Tagging genes with cassette-exchange sites

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles

Human Stem Cell-Derived Neurons: A System to Study Human Tau Function and Dysfunction

Background: Intracellular filamentous deposits containing microtubule-associated protein tau constitute a defining characteristic of many neurodegenerative disorders. Current experimental models to study tau pathology in vitro do not usually recapitulate the tau expression pattern characteristic of adult human brain. In this study, we have investigated whether human embryonic stem cell-derived neurons could be a good model to study human tau distribution, function and dysfunction. Methodology/Principal Findings: Using RT-PCR, immunohistochemistry, western blotting and cell transfections we have investigated whether all 6 adult human brain tau isoforms are expressed in neurons derived from human embryonic and fetal stem cells and whether 4 repeat tau over-expression alone, or with the F3 tau repeat fragment, (amino acid 258–380 of the 2N4R tau isoform with the DK280 mutation) affects tau distribution. We found that the shortest 3 repeat tau isoform, similarly to human brain, is the first to be expressed during neuronal differentiation while the other 5 tau isoforms are expressed later. Over expression of tau with 4 repeats affects tau cellular distribution and the short tau F3 fragment appears to increase tau phosphorylation but this effect does not appear to be toxic for the cell. Conclusions: Our results indicate that human embryonic stem cell-derived neurons express all 6 tau isoforms and are

Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson's disease model.

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD

Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development.This work was supported by CurePSP, The Cambridge Newton Trust, The William Scholl Foundation and the NIHR Cambridge Biomedical Research Centre (BRC). The contribution of the NC3Rs CRACK IT- Alzheimer’s Research UK is also acknowledged. S.A. holds a BBSRC studentship. A.G.-R. holds a Michael Foster Studentship. O.P. acknowledges BBSRC support. L.V. is supported by the ERC starting grant relieve-IMDs. S.O. and T.L. acknowledge support by the Irish Institute of Clinical Neuroscience. R.T.K. is supported by the Welcome Trust grant 091543/Z/10/Z and D.G. and M.dC.V.-H. are supported by Wellcome Trust grant #098051.This is the final version. It was first published by OUP at http://dx.doi.org/10.1093/brain/awv22

Disease-Modifying Therapies and Coronavirus Disease 2019 Severity in Multiple Sclerosis

Objective: This study was undertaken to assess the impact of immunosuppressive and immunomodulatory therapies on the severity of coronavirus disease 2019 (COVID-19) in people with multiple sclerosis (PwMS). Methods: We retrospectively collected data of PwMS with suspected or confirmed COVID-19. All the patients had complete follow-up to death or recovery. Severe COVID-19 was defined by a 3-level variable: mild disease not requiring hospitalization versus pneumonia or hospitalization versus intensive care unit (ICU) admission or death. We evaluated baseline characteristics and MS therapies associated with severe COVID-19 by multivariate and propensity score (PS)-weighted ordinal logistic models. Sensitivity analyses were run to confirm the results. Results: Of 844 PwMS with suspected (n = 565) or confirmed (n = 279) COVID-19, 13 (1.54%) died; 11 of them were in a progressive MS phase, and 8 were without any therapy. Thirty-eight (4.5%) were admitted to an ICU; 99 (11.7%) had radiologically documented pneumonia; 96 (11.4%) were hospitalized. After adjusting for region, age, sex, progressive MS course, Expanded Disability Status Scale, disease duration, body mass index, comorbidities, and recent methylprednisolone use, therapy with an anti-CD20 agent (ocrelizumab or rituximab) was significantly associated (odds ratio [OR] = 2.37, 95% confidence interval [CI] = 1.18-4.74, p = 0.015) with increased risk of severe COVID-19. Recent use (<1 month) of methylprednisolone was also associated with a worse outcome (OR = 5.24, 95% CI = 2.20-12.53, p = 0.001). Results were confirmed by the PS-weighted analysis and by all the sensitivity analyses. Interpretation: This study showed an acceptable level of safety of therapies with a broad array of mechanisms of action. However, some specific elements of risk emerged. These will need to be considered while the COVID-19 pandemic persists

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Examining Predictors of Different ABA Treatments: A Systematic Review

In the recent literature, there is a broad consensus on the effectiveness of Applied Behavior Analysis interventions for autism spectrum disorder (ASD). Despite their proven efficacy, research in clinical settings shows that these treatments are not equally effective for all children and the issue of which intervention should be chosen for an individual remains a common dilemma. The current work systematically reviewed studies on predictors and moderators of response to different types of evidence-based treatment for children with ASD. Specifically, our goal was to critically review the relationships between pre-treatment child characteristics and specific treatment outcomes, covering different aspects of functioning (i.e., social, communicative, adaptive, cognitive, motor, global functioning, play, and symptom severity). Our results questioned the binomial “better functioning-better outcome”, emphasizing the complex interplay between pre-treatment child characteristics and treatment outcomes. However, some pre-treatment variables seem to act as prerequisites for a specific treatment, and the issue of “what works for whom and why” remains challenging. Future research should focus on the definition of evidence-based decision-making models that capture those individual factors through which a specific intervention will exert its effects

F3 fragment induces tau hyperphosphorylation and redistribution.

<p><b>(A)</b> Immunostaining with AT8 antibody in hESC-derived neurons transfected at 35 DIV with the shorter 4R tau isoform (c–d) and empty vector (a–b). The over-expression of 4R tau isoform does not increase tau hyperphosphorylation. <b>(e–h)</b> Immunostaining with AT8 antibody in hESC-derived neurons co-transfected at 35 DIV with F3 fragment and 0N4R tau (g–h) and cells transfected only with F3 fragment (e–f). An increase in AT8 staining is present in cells transfected with only F3 (e,f) compared to controls (a,b). Neurons co-transfected with F3 and 0N4R show tau hyperphosphorylation and more dotted appearance in processes (g,h). <b>(B)</b> Immunostaining with pSer262 anti-tau antibody in hESC-derived neurons co-transfected with F3 fragment and 0N4R tau at 35 DIV: neurons co-transfected with F3 fragment and 0N4R (c–d) show an increase in pSer262 staining compared to control cells (a–b).</p

Tau expression in hESC-derived neurons after 72 days in culture.

<p><b>(A)</b> Immunostaining with anti-4R tau antibodies E10 (a–d) and RD4 (e–h) shows that 4R tau isoforms are present in neuronal cell bodies and axons. Both antibodies are 4R tau isoform-specific. <b>(B)</b> Tau isoform expression in hESC-derived neurons at 56 DIV. The immunoblot (human tau antibody, Dako) shows the tau isoforms before and after alkaline phosphatase (AP) treatment compared to human recombinant tau. Bands corresponding to the 6 tau isoforms can be seen and the shortest 3R tau isoform is the most abundant. De-phosphorylated tau extracted from adult human brain is also shown. The data are representative of at least three independent experiments. Similar results were obtained in both H9 and HuES cell lines.</p

Tau expression in adult brain biopsy:

<p><b>(A)</b> (a–d): immunocytochemistry for 3R tau (green), β-IIItubulin (red) and DAPI (blue) and <b>(B)</b> (e–h) for 4R tau (green), β-IIItubulin (red) and DAPI (blue) in primary culture of biopsy derived human adult neurons. Both isoforms co-localize with β-IIItubulin and are similarly expressed in neuronal cell bodies and axons.</p