Janet Brownlees

Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content

Amyotrophic lateral sclerosis (ALS) is a late-onset neurological disorder characterized by death of motoneurons. Mutations in Cu/Zn superoxide dismutase-1 (SOD1) cause familial ALS but the mechanisms whereby they induce disease are not fully understood. Here, we use time-lapse microscopy to monitor for the first time the effect of mutant SOD1 on fast axonal transport (FAT) of bona fide cargoes in living neurons. We analyzed FAT of mitochondria that are a known target for damage by mutant SOD1 and also of membrane-bound organelles (MBOs) using EGFP-tagged amyloid precursor protein as a marker. We studied FAT in motor neurons derived from SOD1G93A transgenic mice that are a model of ALS and also in cortical neurons transfected with SOD1G93A and three further ALS-associated SOD1 mutants. We find that mutant SOD1 damages transport of both mitochondria and MBOs, and that the precise details of this damage are cargo-specific. Thus, mutant SOD1 reduces transport of MBOs in both anterograde and retrograde directions, whereas mitochondrial transport is selectively reduced in the anterograde direction. Analyses of the characteristics of mitochondrial FAT revealed that reduced anterograde movement involved defects in anterograde motor function. The selective inhibition of anterograde mitochondrial FAT enhanced their net retrograde movement to deplete mitochondria in axons. Mitochondria in mutant SOD1 expressing cells also displayed features of damage. Together, such changes to mitochondrial function and distribution are likely to compromise axonal function. These alterations represent some of the earliest pathological features so far reported in neurons of mutant SOD1 transgenic mice.

Neurofilament heavy chain side arm phosphorylation regulates axonal transport of neurofilaments

Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.

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)-3β activity in the brain, we created transgenic mice harbouring wild-type GSK-3β genes or a mutant GSK-3β 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-3β activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3β activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3β is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3β were obtained and it is possible that high levels of GSK-3β activity are lethal.

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.

p38α stress-activated protein kinase phosphorylates neurofilaments and is associated with neurofilament pathology in amyotrophic lateral sclerosis

Abstract Neurofilament middle and heavy chains (NFM and NFH) are heavily phosphorylated on their carboxy-terminal side-arm domains in axons. The mechanisms that regulate this phosphorylation are complex. Here, we demonstrate that p38α, a member of the stress-activated protein kinase family, will phosphorylate NFM and NFH on their side-arm domains. Aberrant accumulations of neurofilaments containing phosphorylated NFM and NFH side-arms are a pathological feature of amyotrophic lateral sclerosis (ALS) and we also demonstrate that p38α and active forms of p38 family kinases are associated with these accumulations. This is the case for sporadic and familial forms of ALS and also in a transgenic mouse model of ALS caused by expression of mutant superoxide dismutase-1 (SOD1). Thus, p38 kinases may contribute to the aberrant phosphorylation of NFM and NFH side-arms in ALS.

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 Alzheimers 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 Alzheimers 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 Alzheimers disease.

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

Tau is a neuronal microtubule‐associated protein whose function is modulated by phosphorylation. GSK‐3β is a tau kinase. GSK‐3β is part of the wingless signalling pathway and stimulation by wingless is predicted to down‐regulate GSK‐3β 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‐3β‐mediated phosphorylation of tau in transfected CHO cells. We find that co‐transfection with dishevelled‐1 is inhibitory to GSK‐3β‐mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimers disease and the possible relevance of these findings to Alzheimers disease pathogenesis are discussed.

Neuropathological Abnormalities in Transgenic Mice Harbouring a Phosphorylation Mutant Neurofilament Transgene

Abstract: 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.

Gigaxonin is associated with the Golgi and dimerises via its BTB domain

Mutations in the gigaxonin gene cause giant axonal neuropathy. The amino-terminus of gigaxonin contains a BTB domain but no binding partners for this domain have so far been identified. Here, we demonstrate that the gigaxonin BTB domain forms homodimers. Other BTB-bearing proteins have also been shown to dimerise via their BTB domains with the dimers then capable of interacting with other ligands. Thus, the gigaxonin BTB domain may function in a similar manner. We also demonstrate that gigaxonin is expressed in a wide variety of neuronal cell types where a significant proportion exists in cell bodies. Confocal microscope studies of gigaxonin-transfected COS-7 cells and cultured neurones revealed that a proportion of gigaxonin localises to the Golgi and endoplasmic reticulum.

Role(s) of mitogen and stress-activated kinases in neurodegeneration

Neurones respond to extracellular stimuli via a variety of intracellular signaling pathways. Some of the most intensely studied of these are those involving the mitogen-activated protein kinases (MAPKs). MAPKs are serine/threonine protein kinases and their substrates include both cytoplasmic and nuclear targets. The founder members of the MAPK family are the p42/p44 MAPKs or extracellular-regulated kinases (ERKs). In non-neuronal cells, these kinases are mainly activated by mitogenic stimuli, but in neurones, such signals include those emanating from neurotrophic and neurotransmitter receptors.