Innervation of Adipocytes is Limited in Mouse Perivascular Adipose Tissue.

BACKGROUND: Perivascular adipose tissue (PVAT) regulates vascular tone by releasing anticontractile factors. These anticontractile factors are driven by processes downstream of adipocyte stimulation by norepinephrine; however, whether norepinephrine originates from neural innervation or other sources is unknown. The goal of this study was to test the hypothesis that neurons innervating PVAT provide the adrenergic drive to stimulate adipocytes in aortic and mesenteric perivascular adipose tissue (aPVAT, mPVAT), and white adipose tissue (WAT). METHODS: Healthy male and female mice (8-13 weeks) were used in all experiments. Expression of genes associated with synaptic transmission were quantified by qPCR and adipocyte activity in response to neurotransmitters and neuron depolarization was assessed in AdipoqCre+;GCaMP5g-tdTf/WT mice. Immunostaining, tissue clearing, and transgenic reporter lines were used to assess anatomical relationships between nerves and adipocytes. RESULTS: While synaptic transmission components genes are expressed in adipose tissues (aPVAT, mPVAT, WAT), strong nerve stimulation with electrical field stimulation does not significantly trigger calcium responses in adipocytes. However, norepinephrine consistently elicits strong calcium responses in adipocytes from all adipose tissues studied. Bethanechol induces minimal adipocyte responses. Imaging neural innervation using various techniques reveals that nerve fibers primarily run alongside blood vessels and rarely branch into the adipose tissue. CONCLUSION: While nerve fibers are associated with blood vessels in adipose tissue, they demonstrate limited anatomical and functional interactions with adjacent adipocytes, challenging the concept of classical innervation. These findings dispute the significant involvement of neural input in regulating PVAT adipocyte function and emphasize alternative mechanisms governing adrenergic-driven anticontractile functions of PVAT.

Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes.

Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration.Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1ß, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.

Brain-gut axis dysfunction in the pathogenesis of traumatic brain injury.

Traumatic brain injury (TBI) is a chronic and progressive disease, and management requires an understanding of both the primary neurological injury and the secondary sequelae that affect peripheral organs, including the gastrointestinal (GI) tract. The brain-gut axis is composed of bidirectional pathways through which TBI-induced neuroinflammation and neurodegeneration impact gut function. The resulting TBI-induced dysautonomia and systemic inflammation contribute to the secondary GI events, including dysmotility and increased mucosal permeability. These effects shape, and are shaped by, changes in microbiota composition and activation of resident and recruited immune cells. Microbial products and immune cell mediators in turn modulate brain-gut activity. Importantly, secondary enteric inflammatory challenges prolong systemic inflammation and worsen TBI-induced neuropathology and neurobehavioral deficits. The importance of brain-gut communication in maintaining GI homeostasis highlights it as a viable therapeutic target for TBI. Currently, treatments directed toward dysautonomia, dysbiosis, and/or systemic inflammation offer the most promise.

Targeted intestinal deletion of Rho guanine nucleotide exchange factor 7, ßPIX, impairs enterocyte proliferation, villus maturation, and mucosal defenses in mice.

Rho guanine nucleotide exchange factors (RhoGEFs) regulate Rho GTPase activity and cytoskeletal and cell adhesion dynamics. ßPix, a CDC42/RAC family RhoGEF encoded by ARHGEF7, is reported to modulate human colon cancer cell proliferation and postwounding restitution of rat intestinal epithelial monolayers. We hypothesized that ßPix plays a role in maintaining intestinal epithelial homeostasis. To test this hypothesis, we examined ßPix distribution in the human and murine intestine and created mice with intestinal epithelial-selective ßPix deletion [ßPixflox/flox/Tg(villin-Cre); Arhgef7 CKO mice]. Using Arhgef7 conditional knockout (CKO) and control mice, we investigated the consequences of ßPix deficiency in vivo on intestinal epithelial and enteroid development, dextran sodium sulfate-induced mucosal injury, and gut permeability. In normal human and murine intestines, we observed diffuse cytoplasmic and moderate nuclear ßPix immunostaining in enterocytes. Arhgef7 CKO mice were viable and fertile, with normal gross intestinal architecture but reduced small intestinal villus height, villus-to-crypt ratio, and goblet cells; small intestinal crypt cells had reduced Ki67 staining, compatible with impaired cell proliferation. Enteroids derived from control mouse small intestine were viable for more than 20 passages, but those from Arhgef7 CKO mice did not survive beyond 24 h despite addition of Wnt proteins or conditioned media from normal enteroids. Adding a Rho kinase (ROCK) inhibitor partially rescued CKO enteroid development. Compared with littermate control mice, dextran sodium sulfate-treated ßPix-deficient mice lost more weight and had greater impairment of intestinal barrier function, and more severe colonic mucosal injury. These findings reveal ßPix expression is important for enterocyte development, intestinal homeostasis, and resistance to toxic injury.NEW & NOTEWORTHY To explore the role of ßPix, a guanine nucleotide exchange factor encoded by ARHGEF7, in intestinal development and physiology, we created mice with intestinal epithelial cell Arhgef7/ßPix deficiency. We found ßPix essential for normal small intestinal epithelial cell proliferation, villus development, and mucosal resistance to injury. Moreover, Rho kinase signaling mediated developmental arrest observed in enteroids derived from ßPix-deficient small intestinal crypts. Our studies provide insights into the role Arhgef7/ßPix plays in intestinal epithelial homeostasis.

Acute colitis during chronic experimental traumatic brain injury in mice induces dysautonomia and persistent extraintestinal, systemic, and CNS inflammation with exacerbated neurological deficits.

BACKGROUND: Disruptions of brain-gut axis have been implicated in the progression of a variety of gastrointestinal (GI) disorders and central nervous system (CNS) diseases and injuries, including traumatic brain injury (TBI). TBI is a chronic disease process characterized by persistent secondary injury processes which can be exacerbated by subsequent challenges. Enteric pathogen infection during chronic TBI worsened cortical lesion volume; however, the pathophysiological mechanisms underlying the damaging effects of enteric challenge during chronic TBI remain unknown. This preclinical study examined the effect of intestinal inflammation during chronic TBI on associated neurobehavioral and neuropathological outcomes, systemic inflammation, and dysautonomia. METHODS: Dextran sodium sulfate (DSS) was administered to adult male C57BL/6NCrl mice 28 days following craniotomy (Sham) or TBI for 7 days to induce intestinal inflammation, followed by a return to normal drinking water for an additional 7 to 28 days for recovery; uninjured animals (Naïve) served as an additional control group. Behavioral testing was carried out prior to, during, and following DSS administration to assess changes in motor and cognitive function, social behavior, and mood. Electrocardiography was performed to examine autonomic balance. Brains were collected for histological and molecular analyses of injury lesion, neurodegeneration, and neuroinflammation. Blood, colons, spleens, mesenteric lymph nodes (mLNs), and thymus were collected for morphometric analyses and/or immune characterization by flow cytometry. RESULTS: Intestinal inflammation 28 days after craniotomy or TBI persistently induced, or exacerbated, respectively, deficits in fine motor coordination, cognition, social behavior, and anxiety-like behavior. Behavioral changes were associated with an induction, or exacerbation, of hippocampal neuronal cell loss and microglial activation in Sham and TBI mice administered DSS, respectively. Acute DSS administration resulted in a sustained systemic immune response with increases in myeloid cells in blood and spleen, as well as myeloid cells and lymphocytes in mesenteric lymph nodes. Dysautonomia was also induced in Sham and TBI mice administered DSS, with increased sympathetic tone beginning during DSS administration and persisting through the first recovery week. CONCLUSION: Intestinal inflammation during chronic experimental TBI causes a sustained systemic immune response and altered autonomic balance that are associated with microglial activation, increased neurodegeneration, and persistent neurological deficits.

Bidirectional brain-gut interactions and chronic pathological changes after traumatic brain injury in mice.

OBJECTIVES: Traumatic brain injury (TBI) has complex effects on the gastrointestinal tract that are associated with TBI-related morbidity and mortality. We examined changes in mucosal barrier properties and enteric glial cell response in the gut after experimental TBI in mice, as well as effects of the enteric pathogen Citrobacter rodentium (Cr) on both gut and brain after injury. METHODS: Moderate-level TBI was induced in C57BL/6mice by controlled cortical impact (CCI). Mucosal barrier function was assessed by transepithelial resistance, fluorescent-labelled dextran flux, and quantification of tight junction proteins. Enteric glial cell number and activation were measured by Sox10 expression and GFAP reactivity, respectively. Separate groups of mice were challenged with Cr infection during the chronic phase of TBI, and host immune response, barrier integrity, enteric glial cell reactivity, and progression of brain injury and inflammation were assessed. RESULTS: Chronic CCI induced changes in colon morphology, including increased mucosal depth and smooth muscle thickening. At day 28 post-CCI, increased paracellular permeability and decreased claudin-1 mRNA and protein expression were observed in the absence of inflammation in the colon. Colonic glial cell GFAP and Sox10 expression were significantly increased 28days after brain injury. Clearance of Cr and upregulation of Th1/Th17 cytokines in the colon were unaffected by CCI; however, colonic paracellular flux and enteric glial cell GFAP expression were significantly increased. Importantly, Cr infection in chronically-injured mice worsened the brain lesion injury and increased astrocyte- and microglial-mediated inflammation. CONCLUSION: These experimental studies demonstrate chronic and bidirectional brain-gut interactions after TBI, which may negatively impact late outcomes after brain injury.

Cell cycle inhibition reduces inflammatory responses, neuronal loss, and cognitive deficits induced by hypobaria exposure following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) patients in military settings can be exposed to prolonged periods of hypobaria (HB) during aeromedical evacuation. Hypobaric exposure, even with supplemental oxygen to prevent hypoxia, worsens outcome after experimental TBI, in part by increasing neuroinflammation. Cell cycle activation (CCA) after TBI has been implicated as a mechanism contributing to both post-traumatic cell death and neuroinflammation. Here, we examined whether hypobaric exposure in rats subjected to TBI increases CCA and microglial activation in the brain, as compared to TBI alone, and to evaluate the ability of a cyclin-dependent kinase (CDK) inhibitor (CR8) to reduce such changes and improve behavioral outcomes. METHODS: Adult male Sprague Dawley rats were subjected to fluid percussion-induced injury, and HB exposure was performed at 6 h after TBI. Western blot and immunohistochemistry (IHC) were used to assess cell cycle-related protein expression and inflammation at 1 and 30 days after injury. CR8 was administered intraperitoneally at 3 h post-injury; chronic functional recovery and histological changes were assessed. RESULTS: Post-traumatic hypobaric exposure increased upregulation of cell cycle-related proteins (cyclin D1, proliferating cell nuclear antigen, and CDK4) and microglial/macrophage activation in the ipsilateral cortex at day 1 post-injury as compared to TBI alone. Increased immunoreactivity of cell cycle proteins, as well as numbers of Iba-1+ and GFAP+ cells in both the ipsilateral cortex and hippocampus were found at day 30 post-injury. TBI/HB significantly increased the numbers of NADPH oxidase 2 (gp91phox) enzyme-expressing cells that were co-localized with Iba-1+. Each of these changes was significantly reduced by the administration of CR8. Unbiased stereological assessment showed significantly decreased numbers of microglia displaying the highly activated phenotype in the ipsilateral cortex of TBI/HB/CR8 rats compared with TBI/HB/Veh rats. Moreover, treatment with this CDK inhibitor also significantly improved spatial and retention memory and reduced lesion volume and hippocampal neuronal cell loss. CONCLUSIONS: HB exposure following TBI increases CCA, neuroinflammation, and associated neuronal cell loss. These changes and post-traumatic cognitive deficits are reduced by CDK inhibition; such drugs may therefore serve to protect TBI patients requiring aeromedical evacuation.

Sex differences in cell genesis, hippocampal volume and behavioral outcomes in a rat model of neonatal HI.

Hypoxia-ischemia (HI) of the brain in near-term and term infants is a leading cause of infant mortality and lifelong disability but current therapeutic approaches remain limited. Males consistently display greater vulnerability to the deleterious consequences of HI in both humans and animal models. Neurogenesis increases after neonatal HI and offers a potential therapeutic target for recovery. The steroid hormone estradiol has been extensively explored as a neuroprotectant in adult models of stroke but with mixed results. Less consideration has been afforded to this naturally occurring agent in the developing brain, which has unique challenges from the adult. Using a model of term HI in the rat we have explored the impact of this insult on cell genesis in the hippocampus of males and females and the ability of estradiol treatment immediately after insult to restore function. Both short-term (3 days) and long-term (7 days) post-injury were assessed and revealed that only females had markedly increased cell genesis on the short-term but both sexes were increased long-term. A battery of behavioral tests revealed motor impairment in males and compromised episodic memory while both sexes were modestly impaired in spatial memory. Juvenile social play was also depressed in both sexes after HI. Estradiol therapy improved behavioral performance in both sexes but did not reverse a deficit in hippocampal volume ipsilateral to the insult. Thus the effects of estradiol do not appear to be via cell death or proliferation but rather involve other components of neural functioning.

Cyclopropyl-containing positive allosteric modulators of metabotropic glutamate receptor subtype 5.

Positive allosteric modulators (PAMs) binding to the transmembrane (TM) domain of metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for psychiatric disorders and traumatic brain injury (TBI). Novel PAMs based on a trans-2-phenylcyclopropane amide scaffold have been designed and synthesized. Facilitating ligand design and allowing estimation of binding affinities to the mGluR5 TM domain was the novel computational strategy, site identification by ligand competitive saturation (SILCS). The potential protective activity of the new compounds was evaluated using nitric oxide (NO) production in BV2 microglial cell cultures treated with lipopolysaccharide (LPS), and the toxicity of the new compounds tested using a cell viability assay. One of the new compounds, 3a, indicated promising activity with potency of 30 µM, which is 4.5-fold more potent than its lead compound 3,3'-difluorobenzaldazine (DFB), and showed no detectable toxicity with concentrations as high as 1000 µM. Thus this compound represents a new lead for possible development as treatment for TBI and related neurodegenerative disorders.

Acyl-2-aminobenzimidazoles: a novel class of neuroprotective agents targeting mGluR5.

Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for treating traumatic brain injury (TBI). Using computational and medicinal methods, the structure-activity relationship of a class of acyl-2-aminobenzimidazoles (1-26) is reported. The new compounds are designed based on the chemical structure of 3,3'-difluorobenzaldazine (DFB), a known mGluR5 PAM. Ligand design and prediction of binding affinities of the new compounds have been performed using the site identification by ligand competitive saturation (SILCS) method. Binding affinities of the compounds to the transmembrane domain of mGluR5 have been evaluated using nitric oxide (NO) production assay, while the safety of the compounds is tested. One new compound found in this study, compound 22, showed promising activity with an IC50 value of 6.4 µM, which is ∼20 fold more potent than that of DFB. Compound 22 represents a new lead for possible development as a treatment for TBI and related neurodegenerative conditions.

PARP-1 inhibition attenuates neuronal loss, microglia activation and neurological deficits after traumatic brain injury.

Traumatic brain injury (TBI) causes neuronal cell death as well as microglial activation and related neurotoxicity that contribute to subsequent neurological dysfunction. Poly (ADP-ribose) polymerase (PARP-1) induces neuronal cell death through activation of caspase-independent mechanisms, including release of apoptosis inducing factor (AIF), and microglial activation. Administration of PJ34, a selective PARP-1 inhibitor, reduced cell death of primary cortical neurons exposed to N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG), a potent inducer of AIF-dependent cell death. PJ34 also attenuated lipopolysaccharide and interferon-γ-induced activation of BV2 or primary microglia, limiting NF-κB activity and iNOS expression as well as decreasing generation of reactive oxygen species and TNFα. Systemic administration of PJ34 starting as late as 24 h after controlled cortical impact resulted in improved motor function recovery in mice with TBI. Stereological analysis demonstrated that PJ34 treatment reduced the lesion volume, attenuated neuronal cell loss in the cortex and thalamus, and reduced microglial activation in the TBI cortex. PJ34 treatment did not improve cognitive performance in a Morris water maze test or reduce neuronal cell loss in the hippocampus. Overall, our data indicate that PJ34 has a significant, albeit selective, neuroprotective effect after experimental TBI, and its therapeutic effect may be from multipotential actions on neuronal cell death and neuroinflammatory pathways.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGluR5) attenuate microglial activation.

Traumatic brain injury causes progressive neurodegeneration associated with chronic microglial activation. Recent studies show that neurodegeneration and neuroinflammation after traumatic brain injury can be inhibited as late as one month in animals by the activation of the metabotropic glutamate receptor 5 in microglia using (RS)-2-chloro-5- hydroxy-phenylglycine. However, the therapeutic potential of this agonist is limited due to its relatively weak potency and brain permeability. To address such concerns, we evaluated the anti-inflammatory activities of several positive allosteric modulators using various in vitro assays, and found that 3,3'-difluorobenzaldazine, 3-cyano-N-(1,3-diphenyl-1H-pyrazol- 5-yl)benzamide and 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide showed significantly improved potency which makes them potential lead compounds for further development of positive allosteric modulators for the treatment of traumatic brain injury.

Propofol limits microglial activation after experimental brain trauma through inhibition of nicotinamide adenine dinucleotide phosphate oxidase.

BACKGROUND: Microglial activation is implicated in delayed tissue damage after traumatic brain injury (TBI). Activation of microglia causes up-regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, with the release of reactive oxygen species and cytotoxicity. Propofol appears to have antiinflammatory actions. The authors evaluated the neuroprotective effects of propofol after TBI and examined in vivo and in vitro whether such actions reflected modulation of NADPH oxidase. METHODS: Adult male rats were subjected to moderate lateral fluid percussion TBI. Effect of propofol on brain microglial activation and functional recovery was assessed up to 28 days postinjury. By using primary microglial and BV2 cell cultures, the authors examined propofol modulation of lipopolysaccharide and interferon-γ-induced microglial reactivity and neurotoxicity. RESULTS: Propofol improved cognitive recovery after TBI in novel object recognition test (48 ± 6% for propofol [n = 15] vs. 30 ± 4% for isoflurane [n = 14]; P = 0.005). The functional improvement with propofol was associated with limited microglial activation and decreased cortical lesion volume and neuronal loss. Propofol also attenuated lipopolysaccharide- and interferon-γ-induced microglial activation in vitro, with reduced expression of inducible nitric oxide synthase, nitric oxide, tumor necrosis factor-α, interlukin-1ß, reactive oxygen species, and NADPH oxidase. Microglial-induced neurotoxicity in vitro was also markedly reduced by propofol. The protective effect of propofol was attenuated when the NADPH oxidase subunit p22 was knocked down by small interfering RNA. Moreover, propofol reduced the expression of p22 and gp91, two key components of NADPH oxidase, after TBI. CONCLUSION: The neuroprotective effects of propofol after TBI appear to be mediated, in part, through the inhibition of NADPH oxidase.

Over-expression of HSP70 attenuates caspase-dependent and caspase-independent pathways and inhibits neuronal apoptosis.

HSP70 is a member of the family of heat-shock proteins that are known to be up-regulated in neurons following injury and/or stress. HSP70 over-expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over-expression by transfection with HSP70-expression plasmids in primary cortical neurons and the SH-SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2-ceramide, and ß-Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease-activating factor 1, as well as apoptosis-inducing factor, key molecules involved in development of caspase-dependent and caspase-independent PCD, respectively. Markers of caspase-dependent PCD, including active caspase-3, caspase-9, and cleaved PARP were attenuated in neurons over-expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase-dependent and caspase-independent PCD pathways.

Inhibition of E2F1/CDK1 pathway attenuates neuronal apoptosis in vitro and confers neuroprotection after spinal cord injury in vivo.

Apoptosis of post-mitotic neurons plays a significant role in secondary tissue damage following traumatic spinal cord injury (SCI). Activation of E2F1-dependent transcription promotes expression of pro-apoptotic factors, including CDK1; this signal transduction pathway is believed to represent an important mechanism for the physiological or pathological neuronal cell death. However, a specific role for this pathway in neuronal apoptosis induced by SCI has not yet been reported. Here we demonstrate up-regulation of the E2F1/CDK1 pathway that is associated with neuronal apoptosis following impact SCI in rats. Expression of E2F1 and CDK1 were robustly up-regulated as early as 15 min after injury and sustained until 3 days post-injury. CDK1 activity and E2F1 downstream targets bim and c-Myb were significantly increased after SCI. Activation of E2F1/CDK1 signaling also was associated with death of neurons in vitro; this was attenuated by shRNA knockdown or pharmacological inhibition of the E2F1/CDK1 pathway. CR8, a novel and potent CDK1 inhibitor, blocked apoptosis of primary cortical neurons at low-micromolar concentrations. Moreover, SCI-induced up-regulation of E2F1/CDK1 and associated neuronal apoptosis was significantly attenuated by systemic injection of CR8 (1 mg/kg, i.p.) at 5 min after injury. CR8 significantly decreased posttraumatic elevation of biochemical markers of apoptosis, such as products of caspase-3 and α-fodrin cleavage, as well as neuronal cell death, as indicated by TUNEL staining. Importantly, CR8 treatment also increased the number of surviving neurons at 5 weeks after injury. Together, these findings indicate that activation of the E2F1/CDK1 pathway contributes to the pathophysiology of SCI and that selective inhibition of this signaling cascade may represent an attractive therapeutic strategy.

Combined inhibition of cell death induced by apoptosis inducing factor and caspases provides additive neuroprotection in experimental traumatic brain injury.

Neuronal programmed cell death (PCD) contributes to delayed tissue damage after traumatic brain injury (TBI). Both caspase-dependent and caspase-independent mechanisms have been implicated, with the latter including apoptosis inducing factor (AIF). The peptidyl-proplyl isomerase Cyclophilin A (CypA) transports AIF from the cytosol to the nucleus, a key step for AIF-dependent cell death. We compared the effects of single versus combined inhibition of caspase and AIF pathways in a mouse controlled cortical impact (CCI) model, by examining the effects of CypA gene knockout (CypA(-/-)), caspase inhibition with a pan-caspase inhibitor (boc-aspartyl(OMe)-fluoromethylketone, BAF), or combined modulation. TBI caused caspase activation as well as translocation of AIF to the nucleus. Markers of caspase activation including caspase-specific fodrin cleavage fragments and number of FLIVO-positive cells were reduced in BAF-treated CypA(+/+) mice, whereas markers of AIF activation including AIF/H2AX interaction and AIF translocation to the nucleus were attenuated in CypA(-/-) mice. Each single intervention, (CypA(-/-) or BAF-treated CypA(+/+)) reduced the number of apoptotic cells (TUNEL-positive) in the cortex and improved long-term sensorimotor function; CypA(-/-) also attenuated microglial activation after injury. Importantly, BAF-treated CypA(-/-) mice, showed greater effects than either intervention alone on multiple outcomes including: reduction in TUNEL-positive cells, decrease in neuroinflammation, improved motor and cognitive recovery, and attenuation of lesion volume and neuronal loss in the hippocampus. Using two in vitro neuronal cell death models known to induce AIF-mediated PCD, we also showed that neurons from CypA(-/-) animals were protected and that effects were unrelated to caspase activation. These data indicate that AIF-mediated and caspase-dependent pathways contribute independently and in parallel to secondary injury after TBI, and suggest that combined therapeutic strategies directed at multiple PCD pathways may provide superior neuroprotection than those directed at single mechanisms.

CR8, a selective and potent CDK inhibitor, provides neuroprotection in experimental traumatic brain injury.

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell cycle activation (CCA), that leads to neuronal death and neurological dysfunction. We recently reported that delayed administration of roscovitine, a relatively selective cyclin-dependent kinase (CDK) inhibitor, inhibits CCA and attenuates neurodegeneration and functional deficits following controlled cortical impact (CCI) injury in mice. Here we evaluated the neuroprotective potential of CR8, a more potent second-generation roscovitine analog, using the mouse CCI model. Key CCA markers (cyclin A and B1) were significantly up-regulated in the injured cortex following TBI, and phosphorylation of CDK substrates was increased. Central administration of CR8 after TBI, at a dose 20 times less than previously required for roscovitine, attenuated CCA pathways and reduced post-traumatic apoptotic cell death at 24 h post-TBI. Central administration of CR8, at 3 h after TBI, significantly attenuated sensorimotor and cognitive deficits, decreased lesion volume, and improved neuronal survival in the cortex and dentate gyrus. Moreover, unlike roscovitine treatment in the same model, CR8 also attenuated post-traumatic neurodegeneration in the CA3 region of the hippocampus and thalamus at 21 days. Furthermore, delayed systemic administration of CR8, at a dose 10 times less than previously required for roscovitine, significantly improved cognitive performance after CCI. These findings further demonstrate the neuroprotective potential of cell cycle inhibitors following experimental TBI. Given the increased potency and efficacy of CR8 as compared to earlier purine analog types of CDK inhibitors, this drug should be considered as a candidate for future clinical trials of TBI.

Cyclin D1 gene ablation confers neuroprotection in traumatic brain injury.

Cell cycle activation (CCA) is one of the principal secondary injury mechanisms following brain trauma, and it leads to neuronal cell death, microglial activation, and neurological dysfunction. Cyclin D1 (CD1) is a key modulator of CCA and is upregulated in neurons and microglia following traumatic brain injury (TBI). In this study we subjected CD1-wild-type (CD1(+/+)) and knockout (CD1(-/-)) mice to controlled cortical impact (CCI) injury to evaluate the role of CD1 in post-traumatic neurodegeneration and neuroinflammation. As early as 24 h post-injury, CD1(+/+) mice showed markers of CCA in the injured hemisphere, including increased CD1, E2F1, and proliferating cell nuclear antigen (PCNA), as well as increased Fluoro-Jade B staining, indicating neuronal degeneration. Progressive neuronal loss in the hippocampus was observed through 21 days post-injury in these mice, which correlated with a decline in cognitive function. Microglial activation in the injured hemisphere peaked at 7 days post-injury, with sustained increases at 21 days. In contrast, CD1(-/-) mice showed reduced CCA and neurodegeneration at 24 h, as well as improved cognitive function, attenuated hippocampal neuronal cell loss, decreased lesion volume, and cortical microglial activation at 21 days post-injury. These findings indicate that CD1-dependent CCA plays a significant role in the neuroinflammation, progressive neurodegeneration, and related neurological dysfunction resulting from TBI. Our results further substantiate the proposed role of CCA in post-traumatic secondary injury, and suggest that inhibition of CD1 may be a key therapeutic target for TBI.

Selective CDK inhibitor limits neuroinflammation and progressive neurodegeneration after brain trauma.

Traumatic brain injury (TBI) induces secondary injury mechanisms, including cell-cycle activation (CCA), which lead to neuronal cell death, microglial activation, and neurologic dysfunction. Here, we show progressive neurodegeneration associated with microglial activation after TBI induced by controlled cortical impact (CCI), and also show that delayed treatment with the selective cyclin-dependent kinase inhibitor roscovitine attenuates posttraumatic neurodegeneration and neuroinflammation. CCI resulted in increased cyclin A and D1 expressions and fodrin cleavage in the injured cortex at 6 hours after injury and significant neurodegeneration by 24 hours after injury. Progressive neuronal loss occurred in the injured hippocampus through 21 days after injury and correlated with a decline in cognitive function. Microglial activation associated with a reactive microglial phenotype peaked at 7 days after injury with sustained increases at 21 days. Central administration of roscovitine at 3 hours after CCI reduced subsequent cyclin A and D1 expressions and fodrin cleavage, improved functional recovery, decreased lesion volume, and attenuated hippocampal and cortical neuronal cell loss and cortical microglial activation. Furthermore, delayed systemic administration of roscovitine improved motor recovery and attenuated microglial activation after CCI. These findings suggest that CCA contributes to progressive neurodegeneration and related neurologic dysfunction after TBI, likely in part related to its induction of microglial activation.

Novel mode for neutrophil protease cathepsin G-mediated signaling: membrane shedding of epidermal growth factor is required for cardiomyocyte anoikis.

Neutrophils are thought to orchestrate myocardial remodeling during the early progression to cardiac failure through the release of reactive oxygen species, antimicrobial peptides, and proteases. Although neutrophil activation may be beneficial at early stages of disease, excessive neutrophil infiltration can induce cardiomyocyte death and tissue damage. The neutrophil-derived serine protease cathepsin G (Cat.G) has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis. However, the involved signaling mechanisms for Cat.G are not well understood. This study identifies epidermal growth factor receptor (EGFR) transactivation as a mechanism whereby Cat.G induces signaling in cardiomyocytes. Cat.G induced a rapid and transient increase in EGFR tyrosine phosphorylation, and inhibition of EGFR kinase activity, either with AG1478 or by expression of kinase inactive EGFR mutants (EGFR-CD533), markedly attenuated EGFR downstream signaling and myocyte anoikis induced by Cat.G. Consistent with this effect of EGFR, high level expression of wild-type EGFR was sufficient to promote myocyte apoptosis. We also found that matrix metalloproteinase-dependent membrane shedding of heparin-binding EGF was involved in Cat.G signaling and that membrane type 1 matrix metalloproteinase activation may constitute a potential target that entails matrix metalloproteinase activation induced by Cat.G. The paradoxical proapoptotic effect of EGFR appeared to be dependent on protein tyrosine phosphatase SHP2 (Src homology domain 2-containing tyrosine phosphatase 2) activation and focal adhesion kinase downregulation. These results show that Cat.G-induced cardiomyocyte apoptosis involves an increase in EGFR-dependent activation of SHP2 that promotes focal adhesion kinase dephosphorylation and subsequent cardiomyocyte anoikis.