Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-12-12
2002-04-23
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007920, C436S501000, C436S503000, C436S504000
Reexamination Certificate
active
06376205
ABSTRACT:
The present invention relates to novel methods for the detection of substances capable of modulating or inhibiting pathological tau-tau protein association and pathological neurofilament aggregation. The methods of the present invention are particularly useful in screening substances for the prophylaxis and treatment of Alzheimer's disease.
Alzheimer's disease (AD) is the most common single cause of dementia in late life (Livingstone (1994) The scale of the problem. In: Dementia (eds. Burns and Levy) Chapman & Hall, London, pp.21-35). Individuals with Alzheimer's disease are characterised by progressive dementia that presents with increasing loss of memory, disturbances in judgement, perception and speech, and global intellectual deterioration (Roth and Iversen (1986) Brit. Med. Bull., 42 (special volume)).
The major pathological hallmarks of Alzheimer's disease are senile plaques and neurofibrillary tangles, both of which contain paired helical filaments (PHFs) of which the microtubule-associated protein tau is a constituent (Wischik et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 4506-4510). Plaques also contain &bgr;-amyloid fibrils derived from an as yet undefined abnormality in the processing of the amyloid precursor protein (APP; Kang et al. (1987) Nature, 325, 733-736).
Studies of Alzheimer's disease have pointed to loss of the normal microtubule associated protein tau (Mukaetova-Ladinska et al. (1993) Am. J. Pathol., 143, 565-578; Wischik et al. (1995a) Neurobiol. Ageing, 16: 409-417; Lai et al. (1995b) Neurobiol. Ageing, 16: 433-445), accumulation of pathological paired helical filaments (PHFs; Mukaetova-Ladinska et al. (1993), loc. cit.; Harrington et al. (1994a) Dementia, 5, 215-228; Harrington et al. (1994b) Am. J. Pathol., 145, 1472-1484; Wischik et al., (1995a), loc. cit.) and loss of synapses in mid-frontal cortex (Terry et al. (1991) Ann. Neurol., 30, 572-580) as strong discriminatory markers for cognitive impairment. Loss of synapses (Terry et al., loc. cit.) and loss of pyramidal cells (Bondareff et al. (1993) Arch. Gen. Psychiatry, 50, 350-356) are both correlated with morphometric measures of tau-reactive neurofibrillary pathology, and this correlates at the molecular level with an almost complete redistribution of the tau protein pool from soluble to polymerised form (PHFs) in Alzheimer's disease (Mukaetova-Ladinska et al. (1993), loc. cit.; Lai et al. (1995), loc. cit.). A possible explanation for these changes is that the pathological redistribution of tau protein into PHFs causes a failure of axonal transport in cortico-cortical association circuits through failure to maintain axonal tubulin in the polymerised state within pyramidal cells (Wischik et al. (1995a), loc. cit.; Wischik et al. (1995b) Neurobiol. Ageing, in press; Wischik et al (1995c) Structure, biochemistry and molecular pathogenesis of paired helical filaments in Alzheimer's disease. Eds. A. Goate and F. Ashall, in press; Lai et al., (1995), loc. cit.). A resulting failure of transport of synaptic constituents from projection soma to distant association neocortex would lead to synaptic loss and cognitive impairment. Further factors include the direct toxicity of PHF accumulation in pyramidal cells (Bondareff et al., (1993), Arch. Gen. Psychiat. 50: 350-356; (1994), J. Neuropath. Exp. Neurol. 53: 158-164), and the possible direct toxicity of truncated tau accumulation impairing cellular function (Mena et al. (1991), J. Neuropath. Exp. Neurol. 50: 474-490).
Although studies of molecular pathogenesis in model systems have emphasised the neurotoxic role of &bgr;-amyloid accumulation (reviewed in Harrington and Wischik (1994) Molecular Pathobiology of Alzheimer's disease. In: Dementia (eds. A. Burns and R. Levy). Chapman & Hall London, pp.211-238), the evidence linking &bgr;-amyloid deposition directly with cognitive impairment in humans is weak. It is more likely that altered processing of APP is only one of several possible factors which might initiate altered processing of tau protein. Other initiating factors include unknown processes associated with apoE4 (Harrington et al. (1994b), loc. cit.), trisomy of chromosome 21 (Mukaetova-Ladinska et al. (1994) Dev. Brain Dysfunct. 7: 311-329), and environmental factors, such as prolonged exposure to sub-toxic levels of aluminium (Harrington et al. (1994c) Lancet, 343, 993-997). Distinct etiological factors are able to initiate a common pattern of disturbance in tau protein processing which includes: C-terminal truncation at Glu-391, formation of PHF tau polymers, loss of soluble tau, and accumulation of abnormally phosphorylated tau species (Wischik et al. (1996) Int. Rev. Psychiat., in press).
The fragment of the microtubule-associated protein tau which has been shown to be an integral constituent of the protease-resistant core structure of the PHF is a 93/95 amino acid residue fragment derived from the microtubule binding domain of tau (Wischik et al. (1988), loc. cit.; Kondo et al. (1988) Neuron, 1, 827-834; Jakes et al. (1991) EMBO J., 10, 2725-2729; Novak et al. (1993) EMBO J., 12, 365-370). Tau protein exists in 6 isoforms of 352-441 amino acid residues in the adult brain (Goedert et al. (1989) Neuron, 3, 519-526). In general structure the tau molecule consists of an extensive N-terminal domain of 252 residues, which projects from the microtubule, a tandem repeat region of 93-125 residues consisting of 3 or 4 tandem repeats and which is the microtubule binding domain, and a C-terminal tail of 64 residues. Each tandem repeat is composed of a 19 residue tubulin binding segment, and 12 residue linker segment (Butner and Kirschner (1991) J. Cell Biol., 115, 717-730; FIG. 1). The major tau constituent which can be extracted from enriched protease-resistant core PHF preparations is a 12 kDa fragment derived from both 3- and 4-repeat isoforms, but restricted to the equivalent of 3 tandem repeats regardless of isoform (Jakes et al., loc. cit.; FIG. 2). The N- and C-terminal boundaries of the fragment define the precise extent of the characteristic protease-resistant core PHF tau unit. It is phase-shifted by {fraction (14/16)} residues with respect to the binder/linker oroanisation of the normal molecule defined by Butner and Kirschner, loc. cit., FIG. 1) and is C-terminally truncated at Glu-391, or at a homologous position in the third repeat of the 4-repeat isoform (Novak et al. (1993), loc. cit.; FIGS. 3A, 3B and 3C). A monoclonal antibody (mAb 423) is available which specifically recognises this C-terminal truncation point, and histological studies using this antibody have shown the presence of tau protein C-terminally truncated at Glu-391 at all stages of neurofibrillary degeneration (Mena et al. (1995) Acta Neuropathol., 89, 50-56; Mena et al. (1996) Acta Neuropathol. (in press)). Thus, a possible post-translation modification implicated in PHF assembly is abnormal proteolysis.
Methods have been developed which permit discrimination between several tau pools found in AD brain tissues: normal soluble tau, phosphorylated tau, and protease-resistant PHFs (Harrington et al. (1990), (1991), (1994a), loc. cit.). These methods have been deployed in studies of severe AD and Down's Syndrome (Mukaetova-Ladinska et al. (1993; 1995), loc. cit.), in prospectively assessed cases at early stage AD (Wischik et al. (1995a), loc. cit.; Lai et al. (1995), loc. cit.) and cases with other neuropathological diagnoses including senile dementia of the Lewy body type and Parkinson's disease (Harrington et al. (1994a), (1994b), loc. cit.). The overall PHF content in brain tissue distinguishes unambiguously between patients with and without dementia of the Alzheimer type. There is overall a 19-fold difference in PHF content, and in temporal cortex the difference reaches 40-fold. Furthermore, apolipoprotein E genotyping of the cortical Lewy body cases showed that the frequency of the E4 allele was raised to a similar extent to that seen in AD. Therefore, the presence of the E4 allele cannot be the sole cause of the characte
Edwards Patricia Carol
Harrington Charles Robert
Klug Aaron
Roth Martin
Wischik Claude Michel
Bacon & Thomas
Duffy Patricia A.
University Court of the University of Aberdeen
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