F. Battaini

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 Alzheimers 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 β-amyloid precursor protein in AD.

Age-related alteration of PKC, a key enzyme in memory processes: physiological and pathological examples.

Brain aging is characterized by a progressive decline of the cognitive and memory functions. It is becoming increasingly clear that protein phosphorylation and, in particular, the activity of the calcium-phospholipid-dependent protein kinase C (PKC) may be one of the fundamental cellular changes associated with memory function. PKC is a multigene family of enzymes highly expressed in brain tissues. The activation of kinase C is coupled with its translocation from the cytosol to different intracellular sites and recent studies have demonstrated the key role played by several anchoring proteins in this mechanism. PKC-phosphorylating activity appears to be impaired during senescence at brain level in a strain-dependent fashion in rodents. Whereas the levels of the various isoforms do not show age-related alterations, the enzyme translocation upon phorbol-ester treatment is deficitary among all strains investigated. Anchoring proteins may contribute to this activation deficit. We discuss also modifications of the PKC system in Alzheimers disease that may be related to pathological alterations in neurotransmission. A better insight of the different factors controlling brain-PKC activation may be important not only for elucidating the molecular basis of neuronal transmission, but also for identifying new approaches for correcting or even preventing age-dependent changes in brain function.

Age-related reduced affinity in [3H]nitrendipine labeling of brain voltage-dependent calcium channels

Literature data indicate a reduced calcium uptake in synaptosomes prepared from old rat brains. On this line, the present paper investigates the binding of ([3H]NDP) to brain synaptic membranes prepared from rats at different ages from birth up to 24 months of age. The binding is undetectable at birth but reaches within 9-18 day the values observed in adults [3H]NDP binding affinity and sensitivity to calcium were decreased in old rats (24 months). Tritiated dihydropyridines are believed to label voltage-dependent calcium channels (VDC). The observed age-related reduction in binding suggests that the characteristics of VDC in the aged brain may change.

Increased natural killer cell cytotoxicity in Alzheimer's disease may involve protein kinase C dysregulation

Increased cytokine-mediated cytotoxic natural killer (NK) cell activity has recently been demonstrated in patients with senile dementia of the Alzheimers type (SDAT). In the present study, we evaluated whether protein-kinase C (PKC), a main regulatory enzyme involved in the mechanism of exocytosis by NK cells, has a role in the cytotoxic response of NK cells (during IL-2 and IFN-beta exposure) from SDAT patients. Our data demonstrate the presence of an increased cytotoxic response by NK cells to IL-2 (mean increase +102%) and IFN-beta (mean increase +132%) in SDAT patients in comparison with healthy elderly subjects (+75% and +88% for IL-2 and IFN-beta, respectively). A smaller suppression of NK cytotoxicity after cortisol was also observed in SDAT (mean decrease -24%) than in the control group (-44%). The NK cell activity of SDAT patients was inversely correlated with the cognitive status as evaluated by the analysis of MMSE (Mini Mental State Examination) score. A comparison of young and elderly healthy subjects revealed no variations in NK cell activity. A physiological decrease in cytosolic PKC activity was demonstrated in healthy old subjects after IL-2 and IFN-beta incubation, but not in SDAT patients, while no variations in kinase activity were observed after cortisol incubation. The decreased activity with cytokines was associated with reduced levels of PKC alpha and betaII isoforms. An alteration in cytokine-mediated NK cell activity associated with PKC dysregulation is therefore suggested to occur in patients with SDAT. These changes may indicate the existence of an immunological component to the pathogenesis and progression of the disease.

Senescence of the Brain: Focus on Cognitive Kinases

Ageing is characterized by alterations in brain anatomy and physiology, finally contributing to an impairment in cognitive functions, such as memory. The most relevant observations indicate that senescent-related cognitive decline is not only due to neuronal loss, instead, functional changes occurring over time play a key role. Overall, these modifications are indeed responsible for an altered interneuronal communication that can represent, rather than morphological modifications, the primum movens leading to cognitive decline. Among the age-induced changes underlying alterations in neuronal communication and synaptic plasticity, those related to neurotransmitter/neurotrophin systems and downstream signalling pathways are of great relevance. In particular, considering that protein kinases play a strategic role aimed to convert the extracellular signals into biological responses, functional alterations on kinases may directly contribute to age-dependent neuronal dysfunctions. Within this context, numerous studies point out on several kinases as positive regulators for memory function and suggest that various memory disturbances are the result of a deficit in kinase signalling pathways. Many kinases associated with synaptic function are indeed age-sensitive; in fact, various studies in senescent animals indicate that a reduction in kinases expression/function in some brain areas correlates with ageing and memory decline. In line with these concepts, pharmacological modulation of kinases may lead to neuroprotective effects that can prevent or counteract age-related memory impairment. This review will mainly focus on the age-induced changes on Protein Kinase C (PKC), Protein Kinase A (PKA), Calcium/calmodulin-dependent Protein Kinase (CaMK), Tyrosine Kinase, widely accepted as key actors in signalling pathways associated with memory.

Effect of a new cognition enhancer, alpha-glycerylphosphorylcholine, on scopolamine-induced amnesia and brain acetylcholine

The present study investigates the effect of the administration of alpha-glycerylphosphorylcholine (alpha-GPC) on scopolamine-induced amnesia and on brain acetylcholine (ACh) levels and release in rats. The results indicate that alpha-GPC, when administered orally, reverses the amnesia caused by scopolamine in passive avoidance. The peak effect is observed using 600 mg/kg IG, 5 h before training. The effect of the drug is long lasting (up 30 h) in accordance with its pharmacokinetic characteristics. Since, alpha-GPC administered IG is cleaved within the gut mucosal cells to glycerophosphate and free choline, it is tempting to speculate that this drug acts by increasing the ACh precursor pool. This view is supported also by the observation that alpha-GPC partially counteracts the decrease of brain ACh levels elicited by scopolamine administration. The effect is observed in the hippocampus and cortex, but not in the striatum. Moreover, in ex vivo experiments, alpha-GPC is able to increase the amount of ACh released by rat hippocampus slices following potassium stimulation.

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 Downs syndrome (DS).We assessed DS subjects at the Hospital Institute of Sospiro, Cremona, and at the Alzheimers 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 Downs 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 Downs 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 Downs fibroblasts the mechanisms controlling APP release are at least quantitatively altered. In addition, these results suggest caution when using information obtained from Downs patients to model Alzheimers disease biochemical defects. NEUROLOGY 1996;47: 1069-1075

Chronic lead treatment affects dopaminergic control of prolactin secretion in rat pituitary.

The effect of lead exposure on dopaminergic mechanisms regulating prolactin (PRL) secretion was studied in rats by measuring dopamine (DA) and dihydroxyphenylacetic acid hypothalamic concentrations and DA receptor density in the hypothalamus and pituitary of lead-exposed animals. [3H]Sulpiride was used as dopamine receptor ligand. A decrease of dihydroxyphenylacetic acid (DOPAC) hypothalamic concentrations and a decrease of DA receptor density in the pituitary are shown. The decreased [3H]sulpiride binding in the pituitary is consistent with the elevated serum PRL concentrations previously described in lead-exposed rats.

Defective Protein Kinase C α Leads to Impaired Secretion of Soluble β‐Amyloid Precursor Protein from Alzheimer's Disease Fibroblastsa

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 Cα 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 β‐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 β‐protein precursor is indeed suggested by literature data on familial AD fibroblasts as well as by the reported results.

L-Type Calcium Channels are Modified in Rat Hippocampus by Short-Term Experimental Ischemia

Increasing evidence suggests a role for calcium ions in the pathophysiology of ischemic brain damage. The major mechanism allowing calcium entry from the extracellular compartment is the opening of voltage-operated calcium channels. In this line, we have explored the hypothesis that the characteristics of central L-type voltage-dependent calcium channels, labeled by the dihydropyridine ligand 3H-PN 200-110, may be modified by experimental ischemia. The results show that short-term mild ischemia, produced in the rat by 1 h of right carotid ligation, induces an increase in the number of 3H-PN 200-110 binding sites in the hippocampus ipsilateral to the side of carotid occlusion, accompanied by an increase in the dissociation constant value, whereas no changes in the kinetic parameters of the binding were observed in the other areas examined, i.e., the cortex and the striatum. The changes in hippocampus are transient: 96 h after the occlusion, binding parameters return to the control range. The modifications of the binding characteristics in the hippocampus may be related to alterations of Ca2+ fluxes through L-type calcium channels.