The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.
A postsynaptic density (PSD) protein, PSD-95, was tagged with green fluorescent protein (GFP-PSD-95) and expressed in cultured hippocampal neurons using recombinant adenoviruses. GFP-PSD-95 was selectively localized to excitatory postsynaptic sites. Time-lapse fluorescence imaging of hippocampal neurons revealed that >20% of GFP-PSD-95 clusters turned over within 24 hours. The appearance rate of clusters was higher than the disappearance rate, and this difference accounted for the gradual increase of the cluster density observed in culture. Dynamics of PSD-95 clusters were also inhibited by blockers of excitatory synaptic transmission. Continual PSD turnover and its regulation by synaptic activity may be important in activity-dependent remodeling of neuronal connections.
Organization and dynamic remodeling of postsynaptic density (PSD) are thought to be critical in postsynaptic signal transduction, but the underlying molecular mechanisms are not well understood. We show here that four major scaffolding molecules, PSD-95, GKAP, Shank, and PSD-Zip45, show distinct instability in total molecular content per synapse. Fluorescence recovery after photobleaching also confirmed their distinct turnover rates. Among the PSD molecules examined, PSD-95 was most stable, but its elimination did not influence the dynamics of its direct binding partner GKAP. Multiple interactions of scaffolding molecules with the actin cytoskeleton have suggested their importance in both maintenance and remodeling of the PSD. Indeed, acute pharmacological disruption of F-actin rapidly eliminated the dynamic fraction of GKAP, Shank, and PSD-Zip45, without changing synaptic localization of PSD-95. GKAP content in synapses increased after pharmacological enhancement of neuronal activity, whereas Shank and PSD-Zip45 content showed reduction. Inhibition of F-actin dynamics prevented activity-dependent redistribution of all three scaffolds. We also assessed involvement of glutamate receptors in the regulation of PSD dynamics. Genetic manipulations eliminating either NMDA receptors or metabotropic glutamate receptors did not primarily influence mobility of their binding scaffolds. These results collectively indicate a critical role of filamentous actin in determining the extent of dynamic reorganization in PSD molecular composition.
Developmental deficits in neuronal connectivity are considered to be present in patients with autism spectrum disorders (ASDs). Here we examine this possibility by using in vivo spine imaging in the early postnatal cortex of ASD mouse models. Spines are classified by the presence of either the excitatory postsynaptic marker PSD-95 or the inhibitory postsynaptic marker gephyrin. ASD mouse models show consistent upregulation in the dynamics of PSD-95-positive spines, which may subsequently contribute to stable synaptic connectivity. In contrast, spines receiving inputs from the thalamus, detected by the presence of gephyrin clusters, are larger, highly stable and unaffected in ASD mouse models. Importantly, two distinct mouse models, human 15q11-13 duplication and neuroligin-3 R451C point mutation, show highly similar phenotypes in spine dynamics. This selective impairment in dynamics of PSD-95-positive spines receiving intracortical projections may be a core component of early pathological changes and be a potential target of early intervention.
Dendritic morphogenesis and formation of synapses at appropriate dendritic locations are essential for the establishment of proper neuronal connectivity. Recent imaging studies provide evidence for stabilization of dynamic distal branches of dendrites by the addition of new synapses. However, molecules involved in both dendritic growth and suppression of synapse maturation remain to be identified. Here we report two distinct functions of doublecortin-like kinases, chimeric proteins containing both a microtubule-binding domain and a kinase domain in postmitotic neurons. First, doublecortin-like kinases localize to the distal dendrites and promote their growth by enhancing microtubule bundling. Second, doublecortin-like kinases suppress maturation of synapses through multiple pathways, including reduction of PSD-95 by the kinase domain and suppression of spine structural maturation by the microtubule-binding domain. Thus, doublecortin-like kinases are critical regulators of dendritic development by means of their specific targeting to the distal dendrites, and their local control of dendritic growth and synapse maturation.
The NMDA receptor regulates spine morphological plasticity by modulating Rho GTPases. However, the molecular mechanisms for NMDA receptor-mediated regulation of Rho GTPases remain elusive. In this study, we show that p250GAP, an NMDA receptor-associated RhoGAP, regulates spine morphogenesis by modulating RhoA activity. Knock-down of p250GAP increased spine width and elevated the endogenous RhoA activity in primary hippocampal neurons. The increased spine width by p250GAP knock-down was suppressed by the expression of a dominant-negative form of RhoA. Furthermore, p250GAP is involved in NMDA receptor-mediated RhoA activation. In response to NMDA receptor activation, exogenously expressed green fluorescent protein (GFP)-tagged p250GAP was redistributed. Thus, these data suggest that p250GAP plays an important role in NMDA receptor-mediated regulation of RhoA activity leading to spine morphological plasticity.
The cells of Blepharisma which possess red pigment (blepharismin) show step‐up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity). Bleaching of the cells by cold shock raised a threshold light intensity for the response, Oxidation of red pigment that produced blue pigment did not raise the threshold for the response. The action spectrum for the step‐up photophobic response of the cells which possess normal red pigment had peaks at about 580, 540 and 490 nm, a value which coincided with peaks of an absorption spectrum of the red pigment. The absorption spectrum of oxidized pigment (blue pigment) shifted 20 nm toward infrared light. The action spectrum for the response of the cells which possess blue pigment also shifted 20 nm toward infrared light. Results suggest that red pigment might be involved in the step‐up photophobic response. Key words. Blepharismin, ciliary reversal, photoreceptors, photoresponse.
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