Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury.

Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal-dependent cognitive deficits induced by TBI in two different injury mouse models-focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma-induced chronic cognitive deficits.

CCR2 antagonism alters brain macrophage polarization and ameliorates cognitive dysfunction induced by traumatic brain injury.

Traumatic brain injury (TBI) is a major risk factor for the development of multiple neurodegenerative diseases. With respect to the increasing prevalence of TBI, new therapeutic strategies are urgently needed that will prevent secondary damage to primarily unaffected tissue. Consistently, neuroinflammation has been implicated as a key mediator of secondary damage following the initial mechanical insult. Following injury, there is uncertainty regarding the role that accumulating CCR2(+) macrophages play in the injury-induced neuroinflammatory sequelae and cognitive dysfunction. Using CX3CR1(GFP/+)CCR2(RFP/+) reporter mice, we show that TBI initiated a temporally restricted accumulation of peripherally derived CCR2(+) macrophages, which were concentrated in the hippocampal formation, a region necessary for learning and memory. Multivariate analysis delineated CCR2(+) macrophages' neuroinflammatory response while identifying a novel therapeutic treatment window. As a proof of concept, targeting CCR2(+) macrophages with CCX872, a novel Phase I CCR2 selective antagonist, significantly reduced TBI-induced inflammatory macrophage accumulation. Concomitantly, there was a significant reduction in multiple proinflammatory and neurotoxic mediators with this treatment paradigm. Importantly, CCR2 antagonism resulted in a sparing of TBI-induced hippocampal-dependent cognitive dysfunction and reduced proinflammatory activation profile 1 month after injury. Thus, therapeutically targeting the CCR2(+) subset of monocytes/macrophages may provide a new avenue of clinical intervention following TBI.

Short- and long-term effects of 56Fe irradiation on cognition and hippocampal DNA methylation and gene expression.

BACKGROUND: Astronauts are exposed to 56Fe ions that may pose a significant health hazard during and following prolonged missions in deep space. We showed previously that object recognition requiring the hippocampus, a structure critical for cognitive function, is affected in 2-month-old mice irradiated with 56Fe ions. Here we examined object recognition in 6-month-old mice irradiated with 56Fe ions, a biological age more relevant to the typical ages of astronauts. Moreover, because the mechanisms mediating the detrimental effects of 56Fe ions on hippocampal function are unclear, we examined changes in hippocampal networks involved in synaptic plasticity and memory, gene expression, and epigenetic changes in cytosine methylation (5mC) and hydroxymethylation (5hmC) that could accompany changes in gene expression. We assessed the effects of whole body 56Fe ion irradiation at early (2 weeks) and late (20 weeks) time points on hippocampus-dependent memory and hippocampal network stability, and whether these effects are associated with epigenetic changes in hippocampal DNA methylation (both 5mC and 5hmC) and gene expression. RESULTS: At the two-week time point, object recognition and network stability were impaired following irradiation at the 0.1 and 0.4 Gy dose, but not following irradiation at the 0.2 Gy dose. No impairments in object recognition or network stability were seen at the 20-week time point at any irradiation dose used. Consistent with this pattern, the significance of pathways for gene categories for 5hmC was lower, though not eliminated, at the 20-week time point compared to the 2-week time point. Similarly, significant changes were observed for 5mC gene pathways at the 2-week time point, but no significant gene categories were observed at the 20-week time point. Only the 5hmC changes tracked with gene expression changes. CONCLUSIONS: Dose- and time-dependent epigenomic remodeling in the hippocampus following 56Fe ion exposure correlates with behavioral changes.

Colony-stimulating factor 1 receptor blockade prevents fractionated whole-brain irradiation-induced memory deficits.

BACKGROUND: Primary central nervous system (CNS) neoplasms and brain metastases are routinely treated with whole-brain radiation. Long-term survival occurs in many patients, but their quality of life is severely affected by the development of cognitive deficits, and there is no treatment to prevent these adverse effects. Neuroinflammation, associated with activation of brain-resident microglia and infiltrating monocytes, plays a pivotal role in loss of neurological function and has been shown to be associated with acute and long-term effects of brain irradiation. Colony-stimulating factor 1 receptor (CSF-1R) signaling is essential for the survival and differentiation of microglia and monocytes. Here, we tested the effects of CSF-1R blockade by PLX5622 on cognitive function in mice treated with three fractions of 3.3 Gy whole-brain irradiation. METHODS: Young adult C57BL/6J mice were given three fractions of 3.3 Gy whole-brain irradiation while they were on diet supplemented with PLX5622, and the effects on periphery monocyte accumulation, microglia numbers, and neuronal functions were assessed. RESULTS: The mice developed hippocampal-dependent cognitive deficits at 1 and 3 months after they received fractionated whole-brain irradiation. The impaired cognitive function correlated with increased number of periphery monocyte accumulation in the CNS and decreased dendritic spine density in hippocampal granule neurons. PLX5622 treatment caused temporary reduction of microglia numbers, inhibited monocyte accumulation in the brain, and prevented radiation-induced cognitive deficits. CONCLUSIONS: Blockade of CSF-1R by PLX5622 prevents fractionated whole-brain irradiation-induced memory deficits. Therapeutic targeting of CSF-1R may provide a new avenue for protection from radiation-induced memory deficits.

TNF-α protein synthesis inhibitor restores neuronal function and reverses cognitive deficits induced by chronic neuroinflammation.

BACKGROUND: Chronic neuroinflammation is a hallmark of several neurological disorders associated with cognitive loss. Activated microglia and secreted factors such as tumor necrosis factor (TNF)-α are key mediators of neuroinflammation and may contribute to neuronal dysfunction. Our study was aimed to evaluate the therapeutic potential of a novel analog of thalidomide, 3,6'-dithiothalidomide (DT), an agent with anti-TNF-α activity, in a model of chronic neuroinflammation. METHODS: Lipopolysaccharide or artificial cerebrospinal fluid was infused into the fourth ventricle of three-month-old rats for 28 days. Starting on day 29, animals received daily intraperitoneal injections of DT (56 mg/kg/day) or vehicle for 14 days. Thereafter, cognitive function was assessed by novel object recognition, novel place recognition and Morris water maze, and animals were euthanized 25 min following water maze probe test evaluation. RESULTS: Chronic LPS-infusion was characterized by increased gene expression of the proinflammatory cytokines TNF-α and IL-1ß in the hippocampus. Treatment with DT normalized TNF-α levels back to control levels but not IL-1ß. Treatment with DT attenuated the expression of TLR2, TLR4, IRAK1 and Hmgb1, all genes involved in the TLR-mediated signaling pathway associated with classical microglia activation. However DT did not impact the numbers of MHC Class II immunoreactive cells. Chronic neuroinflammation impaired novel place recognition, spatial learning and memory function; but it did not impact novel object recognition. Importantly, treatment with DT restored cognitive function in LPS-infused animals and normalized the fraction of hippocampal neurons expressing the plasticity-related immediate-early gene Arc. CONCLUSION: Our data demonstrate that the TNF-α synthesis inhibitor DT can significantly reverse hippocampus-dependent cognitive deficits induced by chronic neuroinflammation. These results suggest that TNF-α is a critical mediator of chronic neuroinflammation-induced neuronal dysfunction and cognitive impairment and targeting its synthesis could provide an effective therapeutic approach to several human neurodegenerative diseases.

Chronic neuroinflammation impacts the recruitment of adult-born neurons into behaviorally relevant hippocampal networks.

Growing evidence suggests that adult-born granule cells integrate into hippocampal networks and are required for proper cognitive function. Although neuroinflammation is involved in many disorders associated with cognitive impairment, it remains unknown whether it impacts the recruitment of adult-born neurons into behaviorally relevant hippocampal networks. Under similar behavioral conditions, exploration-induced expression of the immediate-early gene Arc in hippocampal cells has been linked to cellular activity observed by electrophysiological recording. By detecting exploration-induced Arc protein expression, we investigated whether neuroinflammation alters the recruitment of adult-born neurons into behaviorally relevant hippocampal networks. Neuroinflammation was induced in rats by intra-cerebroventricular infusion of lipopolysaccharide for 28 days. Animals received bromodeoxyuridine injections starting on day 29 (5 days) and were euthanized two months later. Persistent lipopolysaccharide-induced neuroinflammation was reliably detected by microglial activation in the hippocampus. Neuroinflammation did not impact the number of adult-born neurons but did alter their migration pattern through the granule cell layer. There was a positive correlation between the density of activated microglia and alterations in the fraction of existing granule neurons expressing Arc, suggesting that neuroinflammation induced a long-term disruption of hippocampal network activity. The proportion of adult-born neurons expressing behaviorally induced Arc was significantly lower in lipopolysaccharide-treated rats than in controls. This observation supports the fact that neuroinflammation significantly impacts adult-born neurons recruitment into hippocampal networks encoding spatial information.

Delayed-matching-to-place Task in a Dry Maze to Measure Spatial Working Memory in Mice.

The delayed-matching-to-place (DMP) dry maze test is a variant of DMP water maze (Steele and Morris, 1999; Faizi et al., 2012) which measures spatial working/episodic-like learning and memory that depends on both hippocampal and cortical functions (Wang and Morris, 2010; Euston et al., 2012). Using this test we can detect normal aging related spatial working memory decline, as well as trauma induced working memory deficits. Furthermore, we recently reported that fractionated whole brain irradiation does not affect working memory in mice (Feng et al., 2016). Here we describe the experimental setup and procedures of this behavioral test.

Bi-directional and shared epigenomic signatures following proton and 56Fe irradiation.

The brain's response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain's response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome.

Human neural stem cell transplantation provides long-term restoration of neuronal plasticity in the irradiated hippocampus.

For the majority of CNS malignancies, radiotherapy provides the best option for forestalling tumor growth, but is frequently associated with debilitating and progressive cognitive dysfunction. Despite the recognition of this serious side effect, satisfactory long-term solutions are not currently available and have prompted our efforts to explore the potential therapeutic efficacy of cranial stem cell transplants. We have demonstrated that intrahippocampal transplantation of human neural stem cells (hNSCs) can provide long-lasting cognitive benefits using an athymic rat model subjected to cranial irradiation. To explore the possible mechanisms underlying the capability of engrafted cells to ameliorate radiation-induced cognitive dysfunction we analyzed the expression patterns of the behaviorally induced activity-regulated cytoskeleton-associated protein (Arc) in the hippocampus at 1 and 8 months postgrafting. While immunohistochemical analyses revealed a small fraction (4.5%) of surviving hNSCs in the irradiated brain that did not express neuronal or astroglial makers, hNSC transplantation impacted the irradiated microenvironment of the host brain by promoting the expression of Arc at both time points. Arc is known to play key roles in the neuronal mechanisms underlying long-term synaptic plasticity and memory and provides a reliable marker for detecting neurons that are actively engaged in spatial and contextual information processing associated with memory consolidation. Cranial irradiation significantly reduced the number of pyramidal (CA1) and granule neurons (DG) expressing behaviorally induced Arc at 1 and 8 months postirradiation. Transplantation of hNSCs restored the expression of plasticity-related Arc in the host brain to control levels. These findings suggest that hNSC transplantation promotes the long-term recovery of host hippocampal neurons and indicates that one mechanism promoting the preservation of cognition after irradiation involves trophic support from engrafted cells.

Cranial irradiation alters the brain's microenvironment and permits CCR2+ macrophage infiltration.

Therapeutic irradiation is commonly used to treat primary or metastatic central nervous system tumors. It is believed that activation of neuroinflammatory signaling pathways contributes to the development of common adverse effects, which may ultimately contribute to cognitive dysfunction. Recent studies identified the chemokine (C-C motif) receptor (CCR2), constitutively expressed by cells of the monocyte-macrophage lineage, as a mediator of cognitive impairments induced by irradiation. In the present study we utilized a unique reporter mouse (CCR2(RFP/+)CX3CR1(GFP/+)) to accurately delineate the resident (CX3CR1+) versus peripheral (CCR2+) innate immune response in the brain following cranial irradiation. Our results demonstrate that a single dose of 10Gy cranial γ-irradiation induced a significant decrease in the percentage of resident microglia, while inducing an increase in the infiltration of peripherally derived CCR2+ macrophages. Although reduced in percentage, there was a significant increase in F4/80+ activated macrophages in irradiated animals compared to sham. Moreover, we found that there were altered levels of pro-inflammatory cytokines, chemokines, adhesion molecules, and growth factors in the hippocampi of wild type irradiated mice as compared to sham. All of these molecules are implicated in the recruitment, adhesion, and migration of peripheral monocytes to injured tissue. Importantly, there were no measureable changes in the expression of multiple markers associated with blood-brain barrier integrity; implicating the infiltration of peripheral CCR2+ macrophages may be due to inflammatory induced chemotactic signaling. Cumulatively, these data provide evidence that therapeutic levels of cranial radiation are sufficient to alter the brain's homeostatic balance and permit the influx of peripherally-derived CCR2+ macrophages as well as the regional susceptibility of the hippocampal formation to ionizing radiation.

CCR2 deficiency prevents neuronal dysfunction and cognitive impairments induced by cranial irradiation.

Cranial irradiation can lead to long-lasting cognitive impairments in patients receiving radiotherapy for the treatment of malignant brain tumors. Recent studies have suggested inflammation as a major contributor to these deficits; we determined if the chemokine (C-C motif) receptor 2 (CCR2) was a mediator of cognitive impairments induced by irradiation. Two-month-old male Ccr2 knockout (-/-) and wild-type mice received 10 Gy cranial irradiation or sham-treatment. One month after irradiation, bromodeoxyuridine was injected intraperitoneally for seven consecutive days to label newly generated cells. At two months postirradiation, cognitive function was assessed by novel object recognition and Morris water maze. Our results show that CCR2 deficiency prevented hippocampus-dependent spatial learning and memory impairments induced by cranial irradiation. Hippocampal gene expression analysis showed that irradiation induced CCR2 ligands such as CCL8 and CCR2 deficiency reduced this induction. Irradiation reduced the number of adult-born neurons in both wild-type and Ccr2(-/-) mice, but the distribution pattern of the adult-born neurons through the granule cell layer was only altered in wild-type mice. Importantly, CCR2 deficiency normalized the fraction of pyramidal neurons expressing the plasticity-related immediate early gene Arc. These data offer new insight into the mechanism(s) of radiation-injury and suggest that CCR2 is a critical mediator of hippocampal neuronal dysfunction and hippocampal cognitive impairments after irradiation. Targeting CCR2 signaling could conceivably provide an effective approach to reduce or prevent the incidence and severity of this serious side effect of ionizing irradiation.