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 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 did not alter the expression level of tPA in both hippocampi (a) and PFCX (b).

<p>The expression level of tPA in 50 μg post-mitochondrial (P2) fractions prepared from 10 μM NMDA/1 μM glycine or 65 mM K⁺ superfused hippocampal and PFCX slices of P21 prenatal cocaine- and saline-exposed rats were compared by Western blotting. Brain slices (100 μm x 100 μm x 3 mm) were superfused with 0.2 ml/min LMKR for 30 min or with 10 μM NMDA/1 μM glycine in LMKR for 10 min or 65 mM K+-depolarization for 1 min followed by 9 min with LMKR. Slices were collected, homogenized and centrifuged to obtain post-mitochondrial fractions. The blots were stripped and re-probed with anti-β-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in tPA expression level. n = 4 (2 males and 2 females). Data are mean ± s.e.m. of the ratio of tPA to β-actin optical intensities.</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

Prenatal cocaine exposure increased BDNF binding affinity for TrkB.

<p>Prenatal cocaine exposure increased BDNF binding affinity for TrkB by 18- and 15- fold (dotted lines) in biotinylated hippocampal and PFCX synaptic membranes, respectively from P21 prenatal cocaine- and saline-treated rats. In the control membranes, there were two BDNF binding sites, a high-affinity site (KD1) and a low-affinity site (KD2) (solid lines). Prenatal cocaine exposure also increased the low-affinity binding of BDNF by 82- and 12-fold. Nonlinear regression data curve fit was performed using Prism. Data points are the means and vertical bars are the s.e.m. derived from 6 independent rats (3 males and 3 females) in each treatment group.</p

<i>Ex vivo</i> exposure to 50–200 ng/ml BDNF did not alter the expression of full-length (145-KDa) and truncated (95-KDa) TrkB and p75NTR in BDNF-incubated hippocampal and prefrontal cortical slices.

<p>The abundance of 145- and 95-KDa TrkB (a, b) as well as p75NTR (c, d) in 50 μg post-mitochondrial synaptosome-enriched fractions prepared from hippocampal and PFCX slices of P21 prenatal cocaine- and saline-exposed rats following 30-min incubation with 50–200 ng/ml BDNF 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 as well as p75NTR expression levels as a result of incubation with BDNF. n = 4. Data are mean ± s.e.m. of the ratio of 145-, 95-KDa TrkB, p75NTR to β-actin optical intensities.</p

Prenatal cocaine exposure increased 50 ng/ml BDNF-induced TrkB activation in hippocampi and PFCX.

<p>(a) Representative blots showing prenatal cocaine exposure increased 50 ng/ml BDNF-induced TrkB activation in hippocampi and PFCX evidenced by higher activated (tyrosine-phosphorylated [pY]) TrkB. (b) Summary of the densitometric quantification of the pY and total 145- and 95-KDa TrkB. The data are expressed as the ratios of pY- full-length (145-KDa) and truncated TrkB (95-KDa) optical intensity normalized by the optical intensity of total 145- and 95-KDaTrkB, respectively. n = 6 (3 females and 3 males). Data are reported as means ± s.e.m. of the ratio of pY-TrkB to TrkB optical intensities. *p < 0.01, **p < 0.05 compared to respective protein in the saline-treated group.</p