Critical role of KV3.4 Potassium Channel in Aβ oligomers effects on neuron excitability, astrocytes activation and cognitive functions in Alzheimer’s Disease

Voltage gated potassium channels (KV) play a pathogenetic role in many neurodegenerative disorders, including Alzheimer’s Disease (AD). Recently, it has been demonstrated that hippocampal neurons and NGF-differentiated PC-12 cells exposed to Aβ1–42 display a selective over-expression and increased activity of KV3.4 potassium channels. The goal of this study has been to further investigate the role of KV3.4 channels in a transgenic mouse model of AD, Tg2576. Firstly, we observed an over-expression of KV3.4 and its accessory subunit MiRP2 in the hippocampus of young Tg2576 mice (3 months old), whereas no significant modification was observed in elderly Tg2576 mice (14-18 months old). Moreover, Tg2576 hippocampal neurons showed an early increase in KV3.4 channel activity These data suggested that KV3.4 up-regulation play a critical role in the early stages of AD, since it contributes to neuronal hyperexcitability induced by the accumulation of Aβ1-42 dimers and trimers occurring in these stages. The immunocytochemical analysis showed that the early increase in KV3.4 expression was accompanied by an altered subcellular distribution of this protein in Tg2576 hippocampal neurons, in particular we observed a more intense KV3.4 immunosignal localized to the soma plasma membrane. Intriguingly, the over-expression and increase activity of KV3.4 in young Tg2576 mice were accompanied by the activation of caspase-3. Moreover, the KV3.4 silencing in vivo by intracerebroventricular injection, not only strongly reduced KV3.4 expression but also prevented caspase-3 activation. Interestingly, siKV3.4 reduced the Aβ1-42 trimers levels in the same young Tg2576 mice suggesting the possible indirect link between KV3.4 over-expression, caspases activation and Aβ1-42 trimers deposition. Several studies showed that Aβ1-42 trimers are closely related to memory impairment occurring in the early stages of AD. Our studies performed by T-maze spontaneous alternation test indicated that Tg2576 mice (3 months old) exhibited the impairment of exploration ability, spatial learning and memory abilities. Interestingly, Tg2576 mice in the presence of siKV3.4 displayed the amelioration in exploration ability and memory performance. Furthermore, the Open Field test showed that Tg2576 mice (3 months old) were hyperactive since they travelled a greater average distance compared to Wild Type mice. Attractively, Tg2576 in the presence of siKV3.4 displayed a reduction in the distance traveled compared to Tg2576 mice in the absence of siKV3.4. Collectively, these data proposed that the inhibition of KV3.4, ameliorating memory performance and non-cognitive symptoms, could become a new pharmacological target in the care of AD. To this aim we tested a new synthetic compound, BDS1-8, containing the first eight aminoacids from full lenght BDS-I, a well known KV3.4 inhibitor. BDS1-8 was able to prevent both increased KV3.4 activity and caspase-3 activation induced by Aβ1–42 oligomers. At last, we observed an up-regulation of KV3.4 and GFAP protein expression in primary astrocytes exposed to Aβ1-42 peptide. Interestingly, coexpression and co-immunoprecipitation studies revealed a large overlap and a direct binding of KV3.4 with GFAP. Moreover, the in vivo siKV3.4 significantly down-regulated GFAP in Tg2576 mice (3 months old) confirmig the closely link between GFAP and KV3.4. These data suggested that GFAP might promote channel trafficking in precise membrane domains faciliting the accumulation of KV3.4 channel, in order to modulate cellular mechanisms mediated by K+ ions. Collectively, our data suggest a critical role of KV3.4 in Aβ oligomers effects on neuron excitability, astrocytes activation and cognitive functions in AD

Synthesis and Pharmacological Evaluation of a Novel Peptide Based on Anemonia sulcata BDS-I Toxin as a New KV3.4 Inhibitor Exerting a Neuroprotective Effect Against Amyloid-β Peptide

There is increasing evidence that the fast-inactivating potassium current IA, encoded by KV3. 4 channels, plays an important role in Alzheimer's Disease (AD), since the neurotoxic β-amyloid peptide1-42 (Aβ1-42) increases the IA current triggering apoptotic processes. The specific inhibition of KV3.4 by the marine toxin extracted from Anemonia sulcata, named blood depressing substance-I (BDS-I), reverts the Aβ peptide-induced cell death. The aim of the present study was to identify the smallest fragments of BDS-I, obtained by peptide synthesis, able to inhibit KV3.4 currents. For this purpose, whole-cell patch clamp technique was used to evaluate the effects of BDS-I fragments on KV3.4 currents in CHO cells heterologously expressing KV3.4. We found that BDS-I[1-8] fragment, containing the N-terminal octapeptide sequence of full length BDS-I, was able to inhibit KV3.4 currents in a concentration dependent manner, whereas the scrambled sequence of BDS-I[1-8] and all the other fragments obtained from BDS-I full length were ineffective. As we demonstrated in a previous study, BDS-I full length is able to counteract Aβ1-42-induced enhancement of KV3.4 activity, preventing Aβ1-42-induced caspase-3 activation and the abnormal nuclear morphology in NGF-differentiated PC-12 cells. Similarly to BDS-I, we found that BDS-I[1-8] blocking KV3.4 currents prevented Aβ1-42-induced caspase-3 activation and apoptotic processes. Collectively, these results suggest that BDS-I[1-8] could represent a lead compound to be developed as a new drug targeting KV3.4 channels

The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aβ1–42 Oligomers by Restoring [Ca2+]i Transients and ER Ca2+ Signaling

Intracellular calcium concentration ([Ca2+]i) transients in astrocytes represent a highly plastic signaling pathway underlying the communication between neurons and glial cells. However, how this important phenomenon may be compromised in Alzheimer’s disease (AD) remains unexplored. Moreover, the involvement of several K+ channels, including KV3.4 underlying the fast-inactivating currents, has been demonstrated in several AD models. Here, the effect of KV3.4 modulation by the marine toxin blood depressing substance-I (BDS-I) extracted from Anemonia sulcata has been studied on [Ca2+]i transients in rat primary cortical astrocytes exposed to Aβ1–42 oligomers. We showed that: (1) primary cortical astrocytes expressing KV3.4 channels displayed [Ca2+]i transients depending on the occurrence of membrane potential spikes, (2) BDS-I restored, in a dose-dependent way, [Ca2+]i transients in astrocytes exposed to Aβ1–42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aβ1–42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aβ1–42 oligomers, and (5) BDS-I prevented Aβ1–42-induced reactive oxygen species (ROS) production and cell suffering measured as mitochondrial activity and lactate dehydrogenase (LDH) release. Collectively, we proposed that the marine toxin BDS-I, by inhibiting the hyperfunctional KV3.4 channels and restoring [Ca2+]i oscillation frequency, prevented Aβ1–42-induced ER stress and cell suffering in astrocytes

Amyloid β-Induced Upregulation of Nav1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer's Disease Mouse Model

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer's Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-β1-42 (Aβ1-42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aβ1-42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD

Exploring the Therapeutic Potential of Phytochemicals in Alzheimer's Disease: Focus on Polyphenols and Monoterpenes

: Alzheimer's disease (AD) is a chronic, complex neurodegenerative disorder mainly characterized by the irreversible loss of memory and cognitive functions. Different hypotheses have been proposed thus far to explain the etiology of this devastating disorder, including those centered on the Amyloid-β (Aβ) peptide aggregation, Tau hyperphosphorylation, neuroinflammation and oxidative stress. Nonetheless, the therapeutic strategies conceived thus far to treat AD neurodegeneration have proven unsuccessful, probably due to the use of single-target drugs unable to arrest the progressive deterioration of brain functions. For this reason, the theoretical description of the AD etiology has recently switched from over-emphasizing a single deleterious process to considering AD neurodegeneration as the result of different pathogenic mechanisms and their interplay. Moreover, much relevance has recently been conferred to several comorbidities inducing insulin resistance and brain energy hypometabolism, including diabetes and obesity. As consequence, much interest is currently accorded in AD treatment to a multi-target approach interfering with different pathways at the same time, and to life-style interventions aimed at preventing the modifiable risk-factors strictly associated with aging. In this context, phytochemical compounds are emerging as an enormous source to draw on in the search for multi-target agents completing or assisting the traditional pharmacological medicine. Intriguingly, many plant-derived compounds have proven their efficacy in counteracting several pathogenic processes such as the Aβ aggregation, neuroinflammation, oxidative stress and insulin resistance. Many strategies have also been conceived to overcome the limitations of some promising phytochemicals related to their poor pharmacokinetic profiles, including nanotechnology and synthetic routes. Considering the emerging therapeutic potential of natural medicine, the aim of the present review is therefore to highlight the most promising phytochemical compounds belonging to two major classes, polyphenols and monoterpenes, and to report the main findings about their mechanisms of action relating to the AD pathogenesis

ORAI1/STIM1 INTERACTION INTERVENES IN STROKE AND IN NEUROPROTECTION INDUCED BY ISCHEMIC PRECONDITIONING THROUGH ER Ca2+ REFILLING

Disturbance of Ca2+ homeostasis in endoplasmic reticulum (ER) causes neuronal cell injury in stroke. On the other hand ischemic preconditioning (IPC), a brief non-lethal ischemic episode affording tolerance to a subsequent ischemic insult, restores ER Ca2+ homeostasis. Under physiological conditions, ER content is continuously refilled by the interaction between the ER-located Ca2+ sensor stromal interacting molecule 1 named STIM1 and the plasma membrane channel ORAI1, both underlying the store-operated calcium entry (SOCE) mechanism. However, the role played by ORAI1 and STIM1 in stroke and in IPC-induced neuroprotection during stroke remains unknown. Therefore, we explored whether ORAI1 and STIM1 might be involved in stroke pathogenesis and in IPC-induced neuroprotection. To this aim primary cortical neurons were subjected to OGD+reoxygenation (Rx) to reproduce in vitro brain ischemia. Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion (tMCAO). Expression of ORAI1 and STIM1 transcripts and proteins and immunosignals were detected by qRT-PCR, Western blot and immunocytochemistry, respectively. SOCE and Ca2+ release activated Ca2+ (CRAC) currents (ICRAC) were measured by Fura-2AM videoimaging and patch-clamp electrophysiology in whole cell configuration, respectively. The results of the present study showed that STIM1 and ORAI1 protein expression and immunosignals decreased in the ipsilesional temporoparietal cortex of rats subjected to tMCAO followed by reperfusion. Analogously, in primary hypoxic cortical neurons there was a reduction of STIM1 and ORAI1 transcripts and proteins accompanied by a decrease in SOCE and ICRAC. By contrast, IPC induced SOCE and ICRAC upregulation, preventing STIM1 and ORAI1 downregulation induced by OGD+Rx. Interestingly, the silencing of STIM1 or ORAI1 prevented IPC-induced tolerance and caused ER-stress as measured by GRP78 and caspase-3 upregulation. Collectively ORAI1 and STIM1 which participate to SOCE take part to stroke pathophysiology and play an important role in the neuroprotection induced by IPC

The expression and activity of KV3.4 channel subunits are precociously upregulated in astrocytes exposed to Aβ oligomers and in astrocytes of Alzheimer's disease Tg2576 mice

Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel KV3.4 subunit, which underlies the fast-inactivating K(+) currents, has been recognized to be relevant for AD pathogenesis and is emerging as a new target candidate for AD. In the present study, we investigated both in in vitro and in vivo models of AD the expression and functional activity of KV3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of KV3.4 expression and functional activity after exposure to amyloid-β (Aβ) oligomers. Consistently, astrocytic KV3.4 expression was upregulated in the cerebral cortex, hippocampus, and cerebellum of 6-month-old Tg2576 mice. Further, confocal triple labeling studies revealed that in 6-month-old Tg2576 mice, KV3.4 was intensely coexpressed with Aβ in nonplaque associated astrocytes. Interestingly, in the cortical and hippocampal regions of 12-month-old Tg2576 mice, plaque-associated astrocytes much more intensely expressed KV3.4 subunits, but not Aβ. More important, we evidenced that the selective knockdown of KV3.4 expression significantly downregulated both glial fibrillary acidic protein levels and Aβ trimers in the brain of 6-month-old Tg2576 mice. Collectively, our results demonstrate that the expression and function of KV3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aβ oligomers and in reactive astrocytes of AD Tg2576 mice

D-Aspartate treatment attenuates myelin damage and stimulates myelin repair

Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC-neuron synapses. D-Aspartate is a D-amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D-Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D-Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small-diameter myelinated axons. Chronically administered during demyelination, D-Aspartate also attenuated myelin loss and inflammation. Interestingly, D-Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D-Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na+/Ca2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D-Aspartate-induced [Ca2+]i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca2+]i response as well as D-Aspartate-induced inward currents in OPC Our findings reveal that D-Aspartate treatment may represent a novel strategy for promoting myelin recovery

A New Cell-penetrating Peptide That Blocks the Autoinhibitory XIP Domain of NCX1 and Enhances Antiporter Activity

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na(+) ions for 1Ca(2+) ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIPNCX1), represent an autoinhibitory region involved in the Na(+)-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIPNCX1 region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIPNCX1 domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful