Kuo Wu

BDNF acutely increases tyrosine phosphorylation of the NMDA receptor subunit 2B in cortical and hippocampal postsynaptic densities.

While neurotrophins are critical for neuronal survival and differentiation, recent work suggests that they acutely regulate synaptic transmission as well. Brain-derived neurotrophic factor (BDNF) enhances excitatory postsynaptic currents in cultured dissociated hippocampal neurons within 2-3 min through postsynaptic, phosphorylation-dependent mechanisms. Moreover, BDNF modulates hippocampal long-term potentiation, in which postsynaptic NMDA (N-methyl-D-aspartate) receptors (NRs) play a key role. We now report that BDNF acutely increases tyrosine phosphorylation of the specific NMDA receptor subunit NR2B, which has recently been shown to play a role in long-term potentiation. Incubation of BDNF with cortical or hippocampal postsynaptic densities for 5 min increased tyrosine phosphorylation of the NR2B subunits in a dose-dependent manner. A maximal increase to 165% of control phosphorylation occurred at a BDNF concentration of 2 ng/ml. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, had no effect on NR2B phosphorylation. Further, BDNF action was selective, since it did not alter tyrosine phosphorylation of NR2A subunits. Our results suggest that tyrosine phosphorylation of NR2B subunits of the NMDA receptor may contribute to neurotrophin modulation of postsynaptic responsiveness and long-term potentiation.

Functional trkB neurotrophin receptors are intrinsic components of the adult brain postsynaptic density.

Neurotrophins have long been thought to act as target-derived factors that regulate the survival and differentiation of afferent neurons. Recently, brain-derived neurotrophic factor (BDNF) was shown to elicit rapid increases in synaptic activity of cultured hippocampal neurons by enhancing responsiveness to excitatory input. These findings suggest a postsynaptic localization of neurotrophin receptors. In this study, we examined the expression of trkB, a high-affinity receptor for BDNF, in the postsynaptic density (PSD), a proteinaceous specialization of the postsynaptic membrane. Western blot analyses with antibodies to trkB revealed localization to the PSD in adult rat cerebral cortex and hippocampus. Only the full-length, active form of trkB was detected in PSD samples. BDNF treatment of the adult cortical PSD resulted in a 5-fold increase in trkB autophosphorylation, supporting the contention that the PSD contains functional trkB. Truncated trkB, which does not contain the tyrosine kinase signaling domain, though present in membrane fractions, was undetectable in the PSD. The presence of trkB in the PSD is consistent with a role for neurotrophins in the regulation of synaptic activity via direct postsynaptic mechanisms.

NMDA receptor subunits in the postsynaptic density of rat brain : expression and phosphorylation by endogenous protein kinases

N-methyl-D-aspartate (NMDA) receptors (NRs) play critical roles in diverse synaptic processes in the brain. However, subcellular distribution, spatiotemporal expression and regulation of NR subunits in brain synapses are unknown. We report that NR1 and NR2A-2C subunits are all enriched in the postsynaptic density (PSD), which plays critical roles in trophin-mediated synaptic plasticity. Significant expression of NRs was observed the first two weeks after birth, during synaptogenesis, and in adulthood. Functional diversity of NRs, resulting from heterogeneous composition, was supported by the finding that different NR2 subunits were associated in a region-specific manner with NR1. Phosphorylation of NR1, a key subunit of the NMDA receptor-channel complex, was significantly enhanced by activators of calmodulin (CaM) kinases (CKs) or protein kinase C (PKC), but not by those of PKA. Co-immunoprecipitation studies revealed that NR1 was physically associated with functionally active PKCgamma and the major PSD protein (mPSDp) through noncovalent interactions. Our results suggest that NMDA receptors play roles in postsynaptic mechanisms in a subunit-, composition-, brain region- and developmental-specific manner. Our findings also indicate that the PSD is a coherent functional unit containing protein kinases that potentially regulate NMDA receptor function via phosphorylation.

Selective localization of amyloid precursor-like protein 1 in the cerebral cortex postsynaptic density

Senile plaques, a hallmark of Alzheimers disease (AD), contain amyloid beta-peptide (A beta), which is generated from the larger amyloid beta protein precursor (APP). In addition to APP, several APP-related proteins have been recently identified in different organisms, including Drosophila amyloid precursor protein-like protein (APPL). Deficiency of APPL causes behavioral deficits in Drosophila, implicating a role in brain function. Moreover, mouse and human cDNA clones encoding amyloid precursor-like proteins (APLP1 and APLP2) have been identified and exhibit extensive sequence similarity to the APPL and APP genes. To define the potential role of APLP in the mammalian brain, we sought to directly localize APLP1 within the complex cortical synaptic structure. We focused on the postsynaptic density (PSD), which appears to be central to synaptic function. We now report that the 90 kDa APLP1, the first known APLP, is localized to the PSD from rat and human cerebral cortex. APLP1 increased during cortical synaptic development, suggesting a role in synaptogenesis or synaptic maturation. In contrast, APP was predominantly expressed in the synaptic membrane fraction, but was barely detectable in the PSD, including different subcellular distributions of APP and APLP1. Our observations raise the possibility that APLP1, a homologue of APPL, which appears to be necessary for normal behavior in Drosophila, participates in brain synaptic function in mammals.

Brain-derived neurotrophic factor enhances association of protein tyrosine phosphatase PTP1D with the NMDA receptor subunit NR2B in the cortical postsynaptic density

Our recent studies revealed that brain-derived neurotrophic factor (BDNF) rapidly enhances tyrosine phosphorylation and dephosphorylation of the NMDA receptor subunit, NR2B, in the postsynaptic density (PSD), potentially regulating synaptic plasticity. To explore the molecular mechanisms underlying synaptic NR2B signaling, we examined the protein tyrosine phosphatase, PTP1D; BDNF reportedly increases association of PTP1D with tyrosine phosphorylated proteins in cortical neurons and PC 12 cells. We now report that PTP1D is an intrinsic component of the rat cerebrocortical PSD, based on Western blot analysis using specific anti-PTP1D antibodies. In addition, NR2B was co-immunoprecipitated with PTP1D using anti-NR2B antibodies or anti-PTP1D antibodies, indicating physical association of the subunit with PTP1D. Moreover, treatment of the purified PSD with BDNF for 5 min elicited a two-fold increase in the association of NR2B with PTP1D. The BDNF action appeared to be specific, since nerve growth factor, another member of the neurotrophin gene family, did not alter the association. Finally, an overlay assay revealed that BDNF caused a two-fold increase in binding of blotted PSD NR2B proteins to PTP1D-SH2 domains, revealing molecular mechanisms mediating the PTP1D-NR2B binding. Taken together, our results raise the possibility that PTP1D participates in BDNF-mediated NR2B signaling cascades at the postsynaptic site, thereby regulating synaptic plasticity.

Efficient method for generating nuclear fractions from marrow stromal cells.

Stem cells have received significant attention for their envisioned potential to treat currently unapproachable diseases. No less important is the utility of stem cells to serve as model systems of differentiation. Analyses at the transcriptome, miRNA and proteome levels have yielded valuable insights into events underlying stem cell differentiation. Proteomic analysis is often cumbersome, detecting changes in hundreds of proteins that require subsequent identification and validation. Targeted analysis of nuclear constituents would simplify proteomic studies, focusing efforts on transcription factor abundance and modification. To facilitate such studies, a simple and efficient methodology to isolate pure nuclear fractions from Marrow Stromal Cells (MSCs), a clinically relevant stem cell population, has been developed. The modified protocol greatly enhances cell disruption, yielding free nuclei without attached cell body remnants. Light and electron microscopic analysis of purified nuclei demonstrated that preparations contained predominantly intact nuclei with minimal cytoplasmic contamination. Western analysis revealed an approximately eightfold enrichment of the transcription factor CREB in the isolated nuclei over that in the starting homogenates. This simple method for isolation of highly purified nuclear fractions from stem cell populations will allow rigorous examination of nuclear proteins critical for differentiation.