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

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 KPI+) are over expressed in the brains of patients with AD when compared to the non-Kunitz protease inhibitor containing isoforms (APP KPI-). 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 35S to detect the two principal splice variants of APP: APP 695 (KPI-) and APP 751 (KPI+). A prospective brain bank of frozen brain specimens has been established and includes pathologically proven AD (n=15) and other neurodegenerative disorders as controls (n=18). The controls consist of frontal lobe atrophy (n=4), Huntington's disease (n=5), Parkinson's disease (n=4), motor neuron disease (n=2), multi-infarct dementia (n=1), multisystem atrophy (n=1), and subacute sclerosing panencephalitis (n=1). We have observed no significant differences in the expression of APP 695 KPI- mRNA in frontal lobe: 17.49+/-3.26 optical density (OD) units of mRNA expression in AD vs. 16.13+/-1.76 OD units mRNA in controls (P=0.80, linear regression); or temporal lobe: 14.73+/-2.96 in AD vs. 16.49+/-2.15 in controls (P=0.55). No significant differences have been found in APP 751 KPI+ in frontal lobe: 12.86+/-2.98 in AD vs. 13.70+/-2.88 in controls (P=0.97); and temporal lobe: 13.31+/-4.93 in AD vs. 11.07+/-1.99 in controls (P=0. 65). Analysis of the ratios of APP 751 KPI+ OD units of mRNA to APP 695 KPI- mRNA revealed a trend to an increased ratio which did not reach statistical significance: frontal lobe APP 751 KPI+/APP 695 KPI- 1.92+/-1.04 in AD vs. 0.86+/-0.17 in controls (P=0.54); temporal lobe 2.54+/-1.59 in AD vs. 0.96+/-0.11 controls (P=0.34). 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.     

An abnormality of plasma amyloid protein precursor in Alzheimer's disease.

beta A4 amyloid deposition in the brain, which is characteristic of Alzheimer's disease (AD), may result from either overexpression of the amyloid protein precursor (APP) or failure of APP to be correctly processed. A blood marker reflecting this abnormal metabolism would be of diagnostic value and would provide a means of monitoring the efficacy of therapeutic interventions. We analyzed immunoblots of plasma APP enriched by heparin-Sepharose chromatography from patients with moderate to severe AD dementia (n = 34) and control subjects (n = 77) and found an approximately 50% increase in the proportion of 130-kd APP species in patients with AD (p less than 0.001), no difference in the 110-kd form, a 15 to 30% decrease in the 65-kd form (p less than 0.001), and a 20 to 35% decrease in the proportion of 42-kd APP (p less than 0.001). These species of APP were soluble, lacked the carboxyl terminus, and the 110- and 42-kd species were shown to be consistent with degradation products derived from the 130-kd species. A comparison of levels of 130-kd plasma APP from moderately to severely demented patients with AD and control subjects distinguished the two groups with a specificity of 87.0% and a sensitivity of 79.4%.     

Reduced expression of amyloid precursor protein, presenilin-1 and rab3a in cortical brain regions in Alzheimer's disease

To study the role of amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD), the level of APP was analysed by quantitative immunoblotting in 6 AD patients and 6 age-matched controls in 9 brain regions. These were associative cortices (orbital frontal cortex, inferior temporal cortex, inferior parietal cortex), primary cortex (occipital cortex), limbic structures (anterior cingulate gyrus, hippocampus), subcortical structures (putamen, thalamus) and cerebellum. To assess a potential relationship between APP and presenilin-1 (PS-1) and/or synaptic proteins, the levels of PS-1 and rab3a, a specific synaptic vesicle protein, were also determined in the same tissue samples. The level of APP was almost the same in the association cortical regions, primary cortex, and limbic structures and in the subcortical structures, while the lowest level was found in the cerebellum. There were more marked differences in the level of PS-1 and rab3a between different brain regions. The highest levels of PS-1 and rab3a were found in the association cortical areas, while intermediate levels were found in primary cortex, limbic structures and subcortical structures. As for APP, the lowest level was found in cerebellum. We found significantly reduced levels of all three proteins in the association cortices and in hippocampus in AD. Our data show that the protein levels are reduced in specific areas, restricted to neuronal populations that are known to degenerate in AD. Due to the similarity of the expression of APP, PS-1 and rab3a, it is tempting to speculate whether there is a functional relationship between these proteins.     

Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer's disease

The activation of caspase-3 and possibly other caspases during apoptosis may lead to the cleavage of the amyloid precursor protein (APP) and subsequent accumulation of APP cleavage products (cAPP). We examined the association between activated caspase-3 and cAPP in human brain by qualitative and quantitative analysis of in situ immunohistochemistry and Western blots. Frontal cortex and hippocampal tissue from age-matched control and Alzheimer's brains (AD) was used. Both activated caspase-3 and cAPP are increased in AD [Braak and Braak (BB) stage IV-VI] compared to aged control (BB stage 0-1) and transitional (BB stage II-III) cases in the hippocampal and frontal cortex. Caspase-3 activation and the accumulation of APP cleavage fragments appear to either parallel or precede neurofibrillary tangle formation. These findings raise the possibility that the activation of caspase-3 and cleavage of APP may be involved with neuronal degeneration and that pathways characteristic of apoptosis are activated in AD.     

Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease

Beta-secretase cleavage represents the first step in the generation of Abeta polypeptides and initiates the amyloid cascade that leads to neurodegeneration in Alzheimer's disease. By comparative Western blot analysis, we show a 2.7-fold increase in protein expression of the beta-secretase enzyme BACE in the brain cortex of Alzheimer's disease patients as compared to age-matched controls. Similarly, we found the levels of the amyloid precursor protein C-terminal fragment produced by beta-secretase to be increased by nearly twofold in Alzheimer's disease cortex.     

Hereditary Alzheimer's disease with amyloid angiopathy caused by amyloid precursor protein locus

We report a patient with early-onset autosomal dominant dementia. The CSF showed increased levels of tau protein and decreased amyloid beta (ratio 42:40) typical for Alzheimer's disease. Cerebral MRI revealed vascular lesions and white-matter changes around the posterior horns of the ventricles with only moderate atrophy of the brain. Susceptibility-weighted imaging detected multiple small hemorrhagic changes. Gene analysis revealed amyloid precursor protein (APP) locus duplication as the cause of hereditary Alzheimer's dementia. The co-occurrence of CSF changes typical for Alzheimer's disease and MRI findings of cerebral amyloid angiopathy is remarkable, as it is also described for APP locus duplication. In conjunction with a family history suggestive of hereditary dementia, such a constellation should lead to enhanced gene analysis.     

Amyloid precursor protein mRNA levels in Alzheimer's disease brain

Insoluble beta-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(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta 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.     

The amyloid precursor protein of Alzheimer's disease and the Abeta peptide

Alzheimer's disease is characterized by the accumulation of beta amyloid peptides in plaques and vessel walls and by the intraneuronal accumulation of paired helical filaments composed of hyperphosphorylated tau. In this review, we concentrate on the biology of amyloid precursor protein, and on the central role of amyloid in the pathogenesis of Alzheimer's disease. Amyloid precursor protein (APP) is part of a super-family of transmembrane and secreted proteins. It appears to have a number of roles, including regulation of haemostasis and mediation of neuroprotection. APP also has potentially important metal and heparin-binding properties, and the current challenge is to synthesize all these varied activities into a coherent view of its function. Cleavage of amyloid precursor protein by beta-and gamma-secretases results in the generation of the Abeta (betaA4) peptide, whereas alpha-secretase cleaves within the Abeta sequence and prevents formation from APP. Recent findings indicate that the site of gamma-secretase cleavage is critical to the development of amyloid deposits; Abeta1-42 is much more amyloidogenic than Abeta1-40. Abeta1-42 formation is favoured by mutations in the two presenilin genes (PS1 and PS2), and by the commonest amyloid precursor protein mutations. Transgenic mouse models of Alzheimer's disease incorporating various mutations in the presenilin gene now exist, and have shown amyloid accumulation and cognitive impairment. Neurofibrillary tangles have not been reproduced in these models, however. While aggregated Abeta is neurotoxic, perhaps via an oxidative mechanism, the relationship between such toxicity and neurofibrillary tangle formation remains a subject of ongoing research.     

Distribution of amyloid beta protein precursor in the Alzheimer's disease brain

In order to clarify the distribution and pathological changes of the amyloid beta protein precursor (betaAPP), 10 Alzheimer's disease (AD) brains and seven normal control brains were examined by immunocytochemistry and in situ hybridization histochemistry. All betaAPP isoforms were distributed evenly in neuronal cell bodies and their axons and dendrites. The betaAPP-positive neuronal processes showed mesh-like networks. In AD brains, betaAPP-positive neurons and mesh-like networks were generally decreased in spite of some intensely labeled neurons. All betaAPP isoforms accumulated in neuronal processes, dystrophic neurites and senile plaques. In situ hybridization histochemistry confirmed that all isoforms of betaAPP were expressed in neurons in control brains. In AD brains, the betaAPP mRNA signal was generally decreased besides some intense signal neurons corresponding to immunostaining findings. Few astrocytes expressed betaAPP. Thus, uniform expression and distribution of betaAPP were disturbed in AD brains showing uneven decreases or increases of neuronal betaAPP expression in individual neurons and betaAPP accumulation in neurons, neuronal processes and abnormal structures including dystrophic neurites, senile plaques and neurofibrillary tangles.     

Mint2/X11-like colocalizes with the Alzheimer's disease amyloid precursor protein and is associated with neuritic plaques in Alzheimer's disease.

Aberrant metabolism of the amyloid precursor protein (APP) is believed to be at least part of the pathogenic process in Alzheimer's disease. The carboxy-terminus of APP has been shown to interact with the Mint/X11 family of phosphotyrosine binding (PTB) domain-bearing proteins. It is via their PTB domains that the Mints/X11s bind to APP. Here we report the cloning of full-length mouse Mint2 and demonstrate that in primary cortical neurons, Mint2 and APP share highly similar distributions. Mint2 also colocalizes with APP in transfected CHO cells. In Mint2/APP-cotransfected cells, Mint2 reorganizes the subcellular distribution of APP and also increases the steady-state levels of APP. Finally, we demonstrate that Mint2 is associated with the neuritic plaques found in Alzheimer's disease but not with neurofibrillary tangles. These results are consistent with a role for Mint2 in APP metabolism and trafficking, and suggest a possible role for the Mints/X11s in the pathogenesis of Alzheimer's disease.     

Melatonin in Alzheimer's disease and other neurodegenerative disorders

Increased oxidative stress and mitochondrial dysfunction have been identified as common pathophysiological phenomena associated with neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). As the age-related decline in the production of melatonin may contribute to increased levels of oxidative stress in the elderly, the role of this neuroprotective agent is attracting increasing attention. Melatonin has multiple actions as a regulator of antioxidant and prooxidant enzymes, radical scavenger and antagonist of mitochondrial radical formation. The ability of melatonin and its kynuramine metabolites to interact directly with the electron transport chain by increasing the electron flow and reducing electron leakage are unique features by which melatonin is able to increase the survival of neurons under enhanced oxidative stress. Moreover, antifibrillogenic actions have been demonstrated in vitro, also in the presence of profibrillogenic apoE4 or apoE3, and in vivo, in a transgenic mouse model. Amyloid-β toxicity is antagonized by melatonin and one of its kynuramine metabolites. Cytoskeletal disorganization and protein hyperphosphorylation, as induced in several cell-line models, have been attenuated by melatonin, effects comprising stress kinase downregulation and extending to neurotrophin expression. Various experimental models of AD, PD and HD indicate the usefulness of melatonin in antagonizing disease progression and/or mitigating some of the symptoms. Melatonin secretion has been found to be altered in AD and PD. Attempts to compensate for age- and disease-dependent melatonin deficiency have shown that administration of this compound can improve sleep efficiency in AD and PD and, to some extent, cognitive function in AD patients. Exogenous melatonin has also been reported to alleviate behavioral symptoms such as sundowning. Taken together, these findings suggest that melatonin, its analogues and kynuric metabolites may have potential value in prevention and treatment of AD and other neurodegenerative disorders.     

Correlation between platelet amyloid precursor protein isoform ratio and cognition in Alzheimer's disease

This study analyzed whether platelet amyloid beta-protein precursor (AbetaPP) isoform ratio correlates with cognition or cognitive decline in patients with Alzheimer's disease (AD). Platelet AbetaPP isoform ratio was measured, and cognitive assessment was performed using the Mini-Mental State Examination (MMSE) in 66 AD patients at baseline (T0) and in 29 of these patients in a one-year follow-up (T1). There was a significant correlation between the AbetaPP isoform ratios and MMSE scores in the 66 AD patients at T0. The T1 subjects were divided into two groups: 12 "no decliners" (MMSE score, T1-T0 > or = 0) and 17 "decliners" (MMSE score, T1-T0 < 0). The decliners group showed a significantly greater reduction of AbetaPP isoform ratio from T0 to T1 than the no decliners group. However, the decline of the ratio did not correlate with the decline of MMSE score. These findings indicate that AbetaPP isoform ratio correlates with cognition, and reduction in this ratio may be a marker for cognitive decline in AD patients.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein in brain and platelets

Proteolytic processing of the amyloid precursor protein by beta -and gamma-secretases results in the production of Alzheimer's disease (AD) Abeta amyloid peptides. Modulation of secretase activity is being investigated as a potential therapeutic approach. Recent studies with human brain have revealed that the beta-secretase protein, BACE, is increased in cortex of AD patients. Analysis of betaCTF (or C99), the amyloid precursor protein (APP) product of BACE cleavage that is the direct precursor to Abeta, shows it is also elevated in AD, underlying the importance of beta-secretase cleavage in AD pathogenesis. The C-terminal product of gamma-secretase cleavage of APP, epsilonCTF (or AICD), is enriched in human brain cortical nuclear fractions, a subcellular distribution appropriate for a putative involvement of APP cytosolic domain in signal transduction. Analysis of AD cortex samples, particularly that of a carrier of a familial APP mutation, suggests that processing of APP transmembrane domain generates an alternative CTF product. All these particularities observed in the AD brain demonstrate that APP processing is altered in AD. The transgenic mouse model Tg2576 seems to be a promising laboratory tool to test potential modulators of Abeta formation. Indeed, C-terminal products of alpha-, beta-, and gamma-secretase cleavage are readily detectable in the brain of these transgenic mice. Finally, the finding of the same secretase products in platelets and neurons make platelets a potentially useful and easily accessible clinical tool to monitor effects of novel therapies based on inhibition of beta- or gamma-secretase.     

CRMP2 hyperphosphorylation is characteristic of Alzheimer's disease and not a feature common to other neurodegenerative diseases

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that regulates neurite outgrowth. It is phosphorylated by Cdk5 and GSK3, and these modifications are abnormally high in the brains of Alzheimer's disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AβPP and PSEN1, but not AβPP or tau alone, where it is detectable before the appearance of amyloid plaques and neurofibrillary tangles, suggesting it is an early event in AD pathogenesis. Here, we have extended these observations by showing that CRMP2 is not hyperphosphorylated in mice overexpressing mutated PSEN1 alone, or in cultured neurons treated with soluble, oligomeric Aβ42 peptide. Similarly, CRMP2 phosphorylation was not increased in a mouse model of severe neurodegeneration (PMSC-1 knockout) or in cultured neurons subjected to neurotoxic concentrations of NMDA or staurosporine. Most interestingly, CRMP2 phosphorylation was not increased in frontal cortex from patients with frontotemporal lobar degeneration associated with mutations in MAPT or with Pick bodies. Together, these observations are consistent with the hypothesis that abnormal phosphorylation of CRMP2 is specific to AD and occurs downstream of excessive processing of AβPP, but that neither excessive Aβ42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.     

Neurite-outgrowth regulating functions of the amyloid protein precursor of Alzheimer's disease

Many studies have shown that breakdown of the amyloid protein precursor (APP) to produce the amyloid protein is an important step in the pathogenic mechanism which causes Alzheimer's disease (AD). However, little is known about the normal function of APP. Developmental studies show that APP expression increases during the period of brain development when neurite outgrowth and synaptogenesis is maximal. APP is expressed highly within growing neurites and in growth cones, and purified APP has been shown to stimulate neurite outgrowth from cells in culture. Thus APP may regulate neurite outgrowth or synaptogenesis in vivo. APP is actively secreted from many cells, and the C-terminally secreted APP has been shown to associate with components of the extracellular matrix, such as the heparan sulphate proteoglycans (HSPGs). Two putative heparin-binding domains on APP have been reported. Binding of HSPGs to an N-terminal heparin-binding domain (HBD-1) stimulates the effect of substrate-bound APP on neurite outgrowth. In the mature nervous system, APP may play an important role in the regulation of wound repair. It is highly likely that studies on the normal functions of APP will shed further light on aspects of the pathogenesis of AD.     

Platelet amyloid precursor protein processing: a bio-marker for Alzheimer's disease

The amyloid precursor protein (APP) in brain is processed either by an amyloidogenic pathway by beta-secretase and gamma-secretase to yield Abeta (beta-amyloid 4 kDa) peptide or by alpha-secretase within the beta-amyloid domain to yield non-amyloidogenic products. We have studied blood platelet levels of a 22-kDa fragment containing the Abeta (beta-amyloid 4 kDa) peptide, beta-secretase (BACE1), alpha-secretase (ADAM10), and APP isoform ratios of the 120-130 kDa to 110 kDa peptides from 31 Alzheimer's disease (AD) patients and 10 age-matched healthy control subjects. We found increased levels of Abeta4, increased activation of beta-secretase (BACE1), decreased activation of alpha-secretase (ADAM10) and decreased APP ratios in AD patients compared to normal control subjects. These observations indicate that the blood platelet APP is processed by the same amyloidogenic and non-amyloidogenic pathways as utilized in brain and that APP processing in AD patients is altered compared to control subjects and may be a useful bio-marker for the diagnosis of AD, the progression of disease and for monitoring drug responses in clinical trials.