Kwok-Fai Lau

ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43.

Mitochondria and the endoplasmic reticulum (ER) form tight structural associations and these facilitate a number of cellular functions. However, the mechanisms by which regions of the ER become tethered to mitochondria are not properly known. Understanding these mechanisms is not just important for comprehending fundamental physiological processes but also for understanding pathogenic processes in some disease states. In particular, disruption to ER–mitochondria associations is linked to some neurodegenerative diseases. Here we show that the ER-resident protein VAPB interacts with the mitochondrial protein tyrosine phosphatase-interacting protein-51 (PTPIP51) to regulate ER–mitochondria associations. Moreover, we demonstrate that TDP-43, a protein pathologically linked to amyotrophic lateral sclerosis and fronto-temporal dementia perturbs ER–mitochondria interactions and that this is associated with disruption to the VAPB–PTPIP51 interaction and cellular Ca2+ homeostasis. Finally, we show that overexpression of TDP-43 leads to activation of glycogen synthase kinase-3β (GSK-3β) and that GSK-3β regulates the VAPB–PTPIP51 interaction. Our results describe a new pathogenic mechanism for TDP-43.

Phosphorylation of thr668 in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3)

Threonine668 (thr668) within the carboxy‐terminus of the Alzheimers disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr668 is believed to regulate APP function and metabolism. Thr668 precedes a proline, which suggests that it is targeted for phosphorylation by proline‐directed kinase(s). We have investigated the ability of four major neuronally active proline‐directed kinases, cyclin dependent protein kinase‐5, glycogen synthase kinase‐3β, p42 mitogen‐activated protein kinase and stress‐activated protein kinase‐1b, to phosphorylate APPthr668 and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.

The c-Abl Tyrosine Kinase Phosphorylates the Fe65 Adaptor Protein to Stimulate Fe65/Amyloid Precursor Protein Nuclear Signaling

The amyloid precursor protein (APP) is proteolytically processed to release a C-terminal domain that signals to the nucleus to regulate transcription of responsive genes. The APP C terminus binds to a number of phosphotyrosine binding (PTB) domain proteins and one of these, Fe65, stimulates APP nuclear signaling. Fe65 is an adaptor protein that contains a number of protein-protein interaction domains. These include two PTB domains, the second of which binds APP, and a WW domain that binds proline-rich ligands. One ligand for the Fe65WW domain is the tyrosine kinase c-Abl. Here, we show that active c-Abl stimulates APP/Fe65-mediated gene transcription and that this effect is mediated by phosphorylation of Fe65 on tyrosine 547 within its second PTB domain. The homologous tyrosine within the motif Tyr-(Leu/Met)-Gly is conserved in a variety of PTB domains, and this suggests that PTB tyrosine phosphorylation occurs in other proteins. As such, PTB domain phosphorylation may represent a novel mechanism for regulating the function of this class of protein.

Cyclin-Dependent Kinase-5/p35 Phosphorylates Presenilin 1 to Regulate Carboxy-Terminal Fragment Stability

Mutations in the Presenilin 1 gene are the cause of the majority of autosomal dominant familial forms of Alzheimers disease. Presenilin 1 (PS1) is produced as a holoprotein but is then rapidly processed to amino- (N-PS1) and carboxy-terminal (C-PS1) fragments that are incorporated into stable high molecular mass complexes. The mechanisms that control PS1 cleavage and stability are not properly understood but sequences within C-PS1 have been shown to regulate both of these properties. Here we demonstrate that cyclin dependent kinase-5/p35 (cdk5/p35) phosphorylates PS1 on threonine(354) within C-PS1 both in vitro and in vivo. Threonine(354) phosphorylation functions to selectively stabilize C-PS1. Our results demonstrate that cdk5/p35 is a regulator of PS1 metabolism.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

Glycogen synthase kinase-3β-mediated tau phosphorylation in cultured cell lines

To study further the role of glycogen synthase kinase-3beta on tau phosphorylation, glycogen synthase kinase-3beta and tau expression vectors were co-transfected into CHO-K1, COS-7 and SH-SY5Y cell. Tau phosphorylation was assessed by phosphorylation-dependent antibodies AT-8, AT-180, AT-270 and PHF-1. The AT-270 and AT-8 epitopes were consistently phosphorylated by glycogen synthase kinase-3beta in the three cell lines. Phosphorylation on AT-180 epitope was significant in CHO-K1 and SH-SY5Y cells while PHF-1 epitope was hyper-phosphorylated only in SH-SY5Y cells. We also found that lithium induces phosphorylation of the serine 9 residue of glycogen synthase kinase-3beta together with inhibition of tau phosphorylation on PHF-1 epitope in all the three cell lines. This suggests a novel mechanism whereby lithium-mediated inhibition of GSK-3beta activity influences tau phosphorylation.

Expression of the Fe65 adapter protein in adult and developing mouse brain

Fe65 is a multimodular adaptor protein expressed mainly in the nervous system. Fe65 binds to the Alzheimers disease amyloid precursor protein (APP) and the interaction is mediated via a phosphotyrosine binding domain in Fe65 and the carboxy-terminal cytoplasmic domain of APP. Fe65 modulates trafficking and processing of APP, including production of the beta-amyloid peptide that is believed to be central to the pathogenesis of Alzheimers disease. Fe65 also facilitates translocation of a carboxy-terminal fragment of APP to the nucleus and is required for APP-mediated transcription events. In addition, Fe65 functions in regulation of the actin cytoskeleton and cell movement. Here we report the distribution profile of Fe65 immunoreactivity in adult mouse brain. Fe65 expression was found to be widespread in neurones in adult brain. The areas of highest expression included regions of the hippocampus in which the earliest abnormalities of Alzheimers disease are detectable. Fe65 was also highly expressed in the cerebellum, thalamus and selected brain stem nuclei. Fe65 was evident in a sub-set of astrocytes within the stratum oriens and radiatum in the hippocampus. Expression of Fe65 was found to be developmentally regulated with levels reducing after embryonic day 15 and increasing again progressively from post-partum day 10 up to adulthood, a developmental pattern that partially parallels that of APP. These data indicate a widespread distribution of Fe65 in neurones throughout mouse brain and also suggest that Fe65 may have functions independent of APP and any potential role in the pathogenesis of Alzheimers disease.

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11α participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11α, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11α/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathioneS-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11α and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11α inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11α binding to CCS is inhibitory to SOD1 activation. X11α also interacts with another copper-binding protein found in neurons, the Alzheimers disease amyloid precursor protein. Thus, X11α may participate in copper homeostasis within neurons.

X11β rescues memory and long-term potentiation deficits in Alzheimer's disease APPswe Tg2576 mice

Increased production and deposition of amyloid beta-protein (Abeta) are believed to be key pathogenic events in Alzheimers disease. As such, routes for lowering cerebral Abeta levels represent potential therapeutic targets for Alzheimers disease. X11beta is a neuronal adaptor protein that binds to the intracellular domain of the amyloid precursor protein (APP). Overexpression of X11beta inhibits Abeta production in a number of experimental systems. However, whether these changes to APP processing and Abeta production induced by X11beta overexpression also induce beneficial effects to memory and synaptic plasticity are not known. We report here that X11beta-mediated reduction in cerebral Abeta is associated with normalization of both cognition and in vivo long-term potentiation in aged APPswe Tg2576 transgenic mice that model the amyloid pathology of Alzheimers disease. Overexpression of X11beta itself has no detectable adverse effects upon mouse behaviour. These findings support the notion that modulation of X11beta function represents a therapeutic target for Abeta-mediated neuronal dysfunction in Alzheimers disease.

The neuronal adaptor protein Fe65 is phosphorylated by mitogen-activated protein kinase (ERK1/2)

Fe65 is a neuronal adaptor protein that binds a number of ligands and which functions in both gene transcription/nuclear signalling and in the regulation of cell migration and motility. These different functions within the nucleus and at the cell surface are mediated via Fe65s different binding partners. An Fe65/APP/TIP60 complex is transcriptionally active within the nucleus and an Fe65/APP/Mena complex probably regulates actin dynamics in lamellipodia. The mechanisms that regulate these different Fe65 functions are unclear. Here, we demonstrate that Fe65 is a phosphoprotein and, using mass spectrometry sequencing, identify for the first time in vivo phosphorylation sites in Fe65. We also show that Fe65 is a substrate for phosphorylation by the mitogen-activated protein kinases ERK1/2. Our results provide a mechanism by which Fe65 function may be modulated to fulfil its various roles.