bluuebunny/crossref_metadata_2025_split huggingface dataset:
Competing modes of assembly restrict amyloid formation by a neurodegenerative protein, TDP43
Authors: Wu Jianzheng , Halfmann Randal 
Published: April 2020

The accumulation of aberrant deposits of specific protein(s) in the brain is a hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (TDP43) and frontotemporal lobar degeneration (FUS and/or TDP43). These proteins exhibit “prion‐like” low complexity domains (LCDs), with the defining property of templating their own assembly into highly ordered aggregates known as amyloid. Structural biology investigations from other labs have revealed a multitude of TDP‐43 self‐assembly modes in vitro, ranging from cooperatively folding alpha‐helices to amyloid‐like beta‐sheets of varying stability. However, the extent to which these assembly modes occur in the intracellular environment, and their full relationship to physiological and/or pathological self‐assembly, have not yet been feasible to study.To interrogate mechanistic features of amyloid nucleation by the TDP43 LCD in the cellular milieu, I employed a recently developed FRET‐based flow cytometry approach along with rational mutagenesis informed by existing structural models. I observed that the LCD transitions from monomers to small condensates with a prominent phase boundary. I further found that these condensates were qualitatively distinct from amyloid, and that amyloid occurred probabilistically and only in cells harboring pre‐existing amyloids of other pathogenic proteins.Mutations designed to enhance or disrupt alpha‐helicity exhibited corresponding effects on non‐amyloid condensation, confirming the role of this assembly mode in non‐pathogenic aggregation. Remarkably, amyloid formation exhibited the opposite dependence, with strong alpha‐helix‐promoting mutations eliminating amyloid formation altogether. Mutations designed to probe the role of cooperative beta‐sheet formation revealed that amyloid‐like interactions either promote or inhibit amyloid formation depending on their stability. Importantly, I verified that the full‐length protein follows the same aggregation pathway, but does so at lower concentrations due to additional homotypic interactions mediated by non‐LCD domains. In summary, my data suggested the tendency of TDP43 toward amyloid is balanced by competing alternative modes of self‐assembly.Support or Funding InformationNIH Director’s Early Independence Award DP5‐OD009152 (to RH), the University of Kansas Alzheimer’s Disease CenterStowers Institute for Medical Research
Treatment with Sacubitril‐Valsartan Reduces Muscle Sympathetic Nerve Activity and Improves Functional Capacity in Heart Failure Patients with Reduced Ejection Fraction
Authors: Bunsawat Kanokwan , Ratchford Stephen M. , Alpenglow Jeremy K. , Stehlik Josef , Smith Adam , Richardson Russell S. , Wray D. Walter 
Published: April 2020

BackgroundChronic sympathetic overactivity and reduced functional capacity, characteristics of heart failure with reduced ejection fraction (HFrEF), are associated with poor prognosis and an increased risk of mortality in this patient group. Sacubitril‐valsartan, a recently approved angiotensin receptor neprilysin inhibitor (ARNI), reduces the risk of death from cardiovascular causes or hospitalization for heart failure in patients with HFrEF, but the mechanisms underlying its benefits are not fully understood. This study tested the hypothesis that treatment with sacubitril‐valsartan reduces muscle sympathetic nerve system activity and improves functional capacity in patients with HFrEF.MethodsIn nine Class II and III patients with HFrEF (69±3 yrs; 28.6±1.2 kg/m2), measurements of muscle sympathetic nerve activity (MSNA), functional capacity (six‐minute walk test (6MWT) distance), and blood pressure (BP) were performed at baseline prior to being transitioned to sacubitril‐valsartan. The measurements were repeated after two months of treatment with sacubitril‐valsartan.ResultsSacubitril‐valsartan treatment reduced MSNA burst frequency (baseline: 43±3 bursts/min; 2‐month: 36±3 bursts/min, p=0.05) and burst incidence (baseline: 68±5 bursts/min; 2‐month: 55±5 bursts/min, p=0.02), as well as improved 6MWT distance (baseline: 446±26 m; 2‐month: 500±30 m, p<0.001). The changes in MSNA burst frequency and burst incidence were not correlated with the change in 6MWT distance (r=0.13 and r=0.11, respectively; both p>0.05). No changes in BP were observed with the treatment (p>0.05).ConclusionSacubitril‐valsartan reduced sympathetic nervous system activity and improved functional capacity in patients with HFrEF, providing new evidence for the potential favorable effect of this new drug class on autonomic and functional outcomes in this patient group.Support or Funding InformationThis project is funded in part by the National Institutes of Health (HL118313; D.W.W., R56AG057584; R.S.R., T32HL139451; R.S.R. (recipient) and K.B. (trainee)), the U.S. Department of Veterans Affairs (I01RX001311; D.W.W., I01RX001697; R.S.R., I01RX002323; R.S.R., I21RX001433; R.S.R., I01RX000182; R.S.R.), and the American Heart Association (18POST33960192; K.B.).
The Role of Mitochondrial‐derived Reactive Oxygen Species in Non‐Invasive Anterior Cruciate Ligament Injury
Authors: Kwon Oh Sung , Davi Steven M. , White McKenzie S. , Lepley Lindsey K. 
Published: April 2020

BACKGROUNDAnterior cruciate ligament (ACL) injury results in protracted quadriceps atrophy, however the mechanisms that drive atrophic pathways remain undefined. Mounting evidence has revealed that mitochondrial dysfunction and redox disturbances are causal events in the initiation of muscle atrophy, yet the extent to which mitochondria damage play a key role in quadriceps muscle atrophy after ACL injury has yet to be explored.PURPOSEUsing a pre‐clinical animal model of ACL injury, a time course study was performed to investigate the role of mitochondria function and Reactive Oxygen Species (ROS) after ACL injury.METHODS48 Long Evans rats (n=8 per group; 4m/4f) underwent non‐invasive rupture of the right ACL and were euthanized at 1, 3, 7, 14, 28, 56 days post‐injury. 8 rats (4m/4f) served as healthy controls (HC). Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the right quadriceps. ROS production was determined using Amplex Red assays. ROS production was determined using Amplex Red assays. VL weight was normalized to total body mass for measuring muscle mass loss. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P < 0.05).RESULTSReductions in complex I + II state 3 respiration were observed at 7 and 56 days post‐injury (HC: 33.92 ± 4.26 pmol·s−1·mg−1; 7D: 15.95 ± 1.38 pmol·s−1·mg−1; 56D: 18.80 ± 2.15 pmol·s−1·mg−1; F = 5.99, P = 0.002 and P = 0.015 respectively). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased 7 through 56 days post‐injury (HC: 4.00 ± 0.13; 7D: 1.54 ± 0.16; 56D: 1.82 ± 0.20; F = 13.29, P = 0.001 and P = 0.001 respectively) along with increased mitochondrial ROS production 7 through 56 days post‐injury (HC: 10.16 ± 0.41 pmol·s−1·mg−1; 7D: 12.23 ± 0.54 pmol·s−1·mg−1; 56D: 26.83 ± 0.54 pmol·s−1·mg−1; F = 178.05, P = 0.001 and P = 0.001 respectively). VL atrophy was observed at 7 and 14 days post‐injury (HC: 3.52 ± 0.08 mg·g−1; 7D: 3.14 ± 0.07 mg·g−1; 14D: 3.18 ± 0.07 mg·g−1; F = 5.06, P = 0.013 and P = 0.044 respectively).CONCLUSIONThese results demonstrated that mitochondrial dysfunction and redox disturbances contribute to ACL injury‐induced quadriceps atrophy and that mitochondria are an important source of muscular ROS production after ACL injury.Support or Funding InformationNIH grant K01AR071503 from NIAMS
MERS‐CoV infection is associated with downregulation of genes encoding Th1 and Th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract
Authors: Alosaimi Bandar A. 
Published: April 2020

MERS‐CoV, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. Inflammatory responses have a significant impact on MERS‐CoV pathogenesis and disease outcome. However, CD4+ T‐cell induced immune responses during acute MERS‐CoV infection are barely detectable, with potent inhibition of effector T cells and downregulation of antigen presentation. The local pulmonary immune response, particularly the Th1 and Th2‐related immune response during acute severe MERS‐CoV infection is not fully understood. In this study, we offer the first insights into the pulmonary gene expression profile of Th1 and Th2‐related cytokines/chemokines (Th1 & Th2 responses) during acute MERS‐CoV infection using RT2 Profiler PCR Arrays. We also quantified the expression level of primary inflammatory cytokines/chemokines. Our results showed a downregulation of Th2, inadequate (partial) Th1 immune response and high expression levels of inflammatory cytokines IL‐1α and IL‐1β and the neutrophil chemoattractant chemokine IL‐8 (CXCL8) in the lower respiratory tract of MERS‐CoV infected patients. Moreover, we identified a high viral load in all included patients. We also observed a correlation between inflammatory cytokines, Th1, and Th2 downregulation and the case fatality rate. Th1 and Th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ARDS, and a higher case fatality rate. Further study of the molecular mechanisms underlying the Th1 and Th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies.Support or Funding InformationThe authors thank the Research Center at King Fahad Medical City for funding this study (Grant No 017‐066).
Discovery and Characterization of Small Molecule Potentiators of KCC2 Activity
Authors: Prael Francis J. , Delpire Eric , Weaver C. David 
Published: April 2020

Deficits in inhibitory neurotransmission, and the resulting excitatory/inhibitory (E/I) imbalance, are a feature of a host of neurological disorders, including: epilepsy, chronic pain, schizophrenia, and autism. Increased intracellular Cl− ([Cl−]i) has been implicated as an underlying cause of the E/I imbalance via disruption of fast inhibitory neurotransmission. The K+‐Cl− cotransporter 2 (KCC2) is a principal exporter of neuronal Cl−. Owing to the effect of KCC2 on [Cl−]i, pharmacological potentiation of KCC2 activity has become a promising therapeutic approach for these neurological disorders. While there has been substantial progress in discovering pharmacological potentiators of KCC2 activity in recent years, current pharmacological tools targeting KCC2 either act by indirect mechanisms or have some controversy surrounding their activity at KCC2. Although these compounds represent major improvements to KCC2 potentiator pharmacology, the discovery of direct‐acting KCC2 potentiators would still be a substantial contribution to the field because: (a) direct‐acting KCC2 potentiators would be better chemical tools to assess the contribution of KCC2 to a given phenotype, since they would lack the off‐target effects associated with indirect KCC2 potentiators, and (b) de‐risk KCC2 itself as a therapeutic target for drug discovery. To discover direct‐acting KCC2 potentiators, we have developed and validated a suite of high throughput‐compatible assays to measure KCC2 activity. Using a primary assay measuring Cl− export via a genetically‐encoded Cl− sensor, we have iteratively screened over 30,000 compounds for their effect on KCC2 activity. Our iterative screening approach maximized our ability to explore chemical scaffolds that were active across primary and orthogonal assays – allowing us to discover a series of putative KCC2 potentiators, despite screening a relatively small chemical library. From these efforts, we have discovered compounds capable of decreasing [Cl−]i in a KCC2 inhibitor‐sensitive manner, potentiating transport of KCC2 substrate congeners in a KCC2‐dependent fashion, and blocking epileptiform calcium oscillations in cultured neurons. We are currently investigating the mechanism by which these compounds affect KCC2 activity. The compound class we have discovered may be useful as a pharmacological tool for evaluating the therapeutic potential of KCC2 in a host of neurological disorders which feature E/I imbalance.Support or Funding InformationVanderbilt Institute for Clinical and Translational Research Award (VR52556)Chemistry‐Biology Interface T32 (5T32GM065086‐15)
How Do O‐Fucose Glycans Stabilize Thrombospondin Type 1 Repeats?
Authors: Berardinelli Steven J. , Manjunath Rajashri , Eletsky Alexander , Ito Atsuko , Haltiwanger Robert S. 
Published: April 2020

Thrombospondin Type 1 Repeats (TSRs) are small cysteine‐rich domains found in multiple cell‐surface and secreted proteins. TSRs containing the consensus sequence Cxx(S/T)C are typically modified on the serine or threonine with an O‐linked Glcβ1‐3Fuc disaccharide. The O‐fucose is added by Protein O‐fucosyltransferase 2 (POFUT2), which is then elongated by β1‐3glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in early embryonic lethality, and human mutations in B3GLCT cause Peters Plus Syndrome (PPS), a Congenital Disorder of Glycosylation. POFUT2 and B3GLCT are localized in the endoplasmic reticulum (ER) ‐ the folding compartment for proteins in the secretory pathway, and POFUT2 only adds fucose to properly folded TSRs. Thus, POFUT2 appears to function as a folding sensor for TSRs, and both POFUT2 and B3GLCT have been proposed to assist in the folding of TSR‐containing proteins. Interestingly, loss of POFUT2 causes secretion defects for most TSR‐containing proteins, while loss of B3GLCT only effects secretion of a subset of these proteins. The mechanisms behind why POFUT2 is required for section of most TSR‐containing proteins but B3GLCT is required for only a few is not understood. We hypothesize that this unique disaccharide on TSRs is interacting with amino acids in close proximity to the sugars thereby stabilizing the folded state of TSRs and ultimately the protein as a whole, and that the fucose has the major stabilizing effect, while the glucose is only required for some TSRs. To test this hypothesis, we designed a Reductive Unfolding Assay to monitor the effects of the sugars on the stability of TSRs. We have identified amino acids affected by the presence of the fucose or glucose using NMR and molecular dynamics simulations. We have mutated several of these amino acids to determine if they reduce the stabilizing effects of the sugars using the reductive unfolding assay. One such mutant, P547A in TSR3 from human thrombospondin‐1, decreases the sugar‐mediated stabilization of TSR3, providing evidence that the disaccharide stabilizes the folded state by interacting with certain amino acids in close proximity. We are examining the stabilizing effects of the fucose and glucose on several other TSRs as well. These results provide further evidence that POFUT2 and B3GLCT act as quality control enzymes inside the ER.Support or Funding InformationThis work was supported by NIH grant HD090156.
Saturated fatty acid‐enriched diet exacerbate hepatic mitochondrial dysfunction in rats subjected to maternal protein restriction
Authors: Simões-Alves Aiany C. , Costa-Silva João H. , Barros-Junior Idelfonso B. , da Silva-Filho Reginaldo Correia , Vasconcelos Diogo A. A. , Morio Béatrice , Fernandes Mariana P. 
Published: April 2020

The nutritional transition that the western population has undergone is increasingly associated with chronic metabolic diseases. In this context, dietary factors has been linked to oxidative stress, making the body vulnerable to mitochondrial dysfunction and reactive oxygen species. In this work, we evaluated the impact of a saturated fatty acid‐enriched diet in the post‐weaning offspring subjected to maternal protein restriction on the liver mitochondrial bioenergetics. Wistar rats were mated and during gestation and lactation, mothers received control diets (NP, normal protein content 17%) or low protein (LP, 8% protein). After weaning, rats received either NL (normolipidic) or HL (+59% SFA) diets up to 90 days of life. It was verified that all respiratory states of hepatic mitochondria showed a reduction in the LP group subjected to the post‐weaning HL diet (LP‐HL), basal state (−43.7%), state 3 (−37.3%), state 4 (−32.1%) and uncoupled state (−60.4%). Similarly, respiratory control was significantly lower (p<0.01). This group also presented greater mitochondrial swelling compared to controls (p<0.01), potentiated after Ca2+ addition (p <0,01) and prevented in the presence of EGTA (calcium chelator) (p<0.001); and cyclosporin A (mitochondrial permeability transition pore inhibitor) (p<0.0001). These effects were associated to increased liver protein and lipid peroxidation and reduced anti‐oxidant enzymes activities in the LP group fed HL diet after weaning. Additionaly, it was observed increase in PGC‐1α and VDAC and decrease in Tfam gene expressions compared to LP‐NL (p<0.05). Our data suggest that SFA‐enriched HFD induces mitochondrial dysfunction associated with oxidative stress, greater expression of VDAC that may potentiate the opening of the mitochondrial permeability transition pore, and lower mitochondrial biogenesis in the liver of adults rats, with major effects in animals previously submitted to maternal protein restriction.Support or Funding InformationThis study was supported by FACEPE (Brazil), CNPq (Brazil) and CAPES/COFECUB (Brazil/France).
Spinal cord injury leads to increases in apnea frequency and duration in spontaneously breathing mice with and without central nervous system 5HT
Authors: Qiu Qingchao , Kariharan Thiruchelvam , Kuhn Donald , Mateika Jason 
Published: April 2020

PurposeThe present investigation was designed to explore the impact of serotonin (5HT) on apnea severity and duration, arousal frequency and respiratory stability following spinal cord injury (SCI) in mice.MethodsTelemetry transmitters were surgically implanted in wild type mice (Tph2+/+) (n = 4) and tryptophan hydroxylase 2 knockout mice (Tph2−/−) (n = 3) to measure electroencephalography, core body temperature and gross motor activity. Following recovery, the Tph2+/+ and Tph2−/− mice were placed in whole body plethysmographs and measures of apnea severity, coefficient of variation of the duration of each breath, duration index and arousal frequency during non‐rapid eye movement sleep were determined for 6 hours. Following these measures, a C2 hemisection of the spinal cord was completed and the physiological measurements were repeated 4 and 21 days following hemisection.ResultsFollowing SCI the number of apnea events increased in the Tph2+/+ (baseline vs. 21 days: 52.1 ± 17.4 vs. 106.4 ± 21.2 events/hr, p = 0.02) and the Tph2−/− mice (30.7 ± 9.6 vs. 77.5 ± 34.1, p = 0.02). The coefficient of variance of the duration of each breath also increased after SCI (Tph2+/+ 0.25 ± 0.02 vs. 0.30 ± 0.06, Tph2−/− 0.25 ± 0.02 vs. 0.35 ± 0.06, p = 0.07). Likewise, the apnea duration index increased following SCI in the Tph2+/+ (baseline vs. 21 days: 2.4 ± 0.05 vs. 3.2 ± 0.6 events/hr, p = 0.047) and Tph2−/− mice (baseline vs. 21 days: 2.6 ± 0.5 vs. 3.8 ± 0.5 events/hr, p = 0.047). Lastly, the number of arousals increased after SCI in both groups (Tph2+/+ 19.0 ± 4.6 vs. 39.1 ± 13.8, Tph2−/− 5.1 ± 0.9 vs. 35.6 ± 19.4 arousal/hr, p = 0.03).ConclusionsSCI leads to an increase in the frequency and duration of apneic events, which is coupled to an increase in the number of arousals. Likewise, alterations in the coefficient of variation indicate that breathing is more variable when present. However, the role of 5HT in mediating arousals and stabilizing breathing is less certain in this small sample size, since modifications in breathing stability and arousal frequency was not statistically different in the Tph2+/+ and Tph2−/− mice following SCI.Support or Funding InformationDepartment of Veterans Affairs (I01BX003946 & 15SRCS003 JHM)
A fine‐tuned interaction between the ER and mitochondria regulates Ca2+‐dependent after hyperpolarizations in vasopressin supraoptic neurons
Authors: Kirchner Matthew Karl , Stern Javier Eduardo 
Published: April 2020

It is known that vasopressin magnocellular neurons become hyperexcitable in cardiovascular diseases such as heart failure. This hyperexcitability is mediated by shifts in both synaptic and intrinsic mechanisms. One key intrinsic mechanism is the slow afterhyperpolarization (sAHP), a phenomenon underlain by a calcium‐dependent K+ current (IsAHP) most notable for its role in spike frequency adaptation. While features of the sAHP and its effects on firing are well established, a comprehensive understanding of the underlying mechanisms are still unknown. Previous work demonstrated that sAHP activation requires Ca2+ entry via voltage‐gated Ca2+ channels (VGCCs). However, whether in VP neurons Ca2+ entry via these channels directly activates the sAHP, or alternatively, whether it stimulates Ca2+‐induced Ca2+ release from an internal source such as endoplasmic reticulum (ER) or mitochondria remains unknown. Combining patch clamp electrophysiology with simultaneous Ca2+ imaging in transgenic eGFP‐VP rats, we investigated the role of ER and mitochondrial in shaping the sAHP. We measured sAHPs in voltage clamp by evoking a train of action potentials and measuring the resulting outward current. Pre‐incubation of slices with thapsigargin (3μM), an inhibitor of the sarco/ER Ca2+ ATPase inhibited IsAHPs significantly (96%, p< 0.001) while leaving Ca2+ transients unaffected (p > 0.05). This demonstrates that ER Ca2+ release is necessary for sAHP activation. Conversely, blockade of mitochondrial Ca2+ release via blocking mitochondrial Ca2+ exchangers with TPP (20μM) failed to inhibit the IsAHP (p > 0.05). We next evaluated the role of mitochondrial Ca2+ uptake with bath application of the mitochondrial uncoupler CCCP (1μM) or the mitochondrial Ca2+ uniporter (MCU) blocker Ru360 (20μM) in the patch pipette. These manipulations significantly prolonged the decay time constant of both the IsAHP (p< 0.01) and the underlying Ca2+ transient (p< 0.05), demonstrating that the MCU regulates spatiotemporal dynamics of the sAHP. Finally, combining thapsigargin with Ru360 in the pipette showed an initial block of the sAHP, followed by an enhancement over 15 minutes as Ru360 dialyzed into the cell. These results support the ER (but not mitochondria) as a major source of Ca2+ activating the sAHP, as well as a role for the MCU in regulating the magnitude and time course of the sAHP. Taken together, our studies support a fine‐tuned interaction between VGCCs, ER, and mitochondria in modulating the magnitude and time course of the sAHP in VP neurons. Future studies warrant investigation into whether changes in these interactions contribute to altered VP neuronal activity in cardiovascular disease states.Support or Funding InformationR01HL090948