G Gibb

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.

In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase‐3β

Abstract: The two pathological lesions found in the brains of Alzheimers disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule‐associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β‐amyloid protein, a 4‐kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline‐directed protein kinases, glycogen synthase kinase‐3β (GSK‐3β) and mitogen‐activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK‐3β but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK‐3β was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu‐C digestion of APPcyt and peptide sequencing. The ability of GSK‐3β to phosphorylate APPcyt and τ provides a putative link between the two lesions and indicates a critical role of GSK‐3β in the pathogenesis of Alzheimers disease.

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.

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.

Regional Distribution of Amyloid-Bri Deposition and Its Association with Neurofibrillary Degeneration in Familial British Dementia

Familial British dementia (FBD), pathologically characterized by cerebral amyloid angiopathy (CAA), amyloid plaques, and neurofibrillary degeneration, is associated with a stop codon mutation in the BRI gene resulting in the production of an amyloidogenic fragment, amyloid-Bri (ABri). The aim of this study was to assess the distribution of ABri fibrillar and nonfibrillar lesions and their relationship to neurofibrillary pathology, astroglial and microglial response using immunohistochemistry, confocal microscopy, and immunoelectron microscopy in five cases of FBD. Abnormal tau was studied with immunoblotting. We present evidence that ABri is deposited throughout the central nervous system in blood vessels and parenchyma where both amyloid (fibrillar) and pre-amyloid (nonfibrillar) lesions are formed. Ultrastructurally amyloid lesions appear as bundles of fibrils recognized by an antibody raised against ABri, whereas Thioflavin S-negative diffuse deposits consist of amorphous electron-dense material with sparse, dispersed fibrils. In contrast to nonfibrillar lesions, fibrillar ABri is associated with a marked astrocytic and microglial response. Neurofibrillary tangles and neuropil threads occurring mainly in limbic structures, are found in areas affected by all types of ABri lesions whereas abnormal neurites are present around amyloid lesions. Immunoblotting for tau revealed a triplet electrophoretic migration pattern. Our observations confirm a close link between ABri deposition and neurodegeneration in FBD.

The complex relationship between soluble and insoluble tau in tauopathies revealed by efficient dephosphorylation and specific antibodies

Phosphorylated tau is deposited as insoluble inclusion bodies in the tauopathies. We have used a new efficient method to dephosphorylate tau extracted from control and tauopathy brain. In some tauopathies, including Alzheimers disease and progressive supranuclear palsy, the pattern of insoluble tau isoforms reflected that of soluble tau. In contrast, in corticobasal degeneration, Picks disease, and some forms of fronto‐temporal dementia, specific tau isoforms were selectively sequestered into insoluble inclusion‐forming tau. Therefore the overall expression of individual tau isoforms does not predict which tau isoforms are deposited in all tauopathies and different mechanisms must operate that result in the deposition of specific tau isoforms.

Effects of FTDP-17 mutations on the in vitro phosphorylation of tau by glycogen synthase kinase 3β identified by mass spectrometry demonstrate certain mutations exert long-range conformational changes

In vitro phosphorylation of recombinant wild‐type 2N4R tau and FTDP‐17 exonic mutant forms P301L, V337M and R406W by glycogen synthase kinase 3β (GSK3β) was examined by two dimensional phosphopeptide mapping analysis on thin layer cellulose plates. Comparison of these peptide maps with those generated from wild‐type 1N4R tau isoform from which the phosphopeptide constituents and sites of phosphorylation had been determined previously, enabled us to monitor directly changes in phosphorylation of the individual tau proteins. No differences were found in the phosphorylation of wild‐type, P301L or V337M tau by GSK3β but the R406W mutant showed at least two clear differences from the other three tau proteins. The peptides, identified by mass spectrometry corresponding to phosphorylation at both threonine 231 and serine 235 (spot 3), serines 396, 400 and 404 (spot 6a) and serines 195 and 199 (spot 6b) were absent from the R406W peptide map. The findings imply that the R406W mutation in tau exerts long‐range conformational effects on the structure of tau.

Chromosome 13 dementia syndromes as models of neurodegeneration

Two hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the central nervous system and neurodegeneration. The two amyloid proteins, ABri and ADan, are degradation products of the same precursor molecule BriPP bearing different genetic defects, namely a Stop-to-Arg mutation in FBD and a ten-nucleotide duplication-insertion immediately before the stop codon in FDD. Both de novo created amyloid peptides have the same length (34 amino acids) and the same post-translational modification (pyroglutamate) at their N-terminus. Neurofibrillary tangles containing the classical paired helical filaments as well as neuritic components in many instances co-localize with the amyloid deposits. In both disorders, the pattern of hyperphosphorylatedtau immunoreactivity is almost indistinguishable from that seen in Alzheimers disease. These issues argue for the primary importance of the amyloid deposits in the mechanism(s) of neuronal cell loss. We propose FBD and FDD, the chromosome 13 dementia syndromes, as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain.

Differential effects of apolipoprotein E isoforms on phosphorylation at specific sites on tau by glycogen synthase kinase‐3β identified by nano‐electrospray mass spectrometry

Previously published data have shown an allele‐specific variation in the in vitro binding of apolipoprotein E (apoE) to tau, which prompted the hypothesis that apoE binding may protect tau from phosphorylation, apoE3 being more efficient than apoE4. We have, therefore, investigated the effects of apoE on tau phosphorylation in vitro by the proline‐directed kinase, glycogen synthase kinase (GSK)‐3β. The phosphopeptide maps of tau alone, of tau with apoE3 and of tau with apoE4 were very similar. When apoE2 was present a further four spots were evident. Additionally, of the 15 peptides phosphorylated in the presence or absence of apoE, subtle differences, some isoform‐specific, in the relative amounts of phosphorylation were observed.