Loss of intermediate filament IFB-1 reduces mobility, density, and physiological function of mitochondria in Caenorhabditis elegans sensory neurons.

Mitochondria and intermediate filament (IF) accumulations often occur during imbalanced axonal transport leading to various types of neurological diseases. It is still poorly understood whether a link between neuronal IFs and mitochondrial mobility exist. In Caenorhabditis elegans, among the 11 cytoplasmic IF family proteins, IFB-1 is of particular interest as it is expressed in a subset of sensory neurons. Depletion of IFB-1 leads to mild dye-filling and significant chemotaxis defects as well as reduced life span. Sensory neuron development is affected and mitochondrial transport is slowed down leading to reduced densities of these organelles. Mitochondria tend to cluster in neurons of IFB-1 mutants likely independent of the fission and fusion machinery. Oxygen consumption and mitochondrial membrane potential is measurably reduced in worms carrying mutations in the ifb-1 gene. Membrane potential also seems to play a role in transport such as carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone treatment led to increased directional switching of mitochondria. Mitochondria co-localize with IFB-1 in worm neurons and appear in a complex with IFB-1 in pull-down assays. In summary, we propose a model in which neuronal IFs may serve as critical (transient) anchor points for mitochondria during their long-range transport in neurons for steady and balanced transport.

Methods to Quantify and Relate Axonal Transport Defects to Changes in C. elegans Behavior.

Neuronal growth, differentiation, homeostasis, viability, and injury response heavily rely on functional axonal transport (AT). Erroneous and disturbed AT may lead to accumulation of "disease proteins" such as tau, α-synuclein, or amyloid precursor protein causing various neurological disorders. Changes in AT often lead to observable behavioral consequences in C. elegans such as impeded movements, defects in touch response, chemosensation, and even egg laying. Long C. elegans neurons with clear distinguishable axons and dendrites provide an excellent platform to analyze AT. The possibility to relate changes in AT to neuronal defects that in turn lead to quantifiable changes in worm behavior allows for the advancement of neuropathological disease models. Even more, subsequent suppressor screens may aid in identifying genes responsible for observed behavioral changes providing a target for drug development to eventually delay or cure neurological diseases. Thus, in this chapter, we summarize critical methods to identify and quantify defects in axonal transport as well as exemplified behavioral assays that may relate to these defects.

PTP-3 phosphatase promotes intramolecular folding of SYD-2 to inactivate kinesin-3 UNC-104 in neurons.

UNC-104 is the Caenorhabditis elegans homolog of kinesin-3 KIF1A known for its fast shuffling of synaptic vesicle protein transport vesicles in axons. SYD-2 is the homolog of liprin-α in C. elegans known to activate UNC-104; however, signals that trigger SYD-2 binding to the motor remain unknown. Because SYD-2 is a substrate of PTP-3/LAR PTPR, we speculate a role of this phosphatase in SYD-2-mediated motor activation. Indeed, coimmunoprecipitation assays revealed increased interaction between UNC-104 and SYD-2 in ptp-3 knockout worms. Intramolecular FRET analysis in living nematodes demonstrates that SYD-2 largely exists in an open conformation state in ptp-3 mutants. These assays also revealed that nonphosphorylatable SYD-2 (Y741F) exists predominately in folded conformations, while phosphomimicking SYD-2 (Y741E) primarily exists in open conformations. Increased UNC-104 motor clustering was observed along axons likely as a result of elevated SYD-2 scaffolding function in ptp-3 mutants. Also, both motor velocities as well as cargo transport speeds were visibly increased in neurons of ptp-3 mutants. Lastly, epistatic analysis revealed that PTP-3 is upstream of SYD-2 to regulate its intramolecular folding.