Glutamate slows axonal transport of neurofilaments in transfected neurons.

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein-tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.

Distinct responses of the herpes simplex virus and varicella zoster virus immediate early promoters to the cellular transcription factors Brn-3a and Brn-3b.

The related viruses herpes simplex virus (HSV) and varicella zoster virus (VZV) show distinct but related patterns of latent infection and reactivation in human sensory ganglia. The cellular POU family transcription factors Brn-3a and Brn-3b are expressed in sensory ganglia and bind to the TAATGARAT (R stands for purine) regulatory motifs in the immediate-early gene promoters of these viruses. We show that Brn-3a activates the full length HSV IE1 promoter whereas Brn-3b represses its activity. In contrast both Brn-3a and Brn-3b activate the full length VZV IE promoter. The response of the full length VZV promoter to Brn-3b is not observed with a minimal VZV immediate-early promoter lacking any TAATGARAT elements and cannot be restored by addition of either the upstream TAATGARAT-containing region of the HSV IE promoter or a VZV TAATGARAT-like element to this minimal promoter. The unique effect of Brn-3b on the full length VZV immediate early gene promoter may play a key role in the distinct pattern of latent infection and reactivation observed with this virus in vivo.

Overexpression of the mouse dishevelled-1 protein inhibits GSK-3beta-mediated phosphorylation of tau in transfected mammalian cells.

Tau is a neuronal microtubule-associated protein whose function is modulated by phosphorylation. GSK-3beta is a tau kinase. GSK-3beta is part of the wingless signalling pathway and stimulation by wingless is predicted to down-regulate GSK-3beta activity. In Drosophila imaginal disc cells, overexpression of dishevelled, a component of the wingless pathway, mimics the wingless signal. We have therefore studied the effect that overexpression of the murine dishevelled-1 protein has on GSK-3beta-mediated phosphorylation of tau in transfected CHO cells. We find that co-transfection with dishevelled-1 is inhibitory to GSK-3beta-mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimer's disease and the possible relevance of these findings to Alzheimer's disease pathogenesis are discussed.

Tau phosphorylation in transgenic mice expressing glycogen synthase kinase-3beta transgenes.

In order to investigate the effect on tau of manipulating glycogen synthase kinase (GSK)-3beta activity in the brain, we created transgenic mice harbouring wild-type GSK-3beta genes or a mutant GSK-3beta that is predicted to be more active. Transgene-derived mRNAs were detected in the brains of a number of the transgenic mouse lines and several of these transgenic lines displayed transgenic GSK-3beta activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3beta activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3beta is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3beta were obtained and it is possible that high levels of GSK-3beta activity are lethal.

Purification and immunochemical studies of dipeptidyl peptidase IV from bovine kidney.

DPP IV from bovine kidney has been purified and characterized by molecular mass, pI, pH optimum and sensitivity to inhibitors. Polyclonal antibodies to the enzyme protein were used to show the location of dipeptidyl peptidase IV in microvillar membranes of bovine kidney using immunogold labelling by secondary antibody. The same antibodies reacted to DPP IV in Western blots of rat, pig and bovine kidney tissue, but failed to recognise the enzyme in bovine brain. This suggests that dipeptidyl peptidase IV may exist as different organ-specific rather than species-specific proteins.

Neuropathological abnormalities in transgenic mice harbouring a phosphorylation mutant neurofilament transgene.

Ser55 within the head domain of neurofilament light chain (NF-L) is a target for phosphorylation by protein kinase A. To understand further the physiological role(s) of NF-L Ser55 phosphorylation, we generated transgenic mice with a mutant NF-L transgene in which Ser55 was mutated to Asp so as to mimic permanent phosphorylation. Two lines of NF-L(Asp) mice were created and these animals express the transgene in many neurones of the central and peripheral nervous systems. Both transgenic lines display identical, early onset, and robust pathological changes in the brain. These involve the formation of NF-L(Asp)-containing perikaryal neurofilament inclusion bodies and the development of swollen Purkinje cell axons. Development of these pathologies was rapid and fully established in mice as young as 4 weeks of age. The two transgenic lines show no elevation of NF-L, neurofilament middle chain (NF-M), or neurofilament heavy chain (NF-H), and transgenic NF-L(Asp) represents only a minor proportion of total NF-L protein. Because other published transgenic lines expressing higher levels of wild-type NF-L do not exhibit phenotypic changes that in any way resemble those in the NF-L(Asp) mice and because the two different NF-L(Asp) transgenic lines display identical neuropathological changes, it is likely that the pathological alterations observed in the NF-L(Asp) mice are the result of properties of the mutant NF-L. These results support the notion that phosphorylation of Ser55 is a mechanism for regulating neurofilament organisation in vivo.

Axonal transport of neurofilaments in normal and disease states.

Neurofilaments are among the most abundant organelles in neurones. They are synthesised in cell bodies and then transported into and through axons by a process termed 'slow axonal transport' at a rate that is distinct from that driven by conventional fast motors. Several recent studies have now demonstrated that this slow rate of transport is actually the consequence of conventional fast rates of movement that are interrupted by extended pausing. At any one time, most neurofilaments are thus stationary. Accumulations of neurofilaments are a pathological feature of several human neurodegenerative diseases suggesting that neurofilament transport is disrupted in disease states. Here, we review recent advances in our understanding of neurofilament transport in both normal and disease states. Increasing evidence suggests that phosphorylation of neurofilaments is a mechanism for regulating their transport properties, possibly by promoting their detachment from the motor(s). In some neurodegenerative diseases, signal transduction mechanisms involving neurofilament kinases and phosphatases may be perturbed leading to disruption of transport.

Mint2/X11-like colocalizes with the Alzheimer's disease amyloid precursor protein and is associated with neuritic plaques in Alzheimer's disease.

Aberrant metabolism of the amyloid precursor protein (APP) is believed to be at least part of the pathogenic process in Alzheimer's disease. The carboxy-terminus of APP has been shown to interact with the Mint/X11 family of phosphotyrosine binding (PTB) domain-bearing proteins. It is via their PTB domains that the Mints/X11s bind to APP. Here we report the cloning of full-length mouse Mint2 and demonstrate that in primary cortical neurons, Mint2 and APP share highly similar distributions. Mint2 also colocalizes with APP in transfected CHO cells. In Mint2/APP-cotransfected cells, Mint2 reorganizes the subcellular distribution of APP and also increases the steady-state levels of APP. Finally, we demonstrate that Mint2 is associated with the neuritic plaques found in Alzheimer's disease but not with neurofibrillary tangles. These results are consistent with a role for Mint2 in APP metabolism and trafficking, and suggest a possible role for the Mints/X11s in the pathogenesis of Alzheimer's disease.

p35/cdk5 binds and phosphorylates beta-catenin and regulates beta-catenin/presenilin-1 interaction.

The neuronal cyclin-dependent kinase p35/cdk5 comprises a catalytic subunit (cdk5) and an activator subunit (p35). To identify novel p35/cdk5 substrates, we utilized the yeast two-hybrid system to screen for human p35 binding partners. From one such screen, we identified beta-catenin as an interacting protein. Confirmation that p35 binds to beta-catenin was obtained by using glutathione S-transferase (GST)-beta-catenin fusion proteins that interacted with both endogenous and transfected p35, and by showing that beta-catenin was present in p35 immunoprecipitates. p35 and beta-catenin also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that p35/cdk5 phosphorylates beta-catenin. beta-catenin also binds to presenilin-1 and altered beta-catenin/presenilin-1 interactions may be mechanistic in Alzheimer's disease (AD). Abnormal p35/cdk5 activity has also been suggested to contribute to AD. We therefore investigated how modulation of p35/cdk5 activity influenced beta-catenin/presenilin-1 interactions. Inhibition of p35/cdk5 with roscovitine did not alter the steady state levels of either beta-catenin or presenilin-1 but reduced the amount of presenilin-1 bound to beta-catenin. Thus, p35/cdk5 binds and phosphorylates beta-catenin and regulates its binding to presenilin-1. The findings reported here therefore provide a novel molecular framework to connect p35/cdk5 with beta-catenin and presenilin-1 in AD.

Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3).

Threonine(668) (thr(668)) within the carboxy-terminus of the Alzheimer's disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr(668) is believed to regulate APP function and metabolism. Thr(668) 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-3 beta, p42 mitogen-activated protein kinase and stress-activated protein kinase-1b, to phosphorylate APPthr(668) 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.

Phosphorylation of neurofilament heavy chain side-arms by stress activated protein kinase-1b/Jun N-terminal kinase-3.

Neurofilaments comprise three subunit proteins; neurofilament light, middle and heavy chains (NF-L, NF-M and NF-H). The carboxy-terminal domains of NF-M and NF-H form side-arms that project from the filament and that of NF-H contains multiple repeats of the motif lys-ser-pro, the serines of which are targets for phosphorylation. The level of phosphorylation on the lys-ser-pro repeats varies topographically within the cell; in cell bodies and proximal axons, the side-arms are largely non-phosphorylated whereas in more distal regions of axons, the side-arms are heavily phosphorylated. Here we show that stress activated protein kinase 1b (SAPK1b), a major SAPK in neurones will phosphorylate NF-H side-arms both in vitro and in transfected cells. These studies suggest that SAPK1b targets multiple phosphorylation sites within NF-H side-arms. Additionally, we show that glutamate treatment induces activation of SAPK1b in primary cortical neurones and increased phosphorylation of NF-H in cell bodies. This suggests that glutamate causes increased NF-H phosphorylation at least in part by activation of stress activated protein kinases.