Angelique Bordey - US grants

DESCRIPTION (provided by applicant): The postnatal subventricular zone (SVZ) is a germinal zone containing distinct immature cell types, including stem cells and migrating neuronal progenitors. Understanding the in situ physiological properties of SVZ cells and the diffusible factors influencing SVZ cell physiology is critical to allow endogenous activation of stem cells/progenitors and induction of self-repair. In situ SVZ cell physiology and SVZ signaling factors are, however, largely unknown. Because the identity of neural stem cells is still debated, we propose to identify the signaling factors that regulate SVZ cell properties and could in particular influence neuronal progenitor migration. GABA is a likely candidate as a signaling factor because it regulates migration of embryonic cells and its synthetic enzyme is present in the SVZ. We thus hypothesize that GABA is a signaling factor locally released by migrating progenitors at rest resulting in activation of GABA autoreceptors and ion channels known to be involved in cell migration. The two following aims will be addressed with whole-cell and perforated patch-clamp recordings, and with fluorimetric Ca2+ measurements in SVZ slices. The first aim will determine whether GABA activates GABA receptors (GABAR5) on neuronal progenitors and whether GABAR activation leads to Ca2+ increase and Ca2+-dependent K+ (KCa) channel activation, which triggers membrane potential changes. KCa channel activation is known to be a prerequisite for migration of other cell types. The second aim will investigate whether GABARs are activated by transport-mediated GABA efflux from progenitors following membrane potential depolarization. Neuronal progenitors will be recorded from transgenic mice expressing yellow fluorescent protein in such progenitors under the control of the tubulin promoter. Data from these studies will provide the first fundamental insight into the physiology of SVZ cells in situ and exposes a novel concept of cell migration governed by an interplay between GABARs and GABA transporters.

[unreadable] DESCRIPTION (provided by applicant): Bergmann glia, the specialized cerebellar astrocytes, encapsulate synaptic terminals onto Purkinje cells and possess transporters that are precisely located to remove GABA and glutamate from synaptic clefts. Although astrocytes have been shown to sense neuronal activity, very little is known on their ability to influence synaptic transmission. The goal of this application is to determine whether astrocytic GABA/taurine and glutamate transporters contribute to fast GABAergic neurotransmission. Three aims will be addressed with immunocytochemistry and whole-cell patch-clamp recordings in rat cerebellar slices. The first aim will be to verify the hypothesis that Bergmann glia possess functional GABA and taurine transporters that can work in reverse and are activated by synaptically-released GABA. The second aim will be to evaluate the hypothesis that astrocytic GABA transporters can influence neuronal activity by uptake of GABA or by non-vesicular release of GABA or taurine. This aim will require simultaneous recordings of a Purkinje cell and an adjacent Bergmann glial cell encapsulating GABAergic inputs on the recorded Purkinje cell. While monitoring GABAergic synaptic currents, GABA transporters in Bergmann glia will be blocked by internal perfusion of a transport blocker during the recording. Similarly, the effect of non-vesicular release of GABA or taurine will be tested on neuronal baseline currents. Finally, glutamate has been shown to stimulate GABA release by activating presynaptic NMDA receptors on GABAergic terminals. The third aim will be to verify the hypothesis that astrocytic glutamate transporters regulate presynaptic GABA release by limiting glutamate spillover from glutamatergic synapses and thus regulating glutamate levels at GABAergic synapses. These studies will define important functions of astrocytic transporters on synaptic efficacy and neuronal excitability. Because astrocytes from other brain regions also express similar transporters and encapsulate synapses, findings in the cerebellum can serve as a model of the function of astrocytic transporters at GABAergic synapses. These data will also be important for understanding pathological mechanisms affecting astrocytic transporters and GABA signaling

[unreadable] DESCRIPTION (provided by applicant): The postnatal subventricular zone (SVZ) is a germinal center composed of neuronal precursors and glial fibrillary acidic protein positive (GFAP+) stem cells. The neuronal precursors proliferate and differentiate during their migration throughout the SVZ, along the rostral migratory stream (RMS) toward the olfactory bulb. The behavior of precursors is believed to be influenced by interactions with the progenitor environment, including local signals within the SVZ and surrounding cells. Thus, an understanding of the development of neuronal precursors in the SVZ requires an analysis of the signals that influence their behavior. Thus far, the signaling factors and their functions on SVZ progenitors remain largely unknown. However, we recently described the presence of GABA and functional GABAA receptors in postnatal SVZ neuronal progenitors, suggesting a signaling function of GABA in the SVZ. Furthermore, our recent data shows that GABA reduces the speed of neuronal progenitor migration. The goal of this proposal is to determine whether GABAergic signaling promotes the differentiation of neuronal progenitors in the postnatal subventricular zone. Three aims will be addressed with immunocytochemistry, patch clamp recordings, Ca2+imaging, ELISA, immunohistochemistry, RT-PCR and gene microarray in acute, postnatal mouse slices and SVZ/RMS explants. The first aim will test whether GABA promotes spontaneous Ca2+-dependent GABA release from neuronal progenitors and induces GABA receptor activation in surrounding neuronal progenitors. This positive feedback mechanism is hypothesized to stop when ambient GABA levels reach desensitizing levels that prevent GABAAR activation and reduce GABA release. The second aim will determine whether GABA transporters are expressed in GFAP+ cells of the SVZ that form tubes filled with migrating neuronal progenitors. GFAP+ cells may thus create a microenvironment where ambient GABA levels are tightly regulated. The third aim will examine whether ambient GABA promotes progenitor differentiation by stimulating BDNF synthesis in a Ca2+- and MAPK-dependent manner. The proposed experiments will provide important new information regarding the function of GABA in the SVZ. Data from these experiments will also considerably improve our understanding of the mechanisms of intercellular communication and cell differentiation in the SVZ, which is critical for the future therapeutic use of SVZ cells to replace damaged cells. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The postnatal subventricular zone (SVZ) is a germinal center composed of a network of chains of migrating and proliferative neuroblasts ensheathed by glial fibrillary acidic protein immunopositive (GFAP+) cells, which display the characteristics of stem cells. The progenitors migrate throughout the SVZ and along the rostral migratory stream (RMS) toward the olfactory bulb where they continuously replace interneurons throughout life. Identifying the signals influencing the behavior of SVZ progenitors would lead to a better basic understanding of the mechanisms influencing replacement of olfactory bulb interneurons, and is necessary to design strategies to promote neurogenesis and self-repair. The production of progenitors is thought to be regulated by molecules providing communication signals between neuroblasts and GFAP+ cells, yet our knowledge of such signaling remains rudimentary. The central hypothesis of this grant is that glutamatergic signaling from GFAP+ cells to neuroblasts balances GABA's inhibitory function by promoting the proliferation of postnatal SVZ progenitors. Three aims will be addressed. First we will determine whether a fast glutamatergic signaling from GFAP+ cells to neuroblast exists. As neuroblasts signal to GFAP+ cells via GABAergic signaling, glutamatergic signaling would provide a feedback loop necessary for a bidirectional communication between these two cell types. Second, we will determine whether PGE2 released from SVZ cells modulates glutamate release from GFAP+ cells and thus mGluR activation. Finally, we will determine whether glutamate and PGE2's modulation of glutamate release provide a positive control on cell proliferation to counterbalance GABA's inhibitory influence. We will use patch-clamp and Ca2+ imaging techniques, and perform proliferation assays in brain slices and in vivo. We will use CD1 mice and several lines of transgenic mice to address our aims. It is hoped that together the proposed experiments will identify the existence and function of a bidirectional communication between GFAP+ cells and neuroblasts, and further our understanding of how intercellular signaling regulates postnatal neurogenesis.

DESCRIPTION (provided by applicant): This proposal will test the hypothesis that glutamatergic signaling orchestrates early stages of granule cell development necessary for proper cerebellar circuit formation. Glutamate, which has a conserved signaling function across phyla, controls multiple intracellular signaling pathways through diverse receptors. Glutamate receptor expression in the brain is tightly developmentally regulated, suggesting specific functions of each receptor subtype at different stages of cell development. Indeed, emerging evidence implicates glutamate receptors in brain development. However, our understanding of such functions lags behind the known diversity of glutamate receptor subtypes. Previous and preliminary data suggest that granule cell precursors (GCPs) transiently express functional GluK2-containing kainate receptors (KAR) and group I metabotropic glutamate receptor 5 (mGlu5R) in the external germinal layer (EGL) when they exit the cell cycle and extend axons. However, it is not known whether, and if so how, gain or loss of function in GluK2 receptor and mGlu5R alters GCP development. In this proposal, Aim 1 and Aim 2 will examine the function of GluK2 and mGlu5R on GCP proliferation and axon extension, respectively. Aim 3 will explore whether Bergmann glial cells, a specialized astrocytes extending processes through the EGL, release glutamate controlling GCP cell-cycle exit through mGlu5R. We will use a combination of time- lapse confocal microscopy (for calcium imaging, migration, and axon extension) in acute slices and in vivo approaches (drugs, RNA interference via electroporation, and genetic manipulation) in wild-type and transgenic mice, including mGlu5Rfl/fl (fl, floxed) and GluK2- knockout (KO).

DESCRIPTION (provided by applicant): This proposal will test the hypothesis that the direct mTOR effectors and downstream as well as mTOR-independent molecules contribute to cortical neuron hypertrophy and misplacement in Tuberous Sclerosis Complex (TSC). The long term goal of this proposal is to prevent or rescue brain malformations in TSC and other disorders involving the PI3K pathway. TSC is caused by mutations in TSC1 or TSC2 leading to mTOR hyperactivity and developmental malformations associated with seizures and worsening of cognitive and psychiatric deficits. The mTOR inhibitor rapamycin is the only therapeutic option, does not rescue all defects, and has major side-effects emphasizing the need to better understand the molecular basis of TSC and find novel drug targets. We and others have developed mouse models of TSC-associated cortical lesions. Consistent defects found across these models are neuronal misplacement and dendrite hypertrophy. Spine defects remain unclear. Here, we propose to identify some of the molecular players responsible for abnormal placement and morphogenesis (dendrites and spines) of cortical pyramidal neurons. mTOR directly phosphorylates the translational repressor eIF4E binding protein (4E-BP) and p70 ribosomal S6 protein kinase 1 (S6K1). In Aim 1, we will determine whether 4E-BP and/or S6K1 mediate mTOR effects on cortical neuron development. The canonical mTOR activator, Rheb, which is inhibited by the TSC1/TSC2 complex may contribute to some defects independently of mTOR, but this remains speculative. In aim 2, we propose to identify which mTOR- dependent and -independent molecules contribute to cortical defects in TSC. Finally, in aim 3 we will test whether identified molecules (e.g. FLNA) prevent and rescue neuronal defects in TSC. We will use a combination of genetic and pharmacological manipulations as well as translating ribosome affinity purification (TRAP) analysis in collaboration with Dr. Breunig in wild-type and transgenic mice.

? DESCRIPTION (provided by applicant): The precise timing of formation and length of axonal connectivity in the developing brain is essential for proper neural circuit connectivity and function. As such, alterations in axonal connectivity are thought to contribute to neuropsychological impairments in autism spectrum disorder including monogenic disorders associated with autism such as tuberous sclerosis complex (TSC). TSC individuals express a silencing mutation in one TSC1 or TSC2 allele leading to hyperactivity of the mammalian target of rapamycin complex 1 and 40-60% of these individuals display mild to severe autistic traits and other neuropsychological problems that are independent of seizures or gross brain abnormalities. In TSC as well as other disorders associated with autism, the mechanisms leading to altered axonal connectivity remain unclear. One possible mechanism that remains unexplored is the contribution of astrocytes to abnormal axonal growth in TSC. Astrocytes are a subtype of macroglia that play a critical role in regulating neuronal development including axon elongation. Here, we thus propose to test the hypothesis that Tsc1+/- astrocytes contribute to increased axonal growth in TSC. We have two aims. The first aim proposes to assess whether Tsc1+/- astrocytes accelerate axon growth and increase axonal patterning. Using iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomics, we identified several molecules altered in Tsc1+/- astrocyte-conditioned medium, such as apolipoprotein E (apoE) that is a good candidate for controlling axon elongation. The second aim proposes to examine whether reduced apoE levels in Tsc1+/- astrocytes contributes to abnormal axon growth. To address our hypothesis, we will use purified cultures of astrocytes and neurons as well as in vivo approaches using conditional transgenic mice to delete one allele of Tsc1 selectively in astrocytes combined with in utero electroporation to label selective neuronal populations or single cortical neurons for axon tracing.

? DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is an autosomal dominant disorder occurring in 1/6,000 individuals with mutations in TSC1 or TSC2 leading to increased mTOR activity. About 80- 90% of the TSC individuals will experience seizures of different types. Most types of seizures are refractory to treatment and require invasive surgical resection leading to manageable seizures in only 50% of the cases. There is thus a serious need to improve epilepsy treatment in TSC. To develop such treatment, we need to better understand the mechanisms of seizure generation. It is known that focal cortical malformations, called cortical tubers in TSC are associated with epilepsy, but understanding how tubers generate seizures is limited because of the lack of an experimental model of focal cortical malformation-associated epilepsy. Here, we have generated a novel model of focal tuber-associated seizures, and we propose to further characterize it and address some of the possible mechanisms of epileptogenesis/seizures. As in humans, we found that experimental cortical tubers are characterized by loss of cortical lamination, the presence of ectopic, cytomegalic neurons with hypertrophic dendrites, and gliosis. Our overarching hypothesis is preventing one of the alterations may be sufficient to prevent seizure generation. Here, due to the exploratory nature and the short duration of a R21, we focused on two specific alterations, neuronal misplacement which is associated with circuit disorganization, and mTORC1-induced changes in biophysical properties (i.e., abnormal acquisition of pacemaker channel). Our more specific hypothesis is that preventing neuronal misplacement or specific alterations in biophysical properties prevents seizures. To address this hypothesis, we have two aims, one focused on neuronal misplacement and the other one on the biophysical properties of tuber neurons. We will use a combination of approaches including in utero electroporation to increase mTOR activity in developing cortical neurons followed by patch clamp recordings in slices and electroencephalogram (EEG)/behavior recordings to monitor epileptiform discharges in vivo. In addition, we will use conditional approaches as well as engineered receptors to silence neurons in vivo. This combination of approaches is innovative and we are in a unique position to accomplish the proposed work. Finally, TSC is an exemplary disorder of mTOR-dependent focal cortical malformation-associated epilepsy. The other disorder is focal cortical dysplasia (FCD) type II, which share similar molecular and cellular alterations. Our model is thus applicable to FCD type II. Both disorders are the leading cause of focal malformation-associated refractory epilepsy.s

? DESCRIPTION (provided by applicant): This proposal will test the hypothesis that decreasing Filamin A (FLNA) levels or function in tuberous sclerosis complex (TSC) prevents cortical malformations and associated seizure activity. TSC is caused by mutations in TSC1 or TSC2 leading to mTOR complex 1 (mTORC1) hyperactivity and cortical malformations associated with seizures and worsening of cognitive and psychiatric deficits. The mTORC1 inhibitor rapamycin is the only therapeutic option, does not rescue all defects, and has mild to life-threatening complications emphasizing the need to find novel drug targets. We recently reported that the level of the actin cross-linking molecule FLNA is increased in Tsc1null neurons and that this increase is responsible for dendrite abnormalities. In addition, FLNA regulates the migration of cortical neurons during development. These findings are attractive with respect to TSC for the following reasons: (1) Neuronal dysmorphogenesis (including dendritic abnormality) and stalled migration are hallmarks of TSC-related cortical malformations. (2) FLNA increase in Tsc1null neurons as well as in cells expressing a constitutively active Rheb (the mTORC1 canonical activator) was not mTORC1-dependent but rather ERK1/2-dependent, opening a novel pharmacological option for a possible combination therapy, and (3) Our preliminary data show that normalizing FLNA levels using shRNA or administrating the new FLNA modulator, PTI-125, during development prevents neuronal misplacement and dysmorphogenesis in our new model of TSC-related cortical malformations. To address our hypothesis we have the following three aims. In Aim 1, we will determine whether FLNA controls development of cortical pyramidal neurons in vivo and whether decreasing FLNA levels during development prevents cortical malformations. In Aim 2, we will examine whether there is a time-window in neonates for treatments aimed at preventing cortical malformations and reducing or eliminating associated seizure activity. Our new murine model of focal cortical malformations is associated with a high rate of daily convulsive seizures. Finally in Aim 3, we will investigate how increased FLNA levels leads to dysmorphogenesis and stalled migration, which may identify novel FLNA binding partners involved in cortical defects in TSC. Most experiments will use in utero electroporation to selectively manipulate the development of layer 2/3 cortical pyramidal neurons. This is a 2 PIs grant. The Bordey lab will be in charge of Aim 1 and 2, and the Calderwood lab will be in charge of Aim 3. Both labs will heavily interact on a weekly basis due to the need for tool (plasmid) development and the need for cell biology and in vivo experiments in Aim 2 and 3, respectively. Dr. Bordey is an expert on neuronal development and TSC, and Dr. Calderwood is a cell biologist with extensive expertise on FLNA.

Abstract Tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII) are caused by mutations in mTOR pathway genes leading to mTOR hyperactivity, focal malformations of cortical development (fMCD), and seizures in 80-90% of the patients. The current definitive treatments for epilepsy are surgical resection or treatment with everolimus, which inhibits mTOR activity (only approved for TSC). Because both options have severe limitations, there is a major need to better understand the mechanisms leading to seizures to improve life-long epilepsy treatment in TSC and FCDII. To investigate such mechanisms, we recently developed a murine model of fMCD-associated epilepsy that recapitulates the human TSC and FCDII disorders. fMCD are defined by the presence of misplaced, dysmorphic cortical neurons expressing hyperactive mTOR ? for simplicity we will refer to these as ?mutant? neurons. In our model and in human TSC tissue, we made a surprising finding that mutant neurons express HCN4 channels, which are not normally functionally expressed in cortical neurons. These data led us to ask several important questions based on the known biology of HCN4 channels: (1) As HCN4 channels are responsible for the pacemaking activity of the heart, can HCN4 channel expression lead to repetitive firing of mutant neurons resulting in seizures? (2) HCN4 is the most cAMP-sensitive of the four HCN isoforms. Do coincident increases in cAMP (e.g., ?-adrenergic receptors) and hyperpolarization or depolarizations drive HCN4 channel opening and neuronal firing? (3) HCN4 channel mRNA is expressed in cortical neurons. Is the abnormal HCN4 expression in mutant neurons due to increased translation via mTOR? (4) Seizures can start at any age in patients that have been seizure-free for decades, but we do not know why. Can this be explained by worsening of mTOR hyperactivity with age leading to a progressive increase in HCN4 expression until there is enough HCN4 channels to depolarize cells and reach firing threshold upon activation? (5) There is no selective blocker of the HCN4 channel and blocking other HCN channels would have serious central and peripheral side-effects. Identifying the mechanisms responsible for functional HCN4 expression may therefore provide alternative therapeutic targets. Do binding partners and/or post-translational modifications contribute to HCN4 abnormal expression in mutant neurons? We will address these questions in three aims testing our central hypothesis that abnormal mTOR- and translation- dependent expression of HCN4 channels leads to repetitive neuronal firing and seizures in TSC and FCDII. Aim 1: Test the hypothesis that abnormal HCN4 channel expression in murine TSC/FCDII mutant neurons contribute to neuron excitability and seizure activity. Aim2: Test the hypothesis that abnormal HCN4 expression is mTOR- and translation-dependent and increases with age and seizures. Aim 3: Test the hypothesis that HCN4 binding partners and posttranslational modifications are necessary for its functional expression and function. The proposed studies will be performed through a collaborative effort between the Bordey and Calderwood labs that together combine unique and extensive expertise in in vivo neurobiology, and biochemical and protein science.

Abstract Focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC) are two rare neurodevelopmental disorders each occurring in 1/6000 individuals. In both disorders, mutations affect genes involved in the AKT/mTOR signaling pathway and individuals display focal cortical malformations (FCM) that lead to epilepsy. In TSC, approximately 85% of the patients will have seizures, and nearly two-thirds of these patients will be refractory to treatment and will experience seizures for their life, leading to a spectrum of neurocognitive and psychological disabilities. It is anticipated that similar statistics are represented in FCDII patients. Two treatment options are available for TSC and FCDII patients. One is surgical resection for eligible patients. But only about 50% of the patients will effectively manage their seizures. The second one is treatment with the mTOR blocker, everolimus (Novartis), which (at the highest dose tested) reduced seizures by 40% in 40% of the patients and has grade >3-4 adverse events in 38% of the patients. There is thus a serious need to improve epilepsy treatment in TSC and FCDII patients. Recent work from our lab identified increased activity of the MAPK pathway in TSC and FCDII conditions leading to increased filamin A (FLNA) levels. Preliminary proof-of-concept data suggest that treatment with a small molecule modulator of FLNA function, PTI-125, significantly decreased seizure frequency in our mouse model of TSC and FCDII. PTI-125 binds to FLNA with high-affinity, rapidly enters the CNS of adult mice, and is approximately 90% orally bioavailable . We thus propose studies to optimize PTI-125 dosage and treatment paradigms for in vivo efficacy studies on epilepsy in TSC and FCDII. Our proposed drug therapy is fundamentally different from present treatment in TSC and FCDII considering that FLNA has never been considered as a potential therapeutic target in these disorders and is not downstream of mTOR signaling. In addition, we have developed a murine model of FCM-associated epilepsy that fully recapitulates the human TSC and FCDII disorder. We are thus in a unique position to test the proposed drug. PTI-125 has been obtained through a material transfer agreement with Pain Therapeutics, Inc. PTI-125 is in clinical development for Alzheimer?s disease and has completed the IND-enabling studies. Pain Therapeutics has thus already performed pharmacokinetics studies. The phase 61 proposes studies to inform design and refinement of the in vivo efficacy procedures. The R33 phase proposes in vivo efficacy studies of PTI-125 on seizures. .

The goal of this proposal is to identify how abnormalities of brain development in monogenic disorders involving the PI3K-mTOR pathway (e.g., tuberous sclerosis complex [TSC]) lead to alterations in circuit and in information processing. TSC is a classical disorder of the mTOR signaling pathway due to second-hit somatic mutations in TSC1 or TSC2 leading to mTOR hyperactivity and developmental malformations associated with seizures and neurological deficits. 50-60% of children with TSC have severe cognitive deficits and autism accompanied with abnormal sensory processing. Published work from our lab identified a combination of molecular and cellular alterations in murine neurons with hyperactive mTOR. These alterations include neuron misplacement and dysmorphogenesis that are conserved across cortical regions and between mice and humans. However, the impact of these defects on circuit formation and information processing, and the dependence on mTOR and downstream pathways at the circuit level remain unclear. Here, we propose to use the mouse barrel cortex as a well-established model system to start addressing the link between neuronal abnormalities and alterations in circuitry and sensory processing. Building from our previous findings, we hypothesize that dysmorphogenesis of layer (L) 4 barrel cortex neurons in TSC contributes to mTOR-dependent abnormalities in circuitry, functional connectivity, and sensory responses. We have the following two aims: (1) To test the hypothesis that mTOR hyperactivity in L4 neurons alters barrel circuitry and functional connectivity. (2) To test the hypothesis that reducing mTOR activity during a brief critical window and normalizing two downstream pathways prevent abnormalities in circuit and sensory responses in TSC condition. To address these aims, we will use in utero electroporation to express RhebCA and generate humanized Tsc1 mutations using CRISPR/Cas9 in L4 neurons of the barrel cortex.