GD2-targeting CAR T-cell therapy for patients with GD2+ medulloblastoma.

PURPOSE: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation and chemotherapy, is often responsible for cognitive, neurologic and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T-cells directed towards the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. EXPERIMENTAL DESIGN: GD2 expression was evaluated on primary tumor biopsies of MB children by flow-cytometry. GD2 expression in MB cells was evaluated also in response to an EZH2 inhibitor (Tazemetostat). In in vitro, as well as in in vivo models, GD2+MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible-caspase-9. RESULTS: GD2 was expressed in 73.17% of MB tumors. The SHH and G4 subtypes expressed the highest levels of GD2, while the WNT subtype the lowest. In in-vitro co-culture assays, CAR.GD2 T-cells were able to kill GD2+MB cells. Pre-treatment with Tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T-cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T-cells significantly controlled tumor growth, prolonging overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood brain barrier and to eliminate both blood circulating and tumor infiltrating CAR.GD2 T-cells. CONCLUSIONS: Our experimental data indicate the feasibility of CAR.GD2 T-cell therapy. A phase I/II clinical trial will be conducted to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Neutralizing IFNγ improves safety without compromising efficacy of CAR-T cell therapy in B-cell malignancies.

Chimeric antigen receptor T (CAR-T) cell therapy may achieve long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. However, potentially severe and hard-to-manage side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, and the lack of pathophysiological experimental models limit the applicability and development of this form of therapy. Here we present a comprehensive humanized mouse model, by which we show that IFNγ neutralization by the clinically approved monoclonal antibody, emapalumab, mitigates severe toxicity related to CAR-T cell therapy. We demonstrate that emapalumab reduces the pro-inflammatory environment in the model, thus allowing control of severe CRS and preventing brain damage, characterized by multifocal hemorrhages. Importantly, our in vitro and in vivo experiments show that IFNγ inhibition does not affect the ability of CD19-targeting CAR-T (CAR.CD19-T) cells to eradicate CD19+ lymphoma cells. Thus, our study provides evidence that anti-IFNγ treatment might reduce immune related adverse effect without compromising therapeutic success and provides rationale for an emapalumab-CAR.CD19-T cell combination therapy in humans.

Safe and effective off-the-shelf immunotherapy based on CAR.CD123-NK cells for the treatment of acute myeloid leukaemia.

BACKGROUND: Paediatric acute myeloid leukaemia (AML) is characterized by poor outcomes in patients with relapsed/refractory disease, despite the improvements in intensive standard therapy. The leukaemic cells of paediatric AML patients show high expression of the CD123 antigen, and this finding provides the biological basis to target CD123 with the chimeric antigen receptor (CAR). However, CAR.CD123 therapy in AML is hampered by on-target off-tumour toxicity and a long "vein-to-vein" time. METHODS: We developed an off-the-shelf product based on allogeneic natural killer (NK) cells derived from the peripheral blood of healthy donors and engineered them to express a second-generation CAR targeting CD123 (CAR.CD123). RESULTS: CAR.CD123-NK cells showed significant anti-leukaemia activity not only in vitro against CD123+ AML cell lines and CD123+ primary blasts but also in two animal models of human AML-bearing immune-deficient mice. Data on anti-leukaemia activity were also corroborated by the quantification of inflammatory cytokines, namely granzyme B (Granz B), interferon gamma (IFN-γ) and tumour necrosis factor alpha (TNF-α), both in vitro and in the plasma of mice treated with CAR.CD123-NK cells. To evaluate and compare the on-target off-tumour effects of CAR.CD123-T and NK cells, we engrafted human haematopoietic cells (hHCs) in an immune-deficient mouse model. All mice infused with CAR.CD123-T cells died by Day 5, developing toxicity against primary human bone marrow (BM) cells with a decreased number of total hCD45+ cells and, in particular, of hCD34+CD38- stem cells. In contrast, treatment with CAR.CD123-NK cells was not associated with toxicity, and all mice were alive at the end of the experiments. Finally, in a mouse model engrafted with human endothelial tissues, we demonstrated that CAR.CD123-NK cells were characterized by negligible endothelial toxicity when compared to CAR.CD123-T cells. CONCLUSIONS: Our data indicate the feasibility of an innovative off-the-shelf therapeutic strategy based on CAR.CD123-NK cells, characterized by remarkable efficacy and an improved safety profile compared to CAR.CD123-T cells. These findings open a novel intriguing scenario not only for the treatment of refractory/resistant AML patients but also to further investigate the use of CAR-NK cells in other cancers characterized by highly difficult targeting with the most conventional T effector cells.

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-ß (Aß) 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 Aß 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.

The Na+/Ca2+ Exchanger 3 Is Functionally Coupled With the NaV1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca2+ Refilling in a Transgenic Model of Alzheimer's Disease.

The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na+/Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX) in primary neurons treated with the neurotoxic amyloid ß 1-42 (Aß1-42) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX, the functional interaction between NCX and the NaV1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aß1-42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV1.6 channels. In particular, the increased NaV1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons.

Inclusion of the Inducible Caspase 9 Suicide Gene in CAR Construct Increases Safety of CAR.CD19 T Cell Therapy in B-Cell Malignancies.

T cells engineered with chimeric antigen receptor (CAR-T cells) are an effective treatment in patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia or B-cell non-Hodgkin lymphoma. Despite the reported exciting clinical results, the CAR-T cell approach needs efforts to improve the safety profile, limiting the occurrence of adverse events in patients given this treatment. Besides the most common side effects, such as cytokine release syndrome and CAR-T cell-related encephalopathy syndrome, another potential issue involves the inadvertent transduction of leukemia B cells with the CAR construct during the manufacturing process, thus leading to the possibility of a peculiar mechanism of antigen masking and treatment resistance. In this study, we investigated whether the inclusion of the inducible caspase 9 (iC9) suicide gene in the CAR construct design could be an effective safety switch to control malignant CAR+ B cells, ultimately counteracting this serious adverse event. iC9 is a suicide gene able to be activated through binding with an otherwise inert small biomolecule, known as AP1903. The exposure of iC9.CAR.CD19-DAUDI lymphoma and iC9.CAR.CD19-NALM-6 leukemia cells in vitro to 20 nM of AP1903 resulted into the prompt elimination of CAR+ B-leukemia/lymphoma cell lines. The results obtained in the animal model corroborate in vitro data, since iC9.CAR.CD19+ tumor cells were controlled in vivo by the activation of the suicide gene through administration of AP1903. Altogether, our data indicate that the inclusion of the iC9 suicide gene may result in a safe CAR-T cell product, even when manufacturing starts from biological materials characterized by heavy leukemia blast contamination.

Strategy to prevent epitope masking in CAR.CD19+ B-cell leukemia blasts.

Chimeric antigen receptor T-cells (CAR T-cells) for the treatment of relapsing/refractory B-cell precursor acute lymphoblastic leukemia have led to exciting clinical results. However, CAR T-cell approaches revealed a potential risk of CD19-/CAR+ leukemic relapse due to inadvertent transduction of leukemia cells. BACKGROUND: METHODS: We evaluated the impact of a high percentage of leukemia blast contamination in patient-derived starting material (SM) on CAR T-cell drug product (DP) manufacturing. In vitro as well as in vivo models were employed to identify characteristics of the construct associated with better profile of safety in case of inadvertent B-cell leukemia transduction during CAR T-cell manufacturing. RESULTS: The presence of large amounts of CD19+ cells in SM did not affect the transduction level of DPs, as well as the CAR T-cell rate of expansion at the end of standard production of 14 days. DPs were deeply characterized by flow cytometry and molecular biology for Ig-rearrangements, showing that the level of B-cell contamination in DPs did not correlate with the percentage of CD19+ cells in SM, in the studied patient cohort. Moreover, we investigated whether CAR design may affect the control of CAR+ leukemia cells. We provided evidences that CAR.CD19 short linker (SL) prevents complete epitope masking in CD19+CAR+ leukemia cells and we demonstrated in vitro and in vivo that CD19 +CAR(SL)+leukemic cells are killed by CAR.CD19 T-cells. CONCLUSIONS: Taken together, these data suggest that a VL-VH SL may result in a safe CAR-T product, even when manufacturing starts from biological materials characterized by heavy contamination of leukemia blasts.

Innovative and Promising Strategies to Enhance Effectiveness of Immunotherapy for CNS Tumors: Where Are We?

Although there are several immunotherapy approaches for the treatment of Central Nervous System (CNS) tumors under evaluation, currently none of these approaches have received approval from the regulatory agencies. CNS tumors, especially glioblastomas, are tumors characterized by highly immunosuppressive tumor microenvironment, limiting the possibility of effectively eliciting an immune response. Moreover, the peculiar anatomic location of these tumors poses relevant challenges in terms of safety, since uncontrolled hyper inflammation could lead to cerebral edema and cranial hypertension. The most promising strategies of immunotherapy in neuro-oncology consist of the use of autologous T cells redirected against tumor cells through chimeric antigen receptor (CAR) constructs or genetically modified T-cell receptors. Trials based on native or genetically engineered oncolytic viruses and on vaccination with tumor-associated antigen peptides are also under evaluation. Despite some sporadic complete remissions achieved in clinical trials, the outcome of patients with CNS tumors treated with different immunotherapeutic approaches remains poor. Based on the lessons learned from these unsatisfactory experiences, novel immune-therapy approaches aimed at overcoming the profound immunosuppressive microenvironment of these diseases are bringing new hope to reach the cure for CNS tumors.

Synthesis and Characterization of Novel Mono- and Bis-Guanyl Hydrazones as Potent and Selective ASIC1 Inhibitors Able to Reduce Brain Ischemic Insult.

Acid-sensitive ion channels (ASICs) are sodium channels partially permeable to Ca2+ ions, listed among putative targets in central nervous system (CNS) diseases in which a pH modification occurs. We targeted novel compounds able to modulate ASIC1 and to reduce the progression of ischemic brain injury. We rationally designed and synthesized several diminazene-inspired diaryl mono- and bis-guanyl hydrazones. A correlation between their predicted docking affinities for the acidic pocket (AcP site) in chicken ASIC1 and their inhibition of homo- and heteromeric hASIC1 channels in HEK-293 cells was found. Their activity on murine ASIC1a currents and their selectivity vs mASIC2a were assessed in engineered CHO-K1 cells, highlighting a limited isoform selectivity. Neuroprotective effects were confirmed in vitro, on primary rat cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation, and in vivo, in ischemic mice. Early lead 3b, showing a good selectivity for hASIC1 in human neurons, was neuroprotective against focal ischemia induced in mice.

Lavender and coriander essential oils and their main component linalool exert a protective effect against amyloid-ß neurotoxicity.

Alzheimer's disease (AD) is a neurodegenerative disorder leading to cognitive deficits and cognitive decline. Since no cure or preventing therapy is currently available to counteract AD, natural-derived compounds are investigated to find new potential neuroprotective agents for its treatment. In the present study, we tested the neuroprotective effect of lavender and coriander essential oils (EOs) and their main active constituent linalool, against the neurotoxicity elicited by Aß1-42 oligomers, a key molecular factor in the neurodegeneration of AD. Importantly, our findings on neuronally differentiated PC12 cells exposed to Aß1-42 oligomers are in accordance with previous in vivo studies reporting the neuroprotective potential of lavender and coriander EOs and linalool. We found that lavender and coriander EOs at the concentration of 10 µg/mL as well as linalool at the same concentration were able to improve viability and to reduce nuclear morphological abnormalities in cells treated with Aß1-42 oligomers for 24 hours. Lavender and coriander EOs and linalool also showed to counteract the increase of intracellular reactive oxygen species production and the activation of the pro-apoptotic enzyme caspase-3 induced by Aß1-42 oligomers. Our findings provide further evidence that these EOs and their main constituent linalool could be natural agents of therapeutic interest against Aß1-42 -induced neurotoxicity.

CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells.

The prognosis of many patients with chemotherapy-refractory or multiply relapsed CD30+ non-Hodgkin Lymphoma (NHL) or Hodgkin lymphoma (HL) still remains poor, and novel therapeutic approaches are warranted to address this unmet clinical need. In light of this consideration, we designed and pre-clinically validated a Chimeric Antigen Receptor (CAR) construct characterized by a novel anti-CD30 single-chain variable-fragment cassette, linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. We found that CAR.CD30 T-cells exhibit remarkable cytolytic activity in vitro against HL and NHL cell lines, with sustained proliferation and pro-inflammatory cytokine production, even after multiple and sequential lymphoma cell challenges. CAR.CD30 T-cells also demonstrated anti-lymphoma activity in two in vivo xenograft immune-deficient mouse models of metastatic HL and NHL. We observed that administration of CAR.CD30 T-cells, incorporating the CD28.OX40 costimulatory domains and manufactured in the presence of IL7 and IL15, were associated with the best overall survival in the treated mice, along with the establishment of a long-term immunological memory, able to protect mice from further tumor re-challenge. Our data indicate that, in the context of in vivo systemic metastatic xenograft mouse models, the costimulatory machinery of CD28.OX40 is crucial for improving persistence, in vivo expansion and proliferation of CAR.CD30 T-cells upon tumor encounter. CD28.OX40 costimulatory combination is ultimately responsible for the antitumor efficacy of the approach, paving the way to translate this therapeutic strategy in patients with CD30+ HL and NHL.

The Na+/Ca2+exchanger in Alzheimer's disease.

As a pivotal player in regulating sodium (Na+) and calcium (Ca2+) homeostasis and signalling in excitable cells, the Na+/Ca2+ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca2+ and/or Na+ concentrations occurs, including Alzheimer's disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca2+ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na+ elevation occurring in several pathophysiological conditions. Since the alteration of Na+ and Ca2+ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aß), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aß insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy.

Genetic Up-Regulation or Pharmacological Activation of the Na+/Ca2+ Exchanger 1 (NCX1) Enhances Hippocampal-Dependent Contextual and Spatial Learning and Memory.

The Na+/Ca2+ exchanger 1 (NCX1) participates in the maintenance of neuronal Na+ and Ca2+ homeostasis, and it is highly expressed at synapse level of some brain areas involved in learning and memory processes, including the hippocampus, cortex, and amygdala. Furthermore, NCX1 increases Akt1 phosphorylation and enhances glutamate-mediated Ca2+ influx during depolarization in hippocampal and cortical neurons, two processes involved in learning and memory mechanisms. We investigated whether the modulation of NCX1 expression/activity might influence learning and memory processes. To this aim, we used a knock-in mouse overexpressing NCX1 in hippocampal, cortical, and amygdala neurons (ncx1.4over) and a newly synthesized selective NCX1 stimulating compound, named CN-PYB2. Both ncx1.4over and CN-PYB2-treated mice showed an amelioration in spatial learning performance in Barnes maze task, and in context-dependent memory consolidation after trace fear conditioning. On the other hand, these mice showed no improvement in novel object recognition task which is mainly dependent on non-spatial memory and displayed an increase in the active phosphorylated CaMKIIα levels in the hippocampus. Interestingly, both of these mice showed an increased level of context-dependent anxiety.Altogether, these results demonstrate that neuronal NCX1 participates in spatial-dependent hippocampal learning and memory processes.

The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aß1-42 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 Aß1-42 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 Aß1-42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aß1-42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aß1-42 oligomers, and (5) BDS-I prevented Aß1-42-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 Aß1-42-induced ER stress and cell suffering in astrocytes.

Amyloid ß-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-ß1-42 (Aß1-42) 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 Aß1-42 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.

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-ß 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 ß-amyloid peptide1-42 (Aß1-42) 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 Aß 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 Aß1-42-induced enhancement of KV3.4 activity, preventing Aß1-42-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 Aß1-42-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.

Erratum: Author Correction: Na+/Ca2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca2+ regulation during neuronal differentiation.

[This corrects the article DOI: 10.1038/s41420-017-0018-1.].

ORAI1/STIM1 Interaction Intervenes in Stroke and in Neuroprotection Induced by Ischemic Preconditioning Through Store-Operated Calcium Entry.

Background and Purpose- Disturbance of endoplasmic reticulum (ER) Ca2+ homeostasis causes neuronal cell injury in stroke. By contrast, ischemic preconditioning (IPC)-a brief sublethal ischemic episode affording tolerance to a subsequent ischemic insult-restores ER Ca2+ homeostasis. Under physiological conditions, ER calcium content is continuously refilled by the interaction between the ER-located Ca2+ sensor STIM (stromal interacting molecule) 1 and the plasma membrane channel ORAI1 (a structural component of the CRAC calcium channel)-2 key mediators of 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. Methods- Primary cortical neurons were subjected to oxygen and glucose deprivation+reoxygenation to reproduce in vitro brain ischemia. Focal brain ischemia and IPC were induced in rats by transient middle cerebral artery occlusion. Expression of ORAI1 and STIM1 transcripts and proteins and their immunosignals were detected by qRT-PCR, Western blot, and immunocytochemistry, respectively. SOCE and Ca2+ release-activated Ca2+ currents (ICRAC) were measured by Fura-2 AM video imaging and patch-clamp electrophysiology in whole-cell configuration, respectively. Results- STIM1 and ORAI1 protein expression and immunosignals decreased in the ipsilesional temporoparietal cortex of rats subjected to transient middle cerebral artery occlusion followed by reperfusion. Analogously, in primary hypoxic cortical neurons, STIM1 and ORAI1 transcript and protein levels decreased concurrently with SOCE and Ca2+ release-activated Ca2+currents. By contrast, IPC induced SOCE and Ca2+ release-activated Ca2+current upregulation, thereby preventing STIM1 and ORAI1 downregulation induced by oxygen and glucose deprivation+reoxygenation. Silencing of STIM1 or ORAI1 prevented IPC-induced tolerance and caused ER stress, as measured by GRP78 (78-kDa glucose regulated protein) and caspase-3 upregulation. Conclusions- ORAI1 and STIM1, which participate in SOCE, take part in stroke pathophysiology and play an important role in IPC-induced neuroprotection.

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.