Astrocyte focal adhesion kinase reduces passive stress coping by inhibiting ciliary neurotrophic factor only in female mice.

Astrocytes have been implicated in stress responses and produce ciliary neurotrophic factor (CNTF), which we have shown in the mouse medial amygdala (MeA) to promote passive stress coping response only in females. Pharmacological inhibition of focal adhesion kinase (FAK) upregulates CNTF expression. Here, we found that inducible knockout of FAK in astrocytes or systemic treatment with an FAK inhibitor increased passive coping behavior, i.e., immobility, in an acute forced swim stress test in female, but not male, mice. Strikingly, four weeks of chronic unpredictable stress (CUS) did not further increase passive coping in female astrocytic FAK knockout mice, whereas it exacerbated it in female wildtype mice and male mice of both genotypes. These data suggest that astrocyte FAK inhibition is required for chronic stress-induced passive coping in females. Indeed, CUS reduced phospho-FAK and increased CNTF in the female MeA. Progesterone treatment after ovariectomy activated amygdala FAK and alleviated ovariectomy-induced passive coping in wildtype, but not astrocytic FAK knockout females. This suggests that progesterone-mediated activation of FAK in astrocytes reduces female stress responses. Finally, astrocytic FAK knockout or FAK inhibitor treatment increased CNTF expression in the MeA of both sexes, although not in the hippocampus. As mentioned, MeA CNTF promotes stress responses only in females, which may explain the female-specific role of astrocytic FAK inhibition. Together, this study reveals a novel female-specific progesterone-astrocytic FAK pathway that counteracts CNTF-mediated stress responses and points to opportunities for developing treatments for stress-related disorders in women.

Constitutive and evoked release of ATP in adult mouse olfactory epithelium.

In adult olfactory epithelium (OE), ATP plays a role in constant cell turnover and post-injury neuroregeneration. We previously demonstrated that constitutive and ATP-evoked ATP release are present in neonatal mouse OE and underlie continuous cell turn-over and post-injury neuroregeneration, and that activation of purinergic P2X7 receptors is involved in the evoked release. We hypothesized that both releases are present in adult mouse OE. To study the putative contribution of olfactory sensory neurons to ATP release, we used olfactory sensory neuronal-like OP6 cells derived from the embryonic olfactory placode cells. Calcium imaging showed that OP6 cells and primary adult OE cell cultures express functional purinergic receptors. We monitored ATP release from OP6 cells and whole adult OE turbinates using HEK cells as biosensors and luciferin-luciferase assays. Constitutive ATP release occurs in OP6 cells and whole adult mouse OE turbinates, and P2X7 receptors mediated evoked ATP release occurs only in turbinates. The mechanisms of ATP release described in the present study might underlie the constant cell turn-over and post-injury neuroregeneration present in adult OE and thus, further studies of these mechanisms are warranted as it will improve our knowledge of OE tissue homeostasis and post-injury regeneration.

Female-specific neuroprotection after ischemic stroke by vitronectin-focal adhesion kinase inhibition.

We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.

Liver vitronectin release into the bloodstream increases due to reduced vagal muscarinic signaling after cerebral stroke in female mice.

Vitronectin (VTN) is a glycoprotein enriched in the blood and activates integrin receptors. VTN blood levels increase only in female mice 24 h after an ischemic stroke and exacerbate brain injury through IL-6-driven inflammation, but the VTN induction mechanism is unknown. Here, a 30 min middle cerebral artery occlusion (MCAO) in female mice induced VTN protein in the liver (normally the main source) in concert with plasma VTN. Male mice were excluded as VTN is not induced after stroke. MCAO also increased plasma VTN levels after de novo expression of VTN in the liver of VTN-/- female mice, using a hepatocyte-specific (SERPINA1) promoter. MCAO did not affect SERPINA1 or VTN mRNA in the liver, brain, or several peripheral organs, or platelet VTN, compared to sham mice. Thus, hepatocytes are the source of stroke-induced increases in plasma VTN, which is independent of transcription. The cholinergic innervation by the parasympathetic vagus nerve is a potential source of brain-liver signaling after stroke. Right-sided vagotomy at the cervical level led to increased plasma VTN levels, suggesting that VTN release is inhibited by vagal tone. Co-culture of hepatocytes with cholinergic neurons or treatment with acetylcholine, but not noradrenaline (sympathetic transmitter), suppressed VTN expression. Hepatocytes have muscarinic receptors and the M1/M3 agonist bethanechol decreased VTN mRNA and protein release in vitro via M1 receptors. Finally, systemic bethanechol treatment blocked stroke-induced plasma VTN. Thus, VTN translation and release are inhibited by muscarinic signaling from the vagus nerve and presents a novel target for lessening detrimental VTN expression.

Female-specific role of ciliary neurotrophic factor in the medial amygdala in promoting stress responses.

Ciliary neurotrophic factor (CNTF) is produced by astrocytes which have been implicated in regulating stress responses. We found that CNTF in the medial amygdala (MeA) promotes despair or passive coping, i.e., immobility in an acute forced swim stress, in female mice, while having no effect in males. Neutralizing CNTF antibody injected into the MeA of wildtype females reduced activation of downstream STAT3 (Y705) 24 and 48 h later. In concert, the antibody reduced immobility in the swim test in females and only after MeA injection, but not when injected in the central or basolateral amygdala. Antibody injected into the male MeA did not affect immobility. These data reveal a unique role of CNTF in female MeA in promoting despair or passive coping behavior. Moreover, 4 weeks of chronic unpredictable stress (CUS) increased immobility in the swim test and reduced sucrose preference in wildtype CNTF+/+, but not CNTF-/- littermate, females. Following CUS, 10 min of restraint stress increased plasma corticosterone levels only in CNTF+/+ females. In males, the CUS effects were present in both genotypes. Further, CUS increased CNTF expression in the MeA of female, but not male, mice. CUS did not alter CNTF in the female hippocampus, hypothalamus and bed nucleus of stria terminalis. This suggests that MeA CNTF has a female-specific role in promoting CUS-induced despair or passive coping, behavioral anhedonia and neuroendocrine responses. Compared to CNTF+/+ mice, CNTF-/- mice did not show differences in CUS-induced anxiety-like behavior and sensorimotor gating function as measured by elevated T-Maze, open field and pre-pulse inhibition of the acoustic startle response. Together, this study reveals a novel CNTF-mediated female-specific mechanism in stress responses and points to opportunities for developing treatments for stress-related disorders in women.

Inhibition of focal adhesion kinase increases adult olfactory stem cell self-renewal and neuroregeneration through ciliary neurotrophic factor.

Constant neuroregeneration in adult olfactory epithelium maintains olfactory function by basal stem cell proliferation and differentiation to replace lost olfactory sensory neurons (OSNs). Understanding the mechanisms regulating this process could reveal potential therapeutic targets for stimulating adult olfactory neurogenesis under pathological conditions and aging. Ciliary neurotrophic factor (CNTF) in astrocytes promotes forebrain neurogenesis but its function in the olfactory system is unknown. Here, we show in mouse olfactory epithelium that CNTF is expressed in horizontal basal cells, olfactory ensheathing cells (OECs) and a small subpopulation of OSNs. CNTF receptor alpha was expressed in Mash1-positive globose basal cells (GBCs) and OECs. Thus, CNTF may affect GBCs in a paracrine manner. CNTF-/- mice did not display altered GBC proliferation or olfactory function, suggesting that CNTF is not involved in basal olfactory renewal or that they developed compensatory mechanisms. Therefore, we tested the effect of increased CNTF in wild type mice. Intranasal instillation of a focal adhesion kinase (FAK) inhibitor, FAK14, upregulated CNTF expression. FAK14 also promoted GBC proliferation, neuronal differentiation and basal stem cell self-renewal but had no effective in CNTF-/- mice, suggesting that FAK inhibition promotes olfactory neuroregeneration through CNTF, making them potential targets to treat sensorineural anosmia due to OSN loss.

Blood Vitronectin Induces Detrimental Brain Interleukin-6 and Correlates With Outcomes After Stroke Only in Female Mice.

Background and Purpose- Women have worse stroke outcomes than men, especially after menopause. Few studies have focused on female-specific mechanisms, other than hormones. We investigated the role of the blood protein VTN (vitronectin) after ischemic stroke in mice. Methods- Adult male and female VTN knockout and wild-type littermates and C57BL/6 mice received a middle cerebral artery occlusion and the injured brain tissue analyzed 24 hours to 3 weeks later for cell loss and inflammation, as well as neurological function. Blood VTN levels were measured before and after stroke. Results- Intravenously injected VTN leaked extensively from bloodstream into brain infarct and penumbra by 24 hours after stroke. Strikingly, VTN was detrimental in female, but not male, mice, as shown by reduced brain injury (26.2±2.6% versus 13.4±3.8%; P=0.018; n=6 and 5) and forelimb dysfunction in female VTN knockout mice. Stroke increased plasma VTN 2- to 8-fold at 24 hours in females (36±4 versus 145±24 µg/mL; P<0.0001; n=10 and 7), but not males (62±8 versus 68±6; P>0.99; n=10 and 7), and returned to control levels by 7 days. Individually variable VTN levels at 24 hours correlated with stroke-induced brain injury at 7 days only in females. VTN promoted stroke-induced microglia/macrophage activation and leukocyte infiltration in females. Proinflammatory IL (interleukin)-6 greatly increased in the striatum at 24 hours in wild-type mice but was increased ≈60% less in female (739±159 versus 268±111; P=0.02; n=7 and 6), but not male (889±178 versus 1179±295; P=0.73; n=10 and 11), knockout mice. In individual wild-type females, plasma VTN levels correlated with striatal IL-6 expression at 24 hours. The female-specific effect of VTN-induced IL-6 expression following stroke was not due to gonadal hormones, as shown by ovariectomy and castration. Lastly, intrastriatal injection of IL-6 in female mice immediately before stroke reversed the VTN knockout phenotypes of reduced brain injury and microglia/macrophage activation. Conclusions- VTN plays a novel sexually dimorphic detrimental pathophysiological role in females and might ultimately be a therapeutic target to improve stroke outcomes in women.

Correction to: Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.

An amendment to this paper has been published and can be accessed via the original article.

Vitronectin mitigates stroke-increased neurogenesis only in female mice and through FAK-regulated IL-6.

Vitronectin (VTN) is a blood protein produced mainly by the liver. We show that VTN leaks from the bloodstream into the injury site and neighboring subventricular zone (SVZ) following ischemic stroke (middle cerebral artery occlusion, MCAO) in adult mice. MCAO is known to increase neurogenesis after stroke. VTN inhibits this response in females, but not in males, as shown by ~70% more stroke-induced SVZ neurogenesis in female VTN-/- mice at 14 d. In female VTN-/- mice, stroke-induced expression of interleukin-6 (IL-6) at 24 h was reduced in the SVZ. The closely related leukemia inhibitory factor (LIF) or pro-neurogenic ciliary neurotrophic factor (CNTF) were not affected. The female-specific effect of VTN on IL-6 expression was not due to sex hormones, as shown by ovariectomy and castration. IL-6 injection next to the SVZ reversed the MCAO-induced increase in neurogenesis seen in VTN-/- mice. Our in vitro and vivo data suggest that plasma VTN activates focal adhesion kinase (FAK) in the SVZ following MCAO, which reduces IL-6 expression in astrocytes but increases it in other cells such as microglia/macrophages. Inducible conditional astrocytic FAK deletion increased MCAO-induced IL-6 expression in females at 24 h and blocked MCAO-induced neurogenesis at 14 d, confirming a key detrimental role of IL-6. Collectively, these data suggest that leakage of VTN into the SVZ reduces the neurogenic response to stroke in female mice by promoting IL-6 expression. Reducing VTN or VTN signaling may be an approach to promote neurogenesis for neuroprotection and cell replacement after stroke in females.

Vitronectin from brain pericytes promotes adult forebrain neurogenesis by stimulating CNTF.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN-/- mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN-/- mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.

Ciliary neurotrophic factor is a key sex-specific regulator of depressive-like behavior in mice.

Ciliary neurotrophic factor (CNTF) is produced by astrocytes and promotes neurogenesis and neuroprotection. Little is known about the role of CNTF in affective behavior. We investigated whether CNTF affects depressive- and anxiety-like behavior in adult mice as tested in the forced swim, sucrose preference and elevated-T maze tests. Female wild type CNTF+/+ mice more readily developed behavioral despair with increased immobility time and decreased latency to immobility in the forced swim test than male CNTF+/+ littermates. The lack of CNTF in CNTF-/- mice had an opposite effect on depressive-like behavior in female mice (reduced immobility time and increased sucrose preference) vs. male mice (increased immobility time). Female wildtype mice expressed more CNTF in the amygdala than male mice. Ovariectomy increased CNTF expression, as well as immobility time, which was significantly reduced in CNTF-/- mice, suggesting that CNTF mediates overiectomy-induced immobility time, possibly in the amygdala. Progesterone but not 17-ß estradiol inhibited CNTF expression in cultured C6 astroglioma cells. Progesterone treatment also reduced CNTF expression in the amygdala and decreased immobility time in female CNTF+/+ but not in CNTF-/- mice. Castration did not alter CNTF expression in males nor their behavior. Lastly, there were no effects of CNTF on the elevated T-maze, a behavioral test of anxiety, suggesting that a different mechanism may underlie anxiety-like behavior. This study reveals a novel CNTF-mediated mechanism in stress-induced depressive-like behavior and points to opportunities for sex-specific treatments for depression, e.g. progesterone in females and CNTF-stimulating drugs in males.

Inhibition of astrocyte FAK-JNK signaling promotes subventricular zone neurogenesis through CNTF.

Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK-JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF-/- mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF-/- littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK-JNK-CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin-FAK and uPAR signaling.

We defined how blood-derived vitronectin (VTN) rapidly and potently activates leukemia inhibitory factor (LIF) and pro-inflammatory interleukin 6 (IL-6) in vitro and after vascular injury in the brain. Treatment with VTN (but not fibrinogen, fibronectin, laminin-111 or collagen-I) substantially increased LIF and IL-6 within 4 h in C6-astroglioma cells, while VTN-/- mouse plasma was less effective than that from wild-type mice. LIF and IL-6 were induced by intracerebral injection of recombinant human (rh)VTN in mice, but induction seen upon intracerebral hemorrhage was less in VTN-/- mice than in wild-type littermates. In vitro, VTN effects were inhibited by RGD, αvß3 and αvß5 integrin-blocking peptides and antibodies. VTN activated focal adhesion kinase (FAK; also known as PTK2), whereas pharmacological- or siRNA-mediated inhibition of FAK, but not PYK2, reduced the expression of LIF and IL-6 in C6 and endothelial cells and after traumatic cell injury. Dominant-negative FAK (Y397F) reduced the amount of injury-induced LIF and IL-6. Pharmacological inhibition or knockdown of uPAR (also known as PLAUR), which binds VTN, also reduced cytokine expression, possibly through a common target of uPAR and integrins. We propose that VTN leakage into tissues promotes inflammation. Integrin-FAK signaling is therefore a novel IL-6 and LIF regulation mechanism relevant to the inflammation and stem cell fields.

Reduced FAK-STAT3 signaling contributes to ER stress-induced mitochondrial dysfunction and death in endothelial cells.

Excessive endoplasmic reticulum (ER) stress leads to cell loss in many diseases, e.g., contributing to endothelial cell loss after spinal cord injury. Here, we determined whether ER stress-induced mitochondrial dysfunction could be explained by interruption of the focal adhesion kinase (FAK)-mitochondrial STAT3 pathway we recently discovered. ER stress was induced in brain-derived mouse bEnd5 endothelial cells by thapsigargin or tunicamycin and caused apoptotic cell death over a 72h period. In concert, ER stress caused mitochondrial dysfunction as shown by reduced bioenergetic function, loss of mitochondrial membrane potential and increased mitophagy. ER stress caused a reduction in mitochondrial phosphorylated S727-STAT3, known to be important for maintaining mitochondrial function. Normal activation or phosphorylation of the upstream cytoplasmic FAK was also reduced, through mechanisms that involve tyrosine phosphatases and calcium signaling, as shown by pharmacological inhibitors, bisperoxovanadium (bpV) and 2-aminoethoxydiphenylborane (APB), respectively. APB mitigated the reduction in FAK and STAT3 phosphorylation, and improved endothelial cell survival caused by ER stress. Transfection of cells rendered null for STAT3 using CRISPR technology with STAT3 mutants confirmed the specific involvement of S727-STAT3 inhibition in ER stress-mediated cell loss. These data suggest that loss of FAK signaling during ER stress causes mitochondrial dysfunction by reducing the protective effects of mitochondrial STAT3, leading to endothelial cell death. We propose that stimulation of the FAK-STAT3 pathway is a novel therapeutic approach against pathological ER stress.

Integrin-FAK signaling rapidly and potently promotes mitochondrial function through STAT3.

BACKGROUND: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof. METHODS: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts. RESULTS: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvß3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period. CONCLUSION: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Effect of IP3R3 and NPY on age-related declines in olfactory stem cell proliferation.

Losing the sense of smell because of aging compromises health and quality of life. In the mouse olfactory epithelium, aging reduces the capacity for tissue homeostasis and regeneration. The microvillous cell subtype that expresses both inositol trisphosphate receptor type 3 (IP3R3) and the neuroproliferative factor neuropeptide Y (NPY) is critical for regulation of homeostasis, yet its role in aging is undefined. We hypothesized that an age-related decline in IP3R3 expression and NPY signaling underlie age-related homeostatic changes and olfactory dysfunction. We found a decrease in IP3R3(+) and NPY(+) microvillous cell numbers and NPY protein and a reduced sensitivity to NPY-mediated proliferation over 24 months. However, in IP3R3-deficient mice, there was no further age-related reduction in cell numbers, proliferation, or olfactory function compared with wild type. The proliferative response was impaired in aged IP3R3-deficient mice when injury was caused by satratoxin G, which induces IP3R3-mediated NPY release, but not by bulbectomy, which does not evoke NPY release. These data identify IP3R3 and NPY signaling as targets for improving recovery following olfactotoxicant exposure.

An IP3R3- and NPY-expressing microvillous cell mediates tissue homeostasis and regeneration in the mouse olfactory epithelium.

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3(+/-), and IP3R3(-/-) mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3(-/-) mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3(-/-) mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3(-/-) mice. The reductions in both NPY release and number of progenitor cells in IP3R3(-/-) mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.

Mechanisms of constitutive and ATP-evoked ATP release in neonatal mouse olfactory epithelium.

BACKGROUND: ATP is an extracellular signaling molecule with many ascribed functions in sensory systems, including the olfactory epithelium. The mechanism(s) by which ATP is released in the olfactory epithelium has not been investigated. Quantitative luciferin-luciferase assays were used to monitor ATP release, and confocal imaging of the fluorescent ATP marker quinacrine was used to monitor ATP release via exocytosis in Swiss Webster mouse neonatal olfactory epithelial slices. RESULTS: Under control conditions, constitutive release of ATP occurs via exocytosis, hemichannels and ABC transporters and is inhibited by vesicular fusion inhibitor Clostridium difficile toxin A and hemichannel and ABC transporter inhibitor probenecid. Constitutive ATP release is negatively regulated by the ATP breakdown product ADP through activation of P2Y receptors, likely via the cAMP/PKA pathway. In vivo studies indicate that constitutive ATP may play a role in neuronal homeostasis as inhibition of exocytosis inhibited normal proliferation in the OE. ATP-evoked ATP release is also present in mouse neonatal OE, triggered by several ionotropic P2X purinergic receptor agonists (ATP, αßMeATP and Bz-ATP) and a G protein-coupled P2Y receptor agonist (UTP). Calcium imaging of P2X2-transfected HEK293 "biosensor" cells confirmed the presence of evoked ATP release. Following purinergic receptor stimulation, ATP is released via calcium-dependent exocytosis, activated P2X1,7 receptors, activated P2X7 receptors that form a complex with pannexin channels, or ABC transporters. The ATP-evoked ATP release is inhibited by the purinergic receptor inhibitor PPADS, Clostridium difficile toxin A and two inhibitors of pannexin channels: probenecid and carbenoxolone. CONCLUSIONS: The constitutive release of ATP might be involved in normal cell turn-over or modulation of odorant sensitivity in physiological conditions. Given the growth-promoting effects of ATP, ATP-evoked ATP release following injury could lead to progenitor cell proliferation, differentiation and regeneration. Thus, understanding mechanisms of ATP release is of paramount importance to improve our knowledge about tissue homeostasis and post-injury neuroregeneration. It will lead to development of treatments to restore loss of smell and, when transposed to the central nervous system, improve recovery following central nervous system injury.

Neuropeptide Y and extracellular signal-regulated kinase mediate injury-induced neuroregeneration in mouse olfactory epithelium.

In the olfactory epithelium (OE), injury induces ATP release, and subsequent activation of P2 purinergic receptors by ATP promotes neuroregeneration by increasing basal progenitor cell proliferation. The molecular mechanisms underlying ATP-induced increases in OE neuroregeneration have not been established. In the present study, the roles of neuroproliferative factors neuropeptide Y (NPY) and fibroblast growth factor 2 (FGF2), and p44/42 extracellular signal-regulated kinase (ERK) on ATP-mediated increases of neuroregeneration in the OE were investigated. ATP increased basal progenitor cell proliferation in the OE via activation of P2 purinergic receptors in vitro and in vivo as monitored by incorporation of 5'-ethynyl-2'-deoxyuridine, a thymidine analog, into DNA, and proliferating cell nuclear antigen (PCNA) protein levels. ATP induced p44/42 ERK activation in globose basal cells (GBCs) but not horizontal basal cells (HBCs). ATP differentially regulated p44/42 ERK over time in the OE both in vitro and in vivo with transient inhibition (5-15 min) followed by activation (30 min-1 h) of p44/42 ERK. In addition, ATP indirectly activated p44/42 ERK in the OE via ATP-induced NPY release and subsequent activation of NPY Y1 receptors in the basal cells. There were no synergistic effects of ATP and NPY or FGF2 on OE neuroregeneration. These data clearly have implications for the pharmacological modulation of neuroregeneration in the olfactory epithelium.