Understanding the mechanisms that control processing of the amyloid precursor protein (APP) to produce amyloid-β (Aβ) peptide represents a key area of Alzheimer's disease research. Here, we show that siRNA-mediated loss of calsyntenin-1 in cultured neurons alters APP processing to increase production of Aβ. We also show that calsyntenin-1 is reduced in Alzheimer's disease brains and that the extent of this reduction correlates with increased Aβ levels. Calsyntenin-1 is a ligand for kinesin-1 light chains and APP is transported through axons on kinesin-1 molecular motors. Defects in axonal transport are an early pathological feature in Alzheimer's disease and defective APP transport is known to increase Aβ production. We show that calsyntenin-1 and APP are co-transported through axons and that siRNA-induced loss of calsyntenin-1 markedly disrupts axonal transport of APP. Thus, perturbation to axonal transport of APP on calsyntenin-1 containing carriers induces alterations to APP processing that increase production of Aβ. Together, our findings suggest that disruption of calsyntenin-1-associated axonal transport of APP is a pathogenic mechanism in Alzheimer's disease.

A proline-to-serine substitution at position 56 in the gene encoding vesicle-associated membrane protein-associated protein B (VAPB; VAPBP56S) causes some dominantly inherited familial forms of motor neuron disease, including amyotrophic lateral sclerosis (ALS) type-8. Here, we show that expression of ALS mutant VAPBP56S but not wild-type VAPB in neurons selectively disrupts anterograde axonal transport of mitochondria. VAPBP56S-induced disruption of mitochondrial transport involved reductions in the frequency, velocity and persistence of anterograde mitochondrial movement. Anterograde axonal transport of mitochondria is mediated by the microtubule-based molecular motor kinesin-1. Attachment of kinesin-1 to mitochondria involves the outer mitochondrial membrane protein mitochondrial Rho GTPase-1 (Miro1) which acts as a sensor for cytosolic calcium levels ([Ca2+]c); elevated [Ca2+]c disrupts mitochondrial transport via an effect on Miro1. To gain insight into the mechanisms underlying the VAPBP56S effect on mitochondrial transport, we monitored [Ca2+]c levels in VAPBP56S-expressing neurons. Expression of VAPBP56S but not VAPB increased resting [Ca2+]c and this was associated with a reduction in the amounts of tubulin but not kinesin-1 that were associated with Miro1. Moreover, expression of a Ca2+ insensitive mutant of Miro1 rescued defective mitochondrial axonal transport and restored the amounts of tubulin associated with the Miro1/kinesin-1 complex to normal in VAPBP56S-expressing cells. Our results suggest that ALS mutant VAPBP56S perturbs anterograde mitochondrial axonal transport by disrupting Ca2+ homeostasis and effecting the Miro1/kinesin-1 interaction with tubulin.

Defective transport of mitochondria in axons is implicated in the pathogenesis of several age-associated neurodegenerative diseases. However, the regulation and function of axonal mitochondrial motility during normal ageing is poorly understood. Here, we use novel imaging procedures to characterise axonal transport of these organelles in the adult Drosophila wing nerve. During early adult life there is a boost and progressive decline in the proportion of mitochondria that are motile, which is not due to general changes in cargo transport. Experimental inhibition of the mitochondrial transport machinery specifically in adulthood accelerates the appearance of focal protein accumulations in ageing axons, which is suggestive of defects in protein homeostasis. Unexpectedly, lowering levels of Lissencephaly-1 (Lis1), a dynein motor co-factor, augments axonal mitochondrial transport in ageing wing neurons. Lis1 mutations suppress focal protein accumulations in ageing neurons, including those caused by interfering with the mitochondrial transport machinery. Our data provide new insights into the dynamics of mitochondrial motility in adult neurons in vivo, identify Lis1 as a negative regulator of transport of these organelles, and provide evidence of a link between mitochondrial movement and neuronal protein homeostasis.

SummaryKinesin light chain 1 (KLC1) binds to the intracellular cytoplasmic domain of the type-1 membrane-spanning protein calsyntenin-1 (also known as alcadein-a) to mediate transport of a subset of vesicles. Here, we identify serine 460 in KLC1 (KLC1ser460) as a phosphorylation site and show that mutation of KLC1ser460 influences the binding of KLC1 to calsyntenin-1. Mutation of KLC1ser460 to an alanine residue, to preclude phosphorylation, increased the binding of calsyntenin-1, whereas mutation to an aspartate residue, to mimic permanent phosphorylation, reduced the binding. Mutation of KLC1ser460 did not affect the interaction of KLC1 with four other known binding partners: huntingtin-associated protein 1 isoform A (HAP1A), collapsin response mediator protein-2 (CRMP2), c-Jun N-terminal kinase-interacting protein-1 (JIP1) and kinase-D-interacting substrate of 220 kDa (Kidins220). KLC1ser460 is a predicted mitogen-activated protein kinase (MAPK) target site, and we show that extracellular-signal-regulated kinase (ERK) phosphorylates this residue in vitro. We also demonstrate that inhibition of ERK promotes binding of calsyntenin-1 to KLC1. Finally, we show that expression of the KLC1ser460 mutant proteins influences calsyntenin-1 distribution and transport in cultured cells. Thus, phosphorylation of KLC1ser460 represents a mechanism for selectively regulating the binding and trafficking of calsyntenin-1.

A recent genome wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9 but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3β (GSK3β). KLC2 phosphorylation by GSK3β induces release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3β on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-β (TGFβ) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that siRNA loss of LMTK2 not only reduces binding of Smad2 to KLC2 but also inhibits TGFβ-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.