Amyloid precursor protein mRNA levels in Alzheimer's disease brain

doi:10.1016/j.molbrainres.2003.08.022

Molecular Brain Research

Volume 122, Issue 1, 17 March 2004, Pages 1-9

https://doi.org/10.1016/j.molbrainres.2003.08.022 Get rights and content

Abstract

Insoluble β-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K⁺, APP₇₅₁, APP₇₇₀, APRP₃₆₅ and APRP₅₆₃), and those without (K⁻, APP₆₉₅ and APP₇₁₄,). Given the hypothesis that Aβ is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan™ real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI+ to KPI− mRNA isoforms of APP. Three separate probes, designed to recognise only KPI+ mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI+species increased specifically in the AD brains.

Introduction

Insoluble β-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene spans almost 300,000 bases of DNA from which 18 exons can be alternatively spliced [14]. A functional classification can be made into those APP isoforms containing exon 7 that codes for a Kunitz-type serine protease inhibitor domain (K⁺, APP₇₅₁, APP₇₇₀, APRP₃₆₅ and APRP₅₆₃), and those without (K⁻, APP₆₉₅ and APP₇₁₄,). The figures refer to the number of amino acids in each isoform, whilst APRP stands for amyloid precursor related protein. APRPs are spliced from the same gene as APP but lack the Aβ sequence. The mRNAs for APRPs also lack any transmembrane domains and are most likely coding for secreted soluble proteins [8], [18]. These isoforms have been described previously: APP₆₉₅ [23], APP₇₅₁ [40], [49], APP₇₇₀ [24] APRP₅₆₃ [8], APP₇₁₄ [10], [47] and APRP₃₆₅ [18].

The 56 amino acid insert coded by exon 7 of the APP gene was found to be similar to a group of 17 polypeptides that were all members of a family of Kunitz-type serine protease inhibitors [24], [28], [40], [49]. COS-1 cells transfected with APP₇₇₀ showed an increased inhibition of trypsin activity [24]. Subsequent searches of the Protein Identification Resource database revealed that the secreted forms of APP K⁺ are nearly identical to protease-nexin II (PN-II), a cell-secreted protease inhibitor. APP₇₅₁ formed stable, non-covalent, inhibitory complexes with trypsin, whereas APP₆₉₅ did not [36]. Given the hypothesis that Aβ results from aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors may play an active role in the pathology of AD. Either too much or too little expression of APP/APRP mRNA with KPI domains may upset Aβ processing and result in excessive deposition of Aβ.

Since 1987 over 30 studies have tried to establish a differential expression of APP isoforms in the disease, and in particular a change in the KPI^+/− isoforms ratio [1], [5], [6], [10], [12], [13], [17], [19], [20], [21], [22], [24], [25], [26], [27], [31], [32], [33], [34], [35], [37], [38], [39], [40], [43], [44], [45], [46], [47], [48], [49], [50]. A combination of factors, however, have made it difficult to assess APP mRNA levels and as such there is no consensus on whether K⁺/K⁻ mRNA ratios are altered [13], [17]. Reported attempts are difficult to compare and often inconsistent because of heterogeneity in methods used and terms of reference, brain areas assessed, differences in neuropathological criteria and most important of all, though rarely discussed, cross-hybridisation of probes to more than one APP/APRP isoform. The latter is significant since studies between 1988 and 1991 were unaware of the existence of the various APP isoforms and their conclusions were based only on information known at the time. Without discrete measurements of the different isoforms interpretation is difficult and misleading.

To re-assess the K⁺/K⁻ mRNA ratio in AD we extracted poly A from 513 cortical samples taken from 90 AD and 81 control brains. Results were analysed with methods developed previously [3], [4], [41], [42].

Section snippets

Tissue collection

Brain tissue was obtained from the MRC Brain Bank for Neurodegenerative Diseases, Department of Neuropathology, Institute of Psychiatry, King's College London. The AD cases met the clinical criteria for a diagnosis of probable AD [30]. None of the control cases had any history of neurological or psychiatric illness and all were examined neuropathologically as were the AD cases. Tissue was obtained with informed consent of the next-of-kin and according to Local Ethics Committee guidelines. At

Levels of mRNA

All of the genes tested, including reference genes, had lower levels of mRNA in AD relative to control brains, as well as in females relative to males (Fig. 1 and Table 1). Therefore, there was a need for a reference gene against which the test gene could be measured in relative terms. After extensive analysis of reference gene issues [3] we decided to use Analysis of Covariance (ANOCOVA) with three reference genes (β-actin, GAPDH and cyclophilin) as simultaneous covariates. Table 2 and Fig. 2

Acknowledgements

Post-mortem brain tissue was kindly provided by the Brain Bank, Department of Neuropathology, Institute of Psychiatry, King's College London. Many thanks are also due to Brian Bond of Glaxo SmithKline Pharmaceuticals for his extensive advice on statistical issues.

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Research reportAmyloid precursor protein mRNA levels in Alzheimer's disease brain

Abstract

Introduction

Section snippets

Tissue collection

Levels of mRNA

Acknowledgements

Neurosci. Lett.

J. Mol. Biol.

Mol. Brain Res.

Neuron

Neurodegeneration

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Exp. Neurol.

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Biochem. Biophys. Res. Commun.

Brain Res. Mol. Brain Res.

Neuron

Curr. Opin. Biotechnol.

J. Biol. Chem.

Neuron

Neuron

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Mol. Brain Res.

Mol. Brain Res.

J. Biol. Chem.

Neuron

Brain Res. Mol. Brain Res.

Neuroscience

Biochem. Biophys. Res. Commun.

Brain Res. Mol. Brain Res.

Research report
Amyloid precursor protein mRNA levels in Alzheimer's disease brain