Giselle Cheung

Enriched Monolayer Precursor Cell Cultures from Micro-Dissected Adult Mouse Dentate Gyrus Yield Functional Granule Cell-Like Neurons

Background Stem cell cultures are key tools of basic and applied research in Regenerative Medicine. In the adult mammalian brain, lifelong neurogenesis originating from local precursor cells occurs in the neurogenic regions of the hippocampal dentate gyrus. Despite widespread interest in adult hippocampal neurogenesis and the use of mouse models to study it, no protocol existed for adult murine long-term precursor cell cultures with hippocampus-specific differentiation potential. Methodology/Principal Findings We describe a new strategy to obtain serum-free monolayer cultures of neural precursor cells from microdissected dentate gyrus of adult mice. Neurons generated from these adherent hippocampal precursor cell cultures expressed the characteristic markers like transcription factor Prox1 and showed the TTX-sensitive sodium currents of mature granule cells in vivo. Similar to granule cells in vivo, treatment with kainic acid or brain derived neurotrophic factor (BDNF) elicited the expression of GABAergic markers, further supporting the correspondence between the in vitro and in vivo phenotype. When plated as single cells (in individual wells) or at lowest density for two to three consecutive generations, a subset of the cells showed self-renewal and gave rise to cells with properties of neurons, astrocytes and oligodendrocytes. The precursor cell fate was sensitive to culture conditions with their phenotype highly influenced by factors within the media (sonic hedgehog, BMP, LIF) and externally applied growth factors (EGF, FGF2, BDNF, and NT3). Conclusions/Significance We report the conditions required to generate adult murine dentate gyrus precursor cell cultures and to analyze functional properties of precursor cells and their differentiated granule cell-like progeny in vitro.

Bradykinin-Induced Microglial Migration Mediated by B1-Bradykinin Receptors Depends on Ca2+ Influx via Reverse-Mode Activity of the Na+/Ca2+ Exchanger

Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B1 receptor agonists and markedly inhibited by a B1 antagonist, but not by a B2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B2-knock-out mice but not from B1-knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca2+ or by low extracellular Ca2+, implying that Ca2+ influx is prerequisite. Blocking the reverse mode of Na+/Ca2+ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX+/− mice; B1-agonist-induced motility and chemotaxis was decreased compared with that in NCX+/+ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca2+-dependent K+ currents (IK(Ca)) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B1 receptors and IK(Ca). These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo.

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.

Properties of doublecortin-(DCX)-expressing cells in the piriform cortex compared to the neurogenic dentate gyrus of adult mice

The piriform cortex receives input from the olfactory bulb and (via the entorhinal cortex) sends efferents to the hippocampus, thereby connecting the two canonical neurogenic regions of the adult rodent brain. Doublecortin (DCX) is a cytoskeleton-associated protein that is expressed transiently in the course of adult neurogenesis. Interestingly, the adult piriform cortex, which is usually considered non-neurogenic (even though some reports exist that state otherwise), also contains an abundant population of DCX-positive cells. We asked how similar these cells would be to DCX-positive cells in the course of adult hippocampal neurogenesis. Using BAC-generated transgenic mice that express GFP under the DCX promoter, we studied DCX-expression and electrophysiological properties of DCX-positive cells in the mouse piriform cortex in comparison with the dentate gyrus. While one class of cells in the piriform cortex indeed showed features similar to newly generated immature granule neurons, the majority of DCX cells in the piriform cortex was mature and revealed large Na+ currents and multiple action potentials. Furthermore, when proliferative activity was assessed, we found that all DCX-expressing cells in the piriform cortex were strictly postmitotic, suggesting that no DCX-positive “neuroblasts” exist here as they do in the dentate gyrus. We conclude that DCX in the piriform cortex marks a unique population of postmitotic neurons with a subpopulation that retains immature characteristics associated with synaptic plasticity. DCX is thus, per se, no marker of neurogenesis but might be associated more broadly with plasticity.

C1q, the recognition subcomponent of the classical pathway of complement, drives microglial activation.

Microglia, central nervous system (CNS) resident phagocytic cells, persistently police the integrity of CNS tissue and respond to any kind of damage or pathophysiological changes. These cells sense and rapidly respond to danger and inflammatory signals by changing their cell morphology; by release of cytokines, chemokines, or nitric oxide; and by changing their MHC expression profile. We have shown previously that microglial biosynthesis of the complement subcomponent C1q may serve as a reliable marker of microglial activation ranging from undetectable levels of C1q biosynthesis in resting microglia to abundant C1q expression in activated, nonramified microglia. In this study, we demonstrate that cultured microglial cells respond to extrinsic C1q with a marked intracellular Ca2+ increase. A shift toward proinflammatory microglial activation is indicated by the release of interleukin‐6, tumor necrosis factor‐α, and nitric oxide and the oxidative burst in rat primary microglial cells, an activation and differentiation process similar to the proinflammatory response of microglia to exposure to lipopolysaccharide. Our findings indicate 1) that extrinsic plasma C1q is involved in the initiation of microglial activation in the course of CNS diseases with blood–brain barrier impairment and 2) that C1q synthesized and released by activated microglia is likely to contribute in an autocrine/paracrine way to maintain and balance microglial activation in the diseased CNS tissue.

Activity-Dependent Bulk Endocytosis and Clathrin-Dependent Endocytosis Replenish Specific Synaptic Vesicle Pools in Central Nerve Terminals

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

Adaptor Protein Complexes 1 and 3 Are Essential for Generation of Synaptic Vesicles from Activity-Dependent Bulk Endosomes

Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

Modulation of Fate Determinants Olig2 and Pax6 in Resident Glia Evokes Spiking Neuroblasts in a Model of Mild Brain Ischemia

Background and Purpose— Although in vitro studies suggest that non-neurogenic regions of the adult central nervous system potentially contain multipotent parenchymal progenitors, neurons are clearly not replaced in most brain regions after injury. Here, in a well-established model of mild transient brain ischemia, we explored Olig2 antagonism and Pax6 overexpression as potential avenues to redirect endogenous progenitors proliferating in situ toward a neuronal fate. Methods— Retroviral vectors containing either Pax6 or a strong activator form of the repressor Olig2 (Olig2VP16), ie, a functionally dominant negative form of Olig2, were stereotaxically injected into the lateral striatum at 48 hours after 30 minutes middle cerebral artery occlusion (MCAo)/reperfusion. Results— Retroviral modulation of fate determinants resulted in a significant number of infected cells differentiating into Doublecortin (DCX)-expressing immature neurons that were not observed after injection of a control virus. Whole-cell patch-clamp recordings in acute brain slices showed that the percentage of virus-infected cells with Na+ currents was increased by inhibition of the repressor function of Olig2 and by overexpression of Pax6. Furthermore, on retroviral transduction of fate determinants, we detected newly generated cells within the ischemic lesion that were capable of generating single action potentials and that received synaptic input. Conclusions— Taken together, these data show that resident glia in the striatum can be reprogrammed toward functional neuronal differentiation following brain injury.

Synaptic vesicle generation from activity-dependent bulk endosomes requires calcium and calcineurin

Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high-frequency stimulation in central nerve terminals. ADBE generates endosomes direct from the plasma membrane, meaning that high concentrations of calcium will be present in their interior due to fluid phase uptake from the extracellular space. Morphological and fluorescent assays were used to track the generation of SVs from bulk endosomes in primary neuronal culture. This process was functionally uncoupled from both SV exocytosis and plasma membrane retrieval events by intervening only after SV fusion and endocytosis were completed. Either intracellular (BAPTA-AM) or intra-endosomal (Rhod-dextran) calcium chelation inhibited SV generation from bulk endosomes, indicating that calcium efflux from this compartment is critical for this process. The V-type ATPase antagonist bafilomycin A1 also arrested SV generation from bulk endosomes, indicating endosomal acidification may be required for calcium efflux. Finally, pharmacological inhibition of the calcium-dependent protein phosphatase calcineurin blocked endosomal SV generation, identifying it as a key downstream effector in this process. These results reveal a novel and key role for the fluid phase uptake of extracellular calcium and its subsequent efflux in the SV lifecycle.

GABAergic activities enhance macrophage inflammatory protein‐1α release from microglia (brain macrophages) in postnatal mouse brain

Microglial cells (brain macrophages) invade the brain during embryonic and early postnatal development, migrate preferentially along fibre tracts to their final position and transform from an amoeboid to a ramified morphology. Signals by which the invading microglia communicate with other brain cells are largely unknown. Here, we studied amoeboid microglia in postnatal corpus callosum obtained from 6‐ to 8‐day‐old mice. These cells accumulated on the surface of acute brain slices. Whole‐cell patch‐clamp recordings revealed that the specific GABAA receptor agonist muscimol triggered a transient increase in conductance typical for inward rectifying potassium channels in microglia. This current increase was not mediated by microglial GABAA receptors since microglial cells removed from the slice surface no longer reacted and cultured microglia only responded when a brain slice was placed in their close vicinity. Muscimol triggered a transient increase in extracellular potassium concentration ([K+]o) in brain slices and an experimental elevation of [K+]o mimicked the muscimol response in microglial cells. Moreover, in adult brain slices, muscimol led only to a minute increase in [K+]o and microglial cells failed to respond to muscimol. In turn, an increase in [K+]o stimulated the release of chemokine macrophage inflammatory protein‐1α (MIP1‐α) from brain slices and from cultures of microglia but not astrocytes. Our observations indicate that invading microglia in early postnatal development sense GABAergic activities indirectly via sensing changes in [K+]o which results in an increase in MIP1‐α release.