Mapping Connectome in Mammalian Brain: A Novel Approach by Bioengineering Neuro-Glia specific Vectors.

The connectome is the comprehensive map of the brain represented by wiring diagram of the full set of neuro-glia and synapses within entire brain of an organism. Some recent scientific efforts have successfully been made to visualize such map at neuro-glial networking level, however, capturing it as one unit of the entire brain have never been elucidated. Moreover, in order to derive structure-function relationship of different brain regions in response to a defined stimulus, there is a need to elucidate the connectome at single neuro-glial ensemble level after brain is challenged with the known memory function. This needs developing molecular approaches to tag neuro-glial activities in response to a conditioned brain function. Such approaches of using specific molecular tags have been tried to visualize independently neuron and glial specific events in response to a memory function, however, they could not tag the connectome together at single neuro-glia ensemble level. Therefore, there is a need to develop new methods for mapping entire connectome up to a single neuro-glial precision and resolution, with a purpose of tagging specific brain region accountable to execute a special memory formation process. The present hypothetical paper aims to propose a novel molecular method to generate the structural connectome at neuro-glial level in mice brain. Herein, we propose to tag the entire connectome at neuro-glia precision by generating a transgenic mice via transposing and recombining engineered novel "Neuro-Glia specific Vectors" (NGVs: specific to excitatory neurons, inhibitory neurons and glial cells) vis a vis "Transcriptional/ Translational Messenger (TMs: specific to metalloproteinases, MMP-9) coupled with different color protein tags, followed by the Clarity. Herein, the NGVs will be translated via Neuro-glia specific promoters, while TMs will be translated via endogenous MMP-9 promoter in all neuro-glial cells. The viability of all constructs will be verified in cortical/ hippocampal culture by inducing them to undergo chemically induced long term potentionation (cLTP) following visualization of different colored pattern. This will be further confirmed by Immunostaning, Western Blot and RT-PCR analysis. Additionally, in this approach, one can decipher the dynamics of molecular and cellular events associated with MMP-9 seretome by monitoring the trafficking of tagged endogenous MMP-9 protein after neuronal stimulation by cLTP in vitro. However, for visualizing complete connectome, the adult transgenic mice will be challenged with fear consolidation (Fear context and contextual cue) tests followed by Clarity coupled Light Sheet Microscopy to analyze neuro-glia ensemble following whole brain imaging.

Role for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations.

Chronic stress is a risk factor for the development of psychopathologies characterized by cognitive dysfunction and deregulated social behaviours. Emerging evidence suggests a role for cell adhesion molecules, including nectin-3, in the mechanisms that underlie the behavioural effects of stress. We tested the hypothesis that proteolytic processing of nectins by matrix metalloproteinases (MMPs), an enzyme family that degrades numerous substrates, including cell adhesion molecules, is involved in hippocampal effects induced by chronic restraint stress. A reduction in nectin-3 in the perisynaptic CA1, but not in the CA3, compartment is observed following chronic stress and is implicated in the effects of stress in social exploration, social recognition and a CA1-dependent cognitive task. Increased MMP-9-related gelatinase activity, involving N-methyl-D-aspartate receptor, is specifically found in the CA1 and involved in nectin-3 cleavage and chronic stress-induced social and cognitive alterations. Thus, MMP-9 proteolytic processing emerges as an important mediator of stress effects in brain function and behaviour.

Matrix metalloproteinase (MMP) 9 transcription in mouse brain induced by fear learning.

Memory formation requires learning-based molecular and structural changes in neurons, whereas matrix metalloproteinase (MMP) 9 is involved in the synaptic plasticity by cleaving extracellular matrix proteins and, thus, is associated with learning processes in the mammalian brain. Because the mechanisms of MMP-9 transcription in the brain are poorly understood, this study aimed to elucidate regulation of MMP-9 gene expression in the mouse brain after fear learning. We show here that contextual fear conditioning markedly increases MMP-9 transcription, followed by enhanced enzymatic levels in the three major brain structures implicated in fear learning, i.e. the amygdala, hippocampus, and prefrontal cortex. To reveal the role of AP-1 transcription factor in MMP-9 gene expression, we have used reporter gene constructs with specifically mutated AP-1 gene promoter sites. The constructs were introduced into the medial prefrontal cortex of neonatal mouse pups by electroporation, and the regulation of MMP-9 transcription was studied after contextual fear conditioning in the adult animals. Specifically, -42/-50- and -478/-486-bp AP-1 binding motifs of the mouse MMP-9 promoter sequence have been found to play a major role in MMP-9 gene activation. Furthermore, increases in MMP-9 gene promoter binding by the AP-1 transcription factor proteins c-Fos and c-Jun have been demonstrated in all three brain structures under investigation. Hence, our results suggest that AP-1 acts as a positive regulator of MMP-9 transcription in the brain following fear learning.

Chronic gastric ulceration causes matrix metalloproteinases-9 and -3 augmentation: alleviation by melatonin.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degradation of extracellular matrix (ECM) and key player in various inflammatory diseases. We investigated the regulation of MMPs in chronic gastric ulceration in mice. We generated chronic gastric ulcers in mice by indomethacin and examined the activity and expression of MMP-9 and -3 in stomach. Melatonin (N-acetyl-5-methoxytryptamine) treatment has also been applied to mice to characterize the changes in expression and activities of MMPs in gastric tissues. We observed significant upregulation of MMP-9 and -3 expressions and activities in stomach with increasing doses and duration of indomethacin that corroborated with increased activity of activator protein (AP)-1. Substantial damage in gastric epithelial layer was found during chronic ulceration. Melatonin suppressed MMP-9 and -3 expressions and activities during prevention and healing of chronic gastric ulcers. It also suppressed protein oxidation, lipid peroxidation and antioxidant enzymes. Additionally, expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-8 was significantly high in ulcerated stomachs while melatonin treatment blocked them to control level. We found elevated phosphorylation of extracellular-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK) during chronic gastric ulceration, which were significantly reversed by melatonin. Moreover, expression of NF-κB, c-fos and c-jun were inhibited by melatonin resulting down regulation of MMP-9 and -3 expressions. In summary, oxidative stress is preceded by chronic inflammation that enhances the expression of MMP-9 and -3, while melatonin arrests both of them via reduction of AP-1 activity during protection of ulcer.

Curcumin heals indomethacin-induced gastric ulceration by stimulation of angiogenesis and restitution of collagen fibers via VEGF and MMP-2 mediated signaling.

AIM: We examined the molecular mechanism of curcumin in a preventive and therapeutic model of indomethacin-induced gastric ulceration with regard to angiogenic processes. RESULTS: Disrupted blood vessels, reduced collagen matrices, and significant (60%) injury to mucosal cells were observed during ulceration. In addition, ulcerated tissues exhibited decreased matrix metalloproteinase (MMP)-2 and vascular endothelial growth factor (VEGF) expression in blood vessels. Interestingly, curcumin blocked ulceration by induction of collagenization and angiogenesis in gastric tissues via upregulation of MMP-2, membrane type (MT) 1-MMP, VEGF, and transforming growth factor (TGF)-ß at protein and messenger ribonucleic acid (mRNA) levels. To examine the angiogenic properties of curcumin, we employed a chorioallantoic membrane model and Matrigel assay. During healing, curcumin promoted collagenization and angiogenesis as well as enhanced MMP-2 activity via positive MT1-MMP regulation and negative tissue inhibitor of metalloproteinase-2 regulation. INNOVATION: Our study demonstrates that curcumin-mediated healing is associated with increased MMP-2, TGF-ß, and VEGF expression and that it plays a pivotal role as an angiogenic modulator by stimulating vascular sprout formation and collagen fiber restoration in ulcerated tissues. CONCLUSION: We conclude that curcumin remodels gastric tissues by restoring the collagen architecture and accelerating angiogenesis.

Melatonin promotes angiogenesis during protection and healing of indomethacin-induced gastric ulcer: role of matrix metaloproteinase-2.

Matrix metalloproteinase (MMP)-2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N-acetyl-5-methoxy tryptamine), an antioxidant and anti-inflammatory agent, prevents indomethacin-induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP-2-mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin-induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP-2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over-expression of MMP-2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP-2. In addition, upregulation of MMP-2 was parallel to MMP-14 and inverse to tissue inhibitor of metalloprotease (TIMP)-2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP-2 and VEGF over-expression during protection and healing of gastric ulcers. This study highlights for the first time a phase-associated regulation of MMP-2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin-induced gastropathy.

Induction of matrix metalloproteinase-9 and -3 in nonsteroidal anti-inflammatory drug-induced acute gastric ulcers in mice: regulation by melatonin.

The pathogenesis of gastric ulcer is associated with remodeling of extracellular matrix (ECM) by various matrix metalloproteinases (MMPs). However, how MMPs are regulated during nonsteroidal anti-inflammatory drug (NSAID)-induced acute gastric ulceration is not well studied. In this study, different NSAIDs (80 mg/kg b.w.) were applied to generate acute gastric ulcer in the BALB/c mouse and the regulation of MMPs were investigated. NSAIDs caused dose-dependent induction in MMP-9 and -3 activities and expressions in ulcerated gastric tissues along with significant infiltration of inflammatory cells and disruption of gastric mucosal layer. In addition, an increase in tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-8 expression, excessive generation of hydroxyl radical ((*)OH), and protein oxidation and lipid peroxidation were observed in acute ulcerated gastric tissues. In this study, the efficacy of melatonin on activities of MMP-9 and -3 during prevention of gastric ulcers was tested. Melatonin at a dose of 60 mg/kg b.w. downregulated MMP-9 and -3 both at the enzyme and protein levels in mouse gastric tissues during prevention as well as healing of acute gastric ulcers. It also blocked oxidative stress via inhibition of protein oxidation, lipid peroxidation, (*)OH generation and SOD-2 expression. Moreover, it suppressed myeloperoxidase activity and expressions of TNF-alpha, IL-1beta and IL-8. This study documents for the first time that induction of MMP-9 and -3 activities accompany NSAID-induced inflammation and oxidative stress in gastric tissues and indicates that, melatonin may be a preventive or therapeutic remedy for gastric ulcers.

Matrix metalloproteinase-9 activity and expression is reduced by melatonin during prevention of ethanol-induced gastric ulcer in mice.

Matrix metalloproteinases (MMPs) play an important role in degradation of gastric extracellular matrix proteins. However, no reports are available on the relationship between the activity of MMPs and gastric ulceration induced by alcohol. Our objective was to investigate the effect of melatonin (N-acetyl-5-methoxytryptamine) on the regulation of MMP-9 and MMP-2 activities during prevention of ethanol-induced gastric ulcer. Biochemical and zymographic methods were used to analyze MMP-9 and -2 activities in gastric tissues of Balb/c mice following induction of gastric ulcer by ethanol. Our studies reveal that melatonin arrested cell injury, protein carbonyl formation, and lipid peroxidation in mice during gastroprotection. Melatonin dose-dependently reduced proMMP-9 activity that was induced ( approximately 25-fold) during ethanol-induced gastric ulceration. Severity of gastric ulcers were correlated proportionately with increased dose of ethanol and elevated activity of proMMP-9 and -2. The reduced activities of MMP-9 and -2 were associated with reduced expression of TNF-alpha and increased expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). We conclude that melatonin's ability to prevent ethanol-induced gastric ulceration in mice is related to a reduction in proMMP-9 activity and expression.

Novel role of famotidine in downregulation of matrix metalloproteinase-9 during protection of ethanol-induced acute gastric ulcer.

Gastric ulcer is a multifaceted process including acid secretion, reactive oxygen species generation, prostaglandin inhibition, and extracellular matrix (ECM) degradation. Matrix metalloproteinases (MMPs) have the ability to cleave and remodel the ECM. We investigated the activity and expression of MMP-9 and -2 in ethanol-induced acute gastric ulceration in rats. We found that severity of gastric ulcer was strongly correlated with increasing doses of ethanol and increased secretion of proMMP-9. ProMMP-9 was upregulated approximately 25-fold at maximum ulcer index. Increased secretion of proMMP-9 was associated with increased expression of tumor necrosis factor-alpha and interleukin-6. We examined the effect of H(2)-receptor antagonists and antioxidants on proMMP-9 secretion and synthesis during prevention of ethanol-induced gastric ulcer. Our data reveal that famotidine dose dependently blocked increased secretion and synthesis of proMMP-9 during gastroprotection and arrested infiltration of inflammatory cells as well as oxidative stress in rat gastric tissues. Similar to H(2)-receptor antagonists, N-acetylcysteine and dimethyl sulfoxide, well-known antioxidants, inhibited proMMP-9 upregulation to the control level. In conclusion, ethanol-induced gastric ulceration is associated with increased expression of proMMP-9 that can be attenuated by H(2)-receptor antagonists and antioxidants. These findings furnish a novel MMP-9-mediated pathway and its inhibition via proinflammatory cytokines by famotidine in ethanol-induced gastric ulceration.

Hydrogen peroxide-mediated downregulation of matrix metalloprotease-2 in indomethacin-induced acute gastric ulceration is blocked by melatonin and other antioxidants.

Gastric mucosal damage is directly associated with extracellular matrix degradation in which matrix metalloproteinases (MMPs) play a crucial role. Remodeling of connective tissues and loss of tissue integrity due to the action of MMPs are reported in several inflammatory diseases, including gastric ulcer. Indomethacin-induced gastric ulceration involves the generation of reactive oxygen species (ROS) and a reduction in MMP-2 transcription and translation. Our aim was to identify the mechanism for suppression of MMP-2 activity by ROS during acute ulceration and further to examine the possible actions of antioxidants, especially melatonin, during healing. Melatonin (N-acetyl-5-methoxytryptamine) blocked hydroxyl radical and nitrite anion generation, protein oxidation, mucosal cell disruption, and MMP-2 downregulation. In addition, suppression of MMP-2 activity by H2O2 in a dose- and time-dependent manner in vitro is blocked by melatonin, omeprazole, and curcumin. We observed that melatonin and other antioxidants (e.g., curcumin and omeprazole) offered gastroprotection in vivo by upregulation of suppressed MMP-2 expression and activity at the level of secretion and synthesis. Moreover, antioxidants reversed the suppression of MMP-2 expression by upregulation of MT1-MMP and downregulation of TIMP-2. Hence, we hypothesize that antioxidants exerted protection against H2O2-mediated inactivation and downregulation of MMP-2 expression during onset of indomethacin-induced ulceration.

Effect of melatonin on secreted and induced matrix metalloproteinase-9 and -2 activity during prevention of indomethacin-induced gastric ulcer.

Matrix metalloproteinases (MMPs) maintain the crucial role in physiological turnover of extracellular matrix (ECM) proteins in gastric tissues. However, a little is known about the relationship of MMPs with ECM degradation during gastric ulceration and ECM remodeling during healing. Our objective was to investigate the effect of melatonin (N-acetyl-5 methoxytryptamine) on the regulation of MMP-9 and MMP-2 activity during prevention of gastric ulcer. In the present study, biochemical and zymographic methods were used to analyze the mechanism of melatonin in indomethacin-induced gastric ulcer in a rat model. Our studies reveal that melatonin dose-dependently downregulates the expression and secretion of pro-MMP-9 which is induced (approximately 10-fold) during indomethacin-induced gastric ulceration. Furthermore, melatonin prevents gastric ulceration in a dose-dependent manner through upregulation (approximately two- to threefold) of both pro-MMP-2 and active MMP-2 at the level of induction as well as secretion. It also prevents gastric ulcers by blocking glutathione depletion and lipid peroxidation in cytosolic and microsomal fractions. The novel findings of this study are attributed to the attenuation of the pro-MMP-9 and increase of MMP-2 activity by pretreatment with melatonin. The finding defines one of the MMP-mediated pathways for melatonin's action in gastric ulcer.

Curcumin regulates expression and activity of matrix metalloproteinases 9 and 2 during prevention and healing of indomethacin-induced gastric ulcer.

Matrix metalloproteinases (MMPs) are suggested to play a critical role in extracellular matrix degradation and remodeling during inflammation and wound healing processes. However, the role of MMPs in indomethacin-induced gastric ulcer and its healing process are not clearly understood. This study is aimed at determining the regulation of MMP-9 and -2 activities in indomethacin-induced acute gastric ulceration and healing. Indomethacin-ulcerated stomach extracts exhibit significant up-regulation of pro-MMP-9 (92 kDa) activity and moderate reduction of MMP-2 activity, which strongly correlate with indomethacin dose and severity of ulcer. The anti-inflammatory and antioxidant properties of curcumin, an active component of turmeric, suggest that curcumin may exert antiulcer activity through scavenging reactive oxygen species, by regulating MMP activity, or both. To test these possibilities, the effect of curcumin in indomethacin-induced gastric ulcer is examined by biochemical and histological methods. The results show that curcumin exhibits potent antiulcer activity in acute ulcer in rat model by preventing glutathione depletion, lipid peroxidation, and protein oxidation. Denudation of epithelial cells during damage of gastric lumen is reversed by curcumin through re-epithelialization. Furthermore, both oral and intraperitoneal administration of curcumin blocks gastric ulceration in a dose-dependent manner. It accelerates the healing process and protects gastric ulcer through attenuation of MMP-9 activity and amelioration of MMP-2 activity. Omeprazole, an established antiulcer drug does not inhibit MMP-9 while protecting indomethacin-induced gastric ulcer. We conclude that antiulcer activity of curcumin is primarily attributed to MMP-9 inhibition, one of the major path-ways of ulcer healing.