Early-life prefrontal cortex inhibition and early-life stress lead to long-lasting behavioral, transcriptional, and physiological impairments.

Early-life stress has been linked to multiple neurodevelopmental and neuropsychiatric deficits. Our previous studies have linked maternal presence/absence from the nest in developing rat pups to changes in prefrontal cortex (PFC) activity. Furthermore, we have shown that these changes are modulated by serotonergic signaling. Here we test whether changes in PFC activity during early life affect the developing cortex leading to behavioral alterations in the adult. We show that inhibiting the PFC of mouse pups leads to cognitive deficits in the adult comparable to those seen following maternal separation. Moreover, we show that activating the PFC during maternal separation can prevent these behavioral deficits. To test how maternal separation affects the transcriptional profile of the PFC we performed single-nucleus RNA-sequencing. Maternal separation led to differential gene expression almost exclusively in inhibitory neurons. Among others, we found changes in GABAergic and serotonergic pathways in these interneurons. Interestingly, both maternal separation and early-life PFC inhibition led to changes in physiological responses in prefrontal activity to GABAergic and serotonergic antagonists that were similar to the responses of more immature brains. Prefrontal activation during maternal separation prevented these changes. These data point to a crucial role of PFC activity during early life in behavioral expression in adulthood.

Pharmacokinetics and efficacy of PT302, a sustained-release Exenatide formulation, in a murine model of mild traumatic brain injury.

Traumatic brain injury (TBI) is a neurodegenerative disorder for which no effective pharmacological treatment is available. Glucagon-like peptide 1 (GLP-1) analogues such as Exenatide have previously demonstrated neurotrophic and neuroprotective effects in cellular and animal models of TBI. However, chronic or repeated administration was needed for efficacy. In this study, the pharmacokinetics and efficacy of PT302, a clinically available sustained-release Exenatide formulation (SR-Exenatide) were evaluated in a concussive mild (m)TBI mouse model. A single subcutaneous (s.c.) injection of PT302 (0.6, 0.12, and 0.024 mg/kg) was administered and plasma Exenatide concentrations were time-dependently measured over 3 weeks. An initial rapid regulated release of Exenatide in plasma was followed by a secondary phase of sustained-release in a dose-dependent manner. Short- and longer-term (7 and 30 day) cognitive impairments (visual and spatial deficits) induced by weight drop mTBI were mitigated by a single post-injury treatment with Exenatide delivered by s.c. injection of PT302 in clinically translatable doses. Immunohistochemical evaluation of neuronal cell death and inflammatory markers, likewise, cross-validated the neurotrophic and neuroprotective effects of SR-Exenatide in this mouse mTBI model. Exenatide central nervous system concentrations were 1.5% to 2.0% of concomitant plasma levels under steady-state conditions. These data demonstrate a positive beneficial action of PT302 in mTBI. This convenient single, sustained-release dosing regimen also has application for other neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple system atrophy and multiple sclerosis where prior preclinical studies, likewise, have demonstrated positive Exenatide actions.

Blast traumatic brain injury-induced cognitive deficits are attenuated by preinjury or postinjury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4.

INTRODUCTION: Blast traumatic brain injury (B-TBI) affects military and civilian personnel. Presently, there are no approved drugs for blast brain injury. METHODS: Exendin-4 (Ex-4), administered subcutaneously, was evaluated as a pretreatment (48 hours) and postinjury treatment (2 hours) on neurodegeneration, behaviors, and gene expressions in a murine open field model of blast injury. RESULTS: B-TBI induced neurodegeneration, changes in cognition, and genes expressions linked to dementia disorders. Ex-4, administered preinjury or postinjury, ameliorated B-TBI-induced neurodegeneration at 72 hours, memory deficits from days 7-14, and attenuated genes regulated by blast at day 14 postinjury. DISCUSSION: The present data suggest shared pathologic processes between concussive and B-TBI, with end points amenable to beneficial therapeutic manipulation by Ex-4. B-TBI-induced dementia-related gene pathways and cognitive deficits in mice somewhat parallel epidemiologic studies of Barnes et al. who identified a greater risk in US military veterans who experienced diverse TBIs, for dementia in later life.

Incretin mimetics as pharmacologic tools to elucidate and as a new drug strategy to treat traumatic brain injury.

Traumatic brain injury (TBI), either as an isolated injury or in conjunction with other injuries, is an increasingly common event. An estimated 1.7 million injuries occur within the USA each year and 10 million people are affected annually worldwide. Indeed, nearly one third (30.5%) of all injury-related deaths in the USA are associated with TBI, which will soon outpace many common diseases as the major cause of death and disability. Associated with a high morbidity and mortality and no specific therapeutic treatment, TBI has become a pressing public health and medical problem. The highest incidence of TBI occurs in young adults (15-24 years age) and in the elderly (≥75 years of age). Older individuals are particularly vulnerable to these types of injury, often associated with falls, and have shown increased mortality and worse functional outcome after lower initial injury severity. In addition, a new and growing form of TBI, blast injury, associated with the detonation of improvised explosive devices in the war theaters of Iraq and Afghanistan, are inflicting a wave of unique casualties of immediate impact to both military personnel and civilians, for which long-term consequences remain unknown and may potentially be catastrophic. The neuropathology underpinning head injury is becoming increasingly better understood. Depending on severity, TBI induces immediate neuropathologic effects that, for the mildest form, may be transient; however, with increasing severity, these injuries cause cumulative neural damage and degeneration. Even with mild TBI, which represents the majority of cases, a broad spectrum of neurologic deficits, including cognitive impairments, can manifest that may significantly influence quality of life. Further, TBI can act as a conduit to longer term neurodegenerative disorders. Prior studies of glucagon-like peptide-1 (GLP-1) and long-acting GLP-1 receptor agonists have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of chronic neurodegenerative (Alzheimer's and Parkinson's diseases) and acute cerebrovascular (stroke) disorders. In view of the mechanisms underpinning these disorders as well as TBI, we review the literature and recent studies assessing GLP-1 receptor agonists as a potential treatment strategy for mild to moderate TBI.

Changes in mouse cognition and hippocampal gene expression observed in a mild physical- and blast-traumatic brain injury.

Warfare has long been associated with traumatic brain injury (TBI) in militarized zones. Common forms of TBI can be caused by a physical insult to the head-brain or by the effects of a high velocity blast shock wave generated by the detonation of an explosive device. While both forms of trauma are distinctly different regarding the mechanism of trauma induction, there are striking similarities in the cognitive and emotional status of survivors. Presently, proven effective therapeutics for the treatment of either form of TBI are unavailable. To be able to develop efficacious therapies, studies involving animal models of physical- and blast-TBI are required to identify possible novel or existing medicines that may be of value in the management of clinical events. We examined indices of cognition and anxiety-like behavior and the hippocampal gene transcriptome of mice subjected to both forms of TBI. We identified common behavioral deficits and gene expression regulations, in addition to unique injury-specific forms of gene regulation. Molecular pathways presented a pattern similar to that seen in gene expression. Interestingly, pathways connected to Alzheimer's disease displayed a markedly different form of regulation depending on the type of TBI. While these data highlight similarities in behavioral outcomes after trauma, the divergence in hippocampal transcriptome observed between models suggests that, at the molecular level, the TBIs are quite different. These models may provide tools to help define therapeutic approaches for the treatment of physical- and blast-TBIs. Based upon observations of increasing numbers of personnel displaying TBI related emotional and behavioral changes in militarized zones, the development of efficacious therapies will become a national if not a global priority.

Hippocampal differential expression underlying the neuroprotective effect of delta-9-tetrahydrocannabinol microdose on old mice.

Delta-9-tetrahydrocannabinol (THC) is the primary psychoactive compound of the cannabis plant and an exogenous ligand of the endocannabinoid system. In previous studies, we demonstrated that a single microdose of THC (0.002 mg/kg, 3-4 orders of magnitude lower than the standard dose for rodents) exerts distinct, long-term neuroprotection in model mice subjected to acute neurological insults. When administered to old, healthy mice, the THC microdose induced remarkable long-lasting (weeks) improvement in a wide range of cognitive functions, including significant morphological and biochemical brain alterations. To elucidate the mechanisms underlying these effects, we analyzed the gene expression of hippocampal samples from the model mice. Samples taken 5 days after THC treatment showed significant differential expression of genes associated with neurogenesis and brain development. In samples taken 5 weeks after treatment, the transcriptional signature was shifted to that of neuronal differentiation and survival. This study demonstrated the use of hippocampal transcriptome profiling in uncovering the molecular basis of the atypical, anti-aging effects of THC microdose treatment in old mice.

Reversal of age-related cognitive impairments in mice by an extremely low dose of tetrahydrocannabinol.

This study was designed to test our hypothesis that an ultra-low dose of delta-9 tetrahydrocannabinol (THC) reverses age-dependent cognitive impairments in old mice and to examine the possible biological mechanisms that underlie this behavioral effect. Old female mice aged 24 months that had been injected once with 0.002 mg/kg THC (3-4 orders of magnitudes lower than doses that induce the conventional cannabinoid effects in mice) performed significantly better than vehicle-treated old mice and performed similarly to naive young mice aged 2 months, in 6 different behavioral assays that measured various aspects of memory and learning. The beneficial effect of THC lasted for at least 7 weeks. The single injection of THC increased the level of Sirtuin1, an enzyme that has been previously shown to be involved in neuroprotection and neuroplasticity, in the hippocampus and in the frontal cortex of old mice, for at least 7 weeks. Magnetic resonance imaging demonstrated a larger volume and higher tissue density in various regions of the brain of THC-treated old mice. These findings suggest that extremely low doses of THC that are devoid of any psychotropic effect and do not induce desensitization may provide a safe and effective treatment for cognitive decline in aging humans.

The Invisibility of Mild Traumatic Brain Injury: Impaired Cognitive Performance as a Silent Symptom.

The present study was designed to tackle two notorious features of mild traumatic brain injury (mTBI)-heterogeneity and invisibility-by characterizing the full scope of mTBI symptoms. Mice were exposed to brain injuries of different intensities utilizing a weight-drop model (10, 30, 50, and 70 g) and subsequently subjected to a comprehensive battery of behavioral tests at different time points and immunohistochemical examination of cortical slices. Whereas the physiological, neurological, emotional, and motor function of mTBI mice (i.e., their well-being) remained largely intact, cognitive deficits were identified by the y-maze and novel object recognition. Results from these two cognitive tests were combined and a dose-response relationship was established between injury intensity and cognitive impairment, ranging from an 85% decline after a 70-g impact (p < 0.001) to a 20% decline after a 10-g impact (essentially no effect). In addition, higher intensities of injury were accompanied by decreased expression of axonal and synaptic markers. Thus, our mTBI mice showed a clear discrepancy between performance (poor cognitive function) and appearance (healthy demeanor). This is of major concern given that diagnosis of mTBI is established on the presence of clinical symptoms and emphasizes the need for an alternative diagnostic modality.

Exendin-4 attenuates blast traumatic brain injury induced cognitive impairments, losses of synaptophysin and in vitro TBI-induced hippocampal cellular degeneration.

Mild blast traumatic brain injury (B-TBI) induced lasting cognitive impairments in novel object recognition and less severe deficits in Y-maze behaviors. B-TBI significantly reduced the levels of synaptophysin (SYP) protein staining in cortical (CTX) and hippocampal (HIPP) tissues. Treatment with exendin-4 (Ex-4) delivered by subcutaneous micro-osmotic pumps 48 hours prior to or 2 hours immediately after B-TBI prevented the induction of both cognitive deficits and B-TBI induced changes in SYP staining. The effects of a series of biaxial stretch injuries (BSI) on a neuronal derived cell line, HT22 cells, were assessed in an in vitro model of TBI. Biaxial stretch damage induced shrunken neurites and cell death. Treatment of HT22 cultures with Ex-4 (25 to 100 nM), prior to injury, attenuated the cytotoxic effects of BSI and preserved neurite length similar to sham treated cells. These data imply that treatment with Ex-4 may represent a viable option for the management of secondary events triggered by blast-induced, mild traumatic brain injury that is commonly observed in militarized zones.

Mild traumatic brain injury-induced hippocampal gene expressions: The identification of target cellular processes for drug development.

BACKGROUND: Neurological dysfunction after traumatic brain injury (TBI) poses short-term or long-lasting health issues for family members and health care providers. Presently there are no approved medicines to treat TBI. Epidemiological evidence suggests that TBI may cause neurodegenerative disease later in life. In an effort to illuminate target cellular processes for drug development, we examined the effects of a mild TBI on hippocampal gene expression in mouse. METHODS: mTBI was induced in a closed head, weight drop-system in mice (ICR). Animals were anesthetized and subjected to mTBI (30g). Fourteen days after injury the ipsilateral hippocampus was utilized for cDNA gene array studies. mTBI animals were compared with sham-operated animals. Genes regulated by TBI were identified to define TBI-induced physiological/pathological processes. mTBI regulated genes were divided into functional groupings to provide gene ontologies. Genes were further divided to identify molecular/cellular pathways regulated by mTBI. RESULTS: Numerous genes were regulated after a single mTBI event that mapped to many ontologies and molecular pathways related to inflammation and neurological physiology/pathology, including neurodegenerative disease. CONCLUSIONS: These data illustrate diverse transcriptional changes in hippocampal tissues triggered by a single mild injury. The systematic analysis of individual genes that lead to the identification of functional categories, such as gene ontologies and then molecular pathways, illustrate target processes of relevance to TBI pathology. These processes may be further dissected to identify key factors that can be evaluated at the protein level to highlight possible treatments for TBI in human disease and potential biomarkers of neurodegenerative processes.

The neuroprotective effect of salubrinal in a mouse model of traumatic brain injury.

We have previously reported that mild traumatic brain injury (mTBI) induced cognitive deficits as well as apoptotic changes in the brains of mice. Apoptosis may be caused by severe, prolonged accumulation of misfolded proteins, and protein aggregation in the endoplasmic reticulum (ER stress). In an additional study, we have reported that mTBI activated the pro-apoptotic arm of the integrated stress response (ISR). The main goal of the present study was to test the involvement of the adaptive eIF2α/ATF4 pathway in mTBI-affected brains. Head injury was induced with a noninvasive, closed-head weight drop (30 g) to ICR mice. Salubrinal, the selective phosphatase inhibitor of p-eIF2α, was injected immediately and 24 h after mTBI (1 mg/kg, ip). Y-maze and novel object recognition tests to assess spatial and visual memories, respectively, were conducted either 7 or 30 days post-trauma. Salubrinal administration significantly improved memory deficits following mTBI. Slaubrinal also prevented the elevation of degenerating neurons and the reduction of mature neurons in the cortex (as seen by immunofluorescent staining with Fluoro-Jade-B and NeuN antibodies, 72 h and 1 week post-mTBI, respectively). Western blot analysis revealed that salubrinal prevented the significant reduction in eIF2α and ATF4 phosphorylation in mTBI brains 72 h post-injury. Immunofluorescence staining revealed that although the reduction in p-eIF2α did not reach significance, salubrinal administration elevated it dramatically. Our results show that targeting the translational/adaptive arm of the ISR with salubrinal may serve as a therapeutic strategy for brain damage.

Exendin-4 induced glucagon-like peptide-1 receptor activation reverses behavioral impairments of mild traumatic brain injury in mice.

Mild traumatic brain injury (mTBI) represents a major and increasing public health concern and is both the most frequent cause of mortality and disability in young adults and a chief cause of morbidity in the elderly. Albeit mTBI patients do not show clear structural brain defects and, generally, do not require hospitalization, they frequently suffer from long-lasting cognitive, behavioral, and emotional problems. No effective pharmaceutical therapy is available, and existing treatment chiefly involves intensive care management after injury. The diffuse neural cell death evident after mTBI is considered mediated by oxidative stress and glutamate-induced excitotoxicity. Prior studies of the long-acting GLP-1 receptor agonist, exendin-4 (Ex-4), an incretin mimetic approved for type 2 diabetes mellitus treatment, demonstrated its neurotrophic/protective activity in cellular and animal models of stroke, Alzheimer's and Parkinson's diseases, and, consequent to commonalities in mechanisms underpinning these disorders, Ex-4 was assessed in a mouse mTBI model. In neuronal cultures in this study, Ex-4 ameliorated H2O2-induced oxidative stress and glutamate toxicity. To evaluate in vivo translation, we administered steady-state Ex-4 (3.5 pM/kg/min) or saline to control and mTBI mice over 7 days starting 48 h prior to or 1 h post-sham or mTBI (30 g weight drop under anesthesia). Ex-4 proved well-tolerated and fully ameliorated mTBI-induced deficits in novel object recognition 7 and 30 days post-trauma. Less mTBI-induced impairment was evident in Y-maze, elevated plus maze, and passive avoidance paradigms, but when impairment was apparent Ex-4 induced amelioration. Together, these results suggest that Ex-4 may act as a neurotrophic/neuroprotective drug to minimize mTBI impairment.

Cognitive impairments accompanying rodent mild traumatic brain injury involve p53-dependent neuronal cell death and are ameliorated by the tetrahydrobenzothiazole PFT-α.

With parallels to concussive mild traumatic brain injury (mTBI) occurring in humans, anesthetized mice subjected to a single 30 g weight drop mTBI event to the right parietal cortex exhibited significant diffuse neuronal degeneration that was accompanied by delayed impairments in recognition and spatial memory. To elucidate the involvement of reversible p53-dependent apoptosis in this neuronal loss and associated cognitive deficits, mice were subjected to experimental mTBI followed by the systemic administration of the tetrahydrobenzothiazole p53 inactivator, PFT-α, or vehicle. Neuronal loss was quantified immunohistochemically at 72 hr. post-injury by the use of fluoro-Jade B and NeuN within the dentate gyrus on both sides of the brain, and recognition and spatial memory were assessed by novel object recognition and Y-maze paradigms at 7 and 30 days post injury. Systemic administration of a single dose of PFT-α 1 hr. post-injury significantly ameliorated both neuronal cell death and cognitive impairments, which were no different from sham control animals. Cellular studies on human SH-SY5Y cells and rat primary neurons challenged with glutamate excitotoxicity and H2O2 induced oxidative stress, confirmed the ability of PFT-α and a close analog to protect against these TBI associated mechanisms mediating neuronal loss. These studies suggest that p53-dependent apoptotic mechanisms underpin the neuronal and cognitive losses accompanying mTBI, and that these are potentially reversible by p53 inactivation.

Exendin-4, a glucagon-like peptide-1 receptor agonist prevents mTBI-induced changes in hippocampus gene expression and memory deficits in mice.

Traumatic brain injury (TBI) is a global problem reaching near epidemic numbers that manifests clinically with cognitive problems that decades later may result in dementias like Alzheimer's disease (AD). Presently, little can be done to prevent ensuing neurological dysfunctions by pharmacological means. Recently, it has become apparent that several CNS diseases share common terminal features of neuronal cell death. The effects of exendin-4 (Ex-4), a neuroprotective agent delivered via a subcutaneous micro-osmotic pump, were examined in the setting of mild TBI (mTBI). Utilizing a model of mTBI, where cognitive disturbances occur over time, animals were subjected to four treatments: sham; Ex-4; mTBI and Ex-4/mTBI. mTBI mice displayed deficits in novel object recognition, while Ex-4/mTBI mice performed similar to sham. Hippocampal gene expression, assessed by gene array methods, showed significant differences with little overlap in co-regulated genes between groups. Importantly, changes in gene expression induced by mTBI, including genes associated with AD were largely prevented by Ex-4. These data suggest a strong beneficial action of Ex-4 in managing secondary events induced by a traumatic brain injury.