Amyotrophic lateral sclerosis (ALS)-associated VAPB-P56S inclusions represent an ER quality control compartment

BACKGROUND: Protein aggregation and the formation of intracellular inclusions are a central feature of many neurodegenerative disorders, but precise knowledge about their pathogenic role is lacking in most instances. Here we have characterized inclusions formed in transgenic mice carrying the P56S mutant form of VAPB that causes various motor neuron syndromes including ALS8.RESULTS: Inclusions in motor neurons of VAPB-P56S transgenic mice are characterized by the presence of smooth ER-like tubular profiles, and are immunoreactive for factors that operate in the ER associated degradation (ERAD) pathway, including p97/VCP, Derlin-1, and the ER membrane chaperone BAP31. The presence of these inclusions does not correlate with signs of axonal and neuronal degeneration, and axotomy leads to their gradual disappearance, indicating that they represent reversible structures. Inhibition of the proteasome and knockdown of the ER membrane chaperone BAP31 increased the size of mutant VAPB inclusions in primary neuron cultures, while knockdown of TEB4, an ERAD ubiquitin-protein ligase, reduced their size. Mutant VAPB did not codistribute with mutant forms of seipin that are associated with an autosomal dominant motor neuron disease, and accumulate in a protective ER derived compartment termed ERPO (ER protective organelle) in neurons.CONCLUSIONS: The data indicate that the VAPB-P56S inclusions represent a novel reversible ER quality control compartment that is formed when the amount of mutant VAPB exceeds the capacity of the ERAD pathway and that isolates misfolded and aggregated VAPB from the rest of the ER. The presence of this quality control compartment reveals an additional level of flexibility of neurons to cope with misfolded protein stress in the ER

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches

TRIM46 Controls Neuronal Polarity and Axon Specification by Driving the Formation of Parallel Microtubule Arrays

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development