Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

Morphological features of changes in peripheral olfactory structures in SARS-COV-2 coronavirus infection

Coronavirus infection caused by the SARS-CoV-2 virus is an extremely important and urgent problem of modern medicine. It spreads quickly, has a high probability of a severe course and a large number of critical complications in patients from the risk group. The presence of pathognomonic symptoms, one of which is the development of hypo– or anosmia, makes it possible to quickly differentiate coronavirus infection from other acute respiratory viral infections, that is, to isolate the patient on time and begin correct treatment, taking all possible risks into account. The aim is to identify the morphological features of olfactory structural elements in patients with coronavirus disease (COVID-19) for a better understanding of the mechanisms of olfactory disorders development in coronavirus infection. Materials and methods. The basis of the work is a retrospective analysis of autopsy material, namely the mucous membrane of the upper parts of the nasal cavity (olfactory epithelium) and olfactory bulbs of nine deceased (4 women and 5 men) aged from 53 to 79 years with a laboratory-confirmed diagnosis of COVID-19 and anosmia in anamnesis. We used standard hematoxylin and eosin staining and immunohistochemical reactions in accordance with the TermoScientific protocols (USA) with antibodies to neurospecific beta-III tubulin (clone TuJ-1) and RnDsystems protocols with antibodies to olfactory marker protein (OMP) and angiotensin converting enzyme (ACE-2). To compare the results, a control group of 9 deaths (3 women and 6 men) aged from 59 to 68 years with a laboratory-refuted diagnosis of COVID-19 was formed. The causes of death of these patients were complications of diabetes, coronary heart disease and cerebrovascular disorders of the ischemictype. Results. The average age of the deceased with a laboratory-confirmed diagnosis of COVID-19 and a history of anosmia and the control group was 64.67 ± 7.73 and 62.33 ± 6.48 years, respectively. The expression of olfactory marker protein (OMP) and neurospecific beta-III tubulin (clone TuJ-1) was partially positive (40.89 (25.00–52.00) and 42.44 (29.00–55.00) cells in the field of view at a magnification of 200×, respectively) in seven out of nine sections of the olfactory mucous membrane of deaths with a laboratory-confirmed diagnosis of COVID-19 and anosmia in anamnesis. The reaction with antibodies to angiotensin converting enzyme (ACE-2) was focally or subtotally absent (34.33 (14.00–49.00) cells in the field of view at 200× magnification). There was expression of these three markers in control sections of the olfactory mucosa of a deceased with a laboratory excluded diagnosis of COVID-19 and no symptoms of anosmia (Mann–Whitney test, P < 0.05). In sections of olfactory bulbs of patients with COVID-19 weak (Mann–Whitney test, P < 0.05) expression of receptors for angiotensin-converting enzyme (ACE-2) (26.78 (15.00–39.00) cells in field of view at a magnification of 200×) was revealed in contrast to control sections (100.56 (94.00–107.00) cells in the field of view at a magnification of 200×). Conclusions. The development of anosmia in SARS-CoV-2 coronavirus infection has specific features. This may be due to the primary destruction of cells expressing receptors for the angiotensin-converting enzyme (ACE-2-positive: sustentacular cells of the olfactory mucosa, neurons of the olfactory bulbs). Subsequent dysfunction of olfactory cells (OMP- and TuJ-1-positive) is also possible.

Determination of tissue-specific interaction between vitamin C and vitamin E in vivo using senescence marker protein-30 knockout mice as a vitamin C synthesis deficiency model

Vitamin E (α-tocopherol; VE) is known to be regenerated from VE radicals by vitamin C (L-ascorbic acid; VC) in vitro. However, their in vivo interaction in various tissues is still unclear. Therefore, we alternatively examined the in vivo interaction of VC and VE by measurement of their concentrations in various tissues of senescence marker protein-30 (SMP30) knockout (KO) mice as a VC synthesis deficiency model. Male SMP30-KO mice were divided into four groups (VC+/VE+, VC+/VE-, VC-/VE+, and VC-/VE-), fed diets with or without 500 mg/kg VE and given water with or without 1.5 g/L VC ad libitum. Then, VC and VE concentrations in the plasma and various tissues were determined. Further, gene expression levels of transporters associated with VC and VE, such as α-tocopherol transfer protein (α-TTP) and sodium-dependent vitamin C transporters (SVCTs), were examined. These results showed that the VE levels in the VC-depleted (VC-/VE+) group were significantly lower than those in the VC+/VE+ group in the liver and heart; the VC levels in the VE-depleted (VC+/VE-) group were significantly lower than those in the VC+/VE+ group in the kidneys. The α-TTP gene expression in the liver and kidneys were decreased by VC and/or VE depletion. Moreover, SVCT1 gene expression in the liver was decreased by both VC and VE depletion. In conclusion, these results indicate that VC spares VE mainly in the liver and heart, and that VE spares VC in the kidneys of SMP30-KO mice. Thus, interaction between VC and VE is likely to be tissue specific.

Metformin Inhibits the Phenotypic Transformation of Aortic Vascular Smooth Muscle Cells Induced by Hyperglycemia Through Regulation of the Mir-21-mediated PTEN/pAKT/Egr-1 Pathway

Background The phenotypic transformation of arterial vascular smooth muscle cells (VSMC) is one of the key mechanisms in the formation of atherosclerotic plaque. It is unclear whether metformin can inhibit the phenotypic transformation of VSMC. In this study, we observed the effect of metformin on the phenotypic transformation of VSMC in vivo and in vitro and its mechanism.Methods Patients who underwent simple coronary artery bypass grafting (CABG) were divided into non-diabetic(non-DM) group and diabetes (DM) group according to whether diabetes was combined. The miR-21 and VSMC contractile marker protein, smooth muscle contractile protein (αSMA) and synthetic marker protein osteopontin (OPN) were isolated from the internal mammary artery.30 male, clean, and 6 week old wild type C57/BL6J mice were randomly divided into 3 groups:the control group (NC), the diabetes mellitus group (DM) and the metformin intervention group(DM+MET).Diabetic mice model was established by high-fat diet combined with low-dose streptozotocin. Metformin was given by gavage for 8 weeks.The aortic tissue from aortic root to renal artery was retained, miR-21 was determined by real-time PCR, and SMC and OPN were determined by Western Blotting (WB). In vitro, human aortic smooth muscle cells (HA-SMC) were divided into three groups: control group, HG group and HG+ group. The content of miR-21/PTEN/pAKT/Egr-1 protein was determined by WB, the migration ability was determined by MTT and scratch method.Results 1.In vivo, the expression of miR-21 and OPN in the aortic VSMC of DM petients and diabetic mice increased and the intima media thickness increased. Metformin treatment reduced the expression of miR-21 and OPN in the aorta and decreased the thickness of intima media. 2.In vitro, glucose concentration dependently upregulates the expression of miR-21 and osteopontin in HA-SMC.Conclusion Metformin can inhibit the phenotype transformation of VSMC induced by HG, which may inhibit the migration of VSMC through miR-21/PTEN/pAkt/Egr-1.

Detection of Cerebrospinal Fluid Neurofilament Light Chain as a Marker for Alpha-Synucleinopathies

Alpha-synucleinopathies, such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), are a class of neurodegenerative diseases. A diagnosis may be challenging because clinical symptoms partially overlap, and there is currently no reliable diagnostic test available. Therefore, we aimed to identify a suitable marker protein in cerebrospinal fluid (CSF) to distinguish either between different types of alpha-synucleinopathies or between alpha-synucleinopathies and controls. In this study, the regulation of different marker protein candidates, such as alpha-synuclein (a-Syn), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), and total tau (tau) in different types of alpha-synucleinopathies, had been analyzed by using an ultrasensitive test system called single-molecule array (SIMOA). Interestingly, we observed that CSF-NfL was significantly elevated in patients with DLB and MSA compared to patients with PD or control donors. To differentiate between groups, receiver operating characteristic (ROC) curve analysis resulted in a very good diagnostic accuracy as indicated by the area under the curve (AUC) values of 0.87–0.92 for CSF-NfL. Furthermore, we observed that GFAP and tau were slightly increased either in DLB or MSA, while a-Syn levels remained unregulated. Our study suggests NfL as a promising marker to discriminate between different types of alpha-synucleinopathies or between DLB/MSA and controls.

Comparative two-dimensional fluorescence gel electrophoresis

Comparative two-dimensional gel electrophoresis (CoFGE) is a special version of two-dimensional polyacrylamide GE (2D-PAGE) and related to difference GE (2D-DIGE). It provides reproducibility and standardisation for 2D-PAGE by introducing a reference to the experiment. CoFGE uses different fluorescent labels to distinguish analyte and a marker protein mixture. The method allows in silico correction of the assignment of gel-separated proteins based on the co-run references, which form a grid of landmarks across the entire gel. The variability of spot coordinates is reduced to ~1% error and data can thus be compared to results generated independently with the same method. In this way, searchable repositories of gel-separated proteins become feasible. In addition, the CoFGE experimental principle can be used for protein quantification by applying the proteins of the marker grid in different concentrations. Here we present the protocol for conducting a CoFGE experiment, which takes about 2 days to complete for a technician skilled in GE.

Neuronal Induction and Bioenergetics Characterization of Human Forearm Adipose Stem Cells from Parkinson’s Disease Patients and Healthy Controls

Background: Neurodegenerative diseases, such as Parkinson’s disease, are heterogeneous disorders with multifactorial nature involving impaired bioenergetics; that are on the rise with the increasing global population and average lifespan. Without definite therapeutic options, both stem regenerative medicine and bioenergetics have been proposed as promising therapeutic targets in the neurologic field. The rationale of the present study was to assess human derived adipose stem cells (hASC) potential to transdifferentiate into neuronal-like cells (NhASC and neurospheres), as well as to explore hASC bioenergetic profile. Methods: To this purpose, hASC derived from the forearm of both healthy controls and clinical diagnosed Parkinson’s disease patients (PD) were included in this study and transdifferentiated through neuronal induction. Results: Morphological, growth features and neuronal protein expression markers of differentiated neuron-like NhASC and neurospheres were achieved. Increased MAP-2 neuronal marker protein expression upon neuronal induction (p<0.05 from undifferentiated hASC vs. 28 and 36 days of differentiation) and increased bIII tubulin neuronal marker protein expression upon neuronal induction (p<0.05 from undifferentiated hASC vs. 6, 28 and 36 days of differentiation) were found. Bioenergetic profile was detectable through high resolution respirometry approaches in hASC but did not lead to differential oxidative capacity rates in healthy or clinical diagnosed PD hASC. Conclusions: We have confirmed the capability of transdifferentiation to neuronal-like profile of hASC derived from the forearem of human subjects and characterized the bioenergetic oxidative profile of hASC. Despite the latter did not lead to significant differential respiration profiles, trends to suboptimal maximal respiratory capacity in PD were found. The neuronal induction leading to positive neuronal protein expression markers is a relevant issue that encourages the suitability of the NhASC models in neurodegeneration.