Reducing Filamin A Restores Cortical Synaptic Connectivity and Early Social Communication Following Cellular Mosaicism in Autism Spectrum Disorder Pathways.

Communication in the form of nonverbal, social vocalization, or crying is evolutionary conserved in mammals and is impaired early in human infants that are later diagnosed with autism spectrum disorder (ASD). Defects in infant vocalization have been proposed as an early sign of ASD that may exacerbate ASD development. However, the neural mechanisms associated with early communicative deficits in ASD are not known. Here, we expressed a constitutively active mutant of Rheb (RhebS16H), which is known to upregulate two ASD core pathways, mTOR complex 1 (mTORC1) and ERK1/2, in Layer (L) 2/3 pyramidal neurons of the neocortex of mice of either sex. We found that cellular mosaic expression of RhebS16H in L2/3 pyramidal neurons altered the production of isolation calls from neonatal mice. This was accompanied by an expected misplacement of neurons and dendrite overgrowth, along with an unexpected increase in spine density and length, which was associated with increased excitatory synaptic activity. This contrasted with the known decrease in spine density in RhebS16H neurons of 1-month-old mice. Reducing the levels of the actin cross-linking and adaptor protein filamin A (FLNA), known to be increased downstream of ERK1/2, attenuated dendrite overgrowth and fully restored spine properties, synaptic connectivity, and the production of pup isolation calls. These findings suggest that upper-layer cortical pyramidal neurons contribute to communicative deficits in a condition known to affect two core ASD pathways and that these mechanisms are regulated by FLNA.

Cannabinoid regulation of neurons in the dentate gyrus during epileptogenesis: Role of CB1R-associated proteins and downstream pathways.

The hippocampal formation plays a central role in the development of temporal lobe epilepsy (TLE), a disease characterized by recurrent, unprovoked epileptic discharges. TLE is a neurologic disorder characterized by acute long-lasting seizures (i.e., abnormal electrical activity in the brain) or seizures that occur in close proximity without recovery, typically after a brain injury or status epilepticus. After status epilepticus, epileptogenic hyperexcitability develops gradually over the following months to years, resulting in the emergence of chronic, recurrent seizures. Acting as a filter or gate, the hippocampal dentate gyrus (DG) normally prevents excessive excitation from propagating through the hippocampus, and is considered a critical region in the progression of epileptogenesis in pathological conditions. Importantly, lipid-derived endogenous cannabinoids (endocannabinoids), which are produced on demand as retrograde messengers, are central regulators of neuronal activity in the DG circuit. In this review, we summarize recent findings concerning the role of the DG in controlling hyperexcitability and propose how DG regulation by cannabinoids (CBs) could provide avenues for therapeutic interventions. We also highlight possible pathways and manipulations that could be relevant for the control of hyperexcitation. The use of CB compounds to treat epilepsies is controversial, as anecdotal evidence is not always validated by clinical trials. Recent publications shed light on the importance of the DG as a region regulating incoming hippocampal excitability during epileptogenesis. We review recent findings concerning the modulation of the hippocampal DG circuitry by CBs and discuss putative underlying pathways. A better understanding of the mechanisms by which CBs exert their action during seizures may be useful to improve therapies.

Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy.

Hyperactivation of the mTOR pathway during foetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development and intractable epilepsy. Recent evidence suggests a role for dysregulated cap-dependent translation downstream of mTOR signalling in the formation of focal malformation of cortical development and seizures. However, it is unknown whether modifying translation once the developmental pathologies are established can reverse neuronal abnormalities and seizures. Addressing these issues is crucial with regards to therapeutics because these neurodevelopmental disorders are predominantly diagnosed during childhood, when patients present with symptoms. Here, we report increased phosphorylation of the mTOR effector and translational repressor, 4E-BP1, in patient focal malformation of cortical development tissue and in a mouse model of focal malformation of cortical development. Using temporally regulated conditional gene expression systems, we found that expression of a constitutively active form of 4E-BP1 that resists phosphorylation by focal malformation of cortical development in juvenile mice reduced neuronal cytomegaly and corrected several neuronal electrophysiological alterations, including depolarized resting membrane potential, irregular firing pattern and aberrant expression of HCN4 ion channels. Further, 4E-BP1 expression in juvenile focal malformation of cortical development mice after epilepsy onset resulted in improved cortical spectral activity and decreased spontaneous seizure frequency in adults. Overall, our study uncovered a remarkable plasticity of the juvenile brain that facilitates novel therapeutic opportunities to treat focal malformation of cortical development-related epilepsy during childhood with potentially long-lasting effects in adults.

Small Extracellular Vesicles Control Dendritic Spine Development through Regulation of HDAC2 Signaling.

The release of small extracellular vesicles (sEVs) has recently been reported, but knowledge of their function in neuron development remains limited. Using LC-MS/MS, we found that sEVs released from developing cortical neurons in vitro obtained from mice of both sexes were enriched in cytoplasm, exosome, and protein-binding and DNA/RNA-binding pathways. The latter included HDAC2, which was of particular interest, because HDAC2 regulates spine development, and populations of neurons expressing different levels of HDAC2 co-exist in vivo during the period of spine growth. Here, we found that HDAC2 levels decrease in neurons as they acquire synapses and that sEVs from HDAC2-rich neurons regulate HDAC2 signaling in HDAC2-low neurons possibly through HDAC2 transfer. This regulation led to a transcriptional decrease in HDAC2 synaptic targets and the density of excitatory synapses. These data suggest that sEVs provide inductive cell-cell signaling that coordinates the development of dendritic spines via the activation of HDAC2-dependent transcriptional programs.SIGNIFICANCE STATEMENT A role of small extracellular vesicles (sEVs; also called exosomes) in neuronal development is of particular interest, because sEVs could provide a major signaling modality between developing neurons when synapses are not fully functional or immature. However, knowledge of sEVs on neuron, and more precisely spine development, is limited. We provide several lines of evidence that sEVs released from developing cortical neurons regulate the development of dendritic spines via the regulation of HDAC2 signaling. This paracrine communication is temporally restricted during development because of the age-dependent decrease in sEV release as neurons mature and acquire spines.

mTOR Hyperactivity Levels Influence the Severity of Epilepsy and Associated Neuropathology in an Experimental Model of Tuberous Sclerosis Complex and Focal Cortical Dysplasia.

Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are focal malformations of cortical development (FMCDs) that are highly associated with intractable epilepsy. TSC and FCD are mTORopathies caused by a spectrum of pathogenic variants in the mechanistic target of rapamycin (mTOR) pathway genes leading to differential activation of mTOR signaling. However, whether the degree of mTOR hyperactivity influences disease severity remains unclear. Here, we examined the effects of differential mTOR hyperactivity levels on epilepsy and associated neuropathology in a mouse model of TSC and FCD. Constitutively active Rheb (RhebCA), the canonical activator of mTOR complex 1 (mTORC1), was expressed in mouse embryos of either sex via in utero electroporation at low, intermediate, and high concentrations to induce different mTORC1 activity levels in developing cortical neurons. We found that RhebCA expression induced mTORC1 hyperactivation and increased neuronal soma size and misplacement in a dose-dependent manner. No seizures were detected in the low RhebCA mice, whereas the intermediate and high RhebCA mice displayed spontaneous, recurrent seizures that significantly increased with higher RhebCA concentrations. Seizures were associated with a global increase in microglial activation that was notably higher in the regions containing RhebCA-expressing neurons. These data demonstrate that neuronal mTOR hyperactivity levels influence the severity of epilepsy and associated neuropathology in experimental TSC and FCD. Overall, these findings highlight the importance of evaluating the outcome of individual variants on mTOR activity levels and support personalized medicine strategies based on patient variants and mTOR activity level for TSC, FCD, and potentially other mTORopathies.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) are epileptogenic cortical malformations caused by pathogenic variants in mechanistic target of rapamycin (mTOR) pathway genes leading to differential mTOR hyperactivation. Here, we present novel findings that neuronal mTOR hyperactivity levels correlate with the severity of epilepsy and associated neuropathology in a mouse model of TSC and FCD. Our findings suggest the need to evaluate the outcome of individual variants on mTOR activity levels in clinical assessments and support personalized medicine strategies based on patient variants and mTOR activity level. Additionally, we present useful modifications to a previously described mouse model of TSC and FCD that allows for titration of seizure frequency and generation of a mild to severe epilepsy phenotype as applicable for preclinical drug testing and mechanistic studies.

Hypervascularization in mTOR-dependent focal and global cortical malformations displays differential rapamycin sensitivity.

OBJECTIVES: Patients with mammalian target of rapamycin (mTOR)-dependent malformations of cortical development (MCDs) associated with seizures display hyperperfusion and increased vessel density of the dysmorphic cortical tissue. Some studies have suggested that the vascular defect occurred independently of seizures. Here, we further examined whether hypervascularization occurs in animal models of global and focal MCD with and without seizures, and whether it is sensitive to the mTOR blocker, rapamycin, that is approved for epilepsy treatment in tuberous sclerosis complex. METHODS: We used two experimental models of mTOR-dependent MCD consisting of conditional transgenic mice containing Tsc1null cells in the forebrain generating a global malformation associated with seizures and of wild-type mice containing a focal malformation in the somatosensory cortex generated by in utero electroporation (IUE) that does not lead to seizures. Alterations in blood vessels and the effects of a 2-week-long rapamycin treatment on these phenotypes were assessed in juvenile mice. RESULTS: Blood vessels in both the focal and global MCDs of postnatal day 14 mice displayed significant increase in vessel density, branching index, total vessel length, and decreased tissue lacunarity. In addition, rapamycin treatment (0.5 mg/kg, every 2 days) partially rescued vessel abnormalities in the focal MCD model, but it did not ameliorate the vessel abnormalities in the global MCD model that required higher rapamycin dosage for a partial rescue. SIGNIFICANCE: Here, we identified hypervascularization in mTOR-dependent MCD in the absence of seizures in young mice, suggesting that increased angiogenesis occurs during development in parallel to alterations in corticogenesis. In addition, a predictive functional outcome is that dysplastic neurons forming MCD will have better access to oxygen and metabolic supplies via their closer proximity to blood vessels. Finally, the difference in rapamycin sensitivity between a focal and global MCD suggest that rapamycin treatment will need to be titrated to match the type of MCD.

GATORopathies: The role of amino acid regulatory gene mutations in epilepsy and cortical malformations.

The mechanistic target of rapamycin (mTOR) pathway has been implicated in a growing number of malformations of cortical development (MCD) associated with intractable epilepsy. Mutations in single genes encoding mTOR pathway regulatory proteins have been linked to MCD such as focal cortical dysplasia (FCD) types IIa and IIb, hemimegalencephaly (HME), and megalencephaly. Recent studies have demonstrated that the GATOR1 protein complex, comprised of DEPDC5, NPRL3, and NPRL2, plays a pivotal role in regulating mTOR signaling in response to cellular amino acid levels and that mutations in DEPDC5, NPRL3, or NPRL2 are linked to FCD, HME, and seizures. Histopathological analysis of FCD and HME tissue specimens resected from individuals harboring DEPDC5, NPRL3, or NPRL2 gene mutations reveals hyperactivation of mTOR pathway signaling. Family pedigrees carrying mutations in either DEPDC5 or NPRL3 share clinical phenotypes of epilepsy and MCD, as well as intellectual and neuropsychiatric disabilities. Interestingly, some individuals with seizures associated with DEPDC5, NPRL3, or NPRL2 variants exhibit normal brain imaging suggesting either occult MCD or a role for these genes in non-lesional neocortical epilepsy. Mouse models resulting from knockdown or knockout of either Depdc5 or Nprl3 exhibit altered cortical lamination, neuronal dysmorphogenesis, and enhanced neuronal excitability as reported in models resulting from direct mTOR activation through expression of its canonical activator RHEB. The role of the GATOR1 proteins in regulating mTOR signaling suggest plausible options for mTOR inhibition in the treatment of epilepsy associated with mutations in DEPDC5, NPRL3, or NPRL2.

Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration.

Hyperactive mammalian target of rapamycin complex 1 (mTORC1) is a shared molecular hallmark in several neurodevelopmental disorders characterized by abnormal brain cytoarchitecture. The mechanisms downstream of mTORC1 that are responsible for these defects remain unclear. We show that focally increasing mTORC1 activity during late corticogenesis leads to ectopic placement of upper-layer cortical neurons that does not require altered signaling in radial glia and is accompanied by changes in layer-specific molecular identity. Importantly, we found that decreasing cap-dependent translation by expressing a constitutively active mutant of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) prevents neuronal misplacement and soma enlargement, while partially rescuing dendritic hypertrophy induced by hyperactive mTORC1. Furthermore, overactivation of translation alone through knockdown of 4E-BP2 was sufficient to induce neuronal misplacement. These data show that many aspects of abnormal brain cytoarchitecture can be prevented by manipulating a single intracellular process downstream of mTORC1, cap-dependent translation.

Valnoctamide Inhibits Cytomegalovirus Infection in Developing Brain and Attenuates Neurobehavioral Dysfunctions and Brain Abnormalities.

Cytomegalovirus (CMV) is the most common infectious cause of brain defects and neurological dysfunction in developing human babies. Due to the teratogenicity and toxicity of available CMV antiviral agents, treatment options during early development are markedly limited. Valnoctamide (VCD), a neuroactive mood stabilizer with no known teratogenic activity, was recently demonstrated to have anti-CMV potential. However, it is not known whether this can be translated into an efficacious therapeutic effect to improve CMV-induced adverse neurological outcomes. Using multiple models of CMV infection in the developing mouse brain, we show that subcutaneous low-dose VCD suppresses CMV by reducing the level of virus available for entry into the brain and by acting directly within the brain to block virus replication and dispersal. VCD during the first 3 weeks of life restored timely acquisition of neurological milestones in neonatal male and female mice and rescued long-term motor and behavioral outcomes in juvenile male mice. CMV-mediated brain defects, including decreased brain size, cerebellar hypoplasia, and neuronal loss, were substantially attenuated by VCD. No adverse side effects on neurodevelopment of uninfected control mice receiving VCD were detected. Treatment of CMV-infected human fetal astrocytes with VCD reduced both viral infectivity and replication by blocking viral particle attachment to the cell, a mechanism that differs from available anti-CMV drugs. These data suggest that VCD during critical periods of neurodevelopment can effectively suppress CMV replication in the brain and safely improve both immediate and long-term neurological outcomes.SIGNIFICANCE STATEMENT Cytomegalovirus (CMV) can irreversibly damage the developing brain. No anti-CMV drugs are available for use during fetal development, and treatment during the neonatal period has substantial limitations. We studied the anti-CMV actions of valnoctamide (VCD), a psychiatric sedative that appears to lack teratogenicity and toxicity, in the newborn mouse brain, a developmental period that parallels that of an early second-trimester human fetus. In infected mice, subcutaneous VCD reaches the brain and suppresses viral replication within the CNS, rescuing the animals from CMV-induced brain defects and neurological problems. Treatment of uninfected control animals exerts no detectable adverse effects. VCD also blocks CMV replication in human fetal brain cells.

Activating the translational repressor 4E-BP or reducing S6K-GSK3ß activity prevents accelerated axon growth induced by hyperactive mTOR in vivo.

Abnormal axonal connectivity and hyperactive mTOR complex 1 (mTORC1) are shared features of several neurological disorders. Hyperactive mTORC1 alters axon length and polarity of hippocampal neurons in vitro, but the impact of hyperactive mTORC1 on axon growth in vivo and the mechanisms underlying those effects remain unclear. Using in utero electroporation during corticogenesis, we show that increasing mTORC1 activity accelerates axon growth without multiple axon formation. This was prevented by counteracting mTORC1 signaling through p70S6Ks (S6K1/2) or eukaryotic initiation factor 4E-binding protein (4E-BP1/2), which both regulate translation. In addition to regulating translational targets, S6K1 indirectly signals through GSK3ß, a regulator of axogenesis. Although blocking GSK3ß activity did not alter axon growth under physiological conditions in vivo, blocking it using a dominant-negative mutant or lithium chloride prevented mTORC1-induced accelerated axon growth. These data reveal the contribution of translational and non-translational downstream effectors such as GSK3ß to abnormal axon growth in neurodevelopmental mTORopathies and open new therapeutic options for restoring long-range connectivity.

BDNF promotes axon branching of retinal ganglion cells via miRNA-132 and p250GAP.

A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

Voltage-dependent K+ currents contribute to heterogeneity of olfactory ensheathing cells.

The olfactory nerve is permissive for axon growth throughout life. This has been attributed in part to the olfactory ensheathing glial cells that encompass the olfactory sensory neuron fascicles. Olfactory ensheathing cells (OECs) also promote axon growth in vitro and when transplanted in vivo to sites of injury. The mechanisms involved remain largely unidentified owing in part to the limited knowledge of the physiological properties of ensheathing cells. Glial cells rely for many functions on the properties of the potassium channels expressed; however, those expressed in ensheathing cells are unknown. Here we show that OECs express voltage-dependent potassium currents compatible with inward rectifier (Kir ) and delayed rectifier (KDR ) channels. Together with gap junction coupling, these contribute to the heterogeneity of membrane properties observed in OECs. The relevance of K(+) currents expressed by ensheathing cells is discussed in relation to plasticity of the olfactory nerve.

Hypoxia-inducible factor 1a is a Tsc1-regulated survival factor in newborn neurons in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 resulting in hyperactivity of the mammalian target of rapamycin and disabling brain lesions. These lesions contain misplaced neurons enriched in hypoxia-inducible factor 1a (HIF1a). However, the relationship between TSC1/2 and HIF1a and the function of HIF1a in TSC neurons remain unexplored. Here, we examine the degree of HIF1a activity and its function in newborn Tsc1(null) neurons in a mouse model of TSC. Using single cell electroporation in the neurogenic subventricular zone (SVZ) of neonatal mice, we deleted Tsc1 and generated olfactory lesions containing misplaced Tsc1(null) neurons as previously reported. These newborn neurons displayed elevated HIF1a-mediated transcriptional activity when compared with Tsc1 heterozygote neurons and a marked resistance to cell death induced by a HIF1a antagonist. Electroporation of Hif1a targeting short hairpin RNA (shRNA) or dominant negative HIF1a constructs resulted in 80-90% loss of Tsc1(null) newborn neurons although sparing SVZ stem cells. Consistent with this later finding, induction of Hif1a shRNA expression during synaptic integration thus bypassing neuron production also resulted in newborn neuron death. Collectively, these results suggest that HIF1a acts as a molecular determinant of newborn neuron survival and that its TSC1-dependent up-regulation gave Tsc1(null) neurons a survival advantage, despite their misplacement in a novel microenvironment.

Rheb activation in subventricular zone progenitors leads to heterotopia, ectopic neuronal differentiation, and rapamycin-sensitive olfactory micronodules and dendrite hypertrophy of newborn neurons.

Mammalian target of rapamycin (mTOR) hyperactivity in perinatal neural progenitor cells (NPCs) of tuberous sclerosis complex 1 (Tsc1) heterozygote mice leads to heterotopia and abnormal neuronal morphogenesis as seen in patients with tuberous sclerosis. Considering that pathological hyperactive mTOR also occurs in individuals carrying no genetic mutations, we examined whether increasing mTOR activity in neonatal NPCs of wild-type mice would recapitulate the above phenotypes. Electroporation of a plasmid encoding constitutively active Ras-homolog enriched in brain (Rheb(CA)) into subventricular zone NPCs increased mTOR activity in newborn cells. At 19 d post-electroporation (dpe), heterotopia and ectopic cells with a neuronal morphology were observed along the migratory path [rostral migratory stream (RMS)] and in the olfactory bulb (OB). These ectopic cells displayed action potentials and received synaptic inputs identifying them as synaptically integrated neurons. RMS heterotopias contained astrocytes, neurons, and entrapped neuroblasts. Immunostaining at 3 dpe revealed the presence of Mash1(+) Olig2(-) cells in the migratory route accompanied by ectopic neuronal differentiation and altered direction and speed of neuroblast migration at 7 dpe, suggesting a non-cell-autonomous disruption of migration. At >19 dpe, newborn Rheb(CA)-expressing neurons displayed altered distribution and formed micronodules in the OB. In addition, they displayed increased dendritic complexity along with altered membrane biophysics and increased frequency of GABAergic synaptic inputs. OB heterotopia, micronodules, and dendrite hypertrophy were notably prevented by rapamycin treatment, suggesting their mTOR dependence. Collectively, these data show that increasing mTOR activity in neonatal NPCs of wild-type mice recapitulate the pathologies observed in Tsc1 mutant mice. In addition, increased mTOR activity in individuals without known mutations could significantly impact neurogenesis and circuit formation.

Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion.

Neurological symptoms in tuberous sclerosis complex (TSC) and associated brain lesions are thought to arise from abnormal embryonic neurogenesis due to inherited mutations in Tsc1 or Tsc2. Neurogenesis persists postnatally in the human subventricular zone (SVZ) where slow-growing tumors containing Tsc-mutant cells are generated in TSC patients. However, whether Tsc-mutant neurons from the postnatal SVZ contribute to brain lesions and abnormal circuit remodeling in forebrain structures remain unexplored. Here, we report the formation of olfactory lesions following conditional genetic Tsc1 deletion in the postnatal SVZ using transgenic mice or targeted single-cell electroporation. These lesions include migratory heterotopias and olfactory micronodules containing neurons with a hypertrophic dendritic tree. Most significantly, our data identify migrating glial and neuronal precursors that are re-routed and infiltrate forebrain structures (e.g. cortex) and become glia and neurons. These data show that Tsc1-mutant cells from the neonatal and juvenile SVZ generate brain lesions and structural abnormalities, which would not be visible using conventional non-invasive imaging. These findings also raise the hypothesis that micronodules and the persistent infiltration of cells to forebrain structures may contribute to network malfunction leading to progressive neuropsychiatric symptoms in TSC.

The mTOR pathway genes mTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.

Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activators, Rheb or mTOR, or biallelic inactivation of the mTORC1 repressors, Depdc5, Tsc1, or Pten in mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.

The mTOR pathway genes MTOR, Rheb, Depdc5, Pten, and Tsc1 have convergent and divergent impacts on cortical neuron development and function.

Brain somatic mutations in various components of the mTOR complex 1 (mTORC1) pathway have emerged as major causes of focal malformations of cortical development and intractable epilepsy. While these distinct gene mutations converge on excessive mTORC1 signaling and lead to common clinical manifestations, it remains unclear whether they cause similar cellular and synaptic disruptions underlying cortical network hyperexcitability. Here, we show that in utero activation of the mTORC1 activator genes, Rheb or MTOR, or biallelic inactivation of the mTORC1 repressor genes, Depdc5, Tsc1, or Pten in the mouse medial prefrontal cortex leads to shared alterations in pyramidal neuron morphology, positioning, and membrane excitability but different changes in excitatory synaptic transmission. Our findings suggest that, despite converging on mTORC1 signaling, mutations in different mTORC1 pathway genes differentially impact cortical excitatory synaptic activity, which may confer gene-specific mechanisms of hyperexcitability and responses to therapeutic intervention.

Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone.

In the postnatal subventricular zone (SVZ), S phase entry of neural progenitor cells (NPCs) correlates with a local increase in blood flow. However, the cellular mechanism controlling this hemodynamic response remains unknown. We show that a subpopulation of SVZ cells, astrocyte-like cells or B-cells, sends projections ensheathing pericytes on SVZ capillaries in young mice. We examined whether calcium increases in pericytes or B-cells led to a vascular response in acute slices using the P2Y(2/4) receptor (P2Y(2/4)R) agonist UTP, electrical stimulation, or transgenic mice expressing exogenous Gq-coupled receptors (MrgA1) in B-cells. UTP increased calcium in pericytes leading to capillary constrictions. Electrical stimulation induced calcium propagation in SVZ cells followed by capillary constrictions involving purinergic receptors. In transgenic mice, selective calcium increases in B-cells induced P2Y(2/4)R-dependent capillary constrictions, suggesting that B-cells release ATP activating purinergic receptors on pericytes. Interestingly, in the presence of a P2Y(2/4)R blocker, dilation was observed. Intraventricular UTP injection transiently decreased blood flow monitored in vivo using laser Doppler flowmetry. Using neonatal electroporation, we expressed MrgA1 in slow cycling radial glia-derived B1 cells, i.e., NPCs. Intraventricular injection of an MrgA1 ligand increased blood flow in the SVZ. Thus, upon intracellular calcium increases B-cells/NPCs release ATP and vasodilating factors that activate purinergic receptors on pericytes triggering a vascular response and blood flow increase in vivo. Considering that NPCs receive signals from other SVZ cells, these findings further suggest that NPCs act as transducers of neurometabolic coupling in the SVZ.

NKCC1 knockdown decreases neuron production through GABA(A)-regulated neural progenitor proliferation and delays dendrite development.

Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.