TGF-β2 regulates neuronal Ankyrin-G and promotes its interaction with KCC2

Abstract

ABSTRACTThe neuronal K+/Cl-cotransporter 2 (KCC2) is the major Cl-extruder in CNS neurons and responsible for fast hyperpolarizing postsynaptic inhibition in mature neurons. Impaired KCC2 function has been associated with several brain pathologies. KCC2 forms immunocomplexes with several proteins that may regulate KCC2 membrane trafficking, stability and function, thus, tuning important cellular processes, including chloride homeostasis and dendritic spine development. In the brain, the scaffold protein Ankyrin-G, encoded by theAnk3gene, is expressed in several isoforms with distinct spatial and temporal expression patterns, is regulated by TGF-β signalling and is proposed as a KCC2 interaction partner. Moreover,Ank3gene has been implicated in several neuropsychiatric disorders.Here, we investigated a putative impact of transforming growth factor beta 2 (TGF-β2) on KCC2/Ankyrin-G interaction using quantitative RT-PCR, immunoblotting, immunoprecipitation and immunofluorescence in mouse immature and differentiated hippocampal neurons and in forebrain and brainstem tissue fromTgf-β2deficient mice. The results show TGF-β2-dependent downregulation ofAnk3transcripts, as well as KCC2/Ankyrin-G interaction in mouse brainstem tissue at embryonic day (E) 17.5.In vitro, loss ofTgf-β2resulted in significantly reduced axonal and somatic Ankyrin-G in immature neurons and significantly reduced somatic Ankyrin-G abundance in differentiated mouse hippocampal neurons. Membrane abundance of Ankyrin-G was downregulated inTgf-β2mutants as well, a phenotype rescued by application of exogenous TGF-β2. Moreover, the results suggest the presence of a Golgi-associated Ankyrin-G isoform in neurons and significantly impaired membrane KCC2 abundance following knock down ofAnk3. Thus, the present study provides new insights into Ankyrin-G regulation by TGF-β2 in neurons and first evidence of a TGF-β2-regulated interaction of KCC2 with Ankyrin-G. Moreover, these results strengthen the notion for TGF-β2 as pivotal regulator of KCC2 abundance and function.