Roles and mechanisms of synuclein and ataxin-3 spreading in Parkinson and Machado-Joseph diseases (Martin Oheim)

Parkinson’s disease (PD) and Machado Joseph disease (MJD) represent two incurable prototypical neurodegenerative diseases associated with protein aggregation and progressive spread of disease in the brain. Preliminary evidence suggests that both disorders share a common mechanism where autophagosome trafficking and exosome secretion intersect.

While autophagosomes are important intracellular vesicles for routing proteins and organelles for lysosomal degradation, extracellular microvesicles (exosomes) contain collections of proteins, RNA and lipids that are important cell-to-cell carriers for intercellular signaling-molecules.

How this common mechanism causes PD and MJD will be investigated by the Synspread consortium* in neurons and astrocytes derived from PD and MJD patients’ inducible pluripotent stem cells (iPSC) cells, using state-of-the-art super-resolution imaging of autophagosome and exosome formation/secretion and 2-photon whole- brain imaging of cleared tissue to trace the routes of synuclein and ataxin-3 protein spreading. The results will be plugged into a novel computational model of aggregate and exosome propagation in the mouse brain to predict disease progression and identify biochemical pathways underlying progression.

This SynSpread project is expected to generate unique insights into the process of protein and aggregate spreading in PD, MJD and potentially other neurodegenerative disorders, and to lead to identification of new targets and modifiers for therapeutic intervention in a significant step towards treatment of patients.

Collaborations

• Luis Pereira de Almeida, CNC, University of Coimbra, Portugal (Coordinator)
• Jens C. Schwamborn, University of Luxembourg, Luxembourg
• Ronan MT Fleming, University of Luxembourg, Luxembourg

Funding sources

• ANR- A National Research Infrastructure for Biological Imaging: France BioImaging (2013-23)
• EU-JPND (2015-18)

Fast, large-field multi-color imaging of astrocyte organelle interactions at isotropic 100-nm resolution (Martin Oheim)

Dual-color superresolution imaging. TIRF (left) and TIRF-SIM (right) images of a cultured cortical mouse astrocyte expressing ER-EGFP (green), a marker of the endoplasmic reticulum, and labeled with Mito-Tracker-Deep-Red (red), a marker of mitochondria. Images are zooms (2 µm by 2 µm) of a larger field-of-view and were taken sequentially upon 488- and 561-nm excitation, respectively. Exposure time was 200 ms per frame (total time: 3.8 s for the acquisition of the two colors). Pattern period was 180 nm (206 nm) for the green (red) channel, respectively.

Most structured illumination microscopes use a physical or synthetic grating that is projected into the sample plane to  generate a periodic illumination pattern. Albeit simple and cost-effective, this arrangement hampers fast or multi-color acquisition, which is a critical requirement for time-lapse imaging of cellular and sub-cellular dynamics. We designed and implemented an interferometric approach allowing large-field, fast, dual-color imaging at an isotropic 100-nm resolution based on a sub-diffraction fringe pattern generated by the interference of two colliding evanescent waves. Our all-mirror-based system generates illumination patterns of arbitrary orientation and period, limited only by the illumination aperture (NA) of the objective lens, the response time of a fast, piezo-driven tip-tilt mirror and the available fluorescence signal. At low µW laser powers suitable for long-period observation of life cells and with a camera exposure time of 20 ms, our system permits the acquisition of super-resolved 50 µm by 50 µm images at 3.3 Hz. The possibility it offers for rapidly adjusting the pattern between images is particularly advantageous for experiments that require multi-scale and multi-color information. We demonstrated the performance of our instrument by imaging the collective dynamics of mitochondria and the endoplasmic reticulum (ER) in cultured cortical astrocytes. As an illustration of dual-color excitation dual-color detection, we also resolve interaction sites between near-membrane mitochondria and the endoplasmic reticulum. Our TIRF-SIM microscope – now housing five lasers spanning the entire visible wavelength range – provides a versatile, compact and cost-effective arrangement for large-field super-resolution imaging, allowing the investigation of co-localization and dynamic interactions between organelles – important questions in both cell biology and neurophysiology

Collaborations

• D. Abi-Haidar (IMNC – CNRS UMR 8165, Orsay)
• B. Devaux & P. Varlet (CHSA, Sainte-Anne Hospital, Paris)

Funding sources

• National grants: ANR FranceBioImaging
• Région Ile-de-France (DIM C’nano)

Team leader

Members

Recent Publications

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