Three Routes to Suppression of the Neurodegenerative Phenotypes Caused by Kinesin Heavy Chain Mutations

Abstract

Kinesin-1 is a motor protein that moves stepwise along microtubules by employing dimerized kinesin heavy chain (Khc) subunits that alternate cycles of microtubule binding, conformational change, and ATP hydrolysis. Mutations in the Drosophila Khc gene are known to cause distal paralysis and lethality preceded by the occurrence of dystrophic axon terminals, reduced axonal transport, organelle-filled axonal swellings, and impaired action potential propagation. Mutations in the equivalent human gene, Kif5A, result in similar problems that cause hereditary spastic paraplegia (HSP) and Charcot-Marie-Tooth type 2 (CMT2) distal neuropathies. By comparing the phenotypes and the complementation behaviors of a large set of Khc missense alleles, including one that is identical to a human Kif5A HSP allele, we identified three routes to suppression of Khc phenotypes: nutrient restriction, genetic background manipulation, and a remarkable intramolecular complementation between mutations known or likely to cause reciprocal changes in the rate of microtubule-stimulated ADP release by kinesin-1. Our results reveal the value of large-scale complementation analysis for gaining insight into protein structure-function relationships in vivo and point to possible paths for suppressing symptoms of HSP and related distal neuropathies.T O transport organelles and other cargo complexes through cytoplasm over long distances, molecular motor proteins use paired, force-producing subunits to walk along microtubules. This is especially important for the development and maintenance of cells in which biosynthetic processes are asymmetrically located, such as neurons with long axons (Saxton and Hollenbeck 2012). Mutations in microtubule motor proteins are known to cause hereditary forms of spastic paraplegia (HSP) (Reid et al. 2002;Blackstone et al. 2011) and Charcot-Marie-Tooth type 2 (CMT2) (Goizet et al. 2009;Crimella et al. 2011) distal neuropathies and are suspected of contributing to the progression of other neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Huntington's, and Parkinson's (De Vos et al. 2008;Morfini et al. 2009;Perlson et al. 2010). Defining the basic mechanisms of motor-driven cytoplasmic transport in axons and understanding how defects in them contribute to neurodegeneration are important areas for investigation.The structure and mechanochemistry of kinesin-1, a major axonal transport motor, have been the subject of intense study. The holoenzyme is centered around kinesin heavy chain (Khc, or Kif5 in humans), composed of an N-terminal globular head (the motor domain) connected to a long a-helical stalk that terminates in a small C-terminal globular tail (Vale et al. 1985;Yang et al. 1989). Two Khc heads, dimerized via coiled-coil interactions of their stalks, alternate cycles of ATP binding-hydrolysis-release and microtubule binding-release to move stepwise toward microtubule plus ends (reviewed by Vale and Milligan 2000;Sindelar 2011), which in axons are oriented away from the cell...

Pavarotti/MKLP1 Regulates Microtubule Sliding and Neurite Outgrowth in Drosophila Neurons

“…A lateral view of those larvae showed a characteristic curling of the posterior end (the tail-flipping phenotype, Figure S3A). This phenotype is caused by paralysis of dorsal musculature and is typically observed in animals with mutations that cause degeneration of motor neurons [14, 15]. In addition, locomotion of the surviving elav > pav -RNAi third instar larvae was dramatically affected; these larvae were very sluggish and their crawling trajectories were typically circular (Figure S3B and Movie S5).…”

Limited distal organelles and synaptic function in extensive monoaminergic innervation

“…It is possible that synaptic capture of cargos of both motors is not scaled down sufficiently to allow cargo-carrying motors to travel through scores of boutons to reach the most distal points of octopamine neuron innervation. Alternatively, because DCV transport is attenuated, but not eliminated, by loss of kinesin-1 (Djagaeva et al, 2012), the involvement of kinesin-1 in the transport of both mitochondria and DCV provides a potential link that could explain their common problem with distal accumulation. However, because the absence of mitochondria and DCVs is not always coincident in individual boutons, there must not be a tight coupling between the mechanisms governing distribution of these organelles in distal boutons.…”

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide, Microtubule, and Neck-Linker Binding Sites

“…The nucleotide-dependent correlations of these regions imply a potentially important allosteric role for these sites. Intriguingly, mutations at a number of these sites (see Table 1) are associated with deficiencies in the kinesin cycle that lead to pathologies such as spastic paraplegia and Charcot-Marie-Tooth syndrome (46,47).…”