An anti-CAPN5 intracellular antibody acts as an inhibitor of CAPN5-mediated neuronal degeneration.

Yan Wang,Zongming Song,Xiao Zhang,Feng Gu

doi:10.18632/oncotarget.22221

Abstract

CAPN5 has been linked to autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). Activation of CAPN5 may increase proteolysis and degradation of a wide range of substrates to induce degeneration in the retina and the nerve system. Thus, we developed an inhibitory intracellular single chain variable fragment (scFv) against CAPN5 as a potential way to rescue degeneration in ADNIV disease or in neuronal degeneration. We report that overexpression CAPN5 increases the levels of the auto-inflammatory factors toll like receptor 4 (TLR4), interleukin 1 alpha (IL1alpha), tumor necrosis factor alpha (TNFalpha) and activated caspase 3 in 661W photoreceptor-like cells and SHSY5Y neuronal-like cells. Both C4 and C8 scFvs specifically recognize human/mouse CAPN5 in 661W cells and SHSY5Y cells, moreover, both the C4 and C8 scFvs protected cells from CAPN5-induced apoptosis by reducing the levels of activated caspase 3 and caspase 9. The cellular expression C4 scFv reduced levels of the pro-inflammatory factor IL1-alpha activated caspase 3 in cells after CAPN5 overexpression. We suggest that CAPN5 expression has important functional consequences in auto-inflammatory processes, and apoptosis in photoreceptor like cells and neural-like cells. Importantly, the specific intracellular targeting of antibody fragments blocking activation of CAPN5 act as inhibitors of CAPN5 functions in neural like cells, thus, our data provides a novel potential tool for therapy in CAPN5-mediated ADNIV or neurodegenerative diseases.

Highlights

calpain 5calcium-activated cysteine protease (CAPN5) encodes calpain-5, a member of the calcium-activated cysteine protease family [1, 2]
We report that overexpression CAPN5 increases the levels of the auto-inflammatory factors toll like receptor 4 (TLR4), interleukin 1 alpha (IL1alpha), tumor necrosis factor alpha (TNFalpha) and activated caspase 3 in 661W photoreceptor-like cells and SHSY5Y neuronal-like cells
After 60 hours post-transfection, both the CAPN5 and CAPN5 mutant R289W vectors transfection increased the mRNA levels of TLR4/6, IL1alpha and TNFalpha when compared to empty vector transfection, and this was especially pronounced for the mutant CAPN5 R289W expression which increased both caspase 3 activation and IL1alpha levels when compared to CAPN5 wt transfection in both 661W and SHSY5Y Cell lines (Figure 1C, 1D)

Summary

Introduction

CAPN5 encodes calpain-5, a member of the calcium-activated cysteine protease family [1, 2]. CAPN5 has been associated with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) [3,4,5,6], obesity [7], Huntington’s disease [8, 9], and polycystic ovary syndrome [10]. Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function [13]. Pathologic activation of calpain induces the cleavage of substrates that negatively affect neuronal structure and function, leading to inhibition of essential neuronal survival mechanisms [15]. Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an inherited autoimmune uveitis and vitreoretinal degeneration [26]. ADNIV is caused by mutations of the CAPN5 gene which leads to photoreceptor degeneration, autoimmune uveitis, and retinal neovascularization. It has been found that mutations of CAPN5 activated CAPN5 protein that generates the various pathological features involved in blindness and could be therapeutically relevant [27, 28]

Methods

Results

Conclusion