The accumulation and deposition of α-synuclein aggregates in brain tissue is the main event in the pathogenesis of different neurodegenerative disorders grouped under the term of synucleinopathies. They include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. To date, the diagnosis of any of these disorders mainly relies on the recognition of clinical symptoms, when the neurodegeneration is already in an advanced phase. In the last years, several efforts have been carried out to develop new diagnostic tools for early diagnosis of synucleinopathies, with special interest to Parkinson's disease. The Protein-Misfolding Cyclic Amplification (PMCA) and the Real-Time Quaking-Induced Conversion (RT-QuIC) are ultrasensitive protein amplification assays for the detection of misfolded protein aggregates. Starting from the successful application in the diagnosis of human prion diseases, these techniques were recently tested for the detection of misfolded α-synuclein in brain homogenates and cerebrospinal fluid samples of patients affected by synucleinopathies. So far, only a few studies on a limited number of samples have been performed to test PMCA and RT-QuIC diagnostic reliability. Neverthless, these assays have shown very high sensitivity and specificity in detecting synucleinopathies even at the pre-clinical stage. Despite the application of PMCA and RT-QuIC for α-synuclein detection in biological fluids is very recent, these techniques seem to have the potential for identifying subjects that will be likely to develop synucleinopathies.
Enteric glial cells (EGCs) are components of the intestinal epithelial barrier essential for regulating the enteric nervous system. Clostridium difficile is the most common cause of antibiotic-associated colitis, toxin B (TcdB) being the major virulence factor, due to its ability to breach the intestinal epithelial barrier and to act on other cell types. Here we investigated TcdB effects on EGCs and the activated molecular mechanisms. Already at 2 hours, TcdB triggered ROS formation originating from NADPH-oxidase, as demonstrated by their reduction in the presence of the NADPH-oxidase inhibitor ML171. Although EGCs mitochondria support almost completely the cellular ATP need, TcdB exerted weak effects on EGCs in terms of ATP and mitochondrial functionality, mitochondrial ROS production occurring as a late event. ROS activated the JNK signalling and overexpression of the proapoptotic Bim not followed by cytochrome c or AIF release to activate the downstream apoptotic cascade. EGCs underwent DNA fragmentation through activation of the ROS/JNK/caspase-3 axis, evidenced by the ability of ML171, N-acetylcysteine, and the JNK inhibitor SP600125 to inhibit caspase-3 or to contrast apoptosis. Therefore, TcdB aggressiveness towards EGCs is mainly restricted to the cytosolic compartment, which represents a peculiar feature, since TcdB primarily influences mitochondria in other cellular types.
α-Synuclein
(α-syn) is found to be naturally present
in biofluids such as cerebrospinal fluid (CSF) and serum. Human serum
albumin (HSA) is the most abundant protein found in these biofluids,
which, beyond transporting hormones and drugs, also exerts a chaperone-like
activity binding other proteins in blood and inhibiting their aggregation.
Contrasting results are reported in the literature about the effects
of albumin on α-syn aggregation. We characterized the binding
region of HSA on α-syn by high-field solution NMR spectroscopy
and the effect of HSA on α-syn aggregation by thioflavin-T (ThT)
fluorescence under both low-ionic-strength and physiological conditions
at the albumin concentration in serum and CSF. We found that HSA,
at the concentration found in human serum, slows the aggregation of
α-syn significantly. α-Syn interacts with HSA in an ionic
strength- and pH-dependent manner. The binding is driven by hydrophobic
interactions at the N-terminus under physiological experimental conditions
and by electrostatic interactions at the C-terminus at low ionic strength.
This work provides novel information about the proteostasis of α-syn
in biofluids and supports the hypothesis of a chaperone-like behavior
of HSA.
We have explored the role of Tm7sf2 gene, which codifies for 3β-hydroxysterol Δ14-reductase, an endoplasmic reticulum resident protein, in the sensitivity to endoplasmic reticulum stress and in the resulting inflammatory response. We used mouse embryonic fibroblasts, derived from Tm7sf2+/+ and Tm7sf2−/− mice, to determine the in vitro effects of thapsigargin on NF-κB activation. Our results show that the Tm7sf2 gene controls the launch of the unfolded protein response and presides an anti-inflammatory loop thus its absence correlates with NF-κB activation and TNFα up-regulation. Our data also show that Tm7sf2 gene regulates liver X receptor activation and its absence inhibits LXR signalling. By expressing the hTm7sf2 gene in KO MEFs and observing a reduced NF-κB activation, we have confirmed that Tm7sf2 gene is linked to NF-κB activation. Finally we used genetically modified mice in an in vivo model of ER stress and of inflammation. Our results show a significant increase in renal TNFα expression after tunicamycin exposure and in the oedematogenic response in Tm7sf2−/− mice. In conclusion, we have shown that the Tm7sf2 gene, to date involved only in cholesterol biosynthesis, also controls an anti-inflammatory loop thereby confirming the existence of cross talk between metabolic pathways and inflammatory response.