Climbing fiber activity reduces 14-3-3-θ regulated GABAA receptor phosphorylation in cerebellar Purkinje cells

Abstract

Cerebellar adaptive plasticity regulates posture and movement in response to changing conditions of sensory stimulation. Study of adaptive plasticity of cerebellar circuitry in vitro confines experimental interest to mechanisms with a time scale of minutes. However, cerebellar plasticity, measured behaviorally or electrophysiologically in vivo, occurs over a time scale of tens of minutes and hours. Here we investigate how optokinetically-evoked increases in climbing fiber activity influence expression of key subcellular signaling proteins that regulate the accumulation of GABAA receptors (GABAARs) in the cytoplasm of Purkinje cells and their insertion into the plasma membrane. We used long-term horizontal optokinetic stimulation (HOKS) to activate climbing fibers that project to the flocculus of mice. While long-term increases in climbing fiber activity in vivo do not alter the expression of any of the subunits of GABAARs expressed by Purkinje cells, they do influence other subcellular events such as transcription and interaction of signaling proteins. Specifically, increased climbing fiber activity evoked decreased expression of 14-3-3-θ, reduced serine phosphorylation of GABAAγ2 and reduced the interaction of 14-3-3-θ with Protein Kinase C-γ (PKC-γ). Knockdown of 14-3-3-θ in vivo reduced the serine phosphorylation of GABAAγ2. Conversely, treatment of cerebellar lysates with phorbol 12-myristate-13-acetate (PMA), a PKC activator, increased serine phosphorylation of GABAAγ2. Knockdown of 14-3-3-θ or PKC-γ in N2a cells in vitro reduced serine phosphorylation of GABAAγ2 and reduced its cell-surface expression. We interpret these data to mean that a prolonged increase in climbing fiber activity decreases the cell surface expression of GABAARs in Purkinje cells and thereby reduces their sensitivity to GABAergic inhibition. This provides a homeostatic mechanism by which Purkinje cells become less sensitive to stellate cell inhibition also evoked by climbing fiber activity.

Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

“…As a result we identified 46 proteins with elevated levels of interaction with γ 2-GABA A Rs, including 10 proteins that were only found in the DZP treated group (Table 1, not found in vehicle samples, NF-V). Notably, we found a significant increase in γ 2 association with 14-3-3 protein family members, the heat shock protein family A (Hsp70) member 8 (also known as Hsc70) and the GABA A R α 5 subunit, suggesting DZP induced changes in GABA A R surface trafficking (Qian et al, 2012;Nakamura et al, 2016a), gephyrin clustering (Machado et al, 2011) and receptor composition (van Rijnsoever et al, 2004). In contrast, 23 proteins were found to coimmunoprecipitate with γ 2 less in DZP animals relative to control, seven of which were only present in the vehicle treatment group (Table 2, not found in DZP, NF-DZP).…”

Migratory activation of parasitized dendritic cells by the protozoanToxoplasma gondii14-3-3 protein

“…14‐3‐3 proteins form homo‐dimer and/or hetero‐dimer interactions and future research needs to test whether host‐14‐3‐3 and parasite‐14‐3‐3 interactions occur under physiological conditions. Interestingly, GABAergic signalling pathways in DCs are implicated in the hypermigratory phenotype (Fuks et al, ), and recent reports have implicated 14‐3‐3 molecules as regulators of GABA receptor signalling in neurological models (Benke and Zeilhofer, ; Qian et al, ). Thus, this implies a putative hypothetical link to GABA receptor regulation, and future investigations will be important to establish the molecular interplay of Tg14‐3‐3 with putative host counterparts.…”

Altered Purkinje cell miRNA expression and SCA1 pathogenesis

“…It is possible that altered miR-335 expression drives mutant Atxn1 pathogenicity during post-natal development by altering climbing fiber-Purkinje cell physiology. It could accomplish this, for example, by deregulating the expression of its targets Calbindin-1 and 14-3-3θ in Purkinje neurons (Barmack et al, 2010; Qian et al, 2012) during this critical post-natal period. Alternatively, changes in miR-335 expression might ensue as a consequence of altered climbing fiber-Purkinje cell physiology during early stages of disease.…”