Gerardo Morfini

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane‐bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin‐based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule‐stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

Reelin-mediated Signaling Locally Regulates Protein Kinase B/Akt and Glycogen Synthase Kinase 3β

Reelin is a large secreted protein that controls cortical layering by signaling through the very low density lipoprotein receptor and apolipoprotein E receptor 2, thereby inducing tyrosine phosphorylation of the adaptor protein Disabled-1 (Dab1) and suppressing tau phosphorylation in vivo. Here we show that binding of Reelin to these receptors stimulates phosphatidylinositol 3-kinase, resulting in activation of protein kinase B and inhibition of glycogen synthase kinase 3β. We present genetic evidence that this cascade is dependent on apolipoprotein E receptor 2, very low density lipoprotein receptor, and Dab1. Reelin-signaling components are enriched in axonal growth cones, where tyrosine phosphorylation of Dab1 is increased in response to Reelin. These findings suggest that Reelin-mediated phosphatidylinositol 3-kinase signaling in neuronal growth cones contributes to final neuron positioning in the mammalian brain by local modulation of protein kinase B and glycogen synthase kinase 3β kinase activities.

Alzheimer's Presenilin 1 Mutations Impair Kinesin-Based Axonal Transport

Several lines of evidence indicate that alterations in axonal transport play a critical role in Alzheimers disease (AD) neuropathology, but the molecular mechanisms that control this process are not understood fully. Recent work indicates that presenilin 1 (PS1) interacts with glycogen synthase kinase 3β (GSK3β). In vivo, GSK3β phosphorylates kinesin light chains (KLC) and causes the release of kinesin-I from membrane-bound organelles (MBOs), leading to a reduction in kinesin-I driven motility (Morfini et al., 2002b). To characterize a potential role for PS1 in the regulation of kinesin-based axonal transport, we used PS1-/- and PS1 knock-inM146V (KIM146V) mice and cultured cells. We show that relative levels of GSK3β activity were increased in cells either in the presence of mutant PS1 or in the absence of PS1 (PS1-/-). Concomitant with increased GSK3β activity, relative levels of KLC phosphorylation were increased, and the amount of kinesin-I bound to MBOs was reduced. Consistent with a deficit in kinesin-I-mediated fast axonal transport, densities of synaptophysin- and syntaxin-I-containing vesicles and mitochondria were reduced in neuritic processes of KIM146V hippocampal neurons. Similarly, we found reduced levels of PS1, amyloid precursor protein, and synaptophysin in sciatic nerves of KIM146V mice. Thus PS1 appears to modulate GSK3β activity and the release of kinesin-I from MBOs at sites of vesicle delivery and membrane insertion. These findings suggest that mutations in PS1 may compromise neuronal function by affecting GSK-3 activity and kinesin-I-based motility.

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta.

The pathological mechanism by which Aβ causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimers disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric Aβ (oAβ) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oAβ showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oAβ was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oAβ on FAT. Both oAβ and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD.

Fast axonal transport misregulation and Alzheimer's disease.

Pathological alterations in the microtubule-associated protein (MAP) tau are well-established in a number of neurodegenerative disorders, including Alzheimer’s Disease (AD), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), and others. Tau protein and in some cases, neurofilament subunits exhibit abnormal phosphorylation on specific serine and threonine residues in these diseases. A large body of biochemical, genetic, and cell biological evidence implicate two major serine-threonine protein kinases, glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase 5 (CDK5) as major kinases responsible for both normal and pathological phosphorylation of tau protein in vivo. What remains unclear is whether tau phosphorylation and/or neurofibrillary tangle (NFT) formation are causal or secondary to initiation of neuronal pathology. In fact, many studies have indicated that tau misphosphorylation is not the causal event. Interestingly, some of these kinase and phosphatase activities have recently merged as key regulators of fast axonal transport (FAT). Specifically, CDK5 and GSK-3 have been recently shown to regulate kinesin-driven motility. Given the essential role of FAT in neuronal function, an alternate model for pathogenesis can be proposed. In this model, misregulation of FAT induced by an imbalance in specific kinase-phosphatase activities within neurons represents an early and critical step for the initiation of neuronal pathology. Such a model may explain many of the unique characteristics of late onset of neurological diseases such as AD.

Pictures in Cell Biology:GSK-3 and regulation of kinesin function

Kinesins mediate trafficking of membrane-bounded organelles towards microtubule plus-ends. Different proteins and cargos are targeted for different locations, but, until recently, little was known about regulation of kinesin-based motility. Phosphorylation of kinesin light chains by glycogen synthase kinase 3 (GSK-3) [1xGlycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. Morfini, G et al. EMBO J. 2002; 23: 281–293Crossref | Scopus (261)See all References[1] has now been shown to provide one such regulatory mechanism. GSK-3 inhibits anterograde, but not retrograde, transport in squid axoplasm and reduces amounts of kinesin bound to membranes. Kinesin activities other than cargo binding are unaffected by GSK-3 phosphorylation. As predicted, active GSK-3 is localized preferentially in regions known to be sites of membrane delivery.In this set of images (Fig. 1Fig. 1), distributions for tubulin [green; (c)] and total GSK-3 [red; (a)] are compared in a rat hippocampal neuron and oligodendrocyte. Microtubule-containing regions in both neurons and glia co-label for GSK-3 [yellow (b)]. This distribution is similar to that seen for kinesin [1.xGlycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility. Morfini, G et al. EMBO J. 2002; 23: 281–293Crossref | Scopus (261)See all References, 2.xMonoclonal antibodies to kinesin heavy and light chains stain vesicle-like structures, but not microtubules, in cultured cells. Pfister, K.K et al. J. Cell Biol. 1989; 108: 1453–1463Crossref | PubMedSee all References]. However, in locations where membrane proteins are delivered, such as tips of elongating processes (i.e. growth cones) or membrane sheets of oligodendrocytes, GSK-3 immunoreactivity is distinct from that of microtubules. Moreover, the GSK-3 in growth cones and similar regions is preferentially activated, consistent with a role for GSK-3 phosphorylation of kinesin in regulating delivery of some membrane proteins to specific subcellular domains.Fig. 1Localization of glycogen synthase kinase 3 [GSK-3; (a)] and tubulin (c) in a rat hippocampal neuron and oligodendrocyte. In the merged image (b), GSK-3 is shown in red and tubulin in green; colocalization is shown in yellow. Rat hippocampal cultures were a generous gift of Ann Marie Craig, Washington University, St Louis, MO, USA.View Large Image | Download PowerPoint Slide