TGF-β2 Regulates Transcription of the K+/Cl− Cotransporter 2 (KCC2) in Immature Neurons and Its Phosphorylation at T1007 in Differentiated Neurons

Rigkou, Anastasia; Magyar, Attila; Speer, Jan Manuel; Roussa, Eleni

doi:10.3390/cells11233861

Cited by 3 publications

(7 citation statements)

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“…1). This lack of effect can be attributed to the presence of endogenous TGF-β2, which may saturate the signaling pathway, similar to what was previously observed on KCC2 expression (Rigkou et al, 2022). Subsequent loss-of-function experiments in brain areas at different maturation stages in Tgf-β2 mutants and expression analysis of multiple transcript variants of Ank3 , showed no regulation in the forebrain of Tgf-β2 mutants (Fig.…”

Section: Discussionsupporting

confidence: 82%

“…We next investigated whether loss of the TGF-β2 ligand affects Ankyrin-G expression, using Tgf-β2 deficient mice at E17.5. We have previously demonstrated that the impact of TGF-β2 on KCC2 varies between the embryonic forebrain and brainstem, presumably due to differences in neuronal maturation (Rigkou et al, 2022). Here, we followed the same principal to investigate a potential differential regulation of Ankyrin-G by TGF-β2 in forebrain and brainstem.…”

Section: Resultsmentioning

confidence: 99%

“…We have previously introduced TGF-β2 as a key regulator of KCC2 throughout neuronal maturation and identified mechanisms through which TGF-β2 exerts its effect on KCC2: In immature neurons, TGF-β2 regulates Kcc2 expression via binding of AP2β to Kcc2 promoter, whereas in differentiated neurons TGF-β2 regulates phosphorylation of KCC2 at T1007 (Rigkou et al, 2022), as well as KCC2 trafficking to the membrane via Rab11b (Roussa et al, 2016). Here, we provide evidence for a putative novel mechanism of TGF-β2 -dependent regulation of KCC2, by regulating its interaction with the scaffolding protein Ankyrin-G.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously reported multiple modes of regulation of KCC2 by TGF-β2 throughout neuronal development. In immature neurons TGF-β2 regulates Kcc2 transcription via transcription factor AP2β (Rigkou et al, 2022), whereas in differentiating and mature neurons it regulates the (de)phosphorylation of KCC2 at T1007 and membrane trafficking and functionality of KCC2 through a CREB/Rab11b mechanism (Roussa et al, 2016; Rigkou et al, 2022). Another study demonstrated that TGF-β signaling promotes dendritic spine development in cortical neurons by stabilizing Ankyrin-G through interaction with the deubiquitinase Usp9X (Yoon et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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

Rigkou,

Pethe,

Roussa

2023

Preprint

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The 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.

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Section: Discussionsupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Rigkou,

Pethe,

Roussa

2023

Preprint

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“…TGF-β2 enhanced the colocalization and interaction between KCC2 and Rab11b, and impairing CREB1 or Rab11b hindered TGF-β2-mediated trafficking, surface expression, and functionality of KCC2. In a recent study, Rigkou et al examined the effects of TGF-β2 on KCC2 during neuronal maturation [ 92 ]. They found that inhibiting TGF-β/activin signaling decreased KCC2 mRNA expression in immature neurons.…”

Section: Posttranslational Regulatory Mechanisms Of Kcc2mentioning

confidence: 99%

Neuronal K+-Cl- cotransporter KCC2 as a promising drug target for epilepsy treatment

McMoneagle,

Zhou,

Zhang

et al. 2023

Acta Pharmacol Sin

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Epilepsy is a prevalent neurological disorder characterized by unprovoked seizures. γ-Aminobutyric acid (GABA) serves as the primary fast inhibitory neurotransmitter in the brain, and GABA binding to the GABAA receptor (GABAAR) regulates Cl- and bicarbonate (HCO3-) influx or efflux through the channel pore, leading to GABAergic inhibition or excitation, respectively. The neuron-specific K+-Cl- cotransporter 2 (KCC2) is essential for maintaining a low intracellular Cl- concentration, ensuring GABAAR-mediated inhibition. Impaired KCC2 function results in GABAergic excitation associated with epileptic activity. Loss-of-function mutations and altered expression of KCC2 lead to elevated [Cl-]i and compromised synaptic inhibition, contributing to epilepsy pathogenesis in human patients. KCC2 antagonism studies demonstrate the necessity of limiting neuronal hyperexcitability within the brain, as reduced KCC2 functioning leads to seizure activity. Strategies focusing on direct (enhancing KCC2 activation) and indirect KCC2 modulation (altering KCC2 phosphorylation and transcription) have proven effective in attenuating seizure severity and exhibiting anti-convulsant properties. These findings highlight KCC2 as a promising therapeutic target for treating epilepsy. Recent advances in understanding KCC2 regulatory mechanisms, particularly via signaling pathways such as WNK, PKC, BDNF, and its receptor TrkB, have led to the discovery of novel small molecules that modulate KCC2. Inhibiting WNK kinase or utilizing newly discovered KCC2 agonists has demonstrated KCC2 activation and seizure attenuation in animal models. This review discusses the role of KCC2 in epilepsy and evaluates its potential as a drug target for epilepsy treatment by exploring various strategies to regulate KCC2 activity.

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K+/Cl− cotransporter 2 (KCC2) and Na+/HCO3− cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation

Pethe,

Hamze,

Giannaki

et al. 2023

Front. Cell. Neurosci.

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K+/Cl− cotransporter 2 (KCC2) is a major Cl− extruder in mature neurons and is responsible for the establishment of low intracellular [Cl−], necessary for fast hyperpolarizing GABAA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl−], Cl− extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction—a mechanism with putative pathophysiological relevance.

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TGF-β2 Regulates Transcription of the K+/Cl− Cotransporter 2 (KCC2) in Immature Neurons and Its Phosphorylation at T1007 in Differentiated Neurons

Cited by 3 publications

References 57 publications

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

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

Neuronal K+-Cl- cotransporter KCC2 as a promising drug target for epilepsy treatment

K+/Cl− cotransporter 2 (KCC2) and Na+/HCO3− cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation

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