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.

Behavioral and Molecular Responses to Exogenous Cannabinoids During Pentylenetetrazol-Induced Convulsions in Male and Female Rats.

Epilepsy is a disabling, chronic brain disease,affecting ~1% of the World's population, characterized by recurrent seizures (sudden, uncontrolled brain activity), which may manifest with motor symptoms (e.g., convulsions) or non-motor symptoms. Temporal lobe epilepsies (TLE) compromising the hippocampus are the most common form of focal epilepsies. Resistance in ~1/3 of epileptic patients to the first line of treatment, i.e., antiepileptic drugs (AEDs), has been an important motivation to seek alternative treatments. Among these, the plant Cannabis sativa (commonly known as marihuana) or compounds extracted from it (cannabinoids) have gained widespread popularity. Moreover, sex differences have been proposed in epilepsy syndromes and in cannabinoid action. In the hippocampus, cannabinoids interact with the CB1R receptor whose membrane levels are regulated by ß-Arrestin2, a protein that promotes its endocytosis and causes its downregulation. In this article, we evaluate the modulatory role of WIN 55,212-2 (WIN), a synthetic exogenous cannabinoid on behavioral convulsions and on the levels of CB1R and ß-Arrestin2 in female and male adolescent rats after a single injection of the proconvulsant pentylenetetrazol (PTZ). As epilepsies can have a considerable impact on synaptic proteins that regulate neuronal toxicity, plasticity, and cognition, we also measured the levels of key proteins markers of excitatory synapses, in order to examine whether exogenous cannabinoids may prevent such pathologic changes after acute seizures. We found that the exogenous administration of WIN prevented convulsions of medium severity in females and males and increased the levels of phosphorylated CaMKII in the hippocampus. Furthermore, we observed a higher degree of colocalization between CB1R and ß-Arrestin2 in the granule cell layer.

eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects.

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

A Role for mir-26a in Stress: A Potential sEV Biomarker and Modulator of Excitatory Neurotransmission.

Stress is a widespread problem in today's societies, having important consequences on brain function. Among the plethora of mechanisms involved in the stress response at the molecular level, the role of microRNAs (miRNAs) is beginning to be recognized. The control of gene expression by these noncoding RNAs makes them essential regulators of neuronal and synaptic physiology, and alterations in their levels have been associated with pathological conditions and mental disorders. In particular, the excitatory (i.e., glutamate-mediated) neurotransmission is importantly affected by stress. Here, we found that loss of miR-26a-5p (miR-26a henceforth) function in primary hippocampal neurons increased the frequency and amplitude of miniature excitatory currents, as well as the expression levels of the excitatory postsynaptic scaffolding protein PSD95. Incubation of primary hippocampal neurons with corticosterone downregulated miR-26a, an effect that mirrored our in vivo results, as miR-26a was downregulated in the hippocampus as well as in blood serum-derived small extracellular vesicles (sEVs) of rats exposed to two different stress paradigms by movement restriction (i.e., stress by restraint in cages or by complete immobilization in bags). Overall, these results suggest that miR-26a may be involved in the generalized stress response and that a stress-induced downregulation of miR-26a could have long-term effects on glutamate neurotransmission.

Astrocyte-Derived Small Extracellular Vesicles Regulate Dendritic Complexity through miR-26a-5p Activity.

In the last few decades, it has been established that astrocytes play key roles in the regulation of neuronal morphology. However, the contribution of astrocyte-derived small extracellular vesicles (sEVs) to morphological differentiation of neurons has only recently been addressed. Here, we showed that cultured astrocytes expressing a GFP-tagged version of the stress-regulated astrocytic enzyme Aldolase C (Aldo C-GFP) release small extracellular vesicles (sEVs) that are transferred into cultured hippocampal neurons. Surprisingly, Aldo C-GFP-containing sEVs (Aldo C-GFP sEVs) displayed an exacerbated capacity to reduce the dendritic complexity in developing hippocampal neurons compared to sEVs derived from control (i.e., GFP-expressing) astrocytes. Using bioinformatics and biochemical tools, we found that the total content of overexpressed Aldo C-GFP correlates with an increased content of endogenous miRNA-26a-5p in both total astrocyte homogenates and sEVs. Notably, neurons magnetofected with a nucleotide sequence that mimics endogenous miRNA-26a-5p (mimic 26a-5p) not only decreased the levels of neuronal proteins associated to morphogenesis regulation, but also reproduced morphological changes induced by Aldo-C-GFP sEVs. Furthermore, neurons magnetofected with a sequence targeting miRNA-26a-5p (antago 26a-5p) were largely resistant to Aldo C-GFP sEVs. Our results support a novel and complex level of astrocyte-to-neuron communication mediated by astrocyte-derived sEVs and the activity of their miRNA content.

Aberrant expression of PAR bZIP transcription factors is associated with epileptogenesis, focus on hepatic leukemia factor.

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.

Small Extracellular Vesicles in Rat Serum Contain Astrocyte-Derived Protein Biomarkers of Repetitive Stress.

BACKGROUND: Stress precipitates mood disorders, characterized by a range of symptoms present in different combinations, suggesting the existence of disease subtypes. Using an animal model, we previously described that repetitive stress via restraint or immobilization induced depressive-like behaviors in rats that were differentially reverted by a serotonin- or noradrenaline-based antidepressant drug, indicating that different neurobiological mechanisms may be involved. The forebrain astrocyte protein aldolase C, contained in small extracellular vesicles, was identified as a potential biomarker in the cerebrospinal fluid; however, its specific origin remains unknown. Here, we propose to investigate whether serum small extracellular vesicles contain a stress-specific protein cargo and whether serum aldolase C has a brain origin. METHODS: We isolated and characterized serum small extracellular vesicles from rats exposed to restraint, immobilization, or no stress, and their proteomes were identified by mass spectrometry. Data available via ProteomeXchange with identifier PXD009085 were validated, in part, by western blot. In utero electroporation was performed to study the direct transfer of recombinant aldolase C-GFP from brain cells to blood small extracellular vesicles. RESULTS: A differential proteome was identified among the experimental groups, including aldolase C, astrocytic glial fibrillary acidic protein, synaptophysin, and reelin. Additionally, we observed that, when expressed in the brain, aldolase C tagged with green fluorescent protein could be recovered in serum small extracellular vesicles. CONCLUSION: The protein cargo of serum small extracellular vesicles constitutes a valuable source of biomarkers of stress-induced diseases, including those characterized by depressive-like behaviors. Brain-to-periphery signaling mediated by a differential molecular cargo of small extracellular vesicles is a novel and challenging mechanism by which the brain might communicate health and disease states to the rest of the body.

Contribution of early Alzheimer's disease-related pathophysiology to the development of acquired epilepsy.

Aberrant epileptic activity is detectable at early disease stages in Alzheimer's disease (AD) patients and in AD mouse models. Here, we investigated in young ArcticAß mice whether AD-like pathology renders neuronal networks more susceptible to the development of acquired epilepsy induced by unilateral intrahippocampal injection of kainic acid (IHK). In this temporal lobe epilepsy model, IHK induces a status epilepticus followed after two weeks by spontaneous recurrent seizures (SRS). ArcticAß mice exhibited more severe status epilepticus and early onset of SRS. This hyperexcitable phenotype was characterized in CA1 neurons by decreased synaptic strength, increased kainic acid-induced LTP and reduced frequency of spontaneous inhibitory currents. However, no difference in neurodegeneration, neuroinflammation, axonal reorganization or adult neurogenesis was observed in ArcticAß mice compared to wild-type littermates following IHK-induced epileptogenesis. Neuropeptide Y (NPY) expression was reduced at baseline and its IHK-induced elevation in mossy fibres and granule cells was attenuated. However, although this alteration might underlie premature seizure onset, neutralization of soluble Aß species by intracerebroventricular Aß-specific antibody application mitigated the hyperexcitable phenotype of ArcticAß mice and prevented early SRS onset. Therefore, the development of seizures at early stages of AD is mediated primarily by Aß species causing widespread changes in synaptic function.

Astrocytes at the Hub of the Stress Response: Potential Modulation of Neurogenesis by miRNAs in Astrocyte-Derived Exosomes.

Repetitive stress negatively affects several brain functions and neuronal networks. Moreover, adult neurogenesis is consistently impaired in chronic stress models and in associated human diseases such as unipolar depression and bipolar disorder, while it is restored by effective antidepressant treatments. The adult neurogenic niche contains neural progenitor cells in addition to amplifying progenitors, neuroblasts, immature and mature neurons, pericytes, astrocytes, and microglial cells. Because of their particular and crucial position, with their end feet enwrapping endothelial cells and their close communication with the cells of the niche, astrocytes might constitute a nodal point to bridge or transduce systemic stress signals from peripheral blood, such as glucocorticoids, to the cells involved in the neurogenic process. It has been proposed that communication between astrocytes and niche cells depends on direct cell-cell contacts and soluble mediators. In addition, new evidence suggests that this communication might be mediated by extracellular vesicles such as exosomes, and in particular, by their miRNA cargo. Here, we address some of the latest findings regarding the impact of stress in the biology of the neurogenic niche, and postulate how astrocytic exosomes (and miRNAs) may play a fundamental role in such phenomenon.

Endothelial Nitric Oxide Synthase Is Present in Dendritic Spines of Neurons in Primary Cultures.

Nitric oxide exerts important regulatory functions in various brain processes. Its synthesis in neurons has been most commonly ascribed to the neuronal nitric oxide synthase (nNOS) isoform. However, the endothelial isoform (eNOS), which is significantly associated with caveolae in different cell types, has been implicated in synaptic plasticity and is enriched in the dendrites of CA1 hippocampal neurons. Using high resolution microscopy and co-distribution analysis of eNOS with synaptic and raft proteins, we now show for the first time in primary cortical and hippocampal neuronal cultures, virtually devoid of endothelial cells, that eNOS is present in neurons and is localized in dendritic spines. Moreover, eNOS is present in a postsynaptic density-enriched biochemical fraction isolated from these neuronal cultures. In addition, qPCR analysis reveals that both the nNOS as well as the eNOS transcripts are present in neuronal cultures. Moreover, eNOS inhibition in cortical cells has a negative impact on cell survival after excitotoxic stimulation with N-methyl-D-aspartate (NMDA). Consistent with previous results that indicated nitric oxide production in response to the neurotrophin BDNF, we could detect eNOS in immunoprecipitates of the BDNF receptor TrkB while nNOS could not be detected. Taken together, our results show that eNOS is located at excitatory synapses where it could represent a source for NO production and thus, the contribution of eNOS-derived nitric oxide to the regulation of neuronal survival and function deserves further investigations.

BDNF/NF-κB Signaling in the Neurobiology of Depression.

BACKGROUND: Mood disorders, consisting of unipolar and bipolar depression, are complex diseases characterized by depressed mood and anhedonia. These core symptoms are accompanied in a varying manner by anxiety, several neurovegetative symptoms and cognitive impairment. Mood disorders are characterized by decreases in neurogenesis, alteration in synaptic structure and synaptic transmission, all of them regulated by BDNF, a neurotrophin that performs multiple functions in the adult central nervous system. Many evidences show that BDNF is critically decreased in mood disorders and plays an essential role in most anti-depressant treatments. In turn, the transcription factor NF-kB has recently emerged as an important player in the pathophysiology of depression, with roles in neurogenesis, synaptic transmission and plasticity. METHODOLOGY: We review the bidirectional interactions between BDNF and NF-kB signaling pathways. RESULTS AND CONCLUSIONS: We discuss a potential beneficial effect of a positive feedback loop between BDNF and NF-kB activated pathways in antidepressant action. This could be transduced into the identification of downstream NF-kB gene targets able to potentiate antidepressant mechanisms, thus guiding the development of novel and faster acting antidepressant drugs.

Modulatory Effect of 2-(4-Hydroxyphenyl)amino-1,4-naphthoquinone on Endothelial Vasodilation in Rat Aorta.

The vascular endothelium plays an essential role in the control of the blood flow. Pharmacological agents like quinone (menadione) at various doses modulate this process in a variety of ways. In this study, Q7, a 2-phenylamino-1,4-naphthoquinone derivative, significantly increased oxidative stress and induced vascular dysfunction at concentrations that were not cytotoxic to endothelial or vascular smooth muscle cells. Q7 reduced nitric oxide (NO) levels and endothelial vasodilation to acetylcholine in rat aorta. It also blunted the calcium release from intracellular stores by increasing the phenylephrine-induced vasoconstriction when CaCl2 was added to a calcium-free medium but did not affect the influx of calcium from extracellular space. Q7 increased the vasoconstriction to BaCl2 (10-3 M), an inward rectifying K+ channels blocker, and blocked the vasodilation to KCl (10-2 M) in aortic rings precontracted with BaCl2. This was recovered with sodium nitroprusside (10-8 M), a NO donor. In conclusion, Q7 induced vasoconstriction was through a modulation of cellular mechanisms involving calcium fluxes through K+ channels, and oxidative stress induced endothelium damage. These findings contribute to the characterization of new quinone derivatives with low cytotoxicity able to pharmacologically modulate vasodilation.

MiRNAs in Astrocyte-Derived Exosomes as Possible Mediators of Neuronal Plasticity.

Astrocytes use gliotransmitters to modulate neuronal function and plasticity. However, the role of small extracellular vesicles, called exosomes, in astrocyte-to-neuron signaling is mostly unknown. Exosomes originate in multivesicular bodies of parent cells and are secreted by fusion of the multivesicular body limiting membrane with the plasma membrane. Their molecular cargo, consisting of RNA species, proteins, and lipids, is in part cell type and cell state specific. Among the RNA species transported by exosomes, microRNAs (miRNAs) are able to modify gene expression in recipient cells. Several miRNAs present in astrocytes are regulated under pathological conditions, and this may have far-reaching consequences if they are loaded in exosomes. We propose that astrocyte-derived miRNA-loaded exosomes, such as miR-26a, are dysregulated in several central nervous system diseases; thus potentially controlling neuronal morphology and synaptic transmission through validated and predicted targets. Unraveling the contribution of this new signaling mechanism to the maintenance and plasticity of neuronal networks will impact our understanding on the physiology and pathophysiology of the central nervous system.

Partial inactivation of GABAA receptors containing the α5 subunit affects the development of adult-born dentate gyrus granule cells.

Alterations of neuronal activity due to changes in GABAA receptors (GABAA R) mediating tonic inhibition influence different hippocampal functions. Gabra5-null mice and α5 subunit((H105R)) knock-in mice exhibit signs of hippocampal dysfunction, but are capable of improved performance in several learning and memory tasks. Accordingly, alleviating abnormal GABAergic tonic inhibition in the hippocampal formation by selective α5-GABAA R modulators represents a possible therapeutic approach for several intellectual deficit disorders. Adult neurogenesis in the dentate gyrus is an important facet of hippocampal plasticity; it is regulated by tonic GABAergic transmission, as shown by deficits in proliferation, migration and dendritic development of adult-born neurons in Gabra4-null mice. Here, we investigated the contribution of α5-GABAA Rs to granule cell development, using retroviral vectors expressing eGFP for labeling precursor cells in the subgranular zone. Global α5-GABAA R knockout (α5-KO) mice showed no alterations in migration and morphological development of eGFP-positive granule cells. However, upregulation of α1 subunit-immunoreactivity was observed in the hippocampal formation and cerebral cortex. In contrast, partial gene inactivation in α5-heterozygous (α5-het) mice, as well as single-cell deletion of Gabra5 in newborn granule cells from α5-floxed mice, caused severe alterations of migration and dendrite development. In α5-het mice, retrovirally mediated overexpression of Cdk5 resulted in normal migration and dendritic branching, suggesting that Cdk5 cooperates with α5-GABAA Rs to regulate neuronal development. These results show that minor imbalance of α5-GABAA R-mediated transmission may have major consequences for neuronal plasticity; and call for caution upon chronic therapeutic use of negative allosteric modulators acting at these receptors.

Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche.

Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future.

Exosomes as Novel Regulators of Adult Neurogenic Niches.

Adult neurogenesis has been convincingly demonstrated in two regions of the mammalian brain: the sub-granular zone (SGZ) of the dentate gyrus (DG) in the hippocampus, and the sub-ventricular zone (SVZ) of the lateral ventricles (LV). SGZ newborn neurons are destined to the granular cell layer (GCL) of the DG, while new neurons from the SVZ neurons migrate rostrally into the olfactory bulb (OB). The process of adult neurogenesis persists throughout life and is supported by a pool of neural stem cells (NSCs), which reside in a unique and specialized microenvironment known as "neurogenic niche". Neurogenic niches are structured by a complex organization of different cell types, including the NSC-neuron lineage, glial cells and vascular cells. Thus, cell-to-cell communication plays a key role in the dynamic modulation of homeostasis and plasticity of the adult neurogenic process. Specific cell-cell contacts and extracellular signals originated locally provide the necessary support and regulate the balance between self-renewal and differentiation of NSCs. Furthermore, extracellular signals originated at distant locations, including other brain regions or systemic organs, may reach the niche through the cerebrospinal fluid (CSF) or the vasculature and influence its nature. The role of several secreted molecules, such as cytokines, growth factors, neurotransmitters, and hormones, in the biology of adult NSCs, has been systematically addressed. Interestingly, in addition to these well-recognized signals, a novel type of intercellular messengers has been identified recently: the extracellular vesicles (EVs). EVs, and particularly exosomes, are implicated in the transfer of mRNAs, microRNAs (miRNAs), proteins and lipids between cells and thus are able to modify the function of recipient cells. Exosomes appear to play a significant role in different stem cell niches such as the mesenchymal stem cell niche, cancer stem cell niche and pre-metastatic niche; however, their roles in adult neurogenic niches remain virtually unexplored. This review focuses on the current knowledge regarding the functional relationship between cellular and extracellular components of the adult SVZ and SGZ neurogenic niches, and the growing evidence that supports the potential role of exosomes in the physiology and pathology of adult neurogenesis.

GABAergic striatal neurons project dendrites and axons into the postnatal subventricular zone leading to calcium activity.

GABA regulates the behavior of neuroblasts and neural progenitor cells in the postnatal neurogenic subventricular zone (SVZ) through GABAA receptor (GABAAR)-mediated calcium increases. However, the source of GABA necessary for sufficient GABAAR-mediated depolarization and calcium increase has remained speculative. Here, we explored whether GABAergic striatal neurons functionally connect with SVZ cells. Using patch clamp recordings or single cell electroporation, striatal neurons along the SVZ were filled with a fluorescent dye revealing that they send both dendrites and axons into the SVZ. About 93% of the recorded neurons were medium spiny or aspiny GABAergic neurons and each neuron sent 3-4 processes into the SVZ covering ~56 µm. Using calcium imaging, we found that depolarization of striatal neurons led to increased calcium activity in SVZ cells that were mediated by GABAAR activation. Collectively, these findings undercover a novel mode of signaling in the SVZ providing a mechanism of brain activity-mediated regulation of postnatal neurogenesis through GABAergic striatal activity.

A circuitry and biochemical basis for tuberous sclerosis symptoms: from epilepsy to neurocognitive deficits.

Tuberous sclerosis complex (TSC) is an autosomal dominant monogenetic disorder that is characterized by the formation of benign tumors in several organs as well as brain malformations and neuronal defects. TSC is caused by inactivating mutations in one of two genes, TSC1 and TSC2, resulting in increased activity of the mammalian Target of Rapamycin (mTOR). Here, we explore the cytoarchitectural and functional CNS aberrations that may account for the neurological presentations of TSC, notably seizures, hydrocephalus, and cognitive and psychological impairments. In particular, recent mouse models of brain lesions are presented with an emphasis on using electroporation to allow the generation of discrete lesions resulting from loss of heterozygosity during perinatal development. Cortical lesions are thought to contribute to epileptogenesis and worsening of cognitive defects. However, it has recently been suggested that being born with a mutant allele without loss of heterozygosity and associated cortical lesions is sufficient to generate cognitive and neuropsychiatric problems. We will thus discuss the function of mTOR hyperactivity on neuronal circuit formation and the potential consequences of being born heterozygous on neuronal function and the biochemistry of synaptic plasticity, the cellular substrate of learning and memory. Ultimately, a major goal of TSC research is to identify the cellular and molecular mechanisms downstream of mTOR underlying the neurological manifestations observed in TSC patients and identify novel therapeutic targets to prevent the formation of brain lesions and restore neuronal function.

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.

Neonatal subventricular zone electroporation.

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates. Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development. More recently this was applied to study the postnatal rodent forebrain. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.