Stabilization of Hyperdynamic Microtubules Is Neuroprotective in Amyotrophic Lateral Sclerosis

Abstract

Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS)2 is a late-onset, progressive neurodegenerative disease affecting motoneurons (1). The etiology of the majority of ALS cases is unknown, but ϳ20% of familial disease cases are due to mutations in copper-zinc superoxide dismutase1 (SOD1) (2). This led to the development of SOD1 transgenic mice as models of disease (2-5).Physically, motoneurons are unique, representing the longest cells in the body, with axons of some motoneurons in the spinal cord extending a meter or more to reach an end organ. As a result of this morphology, exceptional demands are placed on motoneurons. Active transport along lengthy axons is required to convey newly made materials from the cell body to the farthest nerve endings, and to convey nutrients and metabolites back to the cell body. Microtubules are an essential component of the neuron's scaffold and represent the "roadway," or conveyer belt, that neurons use to transport nutrients (6 -10). Microtubule-based transport is mandatory for survival of motoneurons and muscle cells; changes in slow axonal transport have been linked to neuropathogenesis in mutant SOD1 transgenic mice (11-13). In addition, the assembly and disassembly of microtubule polymers in motoneurons is highly responsive to cellular insults, such as excitotoxic stimuli (8, 14 -16).The relation between dynamics of microtubules and neuronal pathogenesis has not been explored in detail, in part due to limited techniques for measuring microtubule dynamics in vivo. In most non-neuronal cells, tubulin dimers and microtubule polymers exist in rapid dynamic equilibrium, as we have recently shown in vivo by isotopic labeling (17). In neurons, however, this rapid turnover of axonal and dendritic microtubules is believed to be less dynamic due to their interactions with a specific subclass of microtubule-associated proteins (MAPs) (18 -20). This stability of microtubule...

Deficits in axonal transport precede ALS symptoms in vivo

“…The binding of SOD1 G93A to mitochondria (25) may also reduce ATP production, thereby reducing its availability for dyneinmediated transport. This defect may be worsened by the accumulation of hyperdynamic microtubules in presymptomatic SOD1 G93A mice (42). This hypothesis is supported by our work showing deficits in the bidirectional transport of mitochondria in vivo in SOD1 G93A mice at a presymptomatic stage.…”

Hyperdynamic Microtubules, Cognitive Deficits, and Pathology Are Improved in Tau Transgenic Mice with Low Doses of the Microtubule-Stabilizing Agent BMS-241027

“…MT hyperdynamicity, along with deficits in slow axonal transport, has also been measured in a model of amyotrophic lateral sclerosis with this stable isotope-labeling technique (Fanara et al, 2007). No changes in total tubulin or MTs are observed, demonstrating another neurodegeneration model where in vivo changes in MT turnover would not have been detected without stable isotope labeling.…”

“…Altered MT dynamics in tau transgenic mice MT dynamics can be measured in vivo using a novel deuterium labeling procedure (Fanara et al, 2004(Fanara et al, , 2007(Fanara et al, , 2010. Following labeling of body fluids with 2 H 2 O, deuterium becomes incorporated into nonessential amino acids and then into newly synthesized proteins.…”

“…Western blot analyses were performed as described previously (Fanara et al, 2007(Fanara et al, , 2010 using Tau5 to detect all forms of tau, ab5392 to detect MAP-2, and DM1A to detect ␣-tubulin (Sigma-Aldrich). Equal amounts of protein (BCA Protein Assay, Thermo Scientific) were loaded in each lane, and quantitation of ␣-tubulin was done using densitometry of film in the linear range.…”

Interaction of Amyotrophic Lateral Sclerosis (ALS)-related Mutant Copper-Zinc Superoxide Dismutase with the Dynein-Dynactin Complex Contributes to Inclusion Formation

“…8), including competition for dynein transport, disruption of dynein motor activity, physical blockage of dynein transport by mutant SOD1 aggregates, and destabilization of microtubules. A recent study showed that microtubule destabilization is an early event in ALS, and a microtubule stabilization drug had beneficial effects in ALS mice (53). Reduced number of available microtubule tracks in combination with increased levels of misfolded mutant SOD1 bound to dynein could over time lead to severe reduction of transport of other cargos such as neurotrophic factors necessary to promote neuronal survival, thus causing neurotoxicity.…”