Proteomic analysis identifies NPTX1 and HIP1R as potential targets of histone deacetylase-3-mediated neurodegeneration

Abstract

A defining feature of neurodegenerative diseases is the abnormal and excessive loss of neurons. One molecule that is particularly important in promoting neuronal death in a variety of cell culture and in vivo models of neurodegeneration is histone deacetylase-3 (HDAC3), a member of the histone deacetylase family of proteins. As a step towards understanding how HDAC3 promotes neuronal death, we conducted a proteomic screen aimed at identifying proteins that were regulated by HDAC3. HDAC3 was overexpressed in cultured rat cerebellar granule neurons (CGNs) and protein lysates were analyzed by mass spectrometry. Of over 3000 proteins identified in the screen, only 21 proteins displayed a significant alteration in expression. Of these, 12 proteins were downregulated whereas 9 proteins were upregulated. The altered expression of five of these proteins, TEX10, NPTX1, TFG, TSC1, and NFL, along with another protein that was downregulated in the proteomic screen, HIP1R, was confirmed using Western blots and commercially available antibodies. Because antibodies were not available for some of the proteins and since HDAC3 is a transcriptional regulator of gene expression, we conducted RT-PCR analysis to confirm expression changes. In separate analyses, we also included other proteins that are known to regulate neurodegeneration, including HDAC9, HSF1, huntingtin, GAPDH, FUS, and p65/RELA. Based on our proteomic screen and candidate protein approach, we identify three genes, Nptx1, Hip1r, and Hdac9, all known to regulate neurodegeneration that are robustly regulated by HDAC3. Given their suggested roles in regulating neuronal death, these genes are likely to be involved in regulating HDAC3-mediated neurotoxicity.

Impact statement

Neurodegenerative diseases are a major medical, social, and economic problem. Recent studies by several laboratories have indicated that histone deacetylase-3 (HDAC3) plays a key role in promoting neuronal death. But the downstream mediators of HDAC3 neurotoxicity have yet to be identified. We conducted a proteomic screen to identify HDAC3 targets the results of which have been described in this report. Briefly, we identify Nptx1, Hip1r, and Hdac9 as genes whose expression is altered by HDAC3. Investigating how these genes are involved in HDAC3 neurotoxicity could shed valuable insight into neurodegenerative disease and identify molecules that can be targeted to treat these devastating disorders.