A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice.

Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.

Neurons on tape: Automated Tape Collecting Ultramicrotomy-mediated volume EM for targeting neuropathology.

In this chapter, we review Automated Tape Collecting Ultramicrotomy (ATUM), which, among other array tomography methods, substantially simplified large-scale volume electron microscopy (vEM) projects. vEM reveals biological structures at nanometer resolution in three dimensions and resolves ambiguities of two-dimensional representations. However, as the structures of interest-like disease hallmarks emerging from neuropathology-are often rare but the field of view is small, this can easily turn a vEM project into a needle in a haystack problem. One solution for this is correlated light and electron microscopy (CLEM), providing tissue context, dynamic and molecular features before switching to targeted vEM to hone in on the object's ultrastructure. This requires precise coordinate transfer between the two imaging modalities (e.g., by micro computed tomography), especially for block face vEM which relies on physical destruction of sections. With array tomography methods, serial ultrathin sections are collected into a tissue library, thus allowing storage of precious samples like human biopsies and enabling repetitive imaging at different resolution levels for an SEM-based search strategy. For this, ATUM has been developed to reliably collect serial ultrathin sections via a conveyor belt onto a plastic tape that is later mounted onto silicon wafers for serial scanning EM (SEM). The ATUM-SEM procedure is highly modular and can be divided into sample preparation, serial ultramicrotomy onto tape, mounting, serial image acquisition-after which the acquired image stacks can be used for analysis. Here, we describe the steps of this workflow and how ATUM-SEM enables targeting and high resolution imaging of specific structures. ATUM-SEM is widely applicable. To illustrate this, we exemplify the approach by reconstructions of focal pathology in an Alzheimer mouse model and CLEM of a specific cortical synapse.

Cold acclimation has a differential effect on leaf vascular bundle structure and carbon export rates in natural Arabidopsis accessions originating from southern and northern Europe.

Acclimation to low but non-freezing temperature represents an ecologically important process for Arabidopsis thaliana but also for many other plant species from temperate regions. Cold acclimation comprises and affects numerous molecular and physiological processes and the maintenance of sugar supply of sink tissue by photosynthetically active source tissue is essential for plant survival. Here, changes in vascular bundle (VB) structure at the leaf petiole were analysed together with sucrose exudation rates before and after cold acclimation. Six natural Arabidopsis accessions originating from southern and northern Europe were compared. Photosynthetic efficiency, that is, maximum and effective quantum yield of photosystem II, revealed a significant effect of environmental condition. Only for northern accessions was a highly significant negative correlation observed between leaf sucrose exudation rates, xylem, and petiole cross-sectional areas. Furthermore, only for northern accessions was a significant increase of VB and leaf petiole cross-sectional area observed during cold acclimation. In contrast, variance of cross-sectional areas of cold acclimated southern accessions was strongly reduced compared to control plants, while mean areas remained similar under both conditions. In summary, these findings suggest that natural Arabidopsis accessions from northern Europe significantly adjust sink strength and leaf VB structure to maintain plant growth and photosynthesis under low temperature.