Administration of adiponectin receptor agonist AdipoRon relieves cancer cachexia by mitigating inflammation in tumour-bearing mice.

BACKGROUND: Cancer cachexia is a life-threatening, inflammation-driven wasting syndrome that remains untreatable. Adiponectin, the most abundant adipokine, plays an important role in several metabolic processes as well as in inflammation modulation. Our aim was to test whether administration of AdipoRon (AR), a synthetic agonist of the adiponectin receptors, prevents the development of cancer cachexia and its related muscle atrophy. METHODS: The effect of AR on cancer cachexia was investigated in two distinct murine models of colorectal cancer. First, 7-week-old CD2F1 male mice were subcutaneously injected with colon-26 carcinoma cells (C26) or vehicle (CT). Six days after injection, mice were treated for 5 days with AdipoRon (50 mg/kg/day; C26 + AR) or the corresponding vehicle (CT and C26). Additionally, a genetic model, the ApcMin/+ mouse, that develops spontaneously numerous intestinal polyps, was used. Eight-week-old male ApcMin/+ mice were treated with AdipoRon (50 mg/kg/day; Apc + AR) or the corresponding vehicle (Apc) over a period of 12 weeks, with C57BL/6J wild-type mice used as controls. In both models, several parameters were assessed in vivo: body weight, grip strength and serum parameters, as well as ex vivo: molecular changes in muscle, fat and liver. RESULTS: The protective effect of AR on cachexia development was observed in both cachectic C26 and ApcMin/+ mice. In these mice, AR administration led to a significant alleviation of body weight loss and muscle wasting, together with rescued muscle strength (P < 0.05 for all). In both models, AR had a strong anti-inflammatory effect, reflected by lower systemic interleukin-6 levels (-55% vs. C26, P < 0.001 and -80% vs. Apc mice, P < 0.05), reduced muscular inflammation as indicated by lower levels of Socs3, phospho-STAT3 and Serpina3n, an acute phase reactant (P < 0.05 for all). In addition, AR blunted circulating levels of corticosterone (-46% vs. C26 mice, P < 0.001 and -60% vs. Apc mice, P < 0.05), the predominant murine glucocorticoid known to induce muscle atrophy. Accordingly, key glucocorticoid-responsive factors implicated in atrophy programmes were-or tended to be-significantly blunted in skeletal muscle by AR. Finally, AR protected against lipid metabolism alterations observed in ApcMin/+ mice, as it mitigated the increase in circulating triglyceride levels (-38%, P < 0.05) by attenuating hepatic triglyceride synthesis and fatty acid uptake by the liver. CONCLUSIONS: Altogether, these results show that AdipoRon rescued the cachectic phenotype by alleviating body weight loss and muscle atrophy, along with restraining inflammation and hypercorticism in preclinical murine models. Therefore, AdipoRon could represent an innovative therapeutic strategy to counteract cancer cachexia.

Conditional deletion of KCC2 impairs synaptic plasticity and both spatial and nonspatial memory.

The postsynaptic inhibition through GABAA receptors (GABAAR) relies on two mechanisms, a shunting effect due to an increase in the postsynaptic membrane conductance and, in mature neurons, a hyperpolarization effect due to an entry of chloride into postsynaptic neurons. The second effect requires the action of the K+-Cl- cotransporter KCC2 which extrudes Cl- from the cell and maintains its cytosolic concentration very low. Neuronal chloride equilibrium seems to be dysregulated in several neurological and psychiatric conditions such as epilepsy, anxiety, schizophrenia, Down syndrome, or Alzheimer's disease. In the present study, we used the KCC2 Cre-lox knockdown system to investigate the role of KCC2 in synaptic plasticity and memory formation in adult mice. Tamoxifen-induced conditional deletion of KCC2 in glutamatergic neurons of the forebrain was performed at 3 months of age and resulted in spatial and nonspatial learning impairment. On brain slices, the stimulation of Schaffer collaterals by a theta burst induced long-term potentiation (LTP). The lack of KCC2 did not affect potentiation of field excitatory postsynaptic potentials (fEPSP) measured in the stratum radiatum (dendrites) but increased population spike (PS) amplitudes measured in the CA1 somatic layer, suggesting a reinforcement of the EPSP-PS potentiation, i.e., an increased ability of EPSPs to generate action potentials. At the cellular level, KCC2 deletion induced a positive shift in the reversal potential of GABAAR-driven Cl- currents (EGABA), suggesting an intracellular accumulation of chloride subsequent to the downregulation of KCC2. After treatment with bumetanide, an antagonist of the Na+-K+-Cl- cotransporter NKCC1, spatial memory impairment, chloride accumulation, and EPSP-PS potentiation were rescued in mice lacking KCC2. The presented results emphasize the importance of chloride equilibrium and GABA-inhibiting ability in synaptic plasticity and memory formation.

Inhibitory synapse dysfunction and epileptic susceptibility associated with KIF2A deletion in cortical interneurons.

Malformation of cortical development (MCD) is a family of neurodevelopmental disorders, which usually manifest with intellectual disability and early-life epileptic seizures. Mutations in genes encoding microtubules (MT) and MT-associated proteins are one of the most frequent causes of MCD in humans. KIF2A is an atypical kinesin that depolymerizes MT in ATP-dependent manner and regulates MT dynamics. In humans, single de novo mutations in KIF2A are associated with MCD with epileptic seizures, posterior pachygyria, microcephaly, and partial agenesis of corpus callosum. In this study, we conditionally ablated KIF2A in forebrain inhibitory neurons and assessed its role in development and function of inhibitory cortical circuits. We report that adult mice with specific deletion of KIF2A in GABAergic interneurons display abnormal behavior and increased susceptibility to epilepsy. KIF2A is essential for tangential migration of cortical interneurons, their positioning in the cerebral cortex, and for formation of inhibitory synapses in vivo. Our results shed light on how KIF2A deregulation triggers functional alterations in neuronal circuitries and contributes to epilepsy.

Overexpression of wild-type human amyloid precursor protein alters GABAergic transmission.

The function of the amyloid precursor protein (APP) is not fully understood, but its cleavage product amyloid beta (Aß) together with neurofibrillary tangles constitute the hallmarks of Alzheimer's disease (AD). Yet, imbalance of excitatory and inhibitory neurotransmission accompanied by loss of synaptic functions, has been reported much earlier and independent of any detectable pathological markers. Recently, soluble APP fragments have been shown to bind to presynaptic GABAB receptors (GABABRs), subsequently decreasing the probability of neurotransmitter release. In this body of work, we were able to show that overexpression of wild-type human APP in mice (hAPPwt) causes early cognitive impairment, neuronal loss, and electrophysiological abnormalities in the absence of amyloid plaques and at very low levels of Aß. hAPPwt mice exhibited neuronal overexcitation that was evident in EEG and increased long-term potentiation (LTP). Overexpression of hAPPwt did not alter GABAergic/glutamatergic receptor components or GABA production ability. Nonetheless, we detected a decrease of GABA but not glutamate that could be linked to soluble APP fragments, acting on presynaptic GABABRs and subsequently reducing GABA release. By using a specific presynaptic GABABR antagonist, we were able to rescue hyperexcitation in hAPPwt animals. Our results provide evidence that APP plays a crucial role in regulating inhibitory neurotransmission.

DIAPH3 deficiency links microtubules to mitotic errors, defective neurogenesis, and brain dysfunction.

Diaphanous (DIAPH) three (DIAPH3) is a member of the formin proteins that have the capacity to nucleate and elongate actin filaments and, therefore, to remodel the cytoskeleton. DIAPH3 is essential for cytokinesis as its dysfunction impairs the contractile ring and produces multinucleated cells. Here, we report that DIAPH3 localizes at the centrosome during mitosis and regulates the assembly and bipolarity of the mitotic spindle. DIAPH3-deficient cells display disorganized cytoskeleton and multipolar spindles. DIAPH3 deficiency disrupts the expression and/or stability of several proteins including the kinetochore-associated protein SPAG5. DIAPH3 and SPAG5 have similar expression patterns in the developing brain and overlapping subcellular localization during mitosis. Knockdown of SPAG5 phenocopies DIAPH3 deficiency, whereas its overexpression rescues the DIAHP3 knockdown phenotype. Conditional inactivation of Diaph3 in mouse cerebral cortex profoundly disrupts neurogenesis, depleting cortical progenitors and neurons, leading to cortical malformation and autistic-like behavior. Our data uncover the uncharacterized functions of DIAPH3 and provide evidence that this protein belongs to a molecular toolbox that links microtubule dynamics during mitosis to aneuploidy, cell death, fate determination defects, and cortical malformation.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment.

To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.

Myosteatosis rather than sarcopenia associates with non-alcoholic steatohepatitis in non-alcoholic fatty liver disease preclinical models.

BACKGROUND: Non-alcoholic fatty liver (NAFL) disease (NAFLD) is the most common chronic liver disease in the world. While most subjects have 'inert' NAFL, a subset will progress to non-alcoholic steatohepatitis (NASH) and its life-threatening complications. A substantial body of literature supports that a low muscle mass, low strength, and/or muscle fatty infiltration (myosteatosis) are associated with NAFLD severity. Here, we evaluated the muscle compartment in NASH preclinical models to decipher the kinetics of muscle alterations in relation with liver disease progression. METHODS: We developed and validated a micro-computed tomography-based methodology to prospectively study skeletal muscle mass and density in muscle and liver (i.e. reflecting fatty infiltration) in a high-throughput and non-invasive manner in three preclinical NAFLD/NASH rodent models: fat aussie (FOZ) mice fed a high-fat diet (FOZ HF), wild-type (WT) mice fed a high-fat high-fructose diet (WT HFF), and WT mice fed a high-fat diet (WT HF). We compared them with WT mice fed a normal diet (WT ND) used as controls. RESULTS: -FOZ HF with fibrosing NASH had sarcopenia characterized by a reduced muscle strength when compared with WT HF and WT HFF with early NASH and WT ND controls (165.2 ± 5.2 g vs. 237.4 ± 11.7 g, 256 ± 5.7 g, and 242.9 ± 9.3 g, respectively, P 60; 0.001). Muscle mass or strength was not lower in FOZ HF, WT HF, and WT HFF with early NASH than in controls. Myosteatosis was present in FOZ HF with fibrosing NASH, but also in FOZ HF, WT HF, and WT HFF with early NASH (muscle density = 0.50 ± 0.02, 0.62 ± 0.02, 0.70 ± 0.05, and 0.75 ± 0.03, respectively, with P 60; 0.001 when compared with respective controls). Myosteatosis degree was strongly correlated with NAFLD activity score (r = -0.87, n = 67, P 60; 0.001). In multivariate analysis, the association between myosteatosis and NASH was independent from homeostatic model assessment of insulin resistance and visceral fat area (P 60; 0.05). Myosteatosis degree powerfully discriminated NASH from benign NAFL and normal liver (area under the receiver operating characteristic = 0.96, n = 67, P 60; 0.001). CONCLUSIONS: Taken together, our data support that there is no sarcopenia in obese mice with early NASH. In contrast, the severity of myosteatosis reflects on hepatocellular damage and inflammation during early NASH development. This observation prompts us to exploit myosteatosis as a novel non-invasive marker of NASH.

Mechanical activation of TRPV4 channels controls albumin reabsorption by proximal tubule cells.

Defects in protein reabsorption by the proximal tubule are toxic for epithelial cells in the nephron and may result in nephropathy. In this study, we showed that the ion channel TRPV4 modulated the endocytosis of albumin and low-molecular weight proteins in the proximal tubule. TRPV4 was found at the basolateral side of proximal tubule cells, and its mechanical activation by cell stretching induced Ca2+ entry into the cytosol, which promoted endocytosis. Trpv4-/- mice presented with mild proximal tubule dysfunction under basal conditions. To challenge endocytic function, the permeability of the glomerular filter was altered by systemic delivery of angiotensin II. The proteinuria induced by this treatment was more severe in Trpv4-/- than in Trpv4+/+ mice. Injecting antibodies against the glomerular basement membrane to induce glomerulonephritis is a more pathophysiologically relevant method of impairing glomerular filter permeability. Albuminuria was more severe in mice that lacked TRPV4 specifically in the proximal tubule than in control mice. These results emphasize the importance of TRPV4 in sensing pressure in the proximal tubule in response to variations in the amount of ultrafiltrate and unveil a mechanism that controls protein reabsorption.

Author Correction: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of the TRPC1 Channel in Hippocampal Long-Term Depression and in Spatial Memory Extinction.

Group I metabotropic glutamate receptors (mGluR) are involved in various forms of synaptic plasticity that are believed to underlie declarative memory. We previously showed that mGluR5 specifically activates channels containing TRPC1, an isoform of the canonical family of Transient Receptor Potential channels highly expressed in the CA1-3 regions of the hippocampus. Using a tamoxifen-inducible conditional knockout model, we show here that the acute deletion of the Trpc1 gene alters the extinction of spatial reference memory. mGluR-induced long-term depression, which is partially responsible for memory extinction, was impaired in these mice. Similar results were obtained in vitro and in vivo by inhibiting the channel by its most specific inhibitor, Pico145. Among the numerous known postsynaptic pathways activated by type I mGluR, we observed that the deletion of Trpc1 impaired the activation of ERK1/2 and the subsequent expression of Arc, an immediate early gene that plays a key role in AMPA receptors endocytosis and subsequent long-term depression.

Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.

Sex-regulated gene dosage effect of PPARα on synaptic plasticity.

Mechanisms driving cognitive improvements following nuclear receptor activation are poorly understood. The peroxisome proliferator-activated nuclear receptor alpha (PPARα) forms heterodimers with the nuclear retinoid X receptor (RXR). We report that PPARα mediates the improvement of hippocampal synaptic plasticity upon RXR activation in a transgenic mouse model with cognitive deficits. This improvement results from an increase in GluA1 subunit expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, eliciting an AMPA response at the excitatory synapses. Associated with a two times higher PPARα expression in males than in females, we show that male, but not female, PPARα null mutants display impaired hippocampal long-term potentiation. Moreover, PPARα knockdown in the hippocampus of cognition-impaired mice compromises the beneficial effects of RXR activation on synaptic plasticity only in males. Furthermore, selective PPARα activation with pemafibrate improves synaptic plasticity in male cognition-impaired mice, but not in females. We conclude that striking sex differences in hippocampal synaptic plasticity are observed in mice, related to differences in PPARα expression levels.

Genetic deletion of soluble 5'-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet.

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.

Activation of TRPC1 Channel by Metabotropic Glutamate Receptor mGluR5 Modulates Synaptic Plasticity and Spatial Working Memory.

Group I metabotropic glutamate receptors, in particular mGluR5, have been implicated in various forms of synaptic plasticity that are believed to underlie declarative memory. We observed that mGluR5 specifically activated a channel containing TRPC1, an isoform of the canonical family of transient receptor potential (TRPC) channels highly expressed in CA1-3 regions of the hippocampus. TRPC1 is able to form tetrameric complexes with TRPC4 and/or TRPC5 isoforms. TRPC1/4/5 complexes have recently been involved in the efficiency of synaptic transmission in the hippocampus. We therefore used a mouse model devoid of TRPC1 expression to investigate the involvement of mGluR5-TRPC1 pathway in synaptic plasticity and memory formation. Trpc1-/- mice showed alterations in spatial working memory and fear conditioning. Activation of mGluR increased synaptic excitability in neurons from WT but not from Trpc1-/- mice. LTP triggered by a theta burst could not maintain over time in brain slices from Trpc1-/- mice. mGluR-induced LTD was also impaired in these mice. Finally, acute inhibition of TRPC1 by Pico145 on isolated neurons or on brain slices mimicked the genetic depletion of Trpc1 and inhibited mGluR-induced entry of cations and subsequent effects on synaptic plasticity, excluding developmental or compensatory mechanisms in Trpc1-/- mice. In summary, our results indicate that TRPC1 plays a role in synaptic plasticity and spatial working memory processes.

Interruption of lactate uptake by inhibiting mitochondrial pyruvate transport unravels direct antitumor and radiosensitizing effects.

Lactate exchange between glycolytic and oxidative cancer cells is proposed to optimize tumor growth. Blocking lactate uptake through monocarboxylate transporter 1 (MCT1) represents an attractive therapeutic strategy but may stimulate glucose consumption by oxidative cancer cells. We report here that inhibition of mitochondrial pyruvate carrier (MPC) activity fulfils the tasks of blocking lactate use while preventing glucose oxidative metabolism. Using in vitro 13C-glucose and in vivo hyperpolarized 13C-pyruvate, we identify 7ACC2 as a potent inhibitor of mitochondrial pyruvate transport which consecutively blocks extracellular lactate uptake by promoting intracellular pyruvate accumulation. Also, while in spheroids MCT1 inhibition leads to cytostatic effects, MPC activity inhibition induces cytotoxic effects together with glycolysis stimulation and uncompensated inhibition of mitochondrial respiration. Hypoxia reduction obtained with 7ACC2 is further shown to sensitize tumor xenografts to radiotherapy. This study positions MPC as a control point for lactate metabolism and expands on the anticancer potential of MPC inhibition.

Defective adaptive thermogenesis contributes to metabolic syndrome and liver steatosis in obese mice.

Fatty liver diseases are complications of the metabolic syndrome associated with obesity, insulin resistance and low grade inflammation. Our aim was to uncover mechanisms contributing to hepatic complications in this setting. We used foz/foz mice prone to obesity, insulin resistance and progressive fibrosing non-alcoholic steatohepatitis (NASH). Foz/foz mice are hyperphagic but wild-type (WT)-matched calorie intake failed to protect against obesity, adipose inflammation and glucose intolerance. Obese foz/foz mice had similar physical activity level but reduced energy expenditure. Thermogenic adaptation to high-fat diet (HFD) or to cold exposure was severely impaired in foz/foz mice compared with HFD-fed WT littermates due to lower sympathetic tone in their brown adipose tissue (BAT). Intermittent cold exposure (ICE) restored BAT function and thereby improved glucose tolerance, decreased fat mass and liver steatosis. We conclude that failure of BAT adaptation drives the metabolic complications of obesity in foz/foz mice, including development of liver steatosis. Induction of endogenous BAT function had a significant therapeutic impact on obesity, glucose tolerance and liver complications and is a potential new avenue for therapy of non-alcoholic fatty liver disease (NAFLD).

TRPV4 is associated with central rather than nephrogenic osmoregulation.

TRPV4 is a polymodal cation channel expressed in osmosensitive neurons of the hypothalamus and in the mammalian nephron. The segmental distribution and role(s) of TRPV4 in osmoregulation remain debated. We investigated the renal distribution pattern of TRPV4 and the functional consequences of its disruption in mouse models. Using qPCR on microdissected segments, immunohistochemistry, and a LacZ reporter mouse, we found that TRPV4 is abundantly expressed in the proximal tubule, the late distal convoluted tubule, and throughout the connecting tubule and collecting duct, including principal and intercalated cells. TRPV4 was undetectable in the glomeruli and thick ascending limb and weakly abundant in the early distal convoluted tubule. Metabolic studies in Trpv4 (+/+) and Trpv4 (-/-) littermates revealed that the lack of TRPV4 did not influence activity, food and water intake, renal function, and urinary concentration at baseline. The mice showed a similar response to furosemide, water loading and deprivation, acid loading, and dietary NaCl restriction. However, Trpv4 (-/-) mice showed a significantly lower vasopressin synthesis and release after water deprivation, with a loss of the positive correlation between plasma osmolality and plasma vasopressin levels, and a delayed water intake upon acute administration of hypertonic saline. Specific activation of TRPV4 in primary cultures of proximal tubule cells increased albumin uptake, whereas no effect of TRPV4 deletion could be observed at baseline. These data reveal that, despite its abundant expression in tubular segments, TRPV4 does not play a major role in the kidney or is efficiently compensated when deleted. Instead, TRPV4 is critical for the release of vasopressin, the sensation of thirst, and the central osmoregulation.

Activation of phagocytic activity in astrocytes by reduced expression of the inflammasome component ASC and its implication in a mouse model of Alzheimer disease.

BACKGROUND: The proinflammatory cytokine interleukin-1ß (IL-1ß) is overexpressed in Alzheimer disease (AD) as a key regulator of neuroinflammation. Amyloid-ß (Aß) peptide triggers activation of inflammasomes, protein complexes responsible for IL-1ß maturation in microglial cells. Downregulation of NALP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome has been shown to decrease amyloid load and rescue cognitive deficits in a mouse model of AD. Whereas activation of inflammasome in microglial cells has been described in AD, no data are currently available concerning activation of inflammasome in astrocytes, although they are involved in inflammatory response and phagocytosis. Here, by targeting the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain), we investigated the influence of activation of the inflammasome on the phagocytic activity of astrocytes. METHODS: We used an ASC knockout mouse model, as ASC is a central protein in the inflammasome, acting as an adaptor and stabilizer of the complex and thus critical for its activation. Lipopolysaccharide (LPS)-primed primary cultures of astrocytes from newborn mice were utilized to evaluate Aß-induced inflammasome activation by measuring IL-1ß release by ECLIA (electro-chemiluminescence immunoassay). Phagocytosis efficiency was measured by incorporation of bioparticles, and the release of the chemokine CCL3 (C-C motif ligand 3) was measured by ECLIA. ASC mice were crossbred with 5xFAD (familial Alzheimer disease) mice and tested for spatial reference memory using the Morris water maze (MWM) at 7-8 months of age. Amyloid load and CCL3 were quantified by thioflavine S staining and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RESULTS: Cultured astrocytes primed with LPS and treated with Aß showed an ASC-dependent production of IL-1ß resulting from inflammasome activation mediated by Aß phagocytosis and cathepsin B enzymatic activity. ASC+/- astrocytes displayed a higher phagocytic activity as compared to ASC+/+ and ASC -/- cells, resulting from a higher release of the chemokine CCL3. A significant decrease in amyloid load was measured in the brain of 7-8-month-old 5xFAD mice carrying the ASC +/- genotype, correlated with an increase in CCL3 gene expression. In addition, the ASC +/- genotype rescued spatial reference memory deficits observed in 5xFAD mice. CONCLUSIONS: Our results demonstrate that Aß is able to activate astrocytic inflammasome. Downregulation of inflammasome activity increases phagocytosis in astrocytes due to the release of CCL3. This could explain why downregulation of inflammasome activity decreases amyloid load and rescues memory deficits in a mouse model of AD.

Vsx1 Transiently Defines an Early Intermediate V2 Interneuron Precursor Compartment in the Mouse Developing Spinal Cord.

Spinal ventral interneurons regulate the activity of motor neurons, thereby controlling motor activities. Interneurons arise during embryonic development from distinct progenitor domains distributed orderly along the dorso-ventral axis of the neural tube. A single ventral progenitor population named p2 generates at least five V2 interneuron subsets. Whether the diversification of V2 precursors into multiple subsets occurs within the p2 progenitor domain or involves a later compartment of early-born V2 interneurons remains unsolved. Here, we provide evidence that the p2 domain produces an intermediate V2 precursor compartment characterized by the transient expression of the transcriptional repressor Vsx1. These cells display an original repertoire of cellular markers distinct from that of any V2 interneuron population. They have exited the cell cycle but have not initiated neuronal differentiation. They coexpress Vsx1 and Foxn4, suggesting that they can generate the known V2 interneuron populations as well as possible additional V2 subsets. Unlike V2 interneurons, the generation of Vsx1-positive precursors does not depend on the Notch signaling pathway but expression of Vsx1 in these cells requires Pax6. Hence, the p2 progenitor domain generates an intermediate V2 precursor compartment, characterized by the presence of the transcriptional repressor Vsx1, that contributes to V2 interneuron development.

Involvement of adiponectin in the pathogenesis of dystrophinopathy.

BACKGROUND: The hormone adiponectin (ApN) is decreased in the metabolic syndrome, where it plays a key pathogenic role. ApN also exerts some anti-inflammatory effects on skeletal muscles in mice exposed to acute or chronic inflammation. Here, we investigate whether ApN could be sufficiently potent to counteract a severe degenerative muscle disease, with an inflammatory component such as Duchenne muscular dystrophy (DMD). METHODS: Mdx mice (a DMD model caused by dystrophin mutation) were crossed with mice overexpressing ApN in order to generate mdx-ApN mice; only littermates were used. Different markers of inflammation/oxidative stress and components of signaling pathways were studied. Global force was assessed by in vivo functional tests, and muscle injury with Evans Blue Dye (EBD). Eventually, primary cultures of human myotubes were used. RESULTS: Circulating ApN was markedly diminished in mdx mice. Replenishment of ApN strikingly reduced muscle inflammation, oxidative stress, and enhanced the expression of myogenic differentiation markers along with that of utrophin A (a dystrophin analog) in mdx-ApN mice. Accordingly, mdx-ApN mice exhibited higher global force and endurance as well as decreased muscle damage as quantified by curtailed extravasation of EBD in myofibers. These beneficial effects of ApN were recapitulated in human myotubes. ApN mediates its protection via the adiponectin receptor 1 (AdipoR1, the main ApN receptor in muscle) and the AMPK-SIRT1-PGC-1α signaling pathway, leading to downregulation of the nuclear factor kappa B (NF-κB) and inflammatory genes, together with upregulation of utrophin. CONCLUSIONS: Adiponectin proves to be an extremely powerful hormone capable of protecting the skeletal muscle against inflammation and injury, thereby offering novel therapeutic perspectives for dystrophinopathies.