Amyloid precursor protein gene isoforms in Alzheimer’s disease and other neurodegenerative disorders

Journal of the Neurological Sciences 173 (2000) 81–92 www.elsevier.com / locate / jns

Amyloid precursor protein gene isoforms in Alzheimer’s disease and other neurodegenerative disorders Peter K. Panegyres*, K. Zafiris-Toufexis, B.A. Kakulas Department of Neuropathology, Royal Perth Hospital, Perth 6000, Western Australia Received 19 May 1999; received in revised form 20 August 1999; accepted 8 November 1999

Abstract Differential expression of the amyloid precursor protein gene (APP) may be important in the development of amyloidosis in Alzheimer’s disease (AD) and experimentally in the brain’s response to injury. Controversial data suggests that APP isoforms containing the Kunitz protease inhibitor isoform (APP KPI1) are over expressed in the brains of patients with AD when compared to the non-Kunitz protease inhibitor containing isoforms (APP KPI2). We have investigated this hypothesis using a quantitative analysis of gene expression on brain tissue collected at post-mortem. In situ hybridization has been used with synthetic oligonucleotide probes labelled with 35 S to detect the two principal splice variants of APP: APP 695 (KPI2) and APP 751 (KPI1). A prospective brain bank of frozen brain specimens has been established and includes pathologically proven AD (n515) and other neurodegenerative disorders as controls (n518). The controls consist of frontal lobe atrophy (n54), Huntington’s disease (n55), Parkinson’s disease (n54), motor neuron disease (n52), multi-infarct dementia (n51), multisystem atrophy (n51), and subacute sclerosing panencephalitis (n51). We have observed no significant differences in the expression of APP 695 KPI– mRNA in frontal lobe: 17.4963.26 optical density (OD) units of mRNA expression in AD vs. 16.1361.76 OD units mRNA in controls (P50.80, linear regression); or temporal lobe: 14.7362.96 in AD vs. 16.4962.15 in controls (P50.55). No significant differences have been found in APP 751 KPI1 in frontal lobe: 12.8662.98 in AD vs. 13.7062.88 in controls (P50.97); and temporal lobe: 13.3164.93 in AD vs. 11.0761.99 in controls (P50.65). Analysis of the ratios of APP 751 KPI1 OD units of mRNA to APP 695 KPI2 mRNA revealed a trend to an increased ratio which did not reach statistical significance: frontal lobe APP 751 KPI1 /APP 695 KPI2 1.9261.04 in AD vs. 0.8660.17 in controls (P50.54); temporal lobe 2.5461.59 in AD vs. 0.9660.11 controls (P50.34). Our data has not revealed differential expression of APP mRNA isoforms in AD and supports the hypothesis that post-translational events in APP metabolism are important in amyloidogenesis and the pathogenesis of AD.  2000 Elsevier Science B.V. All rights reserved. Keywords: Neurodegeneration; APP; Gene expression; In situ hybridization; Alzheimer’s disease

1. Introduction Alzheimer’s disease (AD) is characterised by neuronal loss, neuritic plaques and neurofibrillary tangles [1]. The neuritic plaque contains an amyloidogenic core surrounded by swollen neurites and glial cells [2]. The major component of the amyloidogenic protein is a 40–42 amino acid *Corresponding author. Tel.: 161-8-9224-2433; fax: 161-8-92242556. E-mail address: [email protected] (P.K. Panegyres)

peptide known as b /A4 of 4–4.2 kDa molecular weight [3,4]. This originates from a larger precursor known as the amyloid precursor protein gene (APP) [5,6]. APP is therefore critical in the development of amyloidogenesis and the pathogenesis of AD: a position strengthened by the identification of a number of rare mutations in APP leading to familial AD [7–9]. APP and b /A4 remain central to therapeutic approaches in AD. Mutations in APP and presenilins in association with interactions involving APOE e4 influence b /A4 deposition through a mechanism involving increased production of b /A4 42 leading to enhanced fibrillisation and

0022-510X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-510X( 99 )00311-1

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

82 Table 1 Brain bank diagnostic categories

2.2. Brain processing

Diagnoses

N

Alzheimer’s disease Huntington’s disease Fronto-temporal dementia a Parkinson’s disease Prion diseases Motor neuron disease Miscellaneous ? Multi-infarct dementia ? Multisystem atrophy ? Subacute sclerosing panencephalitis

15 5 4 4 4 2 3

a

The post-mortem examination and brain removal were performed as soon as possible after death. The brain was divided into two: the left hemisphere for routine neuropathological studies, and fixed in 20% buffered formal saline pH 7.4; the right hemisphere specially prepared for molecular analysis as described below.

2.3. Freezing of brain material for in situ hybridization and storage

One patient with frontal lobe atrophy and motor neuron disease.

amyloidogenesis [10–14]. APP may itself be involved in the pathogenesis of AD through alternative splicing [15] and it is this hypothesis that has been investigated in this study using in situ hybridization to detect major splice variants of APP on post-mortem brain tissue. Here we explore the relationship at a gene expression level measuring the mRNA of the two main isoforms of APP generated through alternative splicing with and without the Kunitz protease inhibitor region (KPI6). Contentious data suggests that APP KPI1 isoforms are increased in the brains of patients with AD, a finding that has therapeutic implications [16,17]. No agreement has emerged regarding changes in APP mRNAs in the brains of patients with AD. Recent studies suggest that at a protein level the APP KPI1 isoforms may be increased [18]. We wished to evaluate changes in the major isoforms of APP at an mRNA level in the light of this knowledge.

2. Materials and methods

2.1. Patient recruitment In 1996 a prospective brain bank was established to investigate the molecular pathogenesis of AD and other neurodegenerative disorders. A project having the approval of the Royal Perth Hospital Ethics Committee (approval number E.C. 390). A statewide mail-out to general practitioners, neurologists, psychiatrists, geriatricians and nursing homes was performed. The diagnostic categories included patients with prion diseases which were not used as controls in the in situ hybridization studies (Table 1).

Cubic blocks measuring 1 cm 3 were obtained from the mid-frontal gyrus, superior temporal gyrus, inferior parietal molecule, hippocampal formation, midbrain including substantia nigra, thalamus, pons, medulla, lentiform nucleus, lateral occipital gyrus, cerebellar hemisphere, and high cervical spinal cord. The blocks were snap frozen in bubbling isopentane on dry ice with the temperature monitored by thermostat to 2308C to 2408C — conditions giving the best tissue preservation and minimising ice crystal artefact. After freezing the blocks were wrapped in Parafilm and then aluminium foil labelled with the case number and anatomical region. The blocks from each patient were placed in individual cardboard boxes and stored in 2708C freezers until used. Slices measuring 2 cm in thickness were then prepared from the remaining hemisphere and frozen on dry ice, placed in plastic bags, and stored in cardboard boxes at 2708C for future research.

2.4. Preparation of brain material for in situ hybridization Sections measuring 12 mm in thickness were prepared on a cryostat set at 2158C from the frontal and temporal lobes from each case. The sections were mounted onto poly-L-lysine coated slides and fixed in 4% paraformaldehyde. They were washed in 13phosphate buffered saline, 70% ethanol and placed in 95% ethanol at 48C until used. The sections were dried at room temperature before in situ hybridization.

2.5. In situ hybridization The oligonucleotide probe sequences were complementary to the two principal isoforms of human APP and were based on published information (Table 2) [19]. The

Table 2 The sequences of oligonucleotide probes Gene

Sequence 59–39

APP 695 APP 751

ACT ACT

GGC GGC

TGC TGC

TGT TGT

TGT TGT

AGG AGG

AAC AAT

TCG GGC

AAC GCT

CAC GCC

CTC ACA

TTC CAC

CAC GGC

A C

No. of nucleotides

Reference

40 40

Sola´ et al. [19] Sola´ et al. [19]

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

83

dried at room temperature in the dark room for up to 3 h. They were placed in light protected boxes with desiccant at 48C for up to 24 weeks. Emulsions were developed using Phenisol (Ilford) and stained with haematoxylin and eosin (H&E).

2.7. Quantification of gene expression using in situ hybridization

Fig. 1. Northern analysis on total mRNA extracted from frontal cortex from a patient with Alzheimer’s disease. Approximately 20 mg of RNA were added to each lane and the probes were labelled with 32 P. Positions of molecular weight standards are indicated. The size of the APP transcripts measured about 3.2–3.8 kb.

specificity and transcript size were confirmed by northern analysis (Fig. 1). Further tests of specificity involved in situ hybridization with sense sequences and an excess of cold antisense probe (Fig. 2). The probes (40 mm) were labelled with 50 m Curies of 59–[alpha– 35 S] d ATP (Amersham) using a 39 end labelling system with terminal deoxynucleotidyl transferase (NEN, Du Pont). The probes were purified on chromatography columns (Bio-Rad). The activity of the probe was adjusted to 3310 5 c.p.m. / 100 ml of in situ hybridization buffer to which dithiothreitol (20 ml / ml) was added. The hybridization buffer was composed of formamide (24 ml), 203sodium chloride sodium phosphate EDTA (SSPE) (10 ml), 503Denhardt’s solution (5 ml), salmon sperm DNA 10 mg / ml (1 ml), polyadenylic acid 5 mg / ml (1 ml), dextran sulphate 5 g. A volume of 100 ml of labelled probe in buffer was placed over the sections on each slide and then covered with sterile plastic film. Air bubbles were removed by gentle compression. Slides were placed flat in hybridization trays (Dako) and incubated in a humidified oven at 428C overnight. Stringency washes were then performed in 13saline sodium citrate solution (SSC): first at room temperature for 0.5 h and then at 558C for 0.5 h on a shaking water bath. Dehydration was then achieved by briefly dipping the slides in 13SSC, 0.13SSC, 70% and 95% ethanol. The sections were air-dried, mounted on filter paper and exposed to autoradiography film (Hyperfilm-bmax, Amersham). The duration of exposure was 8 weeks for all experiments. The in situ hybridization experiments were performed without knowledge of the diagnosis and films developed under identical conditions using D-19 developer.

2.6. Emulsion microautoradiography After in situ hybridization the slides were dipped in molten emulsion at 438C (LM–1, Amersham) for 5 s and

Optical density units (OD) of mRNA expression were determined using videodensitometry with a Quantimet 520 Image Analysis System. The exposed films were placed on the macroscanner and the image captured with a video camera. The camera height above the macroscanner and the illumination were constant for all experiments. The quantification was performed in a darkened room using a standard pixel size of 16316 for all measurements. Shading error to correct for differences in camera illumination was set prior to each run. The system was calibrated using standards such that OD50 with a grey level5230– 240 and a OD51.00 with a grey level50. This ensured that the camera was measuring optical density in its most sensitive range. To enhance standardisation between runs 14 C micro-scales (Amersham) composed of a polymer in which the 14 C is uniformly distributed were used. These microscales had an activity range of 31–883 m Ci / g and were exposed to the film at the same time as the sections and developed in the same way. On each quantification the OD of the microscales was determined in triplicate and compared to the findings from other runs — on each occasion there was no greater than 5% variation at each activity between runs. This enabled us to be confident that the OD findings on one day were comparable to those on another. The sections were quantified in the cerebral cortex of each brain region without knowledge of the diagnosis and read in quadruplicate.

2.8. Statistical analysis The diagnostic code was not broken until the statistical analysis at which time the patients were divided into those with AD and those with other neurodegenerative disorders. Non-parametric methods were used to analyse the demographic differences between the two populations and the Mann–Whitney test employed. The optical densities between the two populations was studied using linear regression analysis. A P value ,0.05 was considered significant.

2.9. Neuropathology The pathological criteria of the diagnosis of Alzheimer’s disease were modified from the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) and were based on the number of neuritic plaques, the presence of neurofibrillary tangles and neuronal loss using routine

84

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

Fig. 2. Photomicrographs of emulsions from in situ hybridization experiments using antisense, sense, and excess of cold antisense probe to demonstrate the specificity of the in situ hybridization reactions in the frontal cortex of a patient with Alzheimer’s disease (96A / 32). Upper left in situ hybridization to APP 695 mRNA using antisense probe, right5excess of cold probe. Lower left in situ hybridization to APP 751 mRNA using antisense probe, right5excess of cold probe, middle5sense probe, right5excess cold probe (3400).

stains and a modified Bielschowsky reaction [20–22]. Frontal lobe atrophy by selective atrophy of the frontal and temporal regions with neuronal loss, gliosis, and varying degrees of tau and ubiquitin immunoreactivity [23]. No Pick bodies or Pick cells were identified in our frontal lobe atrophy patients and the changes of AD were absent. Huntington’s disease was detected by selective atrophy of the caudate and putamen associated with neuronal loss and

gliosis in these regions [24]. Parkinson’s disease was characterised by neuronal loss in the substantia nigra and the presence of Lewy bodies [25]. Motor neuron disease by neuronal loss and gliosis of anterior hom cells in the spinal cord and motor cortex with loss of axons and myelin in anterior corticospinal tracts in spinal cord [26]. Multiinfarct dementia by multiple large infracts with the cerebral cortex without the findings of AD [27]. Multisystem

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

atrophy by neuronal loss and gliosis in the putamen, substantiating pontine nuclei, olives, Purkinje cells with characteristic glial cytoplasmic inclusions [28,29]. Subacute sclerosing panencephalitis by neuronal loss, gliosis, intranuclear inclusions and perivascular inflammatory infiltrates throughout the brain [30].

3. Results There were 15 patients with AD and 18 control patients with a range of non-Alzheimer pathologies (Table 1). The control group consisted of patients with frontal lobe atrophy, motor neuron disease, Huntington’s disease, Par-

85

kinson’s disease, multi-infarct dementia, multiple system atrophy, and subacute sclerosing panencephalitis. The mean age of the AD group was significantly greater than the non-AD group by almost 12 years. There was a greater proportion of females to males in both populations. There were no significant differences in the duration of illness and the time from death to removal of the brain for freezing (Table 3). There was a clear signal of APP mRNA isoforms in the brain regions examined using in situ hybridization (Figs. 2 and 3). The strength of the signal for APP 695 appeared greater than APP 757 and this was confirmed using videodensitometry (Table 4). The predominant neuronal localisation and specificity of the mRNA signal was

Table 3 Demographic data Identification number

Age (years)

Sex

Diagnostic category

Duration of illness (years)

Post mortem delay (h)

96A / 32 96A / 50 96A / 61 96A / 57 96A / 62 96A / 75 96A / 69 97A / 27 97A / 87 97A / 58 97A / 24 98A / 49 98A / 34 98A / 35 97A / 57

72 72 79 90 84 73 82 52 87 85 95 68 87 84 84

M F F M F F F M F F F M F F M

Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s

7 8 8 8 8 15 8 8 8 3 3 8 8 8 3

20 24 96 48 96 50 24 44 72 24 30 72 96 30 22

x¯ 6SEM (n515)

79.5362.75

33% M 66% F

7.460.75

49.8667.51

98A / 17 98A / 37 98A / 23 98A / 2 96A / 16 96X / 338 96A / 20 97A / 47 97A / 42 97A / 84 98A / 18 96A / 28 97A / 17 98A / 27 97X / 905 97A / 30 97A / 6 97A / 83

85 64 77 55 62 73 65 75 43 50 63 60 82 87 79 88 76 22

F F M M F F F F F M M M M M M F F F

8 2 8 6 2 6 12 18 7 8 8 5 4 8 8 8 12 0.6

72 50 48 16 77 48 90 96 84

x¯ 6SEM (n518)

6764.02

44.4% M 55.6% F

7.8161.04

53.7265.89

P value Mann–Whitney test

0.0262

Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease

Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Motor Neuron Disease Motor Neuron Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Multi-Infract Dementia Multiple System Atrophy Subacute Sclerosing Panencephalitis

0.9564

24 26 48 72 48 29 72 19

0.6509

86

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

Fig. 3. In situ hybridization of APP mRNAs from patient 97A / 27 with Alzheimer’s disease (37.8).

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

87

Table 4 Standardised optical density units in mRNA expression of APP isoforms in Alzheimer’s disease and other neurodegenerative disorders (OD units5x¯ 6SEM, n54 measurements at each data point) Identification number

Diagnostic category

96A / 32 96A / 50 96A / 61 96A / 57 96A / 62 96A / 75 96A / 69 97A / 27 97A / 87 97A / 58 97A / 24 98A / 49 98A / 34 98A / 35 97A / 57

Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s

Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease

x¯ 6SEM (n515) 98A / 17 98A / 37 98A / 23 98A / 2 96A / 16 96X / 338 96A / 20 97A / 47 97A / 42 97A / 84 98A / 18 96A / 28 97A / 17 98A / 27 97X / 905 97A / 30 97A / 6 97A / 83 x¯ 6SEM P value Linear Regression

Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Motor Neuron Disease Motor Neuron Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Multi Infarct Dementia Multiple System Atrophy Subacute Sclerosing Panencephalitis

APP695

APP751

Frontal lobe

Temporal lobe

Frontal lobe

Temporal lobe

15.3260.75 14.0460.44 17.0862.32 23.0464.24 7.2160.56 4.9760.11 0.1760.02 23.2761.91 13.3560.58 48.2961.10 19.3461.12 37.2462.51 23.0761.04 8.4160.46 11.1861.42

1.1360.37 9.6260.82 15.7161.19 7.0260.98 13.3161.30 3.7160.14 0.0960.06 19.5061.30 27.2862.91 30.2266.43 21.9260.53 40.5964.29 10.2060.83 13.8660.51 6.6760.33

6.2360.90 9.2860.37 7.1860.55 9.8960.53 5.6460.21 5.2960.22 2.8760.20 7.1860.39 49.1162.14 22.8860.70 8.2660.61 10.3861.21 21.8161.05 14.9161.18 11.8861.30

1.6660.70 5.8860.64 9.0160.92 6.1060.32 8.6560.99 3.5661.16 2.4260.51 7.4660.17 80.3764.71 17.1861.21 7.2061.00 12.8160.34 15.9561.28 11.7960.99 9.5460.50

17.4963.26

14.7362.95

12.8562.98

13.3164.93

18.5761.38 9.1561.47 14.9061.88 8.8460.28 13.1560.96 15.7761.11 12.5760.93 18.1961.56 30.4860.09 21.5761.13 20.9661.16 10.5261.27 14.4060.90 22.7861.25 32.6662.94 11.0160.63 12.3761.95 10.8460.28

22.8460.99 11.3163.08 14.4961.36 25.7061.50 11.4361.40 12.3660.57 15.5860.96 9.2360.50 33.3161.97 25.2761.18 18.1261.15 12.0961.10 11.4060.28 25.0962.66 33.2761.78 6.9360.22 13.3461.36 5.6060.062

15.5161.04 8.6360.32 5.2960.23 27.3860.80 3.8560.40 9.9960.57 4.8460.49 25.5461.14 17.6660.41 7.9260.37 10.5760.42 9.0560.94 16.4760.68 51.1862.08 23.4061.31 3.2460.23 12.4361.03 3.9360.44

15.8560.32 8.1260.99 4.8860.36 19.4860.25 3.6660.44 7.1560.57 3.9360.28 18.7661.55 15.7561.00 8.2060.53 10.8261.10 6.3360.72 15.0760.30 32.2861.64 22.4461.12 0.5160.10 19.3761.25 3.5160.32

16.1361.76 0.8062

16.4962.15 0.5468

13.7062.88 0.9695

11.0761.98 0.6466

established by examination of emulsions performed on the slides used for in situ hybridization (Figs. 2 and 4). There were no significant differences in expression of APP 695 mRNA in frontal lobe: 17.4963.26 OD units of mRNA expression in AD vs. 16.1361.76 in controls (Table 4), and no differences in the temporal lobe: 14.7362.95 in AD vs. 16.4962.15 in controls. The expression of APP 751 mRNA was not significantly different in the frontal or temporal lobe between the two populations: frontal lobe 12.8562.98 vs. 13.762.88; temporal lobe 13.3164.93 vs. 11.0761.98 (Table 4). The ratios of OD units of mRNA expression of APP 751 /APP 695 were increased in the frontal and temporal regions (Table 5). The differences in the ratios did not

achieve statistical significance. The degree of this trend was greatest in the temporal lobe. When the data was synthesised we confirm that there were no significant differences in the expression of APP mRNA isoforms in the frontal and temporal lobes (Table 6). There was a trend to an increased ratio of APP 751 /APP 695 in both brain regions which was not significant (Table 6).

4. Discussion We have not demonstrated selective enhancement of the major Kunitz protease inhibitor isoform (APP 751) in the

88

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

Fig. 4. Photomicrographs of emulsions used from in situ hybridization experiments from the superior temporal gyrus from patient 97A / 27 with Alzheimer’s disease. Upper left is APP 695 mRNA (KPI2) stained with H&E and lower left is the negative contrast image. Upper right is APP 757 (KPI1) and lower right is the negative contrast image (3400).

brains of patients with AD, an observation which implies that amyloidogenesis in AD may be secondary to posttranslational events in APP metabolism and not related to differential gene expression generated through alternative splicing. A result contrary to experimental models of excitotoxicity and neurodegeneration which reveal a selective increase in KPI1 mRNA [31]. Using an RNase protection assay other workers have found an increase in the ratio of KPI1 isoforms in AD in comparison to other neurodegenerative diseases [17]. This study was limited by small numbers, and given the differences in methodology, may explain the discrepancy. Similar considerations may apply to other data which suggests a selective enhancement KPI containing isoforms [16,32,33]. The published experience in this field is summarised in Table 7 and from these reports no clear trend emerges in changes in APP mRNA splice variants in AD. Other studies investigating alternative splicing of APP in AD have reported no differences in the proportion of KPI1 transcripts to other transcripts [34–40]. These studies support our findings but are also limited by small numbers, differences in methodology and brain freezing

techniques. Our study has larger numbers of patients studied with a uniform brain freezing technique, quantification of gene expression using unbiased methodology, and a robust technique to study gene expression and disease controls — considerations which give strength to our findings. A reduction in both APP transcripts in comparison to controls has also been found by other workers [41]. A number of variables may influence the study of gene expression in post-mortem brain tissue and these include duration of illness, post-mortem delay to brain freezing, the agonal state, and the cause of death. These variables did not influence our findings as there were no significant differences between the two populations. The time from death to brain freezing had a mean of around 50 h in the AD and control groups. Ideally shorter periods to time of brain freezing would have been preferred but this was the best we could achieve in our institution after permission from next of kin, transfer from place of death to mortuary, approval from institutional authority, and mobilisation of mortuary staff. It has been revealed that the expression of some genes is stable up to 72 or greater hours and the nature of the gene

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

89

Table 5 Ratio of OD units of APP mRNA expression in Alzheimer’s disease and other neurodegenerative disorders (APP751 /APP695 ) Identification number

Diagnostic category

APP751 /APP695 Frontal lobe

APP751 /APP695 Temporal lobe

96A / 32 96A / 50 96A / 61 96A / 57 96A / 62 96A / 75 96A / 69 97A / 27 97A / 87 97A / 58 97A / 24 98A / 49 98A / 34 98A / 35 97A / 57

Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s Alzheimer’s

0.51 0.65 0.37 0.35 0.74 1.22 14.57 0.29 3.46 0.47 0.47 0.29 1.00 1.93 1.05

1.73 0.65 0.57 0.77 0.67 0.97 12.30 0.34 2.66 0.47 0.38 0.25 1.57 0.93 1.48

Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease Disease

x¯ 6SEM (n515)

1.9261.04

2.5461.59

0.77 0.72 0.31 3.02 0.27 0.61 0.41 1.29 0.59 0.35 0.49 0.71 1.09 2.21 0.68 0.28 0.94 0.41

0.64 0.51 0.31 0.70 0.31 0.63 0.26 2.07 0.46 0.33 0.62 0.55 1.29 1.18 0.66 0.09 1.32 0.74

x¯ 6SEM (n518)

0.8660.17

0.9660.10

P value Linear Regression

0.5417

0.3411

98A / 17 98A / 37 98A / 23 98A / 2 96A / 16 96X / 338 96A / 20 97A / 47 97A / 42 97A / 84 98A / 18 96A / 28 97A / 17 98A / 27 97X / 905 97A / 30 97A / 6 97A / 83

Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Frontal Lobe Atrophy Motor Neuron Disease Motor Neuron Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Huntington’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Parkinson’s Disease Multi Infarct Dementia Multiple System Atrophy Subacute Sclerosing Panencephalitis

Table 6 APP isoforms in Alzheimer’s disease and other neurodegenerative disorders: Summary. OD units mRNA expression APP695

APP751

APP751 /APP695

Frontal Lobe Alzheimer’s Disease Other disorders P value

17.4963.26 13.1361.76 0.8062

12.8562.98 13.7062.88 0.9695

1.9261.04 0.8660.17 0.547

Temporal Lobe Alzheimer’s Disease Other disorders P value

14.7362.95 16.4962.15 0.5468

13.3164.93 11.0761.98 0.6466

2.5461.59 0.9660.10 0.3411

under analysis does influence the data interpretation. Neuropeptide genes, and genes like APP, are stable for prolonged intervals after death in comparison to immediate early genes like c-fos and c-jun which have a short half-life and high turnover [42]. The stability of APP mRNA in post-mortem brain for greater than 36 h has been observed by other workers and is thought not to be the main contributor to differences in RNA expression of this gene [43]. The agonal state may be important but in our study populations we have observed no significant differences between the two groups and agonal effects must have cancelled each other. Tissue pH may in the future help in the selection of brain material for investigation of mRNA activity — this potential factor again negating itself

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

90

Table 7 Published studies of APP mRNA expression in Alzheimer’s disease Author

Year

Method

N

Findings

Palmert et al.

1988

ISH

↑APP KPI2

Johnson et al.

1988

Neve et al.

1988

Johnson et al.

1989

Spillantini et al.

1989

Northern analysis Northern analysis 1ISH Northern analysis ISH

AD511 CTRL57 AD55 CTRL55 AD51 CTRL51

Golde et al.

1990

PCR

Johnson et al.

1990

ISH

Koo et al.

1990

¨ Konig et al.

1991

Oyama et al.

1991

Tanaka et al.

1992

Hyman et al.

1993

Harrison et al.

1994

Northern analysis 1ISH S1 nuclease protection assay S1 nuclease protection assay RNase protection Nonisotopic ISH ISH

Panegyres et al.

1999

ISH

AD55 CTRL55 AD55 CTRL51 AD55 CTRL55 AD56 CTRL54 AD57 CTRL55

↓APP KPI2 ↑APP KPI1 /APP KPI2 No change

↓APP KPI2 ↑APP KPI1 /APP KPI2 ↓APP KPI2 ↑APP KPI1 (white matter) ↑APP KPI2 (meninges) ↑APP KPI1 ↑APP KPI1 /APP KPI2 APP KPI2 no change No change

AD55 CTRL58

No change

AD54 CTRL534

No change

AD58 CTRL57 AD511 CTRL58 AD511 CTRL54 Non AD56 dementia AD515 CTRL518

↑APP KPI1 /APP KPI2 No change ↓APP KPI2 ↓APP KPI1

No change

CTRL5Controls; ISH5In situ hybridization.

between the two experimental populations in this study [44]. The nature of the freezing protocol and storage were also identical between the two groups. Bias may also influence the quantification and in our study, in comparison to those reported above where it is not clear from the methods, we have quantified our gene expression studies without knowledge of the diagnosis. The in situ hybridization was also performed without this information. Our data supports the hypothesis that differential gene expression of APP isoforms does not contribute to the amyloidosis found in AD and that post-translational events may be important. It is possible that differential gene expression of alternatively spliced isoforms of APP may be operational early in the disease during plaque formation and this possibility is not excluded by our studies. It may be possible that differential gene expression may be occurring in proximity to the evolving plaques — a notion that has not been confirmed by an investigation of gene expression in the microenvironment of neuritic plaques [12,45]. APP protein containing KPI in purified material from

the soluble subcellular fraction of AD brains has recently been measured as approximately twice the level in controls [18]. This observation supports the possibility that abnormal protein processing of APP is involved in the pathogenesis of AD and our data is consistent with the possibility. Recent evidence suggests that frameshift mutations may occur at a transcriptional level or that post-transcriptional editing of mRNA might lead to the production of abnormal proteins like APP in neurodegenerative disorders [46]. The altered metabolism of APP may involve impaired catabolism and degradation at a lysosomal level, or in protein synthesis and assembly within the trans Golgi network where the conditions might favour the production of APP KPI1 [47]. These conditions may prevail in diseased cells. If APP KPI1 is more abundant then this may have functional implications as APP KPI1 is more amyloidogenic in vitro [48]. If APP KPI1 is increased in cells then the intracellular conditions shift in balance towards protease inhibition — circumstances which favour

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

the greater production of APP KPI1 and may trigger the evolution and progression of AD. Low density lipoprotein receptor related protein (LRP) is a receptor involved in the internalisation and degradation of APP KPI1 isoforms [49]. This interaction is enhanced if APP is bound to epidermal growth factor binding protein (EGFB) [50]. APP 695 does not bind EGFBP. Alpha 2 macroglubulin (A2M) and apolipoprotein E (APOE) also bind to LRP. Given that these proteins are important risk factors for the development of AD [51], a pathway is suggested by which these factors interface and lead to the development of AD. Firstly, the interaction of APP KPI1 and LRP may be modified by A2M and APOE leading to an increase in APP KPI1 protein possibly through a mechanism involving receptor antagonism. Secondly, the increase in APP KPI1 protein favours protease inhibition, a condition which would encourage further increases in APP KPI1. Thirdly, APP KPI1 protein is amyloidogenic. The elevation in the APP KPI1 protein may be the reason that there is no selective increase in its mRNA by a mechanism involving feedback inhibition. Future therapeutic strategies may result from investigation of LRP and its interaction with APP, A2M and APOE.

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

Acknowledgements We are appreciative of the assistance offered by the mortuary staff, the personnel of the Neuropathology Department and the Department of Medical Physics, Royal Perth Hospital. We thank the NH & MRC, the Medical Research Foundation of Royal Perth Hospital, and The Medical Research Fund of Western Australia for making this work possible.

[16]

[17]

[18]

[19]

References [1] Alzheimer A. Uber eine eigenartige erkrankung der hirnrinde. Allgemcine Zeitsch Psych Psychisch-Gerichtishlicke Med 1907: LXIV: 146–148; translated in Bick K, Armaducci L, Pepeu G, editors. The Early Story of Alzheimers Disease. Italy: Livinia Press. 1987, pp. 1–3. [2] Tomlinson BE. Aging and the dementias. In: Hume Adams J, Duchen LW, editors, Greenfields neuropathology, Kent: Edward Arnold, 1992, pp. 1284–410, 5th ed. [3] Glenner GG, Wong CW. Alzheimers disease: Initial report of the purification and characterisation of a novel cerebro-vascular amyloid peptide. Biochem Biophys Res Commun 1984;120:885–90. [4] Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. Amyloid plaque core protein in Alzheimers disease and Down syndrome. Proc Natl Acad Sci USA 1988;82:4245–9. [5] Kang J, Lemaire HG, Unterbeck A et al. The precursor of Alzheimers disease amyloid A4 protein resembles a cell surface receptor. Nature 1987;325:733–6. [6] Selkoe DJ, Podlisny MB, Joachim CL et al. b-amyloid precursor protein of Alzheimers disease occurs as a 110–135 kilo-Dalton

[20]

[21]

[22]

[23] [24]

91

membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci USA 1988;85:7341–5. Chartier-Harlin MC, Hardy J, Mullan M. Early onset Alzheimers disease caused by mutations at exon 717 of the b-amyloid precursor protein gene. Nature 1991;353:844–6. Goate A, Charter-Harlin MC, Mullan M et al. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimers disease. Nature 1991;349:704–6. Murrell J, Farlow M, Ghetti B, Benson MD. A mutation in the amyloid precursor protein associated with hereditary Alzheimers disease. Science 1991;254:97–9. Citron M, Olterdorf T, Haass C et al. Mutation of the beta-amyloid precursor protein in familial Alzheimers disease causes increased beta amyloid production. Nature 1992;360:672–4. Cai XD, Golde TE, Younkin SG. Excess amyloid beta-protein is released from a mutant amyloid beta protein precursor linked to familial Alzheimers disease. Science 1993;259:514–6. Hyman BT, Wenniger JJ, Tanzi RE. Nonisotopic in situ hybridization of amyloid beta protein precursor in Alzheimers disease: expression in neurofibrillary tangle bearing neurons and in the microenvironment surrounding senile plaques. Mol Brain Res 1993;18:253–8. Scheuner D, Eckman C, Jensen M et al. Secreted amyloid b-protein similar to that in the senile plaques of Alzheimers disease is increased in vivo by the presenilin 1 and 1 and APP mutations linked to familial Alzheimers disease. Nat Med 1996;2:864–70. Gomez-Isla T, West HL, Rebeck GW et al. Clinical and pathological correlates of apolipoprotein E e4 in Alzheimers disease. Ann Neurol 1996;39:62–70. Panegyres PK. The amyloid precursor protein gene: a neuropeptide gene with diverse functions in the central nervous system. Neuropeptides 1997;31:523–35. Johnson SA, McNeill T, Cordell B, Finch CE. Relation of neural APP-751 /APP-695 mRNA ratio and neuritic plaque density in Alzheimers disease. Science 1990;248:854–7. Tanaka S, Liu L, Kimura J et al. Age-related changes in the proportion of amyloid precursor protein mRNAs in Alzheimers disease and other neurological disorders. Mol Brain Res 1992;15:303–10. Moir RD, Lynch T, Bush AI et al. Relative increase in Alzheimers disease of soluble forms of cerebral Ab amyloid protein precursor containing the Kunitz protease inhibitory domain. J Biol Chem 1998;273:5013–9. Sola´ C, Mengod G, Probst A, Palacios KM. Differential regional and cellular distribution of b-amyloid precursor protein messenger RNAs containing and lacking the Kunitz protease inhibitor domain in the brain of human, rat and mouse. Neuroscience 1993;53:267– 95. Mirra SS, Heyman A, McKeel D et al. The Consortium to Establish a Registry for Alzheimers Disease (CERAD): II. Standardisation of the neuropathological assessment of Alzheimers disease. Neurology 1991;41:479–86. Hyman BT, Trojanowski JQ. Editorial on consensus recommendations for the postmortem diagnosis of Alzheimers disease from the National Institute on Aging and the Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimers Disease. J Neuropathol Exp Neurol 1997;56:1095–7. National Institute on Aging and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimers Disease. Consensus recommendations for the postmortem diagnosis of Alzheimers disease. Neurobiol Aging 1997;18(45):51– 53. Jackson M, Lowe J. The new neuropathology of degenerative frontotemporal dementias. Acta Neuropathol 1996;91:127–34. Vonsattel J-P, Myers RH, Stevens TJ, Ferrante RJ, Bird ED. Neuropathological classification of Huntington’s disease. J Neuropath Exp Neurol 1985;44:559–77.

92

P.K. Panegyres et al. / Journal of the Neurological Sciences 173 (2000) 81 – 92

[25] Gelb DJ, Oliver E, Gilman S. Diagnostic criteria for Parkinson disease. Arch Neurol 1999;56:33–9. [26] Brownell B, Oppenheimer DR, Hughes JT. The central nervous system in motor neurone disease. J Neurol Neurosurg Psych 1970;33:338–57. ´ GC, Tatemichi TK, Erkinjuntti T et al. Vascular dementia: [27] Roman Diagnostic criteria for research studies. Neurology 1993;43:250–60. [28] Papp MI, Kahn JE, Lantos PL. Glial cytaplasmic inclusions in the CNS of patients with multiple system atrophy (striatonigral degeneration. olivopantocerebellar atrophy and Shy-Drager syndrome). J Neurol Sci 1989;94:1–3. [29] Quinn N. Multiple system atrophy — the nature of the beast. J Neurol Neurosurg Psychiat 1989;52(Special Suppl):78–89. [30] Corsellis J. Subacute sclerosing leukoencephalitis: Clinical and pathological reports of 2 cases. J Ment Sci 1951;97:570–83. [31] Panegyres PK. The effects of excitotoxicity on the expression of the amyloid precursor protein gene in the brain and its modulation by neuroprotective agents. J Neural Transm 1998;105:463–78. [32] Johnson SA, Pasinetti GM, May PC, Ponte PA, Cordell B, Finch CE. Selective reduction of mRNA for the b-amyloid precursor prtein that lacks a Kunitz type protease inhibitor motif in cortex from Alzheimer brains. Exp Neurol 1988;102:264–8. [33] Johnson SA, Rogers J, Finch CE. APP-695 transcript prevalence is selectively reduced during Alzheimers disease in cortex and hippocampus but not in cerebellum. Neurobiol Aging 1989;10:267–72. ¨ [34] Konig G, Salbaum JM, Wiestler O et al. Alternative splicing of the bA4 amyloid gene of Alzheimers disease in cortex of control and Alzheimers disease patient. Mol Brain Res 1991;9:259–62. [35] Koo EH, Sisodia SS, Cork LC, Unterbeck A, Bayney RM, Price DL. Differential expression of amyloid precursor protein mRNAs in cases of Alzheimers disease and in aged nonhuman primates. Neuron 1990;2:97–104. [36] Neve RL, Finch EA, Dawes LR. Expression of the Alzheimer amyloid precursor gene transcripts in the human brain. Neuron 1988;1:669–77. [37] Oyama F, Shimada M, Oyama R, Titani K, Ihara Y. Differential expression of b amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP 751 and four repeat tau. J Neuropath Exp Neurol 1991;50:560– 78. [38] Palmert MR, Golde TE, Cohen ML et al. Amyloid protein precursor messenger RNAs: Differential expression in Alzheimers disease. Science 1988;241:1080–4. [39] Golde TE, Estus S, Usiak M, Younkin LH, Younkin SG. Expression

[40]

[41]

[42]

[43] [44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

of b amyloid protein precursor mRNAs: Recognition of a novel alternatively spliced form and quantitation in Alzheimers disease using PCR. Neuron 1990;4:253–67. Spillantini MG, Hunt SP, Ulrich J, Goedert M. Expression and cellular localisation of amyloid b-protein precursor transcripts in normal human brain and in Alzheimers disease. Mol Brain Res 1989;6:143–50. Harrison PJ, Barton AJL, Procter AW, Bowen DM, Pearson RCA. The effects of Alzheimers disease. other dementias. and premortem course on b-amyloid precursor protein messenger RNA in frontal cortex. J Neurochem 1994;62:635–44. Harrison PJ, Pearson RCA. In situ hybridization histochemistry and the study of gene expression in the human brain. Prog Neurobiol 1990;34:271–312. Barton AJL, Pearson RCA, Najlerahim A, Harrison PJ. Pre- and post-mortem influences on brain RNA. J Neurochem 1993;61:1–11. Kingsbury AE, Foster OJF, Nisbet AP et al. Tissue pH as an indicator of mRNA preservation in human post-mortem brain. Mol Brain Res 1995;28:311–8. Tanzi RE, Wenniger JJ, Hyman BT. Cellular specificity and regional distribution of amyloid b protein precursor alternative transcripts are unaltered in Alzheimer hippocampal formation. Mol Brain Res 1993;18:246–52. Van Leeuwen FW, de Kleijn DPV, van den Hurk HH et al. Frameshift mutations of b amyloid precursor protein and ubiquitin-b in Alzheimers disease and Down patients. Science 1998;279:242–7. Greenfield JP, Tsai J, Gouras GK et al. Endoplasmic reticulum and trans-Golgi network generate distinct populations of Alzheimer beta-amyloid peptides. Proc Natl Acad Sci USA 1999;19:742–7. Ho L, Fukuchi K-I, Younkin SG. The alternatively spliced Kunitz protease inhibitor domain alters amyloid b protein precursor processing and amyloid b protein production in cultured cells. J Biol Chem 1996;271:30929–34. Kounnas MZ, Moir RD, Rebeck GW et al. LDL receptor related protein, a multifunctional APOE receptor, binds secreted betaamyloid precursor protein and mediates its degradation. Cell 1995;28:331–40. Knauer MF, Orlando RA, Glabe CG. Cell surface APP 751 forms complexes with protease nexin 2 ligands and is internalised via the low density lipoprotein receptor-related protein (LRP). Brain Res 1996;18:6–14. Blacker D, Tanzi RE. The genetics of Alzheimers disease. Arch Neurol 1998;55:294–6.