Anti-Inflammatory Impact of Minocycline in a Mouse Model of Tauopathy

Alzheimer's disease (AD) is characterized by the extracellular deposition of β-amyloid in senile plaques, the intraneuronal accumulation of hyperphosphorylated tau aggregates as neurofibrillary tangles, and progressive neuronal loss leading to the onset of dementia. Increasing evidence suggests that neuroinflammatory processes contribute to the progression of AD. Minocycline is a semi-synthetic tetracycline derivative commonly used in the treatment of acne. Many studies have revealed that minocycline also has potent anti-inflammatory actions that are neuroprotective in rodent models of Huntington's disease, Parkinson's disease and motor neuron disease. Recently, we demonstrated that minocycline reduces the development of abnormal tau species in the htau mouse model of Alzheimer's disease. We have now extended these findings by examining the impact of minocycline on inflammatory processes in htau mice. Immunohistochemical analysis revealed that minocycline treatment resulted in fewer activated astrocytes in several cortical regions of htau mice, but did not affect astrocytosis in the hippocampus. We found htau mice have significantly elevated amounts of several cortical pro-inflammatory cytokines. In addition, we find that minocycline treatment significantly reduced the amounts of several inflammatory factors, including monocyte chemoattractant proteins 1 and 5, interleukins -6 and -10, eotaxin, and I-309. Furthermore, the reduced amounts of these cytokines significantly correlated with the amount of tau phosphorylated at Ser396/404 in the cortex of htau mice. These results may reveal new cytokine targets of minocycline that could be associated with its inhibition of tau pathology development in vivo. It is possible that further investigation of the role of these cytokines in neurodegenerative processes may identify novel therapeutic targets for Alzheimer's disease and related disorders

Analysis of Myogenic and Candidate Disease Biomarkers in FSHD Muscle Cells

The UMMS Wellstone Program is a foundation and NIH-funded cooperative research center focusing on identifying biomarkers for facioscapulohumeral muscular dystrophy (FSHD) to gain insight into the molecular pathology of the disease and to develop potential therapies. FSHD is characterized by progressive wasting of skeletal muscles, with weakness often initiating in facial muscles and muscles supporting the scapula and upper arms. While the genetics associated with FSHD are complex, the major form of the disease, FSHD1, is linked to contraction of the D4Z4 repeat region located at chromosome 4q. Recently, a transcript encoded at the distal end of the repeat region, Dux4-fl, normally expressed in embryonic stem cells and germ cells, was also detected in differentiated muscle cells and biopsies from FSHD subjects, giving rise to the hypothesis that DUX4-FL function contributes to muscle weakness. We established a repository of high quality, well-characterized primary and immortalized muscle cell strains from FSHD and control subjects in affected families to provide biomaterials for cell and molecular studies to the FSHD research community. qPCR and immunostaining analyses demonstrate similar growth and differentiation characteristics in cells from FSHD and control subjects within families. We detected Dux4-fl transcript and protein in FSHD cells as recently described; interestingly, we also detected Dux4-fl in muscle cells from a subset of control individuals, suggesting that any Dux4-fl-mediated myopathy would require additional modifying elements. Microarray analysis of FSHD and control muscle cells demonstrated that several genes were upregulated in FSHD cells, including genes that were concurrently identified as downstream targets of Dux4-fl and as candidate FSHD disease genes. Future studies will further characterize the RNA and protein expression of candidate disease genes in cells from FSHD and control subjects, including nonmanifesting subjects with the D4Z4 lesion but no muscle weakness, and utilizing whole transcriptome sequencing (RNAseq) to identify additional candidates

To sit or stand? A preliminary, cross sectional study to investigate if there is a difference in glenohumeral subluxation in sitting or standing in people following stroke

Background: Glenohumeral subluxation (GHS) is a common symptom following stroke. Many therapists postulate that GHS may be reduced if the base of support (BOS) is reduced and the centre of mass (COM) is raised as this requires greater postural muscle activity. However, there is little empirical evidence to support this practice. Objective: The aim of this preliminary study was to investigate if the amount of GHS alters from sitting to standing. Study design: A cross sectional, within-subject design in a convenience sample of 15 stroke patients with GHS was utilised. Methods: A prospective design was used with a single blinded tester who assessed GHS using the calliper method in sitting, standing and on return to sitting. Friedman and post hoc Wilcoxon tests showed that GHS was significantly reduced in standing compared to sitting (p <0.05) but this reduction was not maintained on return to sitting (p = 0.25). Conclusions: The results of this study are limited by its small size. However, these results indicate that reducing BOS during rehabilitation may improve GHS after stroke. Whilst the maintenance of benefit is not established, these findings suggest that reducing BOS as part of treatment may help patients with GHS. Further research is now required to replicate these results in a larger sample and to directly examine shoulder muscle activity to investigate which muscles may influence GHS in response to changing BOS. Future work could also aim to determine whether the reduction in GHS was directly attributable to a reduced BOS or the effort associated with moving from sitting to standing

Rab-GTPase binding effector protein 2 (RABEP2) is a primed substrate for Glycogen Synthase kinase-3 (GSK3)

Glycogen synthase kinase-3 (GSK3) regulates many physiological processes through phosphorylation of a diverse array of substrates. Inhibitors of GSK3 have been generated as potential therapies in several diseases, however the vital role GSK3 plays in cell biology makes the clinical use of GSK3 inhibitors potentially problematic. A clearer understanding of true physiological and pathophysiological substrates of GSK3 should provide opportunities for more selective, disease specific, manipulation of GSK3. To identify kinetically favourable substrates we performed a GSK3 substrate screen in heart tissue. Rab-GTPase binding effector protein 2 (RABEP2) was identified as a novel GSK3 substrate and GSK3 phosphorylation of RABEP2 at Ser200 was enhanced by prior phosphorylation at Ser204, fitting the known consensus sequence for GSK3 substrates. Both residues are phosphorylated in cells while only Ser200 phosphorylation is reduced following inhibition of GSK3. RABEP2 function was originally identified as a Rab5 binding protein. We did not observe co-localisation of RABEP2 and Rab5 in cells, while ectopic expression of RABEP2 had no effect on endosomal recycling. The work presented identifies RABEP2 as a novel primed substrate of GSK3, and thus a potential biomarker for GSK3 activity, but understanding how phosphorylation regulates RABEP2 function requires more information on physiological roles of RABEP2

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies

Two-Dimensional Electrophoresis of Tau Mutants Reveals Specific Phosphorylation Pattern Likely Linked to Early Tau Conformational Changes

The role of Tau phosphorylation in neurofibrillary degeneration linked to Alzheimer's disease remains to be established. While transgenic mice based on FTDP-17 Tau mutations recapitulate hallmarks of neurofibrillary degeneration, cell models could be helpful for exploratory studies on molecular mechanisms underlying Tau pathology. Here, “human neuronal cell lines” overexpressing Wild Type or mutated Tau were established. Two-dimensional electrophoresis highlights that mutated Tau displayed a specific phosphorylation pattern, which occurs in parallel to the formation of Tau clusters as visualized by electron microscopy. In fact, this pattern is also displayed before Tau pathology onset in a well established mouse model relevant to Tau aggregation in Alzheimer's disease. This study suggests first that pathological Tau mutations may change the distribution of phosphate groups. Secondly, it is possible that this molecular event could be one of the first Tau modifications in the neurofibrillary degenerative process, as this phenomenon appears prior to Tau pathology in an in vivo model and is linked to early steps of Tau nucleation in Tau mutants cell lines. Such cell lines consist in suitable and evolving models to investigate additional factors involved in molecular pathways leading to whole Tau aggregation

The social and scientific values that shape national climate scenarios: a comparison of the Netherlands, Switzerland and the UK

This paper seeks to understand why climate information is produced differently from country to country. To do this, we critically examined and compared the social and scientific values that shaped the production of three national climate scenarios in the Netherlands, Switzerland and the UK. A comparative analysis of documentary materials and expert interviews linked to the climate scenarios was performed. Our findings reveal a new typology of use-inspired research in climate science for decision-making: (i) innovators, where the advancement of science is the main objective; (ii) consolidators, where knowledge exchanges and networks are prioritised; and (iii) collaborators, where the needs of users are put first and foremost. These different values over what constitutes ‘good’ science for decision-making are mirrored in the way users were involved in the production process: (i) elicitation, where scientists have privileged decision-making power; (ii) representation, where multiple organisations mediate on behalf of individual users; and (iii) participation, where a multitude of users interact with scientists in an equal partnership. These differences help explain why climate knowledge gains its credibility and legitimacy differently even when the information itself might not be judged as salient and usable. If the push to deliberately co-produce climate knowledge is not sensitive to the national civic epistemology at play in each country, scientist–user interactions may fail to deliver more ‘usable’ climate information

Dual modification of Alzheimer’s disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach

In sporadic Alzheimer’s disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography–tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation

Phosphoproteomic differences in major depressive disorder postmortem brains indicate effects on synaptic function

There is still a lack in the molecular comprehension of major depressive disorder (MDD) although this condition affects approximately 10% of the world population. Protein phosphorylation is a posttranslational modification that regulates approximately one-third of the human proteins involved in a range of cellular and biological processes such as cellular signaling. Whereas phosphoproteome studies have been carried out extensively in cancer research, few such investigations have been carried out in studies of psychiatric disorders. Here, we present a comparative phosphoproteome analysis of postmortem dorsolateral prefrontal cortex tissues from 24 MDD patients and 12 control donors. Tissue extracts were analyzed using liquid chromatography mass spectrometry in a data-independent manner (LC-MSE). Our analyses resulted in the identification of 5,195 phosphopeptides, corresponding to 802 non-redundant proteins. Ninety of these proteins showed differential levels of phosphorylation in tissues from MDD subjects compared to controls, being 20 differentially phosphorylated in at least 2 peptides. The majority of these phosphorylated proteins were associated with synaptic transmission and cellular architecture not only pointing out potential biomarker candidates but mainly shedding light to the comprehension of MDD pathobiology