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Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD., (© 2022. The Author(s).)

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pereira Sena, Weber, Bayezit, Saup, Incebacak Eltemur, Li, Velic, Jung, Macek, Nguyen, Riess and Schmidt.)

Body weight (BW) loss and reduced body mass index (BMI) are the most common peripheral alterations in Huntington disease (HD) and have been found in HD mutation carriers and HD animal models before the manifestation of neurological symptoms. This suggests that, at least in the early disease stage, these changes could be due to abnormal tissue growth rather than tissue atrophy. Moreover, BW and BMI are reported to be more affected in males than females in HD animal models and patients. Here, we confirmed sex-dependent growth alterations in the BACHD rat model for HD and investigated the associated contributing factors. Our results showed growth abnormalities along with decreased plasma testosterone and insulin-like growth factor 1 (IGF-1) levels only in males. Moreover, we demonstrated correlations between growth parameters, IGF-1, and testosterone. Our analyses further revealed an aberrant transcription of testosterone biosynthesis-related genes in the testes of BACHD rats with undisturbed luteinizing hormone (LH)/cAMP/PKA signaling, which plays a key role in regulating the transcription process of some of these genes. In line with the findings in BACHD rats, analyses in the R6/2 mouse model of HD showed similar results. Our findings support the view that mutant huntingtin may induce abnormal growth in males via the dysregulation of gene transcription in the testis, which in turn can affect testosterone biosynthesis.

Among posttranslational modifications, directed proteolytic processes have the strongest impact on protein integrity. They are executed by a variety of cellular machineries and lead to a wide range of molecular consequences. Compared to other forms of proteolytic enzymes, the class of calcium-activated calpains is considered as modulator proteases due to their limited proteolytic activity, which changes the structure and function of their target substrates. In the context of neurodegeneration and - in particular - polyglutamine disorders, proteolytic events have been linked to modulatory effects on the molecular pathogenesis by generating harmful breakdown products of disease proteins. These findings led to the formulation of the toxic fragment hypothesis , and calpains appeared to be one of the key players and auspicious therapeutic targets in Huntington disease and Machado Joseph disease. This review provides a current survey of the role of calpains in proteolytic processes found in polyglutamine disorders. Together with insights into general concepts behind toxic fragments and findings in polyglutamine disorders, this work aims to inspire researchers to broaden and deepen the knowledge in this field, which will help to evaluate calpain-mediated proteolysis as a unifying and therapeutically targetable posttranslational mechanism in neurodegeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Incebacak Eltemur, Nguyen and Weber.)

Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.

Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by a polyglutamine-encoding trinucleotide repeat expansion in the gene of transcription factor TATA box-binding protein (TBP). While its underlying pathomechanism is elusive, polyglutamine-expanded TBP fragments of unknown origin mediate the mutant protein's toxicity. Calcium-dependent calpain proteases are protagonists in neurodegenerative disorders. Here, we demonstrate that calpains cleave TBP, and emerging C-terminal fragments mislocalize to the cytoplasm. SCA17 cell and rat models exhibited calpain overactivation, leading to excessive fragmentation and depletion of neuronal proteins in vivo. Transcriptome analysis of SCA17 cells revealed synaptogenesis and calcium signaling perturbations, indicating the potential cause of elevated calpain activity. Pharmacological or genetic calpain inhibition reduced TBP cleavage and aggregation, consequently improving cell viability. Our work underlines the general significance of calpains and their activating pathways in neurodegenerative disorders and presents these proteases as novel players in the molecular pathogenesis of SCA17., (© 2022. The Author(s).)

Aberrant O -GlcNAcylation, a protein posttranslational modification defined by the O -linked attachment of the monosaccharide N -acetylglucosamine ( O -GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O -GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O -GlcNAc transferase (OGT), which attaches O -GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O -GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O -GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD., Competing Interests: The authors declare no competing interest.

Huntington's disease (HD) is a monogenetic neurodegenerative disorder characterized by the accumulation of polyglutamine-expanded huntingtin (mHTT). There is currently no cure, and therefore disease-slowing remedies are sought to alleviate symptoms of the multifaceted disorder. Encouraging findings in Alzheimer's and Parkinson's disease on alpha-2 adrenoceptor (α2-AR) inhibition have shown neuroprotective and aggregation-reducing effects in cell and animal models. Here, we analyzed the effect of beditin, a novel α2- adrenoceptor (AR) antagonist, on cell viability and mHTT protein levels in cell models of HD using Western blot, time-resolved Foerster resonance energy transfer (TR-FRET), lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) cytotoxicity assays. Beditin decreases cytotoxicity, as measured by TUNEL staining and LDH release, in a neuronal progenitor cell model (ST Hdh cells) of HD and decreases the aggregation propensity of HTT exon 1 fragments in an overexpression model using human embryonic kidney (HEK) 293T cells. α2-AR is a promising therapeutic target for further characterization in HD models. Our data allow us to suggest beditin as a valuable candidate for the pharmaceutical manipulation of α2-AR, as it is capable of modulating neuronal cell survival and the level of mHTT.

Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe 3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans., (© 2020 The Protein Society.)

Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Premise: Understanding species' responses to climate change is a critical challenge facing biologists today. Though many species are widespread, few studies of climate-driven shifts in flowering time have examined large continuous spatial scales for individual species. And even fewer studies have examined these shifts at time scales greater than a few decades., Methods: We used digitized herbarium specimens and PRISM climate data to produce the spatially and temporally broadest-scale study of flowering time in a single species to date, spanning the contiguous United States and 153 years (1863-2016) for a widespread weedy annual, Triodanis perfoliata (Campanulaceae). We examined factors driving phenological shifts as well as the roles of geographic and temporal scale in understanding these trends., Results: Year was a significant factor in both geospatial and climatic analyses, revealing that flowering time has advanced by ~9 days over the past ~150 years. We found that temperature as well as vapor pressure deficit, an understudied climatic parameter associated with evapotranspiration and water stress, were strongly associated with peak flowering. We also examined how sampling at different spatiotemporal scales influences the power to detect flowering-time shifts, finding that relatively large spatial and temporal scales are ideal for detecting flowering-time shifts in this widespread species., Conclusions: Our results emphasize the importance of understanding the interplay of geospatial factors at different scales to examine how species respond to climate change., (© 2019 Botanical Society of America.)

Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of CAG repeats in the ATXN3 gene leading to an elongated polyglutamine tract in the ataxin-3 protein. Previously, we demonstrated that symptoms of SCA3 are reversible in the first conditional mouse model for SCA3 directing ataxin-3 predominantly to the hindbrain. Here, we report on the effects of transgenic ataxin-3 expression in forebrain regions. Employing the Tet-off CamKII-promoter mouse line and our previously published SCA3 responder line, we generated double transgenic mice (CamKII/MJD77), which develop a neurological phenotype characterized by impairment in rotarod performance, and deficits in learning new motor tasks as well as hyperactivity. Ataxin-3 and ubiquitin-positive inclusions are detected in brains of double transgenic CamKII/MJD77 mice. After turning off the expression of pathologically expanded ataxin-3, these inclusions disappear. However, the observed phenotype could not be reversed, very likely due to pronounced apoptotic cell death in the frontal brain. Our data demonstrate that cerebellar expression is not required to induce a neurological phenotype using expanded ATXN3 as well as the pronounced sensibility of forebrain neurons for toxic ataxin-3., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca 2+ -dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.

Plant breeding systems can vary widely among populations, yet few studies have investigated abiotic factors contributing to variation across a broad geographic range. Here we investigate variation in reproductive traits of Triodanis perfoliata (Campanulaceae), a species that exhibits dimorphic cleistogamy, a condition in which individual plants have both closed (selfing: cleistogamous: CL) and open (selfing or outcrossing: chasmogamous: CH) flowers. Chasmogamous production is theorized to be more costly because CH flowers have a larger exposed surface area and thus are more likely to lose more water than CL flowers. We examine relationships between abiotic conditions (temperature, precipitation and soil characteristics) and variation in breeding systems across 14 widespread populations using ordinary least squares models. We found that a large proportion of breeding system variation was described by climate and soil variables ( R 2 = 0.65-0.92). These results support the hypothesis that variation in the environment drives variation in breeding system allocation. Our broad geographic analyses provide a framework for mechanistic studies of cleistogamy, and employ a novel approach for examining reproductive traits and environmental variation at large scales. Given that two major components of our models were temperature and precipitation, our study further emphasizes the potential for ongoing climate change to alter plant breeding systems.

Alterations in mitochondrial morphology and function have been linked to neurodegenerative diseases, including Parkinson disease, Alzheimer disease and Huntington disease. Metabolic defects, resulting from dysfunctional mitochondria, have been reported in patients and respective animal models of all those diseases. Spinocerebellar Ataxia Type 3 (SCA3), another neurodegenerative disorder, also presents with metabolic defects and loss of body weight in early disease stages although the possible role of mitochondrial dysfunction in SCA3 pathology is still to be determined. Interestingly, the SCA3 disease protein ataxin-3, which is predominantly localized in cytoplasm and nucleus, has also been associated with mitochondria in both its mutant and wildtype form. This observation provides an interesting link to a potential mitochondrial involvement of mutant ataxin-3 in SCA3 pathogenesis. Furthermore, proteolytic cleavage of ataxin-3 has been shown to produce toxic fragments and even overexpression of artificially truncated forms of ataxin-3 resulted in mitochondria deficits. Therefore, we analyzed the repercussions of expressing a naturally occurring N-terminal cleavage fragment of ataxin-3 and the influence of an endogenous expression of the S256 cleavage fragment in vitro and in vivo . In our study, expression of a fragment derived from calpain cleavage induced mitochondrial fragmentation and cristae alterations leading to a significantly decreased capacity of mitochondrial respiration and contributing to an increased susceptibility to apoptosis. Furthermore, analyzing mitophagy revealed activation of autophagy in the early pathogenesis with reduced lysosomal activity. In conclusion, our findings indicate that cleavage of ataxin-3 by calpains results in fragments which interfere with mitochondrial function and mitochondrial degradation processes.

A skin biopsy of a patient with spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease (MJD)) caused by a CAG trinucleotide repeat expansion in the ATXN3 gene, was used to generate an induced pluripotent stem cell line, HIHCNi002-A (iPSC-SCA3). Skin fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC, and hLIN28. The iPSC-SCA3 line exhibits chromosomal stability with conservation of the ATXN3 repeat expansion, expresses pluripotency markers and differentiates into endo-, meso-, and ectodermal cells in vitro., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene which results in progressive neurodegeneration in the striatum, cortex, and eventually most brain areas. Despite being a monogenic disorder, environmental factors influence HD characteristics. Both human and mouse studies suggest that mutant HTT (mHTT) leads to gene expression changes that harbor potential to be modulated by the environment. Yet, the underlying mechanisms integrating environmental cues into the gene regulatory program have remained largely unclear. To better understand gene-environment interactions in the context of mHTT, we employed RNA-seq to examine effects of maternal separation (MS) and environmental enrichment (EE) on striatal gene expression during development of BACHD rats. We integrated our results with striatal consensus modules defined on HTT-CAG length and age-dependent co-expression gene networks to relate the environmental factors with disease progression. While mHTT was the main determinant of expression changes, both MS and EE were capable of modulating these disturbances, resulting in distinctive and in several cases opposing effects of MS and EE on consensus modules. This bivalent response to maternal separation and environmental enrichment may aid in explaining their distinct effects observed on disease phenotypes in animal models of HD and related neurodegenerative disorders.

Premise of Study: In a seminal body of theory, Lloyd showed that the fitness consequences of selfing will depend on its timing in anthesis. Selfing that occurs after opportunities for outcrossing or pollen dispersal can provide reproductive assurance when pollinators are limited and is expected to incur little cost, even when inbreeding depression is high. As a result, delayed selfing is often interpreted as a "best-of-both-worlds" mating system that combines the advantages of selfing and outcrossing., Methods: We surveyed 65 empirical studies of delayed selfing, recording floral mechanisms and examining information on inbreeding depression, autofertility, and other parameters to test the support for delayed selfing as a best-of-both-worlds strategy., Key Results: Phylogenetic distribution of the diverse floral mechanisms suggests that some basic floral structures may predispose plant taxa to evolve delayed selfing. Delayed selfing appears to serve as a best-of-both-worlds strategy in some but not all species. While the capacity for autonomous selfing is often high, it is lower, in some cases, than in related species with earlier modes of selfing. In other delayed-selfers, low inbreeding depression and reduced investment in corollas and pollen suggest limited benefits from outcrossing., Conclusions: Despite a growing literature on the subject, experimental evidence for delayed selfing is limited and major gaps in knowledge remain, particularly with respect to the stability of delayed selfing and the conditions that may favor transitions between delayed and earlier selfing. Finally, we suggest a potential role of delayed selfing in facilitating transitions from self-incompatibility to selfing., (© 2018 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila , and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila , KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3., Competing Interests: The authors declare no conflict of interest.

Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Metformin (Met) is an approved antidiabetic drug currently being explored for repurposing in cancer treatment based on recent evidence of its apparent chemopreventive properties. Met is weakly cationic and targets the mitochondria to induce cytotoxic effects in tumor cells, albeit not very effectively. We hypothesized that increasing its mitochondria-targeting potential by attaching a positively charged lipophilic substituent would enhance the antitumor activity of Met. In pursuit of this question, we synthesized a set of mitochondria-targeted Met analogues (Mito-Mets) with varying alkyl chain lengths containing a triphenylphosphonium cation (TPP(+)). In particular, the analogue Mito-Met10, synthesized by attaching TPP(+) to Met via a 10-carbon aliphatic side chain, was nearly 1,000 times more efficacious than Met at inhibiting cell proliferation in pancreatic ductal adenocarcinoma (PDAC). Notably, in PDAC cells, Mito-Met10 potently inhibited mitochondrial complex I, stimulating superoxide and AMPK activation, but had no effect in nontransformed control cells. Moreover, Mito-Met10 potently triggered G1 cell-cycle phase arrest in PDAC cells, enhanced their radiosensitivity, and more potently abrogated PDAC growth in preclinical mouse models, compared with Met. Collectively, our findings show how improving the mitochondrial targeting of Met enhances its anticancer activities, including aggressive cancers like PDAC in great need of more effective therapeutic options. Cancer Res; 76(13); 3904-15. ©2016 AACR., (©2016 American Association for Cancer Research.)

Olesoxime, a small molecule drug candidate, has recently attracted attention due to its significant beneficial effects in models of several neurodegenerative disorders including Huntington's disease. Olesoxime's neuroprotective effects have been assumed to be conveyed through a direct, positive influence on mitochondrial function. In a long-term treatment study in BACHD rats, the latest rat model of Huntington's disease, olesoxime revealed a positive influence on mitochondrial function and improved specific behavioral and neuropathological phenotypes. Moreover, a novel target of the compound was discovered, as olesoxime was found to suppress the activation of the calpain proteolytic system, a major contributor to the cleavage of the disease-causing mutant huntingtin protein into toxic fragments, and key player in degenerative processes in general. Results from a second model of Huntington's disease, the Hdh (Q111) knock-in mouse, confirm olesoxime's calpain-suppressing effects and support the therapeutic value of olesoxime for Huntington's disease and other disorders involving calpain overactivation.

Premise of the Study: Climate change is a widely accepted threat to biodiversity. Species distribution models (SDMs) are used to forecast whether and how species distributions may track these changes. Yet, SDMs generally fail to account for genetic and demographic processes, limiting population-level inferences. We still do not understand how predicted environmental shifts will impact the spatial distribution of genetic diversity within taxa., Methods: We propose a novel method that predicts spatially explicit genetic and demographic landscapes of populations under future climatic conditions. We use carefully parameterized SDMs as estimates of the spatial distribution of suitable habitats and landscape dispersal permeability under present-day, past, and future conditions. We use empirical genetic data and approximate Bayesian computation to estimate unknown demographic parameters. Finally, we employ these parameters to simulate realistic and complex models of responses to future environmental shifts. We contrast parameterized models under current and future landscapes to quantify the expected magnitude of change., Key Results: We implement this framework on neutral genetic data available from Penstemon deustus. Our results predict that future climate change will result in geographically widespread declines in genetic diversity in this species. The extent of reduction will heavily depend on the continuity of population networks and deme sizes., Conclusions: To our knowledge, this is the first study to provide spatially explicit predictions of within-species genetic diversity using climatic, demographic, and genetic data. Our approach accounts for climatic, geographic, and biological complexity. This framework is promising for understanding evolutionary consequences of climate change, and guiding conservation planning., (© 2016 Botanical Society of America.)

Premise of the Study: Project Baseline is a seed bank that offers an unprecedented opportunity to examine spatial and temporal dimensions of microevolution during an era of rapid environmental change. Over the upcoming 50 years, biologists will withdraw genetically representative samples of past populations from this time capsule of seeds and grow them contemporaneously with modern samples to detect any phenotypic and molecular evolution that has occurred during the intervening time., Methods: We carefully developed this living genome bank using protocols to enhance its experimental value by collecting from multiple populations and species across a broad geographical range in sites that are likely to be preserved into the future. Seeds are accessioned with site and population data and are stored by maternal line under conditions that maximize seed longevity. This open-access resource will be available to researchers at regular intervals to evaluate contemporary evolution., Key Results: To date, the Project Baseline collection includes 100-200 maternal lines of each of 61 species collected from over 831 populations on sites that are likely to be preserved into the future across the United States (∼78,000 maternal lines). Our strategically designed collection circumvents some problems that can cloud the results of "resurrection" studies involving naturally preserved or existing seed collections that are available fortuitously., Conclusions: The resurrection approach can be coupled with long-established and newer techniques over the next five decades to elucidate genetic change and thereby vastly improve our understanding of temporal and spatial changes in phenotype and the evolutionary processes underlying it., (© 2016 Botanical Society of America.)

In an age of rapid global change, it is imperative that we continue to improve our understanding of factors that govern genetic differentiation in plants to inform biologically reasonable predictions for the future and enlighten conservation and restoration practices. In this special issue, we have assembled a set of original research and reviews that employ diverse approaches, both classic and contemporary, to illuminate patterns of phenotypic and genetic variation, probe the underlying evolutionary processes that have contributed to these patterns, build predictive models, and test evolutionary hypotheses. Our goal was to underscore the unique insights that can be obtained through the complementary and distinct studies of plant populations across species' geographic ranges., (© 2016 Botanical Society of America.)

Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Understanding the genetic basis of natural phenotypic variation is of great importance, particularly since selection can act on this variation to cause evolution. We examined expression and allelic variation in candidate flowering time loci in Brassica rapa plants derived from a natural population and showing a broad range in the timing of first flowering. The loci of interest were orthologs of the Arabidopsis genes FLC and SOC1 (BrFLC and BrSOC1, respectively), which in Arabidopsis play a central role in the flowering time regulatory network, with FLC repressing and SOC1 promoting flowering. In B. rapa, there are four copies of FLC and three of SOC1. Plants were grown in controlled conditions in the lab. Comparisons were made between plants that flowered the earliest and latest, with the difference in average flowering time between these groups ∼30 days. As expected, we found that total expression of BrSOC1 paralogs was significantly greater in early than in late flowering plants. Paralog-specific primers showed that expression was greater in early flowering plants in the BrSOC1 paralogs Br004928, Br00393 and Br009324, although the difference was not significant in Br009324. Thus expression of at least 2 of the 3 BrSOC1 orthologs is consistent with their predicted role in flowering time in this natural population. Sequences of the promoter regions of the BrSOC1 orthologs were variable, but there was no association between allelic variation at these loci and flowering time variation. For the BrFLC orthologs, expression varied over time, but did not differ between the early and late flowering plants. The coding regions, promoter regions and introns of these genes were generally invariant. Thus the BrFLC orthologs do not appear to influence flowering time in this population. Overall, the results suggest that even for a trait like flowering time that is controlled by a very well described genetic regulatory network, understanding the underlying genetic basis of natural variation in such a quantitative trait is challenging.

Background: Neuroblastoma is a pediatric cancer of neural crest origin. Despite aggressive treatment, mortality remains at 40 % for patients with high-risk disseminated disease, underscoring the need to test new combinations of therapies. In murine tumor models, our laboratory previously showed that T cell-mediated anti-tumor immune responses improve in the context of lymphopenia. The goal of this study was to incorporate lymphodepletion into an effective immune therapy that can be easily translated into neuroblastoma standard of care. Based on the lymphodepleting effects of cyclophosphamide, we hypothesized that cyclophosphamide would synergize with the TLR9 agonist, CpG oligodeoxynucleotide (ODN), to produce a T cell-mediated anti-neuroblastoma effect., Methods: To test this hypothesis, we used the AgN2a aggressive murine model of neuroblastoma. Mice bearing subcutaneous tumors were treated with cyclophosphamide followed by treatment with tumor cell lysate mixed with CpG ODN injected at the tumor site., Results: Subcutaneous neuroblastoma regressed only in mice that were treated with 100 mg/kg cyclophosphamide prior to receiving treatments of tumor lysate mixed with CpG ODN. The anti-neuroblastoma response was T cell-mediated. Synergy between cyclophosphamide and the tumor lysate/CpG ODN treatment influenced the production of anti-tumor CD8 T cell effectors, and dendritic cell homeostasis. For clinical consideration, an allogeneic tumor lysate was used effectively with this protocol to eliminate AgN2a tumor in vivo., Conclusion: Synergistic immune modulating effects of cyclophosphamide and a treatment containing tumor cell lysate and CpG ODN provide T cell-mediated anti-tumor activity against murine neuroblastoma.

Kin discrimination, the differential treatment of conspecifics based on kinship, occurs across the tree of life, from animals to plants to fungi to bacteria. When kin and nonkin interact, the ability to identify kin enables individuals to increase their inclusive fitness by helping kin, harming nonkin, and avoiding inbreeding. For a given species, the strength of selection for kin discrimination mechanisms is influenced by demographic, ecological, and life-history processes that collectively determine the scope for discrimination and the payoffs from kin-biased behavior. In this review, we explore how these processes drive variation in kin discrimination across taxa, highlighting contributions of recent empirical, comparative, and theoretical work to our understanding of when, how, and why kin discrimination evolves. [ABSTRACT FROM AUTHOR]

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility. [ABSTRACT FROM AUTHOR]

As climate change progresses, we are observing widespread changes in phenotypes in many plant populations. Whether these phenotypic changes are directly caused by climate change, and whether they result from phenotypic plasticity or evolution, are active areas of investigation. Here, we review terrestrial plant studies addressing these questions. Plastic and evolutionary responses to climate change are clearly occurring. Of the 38 studies that met our criteria for inclusion, all found plastic or evolutionary responses, with 26 studies showing both. These responses, however, may be insufficient to keep pace with climate change, as indicated by eight of 12 studies that examined this directly. There is also mixed evidence for whether evolutionary responses are adaptive, and whether they are directly caused by contemporary climatic changes. We discuss factors that will likely influence the extent of plastic and evolutionary responses, including patterns of environmental changes, species' life history characteristics including generation time and breeding system, and degree and direction of gene flow. Future studies with standardized methodologies, especially those that use direct approaches assessing responses to climate change over time, and sharing of data through public databases, will facilitate better predictions of the capacity for plant populations to respond to rapid climate change.

The history of polyglutamine diseases dates back approximately 20 years to the discovery of a polyglutamine repeat in the androgen receptor of SBMA followed by the identification of similar expansion mutations in Huntington's disease, SCA1, DRPLA, and the other spinocerebellar ataxias. This common molecular feature of polyglutamine diseases suggests shared mechanisms in disease pathology and neurodegeneration of disease specific brain regions. In this review, we discuss the main pathogenic pathways including proteolytic processing, nuclear shuttling and aggregation, mitochondrial dysfunction, and clearance of misfolded polyglutamine proteins and point out possible targets for treatment.

We investigated the role of morph-based differences in the expression of inbreeding depression in loss of the mid-styled morph from populations of tristylous Oxalis alpina. The extent of self-compatibility (SC) of reproductive morphs, the degree of self-fertilization, and the magnitude of inbreeding depression were investigated in three populations of O. alpina differing in their tristylous incompatibility relationships. All three populations exhibited significant inbreeding depression. In two populations with highly modified tristylous incompatibility, manifested as increased reciprocal compatibility between short- and long-styled morphs, substantial SC and self-fertilization of mid-styled morphs were detected, and expected to result in expression of inbreeding depression in the progeny of mid-styled morphs in the natural populations. In contrast, significant self-fertility of the mid-styled morph was absent from the population with typical tristylous incompatibility, and no self-fertilization could be detected. Although self-fertilization and expression of inbreeding depression should result in selection against the mid-styled morph in the later stages of the transition from tristyly to distyly, in O. alpina selection against the mid-styled morph in the early phases of the evolution of distyly is likely due to genic selection against mid-alleles associated with modified tristylous incompatibility, rather than expression of inbreeding depression., (No Claims to Original U.S. government works.)

Spinocerebellar Ataxia Type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominantly inherited neurodegenerative disease caused by an expanded polyglutamine stretch in the ataxin-3 protein. A pathological hallmark of the disease is cerebellar and brainstem atrophy, which correlates with the formation of intranuclear aggregates in a specific subset of neurons. Several studies have demonstrated that the formation of aggregates depends on the generation of aggregation-prone and toxic intracellular ataxin-3 fragments after proteolytic cleavage of the full-length protein. Despite this observed increase in aggregated mutant ataxin-3, information on soluble mutant ataxin-3 levels in brain tissue is lacking. A quantitative method to analyze soluble levels will be a useful tool to characterize disease progression or to screen and identify therapeutic compounds modulating the level of toxic soluble ataxin-3. In the present study we describe the development and application of a quantitative and easily applicable immunoassay for quantification of soluble mutant ataxin-3 in human cell lines and brain samples of transgenic SCA3 mice. Consistent with observations in Huntington disease, transgenic SCA3 mice reveal a tendency for decrease of soluble mutant ataxin-3 during disease progression in fractions of the cerebellum, which is inversely correlated with aggregate formation and phenotypic aggravation. Our analyses demonstrate that the time-resolved Förster resonance energy transfer immunoassay is a highly sensitive and easy method to measure the level of soluble mutant ataxin-3 in biological samples. Of interest, we observed a tendency for decrease of soluble mutant ataxin-3 only in the cerebellum of transgenic SCA3 mice, one of the most affected brain regions in Spinocerebellar Ataxia Type 3 but not in whole brain tissue, indicative of a brain region selective change in mutant ataxin-3 protein homeostasis.

Tumors are composed of heterogeneous populations of cells including tumor-initiating cells (TICs) and metastatic precursors. While the origin of these cells is unknown, there is evidence that tumor cells can transdifferentiate from an epithelial to a mesenchymal phenotype, a property referred to as epithelial-to-mesenchymal transition (EMT). This cellular plasticity may explain the heterogeneous nature of tumors and differences in the tumorigenic and invasive properties of cells. Understanding the origin of these cells and the contribution of external factors that influence the acquisition of cellular properties is critical for the development of therapeutics to eradicate cancer. In this study, we show that primary murine tumor cells harvested from FVB/N Tg (MMTV/Neu) spontaneous mammary tumors possess differentiation plasticity and can be enriched to be epithelial or mesenchymal-like using selected culture media conditions, and we show evidence of EMT in a clonal population of primary epithelial tumor cells when cultured in fibroblast growth factor-1 (FGF-1) or transforming growth factor-β (TGF-β). We also determined that in contrast to the identification of mesenchymal-like tumor cells as TICs in orthotopic xenograph models of tumorigenicity, epithelial-enriched murine mammary tumor cells were more tumorigenic as compared to mesenchymal-enriched cells when transplanted back subcutaneously into syngeneic immune competent mice. Together, these data suggest that EMT plasticity can be induced in primary murine mammary tumor cells, and that tumorigenicity of epithelial or mesenchymal-like cells may be influenced by factors such as the site of tumor inoculation or the immune state of the host (xenogenic immune compromised versus syngeneic immune competent).

Premise of the Study: Variation among individuals in levels of inbreeding depression associated with selfing levels could influence mating system evolution by purging deleterious alleles, but empirical evidence for this association is limited., Methods: We investigated the association of family-level inbreeding depression and presumed inbreeding history in a tristylous population of Oxalis alpina (Oxalidaceae)., Key Results: Mid-styled individuals possessed the greatest degree of self-compatibility (SC) and produced more autogamous capsules than short- or long-styled individuals. Offspring of highly self-compatible mid-styled individuals showed reduced inbreeding depression. Mid-styled plants that produced capsules autogamously exhibited reduced stigma-anther separation compared to mid-styled plants that produced no capsules autogamously. Reduced inbreeding depression was not correlated with stigma-anther separation, suggesting that self-compatibility and autogamy evolve before morphological changes in stigma-anther separation., Conclusions: Purging of inbreeding depression occurred in SC mid-styled maternal families. Low inbreeding depression in SC mid-styled plants may lead to retention of the mid-styled morph in populations, despite the occurrence of higher selfing rates in mid-styled relative to short- or long-styled morphs. Variation among individuals in levels of self-fertilization within populations may lead to associations between inbreeding lineages and lower levels of inbreeding depression, influencing the evolution of mating systems.

When fertilization triggers flower senescence, early autonomous selfing may cause flowers to senesce before pollen has dispersed, discounting unused pollen. Selfing-induced flower senescence was examined in Leptosiphon jepsonii, a species that varies in the timing of self-compatibility. In field and greenhouse experiments, fertilization had a large effect on flower senescence; most outcrossed flowers senesced after 1 d whereas emasculated flowers lasted 2-5 d. In a comparison of inbred lines from three populations, longevity of autonomously selfed flowers of early self-compatible individuals was significantly less than that of late self-compatible individuals. In field experiments, autonomously selfed flowers were shorter-lived in a predominantly early-selfing population than in a predominantly late-selfing population. Pollen was available and viable beyond the first day of anthesis, suggesting that reductions in flower longevity caused by autonomous selfing could incur a cost to male outcross fitness. We argue that this effect is likely to be most pronounced under intermediate rates of pollinator visitation. Observed pollinator visitation rates ranged from 0.035-0.775 visits per flower per day, indicating a potential for selfing-induced flower senescence to incur pollen discounting in Leptosiphon jepsonii.

Background: The theory of Condition Dependent Sex predicts that – everything else being equal – less fit individuals would outcross at higher rates compared with fitter ones. Here we used the mixed mating plant Lamium amplexicaule, capable of producing both self-pollinating closed flowers (CL), alongside open flowers (CH) that allow cross pollination to test it. We investigated the effects of abiotic stress – salt solution irrigation – on the flowering patterns of plants and their offspring. We monitored several flowering and vegetative parameters, including the number and distribution of flowers, CH fraction, and plant size. Results: We found that stressed plants show an increased tendency for self-pollination and a deficit in floral and vegetative development. However, when parentally primed, stressed plants show a milder response. Un-stressed offspring of stressed parents show reversed responses and exhibit an increased tendency to outcross, and improve floral and vegetative development. Conclusions: In summary, we found that stress affects the reproduction strategy in the plants that experienced the stress and in subsequent offspring through F2 generation. Our results provide experimental evidence supporting a transgenerational extension to the theories of fitness associate sex and dispersal, where an individual's tendency for sex and dispersal may depend on the stress experienced by its parents. [ABSTRACT FROM AUTHOR]

Introduction: Traditional methods for assessing movement quality rely on subjective standardized scales and clinical expertise. This limitation creates challenges for assessing patients with spinocerebellar ataxia (SCA), in whom changes in mobility can be subtle and varied. We hypothesized that a machine learning analytic system might complement traditional clinician-rated measures of gait. Our objective was to use a video-based assessment of gait dispersion to compare the effects of troriluzole with placebo on gait quality in adults with SCA. Methods: Participants with SCA underwent gait assessment in a phase 3, double-blind, placebo-controlled trial of troriluzole (NCT03701399). Videos were processed through a deep learning pose extraction algorithm, followed by the estimation of a novel gait stability measure, the Pose Dispersion Index, quantifying the frame-by-frame symmetry, balance, and stability during natural and tandem walk tasks. The effects of troriluzole treatment were assessed in mixed linear models, participant-level grouping, and treatment group-by-visit week interaction adjusted for age, sex, baseline modified Functional Scale for the Assessment and Rating of Ataxia (f-SARA), and time since diagnosis. Results: From 218 randomized participants, 67 and 56 participants had interpretable videos of a tandem and natural walk attempt, respectively. At Week 48, individuals assigned to troriluzole exhibited significant (p = 0.010) improvement in tandem walk Pose Dispersion Index versus placebo {adjusted interaction coefficient: 0.584 [95% confidence interval (CI) 0.137 to 1.031]}. A similar, nonsignificant trend was observed in the natural walk assessment [coefficient: 1.198 (95% CI − 1.067 to 3.462)]. Further, lower baseline Pose Dispersion Index during the natural walk was significantly (p = 0.041) associated with a higher risk of subsequent falls [adjusted Poisson coefficient: − 0.356 [95% CI − 0.697 to − 0.014)]. Conclusion: Using this novel approach, troriluzole-treated subjects demonstrated improvement in gait as compared to placebo for the tandem walk. Machine learning applied to video-captured gait parameters can complement clinician-reported motor assessment in adults with SCA. The Pose Dispersion Index may enhance assessment in future research. Trial Registration—ClinicalTrials.gov Identifier: NCT03701399. [ABSTRACT FROM AUTHOR]

Introduction: Vulnerably housed individuals access emergency departments (EDs) more frequently than the general population. Despite Canada's universal public health care system, vulnerably housed persons face structural barriers to care and experience discrimination from healthcare providers. This study examines how vulnerably housed persons perceive their experience of care in the ED and Urgent Care Center (UCC) in Kingston, Ontario and aims to develop strategies for improving care for this group. Methods: As part of a larger mixed-methods study, narratives were collected from participants attending the ED/UCC as well as community-based partner organizations, asking them to describe an experience of a recent ED visit (< 24 months). Participants could identify as members of up to three equity-deserving groups (EDGs) (for example homeless, part of an ethnic minority, having a disability, experiencing mental health issues). Coding and thematic analysis were completed for the experiences of participants who identified as being vulnerably housed (n = 171). Results were presented back to individuals with lived experience and service providers working with clients with unstable housing. Results: Participants reported judgement related to a past or presumed history of mental health or substance use and based on physical appearance. They also often felt unheard and that they were treated as less than human by healthcare providers. Lack of effective communication about the ED process, wait times, diagnosis, and treatment led to negative care experiences. Participants reported positive experiences when their autonomy in care-decision making was respected. Furthermore, having a patient-centered approach to care and addressing specific patient needs, identities and priorities led to positive care experiences. Conclusions: The ED care experiences of vulnerably housed persons may be improved through healthcare provider training related to trauma-informed and patient-centered care and communication strategies in the ED. Another potential strategy to improve care is to have advocates accompany vulnerably housed persons to the ED. Finally, improving access to primary care may lead to reduced ED visits and better longitudinal care for vulnerably housed persons. [ABSTRACT FROM AUTHOR]

Competition affects mixed-mating strategies by limiting available abiotic or biotic resources such as nutrients, water, space, or pollinators. Cleistogamous species produce closed (cleistogamous, CL), obligately selfed, simultaneously with open (chasmogamous, CH), potentially outcrossed flowers. The effects of intraspecific competition on fitness and cleistogamy variation can range from limiting the production of costly CH flowers because of resource limitation, to favouring CH production because of fitness advantages of outcrossed, CH offspring. Moreover, the effects of competition can be altered when it co-occurs with other environmental variations. We grew plants from seven populations of the ruderal Lamium amplexicaule , originating from different climates and habitats, in a common garden experiment combining drought, interspecific competition, and seasonal variation. All these parameters have been shown to influence the degree of cleistogamy in the species on their own. In spring, competition and drought negatively impacted fitness, but the CL proportion only increased when plants were exposed to both treatments combined. We did not observe the same results in autumn, which can be due to non-adaptive phenotypic variation, or to differences in soil compactness between seasons. The observed responses are largely due to phenotypic plasticity, but we also observed phenotypic differentiation between populations for morphological, phenological, and cleistogamy traits, pointing to the existence of different ecotypes. Our data do not support the hypothesis that CL proportion should decrease when resources are scarce, as plants with reduced growth had relatively low CL proportions. We propose that variation in cleistogamy could be an adaptation to pollinator abundance, or to environment-dependent fitness differences between offspring of selfed and outcrossed seeds, two hypotheses worth further investigation. This opens exciting new possibilities for the study of the maintenance of mixed-mating systems using cleistogamous species as models that combine the effects of inbreeding and reproductive costs. [ABSTRACT FROM AUTHOR]

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis. Vesicle transport ensures cargo delivery to cellular targets. Here, Soltani et al. show that Drosophila Evi5, a multiple sclerosis risk gene in humans, controls vesicular transport of iron-bound transferrins and physically interacts with ferritin. [ABSTRACT FROM AUTHOR]

Introduction: Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the Huntingtin gene (HTT). Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of Tyrobp, a microglial protein, ameliorates neuronal dysfunction in Alzheimer's disease amyloidopathy and tauopathy mouse models while decreasing components of the complement subnetwork. Objective: While TYROBP/DAP12-mediated microglial activation is detrimental for some diseases such as peripheral nerve injury, it is beneficial for other diseases. We sought to determine whether the TYROBP network is implicated in HD and whether Tyrobp deletion impacts HD striatal function and transcriptomics. Methods: To test the hypothesis that Tyrobp deficiency would be beneficial in an HD model, we placed the Q175 HD mouse model on a Tyrobp-null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the gene signature in isolated Q175 striatal microglia, with and without Tyrobp. Results: Comprehensive analysis of publicly available human HD transcriptomic data revealed that the TYROBP network is overactivated in the HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the Tyrobp-null background. Gene expression analysis revealed that lack of Tyrobp in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain, including genes identified as detrimental in neurodegenerative diseases, e.g. C1q and members of the Ccr5 signaling pathway. Integration of transcriptomic and proteomic data revealed that astrogliosis and complement system pathway were reduced after Tyrobp deletion, which was further validated by immunofluorescence analysis. Conclusions: Our data provide molecular and functional support demonstrating that Tyrobp deletion prevents many of the abnormalities in the HD Q175 mouse model, suggesting that the Tyrobp pathway is a potential therapeutic candidate for Huntington's disease. [ABSTRACT FROM AUTHOR]

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for l-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington's disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR–Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients. [ABSTRACT FROM AUTHOR]