Spinocerebellar ataxia type 3 (SCA3)is an autosomal dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene resulting in an expanded polyglutamine repeat in the ataxin-3 protein. To study the course of the disease, we generated transgenic mice for SCA3 using full-length ataxin-3 constructs containing 15, 70, or 148 CAG repeats, respectively. Control mice (15 CAGs) were phenotypically normal and had no neuropathological findings. However, mice transgenic for ataxin-3 with expanded polyglutamine repeats were severely affected by a strong neurological phenotype with tremor, behavioral deficits, strongly reduced motor and exploratory activity, a hunchback, and premature death at 3 to 6 months of age. Neuropathological examination by immunohistochemical staining revealed ubiquitin-and ataxin-3-positive intranuclear inclusion bodies in a multitude of neurons. Directing ataxin-3 with 148 CAGs to the nucleus revealed an even more pronounced phenotype with more inclusions and earlier death, whereas mice transgenic with the same construct but attached to a nuclear export signal developed a milder phenotype with less inclusions. These studies indicate that nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3 in vivo.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common genetic cause of Parkinson's disease (PD). However, LRRK2 function and molecular mechanisms causing the parkinsonian phenotype remain widely unknown. Most of LRRK2 knockdown and overexpression models strengthen the relevance of LRRK2 in regulating neurite outgrowth. We have recently identified ARHGEF7 as the first guanine nucleotide exchange factor (GEF) of LRRK2. This GEF is influencing neurite outgrowth through regulation of actin polymerization. Here, we examined the expression profile of neuroblastoma cells with reduced LRRK2 and ARHGEF7 levels to identify additional partners of LRRK2 in this process. Tropomyosins (TPMs), and in particular TPM4, were the most interesting candidates next to other actin cytoskeleton regulating transcripts in this dataset. Subsequently, enhanced neurite branching was shown using primary hippocampal neurons of LRRK2 knockdown animals. Furthermore, we observed an enhanced number of growth cones per neuron and a mislocalization and dysregulation of ARHGEF7 and TPM4 in these neuronal compartments. Our results reveal a fascinating connection between the neurite outgrowth phenotype of LRRK2 models and the regulation of actin polymerization directing further investigations of LRRK2-related pathogenesis.
The protein leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene account for up to 10% of all autosomal dominant forms of familiar and for approximately 1-3% of sporadic PD patients. Although the LRRK2 protein has many functional domains like a leucine-rich repeat domain, a Roc-GTPase domain, a kinase domain of the tyrosine kinase-like subfamily and multiple protein interaction domains (armadillo, ankyrin, WD40), the exact biological role of LRRK2 in the human brain is elusive. To gain more insight into the biological function of this protein, we monitored the changes in the expression profiles of SH-SY5Y cells, a dopaminergic neuroblastoma cell line, induced by a depletion of LRRK2 levels by RNA interference (RNAi) with Affymetrix U133 Plus 2.0 microarrays. A total of 187 genes were differentially regulated by at least a 1.5-fold change with 94 transcripts being upregulated and 93 transcripts being downregulated compared to scrambled control siRNA transfected cells. Key players of the interaction networks were independently verified by qRT-PCR. The differentially expressed gene products are involved in axonal guidance, nervous system development, cell cycle, cell growth, cell differentiation, cell communication, MAPKKK cascade, and Ras protein signal transduction. Defined gene expression networks will now serve to look more closely for candidates affected by LRRK2 reduction and how they might be altered in other forms of familial or sporadic PD.
Objective To examine the positive predictive value (PPV) of cfDNA screening for sex chromosome aneuploidies (SCA) in a large series of over 90 000 patients. Methods Retrospective study based on samples that were sent to Cenata, a private laboratory which uses the Harmony Prenatal Test. The SCA high‐risk results were stratified according to the method of diagnostic testing and according to karyotype result. Results The study population consisted of 144 cases. The CfDNA test indicated monosomy X, XXX, XXY, and XYY in 62, 37, 40, and 5 cases, respectively. The overall PPV was 38.9% (30.9‐47.4), 29.0% (18.2‐42.9) for monosomy X, 29.7% (15.9‐47.9) for 47,XXX, 57.5% (40.9‐73.0) for 47,XXY, and 80.0% (28.4‐99.5) for 47,XYY). A total of 112 (77.8%) women with a high‐risk result for SCAs opted for prenatal karyotyping. In this group, there were significant differences in the PPV if the karyotype was assessed by amniocentesis or by CVS: 29.5% vs 50.0%. This significant difference was driven by the monosomy X result which shows a significantly higher PPV in CVS (54.6% (23.4‐83.3) vs 17.1% (6.6‐33.6)). For the other SCAs, the differences were not significant. Conclusion PPV of an abnormal cfDNA test for SCAs is low, particularly for monosomy X. The confirmation rate depends on the type of confirmatory test.
There are forensic inquiries in which an identification of epithelial cell types would provide important probative evidence. In cancer diagnosis, this information is yielded by histological examination of cytokeratin (Ck). Therefore, we tested 19 antibodies against different Cks (Ck1, Ck2e, Ck4, Ck5-6, Ck7, Ck8, Ck9, CK10, Ck13, Ck14, Ck15, Ck16, Ck17, Ck18, Ck19, Ck20, Ck903, PanCkAE1_3, and CAM5-2) on histological sections of epidermis, buccal mucosa, vaginal mucosa, penis, urogenital tract, and rectum and could identify two antigens unique to buccal-cell and vaginal-cell (Ck4) and skin epithelial-cell (Ck10) cytokeratin. Subsequently, we developed an immunocytological technique for distinguishing swabbed skin and mucosal cells up to at least 1 year after sampling. By the detection of the Ck4 and Ck10 mRNAs in biopsy and laser capture microdissection collected samples via quantitative real-time polymerase chain reaction, we were able to confirm our immunological findings. Hence, this study offers techniques to discriminate between skin and mucosal cells (buccal and vaginal) in forensic casework.
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