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.

Protocol for electroporating and isolating murine (sub)ventricular zone cells for single-nuclei omics.

In vivo genetic modification of neural stem cells is necessary to model the origins and pathogenesis of neurological disorders. Electroporation is a technique that applies a transient electrical field to direct charged molecules into living cells to genetically modify the mouse brain. Here, we provide a protocol to electroporate the neural stem cells surrounding the neonatal ventricles. We describe subsequent steps to isolate and prepare nuclei from the cells and their cellular progeny for single-nuclei omics. For complete details on the use and execution of this protocol, please refer to Riley et al.1.

Tsc2 coordinates neuroprogenitor differentiation.

Neural stem cells (NSCs) of the ventricular-subventricular zone (V-SVZ) generate numerous cell types. The uncoupling of mRNA transcript availability and translation occurs during the progression from stem to differentiated states. The mTORC1 kinase pathway acutely controls proteins that regulate mRNA translation. Inhibiting mTORC1 during differentiation is hypothesized to be critical for brain development since somatic mutations of mTORC1 regulators perturb brain architecture. Inactivating mutations of TSC1 or TSC2 genes cause tuberous sclerosis complex (TSC). TSC patients have growths near the striatum and ventricles. Here, it is demonstrated that V-SVZ NSC Tsc2 inactivation causes striatal hamartomas. Tsc2 removal altered translation factors, translatomes, and translational efficiency. Single nuclei RNA sequencing following in vivo loss of Tsc2 revealed changes in NSC activation states. The inability to decouple mRNA transcript availability and translation delayed differentiation leading to the retention of immature phenotypes in hamartomas. Taken together, Tsc2 is required for translational repression and differentiation.

In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology.

Several neurodevelopmental disorders are associated with increased mTOR activity that results in pathogenic neuronal dysmorphogenesis (i.e., soma and dendrite overgrowth), leading to circuit alterations associated with epilepsy and neurologic disabilities. Although an mTOR analog is approved for the treatment of epilepsy in one of these disorders, it has limited efficacy and is associated with a wide range of side effects. There is a need to develop novel agents for the treatment of mTOR-pathway related disorders. Here, we developed a medium-throughput phenotypic assay to test drug efficacy on neurite morphogenesis of mouse neurons in a hyperactive mTOR condition. Our assay involved in utero electroporation (IUE) of a selective population of cortical pyramidal neurons with a plasmid encoding the constitutively active mTOR activator, Rheb, and tdTomato. Labeled neurons from the somatosensory cortex (SSC) were cultured onto 96-well plates and fixed at various days in vitro or following Torin 1 treatment. Automated systems were used for image acquisition and neuron morphologic measurements. We validated our automated approach using traditional manual methods of neuron morphologic assessment. Both automated and manual analyses showed increased neurite length and complexity over time, and decreased neurite overgrowth and soma size with Torin 1. These data validate the accuracy of our automated approach that takes hours compared with weeks when using traditional manual methods. Taken together, this assay can be scaled to screen 32 compounds simultaneously in two weeks, highlighting its robustness and efficiency for medium-throughput screening of candidate therapeutics on a defined population of wild-type or diseased neurons.

Tsc2 shapes olfactory bulb granule cell molecular and morphological characteristics.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations that inactivate TSC1 or TSC2. Hamartin and tuberin are encoded by TSC1 and TSC2 which form a GTPase activating protein heteromer that inhibits the Rheb GTPase from activating a growth promoting protein kinase called mammalian target of rapamycin (mTOR). Growths and lesions occur in the ventricular-subventricular zone (V-SVZ), cortex, olfactory tract, and olfactory bulbs (OB) in TSC. A leading hypothesis is that mutations in inhibitory neural progenitor cells cause brain growths in TSC. OB granule cells (GCs) are GABAergic inhibitory neurons that are generated through infancy by inhibitory progenitor cells along the V-SVZ. Removal of Tsc1 from mouse OB GCs creates cellular phenotypes seen in TSC lesions. However, the role of Tsc2 in OB GC maturation requires clarification. Here, it is demonstrated that conditional loss of Tsc2 alters GC development. A mosaic model of TSC was created by performing neonatal CRE recombinase electroporation into inhibitory V-SVZ progenitors yielded clusters of ectopic cytomegalic neurons with hyperactive mTOR complex 1 (mTORC1) in homozygous Tsc2 mutant but not heterozygous or wild type mice. Similarly, homozygous Tsc2 mutant GC morphology was altered at postnatal days 30 and 60. Tsc2 mutant GCs had hypertrophic dendritic arbors that were established by postnatal day 30. In contrast, loss of Tsc2 from mature GCs had negligible effects on mTORC1, soma size, and dendrite arborization. OB transcriptome profiling revealed a network of significantly differentially expressed genes following loss of Tsc2 during development that altered neural circuitry. These results demonstrate that Tsc2 has a critical role in regulating neural development and shapes inhibitory GC molecular and morphological characteristics.

The amino acid transporter Slc7a5 regulates the mTOR pathway and is required for granule cell development.

Pathogenic mutations in the solute carrier family 7 member 5 (SLC7A5) gene, which encodes an amino acid transporter cause microcephaly and seizures, yet the mechanisms responsible for these phenotypes are unclear. Models have demonstrated that Slc7a5 deletion is embryonic lethal and that these embryos lack a fully formed telencephalon. This phenotype is similar to that of mammalian target of rapamycin (mTOR) protein kinase deletion or mTOR inhibition. Notably, in many cells, Slc7a5 import of amino acids is required to maintain mTOR activity. Slc7a5 is present within neurogenic regions during embryogenesis, is found in cultured neurons and can modulate neuronal electrophysiological properties. However, Slc7a5 is also highly expressed within endothelial cells of the blood-brain barrier where removal in conditional mice leads to severe behavioral defects and non-cell autonomous changes in neurons. Therefore, the extent that neural Slc7a5 is required for development is unclear. Here, subventricular zone neural stem cells that generate olfactory bulb granule cell neurons were electroporated with SLC7A5 or Slc7a5 short hairpin RNA encoding plasmids. Although early phases of neural development were unaltered, Slc7a5 knockdown effected late phases of GC dendrite maturation and survival. Slc7a5 knockdown also decreased mTOR pathway activity. Ras homolog enriched in brain, an mTOR activator, rescued the effect of Slc7a5 knockdown on mTOR pathway activity and dendrite arbors. The data presented here demonstrate that Slc7a5 is required for GC mTOR pathway activity, maturation and survival, which may help explain why Slc7a5 mutations prevent normal brain development and function.

A transgenic inducible GFP extracellular-vesicle reporter (TIGER) mouse illuminates neonatal cortical astrocytes as a source of immunomodulatory extracellular vesicles.

Extracellular vesicles (EVs) are cellular derived particles found throughout the body in nearly all tissues and bodily fluids. EVs contain biological molecules including small RNAs and protein. EVs are proposed to be transferred between cells, notably, cells of the immune system. Tools that allow for in vivo EV labeling while retaining the ability to resolve cellular sources and timing of release are required for a full understanding of EV functions. Fluorescent EV fusion proteins are useful for the study of EV biogenesis, release, and identification of EV cellular recipients. Among the most plentiful and frequently identified EV proteins is CD9, a tetraspanin protein. A transgenic mouse containing a CRE-recombinase inducible CAG promoter driven CD9 protein fused to Turbo-GFP derived from the copepod Pontellina plumata was generated as an EV reporter. The transgenic inducible GFP EV reporter (TIGER) mouse was electroporated with CAG-CRE plasmids or crossed with tamoxifen inducible CAG-CRE-ERT2 or nestin-CRE-ERT2 mice. CD9-GFP labeled cells included glutamine synthetase and glial fibrillary acidic protein positive astrocytes. Cortical astrocytes released ~136 nm EVs that contained CD9. Intraventricular injected EVs were taken up by CD11b/IBA1 positive microglia surrounding the lateral ventricles. Neonatal electroporation and shRNA mediated knockdown of Rab27a in dorsal subventricular zone NSCs and astrocytes increased the number of CD11b/IBA1 positive rounded microglia. Neonatal astrocyte EVs had a unique small RNA signature comprised of morphogenic miRNAs that induce microglia cytokine release. The results from this study demonstrate that inducible CD9-GFP mice will provide the EV community with a tool that allows for EV labeling in a cell-type specific manner while simultaneously allowing in vivo experimentation and provides evidence that EVs are required immunomodulators of the developing nervous system.

Dendrite growth and the effect of ectopic Rheb expression on cortical neurons.

Ras homology enriched in brain (Rheb) is a GTPase that activates the protein kinase mammalian Target of Rapamycin (mTOR). Rheb mutations cause intellectual delay and megalencephaly. mTOR hyperactivation causes a constellation of neurodevelopmental disorders called "mTOR-opathies" that are frequently accompanied by hyperexcitable cortical malformations. Cortical malformations within the anterior cingulate cortex (ACC) and somatosensory cortex (SSC) frequently colocalize with hyperexcitability. Although Rheb and mTOR are implicated in the formation of cortical lesions, seizure activity, and defects in neuronal migration, the contribution of Rheb to changes in neuron size and dendrite morphology is not well established. Here, in utero electroporation of the developing embryonic brain was used to assess soma and dendrite growth in ACC and SCC layer II/III neurons. We found that between P0 and P21, neuronal soma size increased by 50 and 122 percent in the ACC and SSC, respectively. The increased size was accompanied by an increase in the number of basal dendrites and enhanced dendrite complexity. As an indicator of the involvement of the mTOR pathway in neuron maturation, phosphorylation of the mammalian target of rapamycin (mTOR) substrate S6 was identified in migrating cortical neuroblasts and maturing neurons. Notably, ectopic expression of Rheb caused cortical malformations comprised of ectopically positioned cytomegalic neurons with dendrite hypertrophy. This study provides a direct comparison of neuron maturation across two cortical regions during development, provides evidence for mTOR pathway activity during neuron maturation, and demonstrates that ectopic Rheb expression without mutation is sufficient to induce cortical malformations with cytomegaly and dendrite hypertrophy.