Altered Expression of Matrix Metalloproteinases and Their Endogenous Inhibitors in a Human Isogenic Stem Cell Model of Huntington's Disease.

Swati Naphade,Kuruwitage Lakshika Madushani,Karen L Ring,Alexander Embusch,Lisa M Ellerby

doi:10.3389/fnins.2017.00736

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by a progressive movement disorder, psychiatric symptoms, and cognitive impairments. HD is caused by a CAG repeat expansion encoding a stretch of polyglutamine residues in the N-terminus of mutant huntingtin (mHTT) protein. Proteolytic processing of mHTT yields toxic fragments, which cause neurotoxicity and massive neuronal cell death predominantly in the striatum and cortex. Inhibition of mHTT cleavage reduces neuronal toxicity suggesting mHTT proteolysis contributes to HD pathogenesis. A previously conducted unbiased siRNA screen in our lab for known human proteases identified matrix metalloproteinases (MMPs) as modifiers of mHTT proteolysis and toxicity. To further study MMP activation in HD, isogenic HD, and control corrected (C116) neural stem cells (NSCs) prepared from HD patient-derived induced pluripotent stem cells were used to examine the role of MMPs and their endogenous inhibitors in this highly relevant model system. We found altered expression of MMP-2 and MMP-9 (gelatinases), MMP-3/10, and MMP-14, activity in HD-NSCs when compared to control C116-NSCs. Dysregulation in MMP activity was accompanied with concomitant changes in levels of endogenous inhibitors of MMPs, called tissue inhibitors of matrix metalloproteinases (TIMPs). Specifically, we observed decreased levels of TIMP-1 and TIMP-2 in HD-NSCs, suggesting part of the altered expression and activity of MMPs is due to lower abundance of these endogenous inhibitors. Immunofluorescence analysis revealed increased MMP/TIMP localization in the nucleus or aggregates of HD-NSCs, suggesting potential interaction with mHTT. TIMP-1 was found to associate with mHTT aggregates in discrete punctate structures in HD-NSCs. These events collectively contribute to increased neurotoxicity in HD. Previous characterization of these NSCs revealed transforming growth factor beta (TGF-β) pathway as the top dysregulated pathway in HD. TGF-β was significantly upregulated in HD-NSCs and addition of TGF-β to HD-NSCs was found to be neuroprotective. To determine if TGF-β regulated MMP and TIMP activity, C116- and HD-NSCs were exogenously treated with recombinant TGF-β. TIMP-1 levels were found to be elevated in response to TGF-β treatment, representing a potential mechanism through which elevated TGF-β levels confer neuroprotection in HD. Studying the mechanism of action of MMPs and TIMPs, and their interactions with mHTT in human isogenic patient-derived NSCs elucidates new mechanisms of HD neurotoxicity and will likely provide novel therapeutics for treatment of HD.

Highlights

Characterized as a movement disorder, Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder with cognitive decline, chorea, and emotional disturbances as the disease progresses
matrix metalloproteinase (MMP)-3/10 and MMP-14 Levels Are Altered in HD Our previous studies showed elevated levels of MMP-14 and proteolytically processed active MMP-10 form in mouse striatal Hdh111Q/111Q cells compared to Hdh7Q/7Q cells (Miller et al, 2010)
Despite the reduced overall levels detected by western blot analysis (Figure 3A, 1.7-fold decrease) and room temperature (RT)-PCR analysis (Supplementary Figure 1C, 15.4-fold decrease), MMP-14 expression was found to be strongly nuclear in HD-neural stem cell (NSC) where it possibly associates with nuclear HTT immunoreactivity (Figure 3B)

Summary

Introduction

Characterized as a movement disorder, Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder with cognitive decline, chorea, and emotional disturbances as the disease progresses. MHTT is susceptible to proteolysis at the N-terminus with the resulting short N-terminal fragments contributing to cellular toxicity by inducing apoptotic cell death. In order to identify critical proteases that directly cleave mHTT, an unbiased western blot-based siRNA screen for 514 known human proteases was conducted (Miller et al, 2010). This screen confirmed 11 proteases that, when silenced, reduced toxic N-terminal HTT fragment formation. Three of these eleven modifiers of HTT proteolysis and toxicity belonged to the matrix metalloproteinase (MMP) family (MMP-10, -14, and -23B)

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Conclusion