Low levels of mutant ubiquitin are degraded by the proteasome in vivo

The ubiquitin‐proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin‐ containing proteinaceous deposits. UBB⁺¹, a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB⁺¹ properties shift from a proteasomal ubiquitin‐fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB⁺¹. In these mice, UBB⁺¹ protein is scarcely detectable in the neuronal cell population. Accumulation of UBB⁺¹ commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB⁺¹ protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB⁺¹ is continuously degraded and disease‐related UBB⁺¹ accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB⁺¹ and its role in neurodegenerative disease. © 2010 Wiley‐Liss, Inc.     

Levels of DNAJB family members (HSP40) correlate with disease onset in patients with spinocerebellar ataxia type 3

In polyglutamine disorders, the length of the expanded CAG repeat shows a strong inverse correlation with the age at disease onset, yet up to 50% of the variation in age of onset is determined by other additional factors. Here, we investigated whether variations in the expression of heat shock proteins (HSP) are related to differences in the age of onset in patients with spinocerebellar ataxia (SCA)3. Hereto, we analysed the protein expression levels of HSPA1A (HSP70), HSPA8 (HSC70), DNAJB (HSP40) and HSPB1 (HSP27) in fibroblasts from patients and healthy controls. HSPB1 levels were significantly upregulated in fibroblasts from patients with SCA3, but without relation to age of onset. Exclusively for expression of DNAJB family members, a correlation was found with the age of onset independent of the length of the CAG repeat expansion. This indicates that DNAJB members might be contributors to the variation in age of onset and underlines the possible use of DNAJB proteins as therapeutic targets.     

Modest proteasomal inhibition by aberrant ubiquitin exacerbates aggregate formation in a Huntington disease mouse model

UBB⁺¹, a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB⁺¹ and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB⁺¹ transgenic mouse line expressing UBB⁺¹ within the neurons of the striatum. In these mice lentiviral driven expression of expanded huntingtin constructs in the striatum results in a significant increase in neuronal inclusion formation. Although UBB⁺¹ transgenic mice show neither a decreased lifespan nor apparent neuronal loss, they appear to be more vulnerable to toxic insults like expanded polyglutamine proteins due to a modest proteasome inhibition. These findings underscore the relevance of an efficient ubiquitin–proteasome system in HD.     

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B⁺¹ (UBB⁺¹), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB⁺¹-expressing transgenic mice display widespread labeling for UBB⁺¹ in brain and exhibit behavioral deficits. Here, we show that UBB⁺¹ is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB⁺¹-expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.     

Axonal inclusions in spinocerebellar ataxia type 3

Protein aggregation is a major pathological hallmark of many neurodegenerative disorders including polyglutamine diseases. Aggregation of the mutated form of the disease protein ataxin-3 into neuronal nuclear inclusions is well described in the polyglutamine disorder spinocerebellar ataxia type 3 (SCA3 or Machado–Joseph disease), although these inclusions are not thought to be directly pathogenic. Neuropil aggregates have not yet been described in SCA3. We performed a systematic immunohistochemical study of serial thick sections through brains of seven clinically diagnosed and genetically confirmed SCA3 patients. Using antibodies against ataxin-3, p62, ubiquitin, the polyglutamine marker 1C2 as well as TDP-43, we analyzed neuronal localization, composition and distribution of aggregates within SCA3 brains. The analysis revealed widespread axonal aggregates in fiber tracts known to undergo neurodegeneration in SCA3. Similar to neuronal nuclear inclusions, the axonal aggregates were ubiquitinated and immunopositive for the proteasome and autophagy associated shuttle protein p62, indicating involvement of neuronal protein quality control mechanisms. Rare TDP-43 positive axonal inclusions were also observed. Based on the correlation between affected fiber tracts and degenerating neuronal nuclei, we hypothesize that these novel axonal inclusions may be detrimental to axonal transport mechanisms and thereby contribute to degeneration of nerve cells in SCA3.     

G protein‐coupled receptor 26 immunoreactivity in intranuclear inclusions associated with polyglutamine and intranuclear inclusion body diseases

G protein‐coupled receptor 26 (GPR26) is one of the G‐protein‐coupled receptors (GPCRs), which comprise the largest family of membrane proteins and mediate most of the physiological responses to hormones, neurotransmitters and environmental stimulants. Although GPCRs are considered to play an important role in the pathophysiology of neurodegenerative disorders, it is uncertain whether GPR26 is involved in the pathogenesis of polyglutamine and intranuclear inclusion body diseases. We immunohistochemically examined the brain tissues of patients with four polyglutamine diseases (Huntington's disease, dentatorubral‐pallidoluysian atrophy, and spinocerebellar ataxia types 1 and 3) and intranuclear inclusion body disease, and normal control subjects. In controls, anti‐GPR26 antibody immunolabeled the neuronal cytoplasm in a diffuse granular pattern. Neuronal nuclear inclusions in polyglutamine diseases were immunopositive for GPR26. In intranuclear inclusion body disease, GPR26‐positive nuclear inclusions were found in both neurons and glial cells. Marinesco bodies in aged control subjects were also positive for GPR26. Double immunofluorescence analysis revealed co‐localization of GPR26 with polyglutamine or ubiquitin in these nuclear inclusions. These findings suggest that GPR26 may have a common role in the formation or degradation of intranuclear inclusions in several neurodegenerative diseases.     

Association of moderate polyglutamine tract expansions in the slow calcium-activated potassium channel type 3 with ataxia

BACKGROUND: The small-conductance calcium-activated potassium channel gene (hSKCa3) contains 2 CAG repeats, 1 of which is highly polymorphic. Although this repeat is not pathologically expanded in patients with schizophrenia, some studies have suggested an allelic association with schizophrenia. CAG expansions in other genes such as the alpha1 subunit of a brain-specific P/Q-type calcium channel gene cause spinocerebellar ataxia type 6, whereas the length of the CAG repeat in the RAI1 gene modifies the age of onset of spinocerebellar ataxia type 2. OBJECTIVES: To evaluate expansions in the hSKCa3 polyglutamine domain as causative for ataxia, and to study the association between the length of the polyglutamine repeat and the presence of ataxia. METHODS: We analyzed this repeat in 122 patients with autosomal dominant cerebellar ataxia, or sporadic ataxia, and compared allele distribution with 750 alleles seen in 2 healthy control groups and 172 alleles in patients with Parkinson disease. RESULTS: The distribution of alleles in ataxia patients and controls was significantly different by Wilcoxon rank test (P <.001). Twenty-two or more polyglutamine tracts were more common in ataxia patients compared with controls by chi2 analysis (P<.001). CONCLUSION: Longer stretches of polyglutamines in a human potassium channel are not causative for ataxia, but they are associated with the presence of ataxia. There is no association with the presence of Parkinson disease.     

Presenile onset of spinocerebellar ataxia type 1 presenting with conspicuous psychiatric symptoms and widespread anti‐expanded polyglutamine antibody‐ and fused in sarcoma antibody‐immunopositive pathology

A 50‐year‐old Japanese man showed slowly progressive gait disturbance and dysarthria. Neurological examination 5 years after onset revealed slow eye movement with nystagmus as well as limb and truncal ataxia. Magnetic resonance imaging showed atrophy of the cerebellum and brainstem. Because genetic examination revealed CAG repeat expansion of the ataxin‐1 gene, the patient was diagnosed with spinocerebellar ataxia type 1. Ten years after onset, he showed psychiatric symptoms with cognitive impairment, and antipsychotic drugs were administered. As psychiatric symptoms gradually worsened, particularly with regard to resisting nursing care and shouting, the doses of the drugs were increased. Although the clinicopathologic findings were generally identical to previously reported spinocerebellar ataxia type 1 cases with the exception of the conspicuous psychiatric symptoms, there are two notable immunohistochemical findings. Firstly, numerous anti‐expanded polyglutamine antibody‐immunopositive neuronal inclusions were extensively observed, including in the cerebral cortex and limbic system, but not in the Purkinje cells. Secondly, anti‐fused in sarcoma antibody‐immunopositive intranuclear inclusions were extensively observed. We posit that the anti‐expanded polyglutamine antibody‐immunopositive neuronal inclusions and possibly the anti‐fused in sarcoma antibody‐immunopositive inclusions, particularly those in the neocortex and limbic system, may correspond to the psychiatric symptoms and cognitive impairment that were observed in the patient.     

Degron capability of the hydrophobic C-terminus of the polyglutamine disease protein,ataxin-3

Ataxin-3 is a deubiquitinase and polyglutamine disease protein whose cellular properties and functions are not entirely understood. Mutations in ataxin-3 cause spinocerebellar ataxia type 3 (SCA3), a neurodegenerative disorder that is a member of the polyglutamine family of diseases. Two major isoforms arise from alternative splicing of ATXN3 and are differently toxic in vivo as a result of faster proteasomal degradation of one isoform compared to the other. The isoforms vary only at their C-termini, suggesting that the hydrophobic C-terminus of the more quickly degraded form of ataxin-3 (here referred to as isoform 2) functions as a degron—that is, a peptide sequence that expedites the degradation of its host protein. We explored this notion in this study and present evidence that: (a) the C-terminus of ataxin-3 isoform 2 signals its degradation in a proteasome-dependent manner, (b) this effect from the C-terminus of isoform 2 does not require the ubiquitination of ataxin-3, and (c) the isolated C-terminus of isoform 2 can enhance the degradation of an unrelated protein. According to our data, the C-terminus of ataxin-3 isoform 2 is a degron, increasing overall understanding of the cellular properties of the SCA3 protein.     

Cutaneous accumulation of abnormal polyglutamine proteins of patients with dentatorubral-pallidoluysian atrophy

Background and purpose:  Dentatorubral-pallidoluysian atrophy (DRPLA) is a hereditary spinocerebellar degeneration caused by expansion of a trinucleotide CAG repeat encoding a polyglutamine tract in a disease protein atrophin-1. The clinical features include ataxia, choreoathetosis, and dementia, which result from neural degeneration caused by the mutant atrophin-1.
Methods:  We performed skin biopsy in two patients with DRPLA.
Results:  We found multiple clear cells in the epidermis, which were positive for proteins containing an expanded polyglutamine stretches. The clear cells were p63 (+), S-100 (−), and cytokeratin 20 (−), showing that they were keratinocytes. Negative or weak signals of pan-cytokeratin were consistent with the finding of decreased tonofilaments at the electron microscopic level.
Conclusions:  The presence of clear keratincoytes showed that the mutant proteins interfered in cellular functions not only in neural cells but also in keratinocytes. The skin is accessible by biopsy, making it important in the diagnosis. Furthermore, the polyglutamine staining in the skin may be useful for evaluation of therapeutic modalities for DRPLA and other polyglutamine diseases.     

Genotype‐phenotype correlations,dystonia and disease progression in spinocerebellar ataxia type 14

Background: Spinocerebellar ataxia type 14 is a rare form of autosomal dominant cerebellar ataxia caused by mutations in protein kinase Cγ gene. Clinically, it presents with a slowly progressive, mainly pure cerebellar ataxia. Methods: Using next generation sequencing, we screened 194 families with autosomal dominant cerebellar ataxia and normal polyglutamine repeats. In‐depth phenotyping was performed using validated clinical rating scales neuroimaging and electrophysiological investigations. Results: We identified 25 individuals from 13 families carrying pathogenic mutations in protein kinase Cγ gene. A total of 10 unique protein kinase Cγ gene mutations have been confirmed of which 5 are novel and 5 were previously described. Our data suggest that the age at onset is highly variable; disease course is slowly progressive and rarely associated with severe disability. However, one third of patients presented with a complex ataxia comprising severe focal and/or task‐induced dystonia, peripheral neuropathy, parkinsonism, myoclonus, and pyramidal syndrome. The most complex phenotype is related to a missense mutation in the catalytic domain in exon 11. Conclusion: We present one of the largest genetically confirmed spinocerebellar ataxia type 14 cohorts contributing novel variants and clinical characterisation. We show that although protein kinase Cγ gene mutations present mainly as slowly progressive pure ataxia, more than a third of cases had a complex phenotype. Overall, our case series extends the phenotype and suggests that protein kinase Cγ gene mutations should be considered in patients with slowly progressive autosomal dominant cerebellar ataxia, particularly when myoclonus, dystonia, or mild cognitive impairment are present in the absence of polyglutamine expansion. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.     

Spinocerebellar ataxias: an update

PURPOSE OF REVIEW: Here we discuss recent advances regarding the molecular genetic basis of dominantly inherited ataxias. RECENT FINDINGS: Important recent observations include insights into the mechanisms by which expanded polyglutamine causes cerebellar degeneration; new findings regarding how noncoding expansions may cause disease; the discovery that conventional (i.e. nonrepeat) mutations underlie recently identified ataxias; and growing recognition that multiple biological pathways, when perturbed, can cause cerebellar degeneration. SUMMARY: The dominant ataxias, also known as spinocerebellar ataxias, continue to grow in number. Here we review the major categories of spinocerebellar ataxias: expanded polyglutamine ataxias; noncoding repeat ataxias; and ataxias caused by conventional mutations. After discussing features shared by these disorders, we present recent evidence supporting a toxic protein mechanism for the polyglutamine spinocerebellar ataxias and the recognition that both protein misfolding and perturbations in nuclear events represent key events in pathogenesis. Less is known about pathogenic mechanisms in spinocerebellar ataxias due to noncoding repeats, though a toxic RNA effect remains possible. Newly discovered, conventional mutations in spinocerebellar ataxias suggest a wide range of biological pathways can be disrupted to cause progressive ataxia. Finally, we discuss how new mechanistic insights can drive the push toward preventive treatment.     

Intranuclear aggregation of nonexpanded ataxin-3 in marinesco bodies of the nonhuman primate substantia nigra

Marinesco bodies (MB) are intranuclear inclusion bodies predominantly found in melanin-pigmented neurons of the substantia nigra. MB are demonstrable not only in humans but also in nonhuman primates. In the present study MB of aged rhesus monkeys (Macaca mulatta; n = 15; mean age 16 years) and aged baboons (Papio anubis; n = 13; mean age 25 years) were examined immunohistochemically. MB were found to be immunoreactive for ubiquitin, a protein involved in initiation of proteasome-mediated proteolysis. We also demonstrate that MB in monkeys are intensely immunoreactive for the protein ataxin-3 as detected by using two monoclonal anti-ataxin-3 antibodies (1H9 and 2B6). The abnormally expanded form of this polyglutamine protein is known to be causally involved in spinocerebellar ataxia type 3 or Machado-Joseph disease. The monoclonal antibody 1C2 was employed to examine whether ataxin-3 in MB in monkeys contains such an abnormally expanded polyglutamine stretch. MB were consistently 1C2-immunonegative, indicating that they are composed of normal wild-type ataxin-3. In conclusion MB in nonhuman primates permit experimental examination of mechanisms involved in transnuclear localization, intranuclear aggregation, and ubiquitination of nonexpanded polyglutamine proteins.     

DNAzyme Cleavage of CAG Repeat RNA in Polyglutamine Diseases

CAG repeat expansion is the genetic cause of nine incurable polyglutamine (polyQ) diseases with neurodegenerative features. Silencing repeat RNA holds great therapeutic value. Here, we developed a repeat-based RNA-cleaving DNAzyme that catalyzes the destruction of expanded CAG repeat RNA of six polyQ diseases with high potency. DNAzyme preferentially cleaved the expanded allele in spinocerebellar ataxia type 1 (SCA1) cells. While cleavage was non-allele-specific for spinocerebellar ataxia type 3 (SCA3) cells, treatment of DNAzyme leads to improved cell viability without affecting mitochondrial metabolism or p62-dependent aggresome formation. DNAzyme appears to be stable in mouse brain for at least 1 month, and an intermediate dosage of DNAzyme in a SCA3 mouse model leads to a significant reduction of high molecular weight ATXN3 proteins. Our data suggest that DNAzyme is an effective RNA silencing molecule for potential treatment of multiple polyQ diseases.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01075-w.     

TDP‐43 pathology in polyglutamine diseases: With reference to amyotrphic lateral sclerosis

A nuclear protein, transactivation response (TAR) DNA binding protein 43 kDa (TDP‐43), is the major component of neuronal cytoplasmic inclusions (NCIs) in frontotemporal lobar degeneration with ubiquitin inclusions (FTLD‐U) and sporadic amyotrophic lateral sclerosis (SALS). While initially thought to be relatively specific to FTLD‐U and ALS, TDP‐43 pathology has now been detected in a number of other neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. In such tauopathies and α‐synucleinopathies, occurrence of TDP‐43‐positive neuronal cytoplasmic inclusions may be associated with other distinct molecular pathologic processes primarily involving their own pathological proteins, tau and α–synuclein, respectively (secondary TDP‐43 proteinopathies). On the other hand, in several polyglutamine (polyQ) diseases, TDP‐43 appears to play an important pathomechanistic role. Interestingly, intermediate‐length polyQ expansions (27–33 Qs) in ataxin 2, the causative gene of spinocerebellar ataxia type 2, have recently been reported to be a genetic risk factor for SALS. Here, with a review of the literature, we discuss the relationship between ALS and polyQ diseases from the viewpoint of TDP‐43 neuropathology.     

Mouse models of Machado-Joseph disease and other polyglutamine spinocerebellar ataxias

Machado-Joseph disease (MJD), also called spinocerebellar ataxia type 3, is caused by mutant ataxin-3 with a polyglutamine expansion. Although there is no treatment available at present to cure or delay the onset of MJD, mouse models have been generated to facilitate the development of a therapy. In this review, the published reports on mouse models of MJD and other polyglutamine spinocerebellar ataxias are compared. Based on these studies, the following approaches will be discussed as candidate treatments for MJD: 1) interfering with the formation of the mutant ataxin-3 cleavage fragment and possibly aggregate or inclusions, 2) reducing the disease protein nuclear localization, and 3) decreasing mutant ataxin-3 expression in neurons.     

Cytoplasmic and nuclear polyglutamine aggregates in SCA6 Purkinje cells.

Aggregations of the alpha1A-calcium channel protein have been previously demonstrated in spinocerebellar ataxia type 6 (SCA6). Here the authors show that small aggregates, labeled by a monoclonal antibody 1C2 that preferentially detects expanded polyglutamine larger than that in SCA6 mutation, are present mainly in the cytoplasm but also in the nucleus of Purkinje cells. Although the length of expansion is small in SCA6, the current finding might indicate that SCA6 conforms to the pathogenic mechanism(s) in other polyglutamine diseases.     

Comparison of cerebellar ataxias: A three‐year prospective longitudinal assessment

We quantitatively investigated the clinical severity and progression of diseases with ataxia, as measured with the Scale for the Assessment and Rating of Ataxia, and examined the potential application of the Scale for the Assessment and Rating of Ataxia for future therapeutic trials. Severity of ataxia was assessed in 238 patients with spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 17, multiple system atrophy‐cerebellar variant, or Gerstman‐Sträussler‐Scheinker disease. Among them, 119 (50%) were longitudinally examined three to seven times, in a period of 8 to 38 months, resulting in a total set of 535 assessments. The differences between spinocerebellar ataxia and multiple system atrophy‐cerebellar variant were ascertained cross‐sectionally and longitudinally. Gerstman‐Sträussler‐Scheinker disease had the fastest progression, followed by multiple system atrophy‐cerebellar variant, spinocerebellar ataxia type 17, spinocerebellar ataxia type 3, spinocerebellar ataxia type 2, and spinocerebellar ataxia type 6. Patients with multiple system atrophy‐cerebellar variant had a faster progression in gait, sitting, speech, and total score than patients with spinocerebellar ataxias. For a randomized, case‐control trial, a sample size of 47 for spinocerebellar ataxia and 85 for multiple system atrophy‐cerebellar variant in the treatment or placebo arms would have a sufficient statistical power to demonstrate the efficacy of a new therapy that would retard ataxia progression by 1 point per year as measured by the Scale for the Assessment and Rating of Ataxia. The results will have a significant impact on the planning and implementation of future therapeutic trials of spinocerebellar ataxia and multiple system atrophy‐cerebellar variant. © 2011 Movement Disorder Society     

USP7, a ubiquitin-specific protease,interacts with ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 has been known to associate with elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system, we have found that USP7, a ubiquitin-specific protease, binds to ataxin-1. Further experiments with deletion mutants indicated that the C-terminal region of ataxin-1 was essential for the interaction. Liquid beta-galactosidase assay and coimmunoprecipitation experiments revealed that the strength of the interaction between USP7 and ataxin-1 is influenced by the length of the polyglutamine tract in the ataxin-1; weaker interaction was observed in mutant ataxin-1 with longer polyglutamine tract and USP7 was not recruited to the mutant ataxin-1 aggregates in the Purkinje cells of SCA1 transgenic mice. Our results suggest that altered function of the ubiquitin system can be involved in the pathogenesis of spinocerebellar ataxia type 1.