Jasmina N. Jovanovic

Synapsins as mediators of BDNF-enhanced neurotransmitter release.

We examined enhancement of synaptic transmission by neurotrophins at the presynaptic level. In a synaptosomal preparation, brain-derived neurotrophic factor (BDNF) increased mitogen-activated protein (MAP) kinase-dependent synapsin I phosphorylation and acutely facilitated evoked glutamate release. PD98059, used to inhibit MAP kinase activity, markedly decreased synapsin I phosphorylation and concomitantly reduced neurotransmitter release. The stimulation of glutamate release by BDNF was strongly attenuated in mice lacking synapsin I and/or synapsin II. These results indicate a causal link of synapsin phosphorylation via BDNF, TrkB receptors and MAP kinase with downstream facilitation of neurotransmitter release.

Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability.

The efficacy of GABAergic synaptic inhibition is a principal factor in controlling neuronal activity. We demonstrate here that brain-derived neurotrophic factor modulates the activity of GABAA receptors, the main sites of fast synaptic inhibition in the brain, within minutes of application. Temporally, this comprised an early enhancement in the miniature IPSC amplitude, followed by a prolonged depression. This modulation was concurrent with enhanced PKC-mediated phosphorylation, followed by protein phosphatase 2A (PP2A)-mediated dephosphorylation of the GABAA receptor. Mechanistically, these events were facilitated by differential recruitment of PKC, receptor for activated C-kinase, and PP2A to GABAA receptors, depending on the phosphorylation state of the receptor β3-subunit. Thus, transient formation of GABAA receptor signaling complexes has the potential to provide a basis for acute changes in receptor function underlying GABAergic synaptic plasticity.

Multiple roles of protein kinases in the modulation of γ-aminobutyric acidA receptor function and cell surface expression

gamma-Aminobutyric acid(A) (GABA(A)) receptors are ligand-gated ion channels that mediate the majority of fast synaptic inhibition in the brain and that are also important drug targets for benzodiazepines, barbiturates, and neurosteriods. These receptors are pentameric hetero-oligomers that can be assembled from 7 subunit classes with multiple members: alpha(1-6), beta(1-3), gamma(1-3), delta, epsilon, theta, and pi. Most receptor subtypes in the brain, however, are believed to be composed of alpha-, beta-, and gamma-subunits. Modifications of GABA(A) receptor function are continually implicated in a range of pathologies, including epilepsy, anxiety, insomnia, and substance abuse. Moreover, changes in the efficacy of synaptic inhibition mediated by GABA(A) receptors are believed to be play central roles in certain forms of synaptic plasticity, including rebound potentiation in the cerebellum, and hippocampal long-term potentiation. Given the critical role that GABA(A) receptors play as mediators of synaptic transmission, it is of fundamental importance to understand the endogenous mechanisms used by neurones to control the function of these receptors. This review will focus on the dynamic regulation of GABA(A) receptor phosphorylation state and channel function as mechanisms involved in determining the efficacy of synaptic inhibition. In addition, the possible role of GABA(A) receptor phosphorylation in controlling receptor internalization and recycling will also be explored.

Generation and Regulation of β‐Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β‐amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the “β‐secretase” pathway, which generates β‐amyloid (Aβ1–40/42; ∼4 kDa), and the “α‐secretase” pathway, which generates a smaller fragment, the “p3” peptide (Aβ17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [35S]methionine pulse‐labeled primary neuronal cultures contained 4‐ and 3‐kDa Aβ‐related species. Radiosequencing analysis revealed that the 4‐kDa band corresponded to conventional Aβ beginning at position Aβ(Asp1), whereas both radio‐sequencing and immunoprecipitation‐mass spectrometry analyses indicated that the 3‐kDa species in these conditioned media began with Aβ(Glu11) at the N terminus, rather than Aβ(Leu17) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPPα release and decreased generation of both the 4‐kDa Aβ and the 3‐kDa N‐truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.

GABAA Receptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

GABAA receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABAA receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAA receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABAA receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

Modulation of GABA A Receptor Phosphorylation and Membrane Trafficking by Phospholipase C-related Inactive Protein/Protein Phosphatase 1 and 2A Signaling Complex Underlying Brain-derived Neurotrophic Factor-dependent Regulation of GABAergic Inhibition *

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABAA receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABAA receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABAA receptor β3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABAA receptors. We demonstrate that PRIP associates with phosphatases as well as with β subunits. PRIP was found to colocalize with GABAA receptor clusters in cultured neurons and with recombinant GABAA receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to β subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABAA receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABAA receptors by mediating the specific association between β subunits of these receptors with protein phosphatases.

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons.

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABAAR)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABAAR activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca2+ influx through L-type voltage-gated Ca2+ channels. We further demonstrate that GABAAR activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABAARs. This BDNF/TrkB-dependent increase in surface levels of GABAARs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABAAR surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABAAR activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

Bidirectional changes in synapsin I phosphorylation at MAP kinase-dependent sites by acute neuronal excitation in vivo.

Synapsin I is a synaptic vesicle‐associated protein which is phosphorylated at multiple sites by various kinases. It has been proposed to play a role in the regulation of neurotransmitter release and the organization of cytoskeletal architecture in the presynaptic terminal. To better understand the physiological regulation of its phosphorylation in vivo, we induced acute, reversible neuronal excitation by electroconvulsive treatment (ECT) in rats, and studied its effects on synapsin I phosphorylation at sites 3, 4/5 and 6 by immunoblot analyses of homogenates from hippocampus and parietal cortex using phospho‐site‐specific antibodies. A decrease in phosphorylation at all sites was observed soon after the electrical stimulation, followed by a large increase in phosphorylation at site 4/5 peaking at 5 min and a moderate increase in phosphorylation at site 6 peaking at 20 min. Systemic injection of SL327, a mitogen‐activated protein kinase (MAPK) kinase inhibitor, prior to ECT, suppressed the increase in phospho‐site 4/5 level, as well as that in MAPK activity, but not that in phospho‐site 6 level. Thus, phosphorylation at site 4/5 of synapsin I has been shown to be regulated by MAPK in vivo.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

γ-Aminobutyric acid type A (GABAA) receptors, a family of Cl--permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for γ-aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated by GABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2+]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2+ influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2+ with Ba2+, or Ca2+/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2+ channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2+/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

Dopamine-Dependent Tuning of Striatal Inhibitory Synaptogenesis

Dopaminergic projections to the striatum, crucial for the correct functioning of this brain region in adulthood, are known to be established early in development, but their role is currently uncharacterized. We demonstrate here that dopamine, by activating D1- and/or D2-dopamine receptors, decreases the number of functional GABAergic synapses formed between the embryonic precursors of the medium spiny neurons, the principal output neurons of the striatum, with associated changes in spontaneous synaptic activity. Activation of these receptors reduces the size of postsynaptic GABAA receptor clusters and their overall cell-surface expression, without affecting the total number of clusters or the size or number of GABAergic nerve terminals. These changes result from an increased internalization of GABAA receptors, and are mediated by distinct signaling pathways converging at the level of GABAA receptors to cause a transient PP2A/PP1-dependent dephosphorylation. Thus, tonic D1- and D2-receptor activity limits the extent of collateral inhibitory synaptogenesis between medium spiny neurons, revealing a novel role of dopamine in controlling the development of intrinsic striatal microcircuits.