Ross Jakes

|[alpha]|-Synuclein in Lewy bodies

Lewy bodies, a defining pathological characteristic of Parkinsons disease and dementia with Lewy bodies (DLB), constitute the second most common nerve cell pathology, after the neurofibrillary lesions of Alzheimers disease. Their formation may cause neurodegeneration, but their biochemical composition is unknown. Neurofilaments and ubiquitin are present, but it is unclear whether they are major components of the filamentous material of the Lewy body,. Here we describe strong staining of Lewy bodies from idiopathic Parkinsons disease with antibodies for α-synuclein, a presynaptic protein of unknown function which is mutated in some familial cases of the disease. α-Synuclein may be the main component of the Lewy body in Parkinsons disease. We also show staining for α-synuclein of Lewy bodies from DLB, indicating that the Lewy bodies from these two diseases may have identical compositions.

Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease

We have determined the sequences of isoforms of human tau protein, which differ from previously reported forms by insertions of 29 or 58 amino acids in the amino-terminal region. Complementary DNA cloning shows that the insertions occur in combination with both three and four tandem repeats. RNAase protection assays indicate that transcripts encoding isoforms with the insertions are expressed in an adult-specific manner. Transcripts encoding four tandem repeats are also expressed in an adult-specific manner, whereas mRNAs encoding three tandem repeats are expressed throughout life, including in fetal brain. The levels of transcripts encoding the 29 or 58 amino acid inserts were not significantly changed in cerebral cortex from patients with Alzheimers disease. Antisera raised against synthetic peptides corresponding to these different human tau isoforms demonstrate that multiple tau protein isoforms are incorporated into the neurofibrillary tangles of Alzheimers disease.

Identification of two distinct synucleins from human brain

Two abundant proteins of 140 and 134 amino acids were purified and sequenced from human brain. They were identified through their reactivity on immunoblots with a partially characterised monoclonal antibody that recognises tau protein in a phosphorylation‐dependent manner. The 140 amino acid protein is identical with the precursor of the non‐Aβ component of Alzheimers disease amyloid which in turn is highly homologous to synuclein from Torpedo electroplaques and rat brain. The 134 amino acid protein is the human homologue of bovine phosphoneuroprotein 14; it is 61% identical in sequence to the 140 amino acid protein. The previously unrecognised homology between these two proteins defines a family of human brain synucleins. We refer to the 140 and 134 amino acid proteins as α‐synuclein and β‐synuclein, respectively. Both synucleins are expressed predominantly in brain, where they are concentrated in presynaptic nerve terminals.

Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding.

Abnormally phosphorylated tau proteins (A68) are the building blocks of Alzheimers disease (AD) paired helical filaments. The biological consequences of the conversion of normal adult tau to A68 remain unknown. Here we demonstrate that native A68 does not bind to microtubules (MTs), yet dephosphorylated A68 regains the ability to bind to MTs. Ser396 is phosphorylated in A68, but not in normal adult tau, whereas fetal tau is phosphorylated transiently at this site. Phosphorylation of tau at Ser396 by protein kinases in CHO cells and rat brain produces an electrophoretic mobility similar to that of A68. Using CHO cells transfected with an Ala396 mutant, we show that the phosphorylation of tau at Ser396 reduces its affinity for MTs and its ability to stabilize MTs against nocodazole-induced depolymerization. Our results demonstrate that the abnormal phosphorylation of tau in AD involves Ser396, and we suggest that this may be mediated by the inappropriate activation of fetal kinases or the reduced activity of tau protein phosphatases. Thus, phosphorylation of Ser396 may destabilize MTs in AD, resulting in the degeneration of affected cells.

Filamentous α-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies

α-Synuclein forms the major component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of Parkinsons disease and dementia with Lewy bodies. Here we show that α-synuclein is also the major component of the filamentous inclusions of multiple system atrophy which comprises several neurodegenerative diseases with a shared filamentous pathology in nerve cells and glial cells. These findings provide an unexpected link between multiple system atrophy and Lewy body disorders and establish that α-synucleinopathies constitute a major class of human neurodegenerative disorder.

Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization.

We have expressed six previously cloned isoforms of human microtubule‐associated tau protein in Escherichia coli and purified them to homogeneity in a biologically active form. They range from 352 to 441 amino acids in length and differ from each other by the presence of three or four tandem repeats in the carboxy‐terminal half and by the presence or absence of 29 or 58 amino acid inserts in the amino‐terminus. When mixed together they gave a set of six bands on SDS‐PAGE gels with apparent molecular weights of 48–67 kd and with a characteristic pattern of spacings. Four of these bands aligned with the major tau bands found in adult human cerebral cortex following perchloric acid extraction and alkaline phosphatase treatment. They consisted of isoforms with three repeats and no insertions, four repeats and no amino‐terminal insertions and three‐ and four‐repeat containing isoforms with the 29 amino acid insertion. In fetal human brain extracts treated with alkaline phosphatase one of the two major tau bands aligned with the three‐repeat containing isoform with no insertions, whereas the molecular nature of the second major tau band remains to be established. The recombinant tau isoforms were biologically active at micromolar concentrations, as assessed by their ability to promote microtubule assembly. The rates of assembly were 2.5–3.0 times faster for isoforms containing four repeats when compared with three‐repeat containing isoforms, with no significant contribution by the amino‐terminal insertions.

Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation

The presynaptic protein α-synuclein has been implicated in the pathogenesis of Parkinson’s disease. First, two missense mutations A30P and A53T cause inheritable early onset Parkinson’s disease in some families. Secondly, α-synuclein is present in Lewy bodies of affected nerve cells in the predominant sporadic type of Parkinson’s disease as well as in dementia with Lewy bodies. We demonstrate in the rat optic system that a portion of α-synuclein is carried by the vesicle-moving fast component of axonal transport and that it binds to rat brain vesicles through its amino-terminal repeat region. We find α-synuclein with the A30P mutation of familial Parkinson’s disease devoid of vesicle-binding activity and propose that mutant α-synuclein may accumulate, leading to assembly into Lewy body filaments.

Abundant Tau Filaments and Nonapoptotic Neurodegeneration in Transgenic Mice Expressing Human P301S Tau Protein

The identification of mutations in the Tau gene in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has made it possible to express human tau protein with pathogenic mutations in transgenic animals. Here we report on the production and characterization of a line of mice transgenic for the 383 aa isoform of human tau with the P301S mutation. At 5–6 months of age, homozygous animals from this line developed a neurological phenotype dominated by a severe paraparesis. According to light microscopy, many nerve cells in brain and spinal cord were strongly immunoreactive for hyperphosphorylated tau. According to electron microscopy, abundant filaments made of hyperphosphorylated tau protein were present. The majority of filaments resembled the half-twisted ribbons described previously in cases of FTDP-17, with a minority of filaments resembling the paired helical filaments of Alzheimers disease. Sarkosyl-insoluble tau from brains and spinal cords of transgenic mice ran as a hyperphosphorylated 64 kDa band, the same apparent molecular mass as that of the 383 aa tau isoform in the human tauopathies. Perchloric acid-soluble tau was also phosphorylated at many sites, with the notable exception of serine 214. In the spinal cord, neurodegeneration was present, as indicated by a 49% reduction in the number of motor neurons. No evidence for apoptosis was obtained, despite the extensive colocalization of hyperphosphorylated tau protein with activated MAP kinase family members. The latter may be involved in the hyperphosphorylation of tau.

Hypothesis: Microtubule Instability and Paired Helical Filament Formation in the Alzheimer Disease Brain Are Related to Apolipoprotein E Genotype

A genetic classification of Alzheimer disease(s) (AD) is presented. We describe a potential metabolic process in individuals who inherit apolipoprotein E-epsilon 4 (APOE4, gene; apoE4, protein) alleles, leading to increased risk and earlier age of onset of late-onset Alzheimer disease. Apolipoprotein E-epsilon 3 (apoE3) binds to tau protein, possibly slowing the initial rate of tau phosphorylation and self-assembly into paired helical filaments (PHFs); apoE4 does not bind tau. Tau promotes microtubule assembly and stabilizes microtubules; hyperphosphorylated tau does not bind, thereby destabilizing microtubules. Hyperphosphorylated tau may self-assemble into PHFs. Over time a bias toward destabilization of microtubules and the formation of neurofibrillary tangles may occur in individuals who inherit APOE4 alleles, leading to a shorter functional neuronal life span. This hypothesis focuses attention on two important aspects of AD research design: (1) Although the inheritance of APOE4 is associated with increased risk and decreased age of onset, apoE4 does not directly cause the disease. Our data point to the absence of an important function of apoE3 or apoE2 in individuals who do not inherit these alleles as the genetically relevant metabolic factor. This has important implications for design of experiments directed toward understanding the relevant neuronal metabolism. (2) Should this hypothesis be proven and confirmed, targets for pharmaceutical therapy designed to mimic the metabolic function of apoE3 or apoE2 become a realistic preventive strategy.

Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205

Hyperphosphorylated microtubule-associated protein tau is the major component of the paired helical filament of Alzheimers disease. Phosphorylation-dependent anti-tau antibodies are being used to identify specific amino acids that are phosphorylated in tau from normal brain and Alzheimers disease brain. As such, monoclonal antibody AT8 is widely used. By a combination of site-directed mutagenesis of recombinant tau and in vitro phosphorylation, we show that AT8 requires tau protein to be phosphorylated at both serine 202 and threonine 205 (using the numbering of the longest human brain tau isoform.