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Age and insulin-like growth factor-1 (IGF-1) have an inverse association with cognitive decline and dementia. IGF-1 is known to have important pleiotropic functions beginning in neurodevelopment and extending into adulthood such as neurogenesis. At the cellular level, IGF-1 has pleiotropic signaling mechanisms through the IGF-1 receptor on neurons and neuroglia to attenuate inflammation, promote myelination, maintain astrocytic functions for homeostatic balances, and neuronal synaptogenesis. In preclinical rodent models of aging and transgenic models of IGF-1, increased IGF-1 improves cognition in a variety of behavioral paradigms along with reducing IGF-1 via knockout models being able to induce cognitive impairment. At the clinical levels, most studies highlight that increased levels of IGF-1 are associated with better cognition. This review provides a comprehensive and up-to-date evaluation of the association between IGF-1 and cognition at the cellular signaling levels, preclinical, and clinical levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bioassay-guided fractionation of the essential oil of Santalum album led to the identification of α-santalol ( 1 ) and β-santalol ( 2 ) as new chemotypes of cannabinoid receptor type II (CB 2 ) ligands with K i values of 10.49 and 8.19 μM, respectively. Nine structurally new α-santalol derivatives ( 4a - 4h and 5 ) were synthesized to identify more selective and potent CB 2 ligands. Compound 4e with a piperazine structural moiety demonstrated a K i value of 0.99 μM against CB 2 receptor and did not show binding activity against cannabinoid receptor type I (CB 1 ) at 10 μM. Compounds 1 , 2 , and 4e increased intracellular calcium influx in SH-SY5Y human neuroblastoma cells that were attenuated by CB 2 antagonism or inverse agonism, supporting the results that these compounds are CB 2 agonists. Molecular docking showed that 1 and 4e had similar binding poses, exhibiting a unique interaction with Thr114 within the CB 2 receptor, and that the piperazine structural moiety is required for the binding affinity of 4e . A 200 ns molecular dynamics simulation of CB 2 complexed with 4e confirmed the stability of the complex. This structural insight lays a foundation to further design and synthesize more potent and selective α-santalol-based CB 2 ligands for drug discovery.

Ischemic stroke is a leading cause of death and disability, as therapeutic options for mitigating the long-term deficits precipitated by the event remain limited. Acute administration of the neuroendocrine modulator insulin-like growth factor-1 (IGF-1) attenuates ischemic stroke damage in preclinical models, and clinical studies suggest IGF-1 can reduce the risk of stroke and improve overall outcomes. The cellular mechanism by which IGF-1 exerts this protection is poorly defined, as all cells within the neurovascular unit express the IGF-1 receptor. We hypothesize that the functional regulation of both neurons and astrocytes by IGF-1 is critical in minimizing damage in ischemic stroke. To test this, we utilized inducible astrocyte-specific or neuron-specific transgenic mouse models to selectively reduce IGF-1R in the adult mouse brain prior to photothrombotic stroke. Acute changes in blood brain barrier permeability, microglial activation, systemic inflammation, and biochemical composition of the brain were assessed 3 hours following photothrombosis, and significant protection was observed in mice deficient in neuronal and astrocytic IGF-1R. When the extent of tissue damage and sensorimotor dysfunction was assessed for 3 days following stroke, only the neurological deficit score continued to show improvements, and the extent of improvement was enhanced with additional IGF-1 supplementation. Overall, results indicate that neuronal and astrocytic IGF-1 signaling influences stroke damage but IGF-1 signaling within these individual cell types is not required for minimizing tissue damage or behavioral outcome., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

In the U.S. about half of the HIV-infected individuals are aged 50 and older. In men living with HIV, secondary hypogonadism is common and occurs earlier than in seronegative men, and its prevalence increases with age. While the mechanisms(s) are unknown, the HIV-1 trans-activator of transcription (Tat) protein disrupts neuroendocrine function in mice partly by dysregulating mitochondria and neurosteroidogenesis. We hypothesized that conditional Tat expression in middle-aged male transgenic mice [Tat(+)] would promote age-related comorbidities compared to age-matched controls [Tat(-)]. We expected Tat to alter steroid hormone milieu consistent with behavioral deficits. Middle-aged Tat(+) mice had lower circulating testosterone and progesterone than age-matched controls and greater circulating corticosterone and central allopregnanolone than other groups. Young Tat(+) mice had greater circulating progesterone and estradiol-to-testosterone ratios. Older age or Tat exposure increased anxiety-like behavior (open field; elevated plus-maze), increased cognitive errors (radial arm water maze), and reduced grip strength. Young Tat(+), or middle-aged Tat(-), males had higher mechanical nociceptive thresholds than age-matched counterparts. Steroid levels correlated with behaviors. Thus, Tat may contribute to HIV-accelerated aging.

Current common analgesics are mediated through the mu or kappa opioid receptor agonism. Unfortunately, selective mu or kappa receptor agonists often cause harmful side effects. However, ligands exhibiting dual agonism to the opioid receptors, such as to mu and kappa, or to mu and delta, have been suggested to temper undesirable adverse effects while retaining analgesic activity. Herein we report an introduction of various 6,5-fused rings to C2 of the salvinorin scaffold via an ester linker. In vitro studies showed that many of these compounds have dual agonism on kappa and mu opioid receptors. In vivo studies on the lead dual kappa and mu opioid receptor agonist demonstrated supraspinal thermal analgesic activity while avoiding anxiogenic effects in male mice, thus providing further strong evidence in support of the therapeutic advantages of dual opioid receptor agonists over selective opioid receptor agonists., (© 2022 Wiley-VCH GmbH.)

Purpose: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity., Methods: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 μg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects., Results: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 h, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 h., Conclusions: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 h after dosing., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity., Methods: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 μg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects., Results: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 hours, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 hours., Conclusions: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 hours after dosing.

The age-related reduction in circulating levels of insulin-like growth factor-1 (IGF-1) is associated with increased risk of stroke and neurodegenerative diseases in advanced age. Numerous reports highlight behavioral and physiological deficits in blood-brain barrier function and neurovascular communication when IGF-1 levels are low. Administration of exogenous IGF-1 reduces the extent of tissue damage and sensorimotor deficits in animal models of ischemic stroke, highlighting the critical role of IGF-1 as a regulator of neurovascular health. The beneficial effects of IGF-1 in the nervous system are often attributed to direct actions on neurons; however, glial cells and the cerebrovasculature are also modulated by IGF-1, and systemic reductions in circulating IGF-1 likely influence the viability and function of the entire neuro-glio-vascular unit. We recently observed that reduced IGF-1 led to impaired glutamate handling in astrocytes. Considering glutamate excitotoxicity is one of the main drivers of neurodegeneration following ischemic stroke, the age-related loss of IGF-1 may also compromise neural function indirectly by altering the function of supporting glia and vasculature. In this study, we assess and compare the effects of IGF-1 signaling on glutamate-induced toxicity and reactive oxygen species (ROS)-produced oxidative stress in primary neuron, astrocyte, and brain microvascular endothelial cell cultures. Our findings verify that neurons are highly susceptible to excitotoxicity, in comparison to astrocytes or endothelial cells, and that a prolonged reduction in IGFR activation increases the extent of toxicity. Moreover, prolonged IGFR inhibition increased the susceptibility of astrocytes to glutamate-induced toxicity and lessened their ability to protect neurons from excitotoxicity. Thus, IGF-1 promotes neuronal survival by acting directly on neurons and indirectly on astrocytes. Despite increased resistance to excitotoxic death, both astrocytes and cerebrovascular endothelial cells exhibit acute increases in glutamate-induced ROS production and mitochondrial dysfunction when IGFR is inhibited at the time of glutamate stimulation. Together these data highlight that each cell type within the neuro-glio-vascular unit differentially responds to stress when IGF-1 signaling was impaired. Therefore, the reductions in circulating IGF-1 observed in advanced age are likely detrimental to the health and function of the entire neuro-glio-vascular unit., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hayes, Ashmore, Vijayasankar, Marshall and Ashpole.)

Ischemic strokes are highly prevalent in the elderly population and are a leading cause of mortality and morbidity worldwide. The risk of ischemic stroke increases in advanced age, corresponding with a noted decrease in circulating insulin growth factor-1 (IGF-1). IGF-1 is a known neuroprotectant involved in embryonic development, neurogenesis, neurotransmission, cognition, and lifespan. Clinically, several studies have shown that reduced levels of IGF-1 correlate with increased mortality rate, poorer functional outcomes, and increased morbidities following an ischemic stroke. In animal models of ischemia, administering exogenous IGF-1 using various routes of administration (intranasal, intravenous, subcutaneous, or topical) at various time points prior to and following insult attenuates neurological damage and accompanying behavioral changes caused by ischemia. However, there are some contrasting findings in select clinical and preclinical studies. This review discusses the role of IGF-1 as a determinant factor of ischemic stroke outcomes, both within the clinical settings and preclinical animal models. Furthermore, the review provides insight on the role of IGF-1 in mechanisms and cellular processes that contribute to stroke damage.

Aging is associated with a significant deficiency in circulating insulin-like growth factor-1 (IGF-1), which has an important role in the pathogenesis of age-related vascular cognitive impairment (VCI). Impairment of moment-to-moment adjustment of regional cerebral blood flow via neurovascular coupling (NVC) importantly contributes to VCI. Previous studies established a causal link between circulating IGF-1 deficiency and neurovascular dysfunction. Release of vasodilator mediators from activated astrocytes plays a key role in NVC. To determine the impact of impaired IGF-1 signaling on astrocytic function, astrocyte-mediated NVC responses were studied in a novel mouse model of astrocyte-specific knockout of IGF1R (GFAP-Cre ERT2 /Igf1r f/f ) and accelerated neurovascular aging. We found that mice with disrupted astrocytic IGF1R signaling exhibit impaired NVC responses, decreased stimulated release of the vasodilator gliotransmitter epoxy-eicosatrienoic acids (EETs), and upregulation of soluble epoxy hydrolase (sEH), which metabolizes and inactivates EETs. Collectively, our findings provide additional evidence that IGF-1 promotes astrocyte health and maintains normal NVC, protecting cognitive health.

Hypogonadism is a common comorbidity associated with HIV-1 that is more prevalent among infected individuals over the age of 45. The underlying mechanisms are unknown, but both combined antiretroviral therapeutics and HIV-1 proteins, such as trans-activator of transcription protein (Tat), dysregulate steroid-synthetic mechanisms including lipid storage/synthesis and mitochondrial function. Thus, Tat expression may accelerate age-related comorbidities partly by impairing endocrine function. Few studies exist of Tat-mediated behavioral deficits in aged animals and effects of endocrine status have not been investigated. Accordingly, we tested whether conditional Tat expression in aged (~ 1.5 years old), female, Tat-transgenic [Tat(+)] mice increases anxiety-like behavior, impairs cognition, and augments mechanical allodynia, when compared to age-matched controls that do not express Tat protein [Tat(-)]. We further tested whether aged mice that maintained their endocrine status (pre-estropausal) were more resilient to Tat/age-related comorbidities than peri- or post-estropausal mice. Tat and endocrine aging status exerted separate and interacting effects that influenced anxiety-like and cognitive behaviors. Peri- and post-estropausal mice exhibited greater anxiety-like behavior in the elevated plus-maze and impaired learning in the radial arm water maze compared to pre-estropausal mice. Irrespective of estropause status, Tat(+) mice demonstrated impaired learning, reduced grip strength, and mechanical allodynia compared to Tat(-) mice. Tat exposure reduced circulating estradiol in post-estropausal mice and increased the estradiol-to-testosterone ratio in pre-estropausal mice. Changes in circulating estradiol, testosterone, and progesterone correlated with grip strength. Thus, endocrine status is an important factor in age-related anxiety, cognition, neuromuscular function, and allodynia that can be accelerated by HIV-1 Tat protein.

Flavones are valuable scaffolds in medicinal chemistry, especially as they display activity as antioxidants and neuroprotective agents. The need to incorporate a fluorine atom on flavones has driven much of the recent synthetic work in this area. We now report a route for the production of 3-fluorinated derivatives of 3',4',5'-trihydroxyflavone and 3',4',5'-trimethoxyflavone. Biological evaluation of these agents, along with their non-fluorinated counterparts, demonstrate that antioxidant activity may be enhanced whereas neuroprotective activity is conserved. Also, the 3-fluoro-3',4',5'-trihydroxyflavone can act as an NMR probe to detect structural changes during its action as a radical scavenger., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6- O -desulfated UFH, and 6- O -desulfated enoxaparin with 50% inhibitory concentrations (IC 50 s) of 5.99 μg/liter, 1.08 mg/liter, 1.77 μg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes. IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes., (Copyright © 2021 American Society for Microbiology.)

Current antiepileptic drugs (AEDs) are undesirable for many reasons including the inability to reduce seizures in certain types of epilepsy, such as Dravet syndrome (DS) where in one-third of patients does not respond to current AEDs, and severe adverse effects that are frequently experienced by patients. Epidiolex, a cannabidiol (CBD)-based drug, was recently approved for treatment of DS. While Epidiolex shows great promise in reducing seizures in patients with DS, it is used in conjunction with other AEDs and can cause liver toxicity. To investigate whether other cannabis-derived compounds could also reduce seizures, the antiepileptic effects of CBD, Δ9-tetrahydrocannabinol (THC), cannabidivarin (CBDV), cannabinol (CBN), and linalool (LN) were compared in both a chemically-induced (pentylenetetrazole, PTZ) and a DS (scn1Lab -/- ) seizure models. Zebrafish (Danio rerio) that were either wild-type (Tupfel longfin) or scn1Lab -/- (DS) were exposed to CBD, THC, CBDV, CBN, or LN for 24 h from 5 to 6 days postfertilization. Following exposure, total distance traveled was measured in a ViewPoint Zebrabox to determine if these compounds reduced seizure-like activity. Cannabidiol (0.6 and 1 μM) and THC (1 and 4 μM) significantly reduced PTZ-induced total distance moved. At the highest THC concentration, the significant reduction in PTZ-induced behavior was likely the result of sedation as opposed to antiseizure activity. In the DS model, CBD (0.6 μM), THC (1 μM), CBN (0.6 and 1 μM), and LN (4 μM) significantly reduced total distance traveled. Cannabinol was the most effective at reducing total distance relative to controls. In addition to CBD, other cannabis-derived compounds showed promise in reducing seizure-like activity in zebrafish. Specifically, four of the five compounds were effective in the DS model, whereas in the PTZ model, only CBD and THC were, suggesting a divergence in the mode of action among the cannabis constituents., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic or prophylactic. Like other betacoronaviruses, attachment and entry of SARS-CoV-2 is mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in anti-viral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6- O -desulfated UFH and 6- O -desulfated enoxaparin with an IC 50 of 5.99 μg/L, 1.08 mg/L, 1.77 μg/L, and 5.86 mg/L respectively. The low serum bioavailability of intranasally administered UFH, along with data suggesting that the nasal epithelium is a portal for initial infection and transmission, suggest that intranasal administration of UFH may be an effective and safe prophylactic treatment.

Use of cannabis and cannabinoid-containing substances is increasing among geriatric patients, despite relatively sparse preclinical evidence in aged models. To better understand the effects of exogenous cannabinoids on aging male and female rodents, we compared the age- and dose-dependent physiological and behavioral effects of the synthetic cannabinoid CP55940 in young-adult and aged C57BL/6 mice. Locomotion, body temperature, thermal nociception, and fecal output were measured following CP55940 administration. Our findings indicate that CP55940 is more potent and efficacious in older mice, evidenced by exaggerated antinociception and locomotor inhibition when compared to younger adult mice. In addition, we report that low doses of CP55940 paradoxically stimulate locomotion in young-adult (4 m) mice; however, this hormesis-like response is not as evident in aged animals (21-24 m). These bidirectional effects appear to be mediated via the endocannabinoid CB1 and CB2 receptors., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Increased availability of cannabis and cannabinoid-containing products necessitates the need for an understanding of how these substances influence aging. In this study, zebrafish (Danio rerio) were exposed to different concentrations of THC (0.08, 0.4, 2 μM) during embryonic-larval development and the effects on aging were measured 30 months later and in the offspring of the exposed fish (F1 generation). Exposure to 0.08 μM THC resulted in increased male survival at 30 months of age. As the concentration of THC increased, this protective effect was lost. Treatment with the lowest concentration of THC also significantly increased egg production, while higher concentrations resulted in impaired fecundity. Treatment with the lowest dose of THC significantly reduced wet weight, the incidence of kyphosis, and the expression of several senescence and inflammatory markers (p16 ink4ab , tnfα, il-1β, il-6, pparα and pparγ) in the liver, but not at higher doses indicating a biphasic or hormetic effect. Exposure to THC did not affect the age-related reductions in locomotor behavior. Within the F1 generation, many of these changes were not observed. However, the reduction in fecundity due to THC exposure was worse in the F1 generation because offspring whose parents received high dose of THC were completely unable to reproduce. Together, our results demonstrate that a developmental exposure to THC can cause significant effects on longevity and healthspan of zebrafish in a biphasic manner.

Consumption of cannabinoid-containing products is on the rise, even during pregnancy. Unfortunately, the long-term, age-related consequences of developmental cannabidiol (CBD) exposure remain largely unknown. This is a critical gap given the established Developmental Origins of Health and Disease (DOHaD) paradigm which emphasizes that stressors, like drug exposure, early in life can instigate molecular and cellular changes that ultimately lead to adverse outcomes later in life. Thus, we exposed zebrafish (Danio rerio) to varying concentrations of CBD (0.02, 0.1, 0.5 μM) during larval development and assessed aging in both the F0 (exposed generation) and their F1 offspring 30 months later. F0 exposure to CBD significantly increased survival (~ 20%) and reduced size (wet weight and length) of female fish. While survival was increased, the age-related loss of locomotor function was unaffected and the effects on fecundity varied by sex and dose. Treatment with 0.5 μM CBD significantly reduced sperm concentration in males, but 0.1 μM increased egg production in females. Similar to other model systems, control aged zebrafish exhibited increased kyphosis as well as increased expression markers of senescence, and inflammation (p16 ink4ab , tnfα, il1b, il6, and pparγ) in the liver. Exposure to CBD significantly reduced the expression of several of these genes in a dose-dependent manner relative to the age-matched controls. The effects of CBD on size, gene expression, and reproduction were not reproduced in the F1 generation, suggesting the influence on aging was not cross-generational. Together, our results demonstrate that developmental exposure to CBD causes significant effects on the health and longevity of zebrafish.

Insulin-like Growth Factor-1 (IGF-1) has been studied extensively for its ability to promote neuronal growth and excitability. Declining levels of IGF-1 have been correlated with impaired learning and memory as well as an increased risk of neurodegenerative diseases. While neuronal regulation by IGF-1 is well understood, the role of IGF-1 in influencing astrocyte function requires further exploration. Astrocytes regulate many aspects of the brain microenvironment, including controlling glutamate-glutamine cycling, which ultimately supports neuronal metabolism, neurotransmission, and protection from over stimulation. In this study, we examined whether IGF-1 acts through its cognate receptor, IGFR, to alter astrocytic glutamate handling. We utilized both small molecule IGFR inhibitors and Cre-driven genetic approaches to reduce IGFR in vivo and in cultured rodent astrocytes. When IGFR was knocked out of primary astrocytes derived from igfr f/f mice using AAV5-CMV-Cre, significant reductions in glutamate uptake were observed. Similarly, inhibition of IGFR with picropodophyllotoxin for 2 h, as well as 24 h, reduced glutamate uptake in vitro. Mechanistically, short-term inhibition of IGFR resulted in a significant decrease in glutamate transporter availability on the cell surface, as assessed by biotinylation. Long-term inhibition of IGFR led to significant reductions in mRNA expression of glutamate transport machinery, as assessed with qPCR. Reduced glutamate transporter mRNA was also observed in the brains of astrocyte-specific IGFR-deficient mice, three to four months after knock-out was induced with tamoxifen. Interestingly, long-term IGF-1 inhibition also resulted in an increase in adenosine triphosphate-stimulated glutamate release, though no change in adenosine triphosphate-stimulated calcium flux was observed nor were any changes in purinergic receptor protein expression. Together, these data suggest that reduced IGF-1 signaling will favor an accumulation of extrasynaptic glutamate, which may contribute to neurodegeneration in disease states where IGF-1 levels are low. Cover Image for this issue: doi: 10.1111/jnc.14534., (© 2019 International Society for Neurochemistry.)

Objective: In this study, we evaluated whether exercise prior to memory encoding or during memory consolidation can influence episodic memory function after being exposed to a stressful environment. Methods: We conducted 3 between-group randomized controlled experiments among young adults. We assessed episodic memory (via logic memory task) at the beginning of the experiment and approximately 45 minutes later. Across the 3 experiments, we varied the temporal period (eg, before memory encoding or during consolidation) of the acute bout of exercise (15-minute moderate-intensity exercise) and psychological stress induction. Results: Across all 3 experiments there was a statistically significant main effect for time for memory function, but there were no time x group interaction effects. Conclusion: Memory declined across the 2 assessment periods, but for all 3 experiments, exercise was not associated with memory function after being exposed to a stressful stimulus.

The search for new drugs remains an important focus for the safe and effective treatment of cardiovascular diseases. Previous evidence has shown that choline analogs can offer therapeutic benefit for cardiovascular complications. The current study investigates the effects of 2-(4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethan-1-ol (LQFM032) on cardiovascular function and cholinergic-nitric oxide signaling. Synthesized LQFM032 (0.3, 0.6, or 1.2 mg/kg) was administered by intravenous and intracerebroventricular routes to evaluate the potential alteration of mean arterial pressure, heart rate, and renal sympathetic nerve activity of normotensive and hypertensive rats. Vascular function was further evaluated in isolated vessels, while pharmacological antagonists and computational studies of nitric oxide synthase and muscarinic receptors were performed to assess possible mechanisms of LQFM032 activity. The intravenous and intracerebroventricular administration of LQFM032 elicited a temporal reduction in mean arterial pressure, heart rate, and renal sympathetic nerve activity of rats. The cumulative addition of LQFM032 to isolated endothelium-intact aortic rings reduced vascular tension and elicited a concentration-dependent relaxation. Intravenous pretreatment with L-NAME (nitric oxide synthase inhibitor), atropine (nonselective muscarinic receptor antagonist), pirenzepine, and 4-DAMP (muscarinic M1 and M3 subtype receptor antagonist, respectively) attenuated the cardiovascular effects of LQFM032. These changes may be due to a direct regulation of muscarinic signaling as docking data shows an interaction of choline analog with M1 and M3 but not nitric oxide synthase. Together, these findings demonstrate sympathoinhibitory, hypotensive, and antihypertensive effects of LQFM032 and suggest the involvement of muscarinic receptors.

Numerous aspects of mammalian physiology exhibit cyclic daily patterns known as circadian rhythms. However, studies in aged humans and animals indicate that these physiological rhythms are not consistent throughout the life span. The simultaneous development of disrupted circadian rhythms and age-related impairments suggests a shared mechanism, which may be amenable to therapeutic intervention. Recently, the endocannabinoid system has emerged as a complex signaling network, which regulates numerous aspects of circadian physiology relevant to the neurobiology of aging. Agonists of cannabinoid receptor-1 (CB1) have consistently been shown to decrease neuronal activity, core body temperature, locomotion, and cognitive function. Paradoxically, several lines of evidence now suggest that very low doses of cannabinoids are beneficial in advanced age. One potential explanation for this phenomenon is that these drugs exhibit hormesis-a biphasic dose-response wherein low doses produce the opposite effects of higher doses. Therefore, it is important to determine the dose-, age-, and time-dependent effects of these substances on the regulation of circadian rhythms and other processes dysregulated in aging. This review highlights 3 fields-biological aging, circadian rhythms, and endocannabinoid signaling-to critically assess the therapeutic potential of endocannabinoid modulation in aged individuals. If the hormetic properties of exogenous cannabinoids are confirmed, we conclude that precise administration of these compounds may bidirectionally entrain central and peripheral circadian clocks and benefit multiple aspects of aging physiology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although 4- O -Methylhonokiol (MH) effects on neuronal and immune cells have been established, it is still unclear whether MH can cause a change in the structure and function of the cardiovascular system. The overarching goal of this study was to evaluate the effects of MH, isolated from Magnolia grandiflora , on the development of the heart and vasculature in a Japanese medaka model in vivo to predict human health risks. We analyzed the toxicity of MH in different life-stages of medaka embryos. MH uptake into medaka embryos was quantified. The LC 50 of two different exposure windows (stages 9⁻36 (0⁻6 days post fertilization (dpf)) and 25⁻36 (2⁻6 dpf)) were 5.3 ± 0.1 μM and 9.9 ± 0.2 μM. Survival, deformities, days to hatch, and larval locomotor response were quantified. Wnt 1 was overexpressed in MH-treated embryos indicating deregulation of the Wnt signaling pathway, which was associated with spinal and cardiac ventricle deformities. Overexpression of major proinflammatory mediators and biomarkers of the heart were detected. Our results indicated that the differential sensitivity of MH in the embryos was developmental stage-specific. Furthermore, this study demonstrated that certain molecules can serve as promising markers at the transcriptional and phenotypical levels, responding to absorption of MH in the developing embryo.

Cerebral microcirculation is critical for the preservation of brain health, and vascular impairment is associated with age-related neurodegenerative diseases. Because the retina is a component of the central nervous system, cellular changes that occur in the aging retina are likely relevant to the aging brain, and the retina provides the advantage that the entire vascular bed is visible, en face. In this study, we tested the hypothesis that normal, healthy aging alters the contractile vascular smooth muscle cell (VSMC) coverage of retinal arterioles. We found that aging results in a significant reduction of contractile VSMCs in focal patches along arterioles. Focal loss of contractile VSMCs occurs at a younger age in mice deficient in the senescence-associated protein, caveolin-1. Age-related contractile VSMC loss is not exacerbated by genetic depletion of insulin-like growth factor-1. The patchy loss of contractile VSMCs provides a cellular explanation for previous clinical studies showing focal microirregularities in retinal arteriolar responsiveness in healthy aged human subjects and is likely to contribute to age-related retinal vascular complications., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Reduced circulating levels of IGF-1 have been proposed as a conserved anti-aging mechanism that contributes to increased lifespan in diverse experimental models. However, IGF-1 has also been shown to be essential for normal development and the maintenance of tissue function late into the lifespan. These disparate findings suggest that IGF-1 may be a pleiotropic modulator of health and aging, as reductions in IGF-1 may be beneficial for one aspect of aging, but detrimental for another. We postulated that the effects of IGF-1 on tissue health and function in advanced age are dependent on the tissue, the sex of the animal, and the age at which IGF-1 is manipulated. In this study, we examined how alterations in IGF-1 levels at multiple stages of development and aging influence overall lifespan, healthspan, and pathology. Specifically, we investigated the effects of perinatal, post-pubertal, and late-adult onset IGF-1 deficiency using genetic and viral approaches in both male and female igf f/f C57Bl/6 mice. Our results support the concept that IGF-1 levels early during lifespan establish the conditions necessary for subsequent healthspan and pathological changes that contribute to aging. Nevertheless, these changes are specific for each sex and tissue. Importantly, late-life IGF-1 deficiency (a time point relevant for human studies) reduces cancer risk but does not increase lifespan. Overall, our results indicate that the levels of IGF-1 during development influence late-life pathology, suggesting that IGF-1 is a developmental driver of healthspan, pathology, and lifespan.

Protein tyrosine phosphatase MEG2 (PTP-MEG2) is a unique nonreceptor tyrosine phosphatase associated with transport vesicles, where it facilitates membrane trafficking by dephosphorylation of the N-ethylmaleimide-sensitive fusion factor. In this study, we identify the neurotrophin receptor TrkA as a novel cargo whose transport to the cell surface requires PTP-MEG2 activity. In addition, TrkA is also a novel substrate of PTP-MEG2, which dephosphorylates both Tyr-490 and Tyr-674/Tyr-675 of TrkA. As a result, overexpression of PTP-MEG2 down-regulates NGF/TrkA signaling and blocks neurite outgrowth and differentiation in PC12 cells and cortical neurons., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Epidemiological findings support the concept of Developmental Origins of Health and Disease, suggesting that early-life hormonal influences during a sensitive period of development have a fundamental impact on vascular health later in life. The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in circulating IGF-1 levels during adolescence. The present study was designed to characterize the effect of developmental IGF-1 deficiency on the vascular aging phenotype. To achieve that goal, early-onset endocrine IGF-1 deficiency was induced in mice by knockdown of IGF-1 in the liver using Cre-lox technology (Igf1 f/f mice crossed with mice expressing albumin-driven Cre recombinase). This model exhibits low-circulating IGF-1 levels during the peripubertal phase of development, which is critical for the biology of aging. Due to the emergence of miRNAs as important regulators of the vascular aging phenotype, the effect of early-life IGF-1 deficiency on miRNA expression profile in the aorta was examined in animals at 27 months of age. We found that developmental IGF-1 deficiency elicits persisting late-life changes in miRNA expression in the vasculature, which significantly differed from those in mice with adult-onset IGF-1 deficiency (TBG-Cre-AAV8-mediated knockdown of IGF-1 at 5 month of age in Igf1 f/f mice). Using a novel computational approach, we identified miRNA target genes that are co-expressed with IGF-1 and associate with aging and vascular pathophysiology. We found that among the predicted targets, the expression of multiple extracellular matrix-related genes, including collagen-encoding genes, were downregulated in mice with developmental IGF-1 deficiency. Collectively, IGF-1 deficiency during a critical period during early in life results in persistent changes in post-transcriptional miRNA-mediated control of genes critical targets for vascular health, which likely contribute to the deleterious late-life cardiovascular effects known to occur with developmental IGF-1 deficiency., Competing Interests: Compliance with ethical standards All procedures were approved by and followed the guidelines of the Institutional Animal Care and Use Committee of OUHSC in accordance with the ARRIVE guidelines. Conflict of interest The authors declare that they have no conflict of interest.

Strong epidemiological and experimental evidence indicate that both age and hypertension lead to significant functional and structural impairment of the cerebral microcirculation, predisposing to the development of vascular cognitive impairment (VCI) and Alzheimer's disease. Preclinical studies establish a causal link between cognitive decline and microvascular rarefaction in the hippocampus, an area of brain important for learning and memory. Age-related decline in circulating IGF-1 levels results in functional impairment of the cerebral microvessels; however, the mechanistic role of IGF-1 deficiency in impaired hippocampal microvascularization remains elusive. The present study was designed to characterize the additive/synergistic effects of IGF-1 deficiency and hypertension on microvascular density and expression of genes involved in angiogenesis and microvascular regression in the hippocampus. To achieve that goal, we induced hypertension in control and IGF-1 deficient mice (Igf1 f/f  + TBG-Cre-AAV8) by chronic infusion of angiotensin II. We found that circulating IGF-1 deficiency is associated with decreased microvascular density and exacerbates hypertension-induced microvascular rarefaction both in the hippocampus and the neocortex. The anti-angiogenic hippocampal gene expression signature observed in hypertensive IGF-1 deficient mice in the present study provides important clues for subsequent studies to elucidate mechanisms by which hypertension may contribute to the pathogenesis and clinical manifestation of VCI. In conclusion, adult-onset, isolated endocrine IGF-1 deficiency exerts deleterious effects on the cerebral microcirculation, leading to a significant decline in cortical and hippocampal capillarity and exacerbating hypertension-induced cerebromicrovascular rarefaction. The morphological impairment of the cerebral microvasculature induced by IGF-1 deficiency and hypertension reported here, in combination with neurovascular uncoupling, increased blood-brain barrier disruption and neuroinflammation reported in previous studies likely contribute to the pathogenesis of vascular cognitive impairment in elderly hypertensive humans., Competing Interests: The authors declare that they have no competing interests.

Advanced aging is associated with the loss of structural and biomechanical properties in bones, which increases the risk for bone fracture. Aging is also associated with reductions in circulating levels of the anabolic signaling hormone, insulin-like growth factor (IGF)-1. While the role of IGF-1 in bone development has been well characterized, the impact of the age-related loss of IGF-1 on bone aging remains controversial. Here, we describe the effects of reducing IGF-1 at multiple time points in the mouse life span--early in postnatal development, early adulthood, or late adulthood on tibia bone aging in both male and female igf (f/f) mice. Bone structure was analyzed at 27 months of age using microCT. We find that age-related reductions in cortical bone fraction, cortical thickness, and tissue mineral density were more pronounced when IGF-1 was reduced early in life and not in late adulthood. Three-point bone bending assays revealed that IGF-1 deficiency early in life resulted in reduced maximum force, maximum bending moment, and bone stiffness in aged males and females. The effects of IGF-1 on bone aging are microenvironment specific, as early-life loss of IGF-1 resulted in decreased cortical bone structure and strength along the diaphysis while significantly increasing trabecular bone fraction and trabecular number at the proximal metaphysis. The increases in trabecular bone were limited to males, as early-life loss of IGF-1 did not alter bone fraction or number in females. Together, our data suggest that the age-related loss of IGF-1 influences tibia bone aging in a sex-specific, microenvironment-specific, and time-dependent manner., Competing Interests: Compliance with ethical standards All procedures were approved by and followed the guidelines of the Institutional Animal Care and Use Committee and veterinarians at University of Oklahoma Health Sciences Center (OUHSC).

Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin-like growth factor (IGF-1). Studies have suggested that the reduction in IGF-1 compromises healthspan, whereas others report that loss of IGF-1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF-1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF-1 on vertebral bone aging in male and female Igf(f/f) mice. IGF-1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin-cyclic recombinase [Cre] mice with Igf(f/f) mice); and in early adulthood and in late adulthood using hepatic-specific viral vectors (AAV8-TBG-Cre). Vertebrae bone structure was analyzed at 27 months of age using micro-computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age-related reductions in vertebral bone structure. In male mice, reduction of circulating IGF-1 induced at any age did not diminish vertebral bone loss. Interestingly, early-life loss of IGF-1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early-life IGF-1-deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor-activator of NF-κB-ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF-1, there was a 2.2-fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF-1. Together, these data suggest the age-related loss of vertebral bone density in females can be reduced by modifying circulating IGF-1 levels early in life., (© 2015 American Society for Bone and Mineral Research.)

Aging is associated with marked deficiency in circulating IGF-1, which has been shown to contribute to age-related cognitive decline. Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of age-related cognitive impairment. To establish the link between IGF-1 deficiency and cerebromicrovascular impairment, neurovascular coupling mechanisms were studied in a novel mouse model of IGF-1 deficiency (Igf1(f/f) -TBG-Cre-AAV8) and accelerated vascular aging. We found that IGF-1-deficient mice exhibit neurovascular uncoupling and show a deficit in hippocampal-dependent spatial memory test, mimicking the aging phenotype. IGF-1 deficiency significantly impaired cerebromicrovascular endothelial function decreasing NO mediation of neurovascular coupling. IGF-1 deficiency also impaired glutamate-mediated CBF responses, likely due to dysregulation of astrocytic expression of metabotropic glutamate receptors and impairing mediation of CBF responses by eicosanoid gliotransmitters. Collectively, we demonstrate that IGF-1 deficiency promotes cerebromicrovascular dysfunction and neurovascular uncoupling mimicking the aging phenotype, which are likely to contribute to cognitive impairment., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 μM) and ZL006 (EC50: 12.88 μM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund's adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Growth hormone (GH) and insulin-like growth factor (IGF)-1 regulate the development and function of cells throughout the body. Several clinical diseases that result in a decline in physical and mental functions are marked by mutations that disrupt GH or IGF-1 signaling. During the lifespan there is a robust decrease in both GH and IGF-1. Because GH and IGF-1 are master regulators of cellular function, impaired GH and IGF-1 signaling in aging/disease states leads to significant alterations in tissue structure and function, especially within the brain. This review is intended to highlight the effects of the GH and IGF-1 on neuronal structure, function, and plasticity. Furthermore, we address several potential mechanisms through which the age-related reductions in GH and IGF-1 affect cognition. Together, the studies reviewed here highlight the importance of maintaining GH and IGF-1 signaling in order to sustain proper brain function throughout the lifespan., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Stability of myogenic tone in middle cerebral arteries (MCA) is essential for adequate control over penetration of pressure waves into the distal portion of the cerebral microcirculation. Because the increased pulse pressure observed in advanced aging is associated with cerebromicrovascular injury, the effect of aging on myogenic response of mouse MCAs was determined. Aging did not affect the myogenic constriction in response to static increases in pressure, whereas it significantly impaired pulsatile pressure-induced myogenic tone. Impaired myogenic adaptation of MCAs to pulsatile pressure may allow high pressure to penetrate the distal portion of the cerebral microcirculation, contributing to microvascular damage.

Aging is a major risk factor for cerebrovascular disease. Growth hormone (GH) and its anabolic mediator, insulin-like growth factor (IGF)-1, decrease with advancing age and this decline has been shown to promote vascular dysfunction. In addition, lower GH/IGF-1 levels are associated with higher stroke mortality in humans. These results suggest that decreased GH/IGF-1 level is an important factor in increased risk of cerebrovascular diseases. This study was designed to assess whether GH/IGF-1-deficiency influences the outcome of cerebral ischemia. We found that endothelin-1-induced middle cerebral artery occlusion resulted in a modest but nonsignificant decrease in cerebral infarct size in GH/IGF-1 deficient dw/dw rats compared with control heterozygous littermates and dw/dw rats with early-life GH treatment. Expression of endothelin receptors and endothelin-1-induced constriction of the middle cerebral arteries were similar in the three experimental groups. Interestingly, dw/dw rats exhibited reduced brain edema and less astrocytic infiltration compared with their heterozygous littermates and this effect was reversed by GH-treatment. Because reactive astrocytes are critical for the regulation of poststroke inflammatory processes, maintenance of the blood-brain barrier and neural repair, further studies are warranted to determine the long-term functional consequences of decreased astrocytic activation in GH/IGF-1 deficient animals after cerebral ischemia., (Published by Oxford University Press on behalf of the Gerontological Society of America 2014.)

Whole brain radiation therapy (WBRT) induces profound cerebral microvascular rarefaction throughout the hippocampus. Despite the vascular loss and localized cerebral hypoxia, angiogenesis fails to occur, which subsequently induces long-term deficits in learning and memory. The mechanisms underlying the absence of vessel recovery after WBRT are unknown. We tested the hypotheses that vascular recovery fails to occur under control conditions as a result of loss of angiogenic drive in the circulation, chronic tissue inflammation, and/or impaired endothelial cell production/recruitment. We also tested whether systemic hypoxia, which is known to promote vascular recovery, reverses these chronic changes in inflammation and endothelial cell production/recruitment. Ten-week-old C57BL/6 mice were subjected to a clinical series of fractionated WBRT: 4.5-Gy fractions 2 times/wk for 4 wk. Plasma from radiated mice increased in vitro endothelial cell proliferation and adhesion compared with plasma from control mice, indicating that WBRT did not suppress the proangiogenic drive. Analysis of cytokine levels within the hippocampus revealed that IL-10 and IL-12(p40) were significantly increased 1 mo after WBRT; however, systemic hypoxia did not reduce these inflammatory markers. Enumeration of endothelial progenitor cells (EPCs) in the bone marrow and circulation indicated that WBRT reduced EPC production, which was restored with systemic hypoxia. Furthermore, using a bone marrow transplantation model, we determined that bone marrow-derived endothelial-like cells home to the hippocampus after systemic hypoxia. Thus, the loss of production and homing of EPCs have an important role in the prolonged vascular rarefaction after WBRT., (Copyright © 2014 the American Physiological Society.)

Moment-to-moment adjustment of cerebral blood flow (CBF) to neuronal activity via neurovascular coupling is essential for the maintenance of normal neuronal function. Increased oxidative stress that occurs with aging was shown to impair neurovascular coupling, which likely contributes to a significant age-related decline in higher cortical function, increasing the risk for vascular cognitive impairment. Resveratrol is a polyphenolic compound that exerts significant antiaging protective effects in large vessels, but its effects on the cerebromicrovasculature remain poorly defined. The present study was undertaken to investigate the capacity of resveratrol to improve neurovascular coupling in aging. In aged (24-mo-old) C57BL/6 mice N(ω)-nitro-l-arginine methyl ester-sensitive, nitric oxide-mediated CBF responses to whisker stimulation and to the endothelium-dependent dilator acethylcholine (ACh) were impaired compared with those in young (3-mo-old) mice. Treatment of aged mice with resveratrol rescued neurovascular coupling and ACh-induced responses, which was associated with downregulation of cortical expression of NADPH oxidase and decreased levels of biomarkers of oxidative/nitrative stress (3-nitrotyrosine, 8-isoprostanes). Resveratrol also attenuated age-related increases in reactive oxygen species (ROS) production in cultured cerebromicrovascular endothelial cells (DCF fluorescence, flow cytometry). In conclusion, treatment with resveratrol rescues cortical neurovascular coupling responses to increased neuronal activity in aged mice, likely by restoring cerebromicrovascular endothelial function via downregulation of NADPH oxidase-derived ROS production. Beneficial cerebromicrovascular effects of resveratrol may contribute to its protective effects on cognitive function in aging.

The extent of calcium/calmodulin-dependent protein kinase II (CaMKII) inactivation in the brain after ischemia correlates with the extent of damage. We have previously shown that a loss of CaMKII activity in neurons is detrimental to neuronal viability by inducing excitotoxic glutamate release. In the current study we extend these findings to show that the ability of astrocytes to buffer extracellular glutamate is reduced when CaMKII is inhibited. Furthermore, CaMKII inhibition in astrocytes is associated with the rapid onset of intracellular calcium oscillations. Surprisingly, this rapid calcium influx is blocked by the N-type calcium channel antagonist, ω-conotoxin. Although the function of N-type calcium channels within astrocytes is controversial, these voltage-gated calcium channels have been linked to calcium-dependent vesicular gliotransmitter release. When extracellular glutamate and ATP levels are measured after CaMKII inhibition within our enriched astrocyte cultures, no alterations in glutamate levels are observed, whereas ATP levels in the extracellular environment significantly increase. Extracellular ATP accumulation associated with CaMKII inhibition contributes both to calcium oscillations within astrocytes and ultimately cortical neuron toxicity. Thus, a loss of CaMKII signaling within astrocytes dysregulates glutamate uptake and supports ATP release, two processes that would compromise neuronal survival after ischemic/excitotoxic insults.

The deletion of phenylalanine 1486 (F1486del) in the human cardiac voltage-gated sodium channel (hNav1.5) is associated with fatal long QT (LQT) syndrome. In this study we determined how F1486del impairs the functional properties of hNav1.5 and alters action potential firing in heterologous expression systems (human embryonic kidney (HEK) 293 cells) and their native cardiomyocyte background. Cells expressing hNav1.5-F1486del exhibited a loss-of-function alteration, reflected by an 80% reduction of peak current density, and several gain-of-function alterations, including reduced channel inactivation, enlarged window current, substantial augmentation of persistent late sodium current and an increase in ramp current. We also observed substantial action potential duration (APD) prolongation and prominent early afterdepolarizations (EADs) in neonatal cardiomyocytes expressing the F1486del channels, as well as in computer simulations of myocyte activity. In addition, lidocaine sensitivity was dramatically reduced, which probably contributed to the poor therapeutic outcome observed in the patient carrying the hNav1.5-F1486del mutation. Therefore, despite the significant reduction in peak current density, the F1486del mutation also leads to substantial gain-of-function alterations that are sufficient to cause APD prolongation and EADs, the predominant characteristic of LQTs. These data demonstrate that hNav1.5 mutations can have complex functional consequences and highlight the importance of identifying the specific molecular defect when evaluating potential treatments for individuals with prolonged QT intervals.

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I(Na) gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(C)-CaMKII in HEK293 cells, and I(Na) was recorded. As observed in myocytes, CaMKII shifted WT I(Na) availability to a more negative membrane potential and enhanced accumulation of I(Na) into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted I(Na) availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Aberrant glutamate and calcium signalings are neurotoxic to specific neuronal populations. Calcium/calmodulin-dependent kinase II (CaMKII), a multifunctional serine/threonine protein kinase in neurons, is believed to regulate neurotransmission and synaptic plasticity in response to calcium signaling produced by neuronal activity. Importantly, several CaMKII substrates control neuronal structure, excitability, and plasticity. Here, we demonstrate that CaMKII inhibition for >4 h using small molecule and peptide inhibitors induces apoptosis in cultured cortical neurons. The neuronal death produced by prolonged CaMKII inhibition is associated with an increase in TUNEL staining and caspase-3 cleavage and is blocked with the translation inhibitor cycloheximide. Thus, this neurotoxicity is consistent with apoptotic mechanisms, a conclusion that is further supported by dysregulated calcium signaling with CaMKII inhibition. CaMKII inhibitory peptides also enhance the number of action potentials generated by a ramp depolarization, suggesting increased neuronal excitability with a loss of CaMKII activity. Extracellular glutamate concentrations are augmented with prolonged inhibition of CaMKII. Enzymatic buffering of extracellular glutamate and antagonism of the NMDA subtype of glutamate receptors prevent the calcium dysregulation and neurotoxicity associated with prolonged CaMKII inhibition. However, in the absence of CaMKII inhibition, elevated glutamate levels do not induce neurotoxicity, suggesting that a combination of CaMKII inhibition and elevated extracellular glutamate levels results in neuronal death. In sum, the loss of CaMKII observed with multiple pathological states in the central nervous system, including epilepsy, brain trauma, and ischemia, likely exacerbates programmed cell death by sensitizing vulnerable neuronal populations to excitotoxic glutamate signaling and inducing an excitotoxic insult itself.

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.

Ischemic insults on neurons trigger excessive, pathological glutamate release that causes Ca²⁺ overload resulting in neuronal cell death (excitotoxicity). The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major mediator of physiological excitatory glutamate signals underlying neuronal plasticity and learning. Glutamate stimuli trigger autophosphorylation of CaMKII at T286, a process that makes the kinase "autonomous" (partially active independent from Ca²⁺ stimulation) and that is required for forms of synaptic plasticity. Recent studies suggested autonomous CaMKII activity also as potential drug target for post-insult neuroprotection, both after glutamate insults in neuronal cultures and after focal cerebral ischemia in vivo. However, CaMKII and other members of the CaM kinase family have been implicated in regulation of both neuronal death and survival. Here, we discuss past findings and possible mechanisms of CaM kinase functions in excitotoxicity and cerebral ischemia, with a focus on CaMKII and its regulation.

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2-mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

Aberrant calcium signaling is a common feature of ischemia and multiple neurodegenerative diseases. While activation of calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is a key event in calcium signaling, its role in excitotoxicity is controversial. Our findings demonstrate neuroprotection in neuronal cultures treated with the small molecule (KN-93) and peptide (tat-AIP and tat-CN21) inhibitors of CaMKII immediately prior to excitotoxic glutamate/glycine insult. Unlike KN-93 which blocks CaMKII activation, but not constitutively active forms of CaMKII, tat-CN21 and tat-AIP significantly reduced excitotoxicity in cultured neurons when applied post-insult. We observed that the neuroprotective effects of tat-CN21 are greatest when applied before the toxic glutamate challenge and diminish with time, with the neuroprotection associated with CaMKII inhibition diminishing back to control 3h post glutamate insult. Mechanistically, tat-CN21 inhibition of CaMKII resulted in an increase in CaMKII activity and the percentage of soluble αCaMKII observed in neuronal lysates 24h following glutamate stimulation. To address the impact of prolonged CaMKII inhibition prior to excitotoxic insult, neuronal cultures were treated with CaMKII inhibitors overnight and then subjected to a sub-maximal excitotoxic insult. In this model, CaMKII inhibition prior to insult exacerbated neuronal death, suggesting that a loss of CaMKII enhances neuronal vulnerability to glutamate. Although changes in αCaMKII or NR2B protein levels are not responsible for this enhanced glutamate vulnerability, this process is blocked by the protein translation inhibitor cycloheximide. In total, the neuroprotection afforded by CaMKII inhibition can be seen as neuroprotective immediately surrounding the excitotoxic insult, whereas sustained CaMKII inhibition produced by excitotoxicity leads to neuronal death by enhancing neuronal vulnerability to glutamate., (Copyright © 2010 Elsevier Inc. All rights reserved.)