Flaminio Cattabeni

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.

A Critical Interaction between NR2B and MAGUK in l-DOPA Induced Dyskinesia

Abnormal function of NMDA receptor has been suggested to be correlated with the pathogenesis of Parkinson’s disease (PD) as well as with the development of l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia. Here we show that NMDA receptor NR2 subunits display specific alterations of their subcellular distribution in striata from unilateral 6-hydroxydopamine-lesioned, l-DOPA-treated dyskinetic, and l-DOPA-treated nondyskinetic rats. Dyskinetic animals have significantly higher levels of NR2A subunit in the postsynaptic compartment than all other experimental groups, whereas NR2B subunit shows a significant reduction in both dopamine-denervated and dyskinetic rats. These events are paralleled by profound modifications of NMDA receptor NR2B subunit association with interacting elements, i.e., members of the membrane-associated guanylate kinase (MAGUK) protein family postsynaptic density-95, synapse-associated protein-97 and synapse-associated protein-102. Treatment of nondyskinetic animals with a synthetic peptide (TAT2B) able to affect NR2B binding to MAGUK proteins as well as synaptic localization of this subunit in nondyskinetic rats was sufficient to induce a shift of treated rats toward a dyskinetic motor behavior. These data indicate abnormal NR2B redistribution between synaptic and extrasynaptic membranes as an important molecular disturbance of the glutamatergic synapse involved in l-DOPA-induced dyskinesia.

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.

Synapse-Associated Protein-97 Mediates α-Secretase ADAM10 Trafficking and Promotes Its Activity

Alzheimers disease (AD) is a chronic neurodegenerative disorder caused by a combination of events impairing normal neuronal function. Here we found a molecular bridge between key elements of primary and secondary pathogenic events in AD, namely the elements of the amyloid cascade and synaptic dysfunction associated with the glutamatergic system. In fact, we report that synapse-associated protein-97 (SAP97), a protein involved in dynamic trafficking of proteins to the excitatory synapse, is responsible for driving ADAM10 (a disintegrin and metalloproteinase 10, the most accredited candidate for α-secretase) to the postsynaptic membrane, by a direct interaction through its Src homology 3 domain. NMDA receptor activation mediates this event and positively modulates α-secretase activity. Furthermore, perturbing ADAM10/SAP97 association in vivo by cell-permeable peptides impairs ADAM10 localization in postsynaptic membranes and consequently decreases the physiological amyloid precursor protein (APP) metabolism. Our findings indicate that glutamatergic synapse activation through NMDA receptor promotes the non-amyloidogenic APP cleavage, strengthening the correlation between APP metabolism and synaptic plasticity.

Significance of dopamine metabolites in the evaluation of drugs acting on dopaminergic neurones

The effect of various drugs was studied on 3-methoxytyramine (3-MT) concentrations in rat striatum. The drugs were chosen for their ability to interfere with the dopaminergic system at different levels. Dopamine (DA) acidic metabolites, i.e. homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), were also measured. Changes of 3-MT, unlike those of DOPAC and HVA, seem to reflect the functional activity of dopaminergic neurons. In fact drugs believed to increase or decrease DA content in the synaptic cleft produce predictable changes of striatal 3-MT. Thus cocaine, nomifensine and d-amphetamine increase 3-MT concentrations while gamma-butyrolactone, alpha-methyltyrosine and apomorphine decrease it.

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimers Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well‐known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha‐secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH‐SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH‐SY5Y cells released significantly more sAPPα into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPα levels. Pre‐incubation with atropine decreased release of sAPPα significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.

Platelet APP, ADAM 10 and BACE alterations in the early stages of Alzheimer disease

Amyloid precursor protein (APP), ADAM 10, and β-site-APP cleaving enzyme (BACE) alterations were evaluated in platelets of 31 patients with Alzheimer disease (AD) and 15 age-matched controls. A significant modification of these proteins and enzymes involved in the amyloid cascade was detected from the earliest clinically detectable disease stage. This observation suggests that AD is associated with an early metabolic derangement toward amyloidogenic pathways and supports the potential value of APP and secretase measurements for early diagnosis of AD.

NMDA receptor subunits are modified transcriptionally and post-translationally in the brain of streptozotocin-diabetic rats

Aims/hypothesis. Moderate disturbances of learning and memory were recognized as a complication of diabetes mellitus in patients. The streptozotocin-diabetic rat, an animal model of insulin-dependent diabetes, shows impairments in spatial memory and in long-term potentiation expression. We have studied the effect of experimental diabetes on expression of post-synaptic glutamate N-Methyl-D-Aspartate ionotropic receptors and of other key proteins regulating synaptic transmission at the post-synaptic compartment. Methods. In situ hybridization and Western blot analysis were used to assess expression and protein concentration of N-Methyl-D-Aspartate receptors and α-calcium-calmodulin-dependent kinase II. Receptor subunits αCaMKII-dependent phosphorylation was studied in post-synaptic densities obtained from the hippocampus and cortex of control, streptozotocin-diabetic and insulin-treated rats. Results. The transcript levels of NR1 and NR2A subunits of N-Methyl-D-Aspartate were unchanged in rats with a diabetic duration of 3 months when compared with age-matched control rats. Accordingly, NR1 and NR2A as well as GluR1, GluR2/3, PSD-95 and αCaMKII protein concentrations in post-synaptic densities were the same in both control and diabetic rats, whereas the immunoreactivity for NR2B was reduced by about 40 %. In addition, the activity of αCaMKII on exogenous substrates, such as syntide-2, and the phosphorylation of NR2A/B subunits of N-Methyl-D-Aspartate receptor was reduced in hippocampal post-synaptic densities of streptozotocin-diabetic rats as compared with control rats. Furthermore, we show that insulin intervention for 3 months after diabetic duration partially restored both αCaMKII activity and NR2B levels. Conclusion/interpretation. N-Methyl-D-Aspartate receptor expression and phosphorylation is possibly involved in behavioural and electrophysiological abnormalities observed in streptozotocin-diabetic rats. [Diabetologia (1999) 42: 693–701]

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats

In animal models of diabetes mellitus, such as the streptozotocin‐diabetic rat (STZ‐rat), spatial learning impairments develop in parallel with a reduced expression of long‐term potentiation (LTP) and enhanced expression of long‐term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ‐diabetes and insulin treatment on the hippocampal post‐synaptic glutamate N‐methyl‐d‐aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post‐synaptic density (PSD) fraction. In addition, the functional properties of the NMDA‐receptor complex were examined. One month of STZ‐diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr‐dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and αCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ‐rats compared with age‐matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ‐rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII–NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post‐synaptic glutamatergic transmission is␣related to deficits in learning and plasticity in this animal model.

Decreased NR2B Subunit Synaptic Levels Cause Impaired Long-Term Potentiation But Not Long-Term Depression

The discovery of the molecular mechanisms regulating the abundance of synaptic NMDA receptors is essential for understanding how synaptic plasticity, as well as excitotoxic events, are regulated. However, a complete understanding of the precise molecular mechanisms regulating the composition of the NMDA receptor complex at hippocampal synapse is still missing. Here, we show that 2 h of CaMKII inhibition leads to a specific reduction of synaptic NR2B-containing NMDA receptors without affecting localization of the NR2A subunit; this molecular event is accompanied by a dramatic reduction in the induction of long-term potentiation (LTP), while long-term depression induction is unaffected. The same molecular and functional results were obtained by disrupting NR2B/PSD-95 complex with NR2B C-tail cell permeable peptide (TAT-2B). These data indicate that NR2B redistribution between synaptic and extrasynaptic membranes represents an important molecular disturbance of the glutamatergic synapse and affects the correct induction of LTP.