14-3-3 mitigates alpha-synuclein aggregation and toxicity in the in vivo preformed fibril model

AbstractAlpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies that pathologically define a group of disorders known as synucleinopathies, including Parkinson’s Disease (PD) and Dementia with Lewy Bodies. αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal counts in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and reduction in NECAB1-positive neuron counts induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced reduction in tyrosine hydroxylase (TH)-positive dopaminergic cells in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and enhanced PFF-induced reduction in TH-positive dopaminergic cells. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.

Download Full-text

14-3-3 mitigates alpha-synuclein aggregation and toxicity in the in vivo preformed fibril model

10.1101/2020.11.17.387415 ◽

2020 ◽

Author(s):

Rachel Underwood ◽

Mary Gannon ◽

Aneesh Pathak ◽

Navya Kapa ◽

Sidhanth Chandra ◽

...

Keyword(s):

Social Dominance ◽

Lewy Bodies ◽

Neuronal Loss ◽

Cell Loss ◽

Brain Regions ◽

Alpha Synuclein ◽

Dopaminergic Cell ◽

The Impact

AbstractAlpha-synuclein (αsyn) is the key component of proteinaceous aggregates termed Lewy Bodies (LBs) that pathologically define a group of disorders known as synucleinopathies, including Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is hypothesized to misfold and spread throughout the brain in a prion-like fashion. Transmission of αsyn necessitates the release of misfolded αsyn from one cell and the uptake of that αsyn by another, in which it can template the misfolding of endogenous αsyn upon cell internalization. 14-3-3 proteins are a family of highly expressed brain proteins that are neuroprotective in multiple PD models. We have previously shown that 14-3-3θ acts as a chaperone to reduce αsyn aggregation, cell-to-cell transmission, and neurotoxicity in the in vitro pre-formed fibril (PFF) model. In this study, we expanded our studies to test the impact of 14-3-3s on αsyn toxicity in the in vivo αsyn PFF model. We used both transgenic expression models and adenovirus associated virus (AAV)-mediated expression to examine whether 14-3-3 manipulation impacts behavioral deficits, αsyn aggregation, and neuronal loss in the PFF model. 14-3-3θ transgene overexpression in cortical and amygdala regions rescued social dominance deficits induced by PFFs at 6 months post injection, whereas 14-3-3 inhibition by transgene expression of the competitive 14-3-3 peptide inhibitor difopein in the cortex and amygdala accelerated social dominance deficits. The behavioral rescue by 14-3-3θ overexpression was associated with delayed αsyn aggregation induced by PFFs in these brain regions. Conversely, 14-3-3 inhibition by difopein in the cortex and amygdala accelerated αsyn aggregation and cortical pyramidal neuron loss induced by PFFs. 14-3-3θ overexpression by AAV in the substantia nigra (SN) also delayed αsyn aggregation in the SN and partially rescued PFF-induced dopaminergic cell loss in the SN. 14-3-3 inhibition in the SN accelerated nigral αsyn aggregation and increased PFF-induced dopaminergic cell loss. These data indicate a neuroprotective role for 14-3-3θ against αsyn toxicity in vivo.

Download Full-text

Rab27 GTPases regulate alpha-synuclein uptake, cell-to-cell transmission, and toxicity

10.1101/2020.11.17.387449 ◽

2020 ◽

Author(s):

Rachel Underwood ◽

Bing Wang ◽

Aneesh Pathak ◽

Laura Volpicelli-Daley ◽

Talene A. Yacoubian

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Neuronal Loss ◽

Alpha Synuclein ◽

Rab Gtpases ◽

Double Knockout ◽

The Impact

SUMMARYParkinson’s disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein α-synuclein. α-Synuclein is hypothesized to have prion-like properties, by which misfolded α-synuclein induces the pathological aggregation of endogenous α-synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate α-synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic α-synuclein. Double knockout of both Rab27 isoforms eliminated α-synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary α-synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced α-synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled α-synuclein fibrils revealed that Rab27a/b knockout prevented α-synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of α-synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson’s disease and identifies Rab27s as key players in the progression of synucleinopathies.

Download Full-text

Modulation of the extrinsic cell death signaling pathway by viral Flip induces acute-death mediated liver failure

Cell Death and Disease ◽

10.1038/s41419-019-2115-y ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 1

Author(s):

Miriam Bittel ◽

Andreas E. Kremer ◽

Michael Stürzl ◽

Stefan Wirtz ◽

Iris Stolzer ◽

...

Keyword(s):

Cell Death ◽

Viral Infections ◽

Tissue Injury ◽

Multiple Organ ◽

Severe Liver ◽

Transgenic Expression ◽

Effector Caspase ◽

The Impact

AbstractDuring viral infections viruses express molecules that interfere with the host-cell death machinery and thus inhibit cell death responses. For example the viral FLIP (vFLIP) encoded by Kaposi’s sarcoma-associated herpesvirus interacts and inhibits the central cell death effector, Caspase-8. In order to analyze the impact of anti-apoptotic viral proteins, like vFlip, on liver physiology in vivo, mice expressing vFlip constitutively in hepatocytes (vFlipAlbCre+) were generated. Transgenic expression of vFlip caused severe liver tissue injury accompanied by massive hepatocellular necrosis and inflammation that finally culminated in early postnatal death of mice. On a molecular level, hepatocellular death was mediated by RIPK1-MLKL necroptosis driven by an autocrine TNF production. The loss of hepatocytes was accompanied by impaired bile acid production and disruption of the bile duct structure with impact on the liver-gut axis. Notably, embryonic development and tissue homeostasis were unaffected by vFlip expression. In summary our data uncovered that transgenic expression of vFlip can cause severe liver injury in mice, culminating in multiple organ insufficiency and death. These results demonstrate that viral cell death regulatory molecules exhibit different facets of activities beyond the inhibition of cell death that may merit more sophisticated in vitro and in vivo analysis.

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

Rab27b regulates the release, autophagic clearance, and toxicity of alpha-synuclein

10.1101/2020.03.09.984096 ◽

2020 ◽

Author(s):

Rachel Underwood ◽

Bing Wang ◽

Christine Carico ◽

Robert H. Whitaker ◽

William J. Placzek ◽

...

Keyword(s):

Lewy Bodies ◽

Alpha Synuclein ◽

Primary Component ◽

Size Exclusion ◽

Autophagic Flux ◽

Postmortem Human Brain ◽

Protein Levels ◽

Exclusion Chromatography ◽

The Impact

ABSTRACTAlpha synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy Bodies that pathologically define synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The release and uptake of αsyn from cell to cell are considered important processes for this prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance but has yet to be characterized in its role in αsyn spread. In this study, we used a paracrine αsyn in vitro model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. While Rab27b reduced αsyn release primarily through non-exosomal pathways, the αsyn released under KD conditions was of higher molecular weight species by size exclusion chromatography. Rab27b KD increased intracellular insoluble αsyn levels and led to an accumulation of endogenous LC3 positive puncta. Rab27b KD also decreased LC3 turnover with chloroquine treatment, indicating a defect in autophagic flux. Rab27b protein levels were increased in postmortem human brain lysates from PD and DLB subjects compared to healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and ultimately in the pathogenesis of synucleinopathies.

Download Full-text

Doxycycline inhibits α-synuclein-associated pathologies in vitro and in vivo

10.1101/2020.11.06.371229 ◽

2020 ◽

Author(s):

Antonio Dominguez-Meijide ◽

Valeria Parrales ◽

Eftychia Vasili ◽

Florencia González-Lizárraga ◽

Annekatrin König ◽

...

Keyword(s):

Lewy Bodies ◽

Alpha Synuclein ◽

Oxygen Species ◽

Additional Mechanism ◽

Neuroprotective Effects ◽

Free System ◽

Doxycycline Treatment ◽

A Cell

AbstractParkinson’s disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders characterized by the misfolding and aggregation of alpha-synuclein (aSyn). Doxycycline, a tetracyclic antibiotic shows neuroprotective effects, initially proposed to be due to its anti-inflammatory properties. More recently, an additional mechanism by which doxycycline may exert its neuroprotective effects has been proposed as it has been shown that it inhibits amyloid aggregation. Here, we studied the effects of doxycycline on aSyn aggregation in vivo, in vitro and in a cell free system using real-time quaking induced conversion (RT-QuiC). Our results show that doxycycline decreases the number and size of aSyn aggregates in cells. In addition, doxycycline inhibits the aggregation and seeding of recombinant aSyn, and attenuates the production of mitochondrial-derived reactive oxygen species. Finally, we found doxycycline induces a cellular redistribution of the aggregates in an animal model of PD that is associated with a recovery of dopaminergic function. In summary, we provide strong evidence that doxycycline treatment may be an effective strategy against synucleinopathies.

Download Full-text

Amyloid-Beta Peptides Trigger Aggregation of Alpha-Synuclein In Vitro

Molecules ◽

10.3390/molecules25030580 ◽

2020 ◽

Vol 25 (3) ◽

pp. 580 ◽

Cited By ~ 6

Author(s):

Janett Köppen ◽

Anja Schulze ◽

Lisa Machner ◽

Michael Wermann ◽

Rico Eichentopf ◽

...

Keyword(s):

Lewy Bodies ◽

Alpha Synuclein ◽

Amyloid Beta Peptides ◽

Amyloid Peptides ◽

Amyloid Deposits ◽

Transmission Electron ◽

Number Of Patients ◽

First Time

Alzheimer’s disease (AD) and Parkinson’s disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aβ(1–42) and pGlu-Aβ(3–42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aβ(1–42) and pGlu-Aβ(3–42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aβ in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aβ and pGlu-Aβ, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aβ species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.

Download Full-text

Criticality as a measure of developing proteinopathy in engineered human neural networks

10.1101/2020.05.03.074666 ◽

2020 ◽

Author(s):

Vibeke Devold Valderhaug ◽

Kristine Heiney ◽

Ola Huse Ramstad ◽

Geir Braathen ◽

Wei-Li Kuan ◽

...

Keyword(s):

Neural Networks ◽

Lewy Bodies ◽

Functional Trait ◽

Alpha Synuclein ◽

Network Activity ◽

Multielectrode Arrays ◽

Lewy Neurites ◽

Network Function

A patterned spread of proteinopathy represents a common characteristic of many neurodegenerative diseases. In Parkinson's disease (PD), misfolded forms of alpha-synuclein proteins aggregate and accumulate in hallmark pathological inclusions termed Lewy bodies and Lewy neurites, which seems to affect selectively vulnerable neuronal populations and propagate within interconnected neuronal networks. Research findings suggest that these proteinopathic inclusions are present at very early timepoints in disease development, even before strong behavioural symptoms of dysfunction arise, but that these underlying pathologies might be masked by homeostatic processes working to maintain the function of the degenerating neural circuits. This study investigates whether inducing the PD-related alpha-synuclein pathology in engineered human neural networks can be associated with changes in network function, and particularly with network criticality states. Self-organised criticality represents the critical point between resilience against perturbation and adaptational flexibility, which appears to be a functional trait in self-organising neural networks, both in vitro and in vivo. By monitoring the developing neural network activity through the use of multielectrode arrays (MEAs) for a period of three weeks following proteinopathy induction, we show that although this developing pathology is not clearly manifest in standard measurements of network function, it may be discerned by differences in network criticality states.

Download Full-text

A novel approach to evaluate alpha-synuclein seeding shows a wide heterogeneity in multiple system atrophy

10.1101/2021.08.10.455800 ◽

2021 ◽

Author(s):

Ivan Martinez-Valbuena ◽

Naomi P. Visanji ◽

Ain Kim ◽

Heather H. C. Lau ◽

Raphaella W. L. So ◽

...

Keyword(s):

Ionic Strength ◽

Multiple System Atrophy ◽

Parkinson Disease ◽

Real Time ◽

Brain Regions ◽

Alpha Synuclein ◽

Novel Approach ◽

Reliable Detection

Several in vitro and in vivo findings have consistently shown that alpha-synuclein derived from multiple system atrophy (MSA) subjects has more seeding capacity than Parkinson disease-derived alpha-synuclein. However, reliable detection of alpha-synuclein derived from MSA using seeded amplification assays, such as the Real-Time Quaking-induced Conversion, has remained challenging. Here we demonstrate that the interaction of the Thioflavin T dye with alpha-synuclein from MSA and Parkinson disease patients can be modulated by the type of salt, pH, and ionic strength used to generate strain-specific reaction buffers. Employing this novel approach, we have generated a streamlined Real-Time Quaking-induced Conversion assay capable of categorizing MSA brains according to their alpha-synuclein seeding behavior, and to unravel a previously unrecognized heterogeneity in seeding activity between different brain regions of a given individual that goes beyond immunohistochemical observations and provide a framework for future molecular subtyping of MSA.

Download Full-text