Prenatal Cocaine Exposure Upregulates BDNF-TrkB Signaling

Prenatal cocaine exposure causes profound changes in neurobehavior as well as synaptic function and structure with compromised glutamatergic transmission. Since synaptic health and glutamatergic activity are tightly regulated by brain-derived neurotrophic factor (BDNF) signaling through its cognate tyrosine receptor kinase B (TrkB), we hypothesized that prenatal cocaine exposure alters BDNF-TrkB signaling during brain development. Here we show prenatal cocaine exposure enhances BDNF-TrkB signaling in hippocampus and prefrontal cortex (PFCX) of 21-day-old rats without affecting the expression levels of TrkB, P75NTR, signaling molecules, NMDA receptor—NR1 subunit as well as proBDNF and BDNF. Prenatal cocaine exposure reduces activity-dependent proBDNF and BDNF release and elevates BDNF affinity for TrkB leading to increased tyrosine-phosphorylated TrkB, heightened Phospholipase C-γ1 and N-Shc/Shc recruitment and higher downstream PI3K and ERK activation in response to ex vivo BDNF. The augmented BDNF-TrkB signaling is accompanied by increases in association between activated TrkB and NMDARs. These data suggest that cocaine exposure during gestation upregulates BDNF-TrkB signaling and its interaction with NMDARs by increasing BDNF affinity, perhaps in an attempt to restore the diminished excitatory neurotransmission

Prenatal Cocaine Exposure Uncouples mGluR1 from Homer1 and Gq Proteins

Cocaine exposure during gestation causes protracted neurobehavioral changes consistent with a compromised glutamatergic system. Although cocaine profoundly disrupts glutamatergic neurotransmission and in utero cocaine exposure negatively affects metabotropic glutamate receptor-type 1 (mGluR1) activity, the effect of prenatal cocaine exposure on mGluR1 signaling and the underlying mechanism responsible for the prenatal cocaine effect remain elusive. Using brains of the 21-day-old (P21) prenatal cocaine-exposed rats, we show that prenatal cocaine exposure uncouples mGluR1s from their associated synaptic anchoring protein, Homer1 and signal transducer, Gq/11 proteins leading to markedly reduced mGluR1-mediated phosphoinositide hydrolysis in frontal cortex (FCX) and hippocampus. This prenatal cocaine-induced effect is the result of a sustained protein kinase C (PKC)-mediated phosphorylation of mGluR1 on the serine residues. In support, phosphatase treatment of prenatal cocaine-exposed tissues restores whereas PKC-mediated phosphorylation of saline-treated synaptic membrane attenuates mGluR1 coupling to both Gq/11 and Homer1. Expression of mGluR1, Homer1 or Gα proteins was not altered by prenatal cocaine exposure. Collectively, these data indicate that prenatal cocaine exposure triggers PKC-mediated hyper-phosphorylation of the mGluR1 leading to uncoupling of mGluR1 from its signaling components. Hence, blockade of excessive PKC activation may alleviate abnormalities in mGluR1 signaling and restores mGluR1-regulated brain functions in prenatal cocaine-exposed brains

Prenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP

Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308–6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects

Two Thalamic Regions Screened Using Laser Capture Microdissection with Whole Human Genome Microarray in Schizophrenia Postmortem Samples

We used whole human genome microarray screening of highly enriched neuronal populations from two thalamic regions in postmortem samples from subjects with schizophrenia and controls to identify brain region-specific gene expression changes and possible transcriptional targets. The thalamic anterior nucleus is reciprocally connected to anterior cingulate, a schizophrenia-affected cortical region, and is also thought to be schizophrenia affected; the other thalamic region is not. Using two regions in the same subject to identify disease-relevant gene expression differences was novel and reduced intersubject heterogeneity of findings. We found gene expression differences related to miRNA-137 and other SZ-associated microRNAs, ELAVL1, BDNF, DISC-1, MECP2 and YWHAG associated findings, synapses, and receptors. Manual curation of our data may support transcription repression

Prenatal cocaine exposure did not alter the expression levels of full-length (145-KDa) and truncated (95-KDa) TrkB, p75^NTR, pro-BDNF and BDNF, Akt1 and ERK2, N-Shc and Shc, as well as NR1 and PLC-γ1 in both hippocampus and prefrontal cortex.

<p>The expression levels of 145- and 95-KDa TrkB (a). P75NTR (b), pro-BDNF and BDNF (c) as well as Akt1 and ERK2(d), Shc and N-Shc (e), NR1 and PLC-γ1(f), in 50 μg post-mitochondrial synaptosome-enriched fractions prepared from hippocampi and PFCX of P21 rats exposed to saline or cocaine <i>in utero</i> were compared by Western blotting. The blots were stripped and re-probed with anti-β-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in 145- and 95-KDa TrkB, P75NTR, proBDNF and BDNF, Akt1, ERK2, Shc and N-Shc, NR1 and PLC-γ1 expression levels. n = 4. Data are mean ± s.e.m. of the ratio of 145-, 95-KDa TrkB, p75^NTR, proBDNF, BDNF, Akt1, ERK2, N-Shc, Shc, NR1 or PLC-γ1to β-actins optical intensities.</p

Prenatal cocaine exposure reduced NMDA/Glycine and K⁺-depolarization induced BDNF and proBDNF release in hippocampi (a) and PFCX (b).

<p>Hippocampal and PFCX slices prepared from P21 prenatal cocaine- and saline-treated rats were used to determine spontaneous BDNF/proBDNF efflux as well as BDNF and proBDNF release induced by 10-min 10 μM NMDA/1 μM glycine in LMKR or 1-min 65 mM K⁺-depolarization in a superfusion system. BDNF and proBDNF in the perfusate were then immunoprecipitated with immobilized anti- BDNF and determined by Western blotting with anti-BDNF. The brain slices were collected, homogenized and solubilized and the level of β-actin in the brain slices was determined by Western blotting to illustrate equal quantities of tissues. The blots were quantified by densitometric scanning. Data are expressed as means ± s.e.m. of the ratios of BDNF or proBDNF optical intensity to the optical intensity of β-actin. n = 6. **p < 0.05, *p < 0.01 compared to LMKR-treated in the same group. ##p < 0.05, #p < 0.01 compared to respective protein in the saline-treated group.</p

Prenatal cocaine exposure reduced proBDNF release in hippocampi (top) and PFCX (bottom).

<p>Brain slices treated with 10 μM of MMP-9 inhibitor I and tPA inhibitor were used to assess proBDNF released spontaneously and induced by 10-min 10 μM NMDA/1 μM glycine or 1-min 65 mM K⁺-depolarization. ProBDNF in the perfusate were then immunoprecipitated with immobilized anti-BDNF and the level of proBDNF was determined by Western blotting with specific anti-proBDNF. The brain slices were collected, homogenized, and solubilized and the level of β-actin in the brain slices was determined by Western blotting. The blots were quantified by densitometric scanning. Data are means ± s.e.m. of the ratios of proBDNF optical intensity to the optical intensity of β-actin that serves to verify equal amounts of tissues. n = 5 (3 males and 2 females). **p < 0.05, *p < 0.01 compared to LMKR-treated in the same group. ##p < 0.05, #p < 0.01 compared to respective protein in the saline-treated group.</p

Prenatal cocaine exposure altered 145-KDa TrkB conformation in hippocampi (top) and PFCX (bottom).

<p>The conformational states of the immunopurified 145-KDa TrkB was analyzed by separating on pH3-10 isoelectric focusing gels and then Western blotted with anti-TrkB. Blots were quantified by densitometric scanning. Data are means ± s.e.m. of the pI 6.1 and pI 6.9. n = 5 (3 males and 2 females). p < 0.01 compared to respective protein in the saline-treated group.</p

The concentration-response relationships of BDNF-induced TrkB and p75^NTR activation.

<p>Concentration-response relationships of BDNF-induced TrkB and p75^NTR activation indicate that p75^NTR is far less sensitive to BDNF than TrkB. (a) The magnitudes of TrkB and p75NTR activation induced by a 30-min incubation with 50–200 ng/ml BDNF were assessed in hippocampal and PFCX slices prepared from naïve P21 rats using pY TrkB and TRAF2/6 recruitment to p75^NTR, respectively, as the guides. (b) Densitometric quantification of pY 145- and 95-KDa TrkB and TRAF2/6 blots revealed that while TrkB was activated by 50–200 ng/ml of BDNF in a dose-dependent manner, p75NTR is activated only by 200 ng/ml of BDNF in both hippocampus and PFCX. n = 4. Data are mean ± s.e.m. of the ratios of pY to total 145-, 95-KDa TrkB or TRAF2 and TRAF6 to p75^NTR optical intensities. *p < 0.01 compared to the basal level of respective protein.</p