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Biomedical subjects

I Lieberburg

Publications and source records attributed to I Lieberburg.

At least 37 records · Page 2Linked to original sources
PubMed1994

Channel formation in planar lipid bilayers by a neurotoxic fragment of the beta-amyloid peptide.

Alzheimer's disease (AD) pathology is characterized by plaques, tangles, and neuronal cell loss. The main constituent of plaques is beta-amyloid peptide (A beta), a 39-42 residue peptide which has been linked to disruption of calcium homeostasis and neurotoxicity in vitro. We demonstrate that a neurotoxic fragment of A beta, A beta (25-35) spontaneously inserted into planar lipid membranes to form weakly selective, voltage dependent, ion-permeable channels. We suggest that channel formation may be involved in the pathogenesis of AD and that A beta (25-35) may be the active channel forming segment.

Amino Acid Sequence↗
PubMed1994

Identification of secreted beta-amyloid precursor protein binding sites on intact human fibroblasts.

Based upon recent evidence that the secreted form of APP can cause the release of cytokines and elicit other biological activities, we sought to identify whether a receptor could be identified on the surface of cells. The secreted amyloid precursor protein containing the Kunitz domain (scAPP751) is identical to protease nexin II, a protease inhibitor which has been shown to form complexes with labeled EGF binding protein that subsequently binds to cells. Results of [125I]scAPP751-trypsin complex incubated with intact fibroblast cells show that the complex appears to bind in a saturable time-dependent and reversible manner. The kinetic constants from the binding studies demonstrate a k1 = 2.5 x 10(7) M-1 s-1 and k2 = 4.7 x 10(-4) s-1 and thus a KD (= k2/k1) = 20 pM. Furthermore, the complex formation of [125I]scAPP751 with a protease appears to be a requirement for optimal binding. The binding affinity of secreted APP demonstrated in this study is consistent with its potency in eliminating a range of biological efforts that have been documented.

Alzheimer Disease↗
PubMed1994

Secondary structure of amyloid beta peptide correlates with neurotoxic activity in vitro.

Amyloid beta peptide (A beta), the major protein constituent of senile plaques in patients with Alzheimer's disease, is believed to facilitate the progressive neurodegeneration that occurs in the latter stages of this disease. Early attempts to characterize the structure-activity relationship of A beta toxicity in vitro were compromised by the inability to reproducibly elicit A beta-dependent toxicity across different lots of chemically equivalent peptides. In this study we used CD spectroscopy to demonstrate that A beta secondary structure is an important determinant of A beta toxicity. Solubilized A beta was maximally toxic when the peptide adopted a beta-sheet conformation. Three of the four A beta lots tested had a random coil conformation and were weakly toxic or inactive, whereas the single A beta lot exhibiting toxic activity at low peptide concentrations had significant beta-sheet structure. Incubation of the weakly toxic A beta lots in aqueous stock solutions for several days before use induced a time-dependent conformational transition from random coil to beta-sheet and increased A beta toxicity in three different toxicity assays. Furthermore, the secondary structure of preincubated A beta was dependent upon peptide concentration and pH, so that beta-sheet structures were attenuated when peptide solutions were diluted or buffered at neutral and basic pH. Our data could explain some of the variable toxic activity that has been associated with A beta in the past and provide additional support for the hypothesis that A beta can have a causal role in the molecular neuropathology of Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1993

Serine phosphorylation of the secreted extracellular domain of APP.

The phosphorylation status of full-length APP (FL-APP) and secreted APP (s-APP) was investigated in stably transfected cells. 32P incorporation was detected in the mature full-length APP both in the absence and presence of phorbol ester. Surprisingly, 32P-phosphate was incorporated in the secreted ectodomain, and this was stable to treatment of the [32P]-phospho-s-APP with a large excess of PNGase F, suggesting that N-linked oligosaccharide sites do not account for phosphate incorporation. Phosphoamino acid analysis of the [32P]-phospho-s-APP resulted in the recovery of [32P]-phosphoserine as the preponderant species. Brefeldin A completely inhibited the release of [32P]-phospho s-APP, but did not inhibit the incorporation of 32P into the FL-APP, suggesting that phosphorylation occurs early in the central vacuolar pathway. It is possible that ectodomain phosphorylation by a novel luminal or extracellular protein kinase may play a role in regulating the metabolic fate of APP.

Amino Acids↗
PubMed1993

Altered calcium signaling and neuronal injury: stroke and Alzheimer's disease as examples.

Several cellular signaling systems have been implicated in the neuronal death that occurs both in development ("natural" cell death) or in pathological conditions such as stroke and Alzheimer's disease (AD). Here we consider the possibility that neuronal degeneration in an array of disorders including stroke and AD arises from one or more alterations in calcium-regulating systems that result in a loss of cellular calcium homeostasis. A long-standing hypothesis of neuronal injury, the excitatory amino acid (EAA) hypothesis, is revisited in light of new supportive data concerning the roles of EAAs in stroke and the neurofibrillary degeneration in AD. Two quite new concepts concerning mechanisms of neuronal injury and death are presented, namely: 1) growth factors normally "stabilize" intracellular free calcium levels ([Ca2+]i) and protect neurons against ischemic/excitotoxic injury, and 2) aberrant processing of beta-amyloid precursor protein (APP) can cause neurodegeneration by impairing a neuroprotective function of secreted forms of APP (APPs) which normally regulate [Ca2+]i. Altered APP processing also results in the accumulation of beta-amyloid peptide which contributes to neuronal damage by destabilizing calcium homeostasis; in AD beta-amyloid peptide may render neurons vulnerable to excitotoxic conditions that accrue with increasing age (e.g., altered glucose metabolism, ischemia). Growth factors may normally protect neurons against the potentially damaging effects of calcium influx resulting from energy deprivation and overexcitation. For example, bFGF, NGF and IGFs can protect neurons from several brain regions against excitotoxic/ischemic insults. Growth factors apparently stabilize [Ca2+]i by several means including: a reduction in calcium influx; enhanced calcium extrusion or buffering; and maintenance or improvement of mitochondrial function. For example, bFGF can suppress the expression of a N-methyl-D-aspartate (NMDA) receptor protein that mediates excitotoxic damage in hippocampal neurons. Growth factors may also prevent the loss of neuronal calcium homeostasis and the increased vulnerability to neuronal injury caused by beta-amyloid peptide. Since elevated [Ca2+]i can elicit cytoskeletal alterations similar to those seen in AD neurofibrillary tangles, we propose that neuronal damage in AD results from a loss of calcium homeostasis. The data indicate that a variety of alterations in [Ca2+]i regulation may contribute to the neuronal damage in stroke and AD, and suggest possible means of preventing neuronal damage in these disorders.

Alzheimer Disease↗
PubMed1993

beta-Amyloid precursor protein metabolites and loss of neuronal Ca2+ homeostasis in Alzheimer's disease.

Recent findings link altered processing of beta-amyloid precursor protein (beta APP) to disruption of neuronal Ca2+ homeostasis and an excitotoxic mechanism of cell death in Alzheimer's disease. A major pathway of beta APP metabolism results in the release of secreted forms of beta APP, APPss. These secreted forms are released in response to electrical activity and can modulate neuronal responses to glutamate, suggesting roles in developmental and synaptic plasticity. beta APP is upregulated in response to neural injury and APPss can protect neurons against excitotoxic or ischemic insults by stabilizing the intracellular Ca2+ concentration [Ca2+]i. An alternative beta APP processing pathway liberates intact beta-amyloid peptide, which can form aggregates that disrupt Ca2+ homeostasis and render neurons vulnerable to metabolic or excitotoxic insults. Genetic abnormalities (e.g. certain beta APP mutations or Down syndrome) and age-related changes in brain metabolism (e.g. reduced energy availability or increased oxidative stress) may favor accumulation of [Ca2+]i-destabilizing beta-amyloid peptide and diminish the release of [Ca2+]i-stabilizing, neuroprotective APPss.

Alzheimer Disease↗
PubMed1993

Regulated cleavage of Alzheimer beta-amyloid precursor protein in the absence of the cytoplasmic tail.

Alzheimer beta-amyloid precursor protein can be phosphorylated on residues Thr654, Ser655 and Thr668 on its cytoplasmic domain. Proteolytic cleavage of the amyloid precursor protein and release of the amyloid precursor protein ectodomain into the medium of cultured cells can be activated by phorbol esters which stimulate protein kinase C. In the present study, using mutated amyloid precursor protein, we show that phosphorylation of cytoplasmic residues is not required for the phorbol ester-activated cleavage and release of the amyloid precursor protein ectodomain. Remarkably, deletion of the entire amyloid precursor protein cytoplasmic tail had no effect on the phorbol ester-activated cleavage/release. The results indicate that activation of amyloid precursor protein cleavage/release by protein kinase C involves phosphorylation of some component of the processing pathway, instead of or in addition to the cytoplasmic tail of the amyloid precursor protein.

Amino Acid Sequence↗
PubMed1993

Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the beta-amyloid precursor protein.

The beta-amyloid precursor protein (beta APP) is a membrane-spanning glycoprotein that is the source of the beta-amyloid peptide (beta AP) which accumulates as senile plaques in the brains of patients with Alzheimer's disease. beta APP is normally processed such that a cleavage occurs within the beta AP, liberating secreted forms of beta APP (APPss) from the cell. The neuronal functions of these forms are unknown. We now report that APPss have a potent neuroprotective action in cultured rat hippocampal and septal neurons and in human cortical neurons. APPs695 and APPs751 protected neurons against hypoglycemic damage, and the neuroprotection was abolished by antibodies to a specific region common to both APPs695 and APPs751. APPss caused a rapid and prolonged reduction in [Ca2+]i and prevented the rise in [Ca2+]i that normally mediated hypoglycemic damage. APPss also protected neurons against glutamate neurotoxicity, effectively raising the excitotoxic threshold. APPss may normally play excitoprotective and neuromodulatory roles. Alternative processing of APPss in Alzheimer's disease may contribute to neuronal degeneration by compromising the normal function of APPss and by promoting the deposition of beta AP.

Amyloid beta-Protein Precursor↗
PubMed1993

Cells with a familial Alzheimer's disease mutation produce authentic beta-peptide.

Cells overexpressing the beta-amyloid precursor protein possessing a mutation found in familial Alzheimer's disease overproduce beta-amyloid peptide (A beta). Because these findings were based on immunological identification, we have chemically characterized the peptides produced. Purified A beta fragments from the conditioned media of these cells were found to have N-terminal sequence consistent with the A beta found in cerebral plaques. Mass spectrometric data demonstrated a series of A beta fragments consistent with those found in Alzheimer's disease (AD); the major species corresponding to A beta(1-40). Significantly, a longer fragment corresponding to A beta(1-42) was found. These findings suggest that this cellular system may be useful for mechanistic studies of A beta generation and possibly for the development of therapeutic agents to treat AD.

Alzheimer Disease↗
PubMed1993

Characterization of beta-amyloid peptide from human cerebrospinal fluid.

beta-Amyloid peptide (A beta) is one of the main components of senile plaques in the brain tissue of Alzheimer's disease (AD) patients. A beta is proteolytically cleaved from the amyloid precursor protein (APP), an integral membrane protein possessing a large extracellular N-terminal domain followed by a single membrane-spanning region and a short cytoplasmic C-terminal tail. A beta has been isolated from senile plaques and cerebral vascular tissue of AD brain and characterized as a heterogeneous peptide containing 28-43 amino acids whose sequence begins in the extracellular domain of APP and extends into the putative transmembrane sequence. It has long been speculated that A beta may also be present in body fluids, such as CSF, that contact neuritic plaques. Recently using a specific enzyme-linked immunosorbent assay we were able to quantify one form of A beta in CSF. In this report, using one of these antibodies covalently bound as an affinity matrix, multiple complex forms of A beta have been isolated and characterized from CSF derived from patients with either meningitis or other neurological disorders. Amino acid sequencing reveals A beta species with N-termini of Asp1, Glu3, His6, Glu11, and Val12, although on a molar basis, Asp1 represents the predominant aminoterminus. Laser desorption mass spectrometry confirmed the presence in CSF of A beta species containing 27, 28, 30, 34, 35, 40, 42, and 43 amino acids, all beginning at Asp1; two stable trimers, (Asp1-Met35)3 and (His6-Ala42)3; and one stable dimer containing (Asp1-Val40)2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Microinjection of synthetic amyloid beta-protein in monkey cerebral cortex fails to produce acute neurotoxicity.

The cerebral deposition of amyloid beta protein (A beta P) is an early pathogenetic event in Alzheimer's disease (AD). Recent studies suggest both neurotoxic and neurotrophic effects of A beta P in vitro. Because progressive A beta P deposition and surrounding neuritic dystrophy occur spontaneously in primates, we evaluated the in vivo effects of synthetic A beta P in monkey cortex. Experimental and control (reverse or substituted) peptides were stereotactically injected into multiple neocortical sites of adult rhesus monkeys in a vehicle of either artificial cerebrospinal fluid or acetonitrile. After 2 weeks, all injection sites were identified and characterized. A beta P antibodies specifically detected the injected A beta P1-40 peptide. Serial sections stained with silver and antineurofilament protein demonstrated comparable degrees of degenerating neurons, dystrophic neurites, and axonal spheroids associated with both experimental and control peptide injections. Alz 50 staining was sparse or absent in all sites. Similar results were obtained in an animal killed 3 months after injection. We conclude that specific cellular changes closely resembling the pathology of Alzheimer's disease were not detected in these acute experiments, and that control and experimental A beta P peptides produced indistinguishable effects.

Alzheimer Disease↗
PubMed1992

Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.

Alzheimer Disease↗
PubMed1992

Amyloid beta-peptide is produced by cultured cells during normal metabolism.

Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid beta-peptide (A beta), a fragment, of about 40 amino acids in length, of the integral membrane protein beta-amyloid precursor protein (beta-APP). The mechanism of extracellular accumulation of A beta in brain is unknown and no simple in vitro or in vivo model systems that produce extracellular A beta have been described. We report here the unexpected identification of the 4K (M(r) 4,000) A beta and a truncated form of A beta (approximately 3K) in media from cultures of primary cells and untransfected and beta-APP-transfected cell lines grown under normal conditions. These peptides were immunoprecipitated readily from culture medium by A beta-specific antibodies and their identities confirmed by sequencing. The concept that pathological processes are responsible for the production of A beta must not be reassessed in light of the observation that A beta is produced in soluble form in vitro and in vivo during normal cellular metabolism. Further, these findings provide the basis for using simple cell culture systems to identify drugs that block the formation or release of A beta, the primary protein constituent of the senile plaques of Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1992

Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate.

We have analyzed the metabolic pathway of maturation of APP751 in stably transfected 293 cells, in the presence of either of the cysteine protease inhibitors leupeptin or E-64. Metabolic labeling, followed by immunoprecipitation at various times in the chase with a rabbit polyclonal antibody (anti-BX6) specific to the carboxyl-terminal end of amyloid precursor protein (APP), revealed the accumulation of a novel approximately 22-kDa carboxyl-terminal fragment (22-CTF) in the inhibitor-treated cells. This fragment, which was not detectable in untreated cells, was immunoprecipitated by four separate antibodies to the carboxyl-terminal region of APP as well as by polyclonal and monoclonal antibodies specific to the first 16 amino acids of the beta-peptide domain. Antibodies to the amino-terminal end of APP do not, however, recognize the fragment. Co-treatment of the inhibitor-treated cells with either of the lysosomotropic agents chloroquine or ammonium chloride completely blocked the generation of this fragment but did not significantly affect APP maturation or secretion. All, however, slowed the intracellular turnover of the cell-associated, approximately 9-kDa carboxyl-terminal fragment (c-CTF) produced during constitutive secretion. Densitometric analyses of these results suggest that this non-secretory pathway of APP degradation, mediated by cysteine proteases in an intracellular acidic compartment, accounts for approximately 70% of total APP metabolism and that a key processing intermediate in this pathway is a 22-kDa, beta-peptide-containing APP carboxyl-terminal fragment. It is possible that inefficient degradation of such an intermediate leads to the formation of aggregating beta-peptide.

Amyloid beta-Protein Precursor↗
PubMed1992

Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

Altered proteolysis of the beta-amyloid precursor protein (beta APP) resulting in release of the approximately 40-residue amyloid beta-protein (A beta P) may be a seminal pathogenetic event in Alzheimer disease. Using region-specific beta APP antibodies, we searched for stable proteolytic intermediates containing the intact A beta P region in brain tissue. A 22-kDa beta APP fragment was selectively detected in microvessels purified from cerebral cortex and other brain regions. On immunoblots, the 22-kDa band is labeled by five distinct antisera to beta APP carboxyl-terminal peptides and by affinity-purified antibodies to the recombinant proteins beta APP444-592 and beta APP592-695, which flank the A beta P region. The protein is virtually undetectable in whole-brain homogenates or microvessel-free fractions of brain. The protein is extractable from microvessels in Triton X-100 and other detergents, indicating its membrane association. In comparison with cortical microvessels, microvessels purified from white matter, cerebellum, and nonneural tissues contain lower amounts of the 22-kDa protein. The protein is found in microvessels of both normal and Alzheimer disease brains and occurs in low amounts in microvessels from fresh bovine brain. The size and specific immunoreactivity of the 22-kDa protein indicate that it is a stable fragment of beta APP containing the intact A beta P. The occurrence of this potentially amyloidogenic intermediate in microvessels is consistent with a vascular or hematogenous origin for some A beta P deposits in Alzheimer disease.

Age Factors↗
PubMed1992

Beta/A4 domain of APP: antigenic differences between cell lines.

The expression of amyloid precursor protein (APP) in olfactory neuroblasts has been examined with a panel of antibodies directed against varied regions of the APP molecule. The pattern of reactivity was compared to that in the transformed human glial cell line SVG, human cortical brain tissue, and in kidney epithelial 293 cells containing stably transfected and overexpressed human APP751. Antibodies directed against the C-terminus and extracellular domains of amyloid precursor protein (APP) react more strongly on immunoblot with transfected 293 cells and brain tissue than with olfactory neuroblasts (ON) or SVG cells. Antibodies directed against the beta/A4 region of APP show a contrasting pattern of reactivity, yielding greater reactivity with ON and SVG cells than with transfected 293 cells or brain tissue. Analysis of the APP transcripts using polymerase chain reaction indicates that ON and SVG both make predominantly APP770 and 751, as does the transfected 293 cell line. In the absence of any differences in APP transcripts among the cell lines, the difference in availability of the beta/A4 region appears likely to be due to posttranslational modification. These data therefore indicate that processing of APP varies among cell lines and thus may vary from tissue to tissue.

Amyloid beta-Protein Precursor↗
PubMed1992

beta-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity.

In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta-amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1-38 or 25-35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.

Amino Acid Sequence↗