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.

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.

From seed to flower: blossoming of microglia in development and brain repair.

Physiological functions require coordination of processes between diverse organs, tissues, and cells. This integrative view of science has reemerged complementary to the reductionist philosophy of studying individual cell types. An integrative approach has proven particularly powerful within the field of neuroscience where, intermingled among the most numerous neural cell types of the brain, are immune cells called microglia. Microglia act as a line of defense in the CNS by phagocytizing harmful pathogens and cellular debris and by releasing a variety of factors that mediate immune responses. However, microglia are also appreciated as critical mediators of neurophysiology making them a desired target to rectify neuropathological states. The goal of this review is to discuss microglia ontogenesis, referred to as microgliogenesis, a term that encompasses the events that drive the production, differentiation, migration, and maturation of microglia and opportunities to target microglia for brain repair.

The Neurodevelopmental Pathogenesis of Tuberous Sclerosis Complex (TSC).

Tuberous sclerosis complex (TSC) is a model disorder for understanding brain development because the genes that cause TSC are known, many downstream molecular pathways have been identified, and the resulting perturbations of cellular events are established. TSC, therefore, provides an intellectual framework to understand the molecular and biochemical pathways that orchestrate normal brain development. The TSC1 and TSC2 genes encode Hamartin and Tuberin which form a GTPase activating protein (GAP) complex. Inactivating mutations in TSC genes (TSC1/TSC2) cause sustained Ras homologue enriched in brain (RHEB) activation of the mammalian isoform of the target of rapamycin complex 1 (mTORC1). TOR is a protein kinase that regulates cell size in many organisms throughout nature. mTORC1 inhibits catabolic processes including autophagy and activates anabolic processes including mRNA translation. mTORC1 regulation is achieved through two main upstream mechanisms. The first mechanism is regulation by growth factor signaling. The second mechanism is regulation by amino acids. Gene mutations that cause too much or too little mTORC1 activity lead to a spectrum of neuroanatomical changes ranging from altered brain size (micro and macrocephaly) to cortical malformations to Type I neoplasias. Because somatic mutations often underlie these changes, the timing, and location of mutation results in focal brain malformations. These mutations, therefore, provide gain-of-function and loss-of-function changes that are a powerful tool to assess the events that have gone awry during development and to determine their functional physiological consequences. Knowledge about the TSC-mTORC1 pathway has allowed scientists to predict which upstream and downstream mutations should cause commensurate neuroanatomical changes. Indeed, many of these predictions have now been clinically validated. A description of clinical imaging and histochemical findings is provided in relation to laboratory models of TSC that will allow the reader to appreciate how human pathology can provide an understanding of the fundamental mechanisms of development.

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.

Isolation of Extracellular Vesicles from Subventricular Zone Neural Stem Cells.

The neonatal subventricular zone (SVZ) is a neurogenic niche that contains neural stem cells (NSCs). NSCs release particles called extracellular vesicles (EVs) that contain biological material. EVs are transferred to cells, including immune cells in the brain called microglia. A standard approach to identify EV functions is to isolate and transplant EVs. Here, a detailed protocol is provided that will allow one to culture neonatal SVZ NSCs and to isolate, label, and transplant EVs. The protocol will permit careful and thorough examination of EVs in a wide range of physiological and pathophysiological conditions.

Neonatal Subventricular Zone Neural Stem Cells Release Extracellular Vesicles that Act as a Microglial Morphogen.

Subventricular zone (SVZ) neural stem cells (NSCs) are the cornerstone of the perinatal neurogenic niche. Microglia are immune cells of the nervous system that are enriched in the neonatal SVZ. Although microglia regulate NSCs, the extent to which this interaction is bi-directional is unclear. Extracellular vesicles (EVs) are cell-derived particles that encase miRNA and proteins. Here, we demonstrate that SVZ NSCs generate and release EVs. Neonatal electroporated fluorescent EV fusion proteins were released by NSCs and subsequently cleared from the SVZ. EVs were preferentially targeted to microglia. Small RNA sequencing identified miRNAs within the EVs that regulate microglia physiology and morphology. EVs induced a transition to a CD11b/Iba1 non-stellate microglial morphology. The transition accompanied a microglial transcriptional state characterized by Let-7-regulated cytokine release and a negative feedback loop that controlled NSC proliferation. These findings implicate an NSC-EV-microglia axis and provide insight to normal and pathophysiological brain development.

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.

Neurovesicles in Brain Development.

Long before the nervous system is organized into electrically active neural circuits, connectivity emerges between cells of the developing brain through extracellular signals. Extracellular vesicles that shuttle RNA, proteins, and lipids from donor cells to recipient cells are candidates for mediating connectivity in the brain. Despite the abundance of extracellular vesicles during brain development, evidence for their physiological functions is only beginning to materialize. Here, we review evidence of the existence, content, and functions of extracellular vesicles in brain development.

Switching on mTORC1 induces neurogenesis but not proliferation in neural stem cells of young mice.

Recent evidence reported that activation of the mechanistic target of rapamycin complex 1 (mTORC1) induces terminal differentiation of neural stem cells (NSCs) in the neonatal subventricular zone (SVZ), but did not affect their proliferation. Here, we investigated whether such an effect of hyperactive mTORC1 would be recapitulated in young adults following removal of the negative mTORC1 regulator TSC1as seen in the neurological disorder tuberous sclerosis complex, TSC. Conditional mTORC1 activation in NSCs of 3-4 weeks old mice resulted in the generation of proliferative (Ki67+) cells and newborn neuroblasts. However, hyperactive mTORC1 did not induce NSCs to proliferate, consistent with the findings that mTORC1 induces symmetric division and differentiation of slow-cycling NSCs into proliferative daughter cells. Taken together these data suggest that hyperactivity of mTORC1 could lead to the progressive loss of NSCs over time.

Hypoxia-inducible factor-1a contributes to dendritic overgrowth in tuberous sclerosis.

Expression of hypoxia-inducible factor 1a (HIF1a) is increased under several pathological conditions such as hyperactive mechanistic target of rapamycin complex 1 (mTORC1) in tuberous sclerosis complex (TSC). Hyperactive mTORC1 and the resulting increased dendritic complexity of neurons are shared molecular and cellular alterations in several neurological disorders associated with cognitive disabilities. Despite some evidence that HIF1a contributes to dendritic overgrowth in vitro, it remains unknown whether increased HIF1a in TSC neurons could contribute to their increased dendritic complexity. To address this use in vivo, we generated TSC neurons by deleting Tsc1 in newborn olfactory bulb (OB) neurons of conditional Tsc1 transgenic mice using neonatal electroporation. In addition to their increased dendritic complexity, Tsc1(null) neurons have been reported to display increased Hif1a mRNA level and HIF1a transcriptional activity. We found that Tsc1(null)-dependent dendritic overgrowth was prevented by knocking down HIF1a or expressing a dominant negative HIF1a. In addition, overexpressing HIF1a in wild-type developing neurons resulted in increased dendritic complexity in vivo. These data highlight that an increase in HIF1a levels contributes to abnormal dendritic patterning in developing neurons under normal conditions and hyperactive mTORC1 conditions as in TSC.

Noncanonical Sites of Adult Neurogenesis in the Mammalian Brain.

Two decades after the discovery that neural stem cells (NSCs) populate some regions of the mammalian central nervous system (CNS), deep knowledge has been accumulated on their capacity to generate new neurons in the adult brain. This constitutive adult neurogenesis occurs throughout life primarily within remnants of the embryonic germinal layers known as "neurogenic sites." Nevertheless, some processes of neurogliogenesis also occur in the CNS parenchyma commonly considered as "nonneurogenic." This "noncanonical" cell genesis has been the object of many claims, some of which turned out to be not true. Indeed, it is often an "incomplete" process as to its final outcome, heterogeneous by several measures, including regional location, progenitor identity, and fate of the progeny. These aspects also strictly depend on the animal species, suggesting that persistent neurogenic processes have uniquely adapted to the brain anatomy of different mammals. Whereas some examples of noncanonical neurogenesis are strictly parenchymal, others also show stem cell niche-like features and a strong link with the ventricular cavities. This work will review results obtained in a research field that expanded from classic neurogenesis studies involving a variety of areas of the CNS outside of the subventricular zone (SVZ) and subgranular zone (SGZ). It will be highlighted how knowledge concerning noncanonical neurogenic areas is still incomplete owing to its regional and species-specific heterogeneity, and to objective difficulties still hampering its full identification and characterization.

Cerebrospinal fluid extracellular vesicles undergo age dependent declines and contain known and novel non-coding RNAs.

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.

MEK-ERK1/2-dependent FLNA overexpression promotes abnormal dendritic patterning in tuberous sclerosis independent of mTOR.

Abnormal dendritic complexity is a shared feature of many neurodevelopmental disorders associated with neurological defects. Here, we found that the actin-crosslinking protein filamin A (FLNA) is overexpressed in tuberous sclerosis complex (TSC) mice, a PI3K-mTOR model of neurodevelopmental disease that is associated with abnormal dendritic complexity. Both under- and overexpression of FLNA in wild-type neurons led to more complex dendritic arbors in vivo, suggesting that an optimal level of FLNA expression is required for normal dendritogenesis. In Tsc1(null) neurons, knocking down FLNA in vivo prevented dendritic abnormalities. Surprisingly, FLNA overexpression in Tsc1(null) neurons was dependent on MEK1/2 but not mTOR activity, despite both pathways being hyperactive. In addition, increasing MEK-ERK1/2 activity led to dendritic abnormalities via FLNA, and decreasing MEK-ERK1/2 signaling in Tsc1(null) neurons rescued dendritic defects. These data demonstrate that altered FLNA expression increases dendritic complexity and contributes to pathologic dendritic patterning in TSC in an mTOR-independent, ERK1/2-dependent manner.

Embryonic cerebrospinal fluid nanovesicles carry evolutionarily conserved molecules and promote neural stem cell amplification.

During brain development, neural stem cells (NSCs) receive on-or-off signals important for regulating their amplification and reaching adequate neuron density. However, how a coordinated regulation of intracellular pathways and genetic programs is achieved has remained elusive. Here, we found that the embryonic (e) CSF contains 10¹² nanoparticles/ml (77 nm diameter), some of which were identified as exosome nanovesicles that contain evolutionarily conserved molecules important for coordinating intracellular pathways. eCSF nanovesicles collected from rodent and human embryos encapsulate protein and microRNA components of the insulin-like growth factor (IGF) signaling pathway. Supplementation of eCSF nanovesicles to a mixed culture containing eNSCs activated the IGF-mammalian target of rapamycin complex 1 (mTORC1) pathway in eNSCs and expanded the pool of proliferative eNSCs. These data show that the eCSF serves as a medium for the distribution of nanovesicles, including exosomes, and the coordinated transfer of evolutionary conserved molecules that regulate eNSC amplification during corticogenesis.