Biomolecular Condensates as Emerging Biomaterials: Functional Mechanisms and Advances in Computational and Experimental Approaches.

Biomolecular condensates, a ubiquitous class of biomaterials found in living cells, have been shown to be responsible for key physiological processes, such as gene regulation, signal transduction, and stress response. Since their discovery, extensive efforts have been devoted to this field to better understand the underlying mechanisms using both computational and experimental techniques. While great progress has been achieved, the key challenges still exist.

In-Gel Zymography of Amyotrophic Lateral Sclerosis-Associated Variants of Superoxide Dismutase-1.

In-gel zymography allows the separation of protein via electrophoresis and subsequent measurement of enzymatic activity of enzymes from biological mixtures. The antioxidant enzyme superoxide dismutase 1 (SOD1) is an important cytosolic oxygen radical scavenging enzyme that has been implicated in a range of pathologies, including cancer, metabolic and neurodegenerative diseases, most notably amyotrophic lateral sclerosis (ALS). Here, we describe a method to detect and compare SOD1 activity from both cell and tissue samples.

A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis.

Background: SOD1 mutations are a significant contributor of familial amyotrophic lateral sclerosis (ALS) cases. SOD1 mutations increase the propensity for the protein to misfold and aggregate into insoluble proteinaceous deposits within motor neurons and neighbouring cells. The small molecule, CuATSM, has repeatedly shown in mouse models to be a promising therapeutic treatment for SOD1-associated ALS and is currently in Phase II/III clinical trials for the treatment of ALS.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS).

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients.

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure.

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.

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 SOD1-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish.

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.

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.

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.

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.

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.

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

CuATSM has repeatedly demonstrated to be therapeutically effective in 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, 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.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1 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.

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.

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.

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.

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.

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.

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 (Aβ) 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 Aβ deposition within the vasculature and attenuates endothelial inflammation as measured by monocyte binding.

Results: We demonstrate that HDL reduces vascular Aβ accumulation independently of its principal binding protein, scavenger receptor (SR)-BI, in contrast to the SR-BI-dependent mechanism by which HDL prevents Aβ-induced vascular inflammation.

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

[This corrects the article DOI: 10.3389/fnmol.2019.

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.

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.

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.