Michelle A. Utton

Stress‐Activated Protein Kinase/c‐Jun N‐Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule‐associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimers patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress‐activated protein kinase/c‐Jun N‐terminal kinase, a proline‐directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation‐dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase‐3, the c‐Jun N‐terminal kinase readily phosphorylates Thr205 and Ser422, which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase‐3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress‐activated/c‐Jun N‐terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline‐directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.

Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies.

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in the tauopathies, which include Alzheimers disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17), progressive supranuclear palsy, corticobasal degeneration and Picks disease. Tau isoform composition and cellular and regional distribution as well as morphology of these inclusions vary in each disorder. Recently, several pathological missense and exon 10 splice‐donor site mutations of the tau gene were iden‐tified in FTDP‐17. Exon 10 codes for the second of four microtubule‐binding repeat domains. The splice‐site mutations result in increased inclusion of exon 10 which causes a relative increase in tau isoforms containing four microtubule‐binding repeat domains over those containing three repeat domains. This could be a central aetiological mechanism in FTDP‐17 and, perhaps, other related tauopathies. We have investigated changes in the ratio and distribution of three‐repeat and four‐repeat tau in the different tauopathies as a basis of the phenotypic range of these disorders and the selective vulnerability of different subsets of neurones. In this study, we have developed two monoclonal antibodies, RD3 and RD4 that effectively distinguish these closely related tau isoforms. These new isoform‐specific antibodies are useful tools for analysing tau isoform expression and distribution as well as pathological changes in the human brain.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons

α-Synuclein is a major protein constituent of Lewy bodies and mutations in α-synuclein cause familial autosomal dominant Parkinsons disease. One explanation for the formation of perikaryal and neuritic aggregates of α-synuclein, which is a presynaptic protein, is that the mutations disrupt α-synuclein transport and lead to its proximal accumulation. We found that mutant forms of α-synuclein, either associated with Parkinsons disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, α-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinsons disease-associated α-synuclein mutants examined in this study might contribute to perikaryal accumulation of α-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

Reactivating Kinase/p38 Phosphorylates τ Protein In Vitro

Abstract: Neurofibrillary tangles, one of the major pathological hallmarks of Alzheimer‐diseased brains, consist primarily of aggregated paired helical filaments (PHFs) of hyperphosphorylated τ protein. τ from normal brain and especially from foetal brain is also phosphorylated on some of the sites phosphorylated in PHFs, mainly at serines or threonines followed by prolines. A number of protein kinases can phosphorylate τ in vitro; those that require or accept prolines include GSK3 and members of the mitogen‐activated protein (MAP) kinase family, ERK1, ERK2, and SAP kinase‐β/JNK. In this report, we show that another member of the MAP kinase family, the stress‐activated kinase p38/RK, can phosphorylate τ in vitro. Western blots with phosphorylation‐sensitive antibodies showed that p38, like ERK2 and SAP kinase‐β/JNK, phosphorylated τ at sites found phosphorylated physiologically (Thr181, Ser202, Thr205, and Ser396) and also at Ser422, which is phosphorylated in neurofibrillary tangles but not in normal adult or foetal brain. These findings support the possibility that cellular stress might contribute to τ hyperphosphorylation during the formation of PHFs, and hence, to the development of τ pathology.

Molecular motors implicated in the axonal transport of tau and alpha-synuclein.

Tau and α-synuclein are both proteins implicated in the pathology of neurodegenerative disease. Here we have investigated the mechanisms of axonal transport of tau and α-synuclein, because failure of axonal transport has been implicated in the development of several neurodegenerative disorders. We found that the transport of both of these proteins depend on an intact microtubule- but not actin-cytoskeleton, and that tau and α-synuclein both move at overall slow rates of transport. We used time-lapse video microscopy to obtain images of live neurons that had been transfected with plasmids expressing proteins tagged with enhanced green fluorescent protein. We found that particulate structures containing tau or α-synuclein travel rapidly when moving along axons but spend the majority of the time paused, and these structures have similar characteristics to those previously observed for neurofilaments. The motile particles containing tau or α-synuclein colocalise with the fast-transporting molecular motor kinesin-1 in neurons. Co-immunoprecipitation experiments demonstrate that tau and α-synuclein are each associated with complexes containing kinesin-1, whereas only α-synuclein appears to interact with dynein-containing complexes. In vitro glutathione S-transferase-binding assays using rat brain homogenate or recombinant protein as bait reveals a direct interaction of kinesin-1 light chains 1 and 2 with tau, but not with α-synuclein. Our findings suggest that the axonal transport of tau occurs via a mechanism utilising fast transport motors, including the kinesin family of proteins, and that α-synuclein transport in neurons may involve both kinesin and dynein motor proteins.

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.