The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum.

The Moonwalker (Mwk) mouse is a model of dominantly inherited cerebellar ataxia caused by a gain-of-function mutation in the transient receptor potential (TRP) channel TRPC3. Here, we report impairments in dendritic growth and synapse formation early on during Purkinje cell development in the Mwk cerebellum that are accompanied by alterations in calcium signaling. To elucidate the molecular effector pathways that regulate Purkinje cell dendritic arborization downstream of mutant TRPC3, we employed transcriptomic analysis of developing Purkinje cells isolated by laser-capture microdissection. We identified significant gene and protein expression changes in molecules involved in lipid metabolism. Consistently, lipid homeostasis in the Mwk cerebellum was found to be disturbed, and treatment of organotypic cerebellar slices with ceramide significantly improved dendritic outgrowth of Mwk Purkinje cells. These findings provide the first mechanistic insights into the TRPC3-dependent mechanisms, by which activated calcium signaling is coupled to lipid metabolism and the regulation of Purkinje cell development in the Mwk cerebellum.

Contactin-associated protein-2 antibodies in non-paraneoplastic cerebellar ataxia.

BACKGROUND: Relatively few studies have searched for potentially pathogenic antibodies in non-paraneoplastic patients with cerebellar ataxia. METHODS AND RESULTS: We first screened sera from 52 idiopathic ataxia patients for binding of serum IgG antibodies to cerebellar neurons. One strong-binding serum was selected for immunoprecipitation and mass spectrometry, which resulted in the identification of contactin-associated protein 2 (CASPR2) as a major antigen. CASPR2 antibodies were then found by a cell-based assay in 9/88 (10%) ataxia patients, compared to 3/144 (2%) multiple sclerosis or dementia controls (p=0.011). CASPR2 is strongly expressed in the cerebellum, only partly in association with voltage-gated potassium channels. CONCLUSIONS: Prospective studies are now needed to see whether identification of CASPR2 antibodies has relevance for the diagnosis and treatment of idiopathic cerebellar ataxia.

Candidate screening of the TRPC3 gene in cerebellar ataxia.

The hereditary cerebellar ataxias are a diverse group of neurodegenerative disorders primarily characterised by loss of balance and coordination due to dysfunction of the cerebellum and its associated pathways. Although many genetic mutations causing inherited cerebellar ataxia have been identified, a significant percentage of patients remain whose cause is unknown. The transient receptor potential (TRP) family member TRPC3 is a non-selective cation channel linked to key signalling pathways that are affected in cerebellar ataxia. Furthermore, genetic mouse models of TRPC3 dysfunction display cerebellar ataxia, making the TRPC3 gene an excellent candidate for screening ataxic patients with unknown genetic aetiology. Here, we report a genetic screen for TRPC3 mutations in a cohort of 98 patients with genetically undefined late-onset cerebellar ataxia and further ten patients with undefined episodic ataxia. We identified a number of variants but no causative mutations in TRPC3. Our findings suggest that mutations in TRPC3 do not significantly contribute to the cause of late-onset and episodic human cerebellar ataxias.

A gene expression signature in developing Purkinje cells predicts autism and intellectual disability co-morbidity status.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disease whose underpinning molecular mechanisms and neural substrates are subject to intense scrutiny. Interestingly, the cerebellum has emerged as one of the key brain regions affected in ASD. However, the genetic and molecular mechanisms that link the cerebellum to ASD, particularly during development, remain poorly understood. To gain insight into the genetic and molecular mechanisms that might link the cerebellum to ASD, we analysed the transcriptome dynamics of a developing cell population highly enriched for Purkinje cells of the mouse cerebellum across multiple timepoints. We identified a single cluster of genes whose expression is positively correlated with development and which is enriched for genes associated with ASD. This ASD-associated gene cluster was specific to developing Purkinje cells and not detected in the mouse neocortex during the same developmental period, in which we identified a distinct temporally regulated ASD gene module. Furthermore, the composition of ASD risk genes within the two distinct clusters was significantly different in their association with intellectual disability (ID), consistent with the existence of genetically and spatiotemporally distinct endophenotypes of ASD. Together, our findings define a specific cluster of ASD genes that is enriched in developing PCs and predicts co-morbidity status.

Affinity peptidomics: Peptide selection and affinity capture on hydrogels and microarrays.

Affinity peptidomics relies on the successfully proven approach used widely in mass-spectrometry-based protein analysis, where protein samples are proteolytically digested prior to the analysis. Unlike traditional proteomic analyses, affinity peptidomics employs affinity detection instead of, or in addition to, the mass-spectrometry detection. Affinity peptidomics, therefore, bridges the gap between protein microarrays and mass spectrometry and can be used for the detection, identification and quantification of endogenous or proteolytic peptides on microarrays and by MALDI-MS. Phage display technology is a widely applicable generic molecular display method suitable for studying protein-protein or protein-peptide interactions and the development of recombinant affinity reagents. Phage display complements the affinity peptidomics approach when the latter is used, e.g. to characterise a repertoire of antigenic determinants of polyclonal, monoclonal antibodies or other recombinantly obtained affinity reagents or in studying protein-protein interactions. 3D materials such as membrane-based porous substrates and acrylamide hydrogels provide convenient alternatives and are superior to many 2D surfaces in maintaining protein conformation and minimising non-specific interactions. Hydrogels have been found to be advantageous in performing antibody affinity assays and peptide-binding assays. Here we report a range of peptide selection and peptide-binding assays used for the detection, quantification or validation of peptide targets using array-based techniques and fluorescent or MS detection.