Ina Schmitt

Neurodegeneration and Motor Dysfunction in a Conditional Model of Parkinson's Disease

α-Synuclein (α-syn) has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinsons disease. These disorders are characterized by various neurological and psychiatric symptoms based on progressive neuropathological alterations. Whether the neurodegenerative process might be halted or even reversed is presently unknown. Therefore, conditional mouse models are powerful tools to analyze the relationship between transgene expression and progression of the disease. To explore whether α-syn solely originates and further incites these alterations, we generated conditional mouse models by using the tet-regulatable system. Mice expressing high levels of human wild-type α-syn in midbrain and forebrain regions developed nigral and hippocampal neuropathology, including reduced neurogenesis and neurodegeneration in absence of fibrillary inclusions, leading to cognitive impairment and progressive motor decline. Turning off transgene expression in symptomatic mice halted progression but did not reverse the symptoms. Thus, our data suggest that approaches targeting α-syn-induced pathological pathways might be of benefit rather in early disease stages. Furthermore, α-syn-associated cytotoxicity is independent of filamentous inclusion body formation in our conditional mouse model.

Reversibility of symptoms in a conditional mouse model of spinocerebellar ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a CAG repeat tract that affects the MJD1 gene which encodes the ataxin-3 protein. In order to analyze whether symptoms caused by ataxin-3 with an expanded repeat are reversible in vivo, we generated a conditional mouse model of SCA3 using the Tet-Off system. We used a full-length human ataxin-3 cDNA with 77 repeats in order to generate the responder mouse line. After crossbreeding with a PrP promoter mouse line, double transgenic mice developed a progressive neurological phenotype characterized by neuronal dysfunction in the cerebellum, reduced anxiety, hyperactivity, impaired Rotarod performance and lower body weight gain. When ataxin-3 expression was turned off in symptomatic mice in an early disease state, the transgenic mice were indistinguishable from negative controls after 5 months of treatment. These results show that reducing the production of pathogenic ataxin-3 indeed may be a promising approach to treat SCA3, provided that such treatment is applied before irreversible damage has taken place and that it is continued for a sufficiently long time.

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.

Polymorphisms of the alpha-synuclein promoter: expression analyses and association studies in Parkinson's disease.

Summary. Mutations of the α-synuclein gene have shown to be relevant in some rare families with autosomal dominant Parkinsons disease (PD). Furthermore, α-synuclein protein is a major component of the Lewy bodies also in sporadic PD patients. Increased levels of wildtype α-synuclein in the cell leads to increased intracellular hydrogen peroxide levels and causes death of dopaminergic neurons in rat primary culture. Subsequently, oxidative stress has been directly linked with α-synuclein aggregation in vitro. This raises the question whether increased α-synuclein expression might be linked to higher susceptibility to PD and whether α-synuclein promoter polymorphisms are associated with PD. Here, two polymorphisms (−116C>G and −668T>C) of the α-synuclein promoter defining four haplotypes have been characterized in 315 German PD patients. The influence of the four haplotypes on gene expression was studied by CAT reporter gene assays in neuronal SK-N-AS cells. The −668C/−116G haplotype revealed significant higher CAT expression than the −668T/−116G or the −668T/−116C haplotype, respectively. Although the −668C/−116G haplotype was more common in PD patients, this difference was not significant.

N-terminal ataxin-3 causes neurological symptoms with inclusions, endoplasmic reticulum stress and ribosomal dislocation

Mutant ataxin-3 is aberrantly folded and proteolytically cleaved in spinocerebellar ataxia type 3. The C-terminal region of the protein includes a polyglutamine stretch that is expanded in spinocerebellar ataxia type 3. Here, we report on the analysis of an ataxin-3 mutant mouse that has been obtained by gene trap integration. The ataxin-3 fusion protein encompasses 259 N-terminal amino acids including the Josephin domain and an ubiquitin-interacting motif but lacks the C-terminus with the polyglutamine stretch, the valosin-containing protein binding region and part of the ubiquitin-interacting motif 2. Homozygous ataxin-3 mutant mice were viable and showed no apparent anatomical defects at birth. However, at the age of 9 months, homozygous and heterozygous mutant mice revealed significantly altered behaviour and progressing deficits of motor coordination followed by premature death at ∼12 months. At this time, prominent extranuclear protein aggregates and neuronal cell death was found in mutant mice. This was associated with disturbances of the endoplasmic reticulum-mediated unfolded protein response, consistent with the normal role of ataxin-3 in endoplasmic reticulum homeostasis. Thus, the ataxin-3 gene trap model provides evidence for a contribution of the non-polyglutamine containing ataxin-3 N-terminus, which mimics a calpain fragment that has been observed in spinocerebellar ataxia type 3. Consistent with the disease in humans, gene trap mice develop cytoplasmic inclusion bodies and implicate impaired unfolded protein response in the pathogenesis of spinocerebellar ataxia type 3.

Definite multiple system atrophy in a German family

Multiple system atrophy (MSA) is a presumed sporadic neurodegenerative disorder of unknown aetiology, clinically characterised by poorly levodopa responsive parkinsonism and/or cerebellar dysfunction in combination with autonomic failure.1 We previously identified a family with a typical MSA phenotype in two successive generations.2 One affected patient of that pedigree, now deceased at age 82, and postmortem examination of the brain revealed findings typical of definite MSA.3 Severe atrophy of the putamen (fig 1A) and depigmentation of the substantia nigra and pontine and cerebellar atrophy were found upon macroscopic …

A transgenic mouse model of spinocerebellar ataxia type 3 resembling late disease onset and gender-specific instability of CAG repeats

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is caused by the expansion of a polyglutamine repeat in the ataxin-3 protein. We generated a mouse model of SCA3 expressing ataxin-3 with 148 CAG repeats under the control of the huntingtin promoter, resulting in ubiquitous expression throughout the whole brain. The model resembles many features of the disease in humans, including a late onset of symptoms and CAG repeat instability in transmission to offspring. We observed a biphasic progression of the disease, with hyperactivity during the first months and decline of motor coordination after about 1 year of age; however, intranuclear aggregates were not visible at this age. Few and small intranuclear aggregates appeared first at the age of 18 months, further supporting the claim that neuronal dysfunction precedes the formation of intranuclear aggregates.

Structural modeling of ataxin-3 reveals distant homology to adaptins

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin‐3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure‐based functions for ataxin‐3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin‐3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin‐3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin‐3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin‐3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin‐3. Proteins 2003;50:355–370.

L‐dopa increases α‐synuclein DNA methylation in Parkinson's disease patients in vivo and in vitro

Increasing gene dosages of α‐synuclein induce familial Parkinsons disease (PD); thus, the hypothesis has been put forward that regulation of gene expression, in particular altered α‐synuclein gene methylation, might be associated with sporadic PD and could be used as a biological marker.

DNA methylation in Parkinson's disease.

Epigenetic processes control the embryonic development into multicellular organisms and determine the functional differences of genetically identical cells and individuals. They are also involved in a variety of complex functions such as learning and memory consolidation and have been implicated in aging processes. Beyond the actual genetic information encoded in the DNA sequence, epigenetic modifications in particular DNA methylation and various histone modifications shape the chromatin into a transcriptional permissive or repressive state. DNA methylation patterns are altered by environmental conditions and can be carried forward through mitosis and meiosis. Hence, DNA methylation probably mediates complex environment–gene interactions, determines individual disease characteristics, and contributes to effects and side effects of drugs. In addition to classic monogenic epigenetic diseases, i.e., Prader‐Willi and Rett syndrome, recent data point to an epigenetic component also in apparent sporadic neuro‐psychiatric disorders and increasing evidence suggests a role for altered DNA methylation in Parkinsons disease. Epigenetic alterations, DNA methylation in particular, may account for the yet unexplained individual susceptibility and the variability in the course of Parkinsons disease and could provide hints toward the development of novel therapeutic targets.