Characterization of Synphilin-1/alpha-Synuclein Double Transgenic Mice

Parkinson's disease (PD) is a sporadic and progressive neurodegenerative disease characterized by dopaminergic neuronal cell death in the substantia nigra pars compacta and by the presence of characteristic Lewy bodies (LB) and Lewy neuritic (LN) inclusions. These inclusions are mainly constituted of the alpha-synuclein (a-syn) protein, suggesting a role of a-syn in the neuropathophysiology of PD. Synphilin-1 (sph1) is described as an interacting partner of a-syn, present in LBs and sharing the same intracellular compartments as a-syn. Sph1 was shown to induce the formation of a-syn positive inclusions in vivo and in vitro as well as to promote clearance of protein inclusions by autophagy. But the impact of sph1 in a-syn-mediated toxicity is inconsistent, presumably due to the variety of models used to model synucleinopathy, the variety of toxicity markers analyzed and the poor description of aggregates found in these studies. Furthermore, the normal function of sph1 needs still to be explored. The overall aim of this study is to characterize the effects of sph1 on a-syn aggregation and on the development of synucleinopathy in vivo. Therefore, we cross-bred mice overexpressing the human sph1 under the mouse PrP promoter with a synucleinopathy mouse model overexpressing the human A30P mutated a-syn under the Thy-1 promoter. Using different fractionation protocols of brain lysates, we did not observe in mice coexpressing sph1 an increased amount of soluble a-syn nor detergent-insoluble a-syn. At the histological level, we observed that the coexpression of sph1 drastically decreased levels of proteinase K resistant a-syn inclusions as well as thioflavin S positive structures. These changes were accompanied by the formation of aggresome-like structures positive for a-syn, K63 polyubiquitinated proteins and gamma-tubulin in double transgenic animals. We also observed in double transgenic mice a reduced neuropathology reflected by a decreased neuroinflammation and suggesting a protective role of sph1 in synucleinopathy. Accordingly, we demonstrated that sph1 reduces the accumulation of soluble full-length and truncated species of a-syn with aging. Similarly, we also shown that sph1 decreases levels of fibrillar forms of a-syn. Moreover, we also reported increased levels of ubiquitinated proteins in double transgenic mice without measuring an impairment of the ubiquitin-proteasome system activity. Interestingly, we observed an increased autophagic flux in double transgenic mice, suggesting a possible role of the autophagy-lysosome pathway in the reduction of a-syn species. We tracked the evolution of neuropathology and associated behavioral alterations over 18 months. We found that sph1 coexpression in transgenic mice overexpressing the human mutated A30P alpha-synuclein led to a delayed motor phenotype in rotarod when compared to single transgenic mice overexpressing A30P a-syn. A motor phenotype was also observed in single transgenic sph1 mice when using challenging beam walk. Interestingly, mouse gait analysis suggested a different origin in motor impairment in sph1 and A30P single transgenic mice

rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants. Conclusions In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7

Endogenous but not sensory-driven activity controls migration, morphogenesis and survival of adult-born juxtaglomerular neurons in the mouse olfactory bulb.

The development and survival of adult-born neurons are believed to be driven by sensory signaling. Here, in vivo analyses of motility, morphology and Ca2+ signaling, as well as transcriptome analyses of adult-born juxtaglomerular cells with reduced endogenous excitability (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), revealed a pronounced impairment of migration, morphogenesis, survival, and functional integration of these cells into the mouse olfactory bulb, accompanied by a reduction in cytosolic Ca2+ fluctuations, phosphorylation of CREB and pCREB-mediated gene expression. Moreover, K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in an immature and vulnerable state. Surprisingly, cells deprived of sensory-driven activity developed normally. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as enhanced Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons

Distinct Stress Response and Altered Striatal Transcriptome in Alpha-Synuclein Overexpressing Mice

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with motor symptoms and a plethora of non-motor and neuropsychiatric features that accompany the disease from prodromal to advanced stages. While several genetic defects have been identified in familial forms of PD, the predominance of cases are sporadic and result from a complex interplay of genetic and non-genetic factors. Clinical evidence, moreover, indicates a role of environmental stress in PD, supported by analogies between stress-induced pathological consequences and neuronal deterioration observed in PD. From this perspective, we set out to investigate the effects of chronic stress exposure in the context of PD by using a genetic mouse model that overexpresses human wildtype SNCA. Mimicking chronic stress was achieved by adapting a chronic unpredictable mild stress protocol (CUMS) comprising eight different stressors that were applied randomly over a period of eight weeks starting at an age of four months. A distinctive stress response with an impact on anxiety-related behavior was observed upon SNCA overexpression and CUMS exposure. SNCA-overexpressing mice showed prolonged elevation of cortisol metabolites during CUMS exposure, altered anxiety-related traits, and declined motor skills surfacing with advanced age. To relate our phenotypic observations to molecular events, we profiled the striatal and hippocampal transcriptome and used a 2 × 2 factorial design opposing genotype and environment to determine differentially expressed genes. Disturbed striatal gene expression and minor hippocampal gene expression changes were observed in SNCA-overexpressing mice at six months of age. Irrespective of the CUMS-exposure, genes attributed to the terms neuroinflammation, Parkinson’s signaling, and plasticity of synapses were altered in the striatum of SNCA-overexpressing mice

Comprehensive phenotyping revealed transient startle response reduction and histopathological gadolinium localization to perineuronal nets after gadodiamide administration in rats

Gadolinium based contrast agents (GBCAs) are widely used in clinical MRI since the mid-1980s. Recently, concerns have been raised that trace amounts of Gadolinium (Gd), detected in brains even long time after GBCA application, may cause yet unrecognized clinical consequences. We therefore assessed the behavioral phenotype, neuro-histopathology, and Gd localization after repeated administration of linear (gadodiamide) or macrocyclic (gadobutrol) GBCA in rats. While most behavioral tests revealed no difference between treatment groups, we observed a transient and reversible decrease of the startle reflex after gadodiamide application. Residual Gd in the lateral cerebellar nucleus was neither associated with a general gene expression pathway deregulation nor with neuronal cell loss, but in gadodiamide-treated rats Gd was associated with the perineuronal net protein aggrecan and segregated to high molecular weight fractions. Our behavioral finding together with Gd distribution and speciation support a substance class difference for Gd presence in the brain after GBCA application

Accurate long-read sequencing identified GBA1 as major risk factor in the Luxembourgish Parkinson's study.

peer reviewedHeterozygous variants in the glucocerebrosidase GBA1 gene are an increasingly recognized risk factor for Parkinson's disease (PD). Due to the GBAP1 pseudogene, which shares 96% sequence homology with the GBA1 coding region, accurate variant calling by array-based or short-read sequencing methods remains a major challenge in understanding the genetic landscape of GBA1-associated PD. We analyzed 660 patients with PD, 100 patients with Parkinsonism and 808 healthy controls from the Luxembourg Parkinson's study, sequenced using amplicon-based long-read DNA sequencing technology. We found that 12.1% (77/637) of PD patients carried GBA1 variants, with 10.5% (67/637) of them carrying known pathogenic variants (including severe, mild, risk variants). In comparison, 5% (34/675) of the healthy controls carried GBA1 variants, and among them, 4.3% (29/675) were identified as pathogenic variant carriers. We found four GBA1 variants in patients with atypical parkinsonism. Pathogenic GBA1 variants were 2.6-fold more frequently observed in PD patients compared to controls (OR = 2.6; CI = [1.6,4.1]). Three novel variants of unknown significance (VUS) were identified. Using a structure-based approach, we defined a potential risk prediction method for VUS. This study describes the full landscape of GBA1-related parkinsonism in Luxembourg, showing a high prevalence of GBA1 variants as the major genetic risk for PD. Although the long-read DNA sequencing technique used in our study may be limited in its effectiveness to detect potential structural variants, our approach provides an important advancement for highly accurate GBA1 variant calling, which is essential for providing access to emerging causative therapies for GBA1 carriers.R-AGR-0592 - FNR - NCER-PD Phase II Coordination (01/06/2015 - 30/11/2023) - KRÜGER Rejko3. Good health and well-bein

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Single hadron response measurement and calorimeter jet energy scale uncertainty with the ATLAS detector at the LHC

The uncertainty on the calorimeter energy response to jets of particles is derived for the ATLAS experiment at the Large Hadron Collider (LHC). First, the calorimeter response to single isolated charged hadrons is measured and compared to the Monte Carlo simulation using proton-proton collisions at centre-of-mass energies of sqrt(s) = 900 GeV and 7 TeV collected during 2009 and 2010. Then, using the decay of K_s and Lambda particles, the calorimeter response to specific types of particles (positively and negatively charged pions, protons, and anti-protons) is measured and compared to the Monte Carlo predictions. Finally, the jet energy scale uncertainty is determined by propagating the response uncertainty for single charged and neutral particles to jets. The response uncertainty is 2-5% for central isolated hadrons and 1-3% for the final calorimeter jet energy scale.Comment: 24 pages plus author list (36 pages total), 23 figures, 1 table, submitted to European Physical Journal