Alzheimer's Disease Fibroblasts Research Articles

Defective phorbol ester-stimulated secretion of β-amyloid precursor protein from Alzheimer's disease fibroblasts

The present study shows that cultured fbroblasts from sporadic Alzheimer's disease patients are deficient in protein kinase C-regulated secretion of amyloid precursor protein. In particular, Alzheimer fibroblasts show a reduced basal secretion and a reduced response at low concentrations of phorbol-12,13-dibutyrate, with an EC 50 twofold higher than control fibroblasts. Furthermore, we observed that such defective regulation of the amyloid precursor secretion can possibly be correlated to a specific defect in protein kinase Cα in fibroblasts from Alzheimer patients.

Increased Inositol 1,4,5‐Trisphosphate Accumulation Correlates with an Up‐Regulation of Bradykinin Receptors in Alzheimer's Disease

An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished beta-adrenergic-mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross-talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age-matched controls responded to bradykinin (BK) and released inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in a time- and dose-dependent manner. The level of Ins(1,4,5)P3 increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116-132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P3 concentrations in AD fibroblasts were 73 and 89% above levels in the age-matched controls at 5 and 10 s, respectively. Prostaglandin E1 also increased Ins(1,4,5)P3 formation, but this response was not different between the two groups. Although KD (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors (Bmax) was significantly elevated in AD fibroblasts (186.8 +/- 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 +/- 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P3 production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.

Alterations in metabolic properties in fibroblasts in Alzheimer disease.

Alzheimer disease (AD) leads to alterations in several biochemical properties in cultured skin fibroblasts. Because abnormal glucose metabolism has been reported in both in vivo and in vitro studies of the brain, we examined glucose and glutamine oxidation and lactate production in cultured skin fibroblasts from nine patients with familial AD, 19 with sporadic AD, and 20 age-matched controls. The production of CO2 from glucose and glutamine was significantly lower in both groups of Alzheimer fibroblasts compared to controls after 10 min or 1, 2, and 4 h of incubation. The reduction in CO2 production was most evident after 1 h of incubation with either (U-14C)-glucose or (U-14C)-glutamine. Lactate concentration was comparable in all groups at any time of incubation. These findings suggest that processes that require mitochondrial function as glucose or glutamine oxidation are altered in AD and provide evidence that complex metabolic differences are expressed in cultured nonneuronal cells from Alzheimer patients.

Characteristic Transketolase Alterations in Dermal Fibroblasts of Alzheimer Patients Are Modulated by Culture Conditions

Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline p I, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO 2 saturation in the atmosphere and with 3.7 g/liter NaHCO 3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO 2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.

Abnormality of the α‐ketoglutarate dehydrogenase complex in fibroblasts from familial Alzheimer's disease

To test whether previously demonstrated reductions in the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC) in Alzheimer's disease (AD) brain also occur in morphologically normal AD tissues, we examined KGDHC in cultured skin fibroblasts from patients with familial AD (FAD). KGDHC activity was reduced by 44% in the FAD cells (p < 0.002) from the 4 families studied, including a within-kindred comparison of affected and escapee subjects in the chromosome 14q24.3-linked Nova Scotia kindred. The activities of several other glutamate- and glutamine-metabolizing enzymes were normal in the FAD cells, as was the activity of another mitochondrial multienzyme dehydrogenase complex, that for pyruvate. Mixing experiments indicated that the abnormality of KGDHC activity in FAD fibroblasts was not due to an inhibitor or to excess protease activity. KGDHC is a complex of three proteins. Immunoblots for the E2k component under conditions of optimal protease inhibition revealed the expected 46-kd species in both AD and non-AD fibroblasts, but the patient cells also regularly contained an additional 29-kd species that was absent or present in minimal amounts in the controls. Immunoblotting demonstrated no abnormalities in the E1k and E3 components. Other studies indicate that the human gene for E2k residues on chromosome 14q24.3, in a region associated with FAD in a number of families including the KGDHC-deficient Nova Scotia kindred. The persistence of abnormalities in KGDHC and particularly in its E2k component in FAD fibroblasts indicates that abnormalities of this autosomally coded nuclear component are an intrinsic part of the AD process, and the possible role of this abnormality in the pathogenesis of AD is discussed.

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+.

Mitochondria from Alzheimer's fibroblasts show decreased uptake of calcium and increased sensitivity to free radicals

Many of the biological abnormalities seen in samples of the brain and other tissues from patients with Alzheimer's disease, are consistent with an excessive action of oxygenic free radicals. Mitochondria from fibroblasts grown in tissue culture from skin samples taken at autopsy from patients with histologically confirmed Alzheimer's disease, take up significantly less calcium than do fibroblast mitochondria from age matched non-demented controls. Following exposure to oxygenic free radicals generated by adding ferrous ions to the incubation mixture, calcium uptake is increased to a greater extent in Alzheimer's mitochondria than in controls. At the doses used, pretreatment with the lazaroid antioxidant drug U-74500A, or with the iron chelator deferoxamine, prevents the free radical induced increase in calcium uptake by control mitochondria, but provides only partial protection for Alzheimer's mitochondria. These observations suggest that Alzheimer's fibroblast mitochondria have impaired calcium transport processes and show increased sensitivity to oxygenic free radicals.

Peripheral markers and diagnostic criteria in Alzheimer's disease: critical evaluations.

This review analyzes recent developments in diagnostic criteria and peripheral markers used clinically in the definitive diagnosis of Alzheimer's disease (AD), comparing past and current views, together with a discussion of their shortcoming and difficulties of implementation. Consideration is given to studies on the presence of amyloid substances outside the central nervous system: in cerebrospinal fluid, in plasma, in primary cultures, and in continuous cultures of cell lines of neuronal and glial origin. We discuss alterations of cholinesterases and noradrenaline in red blood cells (RBC) in AD and, with relation to the infectious theory, the presence of spirochaetes in patients. The activities of the enzymes leading to the formation of amyloid substances and those reflecting more general alterations of metabolic processes are considered, both in respect to their role in the pathogenesis of the neurodegenerative disorders of AD and of their potential use as markers. Enzymatic changes have been studied comparing AD patients with non AD controls as well as with AD relatives: proteases and their inhibitors; plasminogen activators; transketolases; increases in the activity of Cu-Zn superoxide dismutase in AD patients' RBC, serum, fibroblasts and cortical neurons, pointing to alterations in oxidative processes; and apolipoprotein E epsilon 4 allele, linked to late-onset AD and familial cases. This review presents reasons why the involvement of peripheral markers in AD should advance from hypothesis to accepted fact.

Potassium channel dysfunction in fibroblasts identifies patients with Alzheimer disease.

Since memory loss is characteristic of Alzheimer disease (AD), and since K+ channels change during acquisition of memory in both molluscs and mammals, we investigated K+ channel function as a possible site of AD pathology and, therefore, as a possible diagnostic index as well. A 113-pS tetraethylammonium (TEA)-sensitive K+ channel was consistently absent from AD fibroblasts, while it was often present in young and aged control fibroblasts. A second (166-pS) K+ channel was present in all three groups. Elevated external potassium raised intracellular Ca2+ in all cases. TEA depolarized and caused intracellular Ca2+ elevation in young and aged control fibroblasts but not AD fibroblasts. The invariable absence of a 113-pS TEA-sensitive K+ channel and TEA-induced Ca2+ signal indicate K+ channel dysfunction in AD fibroblasts. These results suggest the possibility of a laboratory method that would diagnostically distinguish AD patients, with or without a family history of AD, from normal age-matched controls and also from patients with non-AD neurological and psychiatric disorders.

Serum- and bradykinin-induced calcium transients in familial Alzheimer's fibroblasts

The calcium-sensitive photoprotein, aequorin, was used to examine serum- and bradykinin-induced transient increases in free cytosolic calcium ions in skin fibroblasts from 10 individuals with early onset familial AD (FAD), including four who were biopsied before their clinical symptoms would allow a diagnosis of AD, 2 individuals with late onset FAD, 8 at-risk but nonsymptomatic individuals, and 13 controls. The data show that (a) among controls, the peaks of the calcium transients increase in height as a function of donor age; (b) transients induced by 10% serum, 10 nM bradykinin (BK) or 100 nM BK were generally lower in FAD fibroblasts, including those from donors in the early stages of the disease, than in age-matched control cells; (c) such transients are reduced in cells from a proportion of the nonsymptomatic, at-risk individuals. Thus, serum- and BK-induced calcium transients are reduced in fibroblasts from both early and more advanced stage FAD donors and perhaps even from donors who are presymptomatic carriers of the defective gene. The data also suggest that changes in calcium transients in FAD fibroblasts neither mimic nor exaggerate the effects of normal aging.

Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors

An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

Antibody to beta-amyloid precursor protein recognizes an intermediate filament-associated protein in Alzheimer's and control fibroblasts.

The expression of beta-amyloid precursor protein (BAPP) and its mRNAs was studied in fibroblasts obtained from patients afflicted with Alzheimer's disease (AD) and age-matched controls. Using reverse transcriptase-polymerase chain reaction (RT-PCR), transcripts corresponding to 770, 751, 714, and 695 amino acids were detected in both AD and control fibroblasts. Antibody 22C11 against BAPP (Boehringer Mannheim) labeled an intracellular protein, specifically localized to the intermediate filament network. In addition to bands of the predicted molecular weights for BAPP (120-135 kDa), Western blotting revealed a 57 kDa band which was not evident in samples of human brain. As cytoskeletal elements are vital in maintaining cellular architecture and various cell interactions, localization of BAPP or a related molecule to the cytoskeleton suggests a possible structural role for this protein within the cell.

Characterization of proteoglycans in alzheimer's disease fibroblasts

Skin fibroblasts lines established from patients with Alzheimer's disease and old normal individuals were cultured with 35S-sodium sulfate and 3H-glucosamine. Proteoglycans were isolated and characterized. Sulfate incorporation into proteoglycans increased in Alzheimer's disease fibroblasts relative to normal controls. These increases changed the ratio of chondroitin sulfate to heparan sulfate proteoglycan from 1.4 to 1.7 (p = 0.0012) and decreased the ratio of cell to medium proteoglycans from 0.32 to 0.26 in normal and Alzheimer fibroblasts (p = 0.006), respectively. HPLC analysis of the disaccharides produced by chondroitinase ABC revealed no differences in composition between proteoglycans of Alzheimer and normal fibroblasts in either the cell or medium fraction. However, analysis of disaccharides produced by heparinase plus heparitinase showed differences in composition in the medium but not the cell fraction. delta UA-GlcNS was increased by 30% while delta UA-GlcNS-6S was reduced by 40% in Alzheimer's disease.

Change in the cttoskeletal system in fibroblasts from patients with familial Alzheimer's disease

1.1. Fibroblasts were cultured from four patients, two patients from two independent family lines, clinically diagnosed as familial Alzheimer's disease.2.2. Adhesion efficiency to the dish was significantly suppressed with fibroblasts from patients with familial Alzheimer's disease compared with the cells from the age-matched control.3.3. Cytoskeletal systems were visualized by immunofluorescent staining with antibodies against tubulin, actin, and vimentin, showing unique dearrangement of vimentin fibers in fibroblasts from familial Alzheimer's disease.4.4. Regrowth of vimentin fibers after colchiclne treatment was slower with fibroblasts from familial Alzheimer's disease than that of the control.5.5. Western blotting analysis showed no change in tubulin, actin, and vimentin, but the size of fodrin in familial Alzheimer fibroblasts were different from that of the control cell.

Abnormalities of transketolase in Alzheimer's disease fibroblasts: S. Sorbi, F. Lolli, P. Piersanti, ∗R.-K. Sheu, S. Piacentini, L. Amaducci. Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy, and ∗ Burke Medical Research Institute, White Plains, NY, USA

Abnormalities of transketolase in Alzheimer's disease fibroblasts: S. Sorbi, F. Lolli, P. Piersanti, ∗R.-K. Sheu, S. Piacentini, L. Amaducci. Department of Neurological and Psychiatric Sciences, University of Florence, Florence, Italy, and ∗ Burke Medical Research Institute, White Plains, NY, USA

Mitochondrial α-ketoglutarate dehydrogenase abnormality in Alzheimer disease fibroblasts: ∗K.-F.R. Sheu, +A.J.L. Cooper, ∗G. Ali, ∗J.P. Blass and #J.G. Lindsay ∗Burke Med Res Inst, Cornell Univ Med College, White Plains, NY 10605, USA; Depts of ∗Neurology and +Dept of Biochemistry, Cornell Univ Med College, New York, NY 10021, USA; and #Dept of Biochemistry, Univ of Glasgow, Glasgow G128QQ UK

Mitochondrial α-ketoglutarate dehydrogenase abnormality in Alzheimer disease fibroblasts: ∗K.-F.R. Sheu, +A.J.L. Cooper, ∗G. Ali, ∗J.P. Blass and #J.G. Lindsay ∗Burke Med Res Inst, Cornell Univ Med College, White Plains, NY 10605, USA; Depts of ∗Neurology and +Dept of Biochemistry, Cornell Univ Med College, New York, NY 10021, USA; and #Dept of Biochemistry, Univ of Glasgow, Glasgow G128QQ UK

Reduced protein kinase C activity in sporadic Alzheimer's disease fibroblasts

A decrease in the protein kinase C immunoreactivity and an altered protein phosphorylation have been reported in patients with Alzheimer's disease, but discordant results have been obtained from determinations of protein kinase C activity. By assaying the calcium- and phospholipid-dependent phosphorylation of a lysine-rich histone after detergent extraction, we have determined the total protein kinase C activity in fibroblasts from patients with sporadic Alzheimer's disease, age-matched controls and young subjects. The activity was not significantly different between young and aged controls, whereas it was significantly lower (0.70 ± 0.12 vs 1.16 ± 0.23 nmol/min/mg protein, P < 0.01) in the patients. The total amount of protein kinase C estimated from the binding of phorbol dibutyrate to intact cells was also significantly lower (1.70 ± 0.41 vs 2.48 ± 0.54 pmol/mg protein, P < 0.01). This decrease in protein kinase C activity suggests that abnormal protein phosphorylation might play a role in the pathogenesis of the disease.

Study of cytoskeletal proteins in fibroblasts cultured from familial Alzheimer's disease

Cytoskeletal proteins of the cultured fibroblasts obtained from Alzheimer's disease patients were studied. Western blotting studies of tubulin, actin, and vimentin showed no difference between Alzheimer and the control fibroblasts. Western blotting studies of vimentin revealed five partial degradation products in 50 K-57 K Da. molecular size region, but no difference in the degradation pattern was noticed between Alzheimer and the control fibroblasts. The size of fodrin molecule, however, was quite different between Alzheimer and the control fibroblasts. Comparing the molecular size of fodrin purified from the bovine brain, it is concluded that fodrin in Alzheimer fibroblasts is not degraded, while significant amount of fodrin in the control fibroblasts is partially degraded resulting in the smaller size of the 160 K and 200 K Da. molecular weight products.

Enhanced proteolytic activities in cultured fibroblasts of Alzheimer patients are revealed by peculiar transketolase alterations

Characteristic alterations of transketolase (TK) in extracts from cultured Alzheimer fibroblasts have previously been reported (Paoletti et al. (1990) Biochem. Biophys. Res. Commun., 172: 396–401). These abnormalities, encountered in 9 out of 13 Alzheimer patients, were revealed following isoelectric focusing and consisted of enzyme forms having unusually high alkaline pI values (alkaline bands). The present work has shown that immunologically detected alkaline bands were progressively expressed when Alzheimer fibroblasts were incubated for three weeks without medium changes. Full expression of the altered enzyme pattern was not linked to relative cell density in the petri dish; rather, it appeared to be dependent directly on the time elapsed since cell confluence was reached. Alkaline bands could artificially be induced also in both crude and pure TK preparations from normal cells by a treatment with commercial proteases, particularly chymotrypsin. Moreover, specific inhibitors of endogenous cysteine-proteases were capable of abolishing TK alkaline bands in Alzheimer fibroblasts thus turning a pathological into a normal enzyme pattern. Results obtained suggest that Alzheimer fibroblasts contain enhanced Ca 2+-independent cysteine-proteolytic activities as compared to normal and other pathological cells. These enzymes, exhibiting chymotrypsin-like activity, might exert their degradative effects at the time of cell extraction using TK and probably other cell components as potential substrates. However, peculiar TK abnormalities represent so far an useful biochemical marker detectable in fibroblasts of living Alzheimer patients and closely associated to this neurological disorder.

Expression of 'Alzheimer Antigens' in Cultured Skin Fibroblasts

When cultured skin fibroblasts were exposed to culture conditions designed to favor the expression of neuronal antigens, cells from each of 19 patients with Alzheimer's disease reacted immunocytochemically with antibodies to paired helical filaments, Alz-50, or both compared with only a small fraction of cells from 19 identically treated age-matched control cultures. Immunoblots confirmed the presence of soluble material reacting with Alz-50 antibody in the Alzheimer fibroblasts. Ultrastructurally, fascicles of 10-nm filaments were seen that occasionally twisted around each other, but no structures were seen that were identical to paired helical filaments. Thus, cultured skin fibroblasts from patients with Alzheimer's disease developed greater immunocytochemical reactivity with antibodies raised to paired helical filaments than did fibroblasts from control subjects, when cultured under the specified conditions.