Donna L. McPhie

Alzheimer's disease : a dysfunction of the amyloid precursor protein

Abstract In this review, we argue that at least one insult that causes Alzheimer’s disease (AD) is disruption of the normal function of the amyloid precursor protein (APP). Familial Alzheimer’s disease (FAD) mutations in APP cause a disease phenotype that is identical (with the exception that they cause an earlier onset of the disease) to that of ‘sporadic’ AD. This suggests that there are molecular pathways common to FAD and sporadic AD. In addition, all individuals with Down syndrome, who carry an extra copy of chromosome 21 and overexpress APP several-fold in the brain, develop the pathology of AD if they live past the age of 40. These data support the primacy of APP in the disease. Although APP is the source of the β-amyloid (Aβ) that is deposited in amyloid plaques in AD brain, the primacy of APP in AD may not lie in the production of Aβ from this molecule. We suggest instead that APP normally functions in the brain as a cell surface signaling molecule, and that a disruption of this normal function of APP is at least one cause of the neurodegeneration and consequent dementia in AD. We hypothesize in addition that disruption of the normal signaling function of APP causes cell cycle abnormalities in the neuron, and that these abnormalities constitute one mechanism of neuronal death in AD. Data supporting these hypotheses have come from investigations of the molecular consequences of neuronal expression of FAD mutants of APP or overexpression of wild type APP, as well as from identification of binding proteins for the carboxyl-terminus (C-terminus) of APP.

Abnormalities in Mitochondrial Structure in Cells from Patients with Bipolar Disorder

Postmortem, genetic, brain imaging, and peripheral cell studies all support decreased mitochondrial activity as a factor in the manifestation of Bipolar Disorder (BD). Because abnormal mitochondrial morphology is often linked to altered energy metabolism, we investigated whether changes in mitochondrial structure were present in brain and peripheral cells of patients with BD. Mitochondria from patients with BD exhibited size and distributional abnormalities compared with psychiatrically-healthy age-matched controls. Specifically, in brain, individual mitochondria profiles had significantly smaller areas, on average, in BD samples (P = 0.03). In peripheral cells, mitochondria in BD samples were concentrated proportionately more within the perinuclear region than in distal processes (P = 0.0008). These mitochondrial changes did not appear to be correlated with exposure to lithium. Also, these abnormalities in brain and peripheral cells were independent of substantial changes in the actin or tubulin cytoskeleton with which mitochondria interact. The observed changes in mitochondrial size and distribution may be linked to energy deficits and, therefore, may have consequences for cell plasticity, resilience, and survival in patients with BD, especially in brain, which has a high-energy requirement. The findings may have implications for diagnosis, if they are specific to BD, and for treatment, if they provide clues as to the underlying pathophysiology of BD.

Discrimination learning alters the distribution of protein kinase C in the hippocampus of rats

Protein kinase C (PKC), an enzyme that plays an essential role in eukaryotic cell regulation (Nishizuka, 1988; Huang et al., 1989), is critical to memory storage processes both in the marine snail Hermissenda crassicornis and in the rabbit (Alkon et al., 1988; Bank et al., 1988; Olds et al., 1989). Specifically, activation of PKC mimics neurobiological correlates of classical conditioning in both Hermissenda and the rabbit, and the distribution of the enzyme within the rabbit hippocampus changes after Pavlovian conditioning. Here, we report that the amount of PKC, as assayed by specific binding of 3H- phorbol-12,13-dibutyrate (3H-PDBU), decreased significantly within the hippocampal CA3 cell region in rats trained to solve a water maze task either by cognitive mapping or by visual discrimination strategies, but not in control rats. Furthermore, hippocampal lesions interfered with acquisition of both of these tasks. We interpret these findings to support the conclusion that distributional changes of PKC within the mammalian hippocampus play a crucial role in memory storage processes.

DNA Synthesis and Neuronal Apoptosis Caused by Familial Alzheimer Disease Mutants of the Amyloid Precursor Protein Are Mediated by the p21 Activated Kinase PAK3

Apoptotic pathways and DNA synthesis are activated in neurons in the brains of individuals with Alzheimer disease (AD). However, the signaling mechanisms that mediate these events have not been defined. We show that expression of familial AD (FAD) mutants of the amyloid precursor protein (APP) in primary neurons in culture causes apoptosis and DNA synthesis. Both the apoptosis and the DNA synthesis are mediated by the p21 activated kinase PAK3, a serine-threonine kinase that interacts with APP. A dominant-negative kinase mutant of PAK3 inhibits the neuronal apoptosis and DNA synthesis; this effect is abolished by deletion of the PAK3 APP-binding domain or by coexpression of a peptide representing this binding domain. The involvement of PAK3 specifically in FAD APP-mediated apoptosis rather than in general apoptotic pathways is suggested by the facts that a dominant-positive mutant of PAK3 does not alone cause neuronal apoptosis and that the dominant-negative mutant of PAK3 does not inhibit chemically induced apoptosis. Pertussis toxin, which inactivates the heterotrimeric G-proteins Go and Gi, inhibits the apoptosis and DNA synthesis caused by FAD APP mutants; the apoptosis and DNA synthesis are rescued by coexpression of a pertussis toxin-insensitive Go. FAD APP-mediated DNA synthesis precedes FAD APP-mediated apoptosis in neurons, and inhibition of neuronal entry into the cell cycle inhibits the apoptosis. These data suggest that a normal signaling pathway mediated by the interaction of APP, PAK3, and Go is constitutively activated in neurons by FAD mutations in APP and that this activation causes cell cycle entry and consequent apoptosis.

Overexpression in Neurons of Human Presenilin-1 or a Presenilin-1 Familial Alzheimer Disease Mutant Does Not Enhance Apoptosis

Programmed cell death, or apoptosis, has been implicated in Alzheimer’s disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer’s disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein

In Alzheimers disease (AD), a progressive decline of cognitive functions is accompanied by neuropathology that includes the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature. We have proposed that a fragment of the Alzheimer amyloid precursor protein (APP) comprising the carboxyl-terminal 100 amino acids of this molecule (APP-C100) plays a crucial role in the neurodegeneration and subsequent cognitive decline in AD. To test this hypothesis, we performed behavioral analyses on transgenic mice expressing APP-C100 in the brain. The results revealed that homozygous APP-C100 transgenic mice were significantly impaired in cued, spatial and reversal performance of a Morris water maze task, that the degree of the impairment in the spatial learning was age-dependent, and that the homozygous mice displayed significantly more degeneration of neurons in Ammons horn of the hippocampal formation than did heterozygous or control mice. Among the heterozygotes, females were relatively more impaired in their spatial learning than were males. These findings show that expression of APP-C100 in the brain can cause age-dependent cognitive impairments that are accompanied by hippocampal degeneration.

Functional implications of a psychiatric risk variant within CACNA1C in induced human neurons.

Psychiatric disorders have clear heritable risk. Several large-scale genome-wide association studies have revealed a strong association between susceptibility for psychiatric disorders, including bipolar disease, schizophrenia and major depression, and a haplotype located in an intronic region of the L-type voltage-gated calcium channel (VGCC) subunit gene CACNA1C (peak associated SNP rs1006737), making it one of the most replicable and consistent associations in psychiatric genetics. In the current study, we used induced human neurons to reveal a functional phenotype associated with this psychiatric risk variant. We generated induced human neurons, or iN cells, from more than 20 individuals harboring homozygous risk genotypes, heterozygous or homozygous non-risk genotypes at the rs1006737 locus. Using these iNs, we performed electrophysiology and quantitative PCR experiments that demonstrated increased L-type VGCC current density as well as increased mRNA expression of CACNA1C in iNs homozygous for the risk genotype, compared with non-risk genotypes. These studies demonstrate that the risk genotype at rs1006737 is associated with significant functional alterations in human iNs, and may direct future efforts at developing novel therapeutics for the treatment of psychiatric disease.

Aβ42 generation is toxic to endothelial cells and inhibits eNOS function through an Akt/GSK-3β signaling-dependent mechanism

The application of beta-amyloid (Abeta) is cytotoxic to endothelial cells, promotes vasoconstriction and impairs nitric oxide (NO) generation or action. However, there is no information on the effect of intracellular Abeta on endothelial cell biology, although recent studies indicate that neuronal Abeta drives Alzheimers disease pathogenesis. Since the serine-threonine kinase Akt is crucial to both neuronal and endothelial cell survival as well as eNOS activation, we investigated the effects of Abeta expression on Akt-signaling in cultured endothelial cells. Virally-encoded Abeta42 was proapoptotic and inhibitory to Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Toxicity was characterized by mitochondrial dysfunction, DNA condensation and activation of caspase-3. Substrates downstream of Akt action, GSK-3beta and eNOS, are underphosphorylated in the presence of Abeta. Constitutive activation of Akt reversed Abeta-induced toxicity and stimulated caspase-3 activity, suggesting that inhibition of Akt signaling is functionally significant. These Abeta effects were mediated, in part, through the derepression of GSK-3beta activation and correlated with reductions in NO production. We conclude that intracellular production of Abeta42 is cytotoxic to endothelial cells and that disruption of the Akt/GSK-3beta cell signaling pathway is involved.

β-Secretase cleavage of the amyloid precursor protein mediates neuronal apoptosis caused by familial Alzheimer’s disease mutations ☆

The amyloid precursor protein (APP) is cleaved by two enzymes, beta-secretase and gamma-secretase, to generate the pathological amyloid beta (Abeta) peptide. Expression of familial Alzheimers disease (FAD) mutants of APP in primary neurons causes both intracellular accumulation of the C-terminal beta-secretase cleavage product of APP and increased secretion of Abeta, and eventually results in apoptotic death of the cells. To determine whether either of these two processing products of APP is involved in this apoptotic pathway, we first modeled experimentally the accumulation of the beta-secretase cleavage product in neurons. The C-terminal 100 amino acids (C100) of APP, with and without a signal peptide, was expressed in cells via recombinant herpes simplex virus (HSV) vectors. Both transgene products were targeted to the membrane, and both caused apoptosis in the neurons, implicating the beta-secretase cleavage product of APP in apoptosis caused by FAD APPs. Expression in neurons of a mutant of FAD APP that inhibited beta-secretase cleavage inhibited its ability to cause apoptosis. However, expression in neurons of a mutant of FAD APP that inhibited gamma-secretase cleavage did not inhibit the ability of this mutant to cause apoptosis. These data suggested that the C-terminal beta-secretase cleavage product of APP, but not Abeta, mediates the apoptosis caused by FAD mutants of APP. Consistent with this hypothesis, C31, which is generated from the beta-secretase cleavage product, itself caused neuronal apoptosis. Inhibitors of caspases 3, 6 and 8, but not of caspase 9, inhibited the apoptosis caused by FAD mutants of APP. It may be inferred from these data that beta-secretase cleavage of FAD mutants of APP allows the appropriate caspase access to its site of action to produce C31, which directly causes neuronal apoptosis.

APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain.

APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons (Chen, Y., D.L. McPhie, J. Hirschberg, and R.L. Neve. 2000. J. Biol. Chem. 275:8929–8935). We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimers disease brain hippocampus, but not in less-affected areas of Alzheimers disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.