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. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Circular Economy in Mountain Value Chains: The Case of Three PDO Cheeses.

The circular economy (CE) has shown promise for achieving several of the UN's Sustainable Development Goals, replacing the linear system and reducing negative impacts on the environment. This research aims to assess the effective adoption of CE principles in three cheeses with geographical indication (GI) through an analysis of the practices identified in their respective value chains. Qualitative interviews show the persistence of historical practices that preserve the heritage behind the product, maintain autonomy in relation to external inputs and save energy or make intelligent use of by-products. Radical adoption of CE principles requires innovation to reduce the use of new inputs and greenhouse gas emissions. GI food products are generally not constrained by standards beyond those set by law, but their specifications can be modified, while respecting practices consistent with the link to the terroir. However, the remoteness of small businesses in deep rural areas, far from research centers, is slowing down the transfer of knowledge and the adoption of the latest technologies, particularly in mountainous areas. More participatory research and innovative initiatives are needed to ensure the transition to a circular economy for traditional mountain products, which are strongly linked to local culinary traditions and cultural identity.

Effect of the COVID-19 pandemic on the popularity of protected areas for mountain biking and hiking in Australia: Insights from volunteered geographic information.

Although the popularity of protected areas for recreation has been increasing, short term changes in visitation occurred during the COVID-19 pandemic. To examine how volunteer geographic information data can be used to monitor such often rapid changes in visitation across multiple locations, data from online fitness platforms for mountain biking (Trailforks) and remote area hiking (Wikiloc) were analysed before (2019) and during (2020-2021) the COVID-19 pandemic for 40 protected areas in Queensland, Australia. Mountain biking was popular with a total of 93,311 routes on Trailforks, with 26,936 routes in 2019, increasing to 37,406 in 2020, and then decreasing to 28,969 in 2021. Approximately 66% of all the routes were from just three urban protected areas out of the 12 with route data. There were 4367 routes for remote area hiking on Wikiloc across 36 protected areas, which increased slightly from 1081 in 2019, to 1421 in 2020 and to 1865 in 2021. Across 18 factors, distance from urban areas and networks of mountain biking trails best predicted popularity for mountain biking based on Generalised Linear Models. In contrast, average slope and large networks of hiking trails best predicted hiking, with similar results for each year. The two sources of online data were correlated with trail counter data, although not consistently. The results highlight how external factors affect visitation, but also how the same types of protected areas remained popular, and that the impacts of COVID-19 pandemic on visitation in South-East Queensland protected areas was less dramatic than for other regions. This study further highlights how volunteered geographic information can be used to assess the popularity of protected areas, including in rapidly changing conditions. Management implications: Rapid changes in visitation can be challenging to monitor and manage, as occurred with the COVID-19 pandemic. The impacts of the COVID-19 pandemic on mountain biking and hiking and factors predicting protected area popularity were examined across different parks. Visitation increased at different stages of the pandemic, with mountain bikers' preferring urban parks with networks of mountain bike trails while some hikers preferred more remote large parks. Managers can expand on traditional methods of visitor monitoring by using volunteered geographic information to monitor rapid and longer-term trends of visitation to protected areas.

Blood loss in total en bloc spondylectomy for primary spinal bone tumours: a comparison of estimated blood loss versus actual blood loss in a single centre over 10 years.

Background: Total en bloc spondylectomy (TES) is a widely accepted surgical technique for primary spinal bone tumours but is frequently accompanied by substantial peri-operative blood loss. Prior studies have reported estimated blood loss (EBL) can reach up to 3,200 mL. The aim of this study is to estimate the blood loss during TES procedures performed in the last ten years at our tertiary referral centre and compare EBL with actual blood loss (ABL). Methods: We performed a retrospective review of all cases managed surgically with TES referred to our centre between 2005 and 2015. We recorded the oncological characteristics of each tumour and surgical management in terms of resection margins, operative duration and instrumentation. Data relating to peri-operative blood loss was also recorded including an estimation of total blood loss, the use of cell salvage where applicable and transfusion rates. Results: A total of 21 patients were found to meet our inclusion criteria. There were 11 men and 10 women, with a median age of 40 years. The mean total ABL was 3,310 mL. Total operation time ranged from 6.53 to 19.7 h. Compared to ABL, in 59% of cases EBL had been underestimated by an average of 78% by volume. The EBL of the remaining 41% cases had been overestimated by 43%. This was not statistically significant (P=0.373). Cell salvage was used in 62% patients with a mean blood loss of 2,845 mL (884-4,939 mL) and transfusion of 3.8 units (0-12 units) versus 4,069 mL (297-8,335 mL) and 9.3 units (0-18 units) in those not managed with cell salvage. There was no significant difference in ABL between the cell salvage and non-cell salvage groups. Conclusions: We report one of the largest case series in TES for primary bone tumours. EBL is not a reliable predictor for ABL. A large blood loss should be anticipated and use of cell salvage is recommended.

Proteostasis impairment and ALS.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disease that results from the loss of both upper and lower motor neurons. It is the most common motor neuron disease and currently has no effective treatment. There is mounting evidence to suggest that disturbances in proteostasis play a significant role in ALS pathogenesis. Proteostasis is the maintenance of the proteome at the right level, conformation and location to allow a cell to perform its intended function. In this review, we present a thorough synthesis of the literature that provides evidence that genetic mutations associated with ALS cause imbalance to a proteome that is vulnerable to such pressure due to its metastable nature. We propose that the mechanism underlying motor neuron death caused by defects in mRNA metabolism and protein degradation pathways converges on proteostasis dysfunction. We propose that the proteostasis network may provide an effective target for therapeutic development in ALS.

The pivotal role of ubiquitin-activating enzyme E1 (UBA1) in neuronal health and neurodegeneration.

Ubiquitin-activating enzyme E1, UBA1, functions at the apex of the enzymatic ubiquitylation cascade, catalysing ubiquitin activation. UBA1 is thus of fundamental importance to the modulation of ubiquitin homeostasis and to all downstream ubiquitylation-dependent cellular processes, including proteolysis through the ubiquitin-proteasome system and selective autophagy. The proteasome-dependent and -independent functions of UBA1 contribute significantly to a range of processes crucial to neuronal health. The significance of UBA1 activity to neuronal health is clear in light of accumulating evidence implicating impaired UBA1 activity in a range of neurodegenerative conditions, including Parkinson's disease, Alzheimer's disease, Huntington's disease and spinal muscular atrophy. Moreover, ubiquitylation-independent functions of UBA1 of importance to neuronal functioning have been proposed. Here, we summarise findings supporting the significant role of UBA1 in regulating neuronal functioning, and discuss the detrimental consequences of UBA1 impairment that contribute to neuronal dysfunction and degeneration.

A Rapid, Mobile Neurocognitive Screening Test to Aid in Identifying Cognitive Impairment and Dementia (BrainCheck): Cohort Study.

BACKGROUND: The US population over the age of 65 is expected to double by the year 2050. Concordantly, the incidence of dementia is projected to increase. The subclinical stage of dementia begins years before signs and symptoms appear. Early detection of cognitive impairment and/or cognitive decline may allow for interventions to slow its progression. Furthermore, early detection may allow for implementation of care plans that may affect the quality of life of those affected and their caregivers. OBJECTIVE: We sought to determine the accuracy and validity of BrainCheck Memory as a diagnostic aid for age-related cognitive impairment, as compared against physician diagnosis and other commonly used neurocognitive screening tests, including the Saint Louis University Mental Status (SLUMS) exam, the Mini-Mental State Examination (MMSE), and the Montreal Cognitive Assessment (MoCA). METHODS: We tested 583 volunteers over the age of 49 from various community centers and living facilities in Houston, Texas. The volunteers were divided into five cohorts: a normative population and four comparison groups for the SLUMS exam, the MMSE, the MoCA, and physician diagnosis. Each comparison group completed their respective assessment and BrainCheck Memory. RESULTS: A total of 398 subjects were included in the normative population. A total of 84 participants were in the SLUMS exam cohort, 51 in the MMSE cohort, 35 in the MoCA cohort, and 18 in the physician cohort. BrainCheck Memory assessments were significantly correlated to the SLUMS exam, with coefficients ranging from .5 to .7. Correlation coefficients for the MMSE and BrainCheck and the MoCA and BrainCheck were also significant. Of the 18 subjects evaluated by a physician, 9 (50%) were healthy, 6 (33%) were moderately impaired, and 3 (17%) were severely impaired. A significant difference was found between the severely and moderately impaired subjects and the healthy subjects (P=.02). We derived a BrainCheck Memory composite score that showed stronger correlations with the standard assessments as compared to the individual BrainCheck assessments. Receiver operating characteristic (ROC) curve analysis of this composite score found a sensitivity of 81% and a specificity of 94%. CONCLUSIONS: BrainCheck Memory provides a sensitive and specific metric for age-related cognitive impairment in older adults, with the advantages of a mobile, digital, and easy-to-use test. TRIAL REGISTRATION: ClinicalTrials.gov NCT03608722; https://clinicaltrials.gov/ct2/show/NCT03608722 (Archived by WebCite at http://www.webcitation.org/76JLoYUGf).

SOD1A4V aggregation alters ubiquitin homeostasis in a cell model of ALS.

A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.This article has an associated First Person interview with the first author of the paper.

Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation.

ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology.

Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.

Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions.

Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.