Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5–10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular condition that afflicts as many as 1 of 350 males and 420 females over the age of 18 (1). In ALS, degeneration of upper and lower motor neurons causes progressive muscle paralysis and spasticity, affecting mobility, speech, swallowing, and respiration (2). Half of affected individuals die within 3 y, and less than 20% survive for more than 5 y (3); 90–95% of ALS cases are sporadic (SALS) in which some apparently facilitating gene mutations, such as repeat expansions in the gene that encodes ataxin-2 (4), have been identified. The remaining 5–10% of ALS cases are familial (FALS) and predominantly associated with Mendelian-inherited mutations in the genes encoding Cu/Zn superoxide dismutase (SOD1), TAR-DNA–binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), C9ORF72, and other genes (reviewed in ref. 3).Despite the profusion of functionally diverse genes implicated in FALS and SALS, clinical and pathological similarities between all forms of ALS suggest the existence of a common pathogenic pathway that could be united by a single gene/protein (5). One of the mechanisms by which a mutant or wild-type (WT) protein can dominate pathogenesis of phenotypically diverse diseases is by propagated protein misfolding, such as that underpinning the prion diseases, which has been increasingly implicated in other neurodegenerative and systemic disorders (6, 7). A role for propagated protein misfolding in ALS is supported by the prion-like spatiotemporal progression of disease through the neuroaxis (8, 9). However, given the disparity in protein inclusion pathology between subtypes of ALS, a single unifying prion-like protein that could explain such a progression remains obscure.Whereas it is generally accepted SOD1 is not found in large perikaryal cytoplasmic inclusions outside of SOD1 FALS cases, misfolded SOD1 has been increasingly identified in SALS and non-SOD1 FALS (5, 10, 11). Indeed, we have reported that misfolded human wild-type SOD1 (HuWtSOD1) can be detected by spinal cord immunohistochemistry (IHC) in FALS secondary to FUS mutation, and in SALS patients with cytosolic WT TDP-43 accumulation (11). Moreover, in cell models, overexpression of WTTDP-43, or expression of mutant FUS or TDP-43, is associated with HuWtSOD1 misfolding (11). Collectively, these data are consistent with SOD1 being a molecular common denominator for all types of ALS. Furthermore, prion-like activity has been described for the cell-to-cell transmission of misfolding of mutant SOD1 (12), and we have reported that mutant SOD1 can confer its misfold on HuWtSOD1 (13). However, mutant SOD1 cannot explain propagation in SALS.To test if HuWtSOD1 participates in cell-to-cell transmission of protein misfolding, we make use of previously developed mouse mAb probes for misfolded/oxidized SOD1, recognizing either full-length human mutant or WT SOD1, generated against regions that are antibody-inaccessible in natively folded SOD1 (13–15). Misfolded SOD1 mAbs used in this work are 10E11C11 and 3H1, directed against an unstructured electrostatic loop [disease-specific epitope-2 (DSE2)], and 10C12, directed against a C-terminal dimer interface peptide in which the cysteine at position 146 is substituted by a cysteic acid residue to mimic oxidation of this residue (DSE1a) (13). The use of such antibody probes have enabled us to unambiguously determine the role of misfolded mutant G127X in the induced misfolding of HuWtSOD1, which upon misfolding acquires a marked increase in sensitivity to protease digestion, consistent with global loosening of structure (13). The finding that misfolded endogenous HuWtSOD1 was observed long after transfected G127X-SOD1 was degraded suggested that HuWtSOD1, once misfolded, is capable of triggering an intracellular propagated misfolding reaction (13). We now report for the first time that misfolded HuWtSOD1 can transit cell to cell both via exosomes, and release of protein aggregates and subsequent uptake in neuronal cells. In addition, misfolded HuWtSOD1 can sustain intercellular propagated misfolding in vitro and is detectable in the spinal cord of all ALS patients tested, regardless of the genetic etiology of the disease. Collectively, these data indicate that HuWtSOD1 is competent to participate in propagated misfolding, suggesting a common pathogenic mechanism linking FALS and SALS.     

Careful examination of thyroid specimen intraoperatively to reduce incidence of inadvertent parathyroidectomy during thyroid surgery

OBJECTIVE: To assess the incidence and clinical relevance of inadvertent parathyroidectomy during thyroidectomy, and the possibility of reducing its occurrence. DESIGN: Retrospective study. SETTING: University hospital. PATIENTS: Consecutive patients who underwent thyroidectomy from 1999 to 2005, divided into 2 groups (group 1, those with inadvertent parathyroidectomy; and group 2, those without inadvertent parathyroidectomy). Patients who underwent surgical procedures for recurrent thyroid disease, intentional parathyroidectomy, and resection of central compartment viscera were excluded. INTERVENTIONS: All pathology reports were reviewed for the presence of any parathyroid tissue in the resected specimen. Age, sex, preoperative diagnosis, thyroid hormonal status, substernal thyroid extension, number of parathyroid glands identified and spared at the time of surgery, autotransplantation of parathyroid gland, and final histologic findings were recorded. MAIN OUTCOME MEASURES: Identification of parathyroid tissue in resected specimens and postoperative symptomatic hypocalcemia. RESULTS: A total of 307 patients were included. Surgical procedures included bilateral or unilateral thyroidectomy (95% and 5% of procedures, respectively). Central neck lymph node dissection was performed in 5% of cases. Pathologic findings showed inadvertent parathyroidectomy in 12% of cases. Of these, 32% were recognized intraoperatively. The parathyroid tissue was found in extracapsular locations in 37% of cases, intracapsular locations in 39%, and intrathyroidal locations in 24%. There was no statistical difference between the 2 groups in terms of sex, preoperative diagnosis, substernal extension, extent of surgery, pathologic diagnosis, and occurrence of postoperative hypocalcemia, except for the presence of thyroiditis. CONCLUSION: Careful examination of the surgical specimen intraoperatively decreases the incidence of inadvertent parathyroidectomy during thyroidectomy.     

Extracellular aggregated Cu/Zn superoxide dismutase activates microglia to give a cytotoxic phenotype

A large body of literature suggests that amyotrophic lateral sclerosis (ALS) pathology is intimately linked with neuroinflammation, specifically activation and recruitment of microglia and astrocytes. The actual cause of gliosis is unclear. Extracellular Cu/Zn superoxide dismutase (SOD1) has recently been shown to activate microglia in a CD14 dependant mechanism providing one potential pathway by which glial cells become activated. As protein inclusions are thought to be an important part of ALS pathology and are associated with all forms of ALS, we sought to determine if aggregated SOD1 would activate microglia. Recombinant SOD1 was aggregated and this, or monomeric forms of SOD1 were then added to EOC.13 microglial cells or primary microglial cells in culture. Although monomeric mutant SOD1 has been shown to promote microglial activation in the past, we found that aggregated SOD1 was able to much more efficiently activate microglia in culture when compared with the unaggregated form of mutant SOD1. In addition to CD14 dependant pathways, aggregated SOD1 also bound to the surface of glial cells and was internalized in a lipid raft and scavenger receptor dependent manner. We have for the first time shown that aggregated mutant SOD1 potently activates microglia. These results suggest that there may be a potential link between protein aggregation and microglial activation in ALS. © 2012 Wiley Periodicals, Inc.