New cell biological explanations for kinesin-linked axon degeneration

Axons are the slender, up to meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organism's lifetime, meaning up to a century in humans. Axon maintenance requires life-sustaining motor protein-driven transport distributing materials and organelles from the distant cell body. It seems logic that impairing this transport causes systemic deprivation linking to axon degeneration. But the key steps underlying these pathological processes are little understood. To investigate mechanisms triggered by motor protein aberrations, we studied more than 40 loss- and gain-of-function conditions of motor proteins, cargo linkers or further genes involved in related processes of cellular physiology. We used one standardised Drosophila primary neuron system and focussed on the organisation of axonal microtubule bundles as an easy to assess readout reflecting axon integrity. We found that bundle disintegration into curled microtubules is caused by the losses of Dynein heavy chain and the Kif1 and Kif5 homologues Unc-104 and Kinesin heavy chain (Khc). Using point mutations of Khc and functional loss of its linker proteins, we studied which of Khc's sub-functions might link to microtubule curling. One cause was emergence of harmful reactive oxygen species through loss of Milton/Miro-mediated mitochondrial transport. In contrast, loss of the Kinesin light chain linker caused microtubule curling through an entirely different mechanism appearing to involve increased mechanical challenge to microtubule bundles through de-inhibition of Khc. The wider implications of our findings for the understanding of axon maintenance and pathology are discussed.

JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1

The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3's interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.

KLC4 shapes axon arbors during development and mediates adult behavior

Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood. Our work implicates one kinesin light chain subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using several live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches and for proper microtubule (MT) dynamics. Furthermore, KLC4 is required for the contact repulsion necessary for tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons, suggesting that KLC4 patterns axonal compartments and helps define axon identity. Finally, we find that klc4 mutant adults show anxiety-like behavior in a novel tank test, implicating klc4 as a novel gene involved in stress response circuits.

A Novel SLC5A5 Variant Reveals the Crucial Role of Kinesin Light Chain 2 in Thyroid Hormonogenesis

STRUCTURED ABSTRACT Context Iodide transport defect (ITD) (Online Mendelian Inheritance in Man #274400) is an uncommon cause of dyshormonogenic congenital hypothyroidism due to loss-of-function variants in the SLC5A5 gene, which encodes the sodium/iodide symporter (NIS), causing deficient iodide accumulation in thyroid follicular cells. Objective To determine the molecular basis of a patient´s ITD clinical phenotype. Patient The propositus was diagnosed with dyshormonogenic congenital hypothyroidism with minimal 99mTc-pertechnetate accumulation in a eutopic thyroid gland. Design Propositus SLC5A5 gene was sequenced. Functional in vitro characterization of the novel NIS variant was performed. Results Sanger sequencing revealed a novel homozygous missense p.G561E NIS variant. Mechanistically, the G561E substitution reduces iodide uptake, because targeting of G561E NIS to the plasma membrane is reduced. Biochemical analyses revealed that G561E impairs the recognition of an adjacent tryptophan-acidic motif by the kinesin-1 subunit kinesin light chain 2 (KLC2), interfering with NIS maturation beyond the endoplasmic reticulum, and reducing iodide accumulation. Structural bioinformatic analysis suggests that G561E shifts the equilibrium of the unstructured tryptophan-acidic motif towards a more structured conformation unrecognizable to KLC2. Consistently, knockdown of Klc2 causes defective NIS maturation and consequently decreases iodide accumulation in rat thyroid cells. Morpholino knockdown of klc2 reduces thyroid hormone synthesis in zebrafish larvae leading to a hypothyroid state as revealed by expression profiling of key genes related to the hypothalamic-pituitary-thyroid axis. Conclusions We report a novel NIS pathogenic variant associated with dyshormonogenic congenital hypothyroidism. Detailed molecular characterization of G561E NIS uncovered the significance of KLC2 in thyroid physiology.

Bbs5 functions as the docking compartment of Bbsome binding to Klc3 regulated by Bbs5-Ser246 PKC-phosphorylation during mouse spermatogenesis

A mitochondrial and a fibrous sheath form the midpiece of the mammalian sperm flagellum encircling most of the axoneme. It has been documented that Kinesin light chain 3 (KLC3) was involved although the formation procedure remains unclear. Yeast-two-hybrid dataset showed an interaction between Klc3 and Bardet-Biedl Syndrome 5 (BBS5) Protein, another molecular associated with cilia and flagella forming. In this study, we presumed that the most conserved IFT complex BBsome was involved in spermatogenesis via the interaction of one of its subunits, Bbs5 with Klc3. Firstly, the interaction between Klc3 and Bbs5 was confirmed with Co-IP. Secondly, we identified PKC phosphorylation sites in vitro by LC-MS/MS, Ser19 and Ser246 of Bbs5, examined the phosphorylation status of Bbs5 Ser19 and Ser246 in mouse testis. Co-IP was performed to find which PKC isoforms phosphorylate Bbs5. In addition, we tried to discuss the roles of Ser19 and Ser246 of Bbs5 in the Klc3-bbs5 interaction and in mouse spermatogenesis based on our early findings.

KIF5A and KLC1 expression in Alzheimer’s disease: relationship and genetic influences

Background: Early disturbances in axonal transport, before the onset of gross neuropathology, occur in a spectrum of neurodegenerative diseases including Alzheimer’s disease. Kinesin superfamily motor proteins (KIFs) are responsible for anterograde protein transport within the axon of various cellular cargoes, including synaptic and structural proteins. Dysregulated KIF expression has been associated with AD pathology and genetic polymorphisms within kinesin-light chain-1 (KLC1) have been linked to AD susceptibility. We examined the expression of KLC1 in AD, in relation to that of the KLC1 motor complex (KIF5A) and to susceptibility genotypes. Methods: We analysed KLC1 and KIF5A gene and protein expression in midfrontal cortex from 47 AD and 39 control brains. Results: We found that gene expression of both KIF5A and KLC1 increased with Braak tangle stage (0-II vs III-IV and V-VI) but was not associated with significant change at the protein level. We found no effect of KLC1 SNPs on KIF5A or KLC1 expression but KIF5A SNPs that had previously been linked to susceptibility in multiple sclerosis were associated with reduced KIF5A mRNA expression in AD cortex. Conclusions: Future in vitro and in vivo studies are required to understand the cause of upregulated KIF5A and KLC-1 gene expression in AD and any potential downstream consequences on pathogenesis, including any contribution of genetic polymorphisms within the KIF5A gene locus.

The influence of kinesin light chain-2 on the radiosensitivity of non-small cell lung cancer cells and the underlying mechanisms.

e14512 Background: Our previous study found upregulated kinesin light chain-2 (KLC2) in non-small cell lung cancer (NSCLC) cell lines and tissues, which is associated with a poor prognosis. KLC2 promotes the migration ability of lung cancer cells. However, the influence of KLC2 on radiosensitivity of NSCLC has not yet been reported. Methods: Sensitivity of lung cancer cells to radiation was analyzed by colony formation, γH2AX immunofluorescent staining assay and neutral comet assay in vitro and a xenograft tumor model in vivo. Gene set enrichment analysis (GSEA), qRT-PCR and western blot assay were performed to predict and validate the potential target genes of KLC2. Results: We found that down-regulation of KLC2 could significantly improve the radio-sensitivity of lung cancer cells, while the overexpression of KLC2 had the opposite effect. GSEA identified p53 signal pathway as differentially enriched with high KLC2 expression, which was validated by western blot assay. Using data from TCGA, we found a positive correlation between the mRNA level of KLC2 and that of HuR. The expression of KLC2 mRNA and protein in lung cancer cells was significantly reduced when downregulating HuR. Interestingly, the overexpression of KLC2 could also significantly decrease the expression of HuR. Conclusions: The above results indicated that HuR could upregulate the expression of KLC2, which might decrease the phosphorylation of p53 and thereby weakened the radio-sensitivity of NSCLC cells. KLC2 could also positively regulate the expression of HuR as a positive feedback. Support: 81572279, 2016J004, LC2016PY016, 2018CR033