Abstract
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.
Highlights
Spinocerebellar ataxia type 3 (SCA3), known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited ataxia which is characterized by deficits in gait, movement, and coordination linked to a CAG repeat expansion in the ATXN3 gene and a concordant polyglutamine expansion in the ataxin-3 protein
In our work, we used Reverse-phase Protein MicroArray (RPMA) analysis to characterize the altered signaling networks observed upon ataxin-3 expansion in both in vitro and in vivo models
We suggest that elucidating these signaling cascades is integral in understanding the function of ataxin-3 in health and disease
Summary
Spinocerebellar ataxia type 3 (SCA3), known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited ataxia which is characterized by deficits in gait, movement, and coordination linked to a CAG repeat expansion in the ATXN3 gene and a concordant polyglutamine expansion in the ataxin-3 protein. Sowa et al Mol Brain (2021) 14:57 post-translational modifications. Reversible protein phosphorylation, especially on serine, threonine or tyrosine residues, is one of the most important and well-studied post-translational regulating protein function and signal transmission [5]. Phosphorylated proteins are notoriously difficult to analyze on a mass scale by traditional methods such as Western blot especially when small amounts of samples need to be tested with high sensitivity. To that end we utilized the Reverse-phase Protein MicroArray (RPMA) technology, a sensitive, quantitative, and high-throughput immunoassay to analyze protein and post-translational modification at the level of total protein abundance or specific phosphorylation. The amount of bound antibody is quantitated using a highly sensitive colorimetric or fluorometric procedure [60]
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