Dopamine D3 receptor deletion increases tissue plasminogen activator (tPA) activity in prefrontal cortex and hippocampus

Abstract

Considerable evidence indicates that dopamine (DA) influences tissue plasminogen activator (tPA)-mediated proteolytic processing of the precursor of brain-derived neurotrophic factor (proBDNF) into mature BDNF (mBDNF). However, specific roles in this process for the dopamine D3 receptor (D3R) and the underlying molecular mechanisms are yet to be fully characterized. In the present study, we hypothesized that D3R deletion could influence tPA activity in the prefrontal cortex and hippocampus. Using D3R knockout (D3(-/-)) mice, we show that receptor inactivation is associated with increased tPA expression/activity both in the prefrontal cortex and, to a greater extent, in the hippocampus. Augmented tPA expression in D3(-/-) mice correlated with increased BDNF mRNA levels, plasmin/plasminogen protein ratio and the conversion of proBDNF into mBDNF, as well as enhanced tPA and mBDNF immunoreactivity, as determined by quantitative real time polymerase chain reaction (qRT-PCR), immunoblot and immunohistochemistry. In addition, when compared to wild-type controls, D3(-/-) mice exhibited increased basal activation of the canonical cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-driven Akt/cAMP-response element-binding protein (CREB) signaling cascade, as determined by the increased Akt phosphorylation both at Thr304 and Ser473 residues, of DA and cAMP-regulated protein of 32kDa (DARPP-32) at Thr34 and a phosphorylation state-dependent inhibition of glycogen synthetase kinase-3β (GSK-3β) at Ser9, a substrate of Akt whose constitutive function impairs normal CREB transcriptional activity through phosphorylation at its Ser129 residue. Accordingly, CREB phosphorylation at Ser133 was significantly increased in D3(-/-) mice, whereas the GSK-3β-dependent phosphorylation at Ser129 was diminished. Altogether, our finding reveals that mice lacking D3Rs show enhanced tPA proteolytic activity on BDNF which may involve, at least in part, a potentiated Akt/CREB signaling, possibly due to hindered GSK-3β activity.

Keywords: BDNF; BSA; CREB; D(3)(−/−); D(3)R, dopamine D(3) receptor; DA; DA and cAMP-regulated protein of 32 kDa; DARPP; EDTA; EGTA; GSK-3β; IR; PKA; PP-1; TBS; TH; WT; bovine serum albumin; cAMP; cAMP-response element-binding protein; cyclic adenosine monophosphate; dopamine; dopamine D(3) receptor; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; glycogen synthase kinase-3β; hippocampus; immunoreactivity; mBDNF; mature brain-derived neurotrophic factor; precursor of BDNF; prefrontal cortex; proBDNF; protein kinase A; protein phosphatase 1; qRT-PCR; quantitative real time polymerase chain reaction; tPA; tissue plasminogen activator; tris–HCl-buffered saline; tyrosine hydroxylase; wild-type.