Aberrant Localization of FUS and TDP43 Is Associated with Misfolding of SOD1 in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1. Methodology/Principal Findings: Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43. Conclusion/Significance: We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization o

Disease mechanisms in ALS: misfolded SOD1 transferred through exosome-dependent and exosome-independent pathways

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1

Transfection of mutant FUS is associated with SOD1 misfolding by immunocytochemistry.

<p>Human neuroblastoma SH-SY5Y cells and primary neural cultures expressing human wtSOD1 were stained for HA-Tag (red), misfolded SOD1 (green), and Hoechst33342 nuclear counterstain (blue). (<b>A</b>, <b>B</b>) Human wild-type FUS localizes in the nucleus and no misfolded SOD1 is detected. (<b>C</b>–<b>F</b>) Both of the truncated variant, R495x-FUS (<b>B</b>, <b>C</b>), and point mutation variant, P525L-FUS (<b>E</b>, <b>F</b>), localize in the cytosol and are associated with misfolding of SOD1 in the same cells, as detected by the immunocytochemistry with the 3H1 SOD1 misfolding-specific mAb. Exogenous FUS was detected using the N-terminal HA-tag. Arrows point to transfected cells. Scale bar, 20µm.</p

Misfolded SOD1 immunohistochemistry in ventral grey matter and corticospinal tracts of cervical spinal cord of ALS patients.

<p>(<b>A</b>) Cases of FALS with SOD1 mutations show extensive accumulation of misfolded SOD1 in swollen and normal sized axons as well as in the perikarya of some lower motor neurons (inset). (<b>B</b>) Cases of SALS with TDP43 pathology had misfolded SOD1 accumulation only in small numbers of normal sized axons (arrows). (<b>C</b>, <b>D</b>) In the case of FALS with FUS mutation misfolded SOD1 accumulated in some swollen axons in the ventral grey matter (<b>C</b>, arrows) and a moderate number of normal sized axons in corticospinal tract (<b>D</b>, arrows). Scale bar, 60 µm (<b>A</b>, <b>C</b>); 30 µm (<b>B</b>, <b>D</b>).</p

Detection of misfolded SOD1 in various structures and regions by immunohistochemistry.

<p>Relative abundance of DSE antibody (10E11C11) staining in various regions and structures of patient spinal cord sections (N = 5 normal control; N = 5 SOD1-FALS; N = 3 non-SOD1 FALS including R521C-FUS; N = 20 non-SOD1 SALS). Neuronal cytoplasmic inclusions, axonal swellings and axons are sub-structures within the ventral grey matter. Positive staining in corticospinal tract, other tracts and motor roots was in axons. –, no staining; +, some staining; ++, moderate staining; +++, abundant staining.</p

SOD1 misfolding in wild-type and ΔNLS-TDP43-transfected SH-SY5Y cells and human wtSOD1 expressing primary neural cells.

<p>SH-SY5Y cells and primary neural cells stained against HA-tag (transfected TDP43; red), misfolded SOD1 (green), and Hoechst33342 (blue) nuclear counterstain. Human wtTDP43 predominantly localizes in the nucleus (<b>A</b>, <b>B</b>) while the mutant ΔNLS-TDP43 localizes in the cytosol (<b>C</b>, <b>D</b>). Both variants of TDP43 are associated with misfolding of the endogenous SOD1 in the same cells, as detected by 3H1 immunoreactivity. Arrows point to transfected cells. Scale bar, 20µm.</p

Expression of transfected wild-type and mutant FUS and TDP43 in SH-SY5Y cells.

<p>The immunoblot on the left was probed with antibody specific to HA-tag, detecting only the exogenously expressed proteins in these samples. The empty vector (ev; pCINeo) control does not display detectable immunoreactivity with the HA-tag antibody, while both exogenous FUS and TDP43 are detected as 43 kDa and 72 kDa bands, respectively. Top and bottom portions of the immunoblot on the right were probed using FUS and TDP43 specific antibodies, respectively, detecting both the endogenous and exogenous proteins. Endogenous FUS and TDP43 are detected in all samples; however, stronger signals, corresponding to the over-expressed protein, are detected in the transfected samples.</p

Quantitative immunoprecipitation of misfolded human SOD1 in SH-SY5Y cells transfected with wild-type or mutant FUS and TDP43.

<p>(<b>A</b>) Representative immunoblots of immunoprecipitations. SOD1 proteins from transfected (48 h) and untransfected SH-SY5Y cell lysates were precipitated using pan-SOD1 antibody, SOD100 (rabbit polyclonal), and SOD1 misfolding-specific mouse monoclonal antibodies, 3H1 and 10C12. rIgG was used as isotype control for SOD100, and mIgG2a was used as isotype control for the DSE antibodies. Blots were probed with pan-SOD1 antibody. Framed bands show FUS, and TDP43 transfection efficiency of the indicated construct, as was detected by probing with antibody to HA-tag. (<b>B</b>–<b>E</b>) Show percentage of immunoprecipitable misfolded SOD1 (out of the total precipitable SOD1) using 3H1 (<b>B</b>, <b>D</b>) and 10C12 (<b>C</b>, <b>E</b>) from lysates of transfected SH-SY5Y cell cultures. Appreciable differences are indicated (*, <i>p</i><0.01). N = 5 for each of wtFUS, R495x- and P525L-FUS. N = 6 for each of ev, wtTDP43 and ΔNLS-TDP43. Error bars represent s.e.m.</p