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Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1) G93A , revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1 G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1 G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1 G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source., (© 2019 Silverman et al.)

We introduce a global, collective coordinate bias into molecular dynamics simulations that partially unfolds a protein, in order to predict misfolding-specific epitopes based on the regions that locally unfold. Several metrics are used to measure local disorder, including solvent exposed surface area (SASA), native contacts ( Q), and root mean squared fluctuations (RMSF). The method is applied to Cu, Zn superoxide dismutase (SOD1). For this protein, the processes of monomerization, metal loss, and conformational unfolding due to microenvironmental stresses are all separately taken into account. Several misfolding-specific epitopes are predicted, and consensus epitopes are calculated. These predicted epitopes are consistent with the "lower-resolution" peptide sequences used to raise disease-specific antibodies, but the epitopes derived from collective coordinates contain shorter, more refined sequences for the key residues constituting the epitope.

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well-characterized. Here, we characterized three recently introduced antimisfolded SOD1 antibodies and compared them with two commercial, antimisfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new antimisfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, antimisfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming prefibrillar SOD1 oligomers.

The prion hypothesis has extended to the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS), as a means to explain the spatiotemporal spread of pathology from one or more focal points through the neuroaxis. About 20% of inheritable cases of ALS are due to mutation in the gene encoding the Cu/Zn superoxide dismutase (SOD1), causing the protein to misfold and form neurotoxic aggregates. Mutant SOD1 has been shown to impart its misfold onto natively folded wild-type SOD1 in living cells. Furthermore, misfolded wild-type SOD1 can itself induce further rounds of propagated SOD1 misfolding. Finally, this prion-like mechanism of propagated SOD1 misfolding can be transmitted from cell to cell in human cell culture. Here, we describe a protocol for the induction of wild-type SOD1 misfolding inside living cells and its subsequent transmission from cell to cell in a prion-like fashion.

Approximately 20 % of familial Amyotrophic Lateral Sclerosis (ALS) is caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several conformation-restricted antibodies have been generated that specifically recognize misfolded SOD1 protein, and have been used as therapeutics in pre-clinical models. Misfolded SOD1 selectively associates with spinal cord mitochondria in SOD1 rodent models. Using the SOD1(G93A) rat model, we find that SOD1 conformational specific antibodies AMF7-63 and DSE2-3H1 labeled a fibrillar network concentrated in the anterior horn; while A5C3, B8H10, C4F6 and D3H5 labeled motor neurons as well as puncta in the neuropil. There is a time-dependent accumulation of misfolded SOD1 at the surface of spinal cord mitochondria with AMF7-63-labeled mitochondria having increased volume in contrast to a mitochondrial subset labeled with B8H10. In spinal cord homogenates and isolated mitochondria, AMF7-63, DSE2-3H1 and B8H10 detect misfolded SOD1 aggregates. SOD1 that lacks its metal cofactors has an increased affinity for naïve mitochondria and misfolded SOD1 antibodies B8H10 and DSE2-3H1 readily detect demetalated mutant and wild-type SOD1. Together, these data suggest that multiple non-native species of misfolded SOD1 may exist, some of which are associated with mitochondrial damage. Conformational antibodies are invaluable tools to identify and characterize the variation in misfolded SOD1 species with regards to biochemical characteristics and toxicity. This information is highly relevant to the further development of these reagents as therapeutics.

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.

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), a fatal adult-onset degenerative neuromuscular disorder with a poorly defined etiology, progresses in an orderly spatiotemporal manner from one or more foci within the nervous system, reminiscent of prion disease pathology. We have previously shown that misfolded mutant Cu/Zn superoxide dismutase (SOD1), mutation of which is associated with a subset of ALS cases, can induce endogenous wild-type SOD1 misfolding in the intracellular environment in a templating fashion similar to that of misfolded prion protein. Our recent observations further extend the prion paradigm of pathological SOD1 to help explain the intercellular transmission of disease along the neuroaxis. It has been 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 released to the extracellular environment on the surface of exosomes secreted from living cells. Furthermore, once propagation of misfolded wild-type SOD1 has been initiated in human cell culture, it continues over multiple passages of transfer and cell growth. Propagation and transmission of misfolded wild-type SOD1 is therefore a potential mechanism in the systematic progression of ALS pathology.

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 of misfolded SOD1 with cytosolic TDP43 or FUS suggests an indirect induction of SOD1 misfolding followed by propagation through template directed misfolding beyond its site of inception. The identification of a final common pathway in the molecular pathogenesis of ALS provides a treatment target for this devastating disease.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and β-strand 3 of the SOD1 β-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.

Prion diseases occur when the normally α-helical prion protein (PrP) converts to a pathological β-structured state with prion infectivity (PrP(Sc)). Exposure to PrP(Sc) from other mammals can catalyze this conversion. Evidence from experimental and accidental transmission of prions suggests that mammals vary in their prion disease susceptibility: Hamsters and mice show relatively high susceptibility, whereas rabbits, horses, and dogs show low susceptibility. Using a novel approach to quantify conformational states of PrP by circular dichroism (CD), we find that prion susceptibility tracks with the intrinsic propensity of mammalian PrP to convert from the native, α-helical state to a cytotoxic β-structured state, which exists in a monomer-octamer equilibrium. It has been controversial whether β-structured monomers exist at acidic pH; sedimentation equilibrium and dual-wavelength CD evidence is presented for an equilibrium between a β-structured monomer and octamer in some acidic pH conditions. Our X-ray crystallographic structure of rabbit PrP has identified a key helix-capping motif implicated in the low prion disease susceptibility of rabbits. Removal of this capping motif increases the β-structure folding propensity of rabbit PrP to match that of PrP from mouse, a species more susceptible to prion disease.

Using a recently developed mesoscopic theory of protein dielectrics, we have calculated the salt bridge energies, total residue electrostatic potential energies, and transfer energies into a low dielectric amyloid-like phase for 12 species and mutants of the prion protein. Salt bridges and self energies play key roles in stabilizing secondary and tertiary structural elements of the prion protein. The total electrostatic potential energy of each residue was found to be invariably stabilizing. Residues frequently found to be mutated in familial prion disease were among those with the largest electrostatic energies. The large barrier to charged group desolvation imposes regional constraints on involvement of the prion protein in an amyloid aggregate, resulting in an electrostatic amyloid recruitment profile that favours regions of sequence between alpha helix 1 and beta strand 2, the middles of helices 2 and 3, and the region N-terminal to alpha helix 1. We found that the stabilization due to salt bridges is minimal among the proteins studied for disease-susceptible human mutants of prion protein.

Mutations in copper/zinc superoxide dismutase (SOD1) are causative for dominantly inherited amyotrophic lateral sclerosis (ALS). Despite high variability in biochemical properties among the disease-causing mutants, a proportion of both dismutase-active and -inactive mutants are stably bound to spinal cord mitochondria. This mitochondrial proportion floats with mitochondria rather than sedimenting to the much higher density of protein, thus eliminating coincidental cosedimentation of protein aggregates with mitochondria. Half of dismutase-active and approximately 90% of dismutase-inactive mutant SOD1 is bound to mitochondrial membranes in an alkali- and salt-resistant manner. Sensitivity to proteolysis and immunoprecipitation with an antibody specific for misfolded SOD1 demonstrate that in all mutant SOD1 models, misfolded SOD1 is deposited onto the cytoplasmic face of the outer mitochondrial membrane, increasing antigenic accessibility of the normally structured electrostatic loop. Misfolded mutant SOD1 binding is both restricted to spinal cord and selective for mitochondrial membranes, implicating exposure to mitochondria of a misfolded mutant SOD1 conformer mediated by a unique, tissue-selective composition of cytoplasmic chaperones, components unique to the cytoplasmic face of spinal mitochondria to which misfolded SOD1 binds, or misfolded SOD1 conformers unique to spinal cord that have a selective affinity for mitochondrial membranes.

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.

Conformational conversion of proteins in disease is likely to be accompanied by molecular surface exposure of previously sequestered amino-acid side chains. We found that induction of beta-sheet structures in recombinant prion proteins is associated with increased solvent accessibility of tyrosine. Antibodies directed against the prion protein repeat motif, tyrosine-tyrosine-arginine, recognize the pathological isoform of the prion protein but not the normal cellular isoform, as assessed by immunoprecipitation, plate capture immunoassay and flow cytometry. Antibody binding to the pathological epitope is saturable and specific, and can be created in vitro by partial denaturation of normal brain prion protein. Conformation-selective exposure of Tyr-Tyr-Arg provides a probe for the distribution and structure of pathologically misfolded prion protein, and may lead to new diagnostics and therapeutics for prion diseases.

Chronic wasting disease (CWD) is a neurodegenerative, protein misfolding disease affecting cervids in North America in epidemic proportions. While the existence of CWD has been known for more than 40 years, risk management efforts to date have not been able to curtail the spread of this condition. An expert elicitation exercise was carried out in May 2011 to obtain the views of international experts on both the etiology of CWD and possible CWD risk management strategies. This study presents the results of the following three components of the elicitation exercise: (1) expert views of the most likely scenarios for the evolution of the CWD among cervid populations in Canada, (2) ranking analyses of the importance of direct and indirect transmission routes, and (3) rating analyses of CWD control measures in farmed and wild cervids. The implications of these findings for the development of CWD risk management strategies are described in a Canadian context. [ABSTRACT FROM AUTHOR]

Protein misfolding diseases have been classically understood as diffuse errors in protein folding, with misfolded protein arising autonomously throughout a tissue due to a pathologic stressor. The field of prion science has provided an alternative mechanism whereby a seed of pathologically misfolded protein, arising exogenously or through a rare endogenous structural fluctuation, yields a template to catalyze misfolding of the native protein. The misfolded protein may then spread intercellularly to communicate the misfold to adjacent areas and ultimately infect a whole tissue. Mounting evidence implicates a prion-like process in the propagation of several neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis, and the tauopathies. However, the parallels between the events observed in these conditions and those in prion disease are often incomplete. The aim of this review was to examine the current state of knowledge concerning the mechanisms of protein misfolding and aggregation for neurodegeneration-associated proteins. In addition, possible methods of intercellular spread are described that focus on the hypothesis that released microvesicles function as misfolded protein delivery vehicles, and the therapeutic options enabled by viewing these diseases from the prion perspective. [ABSTRACT FROM AUTHOR]

Despite extensive investigation, many features of prion protein misfolding remain enigmatic. Physicochemical variables known to influence misfolding are reviewed to help elucidate the mechanism of prionogenesis and identify salient features of PrPSc, the misfolded conformer of the prion protein. Prospective work on refinement of candidate PrPSc models based on thermodynamic considerations will help to complete atomic-scale structural details missing from experimental studies and may explain the basis for the templating activity of PrPSc in disease. [ABSTRACT FROM AUTHOR]

The authors discuss some neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, which are considered as proteinopathies that are caused by pathologic misfolding and aggregation of disease-specific proteins. They explore the prion diseases, including Creutzfeldt-Jakob disease (CJD), that were long considered the only proteinopathies transmissible between individuals by an infection-like mechanism.

The transmissible spongiform encephalopathies could represent a new mode of transmission for infectious diseases-a process more akin to crystallization than to microbial replication. The prion hypothesis proposes that the normal isoform of the prion protein is converted to a disease-specific species by template-directed misfolding. Therapeutic and prophylactic strategies to combat these diseases have emerged from immunological and chemotherapeutic approaches. The lessons learned in treating prion disease will almost certainly have an impact on other diseases that are characterized by the pathological accumulation of misfolded proteins. [ABSTRACT FROM AUTHOR]