Microtubules and Axonal Transport Defects

By Benjamin Musrie, Medical Researcher. Sydney, Australia.

Illustration by Jenny Liu

The cells cytoskeleton is a dynamic network composed of three main components — microfilaments, intermediate filaments and microtubules. The cytoskeleton gives the cell its shape and provides mechanical support that enables the cell to carry out functions such as division and movement. Microtubules are microscopic tubes made up of alpha and beta tubulin heterodimers which undergo continuous polymerisation and depolymerisation. They are involved in cell division, organisation of intracellular structure and intracellular transport. Axonal transport is one of the most critical functions of microtubules and is crucial for neuronal homeostasis. Defects in axonal transport is considered one of the earliest pathophysiological events in ALS and may be a primary cause of motor neuron degeneration.

Axonal transport:

Axonal transport is defined as the process by which proteins, organelles and other molecules synthesised in the neurosome are transported to and from nerve endings. Axonal transport is essential for the growth and survival of neurons. Microtubules extend throughout the neuron and act as the ‘tracks’ for this form of transportation. Kinesin and Dynein motor proteins facilitate the transport of various cargo along these tracks in an anterograde (from cell body to axon tip) and retrograde (from axon tip to cell body) direction, respectively. These motor proteins transport several different cargoes including mitochondria, autophagosomes, neurofilaments and synaptic vesicles. Defects in axonal transport may arise through various mechanisms and have been implicated in neurodegenerative diseases as an early pathological feature.

Axonal transport defects in MND:

A key pathological hallmark of MND is the mislocalization of protein aggregates and the presence of cytoplasmic aggregates in motor neurons and surrounding cells, both of which suggest defects in axonal transport. Electron microscopy studies have demonstrated swellings that occur in degenerated motor neurons in patients with ALS. These various swellings were found to contain accumulations of organelles and materials including neurofilaments, autophagosomes and mitochondria (Sasaki, Maruyama, Yamane, Sakuma, Takeishi, 1990; Corbo and Hays, 1992). Intracellular accumulation of misfolded protein aggregates are toxic to neurons and may play a role in the formation and progression of MND.

Autophagosomes:

An autophagosome is a double-membrane vehicle that contains cellular material scheduled to be degraded by autophagy. They are key to maintaining cell viability and homeostasis. During states of impaired axonal transport, motor neurons are susceptible to the accumulation of damaged autophagosomes. Unlike lysosomes which can only form in the cell body of a neuron, autophagosomes can form anywhere in the cell where there is a build-up of toxic waste. Autophagosomes may then transport the waste to the lysosomes via dynein motor proteins for degradation (Maday, 2016). Hence, when there is a disruption in dynein motor proteins or axonal transport in general, autophagosomes may not form correctly and accumulate along with their contents in neurons disrupting neuronal integrity. In fact, Xie and colleagues showed that the rescue of impaired dynein motor proteins led to motor neuron survival and ameliorated the ALS disease phenotype in SOD1 mice (Xie et al., 2015), which indicates that inducing autophagy countered accumulation of ALS protein aggregates. Also, aggregates of TAR DNA-binding protein 43 (TDP-43) present in ALS neurons can be reduced by inducing autophagy via mTOR inhibition, an effect that promotes autophagosome formation. TDP-43 becomes localised to its proper nuclear compartment following mTOR inhibition (Wang, Tsai and Shen, 2013).

Mitochondria:

Mitochondria are membrane-bound cell organelles found in the cytoplasm and generate most of the cell’s energy supply in the form of ATP. Defective mitochondrial transport may be responsible for the accumulation of damaged mitochondria in motor neuron axons and has been described as playing a role in motor neuron death in ALS. Live microscopy revealed reduced anterograde axonal transport of fluorescently labelled mitochondria in cultured cortical neurons expressing ALS mutant SOD1G93A, A4V, G85R or G37R and in embryonic motor neurons expressing SOD1G93A (De Vos et al., 2007). When researchers looked at motor neurons of early symptomatic SOD1G37R and SOD1G85R transgenic mice in vivo, the number of axonal mitochondria was reduced, and their distribution was skewed in the axon (Vande Velde et al., 2011). As well, in SOD1G93A transgenic mice, mitochondria were found in abnormal clusters along the axon (Magrané et al., 2014). Defects in mitochondrial transport, however, is not only limited to SOD1 related ALS. Overexpression of wild-type TDP-43 and to a greater extent ALS mutant TDP-43Q331K or M337V reduced mitochondrial transport and mitochondrial density in primary motor neurons (Wang et al., 2013). Disruption of axonal mitochondrial transport was also observed in ALS mutant TDP-43A315T transgenic mice in vivo (Magrané et al., 2014). As well, wild-type TDP-43 and mutant TDP-43M337V overexpressing mice exhibited mitochondrial aggregation consistent with transport defects (Xu et al., 2011, Xu et al., 2010).

Neurofilaments:

Aggregation of neurofilaments in the axons of spinal motor neurons have long been recognised as a hallmark of MNDs. Neurofilaments are transported along microtubules by kinesin and dynein and disruption of these motor proteins results in accumulation of neurofilaments in motor neurons. This has been observed in both KIF5A mutant mouse and dynactin-1 mutant mouse (Laird et al., 2008; Xia et al., 2003; Koehnle and Brown, 1999). Mersiyanova and colleagues showed that the A998C transversion mutation in the first exon of neurofilament light chain (a component of the neurofilament) results in Charcot-Marie-Tooth (CMT) type 2 (Mersiyanova et al., 2000) which manifests as sensory and motor neuron degeneration. Also, missense mutations in neurofilament light chain observed in CMT leads to disruption of axonal transport and aggregation of neurofilament light chain in neuronal cells and primary cultured neurons (Perez-Olle, Jones and Liem, 2004).

Microtubules are complex components of the cells cytoskeleton that act as ‘tracks’ that facilitate movement of organelles and materials through neurons. This function is critical for neuronal health and disruption of axonal transport has been implicated in MND as an early pathological hallmark. Given the importance, future therapeutics may wish to focus on restoring axonal transport. In fact, the feasibility of gene therapy to combat transport deficiencies have already been demonstrated in ALS mice (Xie et al., 2015). It seems most likely that restoring axonal transport function will not be the magic bullet however, in combination with other points of focus, like the microbiome as mentioned in our previous blogs, it may prove to be beneficial in the big picture of MND.

References:

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· De Vos, K.J., A.L. Chapman, M.E. Tennant, C. Manser, E.L. Tudor, K.F. Lau, J. Brownlees, S. Ackerley, P.J. Shaw, D.M. McLoughlin, C.E. Shaw, P.N. Leigh, C.C.J. Miller, and A.J. Grierson. 2007. Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content. Hum Mol Genet. 16:2720–2728.

· Koehnle, T.J., and A. Brown. 1999. Slow axonal transport of neurofilament protein in cultured neurons. J Cell Biol. 144:447–458.

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