Formation Of Neuronal Intranuclear Inclusions Research Articles

GGC repeat expansion in NOTCH2NLC induces dysfunction in ribosome biogenesis and translation.

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.

A Coarse-Grained Model for Polyglutamine Aggregation Modulated by Amphipathic Flanking Sequences

The aggregation of proteins with expanded polyglutamine (polyQ) tracts is directly relevant to the formation of neuronal intranuclear inclusions in Huntington’s disease. In vitro studies have uncovered the effects of flanking sequences as modulators of the driving forces and mechanisms of polyQ aggregation in sequence segments associated with HD. Specifically, a seventeen-residue amphipathic stretch (N17) that is directly N-terminal to the polyQ tract in huntingtin decreases the overall solubility, destabilizes nonfibrillar aggregates, and accelerates fibril formation. Published results from atomistic simulations showed that the N17 module reduces the frequency of intermolecular association. Our reanalysis of these simulation results demonstrates that the N17 module also reduces interchain entanglements between polyQ domains. These two effects, which are observed on the smallest lengthscales, are incorporated into phenomenological pair potentials and used in coarse-grained Brownian dynamics simulations to investigate their impact on large-scale aggregation. We analyze the results from Brownian dynamics simulations using the framework of diffusion-limited cluster aggregation. When entanglements prevail, which is true in the absence of N17, small spherical clusters and large linear aggregates form on distinct timescales, in accord with in vitro experiments. Conversely, when entanglements are quenched and a barrier to intermolecular associations is introduced, both of which are attributable to N17, the timescales for forming small species and large linear aggregates become similar. Therefore, the combination of a reduction of interchain entanglements through homopolymeric polyQ and barriers to intermolecular associations appears to be sufficient for providing a minimalist phenomenological rationalization of in vitro observations regarding the effects of N17 on polyQ aggregation.

Lithium Chloride Alleviates Neurodegeneration Partly by Inhibiting Activity of GSK3β in a SCA3 Drosophila Model

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucelotide repeat that encodes an abnormal polyglutamine (PolyQ) tract in the disease protein, ataxin-3. The formation of neuronal intranuclear inclusions in the specific brain regions is one of the pathological hallmarks of SCA3. Acceleration of the degradation of the mutant protein aggregates is proven to produce beneficial effects in SCA3 and other PolyQ diseases. Lithium is known to be neuroprotective in various models of neurodegenerative disease and can reduce the mutant protein aggregates by inducing autophagy. In this study, we explored the therapeutic potential of lithium in a SCA3 Drosophila model. We showed that chronic treatment with lithium chloride at specific doses notably prevented eye depigmentation, alleviated locomotor disability, and extended the median life spans of SCA3 transgenic Drosophila. By means of genetic approaches, we showed that co-expressing the mutant S9E, which mimicked the phosphorylated S9 state of Shaggy as done by lithium, also partly decreased toxicity of gmr-SCA3tr-Q78. Taken together, our findings suggest that lithium is a promising therapeutic agent for the treatment of SCA3 and other PolyQ diseases.

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1*

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.

Amyloid precursor-like protein 2 cleavage contributes to neuronal intranuclear inclusions and cytotoxicity in spinocerebellar ataxia-7 (SCA7)

In spinocerebellar ataxia-7 (SCA7), a polyglutamine (polyQ) expansion in the ataxin-7 protein leads to the formation of neuronal intranuclear inclusions (NIIs) and neurodegeneration. In this study, amyloid precursor-like protein 2 (APLP2) was identified as a partner protein for ataxin-7. APLP2, belonging to the APP gene family, undergoes secretase and caspase cleavages and has been implicated in the pathogenesis of Alzheimer's disease (AD). Activated caspase-3 cleaves APP family proteins to release N-terminal fragments (NTFs) and intracellular C-terminal domains (ICDs), which can translocate into the nucleus and induce neurotoxicity in AD. Here, we report abnormal nuclear relocation of APLP2 and detection of NTFs in NIIs in SCA7. The ICDs generated by caspase-3 cleavage of APLP2 accumulate in nuclei and contribute to a cumulative toxicity when coexpressed with mutated ataxin-7. Our data suggest that the interaction between APLP2 and ataxin-7 and proteolytic processing of APLP2 may contribute to the pathogenesis of SCA7.

Paradoxical delay in the onset of disease caused by super-long CAG repeat expansions in R6/2 mice

Huntington's disease (HD) is caused by an expanded CAG repeat in the HD gene. The pathological threshold for expansion in HD is around 36 CAG repeats, although ‘super-long’ expansions are found in brains of HD patients. We examined the effect of varying the CAG repeat length (from 170 to 450) on behavior and neuropathology of R6/2 mice. Unexpectedly, we found that increasing the repeat length delayed onset of disease and prolonged survival, from around 4 months to over 18 months in mice with the longest repeats. The delay in onset correlated with a delayed appearance of neuronal intranuclear inclusions (NIIs). However, super-long CAG repeats are not neuroprotective. Mice carrying 2 copies of the mutant transgene die earlier than those carrying a single copy. Furthermore, neurodegeneration is present in super-long repeat length mice at mid-stage disease, whereas little neurodegeneration is seen in mice with shorter CAG repeats until end stage. Expanding the CAG repeat beyond the range where NII formation is the dominant pathology has unmasked a slowly progressing neurological phenotype in R6/2 mice with brain pathology, including the identification of a novel form of inclusion, that more closely resembles that seen in adult onset cases of HD. This mouse may represent a better model for adult-onset HD than R6/2 mice with shorter repeats.

Brain‐derived neurotrophic factor over‐expression in the forebrain ameliorates Huntington’s disease phenotypes in mice

Huntington's disease (HD), a dominantly inherited neurodegenerative disorder characterized by relatively selective degeneration of striatal neurons, is caused by an expanded polyglutamine tract of the huntingtin (htt) protein. The htt mutation reduces levels of brain-derived neurotrophic factor (BDNF) in the striatum, likely by inhibiting cortical BDNF gene expression and anterograde transport of BDNF from cortex to striatum. However, roles of the BDNF reduction in HD pathogenesis have not been established conclusively. We reasoned that increasing striatal BDNF through over-expression would slow progression of the disease if BDNF reduction plays a pivotal role in HD pathogenesis. We employed a Bdnf transgene driven by the promoter for the alpha subunit of Ca(2+)/calmodulin-dependent kinase II to over-express BDNF in the forebrain of R6/1 mice which express a fragment of mutant htt with a 116-glutamine tract. The Bdnf transgene increased BDNF levels and TrkB signaling activity in the striatum, ameliorated motor dysfunction, and reversed brain weight loss in R6/1 mice. Furthermore, it normalized DARPP-32 expression of the 32 kDa dopamine and cAMP-regulated phosphoprotein, increased the number of enkephalin-containing boutons, and reduced formation of neuronal intranuclear inclusions in the striatum of R6/1 mice. These results demonstrate crucial roles of reduced striatal BDNF in HD pathogenesis and suggest potential therapeutic values of BDNF to HD.

Comparative analysis of genetic modifiers in Drosophila points to common and distinct mechanisms of pathogenesis among polyglutamine diseases

Spinocerebellar Ataxia type 1 (SCA1) and Huntington's disease (HD) are two polyglutamine disorders caused by expansion of a CAG repeat within the coding regions of the Ataxin-1 and Huntingtin proteins, respectively. While protein folding and turnover have been implicated in polyglutamine disorders in general, many clinical and pathological differences suggest that there are also disease-specific mechanisms. Taking advantage of a collection of genetic modifiers of expanded Ataxin-1-induced neurotoxicity, we performed a comparative analysis in Drosophila models of the two diseases. We show that while some modifier genes function similarly in SCA1 and HD Drosophila models, others have model-specific effects. Surprisingly, certain modifier genes modify SCA1 and HD models in opposite directions, i.e. they behave as suppressors in one case and enhancers in the other. Furthermore, we find that modulation of toxicity does not correlate with alterations in the formation of neuronal intranuclear inclusions. Our results point to potential common therapeutic targets in novel pathways, and to genes and pathways responsible for differences between Ataxin-1 and Huntingtin-induced neurodegeneration.

Expression of Expanded Polyglutamine Proteins Suppresses the Activation of Transcription Factor NFκB

A major pathological hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various transcription factors, chaperones, and proteasome components. However, how the expanded polyglutamine proteins or their aggregates elicit complex pathogenic responses in the neuronal cells is not fully understood. Here, we have demonstrated that the expression of expanded polyglutamine proteins down-regulated the NFkappaB-dependent transcriptional activity. The expression of expanded polyglutamine proteins increased the stability and the levels of IkappaB-alpha and its phosphorylated derivatives. We have also found that various NFkappaB subunits and IkappaB-alpha aberrantly interacted with the expanded polyglutamine proteins and associated with their aggregates. Finally, we have shown that several NFkappaB-dependent genes are down-regulated in the expanded polyglutamine protein-expressing cells and down-regulation of NFkappaB activity enhances expanded polyglutamine protein-induced cell death. Because the NFkappaB pathway plays a very important role in cell survival, altered regulation of this pathway in expanded polyglutamine protein-expressing cells might be linked with the disease pathogenesis.

Polyglutamine disease: Recent advances in the neuropathology of dentatorubral‐pallidoluysian atrophy

Polyglutamine diseases are hereditary neurodegenerative disorders that are caused by the expansion of a CAG repeat in the causative genes. They comprise at least nine disorders, including DRPLA, HD, and Machado-Joseph disease. Initially, the discovery of neuronal intranuclear inclusions (NIIs) in human brains and in a murine model of HD provided a plausible hypothesis that the expression of expanded polyglutamine stretches leads to NII formation, resulting in neuronal cell death in selective brain regions characteristic to each disease. Recent studies, however, suggest that nuclear dysfunction, especially transcriptional abnormalities caused by the diffuse intranuclear accumulation of mutant proteins, plays a pivotal role in the development and progression of clinical symptoms. Polyglutamine diseases have a similarity with neuronal storage disease, and this pathological process might become a target for the establishment of an effective therapy for these diseases.

Cleavage at the Caspase-6 Site Is Required for Neuronal Dysfunction and Degeneration Due to Mutant Huntingtin

Cleavage of huntingtin (htt) has been characterized in vitro, and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington's disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase-3- and caspase-6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6-resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid (QA), and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD.

Curcumin enhances the polyglutamine-expanded truncated N-terminal huntingtin-induced cell death by promoting proteasomal malfunction

Formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various proteasome components is the major hallmark of the polyglutamine diseases. Curcumin is a polyphenolic compound having anti-inflammatory, anti-tumor, and anti-oxidative properties. Recently, curcumin has been reported to suppress the amyloid-β accumulation, oxidative damage, and inflammation in the transgenic mice model of Alzheimer’s disease. Here, we found that the treatment of curcumin increases the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-dependent cell death. Curcumin also causes rapid proteasomal malfunction in the mutant huntingtin expressing cells in comparison with normal glutamine repeat expressing cells. Finally, we show that N-acetyl cysteine (NAC), a potent antioxidant, reverted the curcumin-induced mutant huntingtin aggregation and proteasomal malfunction in the mutant huntingtin expressing cells. NAC also protects curcumin-induced cell death. Our result suggests that curcumin promotes mutant huntingtin-induced cell death by mimicking proteasomal dysfunction.

Co-chaperone CHIP Associates with Expanded Polyglutamine Protein and Promotes Their Degradation by Proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.

Defining a metabolic phenotype in the brain of a transgenic mouse model of spinocerebellar ataxia 3.

Many of the spinocerebellar ataxias (SCAs) are caused by expansions of CAG trinucleotide repeats encoding abnormal stretches of polyglutamine. SCA3 or Machado-Joseph disease (MJD) is the commonest dominant inherited ataxia disease, with pathological phenotypes apparent with a CAG triplet repeat length of 61-84. In this study a mouse model of SCA3 has been examined which was produced using a human yeast artificial chromosome containing the MJD gene with a CAG triplet expansion of 84 repeats. These mice have previously been shown to possess a mild progressive cerebellar deficit. NMR-based metabolomics/metabonomics in conjunction with multivariate pattern recognition identified a number of metabolic perturbations in SCA3 mice. These changes included a consistent increase in glutamine concentration in tissue extracts of the cerebellum and cerebrum and spectra obtained from intact tissue using magic angle spinning (1)H-NMR spectroscopy. Furthermore, these profiles demonstrated metabolic abnormalities were present in the cerebrum, a region not previously implicated in SCA3. As well as an increase in glutamine both brain regions demonstrated decreases in GABA, choline, phosphocholine and lactate (representing the summation of lactate in vivo, and postmortem glycolysis of glucose and glycogen). The metabolic changes are discussed in terms of the formation of neuronal intranuclear inclusions associated with SCA3. This study suggests high-resolution (1)H-NMR spectroscopy coupled with pattern recognition may provide a rapid method for assessing the phenotype of animal models of human disease.

Neuronal intranuclear hyaline inclusion disease.

Neuronal intranuclear hyaline inclusion disease (NIHID) is a rare neurodegenerative disorder characterized pathologically by the presence of eosinophilic intranuclear inclusions in neuronal and glial cells. It has been considered to be a heterogeneous disease entity because the clinical pictures of previously described cases were highly variable. In the present review, reported NIHID cases have been categorized into three clinical subgroups according to onset and disease duration, and the clinical phenotype of each subgroup is discussed. Neuronal intranuclear inclusions (NII) in NIHID are ubiquitinated and their prevalence is inversely correlated with neuronal loss, suggesting that NII formation is a protective mechanism involving the ubiquitin-proteasome-dependent proteolytic pathway. In several polyglutamine diseases, disease-related proteins containing abnormally expanded polyglutamine tracts aggregate in neuronal nuclei, resulting in NII formation. The similarity between NII in NIHID and polyglutamine diseases suggests that they are formed during a common proteolysis-related process that takes place in the nucleus. Although the pathogenetic mechanism underlying NIHID remains unknown, the data reviewed here suggest that it might be related to accumulation of as yet unidentified abnormal proteins or dysfunction of the intranuclear ubiquitin-proteasome pathway.

Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context.

In order to better understand the role of nuclear localization of polyglutamine in the human CAG repeat disorders, gene targeting was used to add either nuclear localization (NLS) or nuclear export (NES) signals to versions of the mouse Hprt protein containing expanded polyglutamine (HprtQ150). The NLS increased levels of nuclear HprtQ150 protein in the mouse brain and hastened both the presentation of neuronal intranuclear inclusions (NIIs) and the onset of behavioral abnormalities. The NES reduced levels of nuclear HprtQ150 protein in mouse brain and delayed both the presentation of NIIs and the onset of behavioral abnormalities. Together these results indicate the nucleus is the primary site of toxicity in HprtQ150 mice. Furthermore, the signals did not alter the relative regional distribution of NIIs, suggesting that factors other than nuclear access dictate the regional specificity of NII formation in this mouse model.

PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases

In polyglutamine diseases, accumulation in the nucleus of mutant proteins induces the formation of neuronal intranuclear inclusions (NIIs). The nucleus is compartmentalized into structural and functional domains, which are involved in NII formation. Promyelocytic leukemia protein (PML), a major component of nuclear bodies, and mSin3A, a component of the transcription co-repressor complex, were used to investigate how the intranuclear domains/sites relate to NII formation in SCA2, SCA3, SCA7, SCA17 and DRPLA brains. We demonstrate that the size of PML-positive intranuclear structures was larger in pathological brains than in control ones and that these structures contained mutant proteins. PML colocalized only with small NIIs, which maintained the ring-like structure of normal nuclear bodies. Enlarged ring-like PML-positive structures, devoid of mutant proteins, were also found and might represent structures where mutant polyglutamine proteins have been successfully processed. These data suggest that NIIs originate from nuclear bodies, where mutant proteins accumulate for degradation.

Cdc42-interacting protein 4 binds to huntingtin: Neuropathologic and biological evidence for a role in Huntington's disease

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction. To identify previously uncharacterized htt-interacting proteins in a simple model system, we used a yeast two-hybrid screen with a Caenorhabditis elegans activation domain library. We found a predicted SH3 domain protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests. A human homolog of K08E3.3b is the Cdc42-interacting protein 4 (CIP4), a protein involved in Cdc42 and Wiskott-Aldrich syndrome protein-dependent signal transduction. CIP4 interacted in vitro with full-length htt from lymphoblastoid cells. Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients and partially colocalized to ubiquitin-positive aggregates. Marked CIP4 overexpression also was observed in Western blot from human HD brain striatum. The overexpression of CIP4 induced the death of striatal neurons. Our data suggest that CIP4 accumulation and cellular toxicity may have a role in HD pathogenesis.

SUMO-1 co-localized with mutant atrophin-1 with expanded polyglutamines accelerates intranuclear aggregation and cell death.

To investigate the implication of small ubiquitin-related modifier-1 (SUMO-1) in the formation of neuronal intranuclear inclusions in polyglutamine diseases, we examined the localization of SUMO-1 in dentatorubral-pallidoluysian atrophy (DRPLA) brain tissues and PC12 cells expressing truncated atrophin-1 with expanded poly-glutamine stretches. SUMO-1 was co-localized with neuronal intranuclear inclusions in DRPLA brain and the DRPLA model cells, which showed that the aggregates formed by expanded polyglutamine stretches were highly SUMOlylated. In addition, to examine the role of SUMO-1 in nuclear aggregate formation and cell death, either SUMO-1 or DeltaSUMO-1, which is a SUMOlylation defective mutant lacking the C-terminal motif, was co-transfected with atrophin-1 with expanded polyglutamine stretches. Co-transfection of DeltaSUMO-1 decreased number of the cells with nuclear aggregates and consequent apoptosis of PC12 cells, both of which were markedly enhanced by co-transfection of SUMO-1 with atrophin-1 with expanded polyglutamine stretches. These results suggest that SUMO-1 is implicated in the pathogenesis of DRPLA and accelerates aggregate formation and cell death.

'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease.

By crossing Huntington's disease (HD) R6/1 transgenic mice with 'tissue' transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2(-/-) compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the 'tissue' transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2(-/-) versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntington's disease.