Pathogenesis of Parkinson's Disease

Podcast

Play an AI-generated podcast conversation about this lesson

Questions and Answers

Which of the following is true about the α-syn-carrying EVs?

They are isolated from red blood cell-derived extracellular vesicles (RBC-EVs)

They are found in both CSF and brains of Parkinson's disease patients

They can promote the formation of α-syn aggregates when introduced into mouse brains

All of the above (correct)

What is the purpose of shaking the flasks containing confluent astrocyte cultures?

To ensure even distribution of cells

To remove microglia from the culture (correct)

To remove debris from the culture

To stimulate cell growth

What is the role of trypsin EDTA in the astrocyte culture preparation?

To promote cell adhesion

To remove unwanted proteins from the culture medium

To separate cells from the flask surface (correct)

To stimulate cell division

What is the approximate concentration of cells in the final suspension after centrifugation?

1 x 10^5 cells/mL (B) Signup and view all the answers

Which of the following is NOT a component of the neuronal dissection media?

Trypsin EDTA (B) Signup and view all the answers

What is the primary purpose of deparaffinizing the tissue sections in human postmortem tissue staining?

To remove the paraffin wax allowing for antibody penetration (B) Signup and view all the answers

What is the role of citric acid during the human postmortem tissue staining?

To promote antigen retrieval by exposing epitopes (B) Signup and view all the answers

What is the purpose of blocking the tissue with normal goat serum (NGS) during immunostaining?

To prevent non-specific binding of antibodies to the tissue (B) Signup and view all the answers

What is the role of GFAP (glial fibrillary acidic protein) in astrocytes?

GFAP is a structural protein that helps maintain the shape and integrity of astrocytes (D) Signup and view all the answers

How does the researchers' observation of oligomeric α-syn co-localization with GFAP in astrocytic processes differ between animal models and human Parkinson's disease?

In human Parkinson's disease, oligomeric α-syn co-localizes with GFAP in astrocyte somata, while in the animal model, it primarily localizes to astrocytic processes. (F) Signup and view all the answers

What is one limitation cited by the authors of the studies regarding LPS-induced systemic inflammation?

LPS-induced systemic inflammation may influence the behavior of microglia, potentially altering the results. (D) Signup and view all the answers

What specific cell structures are being investigated in the research regarding the uptake of RBC-EVs?

Astrocytic processes (A) Signup and view all the answers

What is the evidence suggesting that RBC-EVs are taken up by astrocyte processes?

RBC-EVs were found in close proximity to astrocyte processes, sometimes appearing to surround them. (C) Signup and view all the answers

What specific brain regions show an increase in oligomeric α-syn in GFAP+ astrocytic processes in Parkinson's disease patients?

Cortex, striatum, and cerebellum. (D) Signup and view all the answers

What cellular markers are used to identify astrocytes, neurons, and microglia in the research?

GFAP for astrocytes, MAP2 for neurons, and Iba1 for microglia. (E) Signup and view all the answers

What does the observation of increased oligomeric α-syn in GFAP+ astrocytic processes of Parkinson's disease patients suggest?

RBC-EV-contained oligomeric α-syn is likely promoting the formation of brain aggregates in astrocytes. (F) Signup and view all the answers

Why was it difficult to classify RBC-EVs within a specific cell type in the brain parenchyma?

Their signal did not co-localize with commonly used cell-type markers in the study. (B), Their signal did not co-localize with commonly used cell-type markers in the study. (C), Their signal did not co-localize with commonly used cell-type markers in the study. (F) Signup and view all the answers

What was the purpose of treating cultured astrocytes with RBC-EVs at 37 °C for 3 hours?

To prepare the cells for immunoassays (B) Signup and view all the answers

Which antibody was used to detect α-syn in the Proximity Ligation assay?

Anti-α-syn clone 42 (A) Signup and view all the answers

What was included in the blocking solution used in the Proximity Ligation assay?

5% donkey serum and 2% BSA (A) Signup and view all the answers

Which step follows the washing of the plate three times using the Washing Buffer?

Addition of sulfo-TAG-labelled anti-α-syn (B) Signup and view all the answers

What temperature was the incubation with primary antibodies conducted at?

4 °C (B) Signup and view all the answers

In which kit were secondary antibodies conjugated with oligonucleotides used?

Duolink II fluorescence kit (C) Signup and view all the answers

How was the visualization of immunoreactive bands achieved during the assay?

Using ECL reagents (C) Signup and view all the answers

What role does blocking solution serve in the immunoassay?

To allow antibodies to bind without interference (D) Signup and view all the answers

What is the significance of oligomeric α-synuclein in red blood cells (RBCs) according to the study?

It may be a key factor in Parkinson's disease development. (B) Signup and view all the answers

What was observed about RBC-EVs' effect on astrocytes in the presence of glutamate?

They impair the ability of astrocytes to protect neurons. (D) Signup and view all the answers

How does α-synuclein transmission potentially occur from the periphery to the brain?

Via blood circulation. (D) Signup and view all the answers

What type of gel was used for electrophoresis in the analysis?

4–15% Criterion™ TGX Stain-Free™ Protein Gel (B) Signup and view all the answers

Which regions of the brain were analyzed for levels of oligomeric α-syn after chronic RBC-EV injection?

Cortex, striatum, midbrain, and cerebellum. (D) Signup and view all the answers

What analytical method was used to quantify fluorescence intensity of synaptophysin labeling in brain regions?

One-way ANOVA. (A) Signup and view all the answers

What was used to probe the membrane after transferring proteins?

Primary antibodies (D) Signup and view all the answers

What does the quantification analysis of synaptophysin puncta indicate about the synaptic health of neurons?

Decreased puncta indicate potential synaptic dysfunction. (C) Signup and view all the answers

Which of the following antibodies was NOT mentioned in the immunoprecipitation process?

Published anti-GFAP (A) Signup and view all the answers

What key statistical significance threshold was indicated in the study's analyses?

p < 0.0001. (C) Signup and view all the answers

What was the purpose of the control nonimmune rabbit immunoglobulins (IgG)?

To serve as a negative control (D) Signup and view all the answers

At what temperature and duration were the primary antibodies incubated?

4 °C overnight (B) Signup and view all the answers

In which context is the role of RBC-EVs particularly examined in the research?

Pathological processes related to Parkinson's disease. (B) Signup and view all the answers

What does EAAT2 stand for?

Excitatory amino acid transporter 2 (D) Signup and view all the answers

Which of the following tissues was co-labeled with EAAT2 in the representative images?

Substantia nigra (C) Signup and view all the answers

What is the role of horseradish peroxidase (HRP) in the analysis?

To enable the detection of bound antibodies (D) Signup and view all the answers

Study Notes

Parkinson's Disease Pathogenesis

Parkinson's disease is a neurodegenerative disorder, characterized by the transmission and accumulation of toxic a-synuclein (a-syn) species.
Extracellular vesicles (EVs) are implicated in spreading toxic a-syn species.
Erythrocytes contain significant amounts of a-syn, and erythrocyte-derived EVs from Parkinson's disease patients carry elevated levels of oligomeric a-syn compared to healthy controls.
Erythrocyte-derived EVs readily cross the blood-brain barrier (BBB) and accumulate in astrocyte endfeet.
Accumulation in astrocytic endfeet impairs glutamate uptake, potentially through interaction with the excitatory amino acid transporter 2 (EAAT2) and oligomeric a-syn.
These observations suggest a role for erythrocyte-derived EVs and oligomeric a-syn in Parkinson's disease progression/initiation.

Astrocyte Dysfunction

Astrocytes are important for glutamate homeostasis, and dysfunction can lead to neuronal excitotoxicity.
Astrocytic dysfunction is observed in Parkinson's disease and is associated with BBB (blood-brain barrier) dysfunction.
The relationship between astrocyte dysfunction and a-syn pathology is unclear.
Astrocytes express less a-syn than neurons.
Yet, A-syn positive inclusions are found in processes of astrocytes, particularly in Parkinson's disease.

Blood-Brain Barrier (BBB) Permeability

BBB dysfunction is an early event in Parkinson's disease pathogenesis, observed in transgenic mice expressing human mutant A53T a-syn.
RBC-EVs can cross the BBB into the CNS in A53T mice even in the absence of LPS pre-treatment.
Oligomeric a-syn in RBC-EVs from Parkinson's disease patients is ~30% higher than in healthy control subjects.
Chronic administration of RBC-EVs containing oligomeric a-syn into A53T mice increased a-syn accumulation in the brain.

Oligomeric a-Synuclein and Astrocytes

Astrocytes readily internalize RBC-EVs in the periphery and accumulate oligomeric a-syn in processes.
RBC-EVs increase astrocytic accumulation of oligomeric a-syn in the cortex, striatum, midbrain, and cerebellum of A53T mice.
Astrocyte internalization of RBC-EVs containing oligomeric α-syn may cause dysfunction in glutamate clearance via interaction with EAAT2.
RBC-EVs impair astrocytic protection of neurons from glutamate excitotoxicity in a transwell co-culture model.
Reduced synaptophysin levels in the striatum of mice injected with Parkinson's disease RBC-EVs were observed.
Increased levels of oligomeric a-syn within synaptosomes of the striatum were observed in mice injected with Parkinson's disease RBC-EVs.
EAAT2 and oligomeric a-syn are observed in close proximity in Parkinson's disease brains.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

Description

This quiz explores the intricate pathogenesis of Parkinson's disease, focusing on the role of extracellular vesicles and a-synuclein in the disease's progression. It highlights the impact of erythrocyte-derived vesicles on astrocyte function and glutamate homeostasis, emphasizing how these processes contribute to neurodegeneration.