A double-hit in vivo model of GBA viral microRNA-mediated downregulation and human alpha-synuclein overexpression demonstrates nigrostriatal degeneration

Preclinical and clinical studies support a strong association between mutations in the GBA1 gene that encodes beta-glucocerebrosidase (GCase) (EC 3.2.1.45; glucosylceramidase beta) and Parkinsons disease (PD). Alpha-synuclein (AS), a key player in PD pathogenesis, and GBA1 mutations may independently and synergistically cause lysosomal dysfunction and thus, embody clinically well-validated targets of the neurodegenerative disease process in PD. However, double-hit in vivo models, recapitulating pathological features of PD that can be used to dissect the nature of the complex relationship between GCase and AS on the nigrostriatal axis, the region particularly vulnerable in PD, are direly needed. To address this, we implemented a bidirectional approach in mice to examine the effects of: 1) GCase overexpression (wildtype and mutant N370S) on endogenous AS levels and 2) downregulation of endogenous GCase combined with AS overexpression. Striatal delivery of viral-mediated GCase overexpression revealed minimal effects on cortical and nigrostriatal AS tissue levels and no significant effect on dopaminergic system integrity. On the other hand, microRNA (miR)-mediated GBA1 downregulation (miR GBA), combined with virus-mediated human AS overexpression (+AS), yields decreased GCase activity in the cortex, mimicking levels seen in GBA1 heterozygous carriers (30-40%), increased astrogliosis and microgliosis, decreased striatal dopamine levels (50% compared to controls) and loss of nigral dopaminergic neurons (~33%)- effects that were all reversible with miR rescue. Most importantly, the synergistic neurodegeneration of miR GBA+AS correlated with augmented AS accumulation and extracellular release in the striatum. Collectively, our results suggest that GCase downregulation alone is not sufficient to recapitulate key pathological features of PD in vivo, but its synergistic interplay with AS, via increased AS levels and release, drives nigrostriatal neurodegeneration. Furthermore, we demonstrate a novel double-hit model that can be used to identify putative mechanisms driving PD pathophysiology and can be subsequently used to test novel therapeutic approaches.

Download Full-text

A double-hit in vivo model of GBA viral microRNA-mediated downregulation and human alpha-synuclein overexpression demonstrates nigrostriatal degeneration

10.21203/rs.3.rs-721944/v1 ◽

2021 ◽

Author(s):

Alexia Polissidis ◽

Georgia Nikolopoulou ◽

Effrosyni Koronaiou ◽

Maria Nikatou ◽

Catherine Viel ◽

...

Keyword(s):

Strong Association ◽

Disease Process ◽

Alpha Synuclein ◽

In Vivo Model ◽

Pathological Features ◽

In Vivo Models ◽

System Integrity ◽

Viral Microrna ◽

Double Hit

Abstract Background: Preclinical and clinical studies support a strong association between mutations in the GBA1 gene that encodes beta-glucocerebrosidase (GCase) (EC 3.2.1.45; glucosylceramidase beta) and Parkinson’s disease (PD). Alpha-synuclein (AS), a key player in PD pathogenesis, and GBA1 mutations may independently and synergistically cause lysosomal dysfunction and thus, embody clinically well-validated targets of the neurodegenerative disease process in PD. However, double-hit in vivo models, recapitulating pathological features of PD that can be used to dissect the nature of the complex relationship between GCase and AS on the nigrostriatal axis, the region particularly vulnerable in PD, are direly needed. Methods: To address this, we implemented a bidirectional approach in mice to examine the effects of: 1) GCase overexpression (wild-type and mutant N370S) on endogenous AS levels and 2) downregulation of endogenous GCase combined with AS overexpression. Results: Striatal delivery of viral-mediated GCase overexpression revealed minimal effects on cortical and nigrostriatal AS tissue levels and no significant effect on dopaminergic system integrity. On the other hand, microRNA (miR)-mediated GBA1 downregulation (miR GBA), combined with virus-mediated human AS overexpression (+AS), yields decreased GCase activity in the cortex, mimicking levels seen in GBA1 heterozygous carriers (30-40%), increased astrogliosis and microgliosis, decreased striatal dopamine levels (50% compared to controls) and loss of nigral dopaminergic neurons (~33%)- effects that were all reversible with miR rescue. Most importantly, the synergistic neurodegeneration of miR GBA+AS correlated with augmented AS accumulation and extracellular release in the striatum. Conclusion: Collectively, our results suggest that GCase downregulation alone is not sufficient to recapitulate key pathological features of PD in vivo, but its synergistic interplay with AS, via increased AS levels and release, drives nigrostriatal neurodegeneration. Furthermore, we demonstrate a novel double-hit model that can be used to identify putative mechanisms driving PD pathophysiology and can be subsequently used to test novel therapeutic approaches.

Download Full-text

A Summary of Phenotypes Observed in the In Vivo Rodent Alpha-Synuclein Preformed Fibril Model

Journal of Parkinson s Disease ◽

10.3233/jpd-212847 ◽

2021 ◽

pp. 1-13

Author(s):

Nicole K. Polinski

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Study Design ◽

Alpha Synuclein ◽

Therapeutic Strategies ◽

Rodent Model ◽

In Vivo Model ◽

Nigrostriatal Degeneration ◽

The Brain

The use of wildtype recombinant alpha-synuclein preformed fibrils (aSyn PFFs) to induce endogenous alpha-synuclein to form pathological phosphorylation and trigger neurodegeneration is a popular model for studying Parkinson’s disease (PD) biology and testing therapeutic strategies. The strengths of this model lie in its ability torecapitulatethe phosphorylation/aggregationof aSyn and nigrostriatal degeneration seen in PD, as well as its suitability for studying the progressive nature of PD and the spread of aSyn pathology. Although the model is commonly used and has been adopted by many labs, variability in observed phenotypes exists. Here we provide summaries of the study design and reported phenotypes frompublished reports characterizing the aSyn PFF in vivo model in rodents following injection into the brain, gut, muscle, vein, peritoneum, and eye. These summariesare designed to facilitate an introduction to the use of aSyn PFFs to generate a rodent model of PD—highlighting phenotypes observed in papers that set out to thoroughly characterize the model.This information will hopefully improve the understanding of this model and clarify when the aSyn PFF model may be an appropriate choice for one’s research.

Download Full-text

Alpha-Synuclein Aggregation Induced by Brief Ischemia Negatively Impacts Neuronal Survival in vivo: A Study in [A30P]alpha-Synuclein Transgenic Mouse

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2010.170 ◽

2010 ◽

Vol 31 (3) ◽

pp. 913-923 ◽

Cited By ~ 24

Author(s):

Isin Unal-Cevik ◽

Yasemin Gursoy-Ozdemir ◽

Muge Yemisci ◽

Sevda Lule ◽

Gunfer Gurer ◽

...

Keyword(s):

Transgenic Mice ◽

Neuronal Survival ◽

Alpha Synuclein ◽

In Vivo Model ◽

Neuronal Necrosis ◽

Mca Occlusion ◽

Nitrative Stress ◽

The Impact

Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic.

Download Full-text

Panel Discussion II: Usefulness of in vitro and in vivo Model Systems for Predicting the Adverse Public Health Outcome for Antimicrobial Drug Residues in Food

Microbial Ecology in Health and Disease ◽

10.1080/08910600050216174 ◽

2000 ◽

Vol 12 (3) ◽

pp. 45-53

Author(s):

Andrew Beaulieu ◽

Jacques Boisseau ◽

Carl Cerniglia ◽

Denis Corpet ◽

A. Haydée Fernández ◽

...

Keyword(s):

Public Health ◽

Health Outcome ◽

Panel Discussion ◽

Antimicrobial Drug ◽

Model Systems ◽

In Vivo Model ◽

Drug Residues ◽

Public Health Outcome

Download Full-text

P1735 Arginine supplementation and oxidative processes: in vivo model

European Heart Journal ◽

10.1016/s0195-668x(03)94873-1 ◽

2003 ◽

Vol 24 (5) ◽

pp. 330

Author(s):

A KOPFF

Keyword(s):

In Vivo Model ◽

Oxidative Processes ◽

Arginine Supplementation

Download Full-text

Establishment of an in vivo model for KCNJ5 dependent hyperaldosteronism

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/s-0035-1547718 ◽

2015 ◽

Vol 122 (03) ◽

Cited By ~ 2

Author(s):

U Lichtenauer ◽

PL Schmid ◽

A Oßwald ◽

I Renner-Müller ◽

M Reincke ◽

...

Keyword(s):

In Vivo Model

Download Full-text

An in vivo Model for the Assessment of Acute Fibrinolytic Capacity of the Endothelium

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657722 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1242-1248 ◽

Cited By ~ 68

Author(s):

David E Newby ◽

Robert A Wright ◽

Christopher A Ludlam ◽

Keith A A Fox ◽

Nicholas A Boon ◽

...

Keyword(s):

Blood Flow ◽

Substance P ◽

Sodium Nitroprusside ◽

Plasma Concentrations ◽

In Vivo Model ◽

Forearm Circulation ◽

Von Willebrand ◽

Local Release ◽

Willebrand Factor

SummaryThe effects on blood flow and plasma fibrinolytic and coagulation parameters of intraarterial substance P, an endothelium dependent vasodilator, and sodium nitroprusside, a control endothelium independent vasodilator, were studied in the human forearm circulation. At subsystemic locally active doses, both substance P (2-8 pmol/min) and sodium nitroprusside (2-8 μg/min) caused dose-dependent vasodilatation (p <0.001 for both) without affecting plasma concentrations of PAI-1, von Willebrand factor antigen or factor VIII:C activity. Substance P caused local increases in t-PA antigen and activity (p <0.001) in the infused arm while sodium nitroprusside did not. At higher doses, substance P increased blood flow and t-PA concentrations in the noninfused arm. We conclude that brief, locally active and subsystemic infusions of intraarterial substance P cause a rapid and substantial local release of t-PA which appear to act via a flow and nitric oxide independent mechanism. This model should provide a useful and selective method of assessing the in vivo capacity of the forearm endothelium to release t-PA acutely.

Download Full-text