Post-translational processing of proteins implicated in the pathogenesis of Alzheimer's disease.

CAMPBELL, Emma. (2001). Post-translational processing of proteins implicated in the pathogenesis of Alzheimer's disease. Doctoral, Sheffield Hallam University (United Kingdom)..

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The beta-amyloid peptide characteristic of the lesions of Alzheimer's disease (AD) is derived from the amyloid precursor protein (APP), a single transmembrane-spanning protein with several alternatively-spliced variants, some of which contain a Kunitz protease inhibitory (KPI) domain. Although intracellular localisation of APP has been described in many cell types, it has not been characterised in NTera 2 (NT2) neurones, which are the best available model of human CNS neurones. Here, the subcellular distributions of APP and APLP2 (amyloid precursor-like protein 2) were demonstrated, by indirect immunocytochemistry, to be overlapping but not the same in both NT2 stem cells and neurones. No obvious differences were apparent when comparing the locations of either APP or APLP2 in stem cells versus neurones, APLP2 being restricted to the region of the Golgi apparatus, and APP extending into compartments approaching the cell membrane, including growth cones of neurones. Therefore, no clear differences in intracellular routing of these proteins were identified, immunocytochemically, in human CNS-type neurones compared with stem cells, which represent non-neuronal cells. This work emphasised the need to use antibodies that distinguish between APP and APLP2 (which does not contain the beta-amyloid sequence) in studies of APP processing and amyloidogenesis, since only APP was concentrated in compartments beyond the Golgi apparatus. Heat-shock and no feeding had no immunocytochemically detectable effects on APP or APLP2 distributions or 13-amyloid production in NT2 cells. Although a preliminary investigation to establish a protocol by which NT2 cells can be studied by electron microscopy produced only scant cellular material, more recent publications have shown that slight variations on methods tested here give a successful protocol.The expression of KPI-containing APP in human brains has only been described previously in terms of its mRNA. The ratio of KPI-/non-KPI-APP mRNA appears to be elevated in AD (Tanaka et al., 1989; Johnston et al., 1996). A novel polyclonal antibody (Ab993), specific for the KPI-domain epitope, was characterised for use in immunohistochemistry using paraffin-embedded human brain sections. Immunohistochemical staining was enhanced significantly by reduction of sulphydryl bonds with 2-mercaptoethanol, followed by alkylation of the reduced bonds with sodium iodoacetate. Microwaving of sections also enhanced immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation with 80% formic acid did not increase immunolabelling. KPI-containing protein distribution in normal and AD human brains was characterised by indirect immunohistochemistry. KPI-APP was concentrated mainly in pyramidal cells of the temporal and visual neocortex. In Alzheimer's disease there was a significantly increased incidence of cellular staining for KPI-APP. KPI-containing protein was closely related to the pathology of AD. It was found in association with the tangle-bearing population of neurones, blood vessels including those affected by cerebro-vascular amyloid, within the neuropil and in association with plaques. This evidence corroborates that supplied previously by mRNA data and studies of hAPP transgenic mice in highlighting the importance of this isoform of APP in the pathogenesis of AD.The cytokine transforming growth factor-beta1 (TGF-beta1) is upregulated in AD brains, but its mRNA expression has not been characterised. TGF-beta1 and hAPP(V717F) bigenicmice show accelerated development of AD-like pathology compared with hAPP(V717F) singly transgenic mice (Wyss-Coray et al, 1997). It has been suggested that elevated TGF-beta1 could increase expression of KPI-containing APP isoforms in AD and/or upregulate CNS extracellular matrix proteins that may promote amyloid deposition. TGF-beta1 mRNA in AD and control frozen human brain sections was quantified by in situ hybridization histochemistry. A modest, significant increase in TGF-beta1 mRNA was found in AD temporal cortex and white matter compared to controls, supporting previous immunohistochemical studies.

Item Type: Thesis (Doctoral)
Thesis advisor - Parkinson, David
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 2001.
Research Institute, Centre or Group - Does NOT include content added after October 2018: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:18
Last Modified: 26 Apr 2021 11:33

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