Misfolded SOD1 Associated with Motor Neuron Mitochondria Alters Mitochondrial Shape and Distribution Prior to Clinical Onset

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS

Motor neuron mitochondria are rounder in mutant SOD1 mice.

<p>MitoEGFP (<i>green</i>) expression in spinal cord motor neurons in control MitoEGFP (A) and SOD1^G37R early symptomatic animals (B–D). The control neuron has normal mitochondria of diverse shapes and sizes. In contrast, SOD1^G37R motor neurons have rounded swollen mitochondria with uneven distribution. Arrows indicate possible axon hillocks. Motor neuron boundaries have been outlined with dotted line. Quantification of mitochondrial length (E) and aspect ratio (F) in motor neuron cell bodies of mutant SOD1 animals of various ages and age-matched MitoEGFP control animals. ***, p<0.0005. Scale bars, 10 µm.</p

Generation of a novel transgenic mouse with mitochondria labeled uniquely in motor neurons.

<p>(A) Schematic of Hb9-MitoEGFP transgene. (B) Immunoblot of spinal cord homogenates of Hb9-MitoEGFP founders (F34, F17, F36, F20) and F34 sublines probed for EGFP and tubulin (loading control). (C–G) MitoEGFP <i>(green)</i> expression in spinal cord motor neurons (C & D), sciatic nerve (E), and L5 motor axons (F & G) labeled with SMI32 (C, <i>red</i>), cytochrome <i>c</i> (D & F, <i>red</i>), and Fluoromyelin Red (F & G, <i>blue/red</i>). Note that mitochondria have typical tubular and punctate morphologies and MitoEGFP expression is excluded from Schwann cells. (G & H) 15% of large caliber (>4.5 µm) L5 motor axons express MitoEGFP in 12 months animal. The expected biphasic distribution of axonal caliber in adult C57Bl/6 mice is indicated in the upper right corner. Comparison of axonal and somal mitochondrial length (I), width (J) and aspect ratio (K) in Hb9-MitoEGFP mice. Scale bars, 10 µm.</p