Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS

Abstract

Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) affects motor neurons (MNs) in the brain and spinal cord. Understanding the pathophysiology of this condition seems crucial for therapeutic design, yet few electrophysiological studies in actively degenerating animal models have been reported. Here, we report a novel preparation of acute slices from adult mouse spinal cord, allowing visualized whole cell patch-clamp recordings of fluorescent lumbar MN cell bodies from ChAT-eGFP or superoxide dismutase 1-yellow fluorescent protein (SOD1YFP) transgenic animals up to 6 mo of age. We examined 11 intrinsic electrophysiologic properties of adult ChAT-eGFP mouse MNs and classified them into four subtypes based on these parameters. The subtypes could be principally correlated with instantaneous (initial) and steady-state firing rates. We used retrograde tracing using fluorescent dye injected into fast or slow twitch lower extremity muscle with slice recordings from the fluorescent-labeled lumbar MN cell bodies to establish that fast and slow firing MNs are connected with fast and slow twitch muscle, respectively. In a G85R SOD1YFP transgenic mouse model of ALS, which becomes paralyzed by 5-6 mo, where MN cell bodies are fluorescent, enabling the same type of recording from spinal cord tissue slices, we observed that all four MN subtypes were present at 2 mo of age. At 4 mo, by which time substantial neuronal SOD1YFP aggregation and cell loss has occurred and symptoms have developed, one of the fast firing subtypes that innvervates fast twitch muscle was lost. These results begin to describe an order of the pathophysiologic events in ALS.neurodegeneration | motor neurons | amyotrophic lateral sclerosis | electrophysiology A myotrophic lateral sclerosis (ALS; Lou Gehrig's disease) is a progressive and usually lethal neurodegenerative condition prominently featuring loss of motor neurons (MNs) and muscle denervation (1-3). Inherited forms of ALS, accounting for ∼10% of cases, potentially inform about disease mechanisms, including: protein folding and quality control [e.g., mutant superoxide dismutase 1 (SOD1), ubiquilin2, and VCP]; RNA binding proteins (e.g., TDP43, FUS, and HNRNPA1); or a DNA expansion (C9ORF72 hexanucleotide expansion). The clinical courses of the various heritable forms and the 90% of cases that are considered sporadic are not distinct, however, reflecting a potentially shared progressive loss of MNs and motor circuit dysfunction (4).ALS has been modeled in mice that are transgenic for a variety of mutant forms of SOD1, allowing for the study of the trajectory of the condition at various time points (5, 6). Among the studies conducted to date are a number addressing electrophysiological changes. From these studies, however, there does not appear to be a clear consensus on the changes that occur in MNs before and during the development of symptoms (7). For example, whereas research on the neuromuscular junction has revealed preferential denervation of fast twitch (type IIb) muscle fibers (8-10), t...

Extended survival of misfolded G85R SOD1-linked ALS mice by transgenic expression of chaperone Hsp110

“…Thus, we were unable to directly correlate the reduction or absence of aggregates at 3 mo of age with the greatest prolongation of survival. [Note that the presence of aggregates in ventral horn motor neurons of G85R SOD1YFP mice is prominent between 1 and 3 mo of age but then is substantially reduced thereafter (19)]. However, our observation from six double transgenic animals killed at 2.5 mo of age for morphology analysis was that two animals of the six exhibited strikingly low levels of aggregate formation.…”

“…The transgenic G85R SOD1YFP strain (containing greater than 210 copies of the transgene via homozygosity, referred to as the 737 line) has been described (14). By 3-4 mo of age, hind-limb clenching and rotarod dysfunction is observed (19), and lower extremity paralysis develops by 6.5 mo of age. The strain transgenic for Thy1-human Hsp110 (HspA4L) was produced by injecting B6/SJL zygotes (Yale Transgenic Mouse Service) with a construct described in SI Methods.…”

Reduced high-frequency motor neuron firing, EMG fractionation, and gait variability in awake walking ALS mice

“…This reflects extensive reduction of functional motor units and is morphologically correlated with loss of MNs in the lateral TA/GC motor pool (8), loss of axons in lumbar ventral nerve roots (Fig. 2B, Left), reduced gastrocnemius muscle fiber area (Fig.…”

“…The development of clinical symptomslower extremity tremor, twitchiness or clenching, or reduced performance on the rotarod-is associated with MN loss, presumed to involve the MNs that innervate fast-twitch muscle. In a G85R SOD1YFP model of SOD1-linked ALS mice, which become paralyzed by 6 mo of age, we observed that development of such clinical symptoms by 4 mo corresponds to selective loss of lowresistance, fast-firing MNs in acute spinal cord slices prepared from these mice (8). The resulting lack of fast-firing physiology in the context of an in vitro preparation led us to ask: How does the intact G85R SOD1YFP motor system perform motor tasks after selective cell loss in the context of symptomatic degeneration?…”

“…In a previous study, we observed that transgenic G85R SOD1YFP mice first exhibit clinical symptoms at 3-4 mo of age, usually manifested as clenching of the lower extremities when picked up by the tail, and reduced performance in the rotarod test (8). This was associated with a loss of ∼50% of large choline acetyltransferase (ChAT)-positive spinal cord MN cell bodies from the ventral horns compared with age-matched wtSOD1YFP control mice.…”