Abstract:
BACKGROUND: The role of macrophages in the development of rhabdomyolysis induced acute kidney injury (RM-AKI) has been established, but an in-depth understanding of the changes in the immune landscape could help to improve targeted strategies. Whereas senescence is usually associated with chronic kidney processes, we also wished to explore whether senescence could also occur in AKI and whether senolytics could act on immune cells. METHODS: Single-cell RNA sequencing was used in the murine glycerol-induced RM-AKI model to dissect the transcriptomic characteristics of CD45+ live cells sorted from kidneys 2 days after injury. Public datasets from murine AKI models were reanalyzed to explore cellular senescence signature in tubular epithelial cells (TECs). A combination of senolytics (dasatinib and quercetin, DQ) was administered to mice exposed or not to RM-AKI. RESULTS: Unsupervised clustering of nearly 17,000 single-cell transcriptomes identified 7 known immune cell clusters. Sub-clustering of the mononuclear phagocyte cells (MPC), revealed 9 distinct cell sub-populations differently modified with RM. One macrophage cluster was particularly interesting since it behaved as a critical node in a trajectory connecting one MCHIIhigh cluster only present in control to 2 MCHIIlow clusters only present in RM-AKI. This critical cluster expressed a senescence gene signature, that was very different from that of the TECs. Senolytic DQ treatment blocked the switch from a F4/80highCD11blow to F4/80lowCD11bhigh phenotype, which correlated with prolonged nephroprotection in RM-AKI. CONCLUSIONS: scRNASeq unmasked novel transitional macrophage subpopulation associated with RM-AKI characterized by the activation of cellular senescence processes. This work provides a proof-of-concept that senolytics nephroprotective effects may rely, at least in part, on subtle immune modulation.

Abstract:
BACKGROUND: Multiple system atrophy (MSA) is a sporadic adult-onset rare neurodegenerative synucleinopathy for which counteracting central nervous system insulin resistance bears the potential of being neuroprotective. G-protein-(heterotrimeric guanine nucleotide-binding protein)-coupled receptor kinase 2 (GRK2) is emerging as a physiologically relevant inhibitor of insulin signaling. OBJECTIVES: We tested whether lowering brain GRK2 abundance may reverse insulin-resistance. METHODS: We lowered brain GRK2 abundance through viral-mediated delivery of a GRK2-specific miRNA and quantified the reversion of a developing or an established insulin-resistant phenotype using the transgenic PLP-SYN mouse model of MSA. RESULTS: Viral vector delivery of a GRK2 miRNA demonstrated a neuroprotective capacity when administered (1) in utero intracerebroventricularly in developing PLP-SYN mice and (2) intrastriatally in adult PLP-SYN mice. Decreased striatal GRK2 levels correlated in both designs with neuroprotection of the substantia nigra dopamine neurons, reduction in high-molecular-weight species of α-synuclein, and reduced insulin resistance. CONCLUSIONS: These data support GRK2 as a potential therapeutic target in MSA. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Abstract:
The endocannabinoid system (ECS) regulates energy metabolism, has been implicated in the pathogenesis of metabolic diseases and exerts its actions mainly through the type 1 cannabinoid receptor (CB1). Likewise, autophagy is involved in several cellular processes. It is required for the normal development of muscle mass and metabolism, and its deregulation is associated with diseases. It is known that the CB1 regulates signaling pathways that control autophagy, however, it is currently unknown whether the ECS could regulate autophagy in the skeletal muscle of obese mice. This study aimed to investigate the role of the CB1 in regulating autophagy in skeletal muscle. We found concomitant deregulation in the ECS and autophagy markers in high-fat diet-induced obesity. In obese CB1-KO mice, the autophagy-associated protein LC3 II does not accumulate when mTOR and AMPK phosphorylation levels do not change. Acute inhibition of the CB1 with JD-5037 decreased LC3 II protein accumulation and autophagic flux. Our results suggest that the CB1 regulates autophagy in the tibialis anterior skeletal muscle in both lean and obese mice.

Abstract:
This study investigated in male mice how age modulates the effects of acute 17beta-estradiol (E2) on dorsal CA1 (dCA1)-dependent retention of temporal associations, which are critical for declarative memory. E2 was systemically injected to young (3-4 months old) and aged (22-24 months old) adult mice either (i) 1 h before the acquisition of an auditory trace fear conditioning (TFC) procedure allowing the assessment of temporal memory retention 24 h later or (ii) during in vivo electrophysiological recordings of CA3 to dCA1 synaptic efficacy under anesthesia. In young mice, E2 induced parallel dose-dependent reductions in memory and synaptic efficacy, i.e. an impairment in TFC retention and a long-term (NMDA receptor-dependent) depression of dCA1 synaptic efficacy as assessed by field excitatory postsynaptic potentials. In contrast, E2 tended to improved TFC retention whilst failing to change synaptic efficacy in aged mice. Age-dependent effects of E2 treatment were confirmed by immunohistochemical analyses of TFC acquisition-elicited dCA1 Fos activation. Thus, such an activation was respectively reduced and enhanced in young and aged E2-treated mice, compared to vehicle treatments. Hippocampal mRNA expression of estrogen receptors by RT-PCR analyses revealed an age-related increase in each receptor mRNA expression. In keeping with the key role of the endocannabinoid system in memory processes and CA3 to dCA1 synaptic plasticity, we next examined the role of cannabinoid type 1 receptors (CB(1)-R) in the aforementioned age-dependent effects of E2. Having confirmed that mRNA expression of CB(1)-R diminishes with age, we then observed that the deleterious effects of E2 on both memory and synaptic efficacy were both prevented by the CB(1)-R antagonist Rimonabant whilst being absent in CB(1)-R knock out mice. This study (i) reveals age-dependent effects of acute E2 on temporal memory and CA3 to dCA1 synaptic efficacy and (ii) suggests a key role of CB(1)-R in mediating E2 deleterious effects in young adulthood. Aging-related reductions in CB(1)-R might thus underlie E2 paradoxical effects across age.

Abstract:
Rhinovirus (RV) infection of the bronchial epithelium is implicated in the vast majority of severe asthma exacerbations. Interestingly, the susceptibility of bronchial epithelium to RV infection is increased in persons with asthma. Bronchial smooth muscle (BSM) remodeling is an important feature of severe asthma pathophysiology, and its reduction using bronchial thermoplasty has been associated with a significant decrease in the exacerbation rate. We hypothesized that asthmatic BSM can play a role in RV infection of the bronchial epithelium. Using an original co-culture model between bronchial epithelium and BSM cells, we show that asthmatic BSM cells increase RV replication in bronchial epithelium following RV infection. These findings are related to the increased production of CCL20 by asthmatic BSM cells. Moreover, we demonstrate an original downregulation of the activity of the epithelial protein kinase RNA-activated (PKR) antiviral pathway. Finally, we identify a direct bottom-up effect of asthmatic BSM cells on bronchial epithelium susceptibility to RV infection.

Abstract:
Dysregulations of lipid metabolism in the liver may trigger steatosis progression, leading to potentially severe clinical consequences such as nonalcoholic fatty liver diseases (NAFLDs). Molecular mechanisms underlying liver lipogenesis are very complex and fine-tuned by chromatin dynamics and multiple key transcription factors. Here, we demonstrate that the nuclear factor HMGB1 acts as a strong repressor of liver lipogenesis. Mice with liver-specific Hmgb1 deficiency display exacerbated liver steatosis, while Hmgb1-overexpressing mice exhibited a protection from fatty liver progression when subjected to nutritional stress. Global transcriptome and functional analysis revealed that the deletion of Hmgb1 gene enhances LXRalpha and PPARgamma activity. HMGB1 repression is not mediated through nucleosome landscape reorganization but rather via a preferential DNA occupation in a region carrying genes regulated by LXRalpha and PPARgamma. Together, these findings suggest that hepatocellular HMGB1 protects from liver steatosis development. HMGB1 may constitute a new attractive option to therapeutically target the LXRalpha-PPARgamma axis during NAFLD.

Abstract:
Recruitment of lymphocytes into tumors is critical for anti-tumor immunity and efficacious immunotherapy. We show in murine models that tumor-associated high endothelial venules (TA-HEVs) are major sites of lymphocyte entry into tumors at baseline and upon treatment with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB). TA-HEV endothelial cells (TA-HECs) derive from post-capillary venules, co-express MECA-79(+) HEV sialomucins and E/P-selectins, and are associated with homing and infiltration into tumors of various T cell subsets. Intravital microscopy further shows that TA-HEVs are the main sites of lymphocyte arrest and extravasation into ICB-treated tumors. Increasing TA-HEC frequency and maturation increases the proportion of tumor-infiltrating stem-like CD8(+) T cells, and ameliorates ICB efficacy. Analysis of tumor biopsies from 93 patients with metastatic melanoma reveals that TA-HEVs are predictive of better response and survival upon treatment with anti-PD-1/anti-CTLA-4 combination. These studies provide critical insights into the mechanisms governing lymphocyte trafficking in cancer immunity and immunotherapy.

Abstract:
Down syndrome (DS) or Trisomy 21 is the most common genetic cause of mental retardation with severe learning and memory deficits. DS is due to the complete or partial triplication of human chromosome 21 (HSA21) triggering gene overexpression and protein synthesis alterations responsible for a plethora of mental and physical phenotypes. Among the diverse brain target systems that affect hippocampal-dependent learning and memory deficit impairments in DS, the upregulation of the endocannabinoid system (ECS), and notably the overexpression of the cannabinoid type-1 receptor (CB1), seems to play a major role. Combining various protein and gene expression targeted approaches using western blot, qRT-PCR and FISH techniques, we investigated the expression pattern of ECS components in the hippocampus (HPC) of male Ts65Dn mice. Among all the molecules that constitute the ECS, we found that the expression of the CB1 is altered in the HPC of Ts65Dn mice. CB1 distribution is differentially segregated between the dorsal and ventral part of the HPC and within the different cell populations that compose the HPC. CB1 expression is upregulated in GABAergic neurons of Ts65Dn mice whereas it is downregulated in glutamatergic neurons. These results highlight a complex regulation of the CB1 encoding gene (Cnr1) in Ts65Dn mice that could open new therapeutic solutions for this syndrome.

Abstract:
Memory impairment is one of the disabling manifestations of multiple sclerosis (MS) possibly present from the early stages of the disease and for which there is no specific treatment. Hippocampal synaptic dysfunction and dendritic loss, associated with microglial activation, can underlie memory deficits, yet the molecular mechanisms driving such hippocampal neurodegeneration need to be elucidated. In early-stage experimental autoimmune encephalomyelitis (EAE) female mice, we assessed the expression level of molecules involved in microglia-neuron interactions within the dentate gyrus and found overexpression of genes of the complement pathway. Compared to sham immunized mice, the central element of the complement cascade, C3, showed the strongest and 10-fold upregulation, while there was no increase of downstream factors such as the terminal component C5. The combination of in situ hybridization with immunofluorescence showed that C3 transcripts were essentially produced by activated microglia. Pharmacological inhibition of C3 activity, by daily administration of rosmarinic acid, was sufficient to prevent early dendritic loss, microglia-mediated phagocytosis of synapses in the dentate gyrus, and memory impairment in EAE mice, while morphological markers of microglial activation were still observed. In line, when EAE was induced in C3 deficient mice (C3KO), dendrites and spines of the dentate gyrus as well as memory abilities were preserved. Altogether, these data highlight the central role of microglial C3 in early hippocampal neurodegeneration and memory impairment in EAE and, therefore, pave the way toward new neuroprotective strategies in MS to prevent cognitive deficit using complement inhibitors.

Abstract:
Despite the central role of Rho GTPases in neuronal development, their functions in adult hippocampal neurogenesis remain poorly explored. Here, by using a retrovirus-based loss-of-function approach in vivo, we show that the atypical Rho GTPase Rnd2 is crucial for survival, positioning, somatodendritic morphogenesis, and functional maturation of adult-born dentate granule neurons. Interestingly, most of these functions are specific to granule neurons generated during adulthood since the deletion of Rnd2 in neonatally-born granule neurons only affects dendritogenesis. In addition, suppression of Rnd2 in adult-born dentate granule neurons increases anxiety-like behavior whereas its deletion in pups has no such effect, a finding supporting the adult neurogenesis hypothesis of anxiety disorders. Thus, our results are in line with the view that adult neurogenesis is not a simple continuation of earlier processes from development, and establish a causal relationship between Rnd2 expression and anxiety.