Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid ß1-42 pathologies.

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-ß 1-42 (Aß42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin-proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

Neuronal Activity Drives Astroglial Connexin 30 in Perisynaptic Processes and Shapes Its Functions.

Astrocytes play key roles in brain functions through dynamic interactions with neurons. One of their typical features is to express high levels of connexins (Cxs), Cx43 and Cx30, the gap junction (GJ)-forming proteins. Cx30 is involved in basic cognitive processes and shapes synaptic and network activities, as shown by recent studies in transgenic animals. Yet it remains unknown whether astroglial Cx30 expression, localization, and functions are endogenously and dynamically regulated by neuronal activity and could therefore play physiological roles in neurotransmission. We here show that neuronal activity increased hippocampal Cx30 protein levels via a posttranslational mechanism regulating lysosomal degradation. Neuronal activity also increased Cx30 protein levels at membranes and perisynaptic processes, as revealed by superresolution imaging. This translated at the functional level in the activation of Cx30 hemichannels and in Cx30-mediated remodeling of astrocyte morphology independently of GJ biochemical coupling. Altogether, these data show activity-dependent dynamics of Cx30 expression, perisynaptic localization, and functions.

Design of a Functionalized Metal-Organic Framework System for Enhanced Targeted Delivery to Mitochondria.

Mitochondria play a key role in oncogenesis and constitute one of the most important targets for cancer treatments. Although the most effective way to deliver drugs to mitochondria is by covalently linking them to a lipophilic cation, the in vivo delivery of free drugs still constitutes a critical bottleneck. Herein, we report the design of a mitochondria-targeted metal-organic framework (MOF) that greatly increases the efficacy of a model cancer drug, reducing the required dose to less than 1% compared to the free drug and ca. 10% compared to the nontargeted MOF. The performance of the system is evaluated using a holistic approach ranging from microscopy to transcriptomics. Super-resolution microscopy of MCF-7 cells treated with the targeted MOF system reveals important mitochondrial morphology changes that are clearly associated with cell death as soon as 30 min after incubation. Whole transcriptome analysis of cells indicates widespread changes in gene expression when treated with the MOF system, specifically in biological processes that have a profound effect on cell physiology and that are related to cell death. We show how targeting MOFs toward mitochondria represents a valuable strategy for the development of new drug delivery systems.

Temperature Treatment of Highly Porous Zirconium-Containing Metal-Organic Frameworks Extends Drug Delivery Release.

Utilizing metal-organic frameworks (MOFs) as a biological carrier can lower the amount of the active pharmaceutical ingredient (API) required in cancer treatments to provide a more efficacious therapy. In this work, we have developed a temperature treatment process for delaying the release of a model drug compound from the pores of NU-1000 and NU-901, while taking care to utilize these MOFs' large pore volume and size to achieve exceptional model drug loading percentages over 35 wt %. Video-rate super-resolution microscopy reveals movement of MOF particles when located outside of the cell boundary, and their subsequent immobilization when taken up by the cell. Through the use of optical sectioning structured illumination microscopy (SIM), we have captured high-resolution 3D images showing MOF uptake by HeLa cells over a 24 h period. We found that addition of a model drug compound into the MOF and the subsequent temperature treatment process does not affect the rate of MOF uptake by the cell. Endocytosis analysis revealed that MOFs are internalized by active transport and that inhibiting the caveolae-mediated pathway significantly reduced cellular uptake of MOFs. Encapsulation of an anticancer therapeutic, alpha-cyano-4-hydroxycinnamic acid (α-CHC), and subsequent temperature treatment produced loadings of up to 81 wt % and demonstrated efficacy at killing cells beyond the burst release effect.

The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes.

The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains: sheets and interconnected tubules. These domains undergo dynamic reshaping in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status: The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven repositioning of lysosomes, which leads to both a redistribution of the ER tubules and a change of its global morphology. Therefore, lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.

A waveguide imaging platform for live-cell TIRF imaging of neurons over large fields of view.

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1,2] and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGCs). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.

OptiJ: Open-source optical projection tomography of large organ samples.

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples.

Computer-aided discovery of a metal-organic framework with superior oxygen uptake.

Current advances in materials science have resulted in the rapid emergence of thousands of functional adsorbent materials in recent years. This clearly creates multiple opportunities for their potential application, but it also creates the following challenge: how does one identify the most promising structures, among the thousands of possibilities, for a particular application? Here, we present a case of computer-aided material discovery, in which we complete the full cycle from computational screening of metal-organic framework materials for oxygen storage, to identification, synthesis and measurement of oxygen adsorption in the top-ranked structure. We introduce an interactive visualization concept to analyze over 1000 unique structure-property plots in five dimensions and delimit the relationships between structural properties and oxygen adsorption performance at different pressures for 2932 already-synthesized structures. We also report a world-record holding material for oxygen storage, UMCM-152, which delivers 22.5% more oxygen than the best known material to date, to the best of our knowledge.

Single particle trajectories reveal active endoplasmic reticulum luminal flow.

The endoplasmic reticulum (ER), a network of membranous sheets and pipes, supports functions encompassing biogenesis of secretory proteins and delivery of functional solutes throughout the cell1,2. Molecular mobility through the ER network enables these functionalities, but diffusion alone is not sufficient to explain luminal transport across supramicrometre distances. Understanding the ER structure-function relationship is critical in light of mutations in ER morphology-regulating proteins that give rise to neurodegenerative disorders3,4. Here, super-resolution microscopy and analysis of single particle trajectories of ER luminal proteins revealed that the topological organization of the ER correlates with distinct trafficking modes of its luminal content: with a dominant diffusive component in tubular junctions and a fast flow component in tubules. Particle trajectory orientations resolved over time revealed an alternating current of the ER contents, while fast ER super-resolution identified energy-dependent tubule contraction events at specific points as a plausible mechanism for generating active ER luminal flow. The discovery of active flow in the ER has implications for timely ER content distribution throughout the cell, particularly important for cells with extensive ER-containing projections such as neurons.

C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.