Bernd O. Evert

Excitation-induced ataxin-3 aggregation in neurons from patients with Machado–Joseph disease

Machado–Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that l-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca2+-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na+ and K+ channels as well as ionotropic and voltage-gated Ca2+ channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

An arginine/lysine-rich motif is crucial for VCP/p97-mediated modulation of ataxin-3 fibrillogenesis

Arginine/lysine‐rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin‐3 (Atx‐3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx‐3 in a concentration‐dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx‐3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine‐rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx‐3. Together, these results define the VCP–Atx‐3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Ataxin-3 Represses Transcription via Chromatin Binding, Interaction with Histone Deacetylase 3, and Histone Deacetylation

Ataxin-3 (AT3), the disease protein in spinocerebellar ataxia type 3 (SCA3), has been associated with the ubiquitin–proteasome system and transcriptional regulation. Here we report that normal AT3 binds to target DNA sequences in specific chromatin regions of the matrix metalloproteinase-2 (MMP-2) gene promoter and represses transcription by recruitment of the histone deacetylase 3 (HDAC3), the nuclear receptor corepressor (NCoR), and deacetylation of histones bound to the promoter. Both normal and expanded AT3 physiologically interacted with HDAC3 and NCoR in a SCA3 cell model and human pons tissue; however, normal AT3-containing protein complexes showed increased histone deacetylase activity, whereas expanded AT3-containing complexes had reduced deacetylase activity. Consistently, histone analyses revealed an increased acetylation of total histone H3 in expanded AT3-expressing cells and human SCA3 pons. Expanded AT3 lost the repressor function and displayed altered DNA/chromatin binding that was not associated with recruitment of HDAC3, NCoR, and deacetylation of the promoter, allowing aberrant MMP-2 transcription via the transcription factor GATA-2. For transcriptional repression normal AT3 cooperates with HDAC3 and requires its intact ubiquitin-interacting motifs (UIMs), whereas aberrant transcriptional activation by expanded AT3 is independent of the UIMs but requires the catalytic cysteine of the ubiquitin protease domain. These findings demonstrate that normal AT3 binds target promoter regions and represses transcription of a GATA-2-dependent target gene via formation of histone-deacetylating repressor complexes requiring its UIM-associated function. Expanded AT3 aberrantly activates transcription via its catalytic site and loses the ability to form deacetylating repressor complexes on target chromatin regions.

Peroxisome Proliferator-Activated Receptor γ Control of Dendritic Cell Function Contributes to Development of CD4+ T Cell Anergy

There is increasing evidence that dendritic cell (DC) immunogenicity is not only positively regulated by ligands of pattern recognition receptors, but also negatively by signals that prevent DC activation and full functional maturation. Depending on their activation status, DCs can induce either immunity or tolerance. In this study, we provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a negative regulator of DC maturation and function. Sustained PPARγ activation in murine DCs reduced maturation-induced expression of costimulatory molecules and IL-12, and profoundly inhibited their capacity to prime naive CD4+ T cells in vitro. Using PPARγ-deficient DCs, generated by Cre-mediated ablation of the PPARγ gene, agonist-mediated suppression of maturation-induced functional changes were abrogated. Moreover, absence of PPARγ increased DC immunogenicity, suggesting a constitutive regulatory function of PPARγ in DCs. Adoptive transfer of PPARγ-activated Ag-presenting DCs induced CD4+ T cell anergy, characterized by impaired differentiation resulting in absent Th1 and Th2 cytokine production and failure of secondary clonal expansion upon restimulation. Collectively, our data support the notion that PPARγ is an efficient regulator of DC immunogenicity that may be exploited to deliberately target CD4+ T cell-mediated immune responses.

Mutant huntingtin represses CBP, but not p300, by binding and protein degradation.

Huntingtons disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyl transferases may affect chromatin structure and transcription and thus contribute to neurodegeneration.

Cell death in polyglutamine diseases

Abstract An increasing number of inherited neurodegenerative diseases are known to be caused by trinucleotide repeat expansions in the respective genes. At least nine disorders result from a CAG trinucleotide repeat expansion which is translated into a polyglutamine stretch in the respective proteins: Huntingtons disease (HD), dentatorubral pallidolysian atrophy (DRPLA), spinal bulbar muscular atrophy (SBMA), and several of the spinocerebellar ataxias (SCA1, 2, 3, 6, 7 and 12). Although the molecular steps leading to the specific neuropathology of each disease are unknown and are still under intensive investigation, there is increasing evidence that some CAG repeat disorders involve the induction of apoptotic mechanisms. This review summarizes the clinical and genetic features of each CAG repeat disorder and focuses on the common mechanistic steps involved in the disease progression of these so-called polyglutamine diseases. Among the common molecular features the formation of intranuclear inclusions, the recruitment of interacting polyglutamine-containing proteins, the involvement of the proteasome and molecular chaperones, and the activation of caspases are discussed with regard to their potential implication for the induction of cell death.

Genes involved in hereditary ataxias

The hereditary ataxias are a group of inherited neurodegenerative disorders characterized by progressive ataxia that results from degeneration of the cerebellum and its afferent and efferent connections. Recent molecular research has led not only to the discovery of a number of causative mutations, but also shed light on the likely mechanisms by which these mutations cause the respective phenotypes. In Friedreichs ataxia (FRDA), the most common type of autosomal recessive ataxia, the loss of a mitochondrial protein, frataxin, results in overload of mitochondrial iron and oxidative stress. The autosomal dominant ataxias, spinocerebellar ataxia type I (SCAI), SCA2, SCA3 and SCA7, are caused by inheritance of an unstable, expanded CAG trinucleotide repeat. These disorders are assumed to be due to a novel deleterious function of the extended polyglutamine sequences within the proteins encoded by the respective genes. Recent observations in transgenic mice and in human post-mortem tissue suggest that the extended proteins are transported into the nucleus of neurons where they form intranuclear inclusions that disrupt normal nuclear function. In another group of dominant disorders, episodic ataxia type I and type 2 (EA-I, EA-2) and SCA6, the mutations affect genes that code for ion channels.

Different methylation of the TNF-alpha promoter in cortex and substantia nigra: Implications for selective neuronal vulnerability

Increasing evidence has linked inflammatory processes to neurodegenerative disorders, including Alzheimers and Parkinsons disease (PD). Tumor necrosis factor alpha (TNF-alpha) is a key inflammatory cytokine and several studies linked increased TNF-alpha to dopaminergic cell death in PD. The TNF-alpha promoter sequence contains several CpG dinucleotides located within or next to transcription factor binding sites. To test the hypothesis whether the methylation state of the TNF-alpha promoter contributes to increased expression of TNF-alpha in PD we compared DNA from different brain regions (substantia nigra pars compacta (SNpc) and cortex) of PD patients and neurologically healthy, age and sex matched controls by bisulfite sequencing of the TNF-alpha promoter region. The TNF-alpha promoter DNA from SNpc was significantly less methylated in comparison to DNA from cortex; however both in PD patients and controls. Although there was a tendency for hypomethylation in PD, our analysis of the 10 CpGs in the TNF-alpha core promoter region (-258 to -35 relative to the TSS) revealed no particular pattern in PD patients compared to control and identified no particular hypomethylated position in cortex or SNpc DNA. Electrophoretic mobility shift and luciferase reporter assays showed that methylation of specific solitary CpG in the TNF-alpha promoter resulted in reduced binding of the transcription factors AP-2 and Sp1, respectively, and suppressed TNF-alpha promoter activity. The brain region specific methylation state of solitary CpG in the TNF-alpha promoter thus determines transcription factor binding efficacy and TNF-alpha expression. A lesser degree of methylation of the TNF-alpha promoter in SNpc cells could underlie the increased susceptibility of dopaminergic neurons to TNF-alpha mediated inflammatory reactions.

CK2-Dependent Phosphorylation Determines Cellular Localization and Stability of Ataxin-3

The nuclear presence of the expanded disease proteins is of critical importance for the pathogeneses of polyglutamine diseases. Here we show that protein casein kinase 2 (CK2)-dependent phosphorylation controls the nuclear localization, aggregation and stability of ataxin-3 (ATXN3), the disease protein in spinocerebellar ataxia type 3 (SCA3). Serine 340 and 352 within the third ubiquitin-interacting motif of ATXN3 were particularly important for nuclear localization of normal and expanded ATXN3 and mutation of these sites robustly reduced the formation of nuclear inclusions; a putative nuclear leader sequence was not required. ATXN3 associated with CK2alpha and pharmacological inhibition of CK2 decreased nuclear ATXN3 levels and the formation of nuclear inclusions. Moreover, we found that ATXN3 shifted to the nucleus upon thermal stress in a CK2-dependent manner, indicating a key role of CK2-mediated phosphorylation of ATXN3 in SCA3 pathophysiology.

The molecular biology of the autosomal-dominant cerebellar ataxias

Autosomal‐dominant cerebellar ataxias (ADCA) may present as progressive or paroxysmal disorders. While the progressive ataxias have been named spinocerebellar ataxias (SCA), the paroxysmal disorders are designated episodic ataxias (EA). Until now, three different mutational mechanisms resulting in distinctive pathogenesis have been identified. The first type of mutation present in SCA1, SCA2, SCA3, and SCA7 is an expanded CAG repeat in genes of unknown function that are translated into proteins with expanded polyglutamine tracts. A common ultrastructural feature of these disorders is the formation of neuronal intranuclear inclusions (NII) harboring the expanded disease proteins and a variety of other proteins. The pathogenic role of these inclusions has yet to be clarified. A second group of disorders is the result of mutations in genes that code for ion channels. In EA‐1, a disorder characterized by episodes of ataxia provoked by movement and startle, missense mutations in a potassium channel gene, KCNA1, have been found. Patients with EA‐2, another form of paroxysmal ataxia, carry nonsense mutations of the gene encoding the α1A voltage‐dependent calcium channel subunit, CACNA1A, that are predicted to result in truncated channel proteins. In SCA6, a progressive ataxia, an expanded CAG repeat in the 3` translated region of the CACNA1A gene, has been found. The third type of mutation is an untranslated CTG expansion resembling the mutation found in myotonic dystrophy. It is associated with a progressive ataxia, SCA8.