Pathogenic SCN2A variants cause early-stage dysfunction in patient-derived neurons.

Pathogenic heterozygous variants in SCN2A, which encodes the neuronal sodium channel NaV1.2, cause different types of epilepsy or intellectual disability (ID)/autism without seizures. Previous studies using mouse models or heterologous systems suggest that NaV1.2 channel gain-of-function typically causes epilepsy, whereas loss-of-function leads to ID/autism. How altered channel biophysics translate into patient neurons remains unknown. Here, we investigated iPSC-derived early-stage cortical neurons from ID patients harboring diverse pathogenic SCN2A variants [p.(Leu611Valfs*35); p.(Arg937Cys); p.(Trp1716*)] and compared them with neurons from an epileptic encephalopathy (EE) patient [p.(Glu1803Gly)] and controls. ID neurons consistently expressed lower NaV1.2 protein levels. In neurons with the frameshift variant, NaV1.2 mRNA and protein levels were reduced by ~ 50%, suggesting nonsense-mediated decay and haploinsufficiency. In other ID neurons, only protein levels were reduced implying NaV1.2 instability. Electrophysiological analysis revealed decreased sodium current density and impaired action potential (AP) firing in ID neurons, consistent with reduced NaV1.2 levels. In contrast, epilepsy neurons displayed no change in NaV1.2 levels or sodium current density, but impaired sodium channel inactivation. Single-cell transcriptomics identified dysregulation of distinct molecular pathways including inhibition of oxidative phosphorylation in neurons with SCN2A haploinsufficiency and activation of calcium signaling and neurotransmission in epilepsy neurons. Together, our patient iPSC-derived neurons reveal characteristic sodium channel dysfunction consistent with biophysical changes previously observed in heterologous systems. Additionally, our model links the channel dysfunction in ID to reduced NaV1.2 levels and uncovers impaired AP firing in early-stage neurons. The altered molecular pathways may reflect a homeostatic response to NaV1.2 dysfunction and can guide further investigations.

Insight into the beneficial immunomodulatory mechanism of the sevoflurane metabolite hexafluoro-2-propanol in a rat model of endotoxaemia.

Volatile anaesthetics such as sevoflurane attenuate inflammatory processes, thereby impacting patient outcome significantly. Their inhalative administration is, however, strictly limited to controlled environments such as operating theatres, and thus an intravenously injectable immunomodulatory drug would offer distinct advantages. As protective effects of volatile anaesthetics have been associated with the presence of trifluorinated carbon groups in their basic structure, in this study we investigated the water-soluble sevoflurane metabolite hexafluoro-2-propanol (HFIP) as a potential immunomodulatory drug in a rat model of endotoxic shock. Male Wistar rats were subjected to intravenous lipopolysaccharide (LPS) and thereafter were treated with HFIP. Plasma and tissue inflammatory mediators, neutrophil invasion, tissue damage and haemodynamic stability were the dedicated end-points. In an endotoxin-induced endothelial cell injury model, underlying mechanisms were elucidated using gene expression and gene reporter analyses. HFIP reduced the systemic inflammatory response significantly and decreased endotoxin-induced tissue damage. Additionally, the LPS-provoked drop in blood pressure of animals was resolved by HFIP treatment. Pathway analysis revealed that the observed attenuation of the inflammatory process was associated with reduced nuclear factor kappa B (NF-κΒ) activation and suppression of its dependent transcripts. Taken together, intravenous administration of HFIP exerts promising immunomodulatory effects in endotoxaemic rats. The possibility of intravenous administration would overcome limitations of volatile anaesthetics, and thus HFIP might therefore represent an interesting future drug candidate for states of severe inflammation.

Experimental heart transplantation: effect of cyclosporine on expression and activity of metzincins.

Metzincins, such as matrix metalloproteases (MMP), and extracellular matrix (ECM) proteins are differentially regulated in inflammation. We hypothesised that metzincins are also dysregulated in experimental acute cardiac allograft rejection. We investigated the Dark Agouti-to-Lewis (DA-to-Lew) rat model of acute cardiac allograft rejection. Cyclosporine (CsA) (7.5 mg/kg/d) was given from transplantation to sacrifice (day +5). At that time, mRNA levels were analysed by Affymetrix genechip and quantitative reverse transcription polymerase chain reaction (qRTPCR). MMP protein and activities were analysed by immunohistology, fluorometry, zymography and Western blots. In untreated rejected DA allografts, mRNA levels of MMP-2/-7/-9/-/12-/14, a disintegrin and metalloprotease (ADAM)-17, tissue inhibitor of metalloprotease (TIMP)-1/-3 were increased, whereas MMP-11/-16/-24 and TIMP-2/-4 were lowered compared to native DA hearts. With respect to these untreated allografts, CsA lowered mRNA levels of MMP-7, TIMP-1/-3 (TIMP-2/-4 remained relatively low) and ADAM17, but augmented mRNA levels of MMP-11/-16/-23 and of many ECM genes. Immunohistology showed increased staining of MMP-2 in acute rejection (AR). Overall MMP activity was augmented in both transplanted groups, but CsA reduced MMP-9 activity and MMP-14 production. Taken together, MMP and TIMP were upregulated during acute AR. CsA ameliorated histology of rejection but showed potential pro-fibrotic effects. Thus, MMP and TIMP may play a role in acute cardiac allograft rejection, and beneficial modification of the MMP-ECM balance requires interventions beyond CsA.

PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts.

Primary cells respond to irradiation by activation of the DNA damage response and cell cycle arrest, which eventually leads to senescence or apoptosis. It is not clear in detail which signaling pathways or networks regulate the induction of either apoptosis or senescence. Primary human fibroblasts are able to withstand high doses of irradiation and to prevent irradiation-induced apoptosis. However, the underlying regulatory basis for this phenotype is not well understood. Here, a kinetic network analysis based on reverse phase protein arrays (RPPAs) in combination with extensive western blot and cell culture analyses was employed to decipher the cytoplasmic and nuclear signaling networks and to identify possible antiapoptotic pathways. This analysis identified activation of known DNA damage response pathways (e.g., phosphorylation of MKK3/6, p38, MK2, Hsp27, p53 and Chk1) as well as of prosurvival (e.g., MEK-ERK, cAMP response element-binding protein (CREB), protein kinase C (PKC)) and antiapoptotic markers (e.g., Bad, Bcl-2). Interestingly, PKC family members were activated early upon irradiation, suggesting a regulatory function in the ionizing radiation (IR) response of these cells. Inhibition or downregulation of PKC in primary human fibroblasts caused IR-dependent downregulation of the identified prosurvival (CREB phosphorylation) and antiapoptotic (Bad phosphorylation, Bcl-2) markers and thus lead to a proliferation stop and to apoptosis. Taken together, our analysis suggests that cytoplasmic PKC signaling conditions IR-stressed MRC-5 and IMR-90 cells to prevent irradiation-induced apoptosis. These findings contribute to the understanding of the cellular and nuclear IR response and may thus eventually improve the efficacy of radiotherapy and help overcome tumor radioresistance.