Synergistic hyperactivation of both mTORC1 and mTORC2 underlies the neural abnormalities of PTEN-deficient human neurons and cortical organoids.

Mutations in the phosphatase and tensin homolog (PTEN) gene are associated with severe neurodevelopmental disorders. Loss of PTEN leads to hyperactivation of the mechanistic target of rapamycin (mTOR), which functions in two distinct protein complexes, mTORC1 and mTORC2. The downstream signaling mechanisms that contribute to PTEN mutant phenotypes are not well delineated. Here, we show that pluripotent stem cell-derived PTEN mutant human neurons, neural precursors, and cortical organoids recapitulate disease-relevant phenotypes, including hypertrophy, electrical hyperactivity, enhanced proliferation, and structural overgrowth. PTEN loss leads to simultaneous hyperactivation of mTORC1 and mTORC2. We dissect the contribution of mTORC1 and mTORC2 by generating double mutants of PTEN and RPTOR or RICTOR, respectively. Our results reveal that the synergistic hyperactivation of both mTORC1 and mTORC2 is essential for the PTEN mutant human neural phenotypes. Together, our findings provide insights into the molecular mechanisms that underlie PTEN-related neural disorders and highlight novel therapeutic targets.

Kit Ligand and Kit receptor tyrosine kinase sustain synaptic inhibition of Purkinje cells.

The cell-type-specific expression of ligand/receptor and cell-adhesion molecules is a fundamental mechanism through which neurons regulate connectivity. Here, we determine a functional relevance of the long-established mutually exclusive expression of the receptor tyrosine kinase Kit and the trans-membrane protein Kit Ligand by discrete populations of neurons in the mammalian brain. Kit is enriched in molecular layer interneurons (MLIs) of the cerebellar cortex (i.e., stellate and basket cells), while cerebellar Kit Ligand is selectively expressed by a target of their inhibition, Purkinje cells (PCs). By in vivo genetic manipulation spanning embryonic development through adulthood, we demonstrate that PC Kit Ligand and MLI Kit are required for, and capable of driving changes in, the inhibition of PCs. Collectively, these works in mice demonstrate that the Kit Ligand/Kit receptor dyad sustains mammalian central synapse function and suggest a rationale for the affiliation of Kit mutation with neurodevelopmental disorders.

TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons.

BACKGROUND: Genetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been 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. METHODS: To model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. RESULTS: Cortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified 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. CONCLUSIONS: Taken 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.

TCF4 mutations disrupt synaptic function through dysregulation of RIMBP2 in patient-derived cortical neurons.

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.

mTORC2 Inhibition Improves Morphological Effects of PTEN Loss, But Does Not Correct Synaptic Dysfunction or Prevent Seizures.

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.

Adult-born neurons inhibit developmentally-born neurons during spatial learning.

Ongoing neurogenesis in the dentate gyrus (DG) subregion of the hippocampus results in a heterogenous population of neurons. Immature adult-born neurons (ABNs) have physiological and anatomical properties that may give them a unique role in learning. For example, compared to older granule neurons, they have greater somatic excitability, which could facilitate their recruitment into memory traces. However, recruitment is also likely to depend on interactions with other DG neurons through processes such as lateral inhibition. Immature ABNs target inhibitory interneurons and, compared to older neurons, they receive less GABAergic inhibition. Thus, they may induce lateral inhibition of mature DG neurons while being less susceptible to inhibition themselves. To test this we used a chemogenetic approach to silence immature ABNs as rats learned a spatial water maze task, and measured activity (Fos expression) in ABNs and developmentally-born neurons (DBNs). A retrovirus expressing the inhibitory DREADD receptor, hM4Di, was injected into the dorsal DG of male rats at 6w to infect neurons born in adulthood. Animals were also injected with BrdU to label DBNs or ABNs. DBNs were significantly more active than immature 4-week-old ABNs. Silencing 4-week-old ABNs did not alter learning but it increased activity in DBNs. However, silencing ABNs did not affect activation in other ABNs within the DG. Silencing ABNs also did not alter Fos expression in parvalbumin- and somatostatin-expressing interneurons. Collectively, these results suggest that ABNs may directly inhibit DBN activity during hippocampal-dependent learning, which may be relevant for maintaining sparse hippocampal representations of experienced events.

Disruption of mTORC1 rescues neuronal overgrowth and synapse function dysregulated by Pten loss.

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.

Pten heterozygosity restores neuronal morphology in fragile X syndrome mice.

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.

PTEN Regulates Dendritic Arborization by Decreasing Microtubule Polymerization Rate.

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.

Dreadds: Use and application in behavioral neuroscience.

Technological advances over the last decade are changing the face of behavioral neuroscience research. Here we review recent work on the use of one such transformative tool in behavioral neuroscience research, chemogenetics (or Designer Receptors Exclusively Activated by Designer Drugs, DREADDS). As transformative technologies such as DREADDs are introduced, applied, and refined, their utility in addressing complex questions about behavior and cognition becomes clear and exciting. In the behavioral neuroscience field, remarkable new findings now regularly appear as a result of the ability to monitor and intervene in neural processes with high anatomical precision as animals behave in complex task environments. As these new tools are applied to behavioral questions, individualized procedures for their use find their way into diverse labs. Thus, "tips of the trade" become important for wide dissemination not only for laboratories that are using the tools but also for those who are interested in incorporating them into their own work. Our aim is to provide an up-to-date perspective on how the DREADD technique is being used for research on learning and memory, decision making, and goal-directed behavior, as well as to provide suggestions and considerations for current and future users based on our collective experience. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Striking a balance: PIP2 and PIP3 signaling in neuronal health and disease.

Phosphoinositides are membrane phospholipids involved in a variety of cellular processes like growth, development, metabolism, and transport. This review focuses on the maintenance of cellular homeostasis of phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidylinositol 3,4,5-trisphosphate (PIP3). The critical balance of these PIPs is crucial for regulation of neuronal form and function. The activity of PIP2 and PIP3 can be regulated through kinases, phosphatases, phospholipases and cholesterol microdomains. PIP2 and PIP3 carry out their functions either indirectly through their effectors activating integral signaling pathways, or through direct regulation of membrane channels, transporters, and cytoskeletal proteins. Any perturbations to the balance between PIP2 and PIP3 signaling result in neurodevelopmental and neurodegenerative disorders. This review will discuss the upstream modulators and downstream effectors of the PIP2 and PIP3 signaling, in the context of neuronal health and disease.

The Role of PTEN in Neurodevelopment.

PTEN is a lipid and protein phosphatase that regulates cell growth and survival. Mutations to PTEN are highly penetrant for autism spectrum disorder (ASD). Here, we briefly review the evidence linking PTEN mutations to ASD and the mouse models that have been used to study the role of PTEN in neurodevelopment. We then focus on the cellular phenotypes associated with PTEN loss in neurons, highlighting the role PTEN plays in neuronal proliferation, migration, survival, morphology, and plasticity.

Activity-dependent dendritic elaboration requires Pten.

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.

Restrained Dendritic Growth of Adult-Born Granule Cells Innervated by Transplanted Fetal GABAergic Interneurons in Mice with Temporal Lobe Epilepsy.

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.

Pten loss results in inappropriate excitatory connectivity.

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.

Nuclear Excluded Autism-Associated Phosphatase and Tensin Homolog Mutations Dysregulate Neuronal Growth.

BACKGROUND: Phosphatase and tensin homolog (PTEN) negatively regulates downstream protein kinase B signaling, resulting in decreased cellular growth and proliferation. PTEN is mutated in a subset of children with autism spectrum disorder (ASD); however, the mechanism by which specific point mutations alter PTEN function is largely unknown. Here, we assessed how ASD-associated single-nucleotide variations in PTEN (ASD-PTEN) affect function. METHODS: We used viral-mediated molecular substitution of human PTEN into Pten knockout mouse neurons and assessed neuronal morphology to determine the functional impact of ASD-PTEN. We employed molecular cloning to examine how PTEN's stability, subcellular localization, and catalytic activity affect neuronal growth. RESULTS: We identified a set of ASD-PTEN mutations displaying altered lipid phosphatase function and subcellular localization. We demonstrated that wild-type PTEN can rescue the neuronal hypertrophy, while PTEN H93R, F241S, D252G, W274L, N276S, and D326N failed to rescue this hypertrophy. A subset of these mutations lacked nuclear localization, prompting us to examine the role of nuclear PTEN in regulating neuronal growth. We found that nuclear PTEN alone is sufficient to regulate soma size. Furthermore, forced localization of the D252G and W274L mutations into the nucleus partially restores regulation of soma size. CONCLUSIONS: ASD-PTEN mutations display decreased stability, catalytic activity, and/or altered subcellular localization. Mutations lacking nuclear localization uncover a novel mechanism whereby lipid phosphatase activity in the nucleus can regulate mammalian target of rapamycin signaling and neuronal growth.

A recombinant lentiviral PDGF-driven mouse model of proneural glioblastoma.

Background: Mouse models of glioblastoma (GBM), the most aggressive primary brain tumor, are critical for understanding GBM pathology and can contribute to the preclinical evaluation of therapeutic agents. Platelet-derived growth factor (PDGF) signaling has been implicated in the development and pathogenesis of GBM, specifically the proneural subtype. Although multiple mouse models of PDGF-driven glioma have been described, they require transgenic mice engineered to activate PDGF signaling and/or impair tumor suppressor genes and typically represent lower-grade glioma. Methods: We designed recombinant lentiviruses expressing both PDGFB and a short hairpin RNA targeting Cdkn2a to induce gliomagenesis following stereotactic injection into the dentate gyrus of adult immunocompetent mice. We engineered these viruses to coexpress CreERT2 with PDGFB, allowing for deletion of floxed genes specifically in transduced cells, and designed another version of this recombinant lentivirus in which enhanced green fluorescent protein was coexpressed with PDGFB and CreERT2 to visualize transduced cells. Results: The dentate gyrus of injected mice showed hypercellularity one week post-injection and subsequently developed bona fide tumors with the pathologic hallmarks of GBM leading to a median survival of 77 days post-injection. Transcriptomic analysis of these tumors revealed a proneural gene expression signature. Conclusion: Informed by the genetic alterations observed in human GBM, we engineered a novel mouse model of proneural GBM. While reflecting many of the advantages of transgenic mice, this model allows for the facile in vivo testing of gene function in tumor cells and makes possible the rapid production of large numbers of immunocompetent tumor-bearing mice for preclinical testing of therapeutics.

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

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.

Can fearlessness come in a tiny package?

A molecule called microRNA-153 helps to prevent rats associating new environments with fear.