TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

Edward Pokrishevsky,Neil R Cashman,Leslie I Grad

doi:10.1038/srep22155

Abstract

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.

Highlights

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TAR-DNA binding protein 43 (TDP-43), fused in sarcoma (FUS), C9ORF72, or can occur sporadically without recognized genetic mutations
Our results show that primary spinal cord cultures incubated for 20 h with neural growth media containing the pellet fraction of HEK293-conditioned media from FUSR495x, FUSP525L, wtTDP-43 and TDP-43ΔNLS, but not empty vector control or wtFUS transfected cells, display significant neurite immunoreactivity for misfolded SOD1 by immunocytochemistry (Fig. 1), despite similar transfection efficiency of the various constructs in HEK293 cells (Supplementary Fig. 2)
Background staining observed in primary cultures incubated with conditioned media from empty vector or wtFUS transfected cells could be attributed to newly translated HuWtSOD1, which may take hours to properly fold, become metalated, disulphide-oxidized and dimerized[24]

Summary

Introduction

Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. We used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Multiple studies have detected misfolded forms of human wild-type SOD1 (HuWtSOD1) protein in SALS and FALS in the absence of SOD1 mutations[3,4,5], suggesting that non-native conformers of SOD1 may play a key pathological role in all cases of ALS. We asked whether TDP-43 or FUS-induced misfolded HuWtSOD1 acquires the prion-like property of seeding HuWtSOD1 propagated misfolding that can be passaged from cell culture to cell culture via conditioned media

Methods

Results

Conclusion