Biomedical subjects

D L McPhie

Publications and source records attributed to D L McPhie.

At least 19 recordsLinked to original sources

PubMed2001

beta-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 Alzheimer's 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.

Alzheimer Disease↗

PubMed2001

Beta-amyloid peptide expression is sufficient for myotube death: implications for human inclusion body myopathy.

Inclusion body myositis (sIBM) is the most common disorder of skeletal muscle in aged humans. It shares biochemical features with Alzheimer's disease, including congophilic deposits, which are immunoreactive for beta-amyloid peptide (Abeta) and C'-terminal betaAPP epitopes. However, the etiology of myofiber loss and the role of intracellular Abeta in IBM is unknown. Here we report correlative evidence for apoptotic cell death in myofibers of IBM patients that exhibit pronounced Abeta deposition. HSV-1-mediated gene transfer of Abeta(42) into cultured C2C12 myotubes resulted in a 12.6-fold increase in dUTP-labeled and condensed nuclei over nonexpressing myotubes (P < 0.05). The C'-terminal betaAPP domain C99 also induced myotube apoptosis, but to a significantly lesser extent than Abeta. Apoptosis specific to Abeta-expressing myotubes was also demonstrated through DNA fragmentation, decreased mitochondrial function and the loss of membrane phospholipid polarity. Myotubes laden with Abeta(42), but not other transgene products, developed cytoplasmic inclusions consisting of fibrillar material. Furthermore, injection of normal mouse gastrocnemius muscle with HSV-encoding Abeta cDNA resulted in TUNEL-positive myofibers with pyknotic nuclei. We conclude that Abeta is sufficient to induce apoptosis in myofibers both in vivo and in vitro and suggest it may contribute to myofiber loss and muscle dysfunction in patients with IBM.

Amyloid beta-Peptides↗

PubMed2001

Repeated exposure to rewarding brain stimulation downregulates GluR1 expression in the ventral tegmental area.

There is considerable evidence that drug reward and brain stimulation reward (BSR) share common neural substrates. Although it is known that exposure to drugs of abuse causes a variety of molecular changes in brain reward systems, little is known about the molecular consequences of BSR. We report that repeated exposure to rewarding stimulation of the medial forebrain bundle (MFB) selectively decreases expression of GluR1 (an AMPA receptor subunit) in the VTA, without effect on expression of several other proteins (GluR2, NMDAR1, tyrosine hydroxylase). This effect of BSR on GluR1 expression is opposite of that caused by intermittent exposure to cocaine and morphine, which are known to elevate GluR1 expression in the VTA. Considering that elevated GluR1 expression in the VTA has been associated with increased sensitivity to drug reward, the finding that BSR and drugs of abuse have opposite effects on GluR1 expression in this region may provide an explanation for why the reward-related effects of many drugs (cocaine, morphine, amphetamine, PCP, nicotine) do not sensitize with repeated testing in BSR procedures that quantify reward strength.

Animals↗

PubMed2001

Alzheimer's disease: dysfunction of a signalling pathway mediated by the amyloid precursor protein?

All individuals with Alzheimer's disease (AD) experience a progressive loss of cognitive function, resulting from a neurodegenerative process characterized by the deposition of beta-amyloid (A beta) in plaques and in the cerebrovasculature, and by the formation of neurofibrillary tangles in neurons. The cause of the neuronal death is unknown but it is thought to be linked in some way to the beta-amyloid precursor protein (APP), which is the source of the A beta that accumulates in the AD brain. There are two pieces of supporting data for this: first, APP is overexpressed in Down's syndrome, which leads to AD-like neuropathology by the age of 40 in virtually all affected individuals; secondly, specific point mutations in APP cause some forms of familial AD. Our laboratory has focused on a specific aspect of APP and its connection with the neuronal destruction seen in AD. We have hypothesized that AD results from a progressive dysfunction of APP. In addition, on the basis of recent data generated by our laboratory and others, we propose that in the normal brain a percentage of APP is present as an integral protein of the plasma membrane that mediates the transduction of extracellular signals into the cell via its A beta-containing C-terminal tail. In AD, accumulation of abnormal levels of the C-terminus in the neuron disturbs this signal-transduction function of APP, causing disorders in the cell-cycle machinery and consequent apoptosis. Here, we discuss the key findings that support this hypothesis, and discuss its therapeutic implications for AD.

Alzheimer Disease↗

PubMed2000

Alzheimer's disease: a dysfunction of the amyloid precursor protein(1).

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 beta-amyloid (Abeta) that is deposited in amyloid plaques in AD brain, the primacy of APP in AD may not lie in the production of Abeta 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.

Alzheimer Disease↗

PubMed2000

The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S-M checkpoint and causes apoptosis in neurons.

APP-BP1 binds to the amyloid precursor protein (APP) carboxyl-terminal domain. Recent work suggests that APP-BP1 participates in a novel ubiquitinylation-related pathway involving the ubiquitin-like molecule NEDD8. We show here that, in vivo in mammalian cells, APP-BP1 interacts with hUba3, its presumptive partner in the NEDD8 activation pathway, and that the APP-BP1 binding site for hUba3 is within amino acids 443-479. We also provide evidence that the human APP-BP1 molecule can rescue the ts41 mutation in Chinese hamster cells. This mutation previously has been shown to lead to successive S phases of the cell cycle without intervening G(2), M, and G(1), suggesting that the product of this gene negatively regulates entry into the S phase and positively regulates entry into mitosis. We show that expression of APP-BP1 in ts41 cells drives the cell cycle through the S-M checkpoint and that this function requires both hUba3 and hUbc12. Overexpression of APP-BP1 in primary neurons causes apoptosis via the same pathway. A specific caspase-6 inhibitor blocks this apoptosis. These findings are discussed in the context of abnormalities in the cell cycle that have been observed in Alzheimer's disease.

Animals↗

PubMed1999

Enhanced BK-induced calcium responsiveness in PC12 cells expressing the C100 fragment of the amyloid precursor protein.

Several lines of evidence have implicated the amyloid precursor protein (APP) and its metabolic products as key players in Alzheimer's disease (AD) pathophysiology. The approximately 100 amino acid C-terminal fragment (C100) of APP has been shown to accumulate intracellularly in neurons expressing familial AD (FAD) mutants of APP and to cause neurodegeneration when expressed in transfected neuronal cells. Transgenic animals expressing this fragment in the brain also exhibit some neuropathological and behavioral AD-like deficits. Here, we present evidence that PC12 cells expressing the C100 fragment either via stable transfections or herpes simplex virus-mediated infections show alterations in calcium handling that are similar to those previously shown in fibroblasts from AD patients. This alteration in calcium homeostasis may contribute to the deleterious effects of C100 in PC12 cells. Our data also lend support for a pathophysiological role for C100 since it induces an alteration thought to play an important role in AD pathology.

Alzheimer Disease↗

PubMed1999

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

In Alzheimer's 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 Ammon's 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.

Age Factors↗

PubMed1998

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.

Alzheimer Disease↗

PubMed1998

The neuronal growth-associated protein GAP-43 interacts with rabaptin-5 and participates in endocytosis.

Structural plasticity of nerve cells is a requirement for activity-dependent changes in the brain. The growth-associated protein GAP-43 is thought to be one determinant of such plasticity, although the molecular mechanism by which it mediates dynamic structural alterations at the synapse is not known. GAP-43 is bound by calmodulin when Ca2+ levels are low, and releases the calmodulin when Ca2+ levels rise, suggesting that calmodulin may act as a negative regulator of GAP-43 during periods of low activity in the neurons. To identify the function of GAP-43 during activity-dependent increases in Ca2+ levels, when it is not bound to calmodulin, we sought proteins with which GAP-43 interacts in the presence of Ca2+. We show here that rabaptin-5, an effector of the small GTPase Rab5 that mediates membrane fusion in endocytosis, is one such protein. We demonstrate that GAP-43 regulates endocytosis and synaptic vesicle recycling. Modulation of endocytosis by GAP-43, in association with rabaptin-5, may constitute a common molecular mechanism by which GAP-43 regulates membrane dynamics during its known roles in activity-dependent neurotransmitter release and neurite outgrowth.

Brain Chemistry↗

PubMed1997

Neuronal expression of beta-amyloid precursor protein Alzheimer mutations causes intracellular accumulation of a C-terminal fragment containing both the amyloid beta and cytoplasmic domains.

Five different Alzheimer mutations of the beta-amyloid precursor protein (APP) were expressed in neurons via recombinant herpes simplex virus (HSV) vectors, and the levels of APP metabolites were quantified. The predominant intracellular accumulation product was a C-terminal fragment of APP that co-migrated with the protein product of an HSV recombinant expressing the C-terminal 100 amino acids (C100) of APP, which is known to cause neurodegeneration. Fractionation studies revealed that the C-terminal fragment generated by expression of the Alzheimer mutations, like C100, partitioned into membrane fractions and was particularly enriched in synaptosomes. The processing abnormality caused by expression of the Alzheimer mutations occurs predominantly in neurons. Expression of these mutations or of C100 alone in neurons caused increased secretion of Abeta relative to that of neurons infected with wild type APP recombinant vectors. These data show that expression of APP mutations that cause familial Alzheimer's disease increases the intracellular accumulation of potentially amyloidogenic and neurotoxic C-terminal fragments of APP in neurons.

Alzheimer Disease↗

PubMed1996

Age-dependent neuronal and synaptic degeneration in mice transgenic for the C terminus of the amyloid precursor protein.

The molecular basis for the degeneration of neurons and the deposition of amyloid in plaques and in the cerebrovasculature in Alzheimer's disease (AD) is incompletely understood. We have proposed that one molecule common to these abnormal processes is a fragment of the Alzheimer amyloid precursor protein (APP) comprising the C-terminal 100 amino acids of this molecule (APP-C100). We tested this hypothesis by creating transgenic mice expressing APP-C100 in the brain. We report here that aging (18-28 month) APP-C100 transgenic mice exhibit profound degeneration of neurons and synapses in Ammon's horn and the dentate gyrus of the hippocampal formation. Of the 106 transgenic mice between 8 and 28 months of age that were examined, all of those older than 18 months displayed severe hippocampal degeneration. The numerous degenerating axonal profiles contained increased numbers of neurofilaments, whorls of membrane, and accumulations of debris resembling secondary lysosomes near the cell body. The dendrites of degenerating granule and pyramidal cells contained disorganized, wavy microtubules. Cerebral blood vessels had thickened refractile basal laminae, and microglia laden with debris lay adjacent to larger venous vessels. Mice transgenic for Flag-APP-C100 (in which the hydrophilic Flag tag was fused to the N terminus of APP-C100) showed a similar degree of neurodegeneration in the hippocampal formation as early as 12 months of age. The 45 control mice displayed only occasional necrotic cells and no extensive cell degeneration in the same brain regions. These findings show that APP-C100 is capable of causing some of the neuropathological features of AD.

Age Factors↗

PubMed1994

Lateralization of membrane-associated protein kinase C in rat piriform cortex: specific to operant training cues in the olfactory modality.

Rats were trained on an olfactory and a control modality (auditory or visual) discrimination task and brain membrane-associated protein kinase C (mPKC) was subsequently assessed using quantitative autoradiography of radiolabelled phorbol ester binding. In rats which received olfactory-cued training, mPKC showed a highly significant lateralization in the piriform cortex but not in the hippocampus. Both olfactory-trained rats and control modality rats showed a significant increase in mPKC in the hippocampus when compared to naive rats. Thus, while behavioral training procedures appeared to result in a hippocampal increase in the activated state of this enzyme as has been reported elsewhere, only olfactory learning produced an piriform cortex lateralization in the activated state of the enzyme. While the functional significance of such a change in the distribution of protein kinase C is still unclear, it does suggest that the monitoring of this enzyme's activational state may prove to be a useful tool in the study of memory formation in a wide variety of behavioral contexts.

Animals↗

PubMed1994

Internal Ca2+ mobilization is altered in fibroblasts from patients with Alzheimer disease.

The recent demonstration of K+ channel dysfunction in fibroblasts from Alzheimer disease (AD) patients and past observations of Ca(2+)-mediated K+ channel modulation during memory storage suggested that AD, which is characterized by memory loss and other cognitive deficits, might also involve dysfunction of intracellular Ca2+ mobilization. Bombesin-induced Ca2+ release, which is inositol trisphosphate-mediated, is shown here to be greatly enhanced in AD fibroblasts compared with fibroblasts from control groups. Bradykinin, another activator of phospholipase C, elicits similar enhancement of Ca2+ signaling in AD fibroblasts. By contrast, thapsigargin, an agent that releases Ca2+ by direct action on the endoplasmic reticulum, produced no differences in Ca2+ increase between AD and control fibroblasts. Depolarization-induced Ca2+ influx data previously demonstrated the absence of between-group differences of Ca2+ pumping and/or buffering. There was no correlation between the number of passages in tissue culture and the observed Ca2+ responses. Furthermore, cells of all groups were seeded and analyzed at the same densities. Radioligand binding experiments indicated that the number and affinity of bombesin receptors cannot explain the observed differences. These and previous observations suggest that the differences in bombesin and bradykinin responses in fibroblasts and perhaps other cell types are likely to be due to alteration of inositol trisphosphate-mediated release of intracellular Ca2+.

Adult↗

PubMed1993

Cell specificity of molecular changes during memory storage.

The aeolid nudibranch, Hermissenda crassicornis, exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C(PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B-cell photoreceptor. The present study was designed to further examine learning-specific PKC involvement in identified cellular areas, particularly those in the visual-vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [3H]phorbol-12,13-dibutyrate ([3H]-PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [3H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [3H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.

Animals↗

PubMed1990

Contraction of neuronal branching volume: an anatomic correlate of Pavlovian conditioning.

Associative memory of the mollusc Hermissenda crassicornis, previously correlated with changes of specific K+ currents, protein phosphorylation, and increased synthesis of mRNA and specific proteins, is here shown to be accompanied by macroscopic alteration in the structure of a single identified neuron, the medial type B photoreceptor cell. Four to five days after training, terminal arborizations of B cells iontophoretically injected with Ni2+ ions and then treated with rubeanic acid were measured with charge-coupled device (CCD)-digitized pseudocolor images of optical sections under "blind" conditions. Boundary volumes enclosing medial-type B-cell arborizations from classically conditioned animals were unequivocally reduced compared with volumes for naive animals or those trained with unpaired stimuli. Branch volume magnitude was correlated with input resistance of the medial type B-cell soma. Such associative learning-induced structural changes may share function with "synapse elimination" described in developmental contexts.

Animals↗

PubMed1990

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.

Animals↗

PubMed1989

Imaging of memory-specific changes in the distribution of protein kinase C in the hippocampus.

Activation of protein kinase C (PKC) can mimic the biophysical effects of associative learning on neurons. Furthermore, classical conditioning of the rabbit nictitating membrane (a form of associative learning) produces translocation of PKC activity from the cytosolic to the membrane compartments of the CA1 region of the hippocampus. Evidence is provided here for a significant change in the amount and distribution of PKC within the CA1 cell field of the rabbit hippocampus that is specific to learning. This change is seen at 1 day after learning as focal increments of [3H]phorbol-12,13-dibutyrate binding to PKC in computer-generated images produced from coronal autoradiographs of rabbit brain. In addition, 3 days after learning, the autoradiographs suggest a redistribution of PKC within CA1 from the cell soma to the dendrites.

Animals↗