Electrostatics in the Stability and Misfolding of the Prion Protein: Salt Bridges, Self-Energy, and Solvation

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

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

Copyright 2020 McAlary, Chew, Lum, Geraghty, Yerbury and Cashman. 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

Establishing a national high fidelity cadaveric emergency urology simulation course to increase trainee preparedness for independent on-call practice: a prospective observational study

Background Whilst competence in the management of a wide range of urological emergencies is a requirement for certification in urology, many conditions are uncommon and exposure during training may be limited. This prospective observational study sought to evaluate the feasibility and effectiveness of a standardised cadaveric emergency urology simulation course aimed at improving operative confidence and competence prior to independent on-call practice in the United Kingdom. Methods A two-day cadaveric emergency urology simulation course supported by the British Association of Urological Surgeons (BAUS) was implemented at two pilot centres. All delegates that undertook one of the initial series of courses were invited to complete online pre- and post-course questionnaires relating to prior operative experience, documented competence and perceived confidence in being able to perform specific emergency procedures independently. Primary outcome was a self-reported ‘confidence score’ selected from a linear numeric scale ranging from 1 (not at all confident to perform a given procedure independently) to 10 (fully confident). Statistical analysis was undertaken using SPSS Statistics for Mac Version 25 and the paired student’s t-test used to compare mean pre- and post-course scores. Results One hundred and four delegates undertook the course during the study period. Of these, 85 (81.7%) completed the pre-course survey and 67 (64.4%) completed the post-course survey, with 61 (58.7%) completing both. The greatest proportion of respondents were Speciality Trainees in Urology of ST5 level or higher (equivalent of Resident/Fellows with 4 or more years of surgical training; n = 31, 36.5%). Delegates reported variable pre-course exposure, with most experience reported in loin approach to the kidney (median 10) and least in exploration and packing of a transurethral resection cavity and emergency nephrectomy (median 0). Following course completion, a statistically significant increase in confidence score was observed for each procedure, with the greatest increases seen for shunt for priapism (4.87 to 8.80, p < 0.001), ureteric reimplantation (3.52 to 7.33, p < 0.001) and primary ureteric anastomosis (3.90 to 7.49, p < 0.001). Conclusions A standardised high fidelity cadaveric simulation course is feasible and significantly improves the confidence of trainees in performing a wide range of emergency procedures to which exposure is currently limited

Walking the tightrope: proteostasis and neurodegenerative disease

A characteristic of many neurodegenerative diseases, including Alzheimer\u27s disease (AD), Parkinson\u27s disease (PD), Huntington\u27s disease (HD), and amyotrophic lateral sclerosis (ALS), is the aggregation of specific proteins into protein inclusions and/or plaques in degenerating brains. While much of the aggregated protein consists of disease specific proteins, such as amyloid-β, α-synuclein, or superoxide dismutase1 (SOD1), many other proteins are known to aggregate in these disorders. Although the role of protein aggregates in the pathogenesis of neurodegenerative diseases remains unknown, the ubiquitous association of misfolded and aggregated proteins indicates that significant dysfunction in protein homeostasis (proteostasis) occurs in these diseases. Proteostasis is the concept that the integrity of the proteome is in fine balance and requires proteins in a specific conformation, concentration, and location to be functional. In this review, we discuss the role of specific mechanisms, both inside and outside cells, which maintain proteostasis, including molecular chaperones, protein degradation pathways, and the active formation of inclusions, in neurodegenerative diseases associated with protein aggregation. A characteristic of many neurodegenerative diseases is the aggregation of specific proteins, which alone provides strong evidence that protein homeostasis is disrupted in these disease states. Proteostasis is the maintenance of the proteome in the correct conformation, concentration, and location by functional pathways such as molecular chaperones and protein degradation machinery. Here, we discuss the potential roles of quality control pathways, both inside and outside cells, in the loss of proteostasis during aging and disease

The paradoxical signals of two TrkC receptor isoforms supports a rationale for novel therapeutic strategies in ALS

Full length TrkC (TrkC-FL) is a receptor tyrosine kinase whose mRNA can be spliced to a truncated TrkC.T1 isoform lacking the kinase domain. Neurotrophin-3 (NT-3) activates TrkC-FL to maintain motor neuron health and function and TrkC.T1 to produce neurotoxic TNF-α; hence resulting in opposing pathways. In mouse and human ALS spinal cord, the reduction of miR-128 that destabilizes TrkC.T1 mRNA results in up-regulated TrkC.T1 and TNF-α in astrocytes. We exploited conformational differences to develop an agonistic mAb 2B7 that selectively activates TrkC-FL, to circumvent TrkC.T1 activation. In mouse ALS,2B7 activates spinal cord TrkC-FL signals, improves spinal cord motor neuron phenotype and function, and significantly prolongs life-span. Our results elucidate biological paradoxes of receptor isoforms and their role in disease progression, validate the concept of selectively targeting conformational epitopes in naturally occurring isoforms, and may guide the development of pro-neuroprotective (TrkC-FL) and anti-neurotoxic (TrkC.T1) therapeutic strategies.Fil: Brahimi, Fouad. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Maira, Mario. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Barcelona, Pablo Federico. Mc Gill University. Lady Davis Research Intitute; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Galan, Alba. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Aboulkassim, Tahar. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Teske, Katrina. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Rogers, Mary Louise. Flinders University, Department Of Human Physiology; AustraliaFil: Bertram, Lisa. University of British Columbia; CanadáFil: Wang, Jing. University of British Columbia; CanadáFil: Yousefi, Masoud. University of British Columbia; CanadáFil: Rush, Robert. Flinders University, Department Of Human Physiology; AustraliaFil: Fabian, Marc. Mc Gill University. Lady Davis Research Intitute; CanadáFil: Cashman, Neil. University of British Columbia; CanadáFil: Saragovi, H. Uri. Mc Gill University. Lady Davis Research Intitute; Canad

Expert elicitation on the uncertainties associated with chronic wasting disease

A high degree of uncertainty exists for chronic wasting disease (CWD) transmission factors in farmed and wild cervids. Evaluating the factors is important as it helps to inform future risk management strategies. Expert opinion is often used to assist decision making in a number of health, science, and technology domains where data may be sparse or missing. Using the Classical Model of elicitation, a group of experts was asked to estimate the most likely values for several risk factors affecting CWD transmission. The formalized expert elicitation helped structure the issues and hence provide a rational basis for estimating some transmission risk factors for which evidence is lacking. Considered judgments regarding environmental transmission, latency of CWD transmission, management, and species barrier were provided by the experts. Uncertainties for many items were determined to be large, highlighting areas requiring more research. The elicited values may be used as surrogate values until research evidence becomes available

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis

Regulation of Amyloid Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex

The prion protein (PrPC) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrPC, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrPC and mGluR5 are co-receptors also for -amyloid oligomers (AOs) and have been shown to modulate toxicity and neuronal death in Alzheimer\u27s disease. In the present work, we addressed the potential crosstalk between these two signaling pathways, laminin-PrPC-mGluR5 or AO-PrPC-mGluR5, as well as their interplay. Herein, we demonstrated that an existing complex containing PrPC-mGluR5 has an important role in AO binding and activity in neurons. A peptide mimicking the binding site of laminin onto PrPC (Ln-1) binds to PrPC and induces intracellular Ca2+ increase in neurons via the complex PrPC-mGluR5. Ln-1 promotes internalization of PrPC and mGluR5 and transiently decreases AO biding to neurons; however, the peptide does not impact AO toxicity. Given that mGluR5 is critical for toxic signaling by AOs and in prion diseases, we tested whether mGlur5 knock-out mice would be susceptible to prion infection. Our results show mild, but significant, effects on disease progression, without affecting survival of mice after infection. These results suggest that PrPC-mGluR5 form a functional response unit by which multiple ligands can trigger signaling. We propose that trafficking of PrPC-mGluR5 may modulate signaling intensity by different PrPC ligands