Longitudinal transcriptomic analysis of mouse sciatic nerve reveals pathways associated with age-related muscle pathology.

BACKGROUND: Sarcopenia, the age-associated decline in skeletal muscle mass and strength, has long been considered a disease of muscle only, but accumulating evidence suggests that sarcopenia could originate from the neural components controlling muscles. To identify early molecular changes in nerves that may drive sarcopenia initiation, we performed a longitudinal transcriptomic analysis of the sciatic nerve, which governs lower limb muscles, in aging mice. METHODS: Sciatic nerve and gastrocnemius muscle were obtained from female C57BL/6JN mice aged 5, 18, 21 and 24 months old (n = 6 per age group). Sciatic nerve RNA was extracted and underwent RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were validated using quantitative reverse transcription PCR (qRT-PCR). Functional enrichment analysis of clusters of genes associated with patterns of gene expression across age groups (adjusted P-value < 0.05, likelihood ratio test [LRT]) was performed. Pathological skeletal muscle aging was confirmed between 21 and 24 months by a combination of molecular and pathological biomarkers. Myofiber denervation was confirmed with qRT-PCR of Chrnd, Chrng, Myog, Runx1 and Gadd45ɑ in gastrocnemius muscle. Changes in muscle mass, cross-sectional myofiber size and percentage of fibres with centralized nuclei were analysed in a separate cohort of mice from the same colony (n = 4-6 per age group). RESULTS: We detected 51 significant DEGs in sciatic nerve of 18-month-old mice compared with 5-month-old mice (absolute value of fold change > 2; false discovery rate [FDR] < 0.05). Up-regulated DEGs included Dbp (log2 fold change [LFC] = 2.63, FDR < 0.001) and Lmod2 (LFC = 7.52, FDR = 0.001). Down-regulated DEGs included Cdh6 (LFC = -21.38, FDR < 0.001) and Gbp1 (LFC = -21.78, FDR < 0.001). We validated RNA-seq findings with qRT-PCR of various up- and down-regulated genes including Dbp and Cdh6. Up-regulated genes (FDR < 0.1) were associated with the AMP-activated protein kinase signalling pathway (FDR = 0.02) and circadian rhythm (FDR = 0.02), whereas down-regulated DEGs were associated with biosynthesis and metabolic pathways (FDR < 0.05). We identified seven significant clusters of genes (FDR < 0.05, LRT) with similar expression patterns across groups. Functional enrichment analysis of these clusters revealed biological processes that may be implicated in age-related changes in skeletal muscles and/or sarcopenia initiation including extracellular matrix organization and an immune response (FDR < 0.05). CONCLUSIONS: Gene expression changes in mouse peripheral nerve were detected prior to disturbances in myofiber innervation and sarcopenia onset. These early molecular changes we report shed a new light on biological processes that may be implicated in sarcopenia initiation and pathogenesis. Future studies are warranted to confirm the disease modifying and/or biomarker potential of the key changes we report here.

Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

Genomic testing for copy number and single nucleotide variants in spermatogenic failure.

PURPOSE: To identify clinically significant genomic copy number (CNV) and single nucleotide variants (SNV) in males with unexplained spermatogenic failure (SPGF). MATERIALS AND METHODS: Peripheral blood DNA from 97/102 study participants diagnosed with oligozoospermia, severe oligozoospermia, or non-obstructive azoospermia (NOA) was analyzed for CNVs via array comparative genomic hybridization (aCGH) and SNVs using whole-exome sequencing (WES). RESULTS: Of the 2544 CNVs identified in individuals with SPGF, > 90% were small, ranging from 0.6 to 75 kb. Thirty, clinically relevant genomic aberrations, were detected in 28 patients (~ 29%). These included likely diagnostic CNVs in 3/41 NOA patients (~ 7%): 1 hemizygous, intragenic TEX11 deletion, 1 hemizygous DDX53 full gene deletion, and 1 homozygous, intragenic STK11 deletion. High-level mosaicism for X chromosome disomy (~ 10% 46,XY and ~ 90% 47,XXY) was also identified in 3 of 41 NOA patients who previously tested normal with conventional karyotyping. The remaining 24 CNVs detected were heterozygous, autosomal recessive carrier variants. Follow-up WES analysis confirmed 8 of 27 (30%) CNVs (X chromosome disomy excluded). WES analysis additionally identified 13 significant SNVs and/or indels in 9 patients (~ 9%) including X-linked AR, KAL1, and NR0B1 variants. CONCLUSION: Using a combined genome-wide aCGH/WES approach, we identified pathogenic and likely pathogenic SNVs and CNVs in 15 patients (15%) with unexplained SPGF. This value equals the detection rate of conventional testing for aneuploidies and is considerably higher than the prevalence of Y chromosome microdeletions. Our results underscore the importance of comprehensive genomic analysis in emerging diagnostic testing of complex conditions like male infertility.

Distinct profiles of LRRK2 activation and Rab GTPase phosphorylation in clinical samples from different PD cohorts.

Despite several advances in the field, pharmacodynamic outcome measures reflective of LRRK2 kinase activity in clinical biofluids remain urgently needed. A variety of targets and approaches have been utilized including assessments of LRRK2 itself (levels, phosphorylation), or its substrates (e.g. Rab10 or other Rab GTPases). We have previously shown that intrinsic kinase activity of LRRK2 isolated from PBMCs of G2019S carriers is elevated, irrespective of disease status. In the present study we find that phosphorylation of Rab10 is also elevated in G2019S carriers, but only those with PD. Additionally, phosphorylation of this substrate is also elevated in two separate idiopathic PD cohorts, but not in carriers of the A53T mutation in α-synuclein. In contrast, Rab29 phosphorylation was specifically reduced in urinary exosomes from A53T and idiopathic PD patients. Taken together, our findings highlight the need for the assessment of multiple complimentary targets for a more comprehensive picture of the disease.

[Place of preperitoneal pelvic packing in severe pelvic traumatisms: 20 cases in a French level 1 trauma center]. / Place du packing pré-péritonéal pelvien dans les traumatismes graves du bassin : étude à partir de 20 cas réalisés dans un trauma center de niveau 1 français.

BACKGROUND: The overall mortality of hemodynamically unstable patients with pelvic trauma is high. Their management is controversial concerning places of arterioembolization and pelvic packing associated with pelvic stabilization. The aim of this study was to collect the pre-peritoneal pelvic packing (PPP) performed in our institution over 10years in order to propose a management algorithm. METHOD: From January 2010 to December 2020, all patients with a hemodynamically unstable pelvic fracture who had PPP combined with pelvic stabilization were included. Data were collected prospectively and analyzed retrospectively. The main judgement criteria were early hemorrhage-induced mortality (<24h) and overall mortality (<30d). RESULTS: Twenty patients had PPP out of 287 polytrauma patients with pelvic fracture. The first-line PPP proposed in our algorithm significantly reduced the number of red blood cells (RBCs) (P=0.0231) and improved systolic blood pressure (SBP) (P<0.001) within 24hours of first-line PPP (compared with preoperative). Six patients (30%) were embolized postoperatively for active bleeding not necessarily pelvic. The overall mortality at 30days was 50% (10/20). CONCLUSION: PPP is a fast, easy, effective and safe procedure for venous, bone and sometimes arterial bleeding. PPP is part of damage control surgery and we propose it as a first-line procedure. AE remains complementary in a second step.

Defining (and blocking) neuronal death in Parkinson's disease: Does it matter what we call it?

Parkinson's disease (PD) is the second most common neurodegenerative disease, comprised of both familial and idiopathic forms, behind only Alzheimer's disease (AD). The disease is characterized, regardless of the pathogenesis, primarily by a loss of DA neurons in the ventral midbrain as well as noradrenergic neurons of the locus coeruleus; however, by the time symptoms manifest, considerable neuronal loss in both areas has occurred. Neuroprotective strategies thus have to be paired with more sensitive and specific biomarker assays that can identify early at-risk patients in order to initiate disease-modifying therapies at an earlier stage in the disease. Complicating this is the fact that multiple forms of cell death mediate the neuronal loss; however, with a common underlying element that the cell death is considered a "regulated" form of cell death, in contrast to an un-controlled necrotic cell death process. In this review we focus our discussion on several categories of regulated cell death in the context of PD: apoptosis, necroptosis, pyroptosis, and autophagic cell death. In clinical studies as well as experimental in vivo models of PD, there is evidence for a role of each of these forms of cell death in the loss of midbrain DA neurons, and specific therapeutic strategies have been proposed and tested. What remains unclear however is the relative contributions of these distinct forms of cell death to the overall loss of DA neurons, whether they occur at different stages of the disease, or whether specific sub-regions within the midbrain are more susceptible to specific death triggers and pathways.

Polygenic Risk Scoring is an Effective Approach to Predict Those Individuals Most Likely to Decline Cognitively Due to Alzheimer's Disease.

BACKGROUND: There is a clear need for simple and effective tests to identify individuals who are most likely to develop Alzheimer's Disease (AD) both for the purposes of clinical trial recruitment but also for improved management of patients who may be experiencing early pre-clinical symptoms or who have clinical concerns. OBJECTIVES: To predict individuals at greatest risk of progression of cognitive impairment due to Alzheimer's Disease in individuals from the Alzheimer's Disease Neuroimaging Initiative (ADNI) using a polygenic risk scoring algorithm. To compare the performance of a PRS algorithm in predicting cognitive decline against that of using the pTau/Aß1-42 ratio CSF biomarker profile. DESIGN: A longitudinal analysis of data from the Alzheimer's Disease Neuroimaging Initiative study conducted across over 50 sites in the US and Canada. SETTING: Multi-center genetics study. PARTICPANTS: 515 subjects who upon entry to the study were diagnosed as cognitively normal or with mild cognitive impairment. MEASUREMENTS: Use of genotyping and/or whole genome sequencing data to calculate polygenic risk scores and assess ability to predict subsequent cognitive decline as measured by CDR-SB and ADAS-Cog13 over 4 years. RESULTS: The overall performance for predicting those individuals who would decline by at least 15 ADAS-Cog13 points from a baseline mild cognitive impairment in 4 years was 72.8% (CI:67.9-77.7) AUC increasing to 79.1% (CI: 75.6-82.6) when also including cognitively normal participants. Assessing mild cognitive impaired subjects only and using a threshold of greater than 0.6, the high genetic risk participant group declined, on average, by 1.4 points (CDR-SB) more than the low risk group over 4 years. The performance of the PRS algorithm tested was similar to that of the pTau/Aß1-42 ratio CSF biomarker profile in predicting cognitive decline. CONCLUSION: Calculating polygenic risk scores offers a simple and effective way, using DNA extracted from a simple mouth swab, to select mild cognitively impaired patients who are most likely to decline cognitively over the next four years.

The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson's Disease.

Evidence is mounting that LRRK2 function, particularly its kinase activity, is elevated in multiple forms of Parkinson's disease, both idiopathic as well as familial forms linked to mutations in the LRRK2 gene. However, sensitive quantitative markers of LRRK2 activation in clinical samples remain at the early stages of development. There are several measures of LRRK2 activity that could potentially be used in longitudinal studies of disease progression, as inclusion/exclusion criteria for clinical trials, to predict response to therapy, or as markers of target engagement. Among these are levels of LRRK2, phosphorylation of LRRK2 itself, either by other kinases or via auto-phosphorylation, its in vitro kinase activity, or phosphorylation of downstream substrates. This is advantageous on many levels, in that multiple indices of elevated kinase activity clearly strengthen the rationale for targeting this kinase with novel therapeutic candidates, and provide alternate markers of activation in certain tissues or biofluids for which specific measures are not detectable. However, this can also complicate interpretation of findings from different studies using disparate measures. In this review we discuss the current state of LRRK2-focused biomarkers, the advantages and disadvantages of the current pallet of outcome measures, the gaps that need to be addressed, and the priorities that the field has defined.

Elevated In Vitro Kinase Activity in Peripheral Blood Mononuclear Cells of Leucine-Rich Repeat Kinase 2 G2019S Carriers: A Novel Enzyme-Linked Immunosorbent Assay-Based Method.

BACKGROUND: Leucine-rich repeat kinase 2 kinase inhibitors are being vigorously pursued as potential therapeutic options; however, there is a critical need for sensitive and quantitative assays of leucine-rich repeat kinase 2 function and target engagement. OBJECTIVES: Our objective was to compare collection and storage protocols for peripheral blood mononuclear cells, and to determine the optimal conditions for downstream analyses of leucine-rich repeat kinase 2 in PD cohorts. METHODS: Here, we describe enzyme-linked immunosorbent assay-based assays capable of detecting multiple aspects of leucine-rich repeat kinase 2 function at endogenous levels in human tissues. RESULTS: In peripheral blood mononuclear cells from both healthy and affected carriers of the G2019S mutation in leucine-rich repeat kinase 2, we report, for the first time, significantly elevated in vitro kinase activity, while detecting a significant increase in pS935/leucine-rich repeat kinase 2 in idiopathic PD patients. CONCLUSIONS: Quantitative assays such as these described here could potentially uncover specific markers of leucine-rich repeat kinase 2 function that are predictive of disease progression, aid in patient stratification, and be a critical component of upcoming clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Future of Targeted Gene-Based Treatment Strategies and Biomarkers in Parkinson's Disease.

Biomarkers and disease-modifying therapies are both urgent unmet medical needs in the treatment of Parkinson's disease (PD) and must be developed concurrently because of their interdependent relationship: biomarkers for the early detection of disease (i.e., prior to overt neurodegeneration) are necessary in order for patients to receive maximal therapeutic benefit and vice versa; disease-modifying therapies must become available for patients whose potential for disease diagnosis and prognosis can be predicted with biomarkers. This review provides an overview of the milestones achieved to date in the therapeutic strategy development of disease-modifying therapies and biomarkers for PD, with a focus on the most common and advanced genetically linked targets alpha-synuclein (SNCA), leucine-rich repeat kinase-2 (LRRK2) and glucocerebrosidase (GBA1). Furthermore, we discuss the convergence of the different pathways and the importance of patient stratification and how these advances may apply more broadly to idiopathic PD. The heterogeneity of PD poses a challenge for therapeutic and biomarker development, however, the one gene- one target approach has brought us closer than ever before to an unprecedented number of clinical trials and biomarker advancements.

Culture-independent and dependent evaluation of the equine paranasal sinus microbiota in health and disease.

BACKGROUND: Horses with bacterial sinusitis frequently undergo empirical treatment with antimicrobials, however, in some cases bacterial culture of the affected sinus is used to direct therapy. Data regarding which organisms are part of the commensal microbiota of the equine sinus are lacking making it difficult to interpret culture results and guide empiric antimicrobial selection. OBJECTIVES: Our objectives were to describe the bacterial and fungal microbiota of the paranasal sinuses in clinically normal horses using culture-dependent and independent approaches and to compare the bacterial culture and susceptibility patterns of normal horses with those from horses affected with primary and secondary sinusitis. STUDY DESIGN: Experimental study and descriptive retrospective review of case records. METHODS: Sinus washes were collected from 23 healthy horses. Washes were submitted for routine culture and susceptibility testing and DNA was isolated for next generation sequencing of bacterial and fungal marker genes. For clinical cases of sinusitis, medical records from 2010 to 2017 were reviewed and horses diagnosed with primary and/or secondary sinusitis were included. RESULTS: The paranasal sinus cavity hosts multiple bacterial and fungal organisms. The bacterial microbiota of healthy horses consists largely of uncultivable, aerobic bacteria. While few anaerobes were isolated from normal horses, the majority of clinical cases resulted in growth of anaerobic organisms with no difference in the proportion of aerobic and anaerobic bacteria isolated from clinical cases. MAIN LIMITATIONS: Small sample size in both populations of horses and heterogeneity of the population prevent a more in-depth analysis. CONCLUSIONS: The microbiota of the paranasal sinuses of horses consists primarily of aerobic bacteria and fungal organisms, the majority of which are uncultivable via common clinical methods. Anaerobic bacteria are found in the majority of horses with clinical sinusitis. These findings suggest anaerobic bacteria are associated with sinusitis and their presence should be considered when treating horses with sinusitis.

Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes.

The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S-LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S-LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S-LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.

Polygenic Risk Score Analysis of Alzheimer's Disease in Cases without APOE4 or APOE2 Alleles.

The We and others have previously shown that polygenic risk score analysis (PRS) has considerable predictive utility for identifying those at high risk of developing Alzheimer's disease (AD) with an area under the curve (AUC) of >0.8. However, by far the greatest determinant of this risk is the apolipoprotein E locus with the E4 allele alone giving an AUC of ~0.68 and the inclusion of the protective E2 allele increasing this to ~0.69 in a clinical cohort. An important question is to determine how good PRS is at predicting risk in those who do not carry the E4 allele (E3 homozygotes, E3E2 and E2E2) and in those who carry neither the E4 or E2 allele (i.e. E3 homozygotes). Previous studies have shown that PRS remains a significant predictor of AD risk in clinical cohorts after controlling for APOE ε4 carrier status. In this study we assess the accuracy of PRS prediction in a cohort of pathologically confirmed AD cases and controls. The exclusion of APOE4 carriers has surprisingly little effect on the PRS prediction accuracy (AUC ~0.83 [95% CI: 0.80-0.86]), and the accuracy remained higher than that in clinical cohorts with APOE included as a predictor. From a practical perspective this suggests that PRS analysis will have predictive utility even in E4 negative individuals and may be useful in clinical trial design.

P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity.

Autosomal-dominant, missense mutations in the leucine-rich repeat protein kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson's disease (PD). LRRK2 kinase activity is increased in several pathogenic mutations (N1437H, R1441C/G/H, Y1699C, G2019S), implicating hyperphosphorylation of a substrate in the pathogenesis of the disease. Identification of the downstream targets of LRRK2 is a crucial endeavor in the field to understand LRRK2 pathway dysfunction in the disease. We have identified the signaling adapter protein p62/SQSTM1 as a novel endogenous interacting partner and a substrate of LRRK2. Using mass spectrometry and phospho-specific antibodies, we found that LRRK2 phosphorylates p62 on Thr138 in vitro and in cells. We found that the pathogenic LRRK2 PD-associated mutations (N1437H, R1441C/G/H, Y1699C, G2019S) increase phosphorylation of p62 similar to previously reported substrate Rab proteins. Notably, we found that the pathogenic I2020T mutation and the risk factor mutation G2385R displayed decreased phosphorylation of p62. p62 phosphorylation by LRRK2 is blocked by treatment with selective LRRK2 inhibitors in cells. We also found that the amino-terminus of LRRK2 is crucial for optimal phosphorylation of Rab7L1 and p62 in cells. LRRK2 phosphorylation of Thr138 is dependent on a p62 functional ubiquitin-binding domain at its carboxy-terminus. Co-expression of p62 with LRRK2 G2019S increases the neurotoxicity of this mutation in a manner dependent on Thr138. p62 is an additional novel substrate of LRRK2 that regulates its toxic biology, reveals novel signaling nodes and can be used as a pharmacodynamic marker for LRRK2 kinase activity.

A motif within the armadillo repeat of Parkinson's-linked LRRK2 interacts with FADD to hijack the extrinsic death pathway.

In experimental models, both in vivo and cellular, over-expression of Parkinson's linked mutant leucine-rich repeat kinase 2 (LRRK2) is sufficient to induce neuronal death. While several cell death associated proteins have been linked to LRRK2, either as protein interactors or as putative substrates, characterization of the neuronal death cascade remains elusive. In this study, we have mapped for the first time the domain within LRRK2 that mediates the interaction with FADD, thereby activating the molecular machinery of the extrinsic death pathway. Using homology modeling and molecular docking approaches, we have identified a critical motif within the N-terminal armadillo repeat region of LRRK2. Moreover, we show that co-expression of fragments of LRRK2 that contain the FADD binding motif, or deletion of this motif itself, blocks the interaction with FADD, and is neuroprotective. We further demonstrate that downstream of FADD, the mitochondrial proteins Bid and Bax are recruited to the death cascade and are necessary for neuronal death. Our work identifies multiple novel points within neuronal death signaling pathways that could potentially be targeted by candidate therapeutic strategies and highlight how the extrinsic pathway can be activated intracellularly in a pathogenic context.

Precise test of internal-conversion theory: αK measurements for transitions in nine nuclei spanning 45 ≤ Z ≤ 78.

We have been testing the theory used to calculate internal-conversion coefficients (ICCs) by making a series of measurements of αK values with precision better than ±2%. So far we have measured E3 transitions in three nuclei, 103Rh, 111Cd and 134Cs; and M4 transitions in six nuclei, 119Sn, 125Te, 127Te, 137Ba, 193Ir and 197Pt. Together, these span a wide range of A and Z values. In all cases, the results strongly favor Dirac-Fock calculations in which the final-state electron wave function has been computed in an atomic field that includes the vacancy created by the internal-conversion process.