Chay T. Kuo

Identification of E2/E3 Ubiquitinating Enzymes and Caspase Activity Regulating Drosophila Sensory Neuron Dendrite Pruning

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, we conducted an E2/E3 ubiquitinating enzyme mutant screen, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme, resulted in retention of C4da neuron dendrites during metamorphosis. Further, we found that UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling.

Transcription factor LKLF is sufficient to program T cell quiescence via a c-Myc-dependent pathway.

T lymphocytes circulate in a quiescent state until they encounter cognate antigen bound to the surface of an antigen-presenting cell. The molecular pathways that regulate T cell quiescence remain largely unknown. Here we show that forced expression of the lung Krüppel-like transcription factor (LKLF) in Jurkat T cells is sufficient to program a quiescent phenotype characterized by decreased proliferation, reduced cell size and protein synthesis and decreased surface expression of activation markers. Conversely, LKLF-deficient peripheral T cells produced by gene targeting showed increased proliferation, increased cell size and enhanced expression of surface activation markers in vivo. LKLF appeared to function, at least in part, by decreasing expression of the proto-oncogene encoding c-Myc. Forced expression of LKLF was associated with markedly decreased c-Myc expression. In addition, many effects of LKLF expression were mimicked by expression of the dominant-negative MadMyc protein and rescued by overexpression of c-Myc. Thus, LKLF is both necessary and sufficient to program quiescence in T cells and functions, in part, by negatively regulating a c-Myc–dependent pathway.

Postnatal Deletion of Numb/Numblike Reveals Repair and Remodeling Capacity in the Subventricular Neurogenic Niche

Neural stem cells are retained in the postnatal subventricular zone (SVZ), a specialized neurogenic niche with unique cytoarchitecture and cell-cell contacts. Although the SVZ stem cells continuously regenerate, how they and the niche respond to local changes is unclear. Here we generated nestin-creER(tm) transgenic mice with inducible Cre recombinase in the SVZ and removed Numb/Numblike, key regulators of embryonic neurogenesis from postnatal SVZ progenitors and ependymal cells. This resulted in severe damage to brain lateral ventricle integrity and identified roles for Numb/Numblike in regulating ependymal wall integrity and SVZ neuroblast survival. Surprisingly, the ventricular damage was eventually repaired: SVZ reconstitution and ventricular wall remodeling were mediated by progenitors that escaped Numb deletion. Our results show a self-repair mechanism in the mammalian brain and may have implications for both niche plasticity in other areas of stem cell biology and the therapeutic use of neural stem cells in neurodegenerative diseases.

Drosophila neuroblast asymmetric cell division: recent advances and implications for stem cell biology.

Asymmetric cell division is an evolutionarily conserved mechanism widely used to generate cellular diversity during development. Drosophila neuroblasts have been a useful model system for studying the molecular mechanisms of asymmetric cell division. In this minireview, we focus on recent progress in understanding the role of heterotrimeric G proteins and their regulators in asymmetric spindle geometry, as well as the role of an Inscuteable-independent microtubule pathway in asymmetric localization of proteins in neuroblasts. We also discuss issues of progenitor proliferation and differentiation associated with asymmetric cell division and their broader implications for stem cell biology.

Protective astrogenesis from the SVZ niche after injury is controlled by Notch modulator Thbs4

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular/subependymal zone (SVZ/SEZ) generate Doublecortin (DCX)+ neuroblasts that migrate and integrate into olfactory bulb circuitry1,2. Continuous production of neuroblasts is controlled by SVZ microenvironmental niche3,4. It is generally believed that enhancing neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear if there are conditions that favor astrogenesis over neurogenesis in the SVZ niche, and if astrocytes produced there exhibit different properties from others in the brain. We have uncovered that SVZ-generated astrocytes express high levels of Thrombospondin-4 (Thbs4)5,6, a secreted homopentameric glycoprotein, in contrast to cortical astrocytes which are Thbs4low. We found that localized photothrombotic/ischemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-CreERtm4 lineage-tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not DCX+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation, modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production7. Consequently, Thbs4KO/KO animals showed severe defects in cortical injury-induced SVZ astrogenesis, instead producing cells expressing DCX from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular hemorrhage into the brain parenchyma of Thbs4KO/KO animals. Taken together, these findings have significant implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members play important roles8,9.Postnatal/adult neural stem cells (NSCs) within the rodent subventricular zone (SVZ; also called subependymal zone) generate doublecortin (Dcx)+ neuroblasts that migrate and integrate into olfactory bulb circuitry. Continuous production of neuroblasts is controlled by the SVZ microenvironmental niche. It is generally thought that enhancing the neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear whether there are conditions that favour astrogenesis over neurogenesis in the SVZ niche, and whether astrocytes produced there have different properties compared with astrocytes produced elsewhere in the brain. Here we show in mice that SVZ-generated astrocytes express high levels of thrombospondin 4 (Thbs4), a secreted homopentameric glycoprotein, in contrast to cortical astrocytes, which express low levels of Thbs4. We found that localized photothrombotic/ischaemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-creERtm4 lineage tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not Dcx+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production. Consequently, Thbs4 homozygous knockout mice (Thbs4KO/KO) showed severe defects in cortical-injury-induced SVZ astrogenesis, instead producing cells expressing Dcx migrating from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular haemorrhage into the brain parenchyma of Thbs4KO/KO mice. Taken together, these findings have important implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members have important roles.

Inhibition of Adult Neurogenesis by Inducible and Targeted Deletion of ERK5 Mitogen-Activated Protein Kinase Specifically in Adult Neurogenic Regions Impairs Contextual Fear Extinction and Remote Fear Memory

Although there is evidence suggesting that adult neurogenesis may contribute to hippocampus-dependent memory, signaling mechanisms responsible for adult hippocampal neurogenesis are not well characterized. Here we report that ERK5 mitogen-activated protein kinase is specifically expressed in the neurogenic regions of the adult mouse brain. The inducible and conditional knock-out (icKO) of erk5 specifically in neural progenitors of the adult mouse brain attenuated adult hippocampal neurogenesis. It also caused deficits in several forms of hippocampus-dependent memory, including contextual fear conditioning generated by a weak footshock. The ERK5 icKO mice were also deficient in contextual fear extinction and reversal of Morris water maze spatial learning and memory, suggesting that adult neurogenesis plays an important role in hippocampus-dependent learning flexibility. Furthermore, our data suggest a critical role for ERK5-mediated adult neurogenesis in pattern separation, a form of dentate gyrus-dependent spatial learning and memory. Moreover, ERK5 icKO mice have no memory 21 d after training in the passive avoidance test, suggesting a pivotal role for adult hippocampal neurogenesis in the expression of remote memory. Together, our results implicate ERK5 as a novel signaling molecule regulating adult neurogenesis and provide strong evidence that adult neurogenesis is critical for several forms of hippocampus-dependent memory formation, including fear extinction, and for the expression of remote memory.

Identification of distinct ChAT+ neurons and activity-dependent control of postnatal SVZ neurogenesis

Postnatal and adult subventricular zone (SVZ) neurogenesis is believed to be primarily controlled by neural stem cell (NSC)-intrinsic mechanisms, interacting with extracellular and niche-driven cues. Although behavioral experiments and disease states have suggested possibilities for higher level inputs, it is unknown whether neural activity patterns from discrete circuits can directly regulate SVZ neurogenesis. We identified a previously unknown population of choline acetyltransferase (ChAT)+ neurons residing in the rodent SVZ neurogenic niche. These neurons showed morphological and functional differences from neighboring striatal counterparts and released acetylcholine locally in an activity-dependent fashion. Optogenetic inhibition and stimulation of subependymal ChAT+ neurons in vivo indicated that they were necessary and sufficient to control neurogenic proliferation. Furthermore, whole-cell recordings and biochemical experiments revealed direct SVZ NSC responses to local acetylcholine release, synergizing with fibroblast growth factor receptor activation to increase neuroblast production. These results reveal an unknown gateway connecting SVZ neurogenesis to neuronal activity-dependent control and suggest possibilities for modulating neuroregenerative capacities in health and disease.

Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus.

BATF belongs to the AP‐1/ATF superfamily of transcription factors and forms heterodimers with Jun proteins to bind AP‐1 consensus DNA. Unlike Fos/Jun heterodimers which stimulate gene transcription, BATF/Jun heterodimers are transcriptionally inert and inhibit biological processes that are associated with the overstimulation of AP‐1 activity. Here, we describe the murine BATF cDNA and genomic clones and map the BATF locus to chromosome 12 D2‐3. Using in situ hybridization of BATF mRNA, we show that BATF gene expression is highly restricted, with the most prominent signals detected in the thymus. BATF mRNA levels are regulated differentially during discrete stages of T cell development and are up‐regulated following activation of T cells in the periphery. To demonstrate the impact of BATF on AP‐1 activity in vivo, AP‐1 luciferase reporter mice were crossed to transgenic mice overexpressing BATF exclusively inthymic T cells. Results show that elevated levels of BATF protein correlate with reduced transactivation by AP‐1. Since the differential regulation of AP‐1 activity is linked to key transitions in the developing immune system, our observations support a critical role for BATF in determining the overall level of AP‐1 activity, and thus AP‐1 target gene expression, in specific T cell subtypes.

Inducible and conditional deletion of extracellular signal-regulated kinase 5 disrupts adult hippocampal neurogenesis.

Background: Regulatory mechanisms of adult neurogenesis are not clearly defined. Results: Extracellular signal-regulated Kinase 5 is specifically expressed in adult neurogenic regions, and is critical for adult hippocampal neurogenesis. Conclusion: ERK5 signaling regulates adult hippocampal neurogenesis, a process that may be mediated through Neurogenin 2. Significance: Identification of signaling pathways involved in adult neurogenesis contributes toward delineating the molecular mechanisms regulating adult neurogenesis. Recent studies have led to the exciting idea that adult-born neurons in the dentate gyrus of the hippocampus may play a role in hippocampus-dependent memory formation. However, signaling mechanisms that regulate adult hippocampal neurogenesis are not well defined. Here we report that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, is selectively expressed in the neurogenic regions of the adult mouse brain. We present evidence that shRNA suppression of ERK5 in adult hippocampal neural stem/progenitor cells (aNPCs) reduces the number of neurons while increasing the number of cells expressing markers for stem/progenitor cells or proliferation. Furthermore, shERK5 attenuates both transcription and neuronal differentiation mediated by Neurogenin 2, a transcription factor expressed in adult hippocampal neural progenitor cells. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, promotes neurogenesis in cultured aNPCs and in the dentate gyrus of the mouse brain. Moreover, neurotrophins including NT3 activate ERK5 and stimulate neuronal differentiation in aNPCs in an ERK5-dependent manner. Finally, inducible and conditional deletion of ERK5 specifically in the neurogenic regions of the adult mouse brain delays the normal progression of neuronal differentiation and attenuates adult neurogenesis in vivo. These data suggest ERK5 signaling as a critical regulator of adult hippocampal neurogenesis.

Inducible and Targeted Deletion of the ERK5 MAP Kinase in Adult Neurogenic Regions Impairs Adult Neurogenesis in the Olfactory Bulb and Several Forms of Olfactory Behavior

Although adult-born neurons in the subventricular zone (SVZ) and olfactory bulb (OB) have been extensively characterized at the cellular level, their functional impact on olfactory behavior is still highly controversial with many conflicting results reported in the literature. Furthermore, signaling mechanisms regulating adult SVZ/OB neurogenesis are not well defined. Here we report that inducible and targeted deletion of erk5, a MAP kinase selectively expressed in the adult neurogenic regions of the adult brain, impairs adult neurogenesis in the SVZ and OB of transgenic mice. Although erk5 deletion had no effect on olfactory discrimination among discrete odorants in the habituation/dishabituation assay, it reduced short-term olfactory memory as well as detection sensitivity to odorants and pheromones including those evoking aggression and fear. Furthermore, these mice show impaired acquisition of odor-cued associative olfactory learning, a novel phenotype that had not been previously linked to adult neurogenesis. These data suggest that ERK5 MAP kinase is a critical kinase signaling pathway regulating adult neurogenesis in the SVZ/OB, and provide strong evidence supporting a functional role for adult neurogenesis in several distinct forms of olfactory behavior.