Conformationally altered p53: a novel Alzheimer's disease marker?

The identification of biological markers of Alzheimer's disease (AD) can be extremely useful to improve diagnostic accuracy and/or to monitor the efficacy of putative therapies. In this regard, peripheral cells may be of great importance, because of their easy accessibility. After subjects were grouped according to diagnosis, the expression of conformationally mutant p53 in blood cells was compared by immunoprecipitation or by a cytofluorimetric assay. In total, 104 patients with AD, 92 age-matched controls, 15 patients with Parkinson's disease and 9 with other types of dementia were analyzed. Two independent methods to evaluate the differential expression of a conformational mutant p53 were developed. Mononuclear cells were analyzed by immunoprecipitation or by flow-cytometric analysis, following incubation with a conformation-specific p53 antibody, which discriminates unfolded p53 tertiary structure. Mononuclear cells from AD patients express a higher amount of mutant-like p53 compared to non-AD subjects, thus supporting the study of conformational mutant p53 as a new putative marker to discriminate AD from non-AD patients. We also observed a strong positive correlation between the expression of p53 and the age of patients. The expression of p53 was independent from the length of illness and from the Mini Mental State Examination value.

Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.

BACKGROUND: Skin immunosenescence accounts for increased susceptibility in the elderly to cutaneous infections and malignancies, and decreased contact hypersensitivity and response to vaccination. We have recently shown in immune cells that decreased expression of the receptor for activated C kinase (RACK)-1 underlies defective protein kinase C (PKC) activation and functional immune impairment with ageing. OBJECTIVES: This study was designed to determine if an age-related decline in skin RACK-1 expression was present and whether it correlated with defective tumour necrosis factor (TNF)-alpha production. METHODS: PKC isoforms and RACK-1 expression were evaluated by Western blot analysis and by immunofluorescence in skin obtained from Sprague-Dawley rats of different ages. TNF-alpha release by epidermal cells induced by lipopolysaccharide, 12-O-tetradecanoyl-phorbol-13-acetate and the contact allergen dinitrochlorobenzene was assessed by the L929 biological assay. RESULTS: Skin obtained from old rats (> 18 months) showed decreased RACK-1 immunoreactivity if compared with young rats (< 3 months). RACK-1 preferentially interacts with PKC beta. Despite a similar total skin content of this isoform, the reduced expression of RACK-1 was associated with a decreased translocation of PKC beta in the membrane compartment. The defective PKC beta translocation associated with ageing correlated with decreased TNF-alpha release from epidermal cells following treatment with different inflammatory stimuli. CONCLUSIONS: Overall, we demonstrated for the first time a decrease in RACK-1 expression, defective PKC beta translocation and reduced TNF-alpha release in epidermal cells with ageing. These alterations might be mechanistically significant, and provide a new understanding of the consequences of ageing on skin immunology.

Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus.

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP.

Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus.

A transition of G to A at nucleotide position 279 in exon 1 of the vasopressin gene has been identified in patients with familial central diabetes insipidus. The mutation predicts an amino acid substitution of Thr (ACG) for Ala (GCG) at the COOH terminus of the signal peptide in preprovasopression (preproVP). Translation in vitro of wild-type and mutant mRNAs produced 19-kD preproVPs. When translated in the presence of canine pancreatic rough microsomes, wild-type preproVP was converted to a 21-kD protein, whereas the mutant mRNA produced proteins of 21 kD and 23 kD. NH2-terminal amino acid sequence analysis revealed that the 21-kD proteins from the wild-type and the mutants were proVPs generated by the proteolytic cleavage of the 19-residue signal peptide and the addition of carbohydrate. Accordingly, mutant preproVP was cleaved at the correct site after Thr-19, but the efficiency of cleavage by signal peptidase was < 25% that observed for the wild-type preproVP, resulting in the formation of a predominant glycosylated but uncleaved 23-kD product. These data suggest that inefficient processing of preproVP produced by the mutant allele is possibly involved in the pathogenesis of diabetes insipidus in the affected individuals.

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases.

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.

Rationalizing a pharmacological intervention on the amyloid precursor protein metabolism.

The treatment of Alzheimer's disease remains a major challenge because of our incomplete understanding of the triggering events that lead to the selective neurodegeneration characteristic of Alzheimer's brains. The rational design of a pharmacological intervention is therefore a great theoretical challenge. One approach involves the study of the pharmacological modulation of the amyloid precursor protein metabolism, in which the goal is to reduce the formation of beta-amyloid in the hope of reducing the formation of a potentially neurotoxic peptide. Such an approach has led to the identification of a complex intracellular mechanism that can be regulated by neurotransmitters and other ligands.

Calcium responses in human fibroblasts: a diagnostic molecular profile for Alzheimer's disease.

We have previously identified alterations of K+ channel function, IP3-mediated calcium release, and Cp20 (a memory-associated GTP binding protein) in fibroblasts from AD patients vs. controls. In the present study we introduce a scoring system based on these response alterations that integrates two or more alterations (and their degree) in AD vs. control fibroblasts. This scoring system generates an index that distinguishes AD patients from controls with both high specificity and sensitivity. We also show that low doses of bradykinin elicit intracellular calcium release almost exclusively in AD cell lines in an all or none fashion that provide a clear measurement of enhanced IP3-mediated function in AD vs. controls.

Cytosol protein kinase C downregulation in fibroblasts from Alzheimer's disease patients.

We attempted to determine whether changes in protein kinase C (PKC) activity in Alzheimer's disease (AD) brains are also present in cultured skin fibroblasts from living patients. Biopsies collected from shoulder skin were transferred to culture plates with an appropriate growth medium, and histone-directed PKC activity as well as phorbol ester binding were individually determined in soluble and particulate fractions prepared from AD and non-AD fibroblast cell lines. Binding experiments indicated that PKC was unevenly distributed between cytosol (78%) and particulate (22%). The Bmax values for phorbol ester binding in soluble and particulate fractions were similar in AD and non-AD patients. Kd values in the cytosol were 94% higher in AD patients, indicating lower affinity of the enzyme for the ligand. Accordingly, the soluble PKC activity was 30% lower in AD patients. The data suggest that the changes in PKC phosphorylating activity represent a diffuse cellular defect in AD and are not confined to the brain. The alterations of the enzyme may participate in the disregulation in processing of beta-amyloid precursor protein in AD.

Fibroblasts of patients affected by Down's syndrome oversecrete amyloid precursor protein and are hyporesponsive to protein kinase C stimulation.

The present study investigates the ability of the pharmacologic activation of protein kinase C (PKC) to modulate amyloid precursor protein (APP) secretion in human skin fibroblasts from patients affected by Down's syndrome (DS). We assessed DS subjects at the Hospital Institute of Sospiro, Cremona, and at the Alzheimer's Disease Unit of the Sacred Heart Hospital in Brescia, and we subdivided them into nondemented (NDS) and demented (DDS) patients. All DS patients were trisomy 21 karyotype. DS fibroblasts had an increased content of APP immunoreactive material as revealed by immunocytochemistry analysis. The basal secretion of soluble APP was higher (+94.6%) in Down's cells with respect to controls. The observation on the fibroblasts prepared from DS is consistent with these patients' possessing an extra copy of the APP gene (mapped on chromosome 21) leading to increased APP expression. Phorbol-12,13-dibutyrate (PdBu, 9 to 150 nM) treatment promoted a dose-dependent increase of secreted APP in the conditioned medium of control fibroblasts. The peak response (+102.2%) was attained using 150 nM PdBu. In Down's fibroblasts, PdBu stimulated APP secretion already maximally at low concentrations (9 nM), but the peak response, due to the higher basal release, was lower on a percentage basis (+16.4%) than in control fibroblasts. The results indicate that in Down's fibroblasts the mechanisms controlling APP release are at least quantitatively altered. In addition, these results suggest caution when using information obtained from Down's patients to model Alzheimer's disease biochemical defects.

Ontogenesis of protein kinase C betaII and its anchoring protein RACK1 in the maturation of alveolar macrophage functional responses.

Alveolar macrophages are the resident airway cells primarily responsible for the protection of the lungs against inhaled toxins and other biologically active materials. The purpose of this study was to investigate the maturation with age of alveolar macrophage functional responses. We characterised the ontogenesis of PKC betaII and its anchoring protein RACK1 in correlation with PKC-dependent immune functions, such as TNF-alpha, hydrogen peroxide production and lysozyme release in resident alveolar macrophages obtained from rats 2, 4 and 12 weeks old. Our results show an age-associated increase in the expression of PKC betaII and RACK1, which correlated with a maturation of alveolar macrophage functional responses.

Amyloid beta antagonizes insulin promoted secretion of the amyloid beta protein precursor.

Amyloid beta (Abeta) peptides are direct competitive inhibitors of insulin binding and action [25]. We demonstrate that Abeta peptides can inhibit the effect of insulin on the metabolic processing of the amyloid beta protein precursor (AbetaPP). As evidence emerges concerning the role of insulin and insulin like growth factors (IGFs) in learning and memory, recent findings have suggested that insulin may have a significant role in the pathogenetic pathways leading to Alzheimer's disease (AD). As an example several investigators have demonstrated upregulation of insulin receptors and defective insulin receptor signal transduction in AD brains. Moreover insulin has been shown to positively modulate AbetaPP proteolytic processing. The fact that insulin and Abeta appear to share a common system for degradation and disposal as they are both substrates of the insulin degrading enzyme (IDE) suggested the possibility of a reciprocal interference. Here we report that Abeta can directly interfere with insulin receptor signalling inhibiting the autophosphorylation of partially purified insulin receptors. As a consequence of such interaction we also demonstrate that Abeta blocks the effect of insulin on the release of sAbetaPPalpha in chinese hamster ovaries (CHO) cells transfected with insulin receptors.

Defective protein kinase C alpha leads to impaired secretion of soluble beta-amyloid precursor protein from Alzheimer's disease fibroblasts.

The present study shows that cultured fibroblasts from sporadic AD patients present: a) reduced (-30%) cytosolic protein kinase C (PKC) activity; b) increased KD of phorbol ester binding (+94%) in cytosolic fractions; c) reduced (-30%) soluble protein kinase C alpha immunoreactivity; d) lower (-27.5%) basal soluble APP secretion and e) reduced soluble APP secretion in response to low phorbol ester concentrations (over threefold difference using 9 nM phorbol-12,13-dibutyrate-PdBu). Since the PKC-stimulated secretion of APP leads to the cleavage of the precursor within the amyloidogenic beta-A4 fragment, the reduced PKC activity in AD patients may lead to accumulation of potentially amyloidogenic or toxic APP fragments. A defect in the secretion of soluble amyloid beta-protein precursor is indeed suggested by literature data on familial AD fibroblasts as well as by the reported results.

Differential regulation of adenylyl cyclase in fibroblasts from sporadic and familial Alzheimer's disease cases with PS1 and APP mutations.

beta-Adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were determined in primary skin fibroblasts established from patients with sporadic Alzheimer's disease (AD) and from individuals with familial APP KM670/671NL, PS1 M146V and PS1 H163Y mutations. Our data showed a significantly decreased beta-adrenoceptor-stimulated adenylyl cyclase activity in fibroblasts from sporadic AD compared with age-matched controls (p < 0.001, Student's unpaired t-test). In contrast, both beta-adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were significantly increased in fibroblasts bearing PS1 M146V and PS1 H163Y mutations compared with controls (p < 0.01 and p < 0.05, respectively). No differences were seen between cell lines with and without the Swedish APP KM670/671NL double mutation. We suggest that various gene mutations associated with AD have different consequences for the regulation of adenylyl cyclase signal transduction in this disorder.

beta amyloid-induced disruption of ionic balance: studies on the isolated frog labyrinth.

The beta-amyloid peptide (A beta) is a key player in the pathogenesis of Alzheimer's disease. Although its mechanisms of action are not fully elucidated, a disruption of ionic homeostasis has been suggested, and A beta aggregation in fibrils seems correlated to its toxic potential. In the present work, we studied the effects of different A beta fragments on the activity of frog ampullar nerve fibers. Our results show that A beta fragments are able to reduce ampullar nerve responses, with a potency correlated to their fibrillogenic capability. This study may have clinical implications, since vestibular problems are often reported in Alzheimer patients, and provide a model for the dissection of A beta effects in a simple multicomponent system.

Changes in beta amyloid precursor protein secretion associated with the proliferative status of CNS derived progenitor cells.

We characterized the secretion of amyloid precursor protein (APP) in rat primary neurons and conditionally immortalized central nervous system (CNS) derived progenitor cell lines, to evaluate their suitability as models for studies on APP metabolism. We observed that primary cells dissociated from different regions of the embryonic brain secreted progressively more APP during in vitro maturation. The increase in basal secretion resulted in an offset of the response to protein kinase C (PKC) activated secretion and was correlated with an increased PKC alpha expression. The same type of analysis was performed on CNS derived conditionally immortalized cells whose growth potential becomes restricted upon shifting of the culture conditions to a non-permissive temperature (39 degrees C), an event that coincides with their progression toward differentiation. With a pattern consistent with that observed in primary neurons, conditionally immortalized cells exposed to 39 degrees C showed an offset of the secretory response to activation by PdBu and also an increased expression of PKC alpha. These data suggest that APP secretion and its regulation in CNS derived cells is influenced by the proliferative status of the cells.

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells.

The present study investigates the influence of energy related metabolic stress on amyloid precursor protein (APP) non-amyloidogenic secretory processing in COS cells. The effect of glucose deprivation on soluble APP (sAPP) secretion has been evaluated: incubation of COS cells with 50 mM 2-deoxy-D-glucose (2-DG) in glucose free medium was able to reduce sAPP secretion (-26%). Sodium azide (NaN3), an inhibitor of cytochrome c oxidase (complex IV of the mitochondrial electron transfer chain) decreased sAPP release in a concentration dependent way (maximum -75%). Treatment of COS cells with the antioxidant glutathione (GSH) fully antagonized the inhibitory effect of azide (1 mM) and elicited sAPP release over basal level. These results suggest that the inhibition of energy metabolism can influence APP processing leading to a decreased secretion of non-amyloidogenic fragments of APP.

Specific role for protein kinase C alpha in the constitutive and regulated secretion of amyloid precursor protein in human skin fibroblasts.

Reduced levels of protein kinase C alpha (PKC alpha) seems to be related to an altered amyloid precursor protein (APP) secretion in fibroblasts from Alzheimer's disease (AD) patients. In this report we used a specific inhibitor of PKC alpha (Gö-6976), to investigate the role of PKC alpha in the basal and phorbol esters regulated secretion of soluble APP (sAPP) in human fibroblasts derived from healthy aged volunteers. Treatment with Gö-6976 alone reduced basal secretion by a maximum of 39%, compared to untreated cells, suggesting the partial dependence of constitutive APP secretory pathway on PKC alpha enzyme. Moreover Gö-6976 treatment completely abolished the effect of phorbol-esters mediated PKC stimulation on sAPP release, suggesting that PKC alpha is the only PKC isoform involved in controlling the secretion of sAPP in human fibroblasts.

Fibroblasts from Alzheimer's disease donors do not differ from controls in response to heat shock.

We investigated HSPs mRNA expression in cultured fibroblasts from control and AD patients. Northern blot analysis using probes for HSP70 and HSC73 revealed that HS induced a several fold increase in both mRNA. Under these condition the extent of mRNA increase was similar in controls and AD. HS elicited also a modest increase in sAPP release that was similar in control and AD. The results suggest that the ability of AD fibroblasts to produce a defensive response to HS is preserved.

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

The present study shows that cultured fibroblasts 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 EC50 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 C alpha in fibroblasts from Alzheimer patients.

Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's disease (AD) patients.

Fibroblasts from Alzheimer's disease (AD) patients displayed decreased cytochrome c oxidase (complex IV) activity (P < 0.05). The basal oxygen consumption rate (QO2) and the response to an uncoupler of oxidative phosphorylation did not differ between AD and control fibroblasts. The QO2 of AD fibroblasts was more susceptible (P < 0.05) to inhibition by azide in the range 0.5-5 mM. The basal intracellular pH (pHi) in AD fibroblasts was significantly more acidic than in control ones. The results support the hypothesis that subtle dysfunctions of oxidative energy-producing processes are present in fibroblasts from sporadic AD patients. The alterations observed scantly influence the fibroblasts functioning even in stressful conditions; however in tissues, such as the brain, that rely heavily on oxidative metabolism for their function, similar alterations may trigger molecular mechanisms leading to cell damage.