Tiffany V. Lin

miR-132 Enhances Dendritic Morphogenesis, Spine Density, Synaptic Integration, and Survival of Newborn Olfactory Bulb Neurons

An array of signals regulating the early stages of postnatal subventricular zone (SVZ) neurogenesis has been identified, but much less is known regarding the molecules controlling late stages. Here, we investigated the function of the activity-dependent and morphogenic microRNA miR-132 on the synaptic integration and survival of olfactory bulb (OB) neurons born in the neonatal SVZ. In situ hybridization revealed that miR-132 expression occurs at the onset of synaptic integration in the OB. Using in vivo electroporation we found that sequestration of miR-132 using a sponge-based strategy led to a reduced dendritic complexity and spine density while overexpression had the opposite effects. These effects were mirrored with respective changes in the frequency of GABAergic and glutamatergic synaptic inputs reflecting altered synaptic integration. In addition, timely directed overexpression of miR-132 at the onset of synaptic integration using an inducible approach led to a significant increase in the survival of newborn neurons. These data suggest that miR-132 forms the basis of a structural plasticity program seen in SVZ-OB postnatal neurogenesis. miR-132 overexpression in transplanted neurons may thus hold promise for enhancing neuronal survival and improving the outcome of transplant therapies.

mTORC1 Targets the Translational Repressor 4E-BP2, but Not S6 Kinase 1/2, to Regulate Neural Stem Cell Self-Renewal In Vivo

The mammalian target of rapamycin complex 1 (mTORC1) integrates signals important for cell growth, and its dysregulation in neural stem cells (NSCs) is implicated in several neurological disorders associated with abnormal neurogenesis and brain size. However, the function of mTORC1 on NSC self-renewal and the downstream regulatory mechanisms are ill defined. Here, we found that genetically decreasing mTORC1 activity in neonatal NSCs prevented their differentiation, resulting in reduced lineage expansion and aborted neuron production. Constitutive activation of the translational repressor 4E-BP1, which blocked cap-dependent translation, had similar effects and prevented hyperactive mTORC1 induction of NSC differentiation and promoted self-renewal. Although 4E-BP2 knockdown promoted NSC differentiation, p70 S6 kinase 1 and 2 (S6K1/S6K2) knockdown did not affect NSC differentiation but reduced NSC soma size and prevented hyperactive mTORC1-induced increase in soma size. These data demonstrate a crucial role of mTORC1 and 4E-BP for switching on and off cap-dependent translation in NSC differentiation.

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.

Detecting floristic structure and pattern across topographic and moisture gradients in a mixed species Central African forest using IKONOS and Landsat-7 ETM+ images

Abstract The strengths and limitations of high spatial resolution broadband IKONOS data and Landsat-7 ETM+ data are compared with respect to, distinguishing floristic structure (basal area, stem density) and pattern (diversity indices, species associations) across a topography that exhibits subtle variations in surface hydrology and elevation. Three site types can be described in relation to the topography and hydrology of the study area: (1) seasonally drought stressed; (2) valley streams; and (3) well-drained bottomlands. Non-metric Multidimensional Scaling (NMS) emphasized the importance of seasonal moisture stress in determining floristic structure and species associations in this forest. Forest structure and species data gathered across the three sites of the topographic/hydrologic gradient are related to spectral values and indices gathered from IKONOS and ETM+. Statistical tests of significance at 95% confidence level or higher showed that the IKONOS wavebands and vegetation indices were most sensitive to changes in floristic structure and species composition for images taken during the dry season (October–March) as compared to those for the wet season (April–September). Within the IKONOS data, the near-infrared (NIR) waveband (band 4) was most sensitive to changes in forest structure and species composition across the three site types (seasonally drought stressed; valley streams; and well-drained bottomlands). However, the IKONOS spectral relationships with biotic variables did not exceed an R2 value of 0.34, with an overwhelming number of best regression models having any two waveband combinations; typically combinations of band 1 and 2, or 1 and 3, or 2 and 3. The best relationships were obtained when ETM+ mid-infrared (MIR) band 5 or 7 were involved with R2 values of 0.52 and 0.54 for basal area and stem density respectively, explaining about 20% greater variability compared to IKONOS data. Thereby, the most interesting aspect of the paper was the degree to which the MIR band of the ETM+ added to the strength of regressions used to explain variation in stem density and basal area of rainforests.

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 (RhebCA) 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 RhebCA-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.

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.

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.

Activating the translational repressor 4E-BP or reducing S6K-GSK3β activity prevents accelerated axon growth induced by hyperactive mTOR in vivo

Abnormal axonal connectivity and hyperactive mTOR complex 1 (mTORC1) are shared features of several neurological disorders. Hyperactive mTORC1 alters axon length and polarity of hippocampal neurons in vitro, but the impact of hyperactive mTORC1 on axon growth in vivo and the mechanisms underlying those effects remain unclear. Using in utero electroporation during corticogenesis, we show that increasing mTORC1 activity accelerates axon growth without multiple axon formation. This was prevented by counteracting mTORC1 signaling through p70S6Ks (S6K1/2) or eukaryotic initiation factor 4E-binding protein (4E-BP1/2), which both regulate translation. In addition to regulating translational targets, S6K1 indirectly signals through GSK3β, a regulator of axogenesis. Although blocking GSK3β activity did not alter axon growth under physiological conditions in vivo, blocking it using a dominant-negative mutant or lithium chloride prevented mTORC1-induced accelerated axon growth. These data reveal the contribution of translational and non-translational downstream effectors such as GSK3β to abnormal axon growth in neurodevelopmental mTORopathies and open new therapeutic options for restoring long-range connectivity.

Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration

Significance Increased mammalian target of rapamycin complex 1 (mTORC1) signaling is associated with many neurological and neurodevelopmental disorders, ranging from epilepsy to autism, that feature developmental defects in the organization of the cortex. Despite the fact that much research has focused on mTORC1-related disorders, the downstream effectors that produce these developmental defects are not well understood. To begin filling that void, this study looked at the role of one of the many cellular processes regulated by mTORC1, cap-dependent translation. Surprisingly, normalizing signaling through only one of mTORC1’s targets [eukaryotic initiation factor 4E-binding proteins (4E-BPs)] was able to block ectopic placement of cortical neurons, and mimicking mTORC1 signaling through 4E-BP alone was sufficient to induce neuron misplacement. Hyperactive mammalian target of rapamycin complex 1 (mTORC1) is a shared molecular hallmark in several neurodevelopmental disorders characterized by abnormal brain cytoarchitecture. The mechanisms downstream of mTORC1 that are responsible for these defects remain unclear. We show that focally increasing mTORC1 activity during late corticogenesis leads to ectopic placement of upper-layer cortical neurons that does not require altered signaling in radial glia and is accompanied by changes in layer-specific molecular identity. Importantly, we found that decreasing cap-dependent translation by expressing a constitutively active mutant of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) prevents neuronal misplacement and soma enlargement, while partially rescuing dendritic hypertrophy induced by hyperactive mTORC1. Furthermore, overactivation of translation alone through knockdown of 4E-BP2 was sufficient to induce neuronal misplacement. These data show that many aspects of abnormal brain cytoarchitecture can be prevented by manipulating a single intracellular process downstream of mTORC1, cap-dependent translation.

In utero electroporation-based translating ribosome affinity purification identifies age-dependent mRNA expression in cortical pyramidal neurons

We combined translating ribosome affinity purification (TRAP) with in utero electroporation (IUE), called iTRAP to identify the molecular profile of specific neuronal populations during neonatal development without the need for viral approaches and FACS sorting. We electroporated a plasmid encoding EGFP-tagged ribosomal protein L10a at embryonic day (E) 14-15 to target layer 2-4 cortical neurons of the somatosensory cortex. At three postnatal (P) ages-P0, P7, and P14-when morphogenesis occurs and synapses are forming, TRAP and molecular profiling was performed from electroporated regions. We found that ribosome bound (Ribo)-mRNAs from ∼7300 genes were significantly altered over time and included classical neuronal genes known to decrease (e.g., Tbr1, Dcx) or increase (e.g., Eno2, Camk2a, Syn1) as neurons mature. This approach led to the identification of specific developmental patterns for Ribo-mRNAs not previously reported to be developmentally regulated in neurons, providing rationale for future examination of their role in selective biological processes. These include upregulation of Lynx1, Nrn1, Cntnap1 over time; downregulation of St8sia2 and Draxin; and bidirectional changes to Fkbp1b. iTRAP is a versatile approach that allows researchers to easily assess the molecular profile of specific neuronal populations in selective brain regions under various conditions, including overexpression and knockdown of target genes, and in disease settings.