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⁺/Ca²⁺-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.

The temporal dynamic expression of Sonic Hedgehog (SHH) and signaling during early midbrain dopaminergic (mDA) neuron development is one of the key players in establishing mDA progenitor diversity. However, whether SHH signaling is also required during later developmental stages and in mature mDA neurons is less understood. We study the expression of SHH receptors Ptch1 and Gas1 (growth arrest-specific 1) and of the transcription factors Gli1, Gli2 and Gli3 in mouse midbrain during embryonic development [embryonic day (E) 12.5 onwards)], in newborn and adult mice using in situ hybridization and immunohistochemistry. Moreover, we examine the expression and regulation of dopaminergic neuronal progenitor markers, midbrain dopaminergic neuronal markers and markers of the SHH signaling pathway in undifferentiated and butyric acid-treated (differentiated) MN9D cells in the presence or absence of exogenous SHH in vitro by RT-PCR, immunoblotting and immunocytochemistry. Gli1 was expressed in the lateral mesencephalic domains, whereas Gli2 and Gli3 were expressed dorsolaterally and complemented by ventrolateral expression of Ptch1. Co-localization with tyrosine hydroxylase could not be observed. GAS1 was exclusively expressed in the dorsal mesencephalon at E11.5 and co-localized with Ki67. In contrast, MN9D cells expressed all the genes investigated and treatment of the cells with butyric acid significantly upregulated their expression. The results suggest that SHH is only indirectly involved in the differentiation and survival of mDA neurons and that the MN9D cell line is a valuable model for investigating early development but not the differentiation and survival of mDA neurons.

Transforming growth factor betas are integral molecular components of the signalling cascades defining development and survival of several neuronal groups. Among TGF-β ligands, TGF-β2 has been considered as relatively more important during development. We have generated a conditional knockout mouse of the Tgf-β2 gene with knock-in of an EGFP reporter and subsequently a mouse line with cell-type specific deletion of TGF-β2 ligand from Krox20 expressing cells (i.e., in cells from rhombomeres r3 and r5). We performed a phenotypic analysis of the hindbrain serotonergic system during development and in adulthood, determined the neurochemical profile in hindbrain and forebrain, and assessed behavioural performance of wild type and mutant mice. Mutant mice revealed significantly decreased number of caudal 5-HT neurons at embryonic day (E) 14, and impaired development of caudal dorsal raphe, median raphe, raphe magnus, and raphe obscurus neurons at E18, a phenotype that was largely restored and even overshot in dorsal raphe of mutant adult mice. Serotonin levels were decreased in hindbrain but significantly increased in cortex of adult mutant mice, though without any behavioural consequences. These results highlight differential and temporal dependency of developing and adult neurons on TGF-β2. The results also indicate TGF-β2 being directly or indirectly potent to modulate neurotransmitter synthesis and metabolism. The novel floxed TGF-β2 mouse model is a suitable tool for analysing the in vivo functions of TGF-β2 during development and in adulthood in many organs.

Background: Reperfusion after myocardial ischaemia can lead to deadly arrhythmias, in part due to heterogeneities in electrophysiology (EP) across affected tissue. There is a need to understand the spatiotemporal dynamics of ischaemia-reperfusion arrhythmias (IRA), so that reperfusion strategies to prevent them can be found. Methods: Langendorff-perfused rabbit isolated hearts were loaded with a voltage-sensitive dye. Epifluorescence imaging was used to track action potential propagation across the cardiac surface. The heart was simultaneously perfused ‘globally’ ( via the aorta) and ‘locally’ ( via cannulation of a single coronary artery) with an oxygenated physiological saline solution. Local perfusion was subsequently switched to and from solutions that mimic aspects of ischaemia (acidosis, hypoxia, hyperkalaemia, or a simulated ischaemia solution combining all three) or to no-flow. Subsequently, different reperfusion strategies were tested to reduce IRA re-entries. The most successful strategy for preventing re-entry was tested in Langendorff-perfused isolated pig hearts to assess the clinical relevance of the observed mechanism and treatment strategy. Results: Upon sudden reperfusion of the cannulated coronary artery in rabbit hearts we observed a preferential recovery of electrical excitability along the vessel’s main branch (‘perivascular excitation tunnelling’, PVET). This resulted in re-entry in roughly half of the hearts. Hyperkalaemia and hypoxia, but not acidosis, were sufficient to lead to conduction block, PVET, and re-entry, with both PVET and re-entry more frequently observed after hyperkalaemia than hypoxia. PVET was also present in pigs and PVET-based re-entries were successfully prevented in rabbit and pig hearts by two-step reperfusion, first of the distal majority of the previously ischaemic region, and then of the remaining tissue from the proximal point. With this strategy, any PVET that developed in the distal tissue was blocked by the still inexcitable proximal tissue. Upon reperfusion of the proximal tissue, there was a reduced path length for PVET. As a consequence, the associated excitable gap was too short for re-entrant excitation. Conclusions: We observed a novel arrhythmia mechanism upon coronary reperfusion (PVET), which suggests that preferential recovery of myocardial excitability along the reperfused vessel is an important mechanism underlying IRA formation. PVET-induced re-entry reliably occurred in both rabbit and pig hearts and could be prevented by two-step reperfusion. Graphical

Transforming growth factor betas are integral molecular components of the signaling cascades defining development and survival of several neuronal groups. Among TGF‐β ligands, TGF‐β2 has been considered as relatively more important during development. We have generated a conditional knockout mouse of the Tgf‐β2 gene with knock‐in of an EGFP reporter and subsequently a mouse line with cell‐type specific deletion of TGF‐β2 ligand from Krox20 expressing cells (i.e. in cells from rhombomeres r3 and r5). We performed a phenotypic analysis of the hindbrain serotonergic system during development and in adulthood, determined the neurochemical profile in hindbrain and forebrain, and assessed behavioural performance of wild type and mutant mice. Mutant mice revealed significantly decreased number of caudal 5‐HT neurons at embryonic day (E) 14, and impaired development of caudal dorsal raphe, median raphe, raphe magnus, and raphe obscurus neurons at E18, a phenotype that was restored and/or overshot in adult mice. Serotonin levels were decreased in hindbrain but significantly increased in cortex of adult mutant mice, though without any behavioural consequences. These results highlight differential and temporal dependency of developing and adult neurons on TGF‐β2 and highlight the isoform‐specific effects during development of the serotonergic system. The results also indicate TGF‐β2 being directly or indirectly potent to modulate neurotransmitter synthesis and metabolism. The novel floxed TGF‐β2 mouse model is a suitable tool for analyzing the in vivo functions of TGF‐β2 during development and in adulthood in many organs. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .