GV1001 reduces neurodegeneration and prolongs lifespan in 3xTg-AD mouse model through anti-aging effects.

GV1001, which mimics the activity of human telomerase reverse transcriptase, protects neural cells from amyloid beta (Aß) toxicity and other stressors through extra-telomeric function, as noted in our prior in vitro studies. As per a recent phase II clinical trial, it improves cognitive function in patients with moderate to severe dementia. However, the underlying protective mechanisms remain unclear. This study aimed to investigate the effects of GV1001 on neurodegeneration, senescence, and survival in triple transgenic Alzheimer's disease (3xTg-AD) mice. GV1001 (1 mg/kg) was subcutaneously injected into old 3xTg-AD mice thrice a week until the endpoint for sacrifice, and survival was analysed. Magnetic resonance imaging (MRI) and Prussian blue staining (PBS) were performed to evaluate entry of GV1001 entrance into the brain. Diverse molecular studies were performed to investigate the effect of GV1001 on neurodegeneration and cellular senescence in AD model mice, with a particular focus on BACE, amyloid beta1-42 (Aß1-42), phosphorylated tau, volume of dentate gyrus, ß-galactosidase positive cells, telomere length, telomerase activity, and ageing-associated proteins. GV1001 crossed the blood-brain barrier, as confirmed by assessing the status of ferrocenecarboxylic acid-conjugated GV1001 using magnetic resonance imaging and PBS. GV1001 increased the survival of 3xTg-AD mice. It decreased BACE and Aß1-42 levels, neurodegeneration (i.e., reduced CA1, CA3 and dentate gyrus volume, decreased levels of senescence-associated ß-galactosidase positive cells, and increased telomere length and telomerase activity), and levels of ageing-associated proteins. We suggest that GV1001 exerts anti-ageing effects in 3xTg-AD mice by reducing neurodegeneration and senescence, which contributes to improved survival.

GV1001 modulates neuroinflammation and improves memory and behavior through the activation of gonadotropin-releasing hormone receptors in a triple transgenic Alzheimer's disease mouse model.

GV1001 protects neural cells from amyloid-ß (Aß) toxicity and other stressors in in vitro studies and demonstrates clinically beneficial effects in patients with moderate to severe Alzheimer's disease (AD). Here, we investigated the protective effects and mechanism of action of GV1001 in triple transgenic AD (3xTg-AD) mice. We found that GV1001 improved memory and cognition in middle- and old-aged 3xTg-AD mice. Additionally, it reduced Aß oligomer and phospho-tau (Ser202 and Thr205) levels in the brain, and mitigated neuroinflammation by promoting a neuroprotective microglial and astrocyte phenotype while diminishing the neurotoxic ones. In vitro, GV1001 bound to gonadotropin releasing hormone receptors (GnRHRs) with high affinity. Levels of cyclic adenosine monophosphate, a direct downstream effector of activated GnRHRs, increased after GV1001 treatment. Furthermore, inhibition of GnRHRs blocked GV1001-induced effects. Thus, GV1001 might improve cognitive and memory functions of 3xTg-AD mice by suppressing neuroinflammation and reducing Aß oligomers levels and phospho-tau by activating GnRHRs and their downstream signaling pathways.

Effects of GV1001 on Language Dysfunction in Patients With Moderate-to-Severe Alzheimer's Disease: Post Hoc Analysis of Severe Impairment Battery Subscales.

Background and Purpose: The efficacy and safety of GV1001 have been demonstrated in patients with moderate-to-severe Alzheimer's disease (AD). In this study, we aimed to further demonstrate the effectiveness of GV1001 using subscales of the Severe Impairment Battery (SIB), which is a validated measure to assess cognitive function in patients with moderate-to-severe AD. Methods: We performed a post hoc analysis of data from a 6 month, multicenter, phase 2, randomized, double-blind, placebo-controlled trial with GV1001 (ClinicalTrials.gov, NCT03184467). Patients were randomized to receive either GV1001 or a placebo for 24 weeks. In the current study, nine subscales of SIB-social interaction, memory, orientation, language, attention, praxis, visuospatial ability, construction, and orientation to name- were compared between the treatment (GV1001 1.12 mg) and placebo groups at weeks 12 and 24. The safety endpoints for these patients were also determined based on adverse events. Results: In addition to the considerable beneficial effect of GV1001 on the SIB total score, GV1001 1.12 mg showed the most significant effect on language function at 24 weeks compared to placebo in both the full analysis set (FAS) and per-protocol set (PPS) (p=0.017 and p=0.011, respectively). The rate of adverse events did not differ significantly between the 2 groups. Conclusions: Patients with moderate-to-severe AD receiving GV1001 had greater language benefits than those receiving placebo, as measured using the SIB language subscale.

Anti-N-methyl-D-aspartate receptor encephalitis after coronavirus disease 2019: A case report and literature review.

RATIONALE: Coronavirus disease 2019 (COVID-19) has become a global pandemic and COVID-19-associated anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis may occur through an immune-mediated pathomechanism. PATIENT CONCERNS: A 21-year-old woman with a history of COVID-19 presented to our hospital with memory decline and psychiatric symptoms. DIAGNOSIS: The patient was diagnosed with anti-NMDAR encephalitis. INTERVENTION: Intravenous methylprednisolone (1 g/day over 5 days) followed by immunoglobulin (0.4 g/kg/day over 5 days) were administered. The patient underwent laparoscopic salpingo-oophorectomy to remove an ovarian teratoma. OUTCOMES: The patient was discharged with sequelae of short-term memory impairment, without other neuropsychiatric symptoms. LESSONS: Cases of previously reported anti-NMDAR encephalitis with COVID-19 were reviewed and compared with the present case. Clinicians should be aware of the occurrence of anti-NMDAR encephalitis in patients who present with neuropsychiatric complaints during or after exposure to COVID-19. Further studies are required to determine the causal relationship between the 2 diseases and predict the prognosis of anti-NMDAR encephalitis after COVID-19 exposure.

Neuroprotective Effects of GV1001 in Animal Stroke Model and Neural Cells Subject to Oxygen-Glucose Deprivation/Reperfusion Injury.

BACKGROUND AND PURPOSE: Previous studies have revealed the diverse neuroprotective effects of GV1001. In this study, we investigated the effects of GV1001 on focal cerebral ischemia-reperfusion injury (IRI) in rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in neural stem cells (NSCs) and cortical neurons. METHODS: Focal cerebral IRI was induced by transient middle cerebral artery occlusion (MCAO). Brain diffusion-weighted imaging (DWI) was performed 2 hours after occlusion, and a total of 37 rats were treated by reperfusion with GV1001 or saline 2 hours after occlusion. Fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging, immunohistochemistry, and neurobehavioral function analyses were performed. Additionally, OGD/R-injured NSCs and cortical neurons were treated with different GV1001 concentrations. Cell viability, proliferation, migration, and oxidative stress were determined by diverse molecular analyses. RESULTS: In the stroke model, GV1001 protected neural cells against IRI. The most effective dose of GV1001 was 60 µM/kg. The infarct volume on FLAIR 48 hours after MCAO compared to lesion volume on DWI showed a significantly smaller ratio in the GV1001-treated group. GV1001-treated rats exhibited better behavioral functions than the saline-treated rats. Treatment with GV1001 increased the viability, proliferation, and migration of the OGD/R-injured NSCs. Free radicals were significantly restored by treatment with GV1001. These neuroprotective effects of GV1001 have also been demonstrated in OGD/R-injured cortical neurons. CONCLUSIONS: The results suggest that GV1001 has neuroprotective effects against IRI in NSCs, cortical neurons, and the rat brain. These effects are mediated through the induction of cellular proliferation, mitochondrial stabilization, and anti-apoptotic, anti-aging, and antioxidant effects.

Efficacy and safety of GV1001 in patients with moderate-to-severe Alzheimer's disease already receiving donepezil: a phase 2 randomized, double-blind, placebo-controlled, multicenter clinical trial.

BACKGROUND: Our previous studies showed that GV1001 has various protective effects against ß-amyloid and other stressors. Based on these findings, we hypothesized that GV1001 might have beneficial effects in patients with Alzheimer's disease (AD). METHODS: A phase 2, double-blind, parallel-group, placebo-controlled, 6-month randomized clinical trial was performed to evaluate the safety and efficacy of subcutaneously administered GV1001. Between September 2017 and September 2019, 13 centers in South Korea recruited participants. A total of 106 patients were screened, and 96 patients with moderate-to-severe AD were randomized 1:1:1 to the placebo (group 1, n = 31), GV1001 0.56 mg (group 2, n = 33), and 1.12 mg (group 3, n = 32) groups. GV1001 was administered every week for 4 weeks (4 times), followed by every 2 weeks until week 24 (10 times). The primary endpoint was the change in the Severe Impairment Battery (SIB) score from baseline to week 24. The key secondary efficacy endpoints were the change in the Clinical Dementia Rating Sum of Box (CDR-SOB), Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL), Neuropsychiatric Inventory (NPI), Mini-Mental State Examination, and Global Deterioration Scale scores. The safety endpoints were also assessed based on adverse events, laboratory test results, vital signs, and other observations related to safety. RESULTS: Group 3 showed less decrease in the SIB score at 12 and 24 weeks compared with group 1 (P < 0.05). These were not significantly observed in group 2. Among the secondary endpoints, only the NPI score showed significantly better improvement in group 2 than in group 3 at week 12; however, there were no other significant differences between the groups. Although the ADCS-ADL and CDR-SOB scores showed a pattern similar to SIB scores, a statistically significant result was not found. Adverse events were similar across all three groups. CONCLUSIONS: The results indicate that GV1001 1.12 mg met the primary endpoint of a statistically significant difference. GV1001 was well tolerated without safety concerns. This study warrants a larger clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT03184467 . Registered on June 12, 2017.

Telmisartan Inhibits the NLRP3 Inflammasome by Activating the PI3K Pathway in Neural Stem Cells Injured by Oxygen-Glucose Deprivation.

Angiotensin II receptor blockers (ARBs) have been shown to exert neuroprotective effects by suppressing inflammatory and apoptotic responses. In the present study, the effects of the ARB telmisartan on the NLRP3 inflammasome induced by oxygen-glucose deprivation (OGD) in neural stem cells (NSCs) were investigated, as well as their possible association with the activation of the PI3K pathway. Cultured NSCs were treated with different concentrations of telmisartan and subjected to various durations of OGD. Cell counting, lactate dehydrogenase, bromodeoxyuridine, and colony-forming unit assays were performed to measure cell viability and proliferation. In addition, the activity of intracellular signaling pathways associated with the PI3K pathway and NLRP3 inflammasome was evaluated. Telmisartan alone did not affect NSCs up to a concentration of 10 µM under normal conditions but showed toxicity at a concentration of 100 µM. Moreover, OGD reduced the viability of NSCs in a time-dependent manner. Nevertheless, treatment with telmisartan increased the viability and proliferation of OGD-injured NSCs. Furthermore, telmisartan promoted the expression of survival-related proteins and mRNA while inhibiting the expression of death-related proteins induced by OGD. In particular, telmisartan attenuated OGD-dependent expression of the NLRP3 inflammasome and its related signaling proteins. These beneficial effects of telmisartan were blocked by a PI3K inhibitor. Together, these results indicate that telmisartan attenuated the activation of the NLRP3 inflammasome by triggering the PI3K pathway, thereby contributing to neuroprotection.

Use of methotrexate in the management of recurrent Tolosa-Hunt syndrome: Two case reports.

RATIONALE: Tolosa-Hunt syndrome (THS) is rare condition characterized by painful ophthalmoplegia that usually responds well to corticosteroid. About a half of THS patients experience recurrence within intervals of months to years from initial presentation. Recurrence is more common in younger patients, and can be ipsilateral, contralateral, or bilateral. Cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, infliximab, and radiotherapy can be considered as second-line treatment. However, there is insufficient evidence for treatments preventing recurrence of THS. PATIENT CONCERNS: We experienced two patients with THS that recurred twice while tapering or after ceasing corticosteroid administration. DIAGNOSIS: Both patients were diagnosed as recurrent THS. INTERVENTIONS: Methotrexate was treated with a combination of corticosteroid after THS recurred twice with corticosteroid therapy alone. OUTCOMES: After adding methotrexate to the steroid regimen, their symptoms were successfully regulated and ceased to recur LESSONS:: These cases add to the evidence for the use of methotrexate as a second-line therapeutic agent for those patients with recurrent THS attacks. Further studies are in need to prove the risk and benefits of second-line treatments in THS.

Early increment of soluble triggering receptor expressed on myeloid cells 2 in plasma might be a predictor of poor outcome after ischemic stroke.

Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) is derived from cleavage of TREM2, which is expressed on the cell surface of microlgia and other tissue-specific macrophages. In the present study, the changes in the sTREM2 levels after ischemic stroke (IS) and their association with clinical outcomes were evaluated. A total of 43 patients diagnosed with non-cardioembolic IS between June 2011 and May 2014 were consecutively included in this study. Patients treated with intravenous thrombolysis or intra-arterial thrombectomy were excluded. Plasma samples were collected three times (days 1, 7, and 90) after ictus. The sTREM2 level was measured in the samples using the highly sensitive solid-phase proximity ligation assay (SP-PLA). Among the 43 subjects, higher initial NIH stroke scale (NIHSS) score (P = 0.005), early increment of sTREM2 (P < 0.001), and late decrement of sTREM2 (P = 0.002), were more common in patients with poor outcome. Based on multivariate analysis, initial NIHSS score (P = 0.015) and early increment of sTREM2 (P = 0.032) were independently associated with poor outcome. The results from the present study indicate that increment of sTREM2 level at the early phase was a predictor of poor outcome. Serial follow-up of sTREM2 may aid prognosis after stroke.

Mitochondria damaged by Oxygen Glucose Deprivation can be Restored through Activation of the PI3K/Akt Pathway and Inhibition of Calcium Influx by Amlodipine Camsylate.

Amlodipine, a L-type calcium channel blocker, has been reported to have a neuroprotective effect in brain ischemia. Mitochondrial calcium overload leads to apoptosis of cells in neurologic diseases. We evaluated the neuroprotective effects of amlodipine camsylate (AC) on neural stem cells (NSCs) injured by oxygen glucose deprivation (OGD) with a focus on mitochondrial structure and function. NSCs were isolated from rodent embryonic brains. Effects of AC on cell viability, proliferation, level of free radicals, and expression of intracellular signaling proteins were assessed in OGD-injured NSCs. We also investigated the effect of AC on mitochondrial structure in NSCs under OGD by transmission electron microscopy. AC increased the viability and proliferation of NSCs. This beneficial effect of AC was achieved by strong protection of mitochondria. AC markedly enhanced the expression of mitochondrial biogenesis-related proteins and mitochondrial anti-apoptosis proteins. Together, our results indicate that AC protects OGD-injured NSCs by protecting mitochondrial structure and function. The results of the present study provide insight into the mechanisms underlying the protective effects of AC on NSCs.

LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been reported to play critical roles in the proliferation of various cancer cells. However, the roles of LGR5 in brain tumors and the specific intracellular signaling proteins directly associated with it remain unknown. Expression of LGR5 was first measured in normal brain tissue, meningioma, and pituitary adenoma of humans. To identify the downstream signaling pathways of LGR5, siRNA-mediated knockdown of LGR5 was performed in SH-SY5Y neuroblastoma cells followed by proteomics analysis with 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In addition, the expression of LGR5-associated proteins was evaluated in LGR5-inhibited neuroblastoma cells and in human normal brain, meningioma, and pituitary adenoma tissue. Proteomics analysis showed 12 protein spots were significantly different in expression level (more than two-fold change) and subsequently identified by peptide mass fingerprinting. A protein association network was constructed from the 12 identified proteins altered by LGR5 knockdown. Direct and indirect interactions were identified among the 12 proteins. HSP 90-beta was one of the proteins whose expression was altered by LGR5 knockdown. Likewise, we observed decreased expression of proteins in the hnRNP subfamily following LGR5 knockdown. In addition, we have for the first time identified significantly higher hnRNP family expression in meningioma and pituitary adenoma compared to normal brain tissue. Taken together, LGR5 and its downstream signaling play critical roles in neuroblastoma and brain tumors such as meningioma and pituitary adenoma.

Sublethal Doses of Zinc Protect Rat Neural Stem Cells Against Hypoxia Through Activation of the PI3K Pathway.

Cerebral infarction is one of the major causes of severe morbidity and mortality, and thus, research has focused on developing treatment options for this condition. Zinc (Zn) is an essential element in the central nervous system and has several neuroprotective effects in the brain. In this study, we examined the neuroprotective effects of Zn on neural stem cells (NSCs) exposed to hypoxia. After treatment with several concentrations of Zn, the viability of NSCs under hypoxic conditions was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Trypan blue staining, and a lactate dehydrogenase assay. To evaluate the effect of Zn on the proliferation of NSCs, bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) labeling and colony formation assays were performed. Apoptosis was also examined in NSCs exposed to hypoxia with and without Zn treatment. In addition, a western blot analysis was performed to evaluate the effect of Zn on intracellular signaling proteins. NSC viability and proliferation were decreased under hypoxic conditions, but treatment with sublethal doses of Zn restored viability and proliferation. Sublethal doses of Zn reduced apoptosis caused by hypoxia, increased the expression levels of proteins related to the phosphatidylinositol-3 kinase (PI3K) pathway, and decreased the expression levels of proteins associated with neuronal cell death. These findings confirm that in vivo, sublethal doses of Zn protect NSCs against hypoxia through the activation of the PI3K pathway. Thus, Zn could be employed as a therapeutic option to protect NSCs in ischemic stroke.

Atorvastatin Rejuvenates Neural Stem Cells Injured by Oxygen-Glucose Deprivation and Induces Neuronal Differentiation Through Activating the PI3K/Akt and ERK Pathways.

Oxygen and glucose (OGD) deprivation is one of the most important pathogenic mechanisms in cerebral infarction and is widely used as an in vitro model for ischemic stroke. OGD also damages neural stem cells (NSCs), which are important in brain recovery after cerebral infarction. To enhance recovery, there have been many studies aimed at determining methods to protect NSCs after stroke. Because atorvastatin has diverse protective effects on neural cells, we studied whether it could rejuvenate NSCs injured by OGD. Primary cultured NSCs were exposed to OGD for 8 h, and the main characteristics of stem cells, such as survival, proliferation, migration, and differentiation, were evaluated to confirm the effect of OGD on NSCs. Next, cells were treated with various concentrations of atorvastatin with exposure to OGD for 8 h to confirm whether it could rejuvenate NSCs. OGD significantly affected the survival, proliferation, migration, and differentiation of NSCs. However, treatment with atorvastatin meaningfully restored survival, proliferation, migration, and differentiation of NSCs. These beneficial effects of atorvastatin were blocked by treatment with either a PI3K inhibitor or an ERK inhibitor. In conclusion, OGD damages NSCs and causes them to lose the main characteristics of stem cells so that they cannot contribute to brain recovery after cerebral infarction. However, treatment with atorvastatin after cerebral infarction can effectively rejuvenate NSCs through activating the PI3K and ERK pathways to aid in brain regeneration.

Effects of aspirin and clopidogrel on neural stem cells.

Cerebral infarction causes severe morbidity and mortality. Most patients with cerebral infarction should take antiplatelet drugs daily, so the effects of those drugs on the regeneration of the brain need to be investigated. Aspirin and clopidogrel are the most widely used antiplatelet drugs for the prevention of ischemic stroke. We investigated the effects of aspirin and clopidogrel on neural stem cells (NSCs). NSCs were dissociated from fetal rat cortex and cultured with basic fibroblast growth factor and N2 medium. To measure the effects of aspirin and clopidogrel on NSCs, NSCs were treated with several concentrations of aspirin, clopidogrel bisulfate, and clopidogrel resinate for 24 h. After the treatment, we measured cell viability by cell counting kit-8, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, flow cytometry, and lactate dehydrogenase assay. To evaluate their effects on NSC proliferation, we performed BrdU cell proliferation assay and colony-forming unit assay. We compared the intracellular protein level in the NSCs treated with aspirin and two types of clopidogrel, by proteomics analysis. Various viability tests showed that clopidogrel resinate and clopidogrel bisulfate did not affect the viability and proliferation of NSCs whereas aspirin decreased them even at low concentrations which are clinically relevant. Moreover, through the proteomics, it was confirmed that the toxicity of aspirin to NSCs might be associated with the alteration of several intracellular proteins. Taken together, these results suggest that clopidogrel resinate and clopidogrel bisulfate are safe but aspirin could be toxic to NSCs. Therefore, when these antiplatelet agents are prescribed over the long-term, the finding that aspirin could be toxic to NSCs should be considered.

Tracking and protection of transplanted stem cells using a ferrocenecarboxylic acid-conjugated peptide that mimics hTERT.

In vivo tracking of transplanted stem cells has been a central aim of stem cell therapy. Although many tracking systems have been introduced, no method has yet been validated for clinical applications. We developed a novel sophisticated peptide (GV1001) that mimics hTERT (human telomerase reverse transcriptase) and analysed its ability to track and protect stem cells after transplantation. Ferrocenecarboxylic acid-conjugated GV1001 (Fe-GV1001) efficiently penetrated stem cells with no adverse effects. Moreover, Fe-GV1001 improved the viability, proliferation, and migration of stem cells under hypoxia. After Fe-GV1001-labelled stem cells were transplanted into the brains of rats after stroke, the labelled cells were easily tracked by MRI. Our findings indicate that Fe-GV1001 can be used for the in vivo tracking of stem cells after transplantation into the brain and can improve the efficacy of stem cell therapy by sustaining and enhancing stem cell characteristics under disease conditions.

Candesartan Restores the Amyloid Beta-Inhibited Proliferation of Neural Stem Cells by Activating the Phosphatidylinositol 3-Kinase Pathway.

BACKGROUND AND PURPOSE: Neurogenesis in the adult brain is important for memory and learning, and the alterations in neural stem cells (NSCs) may be an important aspect of Alzheimer's disease (AD) pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to have an important role in neuronal cell survival and is highly involved in adult neurogenesis. Candesartan is an angiotensin II receptor antagonist used for the treatment of hypertension and several studies have reported that it also has some neuroprotective effects. We investigated whether candesartan could restore the amyloid-ß(25-35) (Aß25-35) oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. METHODS: To evaluate the effects of candesartan on the Aß25-35 oligomer-inhibited proliferation of NSCs, the NSCs were treated with several concentrations of candesartan and/or Aß25-35 oligomers, and MTT assay and trypan blue staining were performed. To evaluate the effect of candesartan on the Aß-inhibited proliferation of NSCs, we performed a bromodeoxyuridine (BrdU) labeling assay. The levels of p85α PI3K, phosphorylated Akt (pAkt) (Ser473), phosphorylated glycogen sinthase kinase-3ß (pGSK-3ß) (Ser9), and heat shock transcription factor-1 (HSTF-1) were analyzed by Western blotting. RESULTS: The BrdU assays demonstrated that NSC proliferation decreased with Aß25-35 oligomer treatment; however, a combined treatment with candesartan restored it. Western blotting displayed that candesartan treatment increased the expression levels of p85α PI3K, pAkt (Ser473), pGSK-3ß (Ser9), and HSTF. The NSCs were pretreated with a PI3K inhibitor, LY294002; the effects of candesartan on the proliferation of NSCs inhibited by Aß25-35 oligomers were almost completely blocked. CONCLUSIONS: Together, these results suggest that candesartan restores the Aß25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway.