Lucia Pastorino

Calcium/Calmodulin‐Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca2+/calmodulin‐dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2‐3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either 32P‐autophosphorylated CaMKII or 32P‐NR2A/B and by evaluating the formation of a CaMKII‐NR2A/B complex by means of the cross‐linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.

Amyloid precursor protein in platelets A peripheral marker for the diagnosis of sporadic AD

Background: An altered pattern of amyloid precursor protein (APP) forms consisting in a reduced ratio between the upper (130 kDa) and the lower (106 to 110 kDa) immunoreactivity bands has been described in platelets of patients with AD. Objective: To evaluate the sensitivity and the specificity of platelet APP forms’ ratio (APPr) as a marker for AD. Methods: Eighty-five patients with probable AD and 95 control subjects (CON), including healthy individuals and neurologic patients, entered the study. Platelet APPr was evaluated by means of Western Blot analysis and immunostaining in the whole platelet homogenate, and calculated by the ratio between the optical density (OD) of the upper (130 kDa) and the lower (106 to 110 kDa) APP immunoreactive bands. Results: Mean APPr levels were decreased in AD patients (mean OD ± SD = 0.35 ± 0.18) compared with the CON group (mean OD ± SD = 0.92 ± 0.38) (DF 1, 178, p < 0.0001). Accuracy levels measured by Receiver Operating Curve analysis showed that a cut-off level of 0.57 resulted in a sensitivity of 88.2% and a specificity of 89.4%, with an area under the curve of 0.945. APPr levels were significantly associated with disease severity (mild AD versus moderate AD: p < 0.0001; moderate AD versus severe AD: p < 0.05). Conclusion: Platelet APPr allowed to differentiate AD from normal aging and other dementing disorders with high sensitivity and specificity. These findings suggest that platelet APPr may be of help as an adjunctive diagnostic tool in clinical practice.

Platelets as a peripheral district where to study pathogenetic mechanisms of Alzheimer disease : the case of amyloid precursor protein

Alzheimer disease is a progressive neurodegenerative disease, characterised by a progressive cognitive and memory decline. From a neuropathological point of view, Alzheimer disease is defined by the presence of characteristic lesions, i.e. mature senile plaques, neurofibrillary tangles (NFTs) and amyloid angiopathy. In particular, accumulation of the amyloid beta-peptide in the brain parenchyma and vasculature is an invariant event in the pathogenesis of both sporadic and familial Alzheimer cases. Amyloid beta-peptide originates from a larger precursor, the amyloid precursor protein (APP) ubiquitously expressed. Among the different peripheral cells expressing APP forms, platelets are particularly interesting since they show concentrations of its isoforms equivalent to those found in brain. Moreover, a number of laboratories independently described alterations in APP metabolism/concentration in platelets of Alzheimer patients when compared to control subjects matched for demographic characteristics. These observations defined the frame of our work aimed to investigate if a correlation between levels of platelet APP forms and Alzheimer disease could be detected. We have reported that patients affected by Alzheimer disease show a differential level of platelet APP forms. This observation has several implications: APP processing abnormalities, believed to be a very early change in Alzheimer disease in neuronal compartment, do occur in extraneuronal tissues, such as platelets, thus, suggesting that Alzheimer disease is a systemic disorder; further, our data strongly indicate that a differential level of platelet APP isoforms can be considered a potential peripheral marker of Alzheimer disease allowing for discrimination between Alzheimer and other types of dementia.

Increased Secretion of the Amino‐Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up‐Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APPs) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol‐treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane‐bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up‐regulated, with a concomitant increase in APPs production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up‐regulated. In addition, phorbol esters or H‐7 modulate APPs release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.

ApoE genotype influences the biological effect of donepezil on APP metabolism in Alzheimer disease: evidence from a peripheral model

Three major amyloid precursor protein (APP) forms with apparent molecular weight ranging from 106 to 130 kDa are present in human platelets. Alzheimer disease (AD) is associated with a decreased APP forms ratio (APPr) between the three major forms. A total of 25 mild to moderate AD patients were investigated. Platelet APPr was studied before and after 30 days of acetylcholinesterase-inhibitor treatment (donepezil, 5 mg daily). Patients were grouped into non-epsilon4 carriers and epsilon4 carriers according to apolipoprotein E (ApoE) genotype. At baseline, all patients showed low APPr levels and no significant difference was found between the two ApoE subgroups. After treatment, although a marked improvement in APPr was observed in most patients, non-epsilon4 carriers displayed a higher increase compared to epsilon4 carriers (P<0.0001). The present study provides evidence that donepezil influences APP metabolism in platelets, and suggests that ApoE genotype might be an important modulating factor for drug responsiveness in AD.

CaMKII-dependent phosphorylation of NR2A and NR2B is decreased in animals characterized by hippocampal damage and impaired LTP.

The calcium‐calmodulin‐dependent protein kinase II (CaMKII) subserves activity‐dependent plasticity in central neurons. To examine in vivo the implication of CaMKII activity in synaptic plasticity, we used an animal model characterized by developmentally induced targeted neuronal ablation within the cortex and the hippocampus, and showing, at presynaptic level, molecular alterations leading to facilitation of glutamate release in hippocampal synapses (methylazoxymethanol‐treated rats, MAM‐rats). We report here that at the postsynaptic side, the activity of CaMKII is markedly decreased in MAM‐rats when compared to controls, although the concentration of the enzyme in Post Synaptic Density (PSD) is not altered. This effect is confined to PSD‐associated CaMKII, as enzyme activity tested in the soluble fraction is unchanged in MAM‐rats. In addition, the decreased activity is not due to inhibition by autophosphorylation in specific sites within the calmodulin‐binding domain, as preincubation with purified phosphatases 1 and 2A failed to restore CaMKII activity in PSD of MAM‐rats. The CaMKII‐dependent phosphorylation of NR2A/B subunits of NMDA receptor is lower in MAM‐rats when compared to controls (51.77 ± 7.39% of controls level), as revealed in back‐phosphorylation experiments. In addition, a treatment able to restore long‐term potentiation (LTP) in hippocampal slices from MAM‐rats, e.g. exposure to d‐serine, is able to restore CaMKII activity to the control value.

(+)-MCPG induces PKCε translocation in cortical synaptosomes through a PLD-coupled mGluR

We have tested whether different agonists of metabotropic glutamate receptors could induce translocation of selective protein kinase C isozymes in nerve terminals. In rat cortical synaptosomes 1S,3R‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD; 100 μm) induced an increase in translocation to 124.6 ± 5.7% of basal unstimulated conditions of the Ca++‐independent protein kinase Cε, but not of the Ca++‐dependent isozyme β. This effect was counteracted by 1‐aminoindan‐1,5‐dicarboxylic acid (100 μm), an antagonist of metabotropic glutamate receptor 1. On the other hand, (+)‐α‐methyl‐4‐carboxyphenylglycine [(+)‐MCPG], an antagonist of metabotropic glutamate receptors group I and II, did not antagonize the effect of 1S,3R‐ACPD, and per se induced a translocation of protein kinase Cε of 164 ± 17.7% of basal unstimulated conditions. Because the (+)‐MCPG induction of protein kinase Cε translocation was not antagonized by 1‐aminoindan‐1,5‐dicarboxylic acid, it is suggested that 1S,3R‐ACPD and (+)‐MCPG activate this signal transduction pathway through distinct membrane receptors. Indeed (2‐[2′′‐carboxy‐3′‐phenylcyclopropyl]glycine)‐13 (300 nm), a new compound known to antagonize metabotropic glutamate receptors coupled to phospholipase D, was able to antagonize protein kinase Cε translocation induced by (+)‐MCPG. Moreover (+)‐MCPG directly induced phospholipase D activity, measured as [3H]phosphoethanol production in cortical synaptosomes. These data suggest that in cortical nerve terminals (i) distinct metabotropic glutamate receptors, coupled to different signal transduction pathways, are present, (ii) (+)‐MCPG is able to induce protein kinase Cε translocation, and that (iii) a metabotropic glutamate receptor associated to phospholipase D might influence translocation of protein kinase C in a calcium‐independent manner.

Platelet amyloid precursor protein forms in AD : a peripheral diagnostic tool and a pharmacological target

Alzheimer Disease (AD) is characterized by the progressive deposition of beta-amyloid in the parenchyma and cerebral microvasculature. The beta-amyloid peptide derives from the metabolism of a larger precursor, Amyloid Precursor Protein (APP). This protein is present in central nervous system, but it is also expressed in peripheral tissues such as circulating cells. An alteration of the APP forms pattern in platelets has been recently reported in AD patients when compared to platelets both of control subjects or non AD patients (NADD). The accuracy of the assay to identify AD is high and decreased levels are found throughout the course of AD with a significant association with severity of symptoms. Moreover, a recent study has demonstrated that AD patients on donepezil (5 mg daily) for 4 weeks displayed two-fold increase in their APPr baseline levels up to normal range. Thus, platelet APP ratio (APPr) holds the potential to be a clinical marker, which might be of helpful and adjunctive value in the diagnosis of AD and in tracking the course of illness, also in the early stages when pharmacological treatment has the greatest potential of being effective.

Determination of the endogenous phosphorylation state of B-50/GAP-43 and neurogranin in different brain regions by electrospray mass spectrometry

Electrospray mass spectrometry coupled to liquid chromatography was utilized to measure two PKC neuronal substrates, B‐50/GAP‐43 and neurogranin, in single rat brain areas. Aliquots of perchloric acid extracts were directly injected and mass spectra recorded. At elution times of 14.2 and 27.0 min two molecular species of MW 7450 and 23 602 Da were observed. These values are in excellent agreement for the expected MW for rat neurogranin and B‐50/GAP‐43. The presence of molecular species shifted by 80 mass units in both cases indicates that these proteins are present in phosphorylated forms in cortical and hippocampal extracts.

Differential translocation of protein kinase C isozymes in rats characterized by a chronic lack of LTP induction and cognitive impairment

The translocation of protein kinase C isozymes was investigated in an animal model of cognitive deficit and lack of induction of long‐term potentiation (LTP). In MAM rats, presynaptic α, β, ϵ PKC showed enhanced translocation, while postsynaptic γ PKC displayed decreased translocation when compared to control levels. This imbalance of PKC isozyme translocation between the pre‐ and post‐synaptic compartment might therefore represent a possible molecular cause for the lack of synaptic plasticity observed in these animals.