Mitochondrial hypermetabolism precedes impaired autophagy and synaptic disorganization in App knock-in Alzheimer mouse models.

Accumulation of amyloid ß-peptide (Aß) is a driver of Alzheimer's disease (AD). Amyloid precursor protein (App) knock-in mouse models recapitulate AD-associated Aß pathology, allowing elucidation of downstream effects of Aß accumulation and their temporal appearance upon disease progression. Here we have investigated the sequential onset of AD-like pathologies in AppNL-F and AppNL-G-F knock-in mice by time-course transcriptome analysis of hippocampus, a region severely affected in AD. Strikingly, energy metabolism emerged as one of the most significantly altered pathways already at an early stage of pathology. Functional experiments in isolated mitochondria from hippocampus of both AppNL-F and AppNL-G-F mice confirmed an upregulation of oxidative phosphorylation driven by the activity of mitochondrial complexes I, IV and V, associated with higher susceptibility to oxidative damage and Ca2+-overload. Upon increasing pathologies, the brain shifts to a state of hypometabolism with reduced abundancy of mitochondria in presynaptic terminals. These late-stage mice also displayed enlarged presynaptic areas associated with abnormal accumulation of synaptic vesicles and autophagosomes, the latter ultimately leading to local autophagy impairment in the synapses. In summary, we report that Aß-induced pathways in App knock-in mouse models recapitulate key pathologies observed in AD brain, and our data herein adds a comprehensive understanding of the pathologies including dysregulated metabolism and synapses and their timewise appearance to find new therapeutic approaches for AD.

Deep learning based on susceptibility-weighted MR sequence for detecting cerebral microbleeds and classifying cerebral small vessel disease.

BACKGROUND: Cerebral microbleeds (CMBs) serve as neuroimaging biomarkers to assess risk of intracerebral hemorrhage and diagnose cerebral small vessel disease (CSVD). Therefore, detecting CMBs can evaluate the risk of intracerebral hemorrhage and use its presence to support CSVD classification, both are conducive to optimizing CSVD management. This study aimed to develop and test a deep learning (DL) model based on susceptibility-weighted MR sequence (SWS) to detect CMBs and classify CSVD to assist neurologists in optimizing CSVD management. Patients with arteriolosclerosis (aSVD), cerebral amyloid angiopathy (CAA), and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) treated at three centers were enrolled between January 2017 and May 2022 in this retrospective study. The SWSs of patients from two centers were used as the development set, and the SWSs of patients from the remaining center were used as the external test set. The DL model contains a Mask R-CNN for detecting CMBs and a multi-instance learning (MIL) network for classifying CSVD. The metrics for model performance included intersection over union (IoU), Dice score, recall, confusion matrices, receiver operating characteristic curve (ROC) analysis, accuracy, precision, and F1-score. RESULTS: A total of 364 SWS were recruited, including 336 in the development set and 28 in the external test set. IoU for the model was 0.523 ± 0.319, Dice score 0.627 ± 0.296, and recall 0.706 ± 0.365 for CMBs detection in the external test set. For CSVD classification, the model achieved a weighted-average AUC of 0.908 (95% CI 0.895-0.921), accuracy of 0.819 (95% CI 0.768-0.870), weighted-average precision of 0.864 (95% CI 0.831-0.897), and weighted-average F1-score of 0.829 (95% CI 0.782-0.876) in the external set, outperforming the performance of the neurologist group. CONCLUSION: The DL model based on SWS can detect CMBs and classify CSVD, thereby assisting neurologists in optimizing CSVD management.

A Human Brain Model Mimicking Umbilical Cord Mesenchymal Stem Cells for the Treatment of Hypoxic-Ischemic Brain Injury.

We used an in vitro model of the human brain immune microenvironment to simulate hypoxic-ischemic brain injury (HIBI) and treatment with human umbilical cord mesenchymal stem cells (hUMSCs) to address the transformation barriers of gene differences between animals and humans in preclinical research. A co-culture system, termed hNAME, consisted of human hippocampal neurons (N), astrocytes (A), microglia (M), and brain microvascular endothelial cells (E). Flow cytometry measured the apoptosis rates of neurons and endothelial cells. hNAME-neurons and endothelial cells experienced more severe damage than monolayer cells, particularly after 48 h and 24 h of reoxygenation (OGD48/R24). Western blotting identified neuroinflammatory response markers, including HIF-1α, C1q, C3, TNF-α, and iNOS. Inflammatory factors originated from the glial chamber rather than the neurons and vascular endothelial chambers. A gradual increase in the release of inflammatory factors was observed as the OGD and reoxygenation times increased, peaking at OGD48/R24. The hNAME value was confirmed in human umbilical cord mesenchymal stem cells (hUMSCs). Treatment with hUMSCs resulted in a notable decrease in the severity of neuronal and endothelial cell damage in hNAME. The hNAME is an ideal in vitro model for simulating the immune microenvironment of the human brain because of the interactions between neurons, vessels, astrocytes, and microglia.

Characterization of the bone marrow niche in patients with chronic myeloid leukemia identifies CXCL14 as a new therapeutic option.

Although tyrosine kinase inhibitors (TKIs) are effective in treating chronic myeloid leukemia (CML), they often fail to eradicate the leukemia-initiating stem cells (LSCs), causing disease persistence and relapse. Evidence indicates that LSC persistence may be because of bone marrow (BM) niche protection; however, little is known about the underlying mechanisms. Herein, we molecularly and functionally characterize BM niches in patients with CML at diagnosis and reveal the altered niche composition and function in these patients. Long-term culture initiating cell assay showed that the mesenchymal stem cells from patients with CML displayed an enhanced supporting capacity for normal and CML BM CD34+CD38- cells. Molecularly, RNA sequencing detected dysregulated cytokine and growth factor expression in the BM cellular niches of patients with CML. Among them, CXCL14 was lost in the BM cellular niches in contrast to its expression in healthy BM. Restoring CXCL14 significantly inhibited CML LSC maintenance and enhanced their response to imatinib in vitro, and CML engraftment in vivo in NSG-SGM3 mice. Importantly, CXCL14 treatment dramatically inhibited CML engraftment in patient-derived xenografted NSG-SGM3 mice, even to a greater degree than imatinib, and this inhibition persisted in patients with suboptimal TKI response. Mechanistically, CXCL14 upregulated inflammatory cytokine signaling but downregulated mTOR signaling and oxidative phosphorylation in CML LSCs. Together, we have discovered a suppressive role of CXCL14 in CML LSC growth. CXCL14 might offer a treatment option targeting CML LSCs.

Molecular programming modulates hepatic lipid metabolism and adult metabolic risk in the offspring of obese mothers in a sex-specific manner.

Male and female offspring of obese mothers are known to differ extensively in their metabolic adaptation and later development of complications. We investigate the sex-dependent responses in obese offspring mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Here we show that maternal obesity prior to and during gestation leads to hepatic steatosis and inflammation in male offspring, while female offspring are protected. Females from obese mothers display important changes in hepatic transcriptional activity and triglycerides profile which may prevent the damaging effects of maternal obesity compared to males. These differences are sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive differently transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver, explaining the sexual dimorphism in obesity-associated metabolic risk.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Clinical effects of continuous theta burst stimulation for generalized anxiety disorder and a mechanism involving α oscillations: a randomized controlled trial.

BACKGROUND: Continuous theta burst stimulation (cTBS) is a much more rapid protocol than low-frequency repetitive transcranial magnetic stimulation (rTMS), but no clinical trial has yet investigated the efficacy and mechanisms of cTBS for the treatment of generalized anxiety disorder. The purpose of this study was to compare the clinical effects and α oscillations induced by cTBS versus 1 Hz rTMS as predictors of response, and to assess the underlying mechanisms of the therapeutic effects of cTBS in patients with generalized anxiety disorder. METHODS: We randomly allocated 120 patients with generalized anxiety disorder to receive cTBS (n = 41), 1 Hz rTMS (n = 40) or sham cTBS (n = 39) over the right dorsolateral prefrontal cortex; we also included healthy controls (n = 30) to compare neurophysiological data. We analyzed changes in Hamilton Anxiety Rating Scale scores and α oscillations (frequency and power) at baseline, post-treatment and 1-month follow-up. RESULTS: After 20 sessions of treatment, patients' anxiety had improved and α power had increased in the cTBS and 1 Hz rTMS groups. However, at 1-month follow-up the cTBS group had significantly more responders and remitters, and higher α oscillations than the 1 Hz rTMS group (post hoc analysis: cTBS > rTMS > sham). At baseline, α frequency was inversely correlated with psychological symptom scores on the Hamilton Anxiety Rating Scale (r = -0.613, p < 0.001); post-treatment, this correlation was present only in the cTBS group (r = -0.685, p < 0.001). LIMITATIONS: Electroencephalography data were limited to the α band. CONCLUSION: Our findings provide evidence for the clinical use of cTBS, a novel brain stimulation protocol. Its therapeutic effects may be the result of increasing α frequency, thereby improving the psychological symptoms of generalized anxiety disorder.

Maternal high-fat diet programs white and brown adipose tissue lipidome and transcriptome in offspring in a sex- and tissue-dependent manner in mice.

OBJECTIVE: The prevalence of overweight and obesity among children has drastically increased during the last decades and maternal obesity has been demonstrated as one of the ultimate factors. Nutrition-stimulated transgenerational regulation of key metabolic genes is fundamental to the developmental origins of the metabolic syndrome. Fetal nutrition may differently influence female and male offspring. METHODS: Mice dam were fed either a control diet or a high-fat diet (HFD) for 6-week prior mating and continued their respective diet during gestation and lactation. At weaning, female and male offspring were fed the HFD until sacrifice. White (WAT) and brown (BAT) adipose tissues were investigated in vivo by nuclear magnetic resonance at two different timepoints in life (midterm and endterm) and tissues were collected at endterm for lipidomic analysis and RNA sequencing. We explored the sex-dependent metabolic adaptation and gene programming changes by maternal HFD in visceral AT (VAT), subcutaneous AT (SAT) and BAT of offspring. RESULTS: We show that the triglyceride profile varies between adipose depots, sexes and maternal diet. In female offspring, maternal HFD remodels the triglycerides profile in SAT and BAT, and increases thermogenesis and cell differentiation in BAT, which may prevent metabolic complication later in life. Male offspring exhibit whitening of BAT and hyperplasia in VAT when born from high-fat mothers, with impaired metabolic profile. Maternal HFD differentially programs gene expression in WAT and BAT of female and male offspring. CONCLUSION: Maternal HFD modulates metabolic profile in offspring in a sex-dependent manner. A sex- and maternal diet-dependent gene programming exists in VAT, SAT, and BAT which may be key player in the sexual dimorphism in the metabolic adaptation later in life.

Retinoic acid receptor responder1 promotes development of glomerular diseases via the Nuclear Factor-κB signaling pathway.

Inflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflammation and fibrosis via transcription factor Nuclear Factor-κB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflammation and fibrosis via the Nuclear Factor-κB signaling pathway.

3D heterospecies spheroids of pancreatic stroma and cancer cells demonstrate key phenotypes of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, partly due to the dense desmoplasia and a lack of suitable model systems to study. In the present work, we developed a 3D heterospecies spheroid model to study the microenvironmental interactions between tumor cells and stellate cells which can also be employed to test therapeutic regimens. We set up monospheroids and heterospheroids made up from murine pancreatic stellate cells (mPSCs) and human PDAC cells (Panc1), which allowed for direct isolation of mRNA from a mixed cell population followed by an in silico separation of the RNA-seq reads. Global transcript level changes for cells in heterospheroids versus monospheroids were calculated, followed by gene set enrichment analysis and molecular subtype analysis. We observed an apparent shift of Panc1 from the classical to the squamous/basal-like phenotype upon co-culture with mPSCs. Moreover, mPSCs acquired a different cancer-associated fibroblast-related phenotype upon co-culture with Panc1. We analyzed the tumor cell-specific chemosensitivities towards gemcitabine, paclitaxel and SN38 and compared these to published pharmacotranscriptomic signatures. In conclusion, our heterospecies spheroid model reflected key aspects of PDAC and facilitated the study of intercellular interactions between tumor and stroma while additionally proving to be a good model for studying therapeutic responses.

Obese mother offspring have hepatic lipidic modulation that contributes to sex-dependent metabolic adaptation later in life.

With the increasing prevalence of obesity in women of reproductive age, there is an urgent need to understand the metabolic impact on the fetus. Sex-related susceptibility to liver diseases has been demonstrated but the underlying mechanism remains unclear. Here we report that maternal obesity impacts lipid metabolism differently in female and male offspring. Males, but not females, gained more weight and had impaired insulin sensitivity when born from obese mothers compared to control. Although lipid mass was similar in the livers of female and male offspring, sex-specific modifications in the composition of fatty acids, triglycerides and phospholipids was observed. These overall changes could be linked to sex-specific regulation of genes controlling metabolic pathways. Our findings revised the current assumption that sex-dependent susceptibility to metabolic disorders is caused by sex-specific postnatal regulation and instead we provide molecular evidence supporting in utero metabolic adaptations in the offspring of obese mothers.

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss.

RATIONALE: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. OBJECTIVE: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level and to correlate them with regional heterogeneities in BBB function and vascular phenotype. METHODS AND RESULTS: We reveal transcriptional, morphological, and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses, and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. CONCLUSIONS: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence, and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 (angiopoietin 2) is paradoxical given its wider role as TIE2 (TEK receptor tyrosine kinase) receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain.

The Degree of Cardiac Remodelling before Overload Relief Triggers Different Transcriptome and miRome Signatures during Reverse Remodelling (RR)-Molecular Signature Differ with the Extent of RR.

This study aims to provide new insights into transcriptome and miRome modifications occurring in cardiac reverse remodelling (RR) upon left ventricle pressure-overload relief in mice. Pressure-overload was established in seven-week-old C57BL/6J-mice by ascending aortic constriction. A debanding (DEB) surgery was performed seven weeks later in half of the banding group (BA). Two weeks later, cardiac function was evaluated through hemodynamics and echocardiography, and the hearts were collected for histology and small/bulk-RNA-sequencing. Pressure-overload relief was confirmed by the normalization of left-ventricle-end-systolic-pressure. DEB animals were separated into two subgroups according to the extent of cardiac remodelling at seven weeks and RR: DEB1 showed an incomplete RR phenotype confirmed by diastolic dysfunction persistence (E/e' ≥ 16 ms) and increased myocardial fibrosis. At the same time, DEB2 exhibited normal diastolic function and fibrosis, presenting a phenotype closer to myocardial recovery. Nevertheless, both subgroups showed the persistence of cardiomyocytes hypertrophy. Notably, the DEB1 subgroup presented a more severe diastolic dysfunction at the moment of debanding than the DEB2, suggesting a different degree of cardiac remodelling. Transcriptomic and miRomic data, as well as their integrated analysis, revealed significant downregulation in metabolic and hypertrophic related pathways in DEB1 when compared to DEB2 group, including fatty acid ß-oxidation, mitochondria L-carnitine shuttle, and nuclear factor of activated T-cells pathways. Moreover, extracellular matrix remodelling, glycan metabolism and inflammation-related pathways were up-regulated in DEB1. The presence of a more severe diastolic dysfunction at the moment of pressure overload-relief on top of cardiac hypertrophy was associated with an incomplete RR. Our transcriptomic approach suggests that a cardiac inflammation, fibrosis, and metabolic-related gene expression dysregulation underlies diastolic dysfunction persistence after pressure-overload relief, despite left ventricular mass regression, as echocardiographically confirmed.

Integrin ß3, a RACK1 interacting protein, is critical for porcine reproductive and respiratory syndrome virus infection and NF-κB activation in Marc-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen of porcine reproductive and respiratory syndrome (PRRS), which is one of the most economically harmful diseases in modern pig production worldwide. Receptor of activated protein C kinase 1 (RACK1) was previously shown to be indispensable for the PRRSV replication and NF-κB activation in Marc-145 cells. Here we identified a membrane protein, integrin ß3 (ITGB3), as a RACK1-interacting protein. PRRSV infection in Marc-145 cells upregulated the ITGB3 expression. Abrogation of ITGB3 by siRNA knockdown or antibody blocking inhibited PRRSV infection and NF-κB activation, while on the other hand, overexpression of ITGB3 enhanced PRRSV infection and NF-κB activation. Furthermore, inhibition of ITGB3 alleviated the cytopathic effects and reduced the TCID50 titer in Marc-145 cells. We also showed that RACK1 and ITGB3 were NF-κB target genes during PRRSV infection, and that they regulated each other. Our data indicated that ITGB3, presumably as a co-receptor, played an imperative role during PRRSV infection and NF-κB activation in Marc-145 cells. PRRSV infection activates a positive feedback loop involving the activation of NF-κB and upregulation of ITGB3 and RACK1 in Marc-145 cells. The findings would advance our elaborated understanding of the molecular host-pathogen interaction mechanisms underlying PRRSV infection in swine and suggest ITGB3 and NF-κB signaling pathway as potential therapeutic targets for PRRS control.

Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance.

Obesity and insulin resistance are risk factors for nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. Because no approved medication nor an accurate and noninvasive diagnosis is currently available for NAFLD, there is a clear need to better understand the link between obesity and NAFLD. Lipid accumulation during obesity is known to be associated with oxidative stress and inflammatory activation of liver macrophages (LMs). However, we show that although LMs do not become proinflammatory during obesity, they display signs of oxidative stress. In livers of both humans and mice, antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) was down-regulated with obesity and insulin resistance, yielding an impaired response to lipid accumulation. At the molecular level, a microRNA-targeting NRF2 protein, miR-144, was elevated in the livers of obese insulin-resistant humans and mice, and specific silencing of miR-144 in murine and human LMs was sufficient to restore NRF2 protein expression and the antioxidant response. These results highlight the pathological role of LMs and their therapeutic potential to restore the impaired endogenous antioxidant response in obesity-associated NAFLD.

Selective estrogen receptor (ER)ß activation provokes a redistribution of fat mass and modifies hepatic triglyceride composition in obese male mice.

Estrogen exerts its action through the binding to two major receptors, estrogen receptor (ER)α and ß. Recently, the beneficial role of selective ERß activation in the regulation of metabolic homeostasis in obesity has been demonstrated, but its importance is still controversial. However, no data are available regarding possible gender differences in response to pharmaceutical activation of ERß. Male mice were fed a control diet (CD) or a high fat diet (HFD) before being treated with the ERß selective ligand, 4-(2-(3-5-dimethylisoxazol-4-yl)-1H-indol-3yl)phenol (DIP) in the same conditions as in our recently published paper in female mice. Magnetic resonance imaging and spectroscopy were performed repeatedly in vivo after 6 weeks of diet and after 2 weeks of DIP. Adipose tissue distribution and hepatic triglycerides composition were quantified. HFD-treated males showed a feminization of their fat distribution towards more subcutaneous fat depots and increase total fat content and visceral adipose tissue showed clear browning sites after DIP. Hepatic lipid composition was modified by DIP, with less saturated and more unsaturated lipids and an improved insulin sensitivity. Finally, brown adipose tissue size expended after DIP, due to an increase of the size of the lipid droplets. Our data demonstrate that selective activation of ERß exerts a tissue-specific and sex-dependent response to metabolic adaptation to overfeeding. Most importantly, together with our previously published results in females, the current findings support the concept that sex should be considered in the future development of obesity-moderating drugs.

Cell shape determines gene expression: cardiomyocyte morphotypic transcriptomes.

Cardiomyocytes undergo considerable changes in cell shape. These can be due to hemodynamic constraints, including changes in preload and afterload conditions, or to mutations in genes important for cardiac function. These changes instigate significant changes in cellular architecture and lead to the addition of sarcomeres, at the same time or at a later stage. However, it is currently unknown whether changes in cell shape on their own affect gene expression and the aim of this study was to fill that gap in our knowledge. We developed a single-cell morphotyping strategy, followed by single-cell RNA sequencing, to determine the effects of altered cell shape in gene expression. This enabled us to profile the transcriptomes of individual cardiomyocytes of defined geometrical morphotypes and characterize them as either normal or pathological conditions. We observed that deviations from normal cell shapes were associated with significant downregulation of gene expression and deactivation of specific pathways, like oxidative phosphorylation, protein kinase A, and cardiac beta-adrenergic signaling pathways. In addition, we observed that genes involved in apoptosis of cardiomyocytes and necrosis were upregulated in square-like pathological shapes. Mechano-sensory pathways, including integrin and Src kinase mediated signaling, appear to be involved in the regulation of shape-dependent gene expression. Our study demonstrates that cell shape per se affects the regulation of the transcriptome in cardiac myocytes, an effect with possible implications for cardiovascular disease.

Titin splicing regulates cardiotoxicity associated with calpain 3 gene therapy for limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector-mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 1013 viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

GPRC5b Modulates Inflammatory Response in Glomerular Diseases via NF-κB Pathway.

BACKGROUND: Inflammatory processes play an important role in the pathogenesis of glomerulopathies. Finding novel ways to suppress glomerular inflammation may offer a new way to stop disease progression. However, the molecular mechanisms that initiate and drive inflammation in the glomerulus are still poorly understood. METHODS: We performed large-scale gene expression profiling of glomerulus-associated G protein-coupled receptors (GPCRs) to identify new potential therapeutic targets for glomerulopathies. The expression of Gprc5b in disease was analyzed using quantitative PCR and immunofluorescence, and by analyzing published microarray data sets. In vivo studies were carried out in a podocyte-specific Gprc5b knockout mouse line. Mechanistic studies were performed in cultured human podocytes. RESULTS: We identified an orphan GPCR, Gprc5b, as a novel gene highly enriched in podocytes that was significantly upregulated in common human glomerulopathies, including diabetic nephropathy, IgA nephropathy, and lupus nephritis. Similar upregulation of Gprc5b was detected in LPS-induced nephropathy in mice. Studies in podocyte-specific Gprc5b knockout mice showed that Gprc5b was not essential for normal development of the glomerular filtration barrier. However, knockout mice were partially protected from LPS-induced proteinuria and recruitment of inflammatory cells. Mechanistically, RNA sequencing in Gprc5b knockouts mice and experiments in cultured human podocytes showed that Gpr5cb regulated inflammatory response in podocytes via NF-κB signaling. CONCLUSIONS: GPRC5b is a novel podocyte-specific receptor that regulates inflammatory response in the glomerulus by modulating the NF-κB signaling pathway. Upregulation of Gprc5b in human glomerulopathies suggests that it may play a role in their pathogenesis.