IGF2 prevents dopaminergic neuronal loss and decreases intracellular alpha-synuclein accumulation in Parkinson's disease models.

Parkinson's disease (PD) is the second most common late-onset neurodegenerative disease and the predominant cause of movement problems. PD is characterized by motor control impairment by extensive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). This selective dopaminergic neuronal loss is in part triggered by intracellular protein inclusions called Lewy bodies, which are composed mainly of misfolded alpha-synuclein (α-syn) protein. We previously reported insulin-like growth factor 2 (IGF2) as a key protein downregulated in PD patients. Here we demonstrated that IGF2 treatment or IGF2 overexpression reduced the α-syn aggregates and their toxicity by IGF2 receptor (IGF2R) activation in cellular PD models. Also, we observed IGF2 and its interaction with IGF2R enhance the α-syn secretion. To determine the possible IGF2 neuroprotective effect in vivo we used a gene therapy approach in an idiopathic PD model based on α-syn preformed fibrils intracerebral injection. IGF2 gene therapy revealed a significantly preventing of motor impairment in idiopathic PD model. Moreover, IGF2 expression prevents dopaminergic neuronal loss in the SN together with a decrease in α-syn accumulation (phospho-α-syn levels) in the striatum and SN brain region. Furthermore, the IGF2 neuroprotective effect was associated with the prevention of synaptic spines loss in dopaminergic neurons in vivo. The possible mechanism of IGF2 in cell survival effect could be associated with the decrease of the intracellular accumulation of α-syn and the improvement of dopaminergic synaptic function. Our results identify to IGF2 as a relevant factor for the prevention of α-syn toxicity in both in vitro and preclinical PD models.

Airy beam light sheet microscopy boosted by deep learning deconvolution.

Common light sheet microscopy comes with a trade-off between light sheet width defining the optical sectioning and the usable field of view arising from the divergence of the illuminating Gaussian beam. To overcome this, low-diverging Airy beams have been introduced. Airy beams, however, exhibit side lobes degrading image contrast. Here, we constructed an Airy beam light sheet microscope, and developed a deep learning image deconvolution to remove the effects of the side lobes without knowledge of the point spread function. Using a generative adversarial network and high-quality training data, we significantly enhanced image contrast and improved the performance of a bicubic upscaling. We evaluated the performance with fluorescently labeled neurons in mouse brain tissue samples. We found that deep learning-based deconvolution was about 20-fold faster than the standard approach. The combination of Airy beam light sheet microscopy and deep learning deconvolution allows imaging large volumes rapidly and with high quality.

Imaging three-dimensional brain organoid architecture from meso- to nanoscale across development.

Organoids are stem cell-derived three-dimensional cultures offering a new avenue to model human development and disease. Brain organoids allow the study of various aspects of human brain development in the finest details in vitro in a tissue-like context. However, spatial relationships of subcellular structures, such as synaptic contacts between distant neurons, are hardly accessible by conventional light microscopy. This limitation can be overcome by systems that quickly image the entire organoid in three dimensions and in super-resolution. To that end we have developed a system combining tissue expansion and light-sheet fluorescence microscopy for imaging and quantifying diverse spatial parameters during organoid development. This technique enables zooming from a mesoscopic perspective into super-resolution within a single imaging session, thus revealing cellular and subcellular structural details in three spatial dimensions, including unequivocal delineation of mitotic cleavage planes as well as the alignment of pre- and postsynaptic proteins. We expect light-sheet fluorescence expansion microscopy to facilitate qualitative and quantitative assessment of organoids in developmental and disease-related studies.

Hard-wired lattice light-sheet microscopy for imaging of expanded samples.

Light-sheet fluorescence microscopy (LSFM) helps investigate small structures in developing cells and tissue for three-dimensional localization microscopy and large-field brain imaging in neuroscience. Lattice light-sheet microscopy is a recent development with great potential to improve axial resolution and usable field sizes, thus improving imaging speed. In contrast to the commonly employed Gaussian beams for light-sheet generation in conventional LSFM, in lattice light-sheet microscopy an array of low diverging Bessel beams with a suppressed side lobe structure is used. We developed a facile elementary lattice light-sheet microscope using a micro-fabricated fixed ring mask for lattice light-sheet generation. In our setup, optical hardware elements enable a stable and simple illumination path without the need for spatial light modulators. This setup, in combination with long-working distance objectives and the possibility for simultaneous dual-color imaging, provides optimal conditions for imaging extended optically cleared tissue samples. We here present experimental data of fluorescently stained neurons and neurites from mouse hippocampus following tissue expansion and demonstrate the high homogeneous resolution throughout the entire imaged volume. Utilizing our purpose-built lattice light-sheet microscope, we reached a homogeneous excitation and an axial resolution of 1.2 µm over a field of view of (333 µm)2.