DOI: 10.25378/janelia.20134979
title: Isotropic 3D electron microscopy data of double-mutant ATL1 (KI) /REEP1 (KO) mouse dorsal corticospinal tract (jrc_mus-sc-zp105a)

description: Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1–86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (<i>SPAST</i>; SPG4), the membrane-bound GTPase atlastin-1 (<i>ATL1</i>; SPG3A) and the reticulon-like, microtubule-binding protein REEP1 (<i>REEP1</i>; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, whereas its binding partner Reep1 is knocked out. <i>Atl1</i><sup><em>KI/KI</em></sup><i>/Reep1</i><sup><em>−/−</em></sup> mice exhibit early onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, on the basis of reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in <i>Atl1</i><sup><em>KI/KI</em></sup><i>/Reep1</i><sup><em>−/−</em></sup> spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. <i>Atl1</i><sup><em>KI/KI</em></sup><i>/Reep1</i><sup><em>−/−</em></sup> mice represent an early onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.<br>Sample: Double-mutant ATL1 (knock-in) / REEP1 (knocked out), female, 6-month-old mouse dorsal corticospinal tract tissueProtocol: Mouse was anesthetized and transcardially perfused with 1× PBS followed by freshly made EM fixative (2% glutaraldehyde and 2% paraformaldehyde in 0.1 N cacodylate buffer). Spinal cords was removed and post-fixed in EM fixative overnight. Vibratome slices were treated with 0.2% OsO4 in phosphate buffer for 30 min, mordanted en bloc with 0.25% uranyl acetate overnight at 40°C, dehydrated with ethanol and embedded in Durcupan resin.Dataset ID: jrc_mus-sc-zp105aFinal voxel size (nm): 6 x 6 x 6 (X, Y, Z)Dimensions (µm): 40 x 30 x 75 (X, Y, Z)Acquisition date: 2018-11-10Dataset URL: s3://janelia-cosem-datasets/jrc_mus-sc-zp105a/jrc_mus-sc-zp105a.zarr/recon-1/em/Visualization Website: https://openorganelle.janelia.org/datasets/jrc_mus-sc-zp105aPublication: Zhu et al., Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons, Human Molecular Genetics (2022).