Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis.

AIM: Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). METHODS: We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). In vitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. RESULTS: CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. In vitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. CONCLUSION: We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

Impact of inflammatory preconditioning on murine microglial proteome response induced by focal ischemic brain injury.

Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).

Extra-domain A containing fibronectin in pulmonary hypertension and treatment effects of a function-blocking antibody.

AIMS: Pulmonary vascular and right ventricular remodelling processes are important for development and progression of pulmonary hypertension (PH). The current study analyzed the functional role of the extra domain A containing fibronectin (ED-A+ Fn) for the development of PH by comparing ED-A+ Fn knockout (KO) and wild-type (WT) mice as well as the effects of an antibody-based therapeutical approach in a model of monocrotaline (MCT)-induced PH, which will be validated in a model of Sugen 5416/Hypoxia induced PH. METHODS AND RESULTS: PH was induced using monocrotaline (MCT) (PH mice). 69 mice were divided into the following groups: sham-treated controls (WT: n=7; KO: n=7), PH mice without specific treatment (WT: n=12; KO: n=10), PH mice treated with a dual endothelin receptor antagonist (MAC; WT: n=6; KO: n=11), WT PH mice treated with the F8 antibody, specifically recognizing ED-A+ Fn, (n=8) and WT PH mice treated with an antibody of irrelevant antigen specificity (KSF, n=8). Compared to controls, WT_PH mice showed a significant elevation of the right ventricular systolic pressure (RVPsys, p=0.04) and RV functional impairment including increased basal right ventricular (RVbasal, p=0.016) diameter or tricuspid annular plane systolic excursion (TAPSE, p=0.008). In contrast, KO PH did not show such effects compared to controls (p=n.s.). In WT_PH mice treated with F8, hemodynamic and echocardiographic parameters were significantly improved compared to untreated WT_PH mice or those treated with the KSF antibody (p<0.05). On the microscopic level, KO_PH mice showed significantly less tissue damage compared to the WT_PH mice (p=0.008). Furthermore, lung tissue damage could significantly be reduced after F8 treatment (p=0.04). Additionally, these findings could be verified in the Sugen 5416/Hypoxia mouse model, in which F8 significantly improved echocardiographic, hemodynamic and histologic parameters. CONCLUSION: ED-A+ Fn is of crucial importance for PH pathogenesis representing a promising therapeutic target in PH. We here show a novel therapeutic approach using antibody-mediated functional blockade of ED-A+ Fn capable to attenuate and partially reverse PH-associated tissue remodelling.