Designing stem-cell-based dopamine cell replacement trials for Parkinson's disease

Clinical studies of Parkinson’s disease (PD) using a dopamine cell replacment strategy have been tried for more than 30 years. The outcomes following transplantation of human fetal ventral mesencephalic tissue (hfVM) have been variable, with some patients coming off their anti-PD treatment for many years and others not responding and/or developing significant side effects, including graft-induced dyskinesia. This led to a re-appraisal of the best way to do such trials, which resulted in a new European-Union-funded allograft trial with fetal dopamine cells across several centers in Europe. This new trial, TRANSEURO (NCT01898390), is an open-label study in which some individuals in a large observational cohort of patients with mild PD who were undergoing identical assessments were randomly selected to receive transplants of hfVM. The TRANSEURO trial is currently ongoing as researchers have completed both recruitment into a large multicenter observational study of younger onset early-stage PD and transplantation of hfVM in 11 patients. While completion of TRANSEURO is not expected until 2021, we feel that sharing the rationale for the design of TRANSEURO, along with the lessons we have learned along the way, can help inform researchers and facilitate planning of transplants of dopamine-producing cells derived from human pluripotent stem cells for future clinical trials

Role of Alpha-Synuclein Protein Levels in Mitochondrial Morphology and Cell Survival in Cell Lines

α-Synuclein is highly associated with some neurodegeneration and malignancies. Overexpressing wild-type or mutant α-synuclein promotes neuronal death by mitochondrial dysfunction, the underlying mechanisms of which remain poorly defined. It was recently reported that α-synuclein expression could directly lead to mitochondrial fragmentation in vitro and in vivo, which may be due to α-synuclein localization on mitochondria. Here, we applied a double staining method to demonstrate mitochondrial morphogenetic changes in cells overexpressed with α-synuclein. We show that mitochondrial localization of α-synuclein was increased following its overexpression in three distinct cell lines, including HeLa, SH-SY5Y, and PC12 cells, but no alteration in mitochondrial morphology was detected. However, α-synuclein knockdown prevents MPP+-induced mitochondrial fragmentation in SH-SY5Y and PC12 cells. These data suggest that α-synuclein protein levels hardly affect mitochondrial morphology in normal cell lines, but may have some influence on that under certain environmental conditions

A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications

Amyloid-β Triggers the Release of Neuronal Hexokinase 1 from Mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD

Transplantation of Neuronal-Primed Human Bone Marrow Mesenchymal Stem Cells in Hemiparkinsonian Rodents

Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment

Observation of the Decay Λ0b→Λ+cτ−¯ν

The first observation of the semileptonic b-baryon decay Λb0→Λc+τ-ν¯τ, with a significance of 6.1σ, is reported using a data sample corresponding to 3 fb-1 of integrated luminosity, collected by the LHCb experiment at center-of-mass energies of 7 and 8 TeV at the LHC. The τ- lepton is reconstructed in the hadronic decay to three charged pions. The ratio K=B(Λb0→Λc+τ-ν¯τ)/B(Λb0→Λc+π-π+π-) is measured to be 2.46±0.27±0.40, where the first uncertainty is statistical and the second systematic. The branching fraction B(Λb0→Λc+τ-ν¯τ)=(1.50±0.16±0.25±0.23)% is obtained, where the third uncertainty is from the external branching fraction of the normalization channel Λb0→Λc+π-π+π-. The ratio of semileptonic branching fractions R(Λc+)B(Λb0→Λc+τ-ν¯τ)/B(Λb0→Λc+μ-ν¯μ) is derived to be 0.242±0.026±0.040±0.059, where the external branching fraction uncertainty from the channel Λb0→Λc+μ-ν¯μ contributes to the last term. This result is in agreement with the standard model prediction

Studies of η\eta and η′\eta' production in pppp and ppPb collisions

The production of $\eta$ and $\eta'$ mesons is studied in proton-proton and proton-lead collisions collected with the LHCb detector. Proton-proton collisions are studied at center-of-mass energies of $5.02$ and $13~{\rm TeV}$, and proton-lead collisions are studied at a center-of-mass energy per nucleon of $8.16~{\rm TeV}$. The studies are performed in center-of-mass rapidity regions $2.5<y_{\rm c.m.}<3.5$ (forward rapidity) and $-4.0<y_{\rm c.m.}<-3.0$ (backward rapidity) defined relative to the proton beam direction. The $\eta$ and $\eta'$ production cross sections are measured differentially as a function of transverse momentum for $1.5<p_{\rm T}<10~{\rm GeV}$ and $3<p_{\rm T}<10~{\rm GeV}$, respectively. The differential cross sections are used to calculate nuclear modification factors. The nuclear modification factors for $\eta$ and $\eta'$ mesons agree at both forward and backward rapidity, showing no significant evidence of mass dependence. The differential cross sections of $\eta$ mesons are also used to calculate $\eta/\pi^0$ cross section ratios, which show evidence of a deviation from the world average. These studies offer new constraints on mass-dependent nuclear effects in heavy-ion collisions, as well as $\eta$ and $\eta'$ meson fragmentation.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://lhcbproject.web.cern.ch/Publications/p/LHCb-PAPER-2023-030.html (LHCb public pages

A study of CP violation in the decays B±→[K+K-π+π-]Dh± (h= K, π) and B±→[π+π-π+π-]Dh±

The first study of CP violation in the decay mode B±→[K+K-π+π-]Dh± , with h= K, π , is presented, exploiting a data sample of proton–proton collisions collected by the LHCb experiment that corresponds to an integrated luminosity of 9 \,fb - 1 . The analysis is performed in bins of phase space, which are optimised for sensitivity to local CP asymmetries. CP -violating observables that are sensitive to the angle γ of the Unitarity Triangle are determined. The analysis requires external information on charm-decay parameters, which are currently taken from an amplitude analysis of LHCb data, but can be updated in the future when direct measurements become available. Measurements are also performed of phase-space integrated observables for B±→[K+K-π+π-]Dh± and B±→[π+π-π+π-]Dh± decays