Anna Pannaccione
Anna Pannaccione
affiliation: Università di Napoli Federico II
research area(s): Neuroscience
Course: Neurosciences
University/Istitution: Università di Napoli Federico II
The molecular mechanisms involved in the pathogenesis of the neurotoxic manifestations associated with Alzheimer"s disease (AD) have been intensively investigated, with particular emphasis on the changes of cellular ionic homeostasis. As a matter of fact, it has been reported that neurotoxic β-amyloid peptides (Abs), generated upon processing of the integral membrane amyloid precursor protein, elevate [Ca2+]i and disrupt Ca2+ homeostasis by several mechanisms, including the modulation of voltage gated Ca2+ channels, the formation of Ca2+-permeable pores, and the inhibition of the Na+/Ca2+ exchanger (NCX). NCX plays a relevant role in the physiology and pathophysiology of the hearth and the brain. NCX, coupling the efflux/influx of Ca2+ to the influx/efflux of Na+ ions by operating in a bidirectional way, is the major regulator of Na+ and Ca2+ homeostasis. In the brain, unlike other tissues, this exchanger is present in three different gene products, named NCX1, NCX2 and NCX3, with a distinct distribution pattern in different brain regions. Under physiological conditions, its primary role is to extrude Ca2+ via a forward mode of operation in response to a depolarization or to an increase in [Ca2+]i coupled to a receptor stimulation. However, during neurotoxic conditions, NCX is also able to operate in the reverse mode, thus extruding Na+ while promoting Ca2+ influx. In particular, it has been shown that NCX1 and NCX3 have a relevant role in the genesis of neurodegenerative mechanisms. In addition, under neurodegenerative conditions such as stroke, AD, and SLA the [Ca2+]i and [Na+]i control is crucial, and it has been shown that the activation of NCX1 and NCX3 plays a neuroprotective action. In addition, a crucial role for intracellular potassium (K+) ions in regulating cell cycle progression and apoptotic cell death has been highlighted in several neurodegenerative processes, including AD. Alteration of neuronal K+ channel (KV) function, which is involved in the regulation of membrane excitability leads to remarkable perturbations in membrane excitability and neuronal function. In particular, [K+]i plays a key role in cell survival. A fall in [K+]i, mainly due to an increased activity of plasma-membrane KV, triggers cell death possibly by activating nucleases and caspases. The first objective will be clearly established that Ab accumulation leads to following consecutive downstream events: [Ca2+]i accumulation" oxidative stress" K+ and Ca2+ ionic dysregulation" apoptotic pathway activation. To this aim will be used an integrated electrophysiological (patch-clamp and current-clamp), biochemical, molecular biology, and pharmacological approach in order to clarify the KV and NCX involvement in pathogenesis of neurodegenerative processes associated to AD, and to verify if they may represent a novel pharmacological molecular targets.
Molinaro P, Viggiano D, Nisticò R, Sirabella R, Secondo A, Boscia F, Pannaccione A, Scorziello A, Mehdawy B, Sokolow S, Herchuelz A, Di Renzo GF, Annunziato L (2011) Na+-Ca2+ Exchanger (NCX3) Knock-Out Mice Display an Impairment in Hippocampal Long-Term Potentiation and Spatial Learning and Memory. J Neurosci 31:7312-7321
Secondo A, Molinaro P, Pannaccione A, Esposito A, Cantile M, Lippiello P, Sirabella R, Iwamoto T, Di Renzo G, Annunziato L (2011) Nitric oxide stimulates NCX1 and NCX2 but inhibits NCX3 isoform by three distinct molecular determinants. Mol Pharmacol 79:558-68
Boscia F, Gala R, Pannaccione A, Secondo A, Scorziello A, Di Renzo G, Annunziato L (2009) NCX1 expression and functional activity increase in microglia invading the infarct core. Stroke 40:3608-17
Secondo A, Pannaccione A, Molinaro P, Ambrosino P, Lippiello P, Esposito A, Cantile M, Khatri PR, Melisi D, Di Renzo G, Annunziato L (2009) Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-n-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells. J Pharmacol Exp Ther 331:212-21
Sirabella R, Secondo A, Pannaccione A, Scorziello A, Valsecchi V, Adornetto A, Bilo L, Di Renzo G, Annunziato L (2009) Anoxia-induced NF-kappaB-dependent upregulation of NCX1 contributes to Ca2+ refilling into endoplasmic reticulum in cortical neurons. Stroke 40:922-9
Molinaro P, Cuomo O, Pignataro G, Boscia F, Sirabella R, Pannaccione A, Secondo A, Scorziello A, Adornetto A, Gala R, Viggiano D, Sokolow S, Herchuelz A, Schurmans S, Di Renzo G, Annunziato L (2008) "Target distruption of Na+-Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage". J Neurosc 28:1179-1184
Cuomo O, Gala R, Pignataro G, Boscia F, Secondo A, Scorziello A, Pannaccione A, Viggiano D, Adornetto A, Molinaro P, Li XF, Lytton J, Di Renzo G, Annunziato L (2008) "A critical role for the potassium-dependent sodium-calcium exchanger NCKX2 in protection against focal ischemic brain damage". J Neurosc 28:2053-63
Molinaro P, Cuomo O, Pignataro G, Boscia F, Sirabella R, Pannaccione A, Secondo A, Scorziello A, Adornetto A, Gala R, Viggiano D, Sokolow S, Herchuelz A, Schurmans S, Di Renzo G, Annunziato L (2008) "Targeted disruption of Na+/Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage". J Neurosc 28:1179-1184
Pannaccione A, Boscia F, Scorziello A, Adornetto A, Castaldo P, Sirabella R, Taglialatela M, Di Renzo G, Annunziato L (2007) "Up-regulation and increased activity of KV3.4 channels and its accessory subunit MIRP2 induced by amyloid peptide is involved in apoptotic neuronal death. Mol Pharm 72:665-73
Secondo A, Pannaccione A, Cataldi M, Sirabella R, Formisano L, Di Renzo G, Annunziato L.(2006) Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents.Am J Physiol Cell Physiol. 290(1):C233-43
Pannaccione A, Secondo A, Scorziello A, Calì G, Taglialatela M, Annunziato L (2006)Nuclear factor-kappaB activation by reactive oxygen species mediates voltage-gated K+ current enhancement by neurotoxic beta-amyloid peptides in nerve growth factor-differentiated PC-12 cells and hippocampal neurones. J Neurochem.94:572-86
Project Title:
To study the role played by KV3.4 voltage gated potassium channel in Alzheimer Desease: possible biomarler
A growing body of evidence suggests oxidative stress involvement in AD. However, it remains to be determined whether
oxidative stress is a cause, a result, or an epiphenomenon of the pathological processes. Several studies have indicated that
oxidative stress initially occurs in the disease-specific, site-restricted sources such as the cerebral cortex of AD brain. Subsequent
events which are common in neurons affected from this disease are represented by increased cytosolic Ca2+ levels, resulting
in activation of Ca2+- dependent and -independent enzymes. These enzymes produce reactive oxygen and nitrogen
species (ROS and RNS), which oxidatively modify nucleic acid, lipids, sugar, and proteins leading to nuclear damage, mitochondrial
damage and dysfunction, proteasome inhibition, and endoplasmic reticulum stress. In particular, Hauptmann et al.
(2008) showed that mitochondrial dysfunction, with a consequent membrane potential collapse and ATP reduction, enhanced
apoptosis and sensitivity to oxidative stress in genetic as well as in sporadic AD. Furthermore, mitochondrial stress-induced
apoptotic cell death is triggered by cytochrome c release from mitochondria and, together with Apaf-1 and dATP/ATP, forms
the apoptosome, induces processing of caspase-9, and initiates the caspase cascade that culminate with the activation of caspase
3. Interestingly, it has been shown that there is a correlation between the intracellular K+ concentration ([K+]i) and
apoptosome formation, since a fall in [K+]i triggers caspase 3 activation, thus propagating cell death signals. This effect
seems to be mainly due to an increased ROS production which, in turns, induces an increased activity of plasma-membrane
voltage-gated potassium (KV) channels(Yu et al. 1997; Lauritzen et al. 2003), in particular, we have recently demonstrated
that hippocampal neurons exposed to Ab1-42 display a selective up-regulation of KV3.4 channel subunits through the activation
of the transcriptional factor NF-kB. This activation is mediated by an early increase of a Ca2+-dependent ROS production
(Pannaccione et al. 2005; 2007). This event is associated with an increased K+ current carried by this channel. In addition,
it has been demonstrated that in neurons over-expressing KV3.4 channels an apoptotic process occurs through the activation
of caspase-3 (Pannaccione et al. 2007). The relationship between KV3.4 and apoptotic cell death is demonstrated by
the fact that the blockade of this channel by means of a specific inhibitor BDS-I prevents cell death caused by bA1-42 exposure.
In support of these data, cDNA array analysis has also demonstrated that KV3.4 gene up-regulation also occurs in cerebral
cortex of AD patients both in early and later stages (Angulo et al., 2004). Interestingly, the same up-regulation has been
found in brain extracts of Tg2576 Swedish mice, a well known pre-clinical model of the disease (Angulo et al., 2004). In order
to establish the correlation between KV3.4 channel and caspase-3 activation in a preclinical model of AD, our objectives
will be to characterize the role played by KV3.4 channel in controlling caspase-3 release in Tg2576 Swedish transgenic mice,
which overexpress KV3.4 channel (Angulo et al., 2004) and are object of investigation of Mercuri's group. This objective
will pursued by an in vivo and in vitro approach. The in vivo approach will be addressed to investigate the effect of KV3.4
knock-down, induced by siRNA, on caspase-3 release in CSF and on AD progression. The in vitro approach will be aimed to
demonstrate that knocking down Kv3.4 is able to reduce caspase-3 activation and to improve neuronal survival. This project
can have a relevant transferability to the national health service since the new information derived from the relationship existing
between Kv3.4 over-expression, caspase- 3 activation and neuronal death will help to further validate caspase-3 as AD