SUMMARY The brain produces two brain-derived neurotrophic factor (BDNF) transcripts, with either short or long 3′ untranslated regions (3′UTR). The physiological significance of the two forms of mRNAs encoding the same protein is unknown. Here we show that the short and long 3′UTR BDNF mRNAs are involved in different cellular functions. The short 3′UTR mRNAs are restricted to somata whereas the long 3′UTR mRNAs are also localized in dendrites. In a mouse mutant where the long 3′UTR is truncated, dendritic targeting of BDNF mRNAs is impaired. There is little BDNF in hippocampal dendrites despite normal levels of total BDNF protein. This mutant exhibits deficits in pruning and enlargement of dendritic spines, as well as selective impairment in long-term potentiation in dendrites, but not somata, of hippocampal neurons. These results provide insights into local and dendritic actions of BDNF and reveal a mechanism for differential regulation of subcellular functions of proteins.

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.

Mutations that inactivate negative translation regulators cause autism spectrum disorders (ASD), which predominantly affect males and exhibit social interaction and communication deficits and repetitive behaviors. However, the cells that cause ASD through elevated protein synthesis resulting from these mutations remain unknown. Here we employ conditional overexpression of translation initiation factor eIF4E to increase protein synthesis in specific brain cells. We show that exaggerated translation in microglia, but not neurons or astrocytes, leads to autism-like behaviors in male mice. Although microglial eIF4E overexpression elevates translation in both sexes, it only increases microglial density and size in males, accompanied by microglial shift from homeostatic to a functional state with enhanced phagocytic capacity but reduced motility and synapse engulfment. Consequently, cortical neurons in the mice have higher synapse density, neuroligins, and excitation-to-inhibition ratio compared to control mice. We propose that functional perturbation of male microglia is an important cause for sex-biased ASD.

Modulation of recombinant N-methyl-D-aspar-tate receptor (NMDAR) currents by insulin was studied using the Xenopus oocyte expression system. Insulin (0.8 M, 10 min) regulated NMDAR currents in a subunitspecific manner. Currents from ⑀1/1, ⑀2/1, and ⑀4/1 receptors were variably potentiated, whereas currents from ⑀3/1 receptors were not. Protein tyrosine kinases (PTKs) and protein kinase C were found to be involved in insulin-mediated modulation in an NMDAR subtype-specific way. Pretreatment with a specific PTK inhibitor, lavendustin A, attenuated and blocked the insulin effect on ⑀2/1 and ⑀4/1, respectively. Preincubation with selective protein kinase C inhibitors, staurosporine or calphostin C, depressed the response of ⑀1/1 and ⑀2/1 receptors to insulin. Basal regulation of NMDAR currents by endogenous PTKs and protein tyrosine phosphatases (PTPs) was also investigated. Of the four receptor subtypes, only ⑀1/1 receptor currents were affected by basal PTK inhibition via lavendustin A, whereas PTP inhibition by phenylarsine oxide or orthovanadate enhanced currents from ⑀1/1 and ⑀2/1 receptors. Surprisingly, a stimulatory PTP modulation was observed for ⑀4/1. As NMDAR subunits are differentially expressed in the brain, the observed subtype-specific modulations of NMDAR currents by insulin, PTKs, and PTPs may provide important insights into certain NMDAR-dependent physiological and pathological processes.

Abstract:The NMDA subtype of glutamate receptor is physically associated with the postsynaptic density protein PSD-95 at glutamatergic synapses. The channel activity of NMDA receptors is regulated by different signaling molecules, including protein tyrosine kinases. Because previous results have suggested a role for protein kinase C (PKC) in insulin potentiation of NMDA currents in oocytes, the effects of coexpression of PSD-95 on insulin and PKC potentiation of NMDA currents from these receptors were compared. Another primary objective was to determine if PSD-95 could enable Src to potentiate currents from NR2A/NR1 and NR2B/NR1 receptors expressed in Xenopus oocytes. The results show opposite effects of PSD-95 coexpression on Src and insulin modulation of NR2A/NR1 receptor currents. Src potentiation of mouse NR2A/NR1 currents required PSD-95 coexpression. In contrast, PSD-95 coexpression eliminated insulin-mediated potentiation of NR2A/NR1 receptor currents. PSD-95 coexpression also eliminated PKC potentiation of NR2A/NR1 receptor currents. PSD-95 may therefore play a key role in controlling kinase modulation of NR2A/NR1 receptor currents at glutamatergic synapses. Key Words: Postsynaptic density protein PSD-95-Src-Insulin-Protein kinase C-NMDA receptorXenopus oocyte. J. Neurochem. 75, 282-287 (2000).Glutamate receptors at synapses interact with a raft of proteins involved in signaling. The NMDA subtype interacts with a particular conglomerate of signaling proteins including the postsynaptic density protein PSD-95. PSD-95 is attached to the NR2 subunit C terminus via the last three amino acid residues (S/TXV) but does not interact with NR1 (Kornau et al., 1995;Niethammer et al., 1996;Sheng and Pak, 1999). PSD-95 in turn interacts with other potential signaling molecules such as a neuronal RasGTPase-activating protein and neuronal nitric oxide synthase (Sheng and Pak, 1999). The signal transduction role of PSD-95 was first shown in gene knockout mice that have a lowered threshold for longterm potentiation induction and impaired long-term depression and spatial learning (Migaud et al., 1998). Although Ca 2ϩ entering through the NMDA receptor (NMDAR) and activating either calcium/calmodulin-dependent kinase II or calcineurin is critical for the induction of long-term potentiation/depression, modulation of NMDA currents by other kinases, including Src, may also be required for long-term potentiation (Lu et al., 1998;Huang and Hsu, 1999;Yu and Salter, 1999). Recently, protein kinase C (PKC)-mediated modulation of NMDA currents and Fyn modulation of NMDAR phosphorylation state have been found to be altered by the receptor-associated protein PSD-95. PSD-95 interferes with PKC potentiation of the mouse NR2B/NR1 current in Xenopus oocytes (Yamada et al., 1999). PSD-95 also accentuates NR2A phosphorylation by the Src family protein tyrosine kinase (PTK) Fyn (Tezuka et al., 1999).Therefore, in the present study, we tested the possibility that PSD-95 would have an opposite, facilitatory effect on Src potentiation of NR2A/N...