Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction.

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). Detailed analyses of transcriptomic changes in the brain and adipose tissue were performed to elucidate the interactive effects between high-fat diet-induced obesity (DIO) and TBI. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. High-throughput transcriptomic analysis using Nanostring panels of the total visceral adipose tissue (VAT) and cellular components in the brain, followed by unsupervised clustering, principal component analysis, and IPA pathway analysis were used to determine shifts in gene expression patterns and molecular pathway activity. Cellular populations in the cortex and hippocampus, as well as in VAT, during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in selected gene expression signatures and pathways. Furthermore, combined TBI and HFD caused additive dysfunction in Y-Maze, Novel Object Recognition (NOR), and Morris water maze (MWM) cognitive function tests. These novel data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and VAT, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Thus, targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue immune cell populations, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.

Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction.

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). We performed a detailed analysis of transcriptomic changes in the brain and adipose tissue to examine the interactive effects between high-fat diet-induced obesity (DIO) and TBI in relation to central and peripheral inflammatory pathways, as well as neurological function. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. Combined TBI and HFD resulted in additive dysfunction in the Y-Maze, novel object recognition (NOR), and Morris water maze (MWM) cognitive function tests. We also performed high-throughput transcriptomic analysis using Nanostring panels of cellular compartments in the brain and total visceral adipose tissue (VAT), followed by unsupervised clustering, principal component analysis, and IPA pathway analysis to determine shifts in gene expression programs and molecular pathway activity. Analysis of cellular populations in the cortex and hippocampus as well as in visceral adipose tissue during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in select gene expression signatures and pathways. These data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and visceral adipose tissue macrophages, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue macrophages, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates' cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

Down-Regulation of miR-23a-3p Mediates Irradiation-Induced Neuronal Apoptosis.

Radiation-induced central nervous system toxicity is a significant risk factor for patients receiving cancer radiotherapy. Surprisingly, the mechanisms responsible for the DNA damage-triggered neuronal cell death following irradiation have yet to be deciphered. Using primary cortical neuronal cultures in vitro, we demonstrated that X-ray exposure induces the mitochondrial pathway of intrinsic apoptosis and that miR-23a-3p plays a significant role in the regulation of this process. Primary cortical neurons exposed to irradiation show the activation of DNA-damage response pathways, including the sequential phosphorylation of ATM kinase, histone H2AX, and p53. This is followed by the p53-dependent up-regulation of the pro-apoptotic Bcl2 family molecules, including the BH3-only molecules PUMA, Noxa, and Bim, leading to mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c, which activates caspase-dependent apoptosis. miR-23a-3p, a negative regulator of specific pro-apoptotic Bcl-2 family molecules, is rapidly decreased after neuronal irradiation. By increasing the degradation of PUMA and Noxa mRNAs in the RNA-induced silencing complex (RISC), the administration of the miR-23a-3p mimic inhibits the irradiation-induced up-regulation of Noxa and Puma. These changes result in an attenuation of apoptotic processes such as MOMP, the release of cytochrome c and caspases activation, and a reduction in neuronal cell death. The neuroprotective effects of miR-23a-3p administration may not only involve the direct inhibition of pro-apoptotic Bcl-2 molecules downstream of p53 but also include the attenuation of secondary DNA damage upstream of p53. Importantly, we demonstrated that brain irradiation in vivo results in the down-regulation of miR-23a-3p and the elevation of pro-apoptotic Bcl2-family molecules PUMA, Noxa, and Bax, not only broadly in the cortex and hippocampus, except for Bax, which was up-regulated only in the hippocampus but also selectively in isolated neuronal populations from the irradiated brain. Overall, our data suggest that miR-23a-3p down-regulation contributes to irradiation-induced intrinsic pathways of neuronal apoptosis. These regulated pathways of neurodegeneration may be the target of effective neuroprotective strategies using miR-23a-3p mimics to block their development and increase neuronal survival after irradiation.

Interferon-ß Plays a Detrimental Role in Experimental Traumatic Brain Injury by Enhancing Neuroinflammation That Drives Chronic Neurodegeneration.

DNA damage and type I interferons (IFNs) contribute to inflammatory responses after traumatic brain injury (TBI). TBI-induced activation of microglia and peripherally-derived inflammatory macrophages may lead to tissue damage and neurological deficits. Here, we investigated the role of IFN-ß in secondary injury after TBI using a controlled cortical impact model in adult male IFN-ß-deficient (IFN-ß-/-) mice and assessed post-traumatic neuroinflammatory responses, neuropathology, and long-term functional recovery. TBI increased expression of DNA sensors cyclic GMP-AMP synthase and stimulator of interferon genes in wild-type (WT) mice. IFN-ß and other IFN-related and neuroinflammatory genes were also upregulated early and persistently after TBI. TBI increased expression of proinflammatory mediators in the cortex and hippocampus of WT mice, whereas levels were mitigated in IFN-ß-/- mice. Moreover, long-term microglia activation, motor, and cognitive function impairments were decreased in IFN-ß-/- TBI mice compared with their injured WT counterparts; improved neurological recovery was associated with reduced lesion volume and hippocampal neurodegeneration in IFN-ß-/- mice. Continuous central administration of a neutralizing antibody to the IFN-α/ß receptor (IFNAR) for 3 d, beginning 30 min post-injury, reversed early cognitive impairments in TBI mice and led to transient improvements in motor function. However, anti-IFNAR treatment did not improve long-term functional recovery or decrease TBI neuropathology at 28 d post-injury. In summary, TBI induces a robust neuroinflammatory response that is associated with increased expression of IFN-ß and other IFN-related genes. Inhibition of IFN-ß reduces post-traumatic neuroinflammation and neurodegeneration, resulting in improved neurological recovery. Thus, IFN-ß may be a potential therapeutic target for TBI.SIGNIFICANCE STATEMENT TBI frequently causes long-term neurological and psychiatric changes in head injury patients. TBI-induced secondary injury processes including persistent neuroinflammation evolve over time and can contribute to chronic neurological impairments. The present study demonstrates that TBI is followed by robust activation of type I IFN pathways, which have been implicated in microglial-associated neuroinflammation and chronic neurodegeneration. We examined the effects of genetic or pharmacological inhibition of IFN-ß, a key component of type I IFN mechanisms to address its role in TBI pathophysiology. Inhibition of IFN-ß signaling resulted in reduced neuroinflammation, attenuated neurobehavioral deficits, and limited tissue loss long after TBI. These preclinical findings suggest that IFN-ß may be a potential therapeutic target for TBI.

Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury.

Traumatic brain injury (TBI) activates multiple neuronal cell death mechanisms, leading to post-traumatic neuronal loss and neurological deficits. TBI-induced cell cycle activation (CCA) in post-mitotic neurons causes regulated cell death involving cyclin-dependent kinase (CDK) activation and initiation of an E2F transcription factor-mediated pro-apoptotic program. Here we examine the mechanisms of CCA-dependent neuronal apoptosis in primary neurons in vitro and in mice exposed to controlled cortical impact (CCI). In contrast to our prior work demonstrating robust neuroprotective effects by CDK inhibitors after TBI, examination of neuronal apoptotic mechanisms in E2F1-/-/E2F2-/- or E2F2-/- transgenic mice following CCI suggests that E2F1 and/or E2F2 likely play only a modest role in neuronal cell loss after brain trauma. To elucidate more critical CCA molecular pathways involved in post-traumatic neuronal cell death, we investigated the neuroprotective effects and mechanisms of the potent CDK inhibitor CR8 in a DNA damage model of cell death in primary cortical neurons. CR8 treatment significantly reduced caspase activation and cleavage of caspase substrates, attenuating neuronal cell death. CR8 neuroprotective effects appeared to reflect inhibition of multiple pathways converging on the mitochondrion, including injury-induced elevation of pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins Puma and Noxa, thereby attenuating mitochondrial permeabilization and release of cytochrome c and AIF, with reduction of both caspase-dependent and -independent apoptosis. CR8 administration also limited injury-induced deficits in mitochondrial respiration. These neuroprotective effects may be explained by CR8-mediated inhibition of key upstream injury responses, including attenuation of c-Jun phosphorylation/activation as well as inhibition of p53 transactivation of BH3-only targets.

Resistin-Like Molecule α in Allergen-Induced Pulmonary Vascular Remodeling.

Resistin-like molecule α (RELMα) has mitogenic, angiogenic, vasoconstrictive, and chemokine-like properties and is highly relevant in lung pathology. Here, we used RELMα knockout (Retnla(-/-)) mice to investigate the role of RELMα in pulmonary vascular remodeling after intermittent ovalbumin (OVA) challenge. We compared saline- and OVA-exposed wild-type (WT) mice and found that OVA induced significant increases in right ventricular systolic pressure, cardiac hypertrophy, pulmonary vascular remodeling of intra-alveolar arteries, goblet cell hyperplasia in airway epithelium, and intensive lung inflammation, especially perivascular inflammation. Genetic ablation of Retnla prevented the OVA-induced increase in pulmonary pressure and cardiac hypertrophy seen in WT mice. Histological analysis showed that Retnla(-/-) mice exhibited less vessel muscularization, less perivascular inflammation, reduced medial thickness of intra-alveolar vessels, and fewer goblet cells in upper airway epithelium (250-600 µm) than did WT animals after OVA challenge. Gene expression profiles showed that genes associated with vascular remodeling, including those related to muscle protein, contractile fibers, and actin cytoskeleton, were expressed at a lower level in OVA-challenged Retnla(-/-) mice than in similarly treated WT mice. In addition, bronchoalveolar lavage from OVA-challenged Retnla(-/-) mice had lower levels of cytokines, such as IL-1ß, -1 receptor antagonist, and -16, chemokine (C-X-C motif) ligand 1, -2, -9, -10, and -13, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, TIMP metallopeptidase inhibitor-1, and triggering receptor expressed on myeloid cells-1, than did that from WT mice when analyzed by cytokine array dot blots. Retnla knockout inhibited the OVA-induced T helper 17 response but not the T helper 2 response. Altogether, our results suggest that RELMα is involved in immune response-induced pulmonary vascular remodeling and the associated increase in inflammation typically observed after OVA challenge.