Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1.

Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilizing to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance (VUS) to a clinically actionable likely pathogenic variant.

Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

RNA-binding properties orchestrate TDP-43 homeostasis through condensate formation in vivo.

Insoluble cytoplasmic aggregate formation of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases including Amyotrophic Lateral Sclerosis. TDP-43 localizes predominantly in the nucleus, arranging itself into dynamic condensates through liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, contributing to the transition of liquid-like biomolecular condensates into solid-like aggregates. However, to date it has been a challenge to study the dynamics of this process in vivo. We demonstrate through live imaging that human TDP-43 undergoes nuclear condensation in spinal motor neurons in a living animal. RNA-binding deficiencies as well as post-translational modifications can lead to aberrant condensation and altered TDP-43 compartmentalization. Single-molecule tracking revealed an altered mobility profile for RNA-binding deficient TDP-43. Overall, these results provide a critically needed in vivo characterization of TDP-43 condensation, demonstrate phase separation as an important regulatory mechanism of TDP-43 accessibility, and identify a molecular mechanism of how functional TDP-43 can be regulated.

Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

Protocol for the generation and automated confocal imaging of whole multi-cellular tumor spheroids.

Multi-cellular tumor spheroids (MCTS) have found widespread use in pre-clinical research. However, their complex three-dimensional structure makes immunofluorescent staining and imaging challenging. Here, we present a protocol for whole spheroid staining and automated imaging using laser-scanning confocal microscopy. We describe steps for cell culture, seeding of spheroids and transfer of MCTS, and adhesion to Ibidi chamber slides. We then detail fixation, immunofluorescent staining based on optimized reagent concentrations and incubation times, and confocal imaging facilitated by glycerol-based optical clearing.

ALS-linked CCNF variant disrupts motor neuron ubiquitin homeostasis.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell-derived motor neurons harboring the CCNF  S621G mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.

Assessment of protein inclusions in cultured cells using automated image analysis.

Proteinaceous inclusions are associated with neurodegenerative diseases and cell models are often used to determine genetic and chemical modifiers of their formation. This protocol involves the usage of automated microscopy and machine learning-based image analysis to accurately quantify the levels of protein inclusion formation in cultured cells from fluorescence microscopy images. This protocol is highly scalable and can be applied to a few images or large datasets. For complete details on the use and execution of this protocol, please refer to McAlary et al. (2022).

Cells Overexpressing ALS-Associated SOD1 Variants Are Differentially Susceptible to CuATSM-Associated Toxicity.

CuATSM has repeatedly demonstrated to be therapeutically effective in SOD1 mouse models of amyotrophic lateral sclerosis (ALS), leading to current clinical trials. CuATSM acts to stabilize ALS-associated mutant SOD1 protein by supplying copper. However, in vitro work has demonstrated that CuATSM is only therapeutic for wild-type-like SOD1 mutants, not metal-binding-region mutants, suggesting that CuATSM may have genotype-specific effects. Furthermore, relatively high doses of CuATSM have been shown to produce adverse events in humans and mice. Here, we investigated the genotype-specific therapeutic window of CuATSM. NSC-34 cells transiently expressing copper-binding or non-binding mutations of SOD1 were treated with a broad range of CuATSM concentrations and examined for survival via time-lapse microscopy. Determination of the no-observed-adverse-effect level and the LC50 suggest that CuATSM-associated toxicity is dependent on the amount of copper-depleted SOD1 available as well as the mutant's ability to bind copper. Our results suggest that the particular variant of SOD1 mutant is crucial in not only determining the level of efficacy achieved but also potential adverse events.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with a C57BL/6 background.

The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.

Amyotrophic Lateral Sclerosis: Proteins, Proteostasis, Prions, and Promises.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of the motor neurons that innervate muscle, resulting in gradual paralysis and culminating in the inability to breathe or swallow. This neuronal degeneration occurs in a spatiotemporal manner from a point of onset in the central nervous system (CNS), suggesting that there is a molecule that spreads from cell-to-cell. There is strong evidence that the onset and progression of ALS pathology is a consequence of protein misfolding and aggregation. In line with this, a hallmark pathology of ALS is protein deposition and inclusion formation within motor neurons and surrounding glia of the proteins TAR DNA-binding protein 43, superoxide dismutase-1, or fused in sarcoma. Collectively, the observed protein aggregation, in conjunction with the spatiotemporal spread of symptoms, strongly suggests a prion-like propagation of protein aggregation occurs in ALS. In this review, we discuss the role of protein aggregation in ALS concerning protein homeostasis (proteostasis) mechanisms and prion-like propagation. Furthermore, we examine the experimental models used to investigate these processes, including in vitro assays, cultured cells, invertebrate models, and murine models. Finally, we evaluate the therapeutics that may best prevent the onset or spread of pathology in ALS and discuss what lies on the horizon for treating this currently incurable disease.

Ubiquitin Homeostasis Is Disrupted in TDP-43 and FUS Cell Models of ALS.

A major feature of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitin (Ub) into intracellular inclusions. This sequestration of Ub may reduce the availability of free Ub, disrupting Ub homeostasis and ultimately compromising cellular function and survival. We previously reported significant disturbance of Ub homeostasis in neuronal-like cells expressing mutant SOD1. Here, we show that Ub homeostasis is also perturbed in neuronal-like cells expressing either TDP-43 or FUS. The expression of mutant TDP-43 and mutant FUS led to UPS dysfunction, which was associated with a redistribution of Ub and depletion of the free Ub pool. Redistribution of Ub is also a feature of sporadic ALS, with an increase in Ub signal associated with inclusions and no compensatory increase in Ub expression. Together, these findings suggest that alterations to Ub homeostasis caused by the misfolding and aggregation of ALS-associated proteins play an important role in the pathogenesis of ALS.

Thulium oxide nanoparticles as radioenhancers for the treatment of metastatic cutaneous squamous cell carcinoma.

Metastases from cutaneous squamous cell carcinoma (cSCC) occur in 2%-5% of cases. Surgery is the standard treatment, often combined with adjuvant radiotherapy. Concurrent carboplatin treatment with post-operative radiotherapy may be prescribed, although it has not shown benefit in recent clinical trials in high-risk cSCC patients. The novel high-Z nanoparticle thulium (III) oxide has been shown to enhance radiation dose delivery to brain tumors by specific uptake of these nanoparticles into the cancerous tissue. As the dose-enhancement capacity of thulium oxide nanoparticles following radiotherapy against metastatic cSCC cells is unknown, its efficacy as a radiosensitizer was evaluated, with and without carboplatin. Novel and validated human patient-derived cell lines of metastatic cSCC were used. The sensitivity of the cells to radiation was investigated using short-term proliferation assays as well as clonogenic survival as the radiobiological endpoint. Briefly, cells were irradiated with 125 kVp orthovoltage x-rays (0-6 Gy) with and without thulium oxide nanoparticles (99.9% trace metals basis; 50 µg ml-1) or low dose carboplatin pre-sensitization. Cellular uptake of the nanoparticles was first confirmed by microscopy and found to have no impact on short-term cell survival for the cSCC cells, highlighting the biocompatibility of thulium oxide nanoparticles. Clonogenic cell survival assays confirmed radio-sensitization when exposed to thulium nanoparticles, with the cell sensitivity increasing by a factor of 1.24 (calculated at the 10% survival fraction) for the irradiated cSCC cells. The combination of carboplatin with thulium oxide nanoparticles with irradiation did not result in significant further reductions in survival compared to nanoparticles alone. This is the first study to provide in vitro data demonstrating the independent radiosensitization effect of high-Z nanoparticles against metastatic cSCC with or without carboplatin. Further preclinical investigations with radiotherapy plus high-Z nanoparticles for the management of metastatic cSCC are warranted.

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells.

Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.

Proteome Homeostasis Dysfunction: A Unifying Principle in ALS Pathogenesis.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease but currently has no effective treatment. Growing evidence suggests that proteome homeostasis underlies ALS pathogenesis. Protein production, trafficking, and degradation all shape the proteome. We present a hypothesis that proposes all genetic lesions associated with ALS (including in mRNA-binding proteins) cause widespread imbalance to an already metastable proteome. The impact of such dysfunction is felt across the entire proteome and is not restricted to a small subset of proteins. Proteome imbalance may cause functional defects, such as excitability alterations, and eventually cell death. While this idea is a unifying principle for all of ALS, we propose that stratification will appear that might dictate the efficacy of therapeutics based on the proteostasis network.

Cerebrovascular amyloid Angiopathy in bioengineered vessels is reduced by high-density lipoprotein particles enriched in Apolipoprotein E.

BACKGROUND: Several lines of evidence suggest that high-density lipoprotein (HDL) reduces Alzheimer's disease (AD) risk by decreasing vascular beta-amyloid (Aß) deposition and inflammation, however, the mechanisms by which HDL improve cerebrovascular functions relevant to AD remain poorly understood. METHODS: Here we use a human bioengineered model of cerebral amyloid angiopathy (CAA) to define several mechanisms by which HDL reduces Aß deposition within the vasculature and attenuates endothelial inflammation as measured by monocyte binding. RESULTS: We demonstrate that HDL reduces vascular Aß accumulation independently of its principal binding protein, scavenger receptor (SR)-BI, in contrast to the SR-BI-dependent mechanism by which HDL prevents Aß-induced vascular inflammation. We describe multiple novel mechanisms by which HDL acts to reduce CAA, namely: i) altering Aß binding to collagen-I, ii) forming a complex with Aß that maintains its solubility, iii) lowering collagen-I protein levels produced by smooth-muscle cells (SMC), and iv) attenuating Aß uptake into SMC that associates with reduced low density lipoprotein related protein 1 (LRP1) levels. Furthermore, we show that HDL particles enriched in apolipoprotein (apo)E appear to be the major drivers of these effects, providing new insights into the peripheral role of apoE in AD, in particular, the fraction of HDL that contains apoE. CONCLUSION: The findings in this study identify new mechanisms by which circulating HDL, particularly HDL particles enriched in apoE, may provide vascular resilience to Aß and shed new light on a potential role of peripherally-acting apoE in AD.

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the "strain" of the conformer that is propagating.

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins.

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as ß-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1ß secretion. Both caspase-1 and IL-1ß contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1ß in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1ß cleavage, ASC speck formation, and the secretion of IL-1ß in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1ß secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1ß secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.

Trajectory Taken by Dimeric Cu/Zn Superoxide Dismutase through the Protein Unfolding and Dissociation Landscape Is Modulated by Salt Bridge Formation.

Native mass spectrometry (MS) is a powerful means for studying macromolecular protein assemblies, including accessing activated states. However, much remains to be understood about what governs which regions of the protein (un)folding funnel, which can be explored by activation of protein ions in a vacuum. Here, we examine the trajectory that Cu/Zn superoxide dismutase (SOD1) dimers take over the unfolding and dissociation free energy landscape in a vacuum. We examined wild-type SOD1 and six disease-related point mutants by using tandem MS and ion-mobility MS as a function of collisional activation. For six of the seven SOD1 variants, increasing activation prompted dimers to transition through two unfolding events and dissociate symmetrically into monomers with (as near as possible) equal charges. The exception was G37R, which proceeded only through the first unfolding transition and displayed a much higher abundance of asymmetric products. Supported by the observation that ejected asymmetric G37R monomers were more compact than symmetric G37R ones, we localized this effect to the formation of a gas-phase salt bridge in the first activated conformation. To examine the data quantitatively, we applied Arrhenius-type analysis to estimate the barriers on the corresponding free energy landscape. This reveals a heightening of the barrier to unfolding in G37R by >5 kJ/mol-1 over the other variants, consistent with expectations for the strength of a salt bridge. Our work demonstrates weaknesses in the simple general framework for understanding protein complex dissociation in a vacuum and highlights the importance of individual residues, their local environment, and specific interactions in governing product formation.

Prion-Like Propagation of Protein Misfolding and Aggregation in Amyotrophic Lateral Sclerosis.

The discovery that prion protein can misfold into a pathological conformation that encodes structural information capable of both propagation and inducing severe neuropathology has revolutionized our understanding of neurodegenerative disease. Many neurodegenerative diseases with a protein misfolding component are now classified as "prion-like" owing to the propagation of both symptoms and protein aggregation pathology in affected individuals. The neuromuscular disorder amyotrophic lateral sclerosis (ALS) is characterized by protein inclusions formed by either TAR DNA-binding protein of 43 kDa (TDP-43), Cu/Zn superoxide dismutase (SOD1), or fused in sarcoma (FUS), in both upper and lower motor neurons. Evidence from in vitro, cell culture, and in vivo studies has provided strong evidence to support the involvement of a prion-like mechanism in ALS. In this article, we review the evidence suggesting that prion-like propagation of protein aggregation is a primary pathomechanism in ALS, focusing on the key proteins and genes involved in disease (TDP-43, SOD1, FUS, and C9orf72). In each case, we discuss the evidence ranging from biophysical studies to in vivo examinations of prion-like spreading. We suggest that the idiopathic nature of ALS may stem from its prion-like nature and that elucidation of the specific propagating protein assemblies is paramount to developing effective therapies.

Emerging Developments in Targeting Proteotoxicity in Neurodegenerative Diseases.

The most common neurodegenerative diseases are Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, frontotemporal lobar degeneration, and the motor neuron diseases, with AD affecting approximately 6% of people aged 65 years and older, and PD affecting approximately 1% of people aged over 60 years. Specific proteins are associated with these neurodegenerative diseases, as determined by both immunohistochemical studies on post-mortem tissue and genetic screening, where protein misfolding and aggregation are key hallmarks. Many of these proteins are shown to misfold and aggregate into soluble non-native oligomers and large insoluble protein deposits (fibrils and plaques), both of which may exert a toxic gain of function. Proteotoxicity has been examined intensively in cell culture and in in vivo models, and clinical trials of methods to attenuate proteotoxicity are relatively new. Therapies to enhance cellular protein quality control mechanisms such as upregulation of chaperones and clearance/degradation pathways, as well as immunotherapies against toxic protein conformations, are being actively pursued. In this article, we summarize the common pathophysiology of neurodegenerative disease, and review therapies in early-phase clinical trials that target the proteotoxic component of several neurodegenerative diseases.