Peter Breuer

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.

Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity

The formation of insoluble protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by proteins with expanded polyglutamine (polyQ) repeats. However, the mechanistic relationship between polyQ aggregation and its toxic effects on neurons remains unclear. Two main hypotheses have been put forward for how polyQ expansions may cause cellular dysfunction. In one model neurotoxicity results from the ability of polyQ-expanded proteins to recruit other important cellular proteins with polyQ stretches into the aggregates. In the other model, aggregating polyQ proteins partially inhibit the ubiquitin–proteasome system for protein degradation. These two mechanisms are not exclusive but may act in combination. In general, protein misfolding and aggregation are prevented by the machinery of molecular chaperones. Some chaperones such as the members of the Hsp70 family also modulate polyQ aggregation and suppress its toxicity. These recent findings suggest that an imbalance between the neuronal chaperone capacity and the production of potentially dangerous polyQ proteins may trigger the onset of polyQ disease.

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.

Calpain inhibition is sufficient to suppress aggregation of polyglutamine-expanded ataxin-3

The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.

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.

Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3)

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Josephin domain-containing proteins from a variety of species are active de-ubiquitination enzymes

Abstract The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by the presence of an extended polyglutamine stretch (polyQ) in the unstructured C-terminus of the human ataxin-3 (AT3) protein. The structured N-terminal Josephin domain (JD) of AT3 is conserved within a novel family of potential ubiquitin proteases, the JD-containing proteins, which are sub-divided into two groups termed ataxins and Josephins. These AT3 orthologs are encoded by the genomes of organisms ranging from Plasmodium falciparum to humans, with most species possessing more than one homolog. While Josephins consist of JDs alone, ataxins contain additional functional domains that may influence their enzyme activity. Here, we show that the enzyme activity of human AT3 (hAT3) is not affected by the length of polyQ in its C-terminus, even when it is in the range associated with SCA3. We also show that JDs of all human proteins with homology to AT3 and its homologs from various species possess de-ubiquitination activity. These results establish JD-containing proteins as a novel family of active de-ubiquitination enzymes with wide phylogenic distribution.

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis

&NA; DJ‐1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinsons disease (PD), but growing evidence suggests involvement of DJ‐1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH‐SY5Y neuroblastoma cell lines that DJ‐1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ‐1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ‐1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ‐1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ‐1 complexes. In the KD model, the absence of DJ‐1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re‐expression of DJ‐1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7‐related PD. Notably, oxidised DJ‐1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.

Quo vadis with the Q tracts

Research on polyglutamine diseases is heralding its second decade. Many advances have been made in this fastmoving field since the first identification of an expanded polyglutamine tract in the androgen receptor as the cause of Kennedy’s disease. Since then, several other neurodegenerative disorders, including Huntington’s disease and the various ataxias, have been found to be caused by the elongation of polyQ sequences. These polyQ expansions occur in otherwise unrelated proteins and result in neurotoxicity by a common pathogenetic process. Soon after the isolation and identification of the Huntington’s Disease (HD) gene in 1993 it was Max Perutz who provided seminal insight into the molecular basis of polyQ diseases by realizing the potential of β-strands of poly-L-glutamine to self-associate into so-called polar zippers. Critical experimental support for the polar zipper hypothesis followed with the discovery by Gillian Bates and co-workers that mice expressing a fragment of the HD gene containing the expanded polyQ sequence develop intranuclear aggregates in the brain. Erich Wanker and colleagues then demonstrated that this aggregation process is faithfully reproduced in vitro with recombinant polyQ proteins. It is now generally accepted that a unifying feature of pathologically elongated polyQ tracts is to cause the aggregation of the proteins in which they occur. A new book on the molecular aspects of polyQ diseases edited by Peter Harper and Max Perutz deals with the fascinating questions raised by these insights, including one that is most intensely debated by experts and interested observers: are the polyQ aggregates the direct cause of neurodegeneration, or are they merely an epiphenomenon of a disease process to which polyQ proteins contribute by a so far unknown mechanism? Glutamine Repeats and Neurodegenerative Diseases: Molecular Aspects is a compilation of updated contributions by leading scientists originally submitted on the occasion of a Royal Society discussion meeting in 1998. After an excellent, clinically oriented introduction by Harper on HD, the most prominent polyQ disorder, it starts off with a review of various animal models now available for the study of this disease. Although most of these models utilize transgenic mice, the reader also learns about the potential of the Drosophila system in analysing polyQ-induced neurotoxicity. Indeed, a recent search for suppressors of polyQ-mediated neurotoxicity in transgenic flies revealed the remarkable capacity of certain molecular chaperones to protect neurons against degeneration and death. Other chapters deal with the molecular basis of polyQ toxicity, the biochemistry of polyQ proteins and the genetic mechanisms underlying the pathological expansion of the CAG trinucleotide repeats encoding polyQ sequences. The last part of the book describes how research on HD and other polyQ diseases promises to unify current understanding of the molecular pathology forming the basis of a larger group of neurodegenerative diseases. Alzheimer’s and Parkinson’s disease as well as the prion diseases are all associated with the deposition of amyloid-like protein aggregates in neurons and these deposits have characteristic features in common with the aggregates found in HD. As stated in the preface, the intention of this book is to stimulate further a field that already is very active, and to make it more accessible to clinicians and workers in allied areas of neuroscience. This goal is successfully achieved both by the breadth of coverage and the quality of the individual contributions. Hopefully many readers will be inspired to explore further the intriguing ways of the polyQ tracts. F. Ulrich Hartl and Peter Breuer are in the Max-Planck-Institute of Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. e-mail uhartl@biochem.mpg.de Other Neurodegeneration Books