon heating tolerant and non-tolerant HeLa S3 cells.

HSP72 levels in the cellular and the nuclear (TX-insoluble) fraction before and after heating of heat- and sodium arsenite-induced thermotolerant and non-tolerant HeLa S3 cells have been investigated by 1D- and 2D-electrophoresis, followed by Western blotting and immunostaining, using a newly developed monoclonal antibody that specifically detects HSP72 (Heine et al. 1991). HSP72 was constitutively expressed in HeLa S3 cells and elevated upon heat or arsenite stress. Immediate association of HSP72 with the nuclear fraction was induced by heat but not arsenite. However, at the time of maximal thermotolerance, elevated levels of HSP72 were found associated with nuclei isolated from both heat- and arsenite-induced thermotolerant cells. After (test) heat treatments (0-60 min at 45 degrees C) translocation of HSP72 to the nuclear fraction in all cells was observed, albeit with different kinetics and to different plateau values. When tolerant and non-tolerant cells were allowed to recover from a heat stress (at 37 degrees C) before isolation of the nuclei, no dissociation of HSP72 from the nuclear fraction was observed within a 5 h time period. Our data indicate that association/dissociation of HSP72 with/from the nuclear fraction is not related to the recovery from heat-induced intranuclear protein aggregation (Kampinga et al. 1992), nor to the extent of thermotolerance in the human HeLa S3 cell line.

Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: multiple pathways to induce tolerance?

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44 degrees C) or pretreatment with sodium-arsenite (1 hour at 37 degrees C, 100 microM). Inhibition of overall protein and heat shock protein (HSP) synthesis (greater than 95%) by cycloheximide (25 micrograms/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat-induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium-arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post-heat incubations of the cells at 37 degrees C. The rate of recovery was faster in heat-induced tolerant cells than in nontolerant cells. Arsenite-induced tolerant cells did not show an enhanced rate of recovery from the heat-induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post-heat recovery was enhanced in heat-induced, but not arsenite-induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.