Brain oscillations play a critical role in information processing and may, therefore, be essential to uncovering the mechanisms of cognitive impairment in neurological disease. In Dravet syndrome (DS), a mutation in SCN1A, coding for the voltage-gated sodium channel Nav1.1, is associated with severe cognitive impairment and seizures. While seizure frequency and severity do not correlate with the extent of impairment, the slowing of brain rhythms may be involved. Here we investigate the role of Nav1.1 on brain rhythms and cognition using RNA interference. We demonstrate that knockdown of Nav1.1 impairs fast- and burst-firing properties of neurons in the medial septum in vivo. The proportion of neurons that fired phase-locked to hippocampal theta oscillations was reduced, and medial septal regulation of theta rhythm was disrupted. During a working memory task, this deficit was characterized by a decrease in theta frequency and was negatively correlated with performance. These findings suggest a fundamental role for Nav1.1 in facilitating fast-firing properties in neurons, highlight the importance of precise temporal control of theta frequency for working memory, and imply that Nav1.1 deficits may disrupt information processing in DS via a dysregulation of brain rhythms.

Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway.

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.

Pten, a gene associated with autism spectrum disorder, is an upstream regulator of receptor tyrosine kinase intracellular signaling pathways that mediate extracellular cues to inform cellular development and activity-dependent plasticity. We therefore hypothesized that Pten loss would interfere with activity dependent dendritic growth. We investigated the effects of this interaction on the maturation of retrovirally labeled postnatally generated wild-type and Pten knockout granule neurons in male and female mouse dentate gyrus while using chemogenetics to manipulate the activity of the perforant path afferents. We find that enhancing network activity accelerates the dendritic outgrowth of wild-type, but not Pten knockout, neurons. This was specific to immature neurons during an early developmental window. We also examined synaptic connectivity and physiological measures of neuron maturation. The input resistance, membrane capacitance, dendritic spine morphology, and frequency of spontaneous synaptic events were not differentially altered by activity in wild-type versus Pten knockout neurons. Therefore, Pten and its downstream signaling pathways regulate the activity-dependent sculpting of the dendritic arbor during neuronal maturation.

Genetic studies of hippocampal granule neuron development have been used to elucidate cellular functions of Pten and Fmr1. While mutations in each gene cause neurodevelopmental disorders such as autism and fragile X syndrome, how Pten and Fmr1 function alone or together during normal development is not known. Moreover, Pten mRNA is bound by the fragile X mental retardation protein (FMRP) RNA binding protein, but how this physical interaction impinges on phosphatase and tensin homolog protein (PTEN) expression is not known. To understand the interaction of PTEN and FMRP, we investigated the dentate gyrus granule neuron development in Pten and Fmr1 knockout (KO) mice. Interestingly, heterozygosity of Pten restored Fmr1 KO cellular phenotypes, including dendritic arborization, and spine density, while PTEN protein expression was significantly increased in Fmr1 KO animals. However, complete deletion of both Pten and Fmr1 resulted in a dramatic increase in dendritic length, spine density, and spine length. In addition, overexpression of PTEN in Fmr1 KO Pten heterozygous background reduced dendritic length, arborization, spine density, and spine length including pS6 levels. Our findings suggest that PTEN levels are negatively regulated by FMRP, and some Fmr1 KO phenotypes are caused by dysregulation of PTEN protein. These observations provide evidence for the genetic interaction of PTEN and FMRP and a possible mechanistic basis for the pathogenesis of Fmr1-related fragile X neurodevelopmental disorders.

Hyperactivation of PI3K/PTEN-mTOR signaling during neural development is associated with focal cortical dysplasia (FCD), autism, and epilepsy. mTOR can signal through two major hubs, mTORC1 and mTORC2, both of which are hyperactive following PTEN loss of function (LOF). Here, we tested the hypothesis that genetic inactivation of the mTORC2 complex via deletion of Rictor is sufficient to rescue morphologic and electrophysiological abnormalities in the dentate gyrus caused by PTEN loss, as well as generalized seizures. An established, early postnatal mouse model of PTEN loss in male and female mice showed spontaneous seizures that were not prevented by mTORC2 inactivation. This lack of rescue occurred despite the normalization or amelioration of many morphologic and electrophysiological phenotypes. However, increased excitatory connectivity proximal to dentate gyrus granule neuron somas was not normalized by mTORC2 inactivation. Further studies demonstrated that, although mTORC2 inactivation largely rescued the dendritic arbor overgrowth caused by PTEN LOF, it increased synaptic strength and caused additional impairments of presynaptic function. These results suggest that a constrained increase in excitatory connectivity and co-occurring synaptic dysfunction is sufficient to generate seizures downstream of PTEN LOF, even in the absence of characteristic changes in morphologic properties.SIGNIFICANCE STATEMENT Homozygous deletion of the Pten gene in neuronal subpopulations in the mouse serves as a valuable model of epilepsy caused by mTOR hyperactivation. To better understand the physiological mechanisms downstream of Pten loss that cause epilepsy, as well as the therapeutic potential of targeted gene therapies, we tested whether genetic inactivation of the mTORC2 complex could improve the cellular, synaptic, and in vivo effects of Pten loss in the dentate gyrus. We found that mTORC2 inhibition improved or rescued all morphologic effects of Pten loss in the dentate gyrus, but synaptic changes and seizures persisted. These data suggest that synaptic dysfunction can drive epilepsy caused by hyperactivation of PI3K/PTEN-mTOR, and that future therapies should focus on this mechanistic link.

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.

Phosphatase and tensin homolog (PTEN) is a major negative regulator of the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway. Loss-of-function mutations in PTEN have been found in a subset of patients with macrocephaly and autism spectrum disorder (ASD). PTEN loss in neurons leads to somal hypertrophy, aberrant migration, dendritic overgrowth, increased spine density, and hyperactivity of neuronal circuits. These neuronal overgrowth phenotypes are present on Pten knock-out (KO) and reconstitution with autism-associated point mutations. The mechanism underlying dendritic overgrowth in Pten deficient neurons is unclear. In this study, we examined how Pten loss impacts microtubule (MT) dynamics in both sexes using retroviral infection and transfection strategies to manipulate PTEN expression and tag the plus-end MT binding protein, end-binding protein 3 (EB3). We found Pten KO neurons sprout more new processes over time compared with wild-type (WT) neurons. We also found an increase in MT polymerization rate in Pten KO dendritic growth cones. Reducing MT polymerization rate to the WT level was sufficient to reduce dendritic overgrowth in Pten KO neurons in vitro and in vivo Finally, we found that rescue of dendritic overgrowth via inhibition of MT polymerization was sufficient to improve the performance of Pten KO mice in a spatial memory task. Taken together, our data suggests that one factor underlying PTEN loss dependent dendritic overgrowth is increased MT polymerization. This opens the possibility for an intersectional approach targeting MT polymerization and mTOR with low doses of inhibitors to achieve therapeutic gains with minimal side effects in pathologies associated with loss of neuronal PTEN function.SIGNIFICANCE STATEMENT Loss of Pten function because of genetic deletion or expression of mutations associated with autism spectrum disorder (ASD), results in overgrowth of neurons including increased total dendritic length and branching. We have discovered that this overgrowth is accompanied by increased rate of microtubule (MT) polymerization. The increased polymerization rate is insensitive to acute inhibition of mechanistic target of rapamycin (mTOR)C1 or protein synthesis. Direct pharmacological inhibition of MT polymerization can slow the polymerization rate in Pten knock-out (KO) neurons to rates seen in wild-type (WT) neurons. Correction of the MT polymerization rate rescues increased total dendritic arborization and spatial memory. Our studies suggest that phosphatase and tensin homolog (PTEN) inhibits dendritic growth through parallel regulation of protein synthesis and cytoskeletal polymerization.

Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals.

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

The dentate gyrus (DG) is a region of the adult rodent brain that undergoes continuous neurogenesis. Seizures and loss or dysfunction of GABAergic synapses onto adult-born dentate granule cells (GCs) alter their dendritic growth and migration, resulting in dysmorphic and hyperexcitable GCs. Additionally, transplants of fetal GABAergic interneurons in the DG of mice with temporal lobe epilepsy (TLE) result in seizure suppression, but it is unknown whether increasing interneurons with these transplants restores GABAergic innervation to adult-born GCs. Here, we address this question by birth-dating GCs with retrovirus at different times up to 12 weeks after pilocarpine-induced TLE in adult mice. Channelrhodopsin 2 (ChR2)-enhanced yellow fluorescent protein (EYFP)-expressing medial-ganglionic eminence (MGE)-derived GABAergic interneurons from embryonic day (E)13.5 mouse embryos were transplanted into the DG of the TLE mice and GCs with transplant-derived inhibitory post-synaptic currents (IPSCs) were identified by patch-clamp electrophysiology and optogenetic interrogation. Putative synaptic sites between GCs and GABAergic transplants were also confirmed by intracellular biocytin staining, immunohistochemistry, and confocal imaging. 3D reconstructions of dendritic arbors and quantitative morphometric analyses were carried out in >150 adult-born GCs. GABAergic inputs from transplanted interneurons correlated with markedly shorter GC dendrites, compared to GCs that were not innervated by the transplants. Moreover, these effects were confined to distal dendritic branches and a short time window of six to eight weeks. The effects were independent of seizures as they were also observed in naïve mice with MGE transplants. These findings are consistent with the hypothesis that increased inhibitory currents over a smaller dendritic arbor in adult-born GCs may reduce their excitability and lead to seizure suppression.

A conventional study design among medical and biological experimentalists involves collecting multiple measurements from a study subject. For example, experiments utilizing mouse models in neuroscience often involve collecting multiple neuron measurements per mouse to increase the number of observations without requiring a large number of mice. This leads to a form of statistical dependence referred to as clustering. Inappropriate analyses of clustered data have resulted in several recent critiques of neuroscience research that suggest the bar for statistical analyses within the field is set too low. We compare naïve analytical approaches to marginal, fixed-effect, and mixed-effect models and provide guidelines for when each of these models is most appropriate based on study design. We demonstrate the influence of clustering on a between-mouse treatment effect, a within-mouse treatment effect, and an interaction effect between the two. Our analyses demonstrate that these statistical approaches can give substantially different results, primarily when the analyses include a between-mouse treatment effect. In a novel analysis from a neuroscience perspective, we also refine the mixed-effect approach through the inclusion of an aggregate mouse-level counterpart to a within-mouse (neuron level) treatment as an additional predictor by adapting an advanced modeling technique that has been used in social science research and show that this yields more informative results. Based on these findings, we emphasize the importance of appropriate analyses of clustered data, and we aim for this work to serve as a resource for when one is deciding which approach will work best for a given study.

Pten mutations are associated with autism spectrum disorder. Pten loss of function in neurons increases excitatory synaptic connectivity, contributing to an imbalance between excitation and inhibition. We aimed to determine whether Pten loss results in aberrant connectivity in neural circuits. We compared postnatally generated wild-type and Pten knockout granule neurons integrating into the dentate gyrus using a variety of methods to examine their connectivity. We found that postsynaptic Pten loss provides an advantage to dendritic spines in competition over a limited pool of presynaptic boutons. Retrograde monosynaptic tracing with rabies virus reveals that this results in synaptic contact with more presynaptic partners. Using independently excitable opsins to interrogate multiple inputs onto a single neuron, we found that excess connectivity is established indiscriminately from among glutamatergic afferents. Therefore, Pten loss results in inappropriate connectivity whereby neurons are coupled to a greater number of synaptic partners.

Genetic variation in the transcription factor 4 ( TCF4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of ASD called Pitt Hopkins Syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models is shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. Here we show that cortical neurons derived from patients with TCF4 mutations have deficits in spontaneous synaptic transmission, network excitability and homeostatic plasticity. Transcriptomic analysis indicates these phenotypes result from altered expression of genes involved in presynaptic neurotransmission and identifies the presynaptic binding protein, RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. Together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of AKT/mTOR signaling pathway. Mutations in PTEN are found in patients with autism, epilepsy, or macrocephaly. In mouse models, Pten loss results in neuronal hypertrophy, hyperexcitability, seizures, and ASD-like behaviors. The underlying molecular mechanisms of these phenotypes are not well delineated. We determined which of the Pten loss-driven aberrations in neuronal form and function are orchestrated by downstream mTOR complex 1 (mTORC1). Rapamycin-mediated inhibition of mTORC1 prevented increase in soma size, migration, spine density, and dendritic overgrowth in Pten knockout dentate gyrus granule neurons. Genetic knockout of Raptor to disrupt mTORC1 complex formation blocked Pten loss-mediated neuronal hypertrophy. Electrophysiological recordings revealed that genetic disruption of mTORC1 rescued Pten loss-mediated increase in excitatory synaptic transmission. We have identified an essential role for mTORC1 in orchestrating Pten loss-driven neuronal hypertrophy and synapse formation.