TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

Abstract

Calcium homeostasis is a cellular process required for proper cell function and survival, maintained by the coordinated action of several transporters, among them members of the Na + /Ca 2+ -exchanger family, such as SLC8A3. Transforming growth factor beta (TGF-β) signaling defines neuronal development and survival and may regulate the expression of channels and transporters. We investigated the regulation of SLC8A3 by TGF-β in a conditional knockout mouse with deletion of TGF-β signaling from Engrailed 1-expressing cells, i.e., in cells from the midbrain and rhombomere 1, and elucidated the underlying molecular mechanisms. The results show that SLC8A3 is significantly downregulated in developing dopaminergic and dorsal raphe serotonergic neurons in mutants and that low SLC8A3 abundance prevents the expression of the anti-apoptotic protein Bcl-xL. TGF-β signaling affects SLC8A3 via the canonical and p38 signaling pathway and may increase the binding of Smad4 to the Slc8a3 promoter. Expression of the lipid peroxidation marker malondialdehyde (MDA) was increased following knockdown of Slc8a3 expression in vitro. In neurons lacking TGF-β signaling, the number of MDA-and 4-hydroxynonenal (4-HNE)-positive cells was significantly increased, accompanied with increased cellular 4-HNE abundance. These results suggest that TGF-β contributes to the regulation of SLC8A3 expression in developing dopaminergic and dorsal raphe serotonergic neurons, thereby preventing oxidative stress.Cellular ionic homeostasis is a prerequisite for proper cellular function and survival, whereby TGF-βs are known to regulate several channels and transporters within or outside the central nervous system [6][7][8][9]. Particularly, the link between TGF-βs and Ca 2+ homeostasis has been documented in several cellular paradigms: In cortical neurons, TGF-β regulates L-type Ca 2+ channels through MEK, JNK1/2, and p38 MAPK signaling [10]; it increases store-operated Ca 2+ entry into megakaryocytes [11]; and it enhances Ca 2+ influx pathways and the expression of transient receptor potential canonical channels (TRPCs) in human cardiac fibroblasts. Interestingly, human cardiac fibroblasts express several TRPC-mediated Ca 2+ influx pathways, which activate the reverse mode Na + /Ca 2+ exchanger (NCX) [12].Among the NCX isoformsSLC8A3), the isoform 3 of the Na + /Ca 2+ exchanger (NCX3), is exclusively expressed in excitable cells [13]. It mediates the electrogenic transport of Na + and Ca 2+ , and contributes to the maintenance of Ca 2+ homeostasis. SLC8A3 acts with a stoichiometry of 3:1 and may operate in the forward (Ca 2+ efflux) or reverse (influx of Ca 2+ ) mode. However, the forward mode is the predominant mode of Na + /Ca 2+ exchanger action. Alternative splicing generates two variants, AC and B, which display a tissue-specific distribution in mice. The variant B of Slc8a3 is mostly expressed in the brain, including substantia nigra pars compacta (SNc) and hindbrain raphe nuclei, whereas the variant AC is predominant in skeletal muscle. The function...

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

“…Images were acquired with a Leica TCS SP8 confocal microscope using an HC PL APO CS2 40×/1.30 or 63×/1.40 oil objective lens, and immunofluorescence intensity was analyzed as described previously [ 41 ]. Within each experiment, the confocal microscope settings (laser power, detector gain and amplifier offset) were kept the same for all scans in which the immunofluorescence intensity was compared.…”

<p>Emodin Retarded Renal Fibrosis Through Regulating HGF and TGFβ–Smad Signaling Pathway</p>

“…39,40 Smad4 is at the core of the TGFβ 1 -signaling pathway. 41 In addition, it has been verified that TGFβ 1 can upregulate Smad2, which can affect this signaling pathway, resulting in fibrosis. 42 In the current research, combination treatment further inhibited the expression of p-Smad2 in HK2 cells after TGFβ 1 treatment compared with HGF or emodin alone.…”

Developmental pathways linked to the vulnerability of adult midbrain dopaminergic neurons to neurodegeneration

“…In support of the latter, one recently identified direct or indirect target gene of TGFb signaling in mature mDA neurons was the member of the Na + /Ca 2+ -exchanger family, Slc8a3 (Chleilat et al, 2020). SLC8A3 is expressed in the SNc and was strongly downregulated in the absence of Tgfbr2mediated signaling (Chleilat et al, 2020). Conversely, treatment of mDA-related cell cultures with TGFb strongly increased the SLC8A3 protein levels, but only in mature neurons and not in immature neural precursors.…”

“…These findings indicated that apart from a direct pro-survival function in the mDA neurons themselves, TGFb signaling is also implicated in synaptogenesis and the establishment of the proper mDA connectivity with their afferent inputs and efferent targets. In support of the latter, one recently identified direct or indirect target gene of TGFb signaling in mature mDA neurons was the member of the Na + /Ca 2+ -exchanger family, Slc8a3 (Chleilat et al, 2020). SLC8A3 is expressed in the SNc and was strongly downregulated in the absence of Tgfbr2mediated signaling (Chleilat et al, 2020).…”