Schwann cells (SC) are responsible for myelination in the peripheral nervous system (PNS). Myelin allows saltatory transmission of action potentials along axons and functionally relies on its unique constitution. We previously reported that Cdk7, a regulator of cell cycle progression and transcription, regulates myelin gene expression in oligodendrocytes and contributes to myelin maintenance in the central nervous system. Using mice with conditional Cdk7 knock-out in SCs, we provide evidence that Cdk7 is dispensable for myelin initiation but needed for the correct myelin thickness of larger caliber fibers in young mice, as well as for myelin elongation and rapid nerve conduction throughout age. We report that Cdk7 loss results in disturbed myelin stoichiometry, with significant dysregulation of lipid-related genes in SCs and a reduction in myelin protein zero. Finally, we demonstrate that Rxrγ, a nuclear receptor involved in lipid metabolism, is significantly downregulated in the absence of Cdk7. However, although Cdk7 regulates myelin segment length, our results indicate that this effect occurs independently of Rxrγ in myelinating dorsal root ganglion explants.

Abstract:BackgroundDiaphanous-related formin 3 (DIAPH3) is a member of the formin family, a group of proteins that regulate actin and microtubule dynamics. During mitosis, DIAPH3 localizes specifically to the centrosome. Its loss destabilizes microtubules and disrupts mitotic spindle polarity, leading to multipolar mitoses and abnormal chromosome segregation, which ultimately causes aneuploidy in daughter cells. Methods:We investigated DIAPH3 expression in glioma samples-including low-grade and high-grade gliomas-using publicly available datasets (The Cancer Genome Atlas and a single-cell RNA-seq study). We also explored the impact of DIAPH3 expression on aneuploidy in cultured glioblastoma cells. Results:DIAPH3 expression was specifically increased in grade 4 gliomas. However, its prognostic value did not surpass that of the WHO CNS5 glioma classification. DIAPH3 was predominantly expressed in mitotic cells and showed strong coexpression with genes involved in cell division, particularly those regulating mitotic progression and chromosome segregation. Several transcription factors known to drive proliferation and cancer progression may regulate DIAPH3 expression. In glioblastoma cell lines, we confirmed that DIAPH3 is upregulated during mitosis and that its knockdown increases aneuploidy. Conclusions:These findings confirm the role of DIAPH3 in chromosome segregation in clinical glioma samples and demonstrate its association with high-grade, poor-prognosis gliomas.

Group I metabotropic glutamate receptors (mGluRs), particularly mGluR5, regulate synaptic plasticity via long-term depression (mGluR-LTD), a process implicated in declarative memory. We previously identified TRPC1, a highly expressed hippocampal ion channel, as a key mGluR5 effector. Using a Cre-tamoxifen system, we acutely deleted Trpc1 in a Fragile X syndrome (FXS) mouse model, characterized by mGluR5 hyperactivity, enhanced mGluR-LTD, and social deficits. In FXS neurons, mGluR5-evoked currents were elevated and normalized by Trpc1 deletion. This deletion also improved social behavior and reduced anxiety. Notably, it abolished mGluR-LTD and normalized memory extinction, as shown in behavioral assays. Mechanistically, mGluR5 activation induced ARC protein expression via eEF2K- and ERK1/2-dependent pathways, modulating Arc translation and transcription. These findings highlight TRPC1 as a crucial mediator of pathological plasticity in FXS and a potential therapeutic target.

Neuronal apoptosis is a mechanism used to clear the cells of oxidative stress or DNA damage and refine the final number of neurons for a functional neuronal circuit. The tumor suppressor protein p53 is a key regulator of the cell cycle and serves as a checkpoint for eliminating neurons with high DNA damage, hyperproliferative signals or cellular stress. During development, p53 is largely expressed in progenitor cells. In the adult brain, p53 expression is restricted to the neurogenic niches where it regulates cell proliferation and self-renewal. To investigate the functional consequences of p53 deletion in the cortex and hippocampus, we generated a conditional mutant mouse (p53-cKO) in which p53 is deleted from pallial progenitors and their derivatives. Surprisingly, we did not find any significant change in the number of neurons in the mutant cortex or CA region of the hippocampus compared with control mice. However, p53-cKO mice exhibit more proliferative cells in the subgranular zone of the dentate gyrus and more granule cells in the granular cell layer. Glutamatergic synapses in the CA3 region are more numerous in p53-cKO mice compared with control littermates, which correlates with overexcitability and higher epileptic susceptibility in the mutant mice.

Patients with Duchenne muscular dystrophy (DMD) commonly show specific cognitive deficits in addition to a severe muscle impairment caused by the absence of dystrophin expression in skeletal muscle. These cognitive deficits have been related to the absence of dystrophin in specific regions of the central nervous system, notably cerebellar Purkinje cells (PCs). Dystrophin has recently been involved in GABAA receptors clustering at postsynaptic densities, and its absence, by disrupting this clustering, leads to decreased inhibitory input to PC. We performed an in vivo electrophysiological study of the dystrophin-deficient muscular dystrophy X-linked (mdx) mouse model of DMD to compare PC firing and local field potential (LFP) in alert mdx and control C57Bl/10 mice. We found that the absence of dystrophin is associated with altered PC firing and the emergence of fast (~160-200 Hz) LFP oscillations in the cerebellar cortex of alert mdx mice. These abnormalities were not related to the disrupted expression of calcium-binding proteins in cerebellar PC. We also demonstrate that cerebellar long-term depression is altered in alert mdx mice. Finally, mdx mice displayed a force weakness, mild impairment of motor coordination and balance during behavioural tests. These findings demonstrate the existence of cerebellar dysfunction in mdx mice. A similar cerebellar dysfunction may contribute to the cognitive deficits observed in patients with DMD.

The factors that contribute to pain after nerve injury remain incompletely understood. Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK), two commonly used surgical techniques to correct myopia, transect the distal axons of trigeminal ganglion neurons within the cornea. After surgery, a subset of patients suffer intense and persistent pain, of unknown origin, described by patients as feeling like shards of glass in their eye. In order to investigate the contribution of genetic factors in corneal neuralgia, we used whole-exome sequencing analysis of patients reporting persistent pain after refractive surgery, which revealed multiple variants of ion channels including transient receptor potential (TRP) channels. Here we evaluated a TRPV1 variant, p.V527M, found in a 30-year-old woman who had developed corneal pain after LASIK/PRK surgery, reporting an Ocular Surface Disease Index (OSDI) score of 100. Using patch-clamp and Ca2+ imaging we found that V527M shows an enhanced response to acidic pH. In addition, increasing proton concentration induced a stronger leftward shift in the activation curve of V527M compared to WT, resulting in channel activity of the mutant in acidic pH at more physiological membrane potentials. Finally, comparing the responses to consecutive applications of different agonists, we found in V527M a reduced capsaicin-induced desensitization and an increased sensitization by the arachidonic acid metabolite 12-hydroxyheicosatetraenoic acid (12-HETE). We hypothesize the increased response to protons and enhanced sensitization by 12-HETE in V527M, two inflammatory mediators present in the cornea after tissue damage, may contribute to the pathogenesis of corneal neuralgia after refractive surgery.

The factors that contribute to pain after nerve injury remain incompletely understood. Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) are common surgical techniques to correct refractive errors. After LASIK or PRK, a subset of patients suffers intense and persistent pain, of unknown origin, described by patients as feeling like shards of glass in their eye. Here, we evaluated a TRPV1 variant, p.V527M, found in a 49-y-old woman who developed corneal pain after LASIK and subsequent PRK enhancement, reporting an Ocular Surface Disease Index score of 100. Using patch-clamp and Ca 2+ imaging, we found that the V527M mutation enhances the response to acidic pH. Increasing proton concentration induced a stronger leftward shift in the activation curve of V527M compared to WT, resulting in channel activity of the mutant in acidic pH at more physiological membrane potentials. Finally, comparing the responses to consecutive applications of different agonists, we found in V527M channels a reduced capsaicin-induced desensitization and increased sensitization by the arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). We hypothesize that the increased response in V527M channels to protons and enhanced sensitization by 12-HETE, two inflammatory mediators released in the cornea after tissue damage, may contribute to the pathogenesis of corneal neuralgia after refractive surgery.

A specific plasma membrane distribution of the mechanosensitive ion channel Piezo1 is required for cell migration, but the mechanism remains elusive. Here, we addressed this question using WT and Piezo1-silenced C2C12 mouse myoblasts and WT and Piezo1-KO human kidney HEK293T cells. We showed that cell migration in a cell-free area and through a porous membrane decreased upon Piezo1 silencing or deletion, but increased upon Piezo1 activation by Yoda1, whereas migration towards a chemoattractant gradient was reduced by Yoda1. Piezo1 organized into clusters, which were preferentially enriched at the front. This polarization was stimulated by Yoda1, accompanied by Ca2+ polarization, and abrogated by partial cholesterol depletion. Piezo1 clusters partially colocalized with cholesterol- and GM1 ganglioside-enriched domains, the proportion of which was increased by Yoda1. Mechanistically, Piezo1 activation induced a differential mobile fraction of GM1 associated with domains and the bulk membrane. Conversely, cholesterol depletion abrogated the differential mobile fraction of Piezo1 associated with clusters and the bulk membrane. In conclusion, we revealed, for the first time, the differential implication of Piezo1 depending on the migration mode and the interplay between GM1/cholesterol-enriched domains at the front during migration in a cell-free area. These domains could provide the optimal biophysical properties for Piezo1 activity and/or spatial dissociation from the PMCA calcium efflux pump.

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.

The postsynaptic inhibition through GABA(A) receptors (GABA(A)R) 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 GABA(A)R-driven Cl- currents (E-GABA), 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.

KIF2A is an atypical kinesin that has the capacity to depolymerize microtubules. Patients carrying mutations in KIF2A suffer from progressive microcephaly and mental disabilities. While the role of this protein is well documented in neuronal migration, the relationship between its dysfunction and the pathobiology of brain disorders is unclear. Here, we report that KIF2A is dispensable for embryogenic neurogenesis but critical in postnatal stages for maturation, connectivity, and maintenance of neurons. We used a conditional approach to inactivate KIF2A in cortical progenitors, nascent postmitotic neurons, and mature neurons in mice. We show that the lack of KIF2A alters microtubule dynamics and disrupts several microtubule-dependent processes, including neuronal polarity, neuritogenesis, synaptogenesis, and axonal transport. KIF2A-deficient neurons exhibit aberrant electrophysiological characteristics, neuronal connectivity, and function, leading to their loss. The role of KIF2A is not limited to development, as fully mature neurons require KIF2A for survival. Our results emphasize an additional function of KIF2A and help explain how its mutations lead to brain disorders.

Trigeminal neuralgia (TN) is a unique pain disorder characterized by intense paroxysmal facial pain within areas innervated by the trigeminal nerve. Although most cases of TN are sporadic, familial clusters of TN suggest that genetic factors may contribute to this disorder. Whole-exome sequencing in patients with TN reporting positive family history demonstrated a spectrum of variants of ion channels including TRP channels. Here, we used patch-clamp analysis and Ca2+ and Na+ imaging to assess a rare variant in the TRPM7 channel, p.Ala931Thr, within transmembrane domain 3, identified in a man suffering from unilateral TN. We showed that A931T produced an abnormal inward current carried by Na+ and insensitive to the pore blocker Gd3+. Hypothesizing that replacement of the hydrophobic alanine at position 931 with the more polar threonine destabilizes a hydrophobic ring, near the voltage sensor domain, we performed alanine substitutions of F971 and W972 and obtained results suggesting a role of A931-W972 hydrophobic interaction in S3-S4 hydrophobic cleft stability. Finally, we transfected trigeminal ganglion neurons with A931T channels and observed that expression of this TRPM7 variant lowers current threshold and resting membrane potential, and increases evoked firing activity in TG neurons. Our results support the notion that the TRPM7-A931T mutation located in the S3 segment at the interface with the transmembrane region S4, generates an omega current that carries Na+ influx in physiological conditions. A931T produces hyperexcitability and a sustained Na+ influx in trigeminal ganglion neurons that may underlie pain in this kindred with trigeminal neuralgia.

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.

The function of the amyloid precursor protein (APP) is not fully understood, but its cleavage product amyloid beta (Aβ) 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 Aβ. 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.

Among genetic susceptibility loci associated with late-onset Alzheimer disease (LOAD), genetic polymorphisms identified in genes encoding lipid carriers led to the hypothesis that a disruption of lipid metabolism could promote disease progression. We previously reported that amyloid precursor protein (APP) involved in Alzheimer disease (AD) physiopathology impairs lipid synthesis needed for cortical networks’ activity and that activation of peroxisome proliferator–activated receptor α (PPARα), a metabolic regulator involved in lipid metabolism, improves synaptic plasticity in an AD mouse model. These observations led us to investigate a possible correlation between PPARα function and full-length APP expression. Here, we report that PPARα expression and activation were inversely related to APP expression both in LOAD brains and in early-onset AD cases with a duplication of the APP gene, but not in control human brains. Moreover, human APP expression decreased PPARA expression and its related target genes in transgenic mice and in cultured cortical cells, while opposite results were observed in APP-silenced cortical networks. In cultured neurons, APP-mediated decrease or increase in synaptic activity was corrected by a PPARα-specific agonist and antagonist, respectively. APP-mediated control of synaptic activity was abolished following PPARα deficiency, indicating a key function of PPARα in this process.

Objective To assess the functional effects of a variant, c.89 G > A (p.Arg30Gln), in the transient receptor potential melastatin 8 (TRPM8) cold-sensing, nonselective cation channel, which we have previously identified in a patient with familial trigeminal neuralgia. Methods We carried out Ca2+ imaging and whole-cell patch-clamp recording. Results The TRPM8 mutation enhances channel activation, increases basal current amplitude and intracellular [Ca2+] in cells carrying the mutant channel, and enhances the response to menthol. Conclusions We propose that Arg30Gln confers gain-of-function attributes on TRPM8, which contribute to pathogenesis of trigeminal neuralgia in patients carrying this mutation.

The functions of the lower urinary tract to store and eliminate urine are regulated by complex neural pathways that coordinate the activity of the smooth and striated muscles of the bladder, urethra, and urethral sphincters.1 Besides control by the central nervous system, micturition reflexes are triggered by bladder filling that stretches the bladder wall and by urine flow in the urethra that triggers a urethra-to-bladder reflex facilitating bladder contraction and emptying. Both the urothelium and the mechanically sensitive afferents from the dorsal root ganglia are involved in stretch and pressure detection.

Hyperthermia mediates social defeat stress–induced impairment of adult neurogenesis.

Pulmonary arterial adventitial fibroblasts (PAF), the most abundant cellular constituent of adventitia, act as a key regulator of pulmonary vascular wall structure and function from the outside-in. Previous studies indicate that transient receptor potential vanilloid 4 (TRPV4) channel plays an important role in the development of pulmonary hypertension (PH), but no attention has been given so far to its role in adventitial remodeling. In this study, we thus investigated TRPV4 implication in PAF activation occurring in PH. First, we isolated and cultured PAF from rat adventitial intrapulmonary artery. RT-PCR, Western blot, immunostaining, and calcium imaging (fluo-4/AM) showed that PAF express functional TRPV4 channels. In extension of these results, using pharmacological and siRNA approaches, we demonstrated TRPV4 involvement in PAF proliferation (BrdU incorporation) and migration (wound-healing assay). Then, Western blot experiments revealed that TRPV4 activation upregulates the expression of extracellular matrix protein synthesis (collagen type I and fibronectin). Finally, we explored the role of TRPV4 in the adventitial remodeling occurring in PH. By means of Western blot, we determined that TRPV4 protein expression was upregulated in adventitia from chronically hypoxic and monocrotaline rats, two animal models of PH. Furthermore, morphometric analysis indicated that adventitial remodeling is attenuated in PH-induced trpv4-/- mice. These data support the concept that PAF play an essential role in hypertensive pulmonary vascular remodeling and point out the participation of TRPV4 channel activity in PAF activation leading to excessive adventitial remodeling.