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Structural Studies of Human Brain in Neurodegeneration

English title Structural Studies of Human Brain in Neurodegeneration
Applicant Stahlberg Henning
Number 188548
Funding scheme Project funding (Div. I-III)
Research institution Laboratoire de microscopie électronique biologique EPFL - SB - IPHYS - LBEM
Institution of higher education EPF Lausanne - EPFL
Main discipline Structural Research
Start/End 01.08.2020 - 31.07.2024
Approved amount 1'344'000.00
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All Disciplines (3)

Discipline
Structural Research
Neurophysiology and Brain Research
Biophysics

Keywords (5)

Electron Microscopy; CLEM; Parkinson's Disease; Neurodegeneration; alpha-synuclein

Lay Summary (German)

Lead
In diesem Projekt werden wir die molekularen und zellulären Ursachen und Vorgänge im menschlichen Gehirn in der Parkinson’schen Krankheit untersuchen.
Lay summary

In der Parkinson’schen Krankheit bilden sich im menschlichen Gehirn kugelförmige Ablagerungen, welche eine hohe Konzentration des Proteins Alpha-Synuclein (aSyn) aufweisen. Unsere frühere Forschung hat jedoch auch gezeigt, dass diese Ablagerungen des Weiteren aus dicht gepackten Fragmenten von biologischen Membranen bestehen. aSyn ist ein kleines Protein, welches in den menschlichen Neuronen in hoher Konzentration auftritt, dessen normale Funktion aber immer noch nicht genau bekannt ist. In der Parkinson’schen Krankheit wird vermutet, dass aSyn sich zu nadelförmigen Fibrillen zusammen lagern könnte, und diese Fibrillen dann je nach Form (English: Structural Strain) entweder die Parkinson’sche Krankheit oder Multiple System Atrophie oder Lewi Körper Demenz auslösen könnten.

Unser hauptsächliches Werkzeug für unsere Studien wird korrelative Licht- und Elektronen-Mikroskopie (English: CLEM) sein. In dieser Methode benutzen wir das Lichtmikroskop (LM), um die Lewi Körperchen in mikroskopisch kleinen Gehirnproben von verstorbenen Spendern aus einer Niederländischen Klinik zu finden, und untersuchen diese dann gezielt mit dem Elektronenmikroskop (EM) bei höchster Auflösung. Parallel dazu werden wir mit dem EM das Protein aSyn, sowie auch andere verdächtige Proteine wie z.B. LRRK2 als Einzelmoleküle untersuchen, um zu verstehen, welche Struktur und Konformationen diese Proteine eingehen können, und wie diese Proteine an der Krankheitsbildung beteiligt sind. 

Das Ziel unserer Forschung ist, die molekularen Ursachen der Parkinson’schen Krankheit zu erkennen, und die Vorgänge bei der zellulären Ausbreitung der Krankheit im menschlichen Gehirn zu verstehen. 

Direct link to Lay Summary Last update: 24.07.2020

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Associated projects

Number Title Start Funding scheme
200628 Coherent Electron Diffractive Imaging for Vitrified Single Protein Particles 01.09.2021 Project funding (Div. I-III)
177195 Molecular and Cellular Modulation in Parkinson's Disease 01.01.2018 Sinergia

Abstract

Parkinson's Disease (PD) is characterized by the presence so-called Lewy bodies (LBs) and Lewy neurites in affected brain regions. LBs are large intracellular aggregates, for which Spillantini et al. identified the protein alpha-synuclein (aSyn) as a major protein component. We have applied correlative light and electron microscopy (CLEM) to human post-mortem brain from Parkinson's disease patients, and investigated the structure of Lewy bodies. Contrary to expectations, most LBs that did stain strongly for phosphorylated alpha-synuclein in the light microscope, showed in the electron microscope that they consisted primarily of aggregated membranous material instead of the expected fibrillar content. In parallel, we have determined the atomic structure of in vitro generated alpha-synuclein fibrils by cryo-EM, revealing three different types of atomic structures, termed polymorphs. We here propose to further develop CLEM towards cryo-CLEM, by combining correlative fluorescence light microscopy and cryo-electron microscopy of high-pressure frozen human brain, which would avoid any chemical fixation or heavy-metal staining. This will require developing a protocol to stain human brain prior to high-pressure freezing or sectioning. We will then apply immuno-EM, conventional CLEM, and cryo-CLEM to study human post-mortem brain from patients. We will determine the molecular composition of filamentous structures found in some of the LBs, and investigate the compositions of LBs found in other brain and tissue regions. We will expand our analysis to brain tissue at earlier stages of Parkinson’s disease, and study the LB appearance in sporadic, as well as genetically triggered PD variants from patients who suffered from mutations in LRRK2 and GBA. In addition, we will seek to purify aSyn fibrils from human brain of PD patients, and determine their high-resolution structure by cryo-EM. We expect our analysis of earlier disease (Braak) stages to shed light into the chronologic development of Lewy pathology. The analysis of the ultrastructure of the post-mortem brain of PD patients who were affected by LRRK2 or GBA mutations, might give clues about the mechanism of disease development in these frequently occurring inherited forms of PD. And finally, the structure of aSyn fibrils purified from PD patient brains might allow conclusions about the mechanism of action of aSyn protein in the fibrillar vs. non-fibrillar form. Brain-derived fibril structures could then be compared with our in vitro generated structures, to identify a suitable drug target candidate among our in vitro aSyn fibril samples. In addition to PD, this research might also help in the understanding of other synucleinopathies such as Multiple System Atrophy and Dementia with Lewy Bodies.
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