The role of neutrophil granule proteins in neuroinflammation and Alzheimer's disease.

Neutrophils are the innate immune system's first line of defense. Neutrophils play a critical role in protecting the host against infectious pathogens, resolving sterile injuries, and mediating inflammatory responses. The granules of neutrophils and their constituent proteins are central to these functions. Although neutrophils may exert a protective role upon acute inflammatory conditions or insults, continued activity of neutrophils in chronic inflammatory diseases can contribute to tissue damage. Neutrophil granule proteins are involved in a number of chronic inflammatory conditions and diseases. However, the functions of these proteins in neuroinflammation and chronic neuroinflammatory diseases, including Alzheimer's disease (AD), remain to be elucidated. In this review, we discuss recent findings from our lab and others that suggest possible functions for neutrophils and the neutrophil granule proteins, CAP37, neutrophil elastase, and cathepsin G, in neuroinflammation, with an emphasis on AD. These findings reveal that neutrophil granule proteins may exert both neuroprotective and neurotoxic effects. Further research should determine whether neutrophil granule proteins are valid targets for therapeutic interventions in chronic neuroinflammatory diseases.

The antimicrobial protein, CAP37, is upregulated in pyramidal neurons during Alzheimer's disease.

Inflammation is a well-defined factor in Alzheimer's disease (AD). There is a strong need to identify the molecules contributing to neuroinflammation so that therapies can be designed to prevent immune-mediated neurotoxicity. The cationic antimicrobial protein of 37 kDa (CAP37) is an inflammatory mediator constitutively expressed in neutrophils (PMNs). In addition to antibiotic activity, CAP37 exerts immunomodulatory effects on microglia. We hypothesize that CAP37 mediates the neuroinflammation associated with AD. However, PMNs are not customarily associated with the pathology of AD. This study was therefore designed to identify non-neutrophilic source(s) of CAP37 in brains of AD patients. Brain tissues from patients and age-matched controls were analyzed for CAP37 expression using immunohistochemistry (IHC). To determine factors that induce CAP37 in AD, HCN-1A primary human neurons were treated with tumor necrosis factor-alpha (TNF-α) or amyloid ß1-40 (Aß) and analyzed by IHC. Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to confirm CAP37 expression in neurons and brain tissues. IHC revealed CAP37 in cortical neurons in temporal and parietal lobes as well as CA3 and CA4 hippocampal neurons in patients with AD. CAP37 was found in more neurons in AD patients compared with age-matched controls. qRT-PCR and Western blotting showed an increase in CAP37 transcript and protein in the AD temporal lobe, a brain region that is highly impacted in AD. qRT-PCR observations confirmed CAP37 expression in neurons. TNF-α and Aß increased neuronal expression of CAP37. These findings support our hypothesis that neuronal CAP37 may modulate the neuroinflammatory response in AD.

Alpha-phenyl-N-tert-butylnitrone (PBN) prevents light-induced degeneration of the retina by inhibiting RPE65 protein isomerohydrolase activity.

α-Phenyl-N-tert-butylnitrone (PBN), a free radical spin trap, has been shown previously to protect retinas against light-induced neurodegeneration, but the mechanism of protection is not known. Here we report that PBN-mediated retinal protection probably occurs by slowing down the rate of rhodopsin regeneration by inhibiting RPE65 activity. PBN (50 mg/kg) protected albino Sprague-Dawley rat retinas when injected 0.5-12 h before exposure to damaging light at 2,700 lux intensity for 6 h but had no effect when administered after the exposure. PBN injection significantly inhibited in vivo recovery of rod photoresponses and the rate of recovery of functional rhodopsin photopigment. Assays for visual cycle enzyme activities indicated that PBN inhibited one of the key enzymes of the visual cycle, RPE65, with an IC(50) = 0.1 mm. The inhibition type for RPE65 was found to be uncompetitive with K(i) = 53 µm. PBN had no effect on the activity of other visual cycle enzymes, lecithin retinol acyltransferase and retinol dehydrogenases. Interestingly, a more soluble form of PBN, N-tert-butyl-α-(2-sulfophenyl) nitrone, which has similar free radical trapping activity, did not protect the retina or inhibit RPE65 activity, providing some insight into the mechanism of PBN specificity and action. Slowing down the visual cycle is considered a treatment strategy for retinal diseases, such as Stargardt disease and dry age-related macular degeneration, in which toxic byproducts of the visual cycle accumulate in retinal cells. Thus, PBN inhibition of RPE65 catalytic action may provide therapeutic benefit for such retinal diseases.

Selecting Multitarget Peptides for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial disease with a complex pathogenesis. Developing multitarget drugs could be a powerful strategy to impact the progressive loss of cognitive functions in this disease. The purpose of this study is to select a multitarget lead peptide candidate among a series of peptide variants derived from the neutrophil granule protein cathepsin G. We screened eight peptide candidates using the following criteria: (1) Inhibition and reversion of amyloid beta (Aß) oligomers, quantified using an enzyme-linked immunosorbent assay (ELISA); (2) direct binding of peptide candidates to the human receptor for advanced glycation end-products (RAGE), the Toll-like receptor 4 (TLR4) and the S100 calcium-binding protein A9 (S100A9), quantified by ELISA; (3) protection against Aß oligomer-induced neuronal cell death, using trypan blue to measure cell death in a murine neuronal cell line; (4) inhibition of TLR4 activation by S100A9, using a human TLR4 reporter cell line. We selected a 27-mer lead peptide that fulfilled these four criteria. This lead peptide is a privileged structure that displays inherent multitarget activity. This peptide is expected to significantly impact cognitive decline in mouse models of Alzheimer's disease, by targeting both neuroinflammation and neurodegeneration.

Neutrophil Granule Proteins Inhibit Amyloid Beta Aggregation and Neurotoxicity.

BACKGROUND: A role for neutrophils in the pathogenesis of Alzheimer's disease (AD) is emerging. We previously showed that the neutrophil granule proteins cationic antimicrobial protein of 37 kDa (CAP37), cathepsin G (CG), and neutrophil elastase (NE) directly bind the amyloid-beta peptide Aß1-42, a central player in AD pathogenesis. CAP37, CG, and NE are serine proteases that can cleave Aß1-42 at different sites and with different catalytic activities. OBJECTIVE: In this study, we compared the effects of these three proteins on Aß1-42 fibrillation and neurotoxicity. METHODS: Using mass spectrometry and in vitro aggregation assay, we found that NE and CG efficiently cleave Aß1-42. This cleavage correlates well with the inhibition of Aß1-42 aggregation into fibrils. In contrast, CAP37 did not efficiently cleave Aß1-42, but was still able to inhibit its fibrillation, most likely through a quenching effect. Inhibition of Aß1-42 aggregation by NE and CG neutralized its toxicity measured in cultured neurons. In contrast, inhibition of Aß1-42 aggregation by CAP37 did not inhibit its neurotoxicity. RESULTS: We found that a peptide derived from CAP37 could mimic the quenching and inhibition of Aß1-42 aggregation effects of the full-length protein. Additionally, this peptide was able to inhibit the neurotoxicity of the most toxic Aß1-42 aggregate, an effect that was not found with the full-length CAP37. CONCLUSION: These results shed light on the mechanisms of action of neutrophil granule proteins with regard to inhibition of Aß1-42 aggregation and neurotoxicity and open up a possible strategy for the discovery of new disease-modifying drugs for AD.

Antimicrobial Peptides Derived from the Immune Defense Protein CAP37 Inhibit TLR4 Activation by S100A9.

Purpose: Corneal abrasion is a common eye injury, and its resolution can be seriously complicated by bacterial infection. We showed that topical application of the cationic antimicrobial protein of 37 kDa (CAP37) promotes corneal re-epithelialization in mice, and peptides derived from CAP37 can recapitulate the antibacterial and wound-healing effects of the full-length protein. The current study was designed to identify the molecular mechanisms mediating the wound-healing effect of CAP37 and derived bioactive peptides. Methods: We used a TriCEPS-based, ligand-receptor glycocapture method to identify the binding partners of CAP37 on live human corneal epithelial cells using the hTCEpi cell line. We used an ELISA method to confirm binding with identified partners and test the binding with CAP37-derived peptides. We used a reporter cell line to measure activation of the identified membrane receptor by CAP37 and derived peptides. Results: We pulled down S100 calcium-binding protein A9 (S100A9) as a binding partner of CAP37 and found that CAP37 and four derived peptides encompassing two regions of CAP37 bind S100A9 with high affinities. We found that CAP37 and the S100A9-binding peptides could also directly interact with the Toll-like receptor 4 (TLR4), a known receptor for S100A9. CAP37 and one peptide partially activated TLR4. The other three peptides did not activate TLR4. Finally, we found that CAP37 and all four peptides could inhibit the activation of TLR4 by S100A9. Conclusions: This study identifies a mechanism of action for CAP37 and derived antimicrobial peptides that may restrain inflammatory responses to corneal injury and favor corneal re-epithelialization.

N-acetylcarnosine lubricant eyedrops possess all-in-one universal antioxidant protective effects of L-carnosine in aqueous and lipid membrane environments, aldehyde scavenging, and transglycation activities inherent to cataracts: a clinical study of the new vision-saving drug N-acetylcarnosine eyedrop therapy in a database population of over 50,500 patients.

The antioxidant activity of L-carnosine (beta-alanyl-L-histidine, bioactivated in ocular tissues) versus N-acetylcarnosine (N-acetyl-beta-alanyl-L-histidine, ocular-targeted small dipeptide molecules) was studied in aqueous solution and in a lipid environment, employing liposomes as a model of lipid membranes. Reactive oxygen species (ROS) were generated by an iron/ascorbate promoter system for induction of lipid peroxidation (LPO). L-carnosine, which is stabilized from enzymatic hydrolysis, operates as a universal aldehyde and ROS scavenger in both aqueous and lipid environments and is effective at preventing ROS-induced damage to biomolecules. Second-generation carnosine analogs bearing the histidyl-hydrazide moiety were synthesized and tested versus L-carnosine for their ability to reverse the glycation process, also known as the Maillard reaction, and reverse the stable intermolecular cross-links, monitored in the glucose-ethylamine Schiff base model, ultimately resulting in the formation of the advanced glycation end products (AGEs) from nonenzymatic glycation, accumulating in numerous body tissues and fluids. The obtained data demonstrate the transglycation properties of the ophthalmically stabilized L-carnosine and L-carnosine histidyl-hydrazide derivatives tested and can be used to decrease or predict the occurrence of long-term complications of AGE formation and improve therapeutically the quality of vision and length of life for diabetes mellitus patients and survivors with early aging. Scientists at Innovative Vision Products, Inc. (IVP), developed lubricant eyedrops designed as a sustained-release 1% N-acetylcarnosine prodrug of L-carnosine. The eyedrops contain a mucoadhesive cellulose-based compound combined with corneal absorption promoters and glycerine in a drug-delivery system. Anti-aging therapeutics with the ophthalmic drug eyedrop formula including N-acetylcarnosine showed efficacy in the nonsurgical treatment of age-related cataracts for enrolled participants in the prospective, randomized, double-masked, placebo-controlled crossover clinical trial after controlling for age, gender, and daily activities. In a cohort in excess of 50,500 various patients seeking cutting-edge medical care, the N-acetylcarnosine topical eyedrops target therapy was demonstrated to have significant efficacy, safety, and good tolerability for the prevention and treatment of visual impairment in this older population with relatively stable patterns of causes for blindness and visual impairment. Overall, accumulated study data demonstrate that the IVP-designed new vision-saving drugs, including N-acetylcarnosine eyedrops, promote health vision and prevent vision disability from senile cataracts, primary open-angle glaucoma, age-related macular degeneration, diabetic retinopathy, and aging. N-acetylcarnosine eyedrop therapy is the crown jewel of the anti-aging medical movement and revolutionizes early detection, treatment, and rejuvenation of aging-related eye-disabling disorders. N-acetylcarnosine, as an innovative medical science tool and component of the home medicine and alternative medicine approaches, has the potential to alleviate visual impairment and its associated social, economic, and political woes for an aging population.

Retinol dehydrogenases RDH11 and RDH12 in the mouse retina: expression levels during development and regulation by oxidative stress.

PURPOSE: RDH11 and RDH12 are closely related retinol dehydrogenases expressed in the retina. RDH12 has been linked to the early-onset retinal dystrophy Leber congenital amaurosis, whereas RDH11 has not been associated with human disease. To understand their physiological roles, the authors investigated their expression during development and their regulation by light-induced oxidative stress in mouse retina. METHODS: Quantitative RT-PCR and immunoblot analysis were used for quantification of RDH11 and RDH12 during development and oxidative stress. Expression during development was measured between embryonic day (E) 12 and postnatal day (P) 210 (7 months) in C57BL/6 mouse eyes. Expression during light-induced oxidative stress was measured between 2 and 24 hours of exposure to light in BALB/c mouse retina. RESULTS: The RDH11 level was low and remarkably constant during development and oxidative stress. RDH12 expression started at P7 and increased until P30 to approximately sevenfold higher than RDH11. Oxidative stress induced by exposure to constant bright light led to a rapid and significant decrease of RDH12 protein. CONCLUSIONS: The low and constant expression of RDH11 suggested a housekeeping function for this enzyme. The onset of RDH12 expression during the maturation of photoreceptor cells suggested a function related to the visual process. The light-induced rapid decrease of RDH12 protein, preceding the decrease of the mRNA, suggested a specific degradation of the protein rather than a regulation of gene expression.

Bioactive Antimicrobial Peptides as Therapeutics for Corneal Wounds and Infections.

Significance: More than 2 million eye injuries and infections occur each year in the United States that leave civilians and military members with reduced or complete vision loss due to the lack of effective therapeutics. Severe ocular injuries and infections occur in varied settings including the home, workplace, and battlefields. In this review, we discuss the potential of developing antimicrobial peptides (AMPs) as therapeutics for the treatment of corneal wounds and infections for which the current treatment options are inadequate. Recent Advances: Standard-of-care employs the use of fluorescein dye for the diagnosis of ocular defects and is followed by the use of antibiotics and/or steroids to treat the infection and reduce inflammation. Recent advances for treating corneal wounds include the development of amniotic membrane therapies, wound chambers, and drug-loaded hydrogels. In this review, we will discuss an innovative approach using AMPs with the dual effect of promoting corneal wound healing and clearing infections. Critical Issues: An important aspect of treating ocular injuries is that treatments need to be effective and administered expeditiously. This is especially important for injuries that occur during combat and in individuals who demonstrate delayed wound healing. To overcome gaps in current treatment modalities, bioactive peptides based on naturally occurring cationic antimicrobial proteins are being investigated as new therapeutics. Future Directions: The development of new therapeutics that can treat ocular infections and promote corneal wound healing, including the healing of persistent corneal epithelial defects, would be of great clinical benefit.

Effect of Cationic Antimicrobial Protein CAP37 on Cytokine Profile during Corneal Wound Healing.

The cationic antimicrobial protein of 37 kDa (CAP37) mediates proliferation, migration, and adhesion of human corneal epithelial cells and promotes corneal re-epithelialization in mouse. The purpose of this study was to investigate the cytokine profile following abrasion of the corneal epithelium, and to identify the cytokines modulated by topical treatment with CAP37 to determine the mechanism by which CAP37 contributes to the recruitment of inflammatory cells and healing of the cornea. The corneal epithelium in mouse eyes was removed and wounds were treated with a saline vehicle or human recombinant CAP37. Wounds were visualized with fluoresce in staining at 0, 16, 24 and 48 h. Mouse corneas were excised at 0, 6, 16, 24 and 48 h post corneal abrasion. The excised corneas were analyzed by immunohistochemistry for re-epithelialization and infiltration of inflammatory cells while the expression profiles of thirty-two cytokines were investigated by multiplex analysis. Results corroborating previous studies showed accelerated wound closure in corneas treated with CAP37 compared to those treated with the saline vehicle. Immunohistochemistry revealed less neutrophil infiltration in CAP37-treated corneas when compared to controls at 24 h. By 48 h post-wounding, histological analysis revealed more staining for neutrophils than the staining observed in the controls. Modulation of cytokine expression occurred for the majority of the cytokines tested at the time of corneal abrasion, during re-epithelialization, and/or by CAP37 treatment. Cytokines monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) were induced during re-epithelialization, at the early 16 h time point. Interleukin 6 (IL-6), leukemia inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), IL-12p70, macrophage inflammatory protein 1 beta (MIP-1ß), and interferon gamma-induced protein 10 (IP-10) were induced at 24 h and unchanged during CAP37 treatment. By contrast, IL-15, monokine induced by gamma interferon (MIG), keratinocyte-derived cytokine (KC), tumor necrosis factor alpha (TNF-α), MIP-1α, IL-1ß, and macrophage colony-stimulating factor (M-CSF) were modulated by CAP37 treatment. In general, CAP37 appeared to decrease pro-inflammatory cytokines at 24 h and increase them at 48 h when compared to the control group. These data demonstrate that CAP37 modulates the production of cytokines in the cornea and suggest that limiting the number of neutrophils recruited during the early inflammatory phase may support corneal re-epithelialization.

The Role of Neutrophil Proteins on the Amyloid Beta-RAGE Axis.

We previously showed an elevated expression of the neutrophil protein, cationic antimicrobial protein of 37kDa (CAP37), in brains of patients with Alzheimer's disease (AD), suggesting that CAP37 could be involved in AD pathogenesis. The first step in determining how CAP37 might contribute to AD pathogenesis was to identify the receptor through which it induces cell responses. To identify a putative receptor, we performed GAMMA analysis to determine genes that positively correlated with CAP37 in terms of expression. Positive correlations with ligands for the receptor for advanced glycation end products (RAGE) were observed. Additionally, CAP37 expression positively correlated with two other neutrophil proteins, neutrophil elastase and cathepsin G. Enzyme-linked immunosorbent assays (ELISAs) demonstrated an interaction between CAP37, neutrophil elastase, and cathepsin G with RAGE. Amyloid beta 1-42 (Aß1-42), a known RAGE ligand, accumulates in AD brains and interacts with RAGE, contributing to Aß1-42 neurotoxicity. We questioned whether the binding of CAP37, neutrophil elastase and/or cathepsin G to RAGE could interfere with Aß1-42 binding to RAGE. Using ELISAs, we determined that CAP37 and neutrophil elastase inhibited binding of Aß1-42 to RAGE, and this effect was reversed by protease inhibitors in the case of neutrophil elastase. Since neutrophil elastase and cathepsin G have enzymatic activity, mass spectrometry was performed to determine the proteolytic activity of all three neutrophil proteins on Aß1-42. All three neutrophil proteins bound to Aß1-42 with different affinities and cleaved Aß1-42 with different kinetics and substrate specificities. We posit that these neutrophil proteins could modulate neurotoxicity in AD by cleaving Aß1-42 and influencing the Aß1-42 -RAGE interaction. Further studies will be required to determine the biological significance of these effects and their relevance in neurodegenerative diseases such as AD. Our findings identify a novel area of study that underscores the importance of neutrophils and neutrophil proteins in neuroinflammatory diseases such as AD.

A multifunctional peptide based on the neutrophil immune defense molecule, CAP37, has antibacterial and wound-healing properties.

CAP37, a protein constitutively expressed in human neutrophils and induced in response to infection in corneal epithelial cells, plays a significant role in host defense against infection. Initially identified through its potent bactericidal activity for Gram-negative bacteria, it is now known that CAP37 regulates numerous host cell functions, including corneal epithelial cell chemotaxis. Our long-term goal is to delineate the domains of CAP37 that define these functions and synthesize bioactive peptides for therapeutic use. We report the novel finding of a multifunctional domain between aa 120 and 146. Peptide analogs 120-146 QR, 120-146 QH, 120-146 WR, and 120-146 WH were synthesized and screened for induction of corneal epithelial cell migration by use of the modified Boyden chamber assay, antibacterial activity, and LPS-binding activity. In vivo activity was demonstrated by use of mouse models of sterile and infected corneal wounds. The identity of the amino acid at position 132 (H vs. R) was important for cell migration and in vivo corneal wound healing. All analogs demonstrated antimicrobial activity. However, analogs containing a W at position 131 showed significantly greater antibacterial activity against the Gram-negative pathogen Pseudomonas aeruginosa. All analogs bound P. aeruginosa LPS. Topical administration of analog 120-146 WH, in addition to accelerating corneal wound healing, effectively cleared a corneal infection as a result of P. aeruginosa. In conclusion, we have identified a multifunctional bioactive peptide, based on CAP37, that induces cell migration, possesses antibacterial and LPS-binding activity, and is effective at healing infected and noninfected corneal wounds in vivo.

Regulation and functional roles of Grb14.

Grb14 is the last described member of the Grb7 family of adaptors, containing Grb7, Grb10 and Grb14. These proteins share a series of conserved domains involved in protein-protein and protein-lipid interactions: an amino terminal proline-rich region, a C-terminal SH2 domain, and a central GM region containing a RA, a PH domain, and a newly described PIR (BPS) region. As shown for the other members of the Grb7/10/14 family, Grb14 binds to various receptor tyrosine kinases (RTKs) under ligand induction. This interaction involves the SH2 and PIR domains, and the respective participation of these domains is likely to be a determinant in the specificity of action of Grb14. At the present time, a role for this Grb14-RTK interaction was established only for insulin (IR) and FGF receptors (FGFR). Grb14, through its PIR, is an inhibitor of IR tyrosine kinase activity and thus of insulin effects. Grb14 also decreases FGF signaling, but more probably by interfering with cellular effectors downstream from the receptor. Only a few cytosolic partners of Grb14 are identified. One of them, the adaptor ZIP, allows phosphorylation of Grb14, and regulation of its inhibitory action on IR signaling. The identification of further proteins interacting with Grb14 is required to elucidate the biological role of this protein.

Corneal wound healing, a newly identified function of CAP37, is mediated by protein kinase C delta (PKCδ).

PURPOSE: The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway. METHODS: The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA. RESULTS: We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA. CONCLUSIONS: These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.

Novel neuroendocrine and metabolic mechanism provides the patented platform for important rejuvenation therapies: targeted therapy of telomere attrition and lifestyle changes of telomerase activity with the timing of neuron-specific imidazole-containing dipeptide-dominant pharmaconutrition provision.

Telomere length is emerging as a biomarker for aging and survival is paternally inherited and associated with parental lifespan. Telomere-associated cellular senescence may contribute to certain age-related disorders, including an increase in cancer incidence, wrinkling and diminished skin elasticity, atherosclerosis, osteoporosis, weight loss, age-related cataract, glaucoma and others. Shorter telomere length in leukocytes was associated cross-sectionally with cardiovascular disorders and its risk factors, including pulse pressure and vascular aging, obesity, vascular dementia, diabetes, coronary artery disease, myocardial infarction (although not in all studies), cellular turnover and exposure to oxidative and inflammatory damage in chronic obstructive pulmonary disease. Effective regulation of abnormal therapeutic targets of an age-related disease requires the alteration of either the topological structure or dynamic characteristics of telomeres which are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation, including those as the metabolic super-achievers in the body, organ systems in a given target network of a disease and aging. In order to manage and control the complex direct and indirect target hubs, in this paper, a review of the recent patents is made analyzing techniques, new approaches developed during the last years in adaptive pharmacology directed at slowing and preventing the loss of telomere length that may slow aging using pharmaceutical and nutritional module-based designs, such as with regard to the timing of administration of imidazole-containing dipeptides. We discuss our recent identification of the role of neuron-specific imidazole- containing dipeptide based compounds (L-carnosine, N-acetylcarnosine, carcinine) that regulate and therapeutically control telomere shortening, telomerase activity and cellular senescence. We support a therapeutic concept of using nonhydrolyzed forms of naturally occurring imidazole-dipeptide based compounds carnosine and carcinine, making it clinically possible that slowing down the rate of telomere shortening could slow down the human aging process in specific tissues where proliferative senescence is known to occur with the demonstrated evidence of telomere shortening appeared to be a hallmark of oxidative stress and disease. The preliminary longitudinal studies of elderly individuals suggest that longer telomeres are associated with better survival and an advanced oral pharmaconutrition provision with non-hydrolyzed carnosine (or carcinine and patented compositions thereof) is a useful therapeutic tool of a critical telomere length maintenance (allowing indirectly to manipulate with telomerase activity) that may fundamentally be applied in the therapeutic treatment of agerelated sight-threatening eye disorders, Diabetes mellitus, sarcopenia (that is the gradual loss of muscle mass) that can affect elderly people and subjects under the effect of exhausting exercises and physical load, prolong life expectancy, increase survival and chronological age of an organism in health control, smoking behavior, metabolic syndrome increasing the risk of developing cardio-vascular diseases, age-related neurodegenerative diseases, including Alzheimer's disease and cognitive impairment.

CAP37 activation of PKC promotes human corneal epithelial cell chemotaxis.

PURPOSE: The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis. METHODS: Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 µM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr(505) phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively. RESULTS: Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr(505). Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay. CONCLUSIONS: These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.

Novel intraocular and systemic absorption drug delivery and efficacy of N-acetylcarnosine lubricant eye drops or carcinine biologics in pharmaceutical usage and therapeutic vision care.

The latest estimates of the World Health Organization indicate that there are 161 million visually impaired individuals worldwide, 37 million of whom are blind, with a yearly increase of 1-2 million. The scientists developed and patented the lubricant eye drops formulated as 1%N-acetylcarnosine prodrug of l-carnosine containing a mucoadhesive cellulose-based compound combined with corneal absorption promoters in an ocular drug delivery system. Carcinine is suitable for the systemic administration (per oral) for ophthalmic therapeutic indications. The HPLC analysis was developed to search the pathways of ocular metabolic activities of 1%N-acetylcarnosine and the bioactivation of this drug molecule promoting transcorneal uptake of l-carnosine in the aqueous humor. A meta-analysis of phase 2 randomized double-blind placebo-controlled clinical trial data was conducted. The intraocular absorbed l-carnosine demonstrated a number of pharmacological mechanisms of prevention and reversal of cataracts. Results of systemic absorption of l-carnosine provide tuberomammillary activation that regulates neuronal functions such as hypothalamic control promoting sensory input in the primary vision perceptual pathway. The parabulbar, subconjunctival, and intravitreal injection of carcinine with most of the vehicle removed is not toxic to intraocular structures, reduces postoperative intraocular inflammation, is a potentially useful tool in the treatment of proliferative vitreoretinopathy as well as considered as the antiapoptotic drug for the protection of photoreceptor cells from oxidative light-induced stress. The discovery of naturally occurring carnosine derivatives introduces N-acetylcarnosine and carcinine as effective medical treatment for sight-threatening eye disorders.

Carcinine has 4-hydroxynonenal scavenging property and neuroprotective effect in mouse retina.

PURPOSE: Oxidative stress induces retinal damage and contributes to vision loss in progressive retinopathies. Carcinine (ß-alanyl-histamine) is a natural imidazole-containing peptide derivative with antioxidant activity. It is predicted to scavenge 4-hydroxynonenal (4-HNE), a toxic product of lipid oxidation. The aim of this study was to confirm the 4-HNE scavenging effect and evaluate the neuroprotective effect of carcinine in mouse retina subjected to oxidative stress. METHODS: HPLC coupled with mass spectrometry was used to analyze carcinine and 4-HNE-carcinine adduct. Protection of retinal proteins from modification by 4-HNE was tested by incubating carcinine with retinal protein extract and 4-HNE. Modified retinal proteins were quantified by dot-blot analysis. Mice were treated with carcinine (intravitreal injection and gavage) and exposed to bright light to induce oxidative damage in the retina. Photoreceptor degeneration was measured by histology and electroretinography. Retinal levels of retinol dehydrogenase 12 (RDH12) were measured by immunoblot analysis, after exposure to bright light and in retinal explants after exposure to 4-HNE. RESULTS: The ability of carcinine to form an adduct with 4-HNE, as well as to prevent and even reverse the adduction of retinal proteins by the toxic aldehyde was demonstrated in vitro. Carcinine, administered by intravitreal injection or gavage, strongly protected mouse retina against light-induced photoreceptor degeneration and had a protective effect on RHD12, a protein found specifically in photoreceptor cells. CONCLUSIONS: This study suggests that carcinine can be administered noninvasively to efficiently protect photoreceptor cells from oxidative damage. Carcinine could be administered daily to prevent vision loss in progressive retinopathies.

Retinol dehydrogenase 12 detoxifies 4-hydroxynonenal in photoreceptor cells.

Mutations of the photoreceptor retinol dehydrogenase 12 (RDH12) gene cause the early onset retinal dystrophy Leber congenital amaurosis (LCA) by mechanisms not completely resolved. Determining the physiological role of RDH12 in photoreceptors is the focus of this study. Previous studies showed that RDH12, and the closely related retinol dehydrogenase RDH11, can enzymatically reduce toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE), in vitro. To explore the significance of this activity, we investigated the ability of RDH11 and RDH12 to protect stably transfected HEK-293 cells against the toxicity of 4-HNE. Both enzymes protected against 4-HNE modification of proteins and 4-HNE-induced apoptosis in HEK-293 cells. In the retina, exposure to bright light induced lipid peroxidation, 4-HNE production, and 4-HNE modification of proteins in photoreceptor inner segments, where RDH11 and RDH12 are located. In mouse retina, RDH12-but not RDH11-protected against adduct formation, suggesting that 4-HNE is a physiological substrate of RDH12. RDH12-but not RDH11-also protected against light-induced apoptosis of photoreceptors. We conclude that in mouse retina RDH12 reduces 4-HNE to a nontoxic alcohol, protecting cellular macromolecules against oxidative modification and protecting photoreceptors from light-induced apoptosis. This activity is of particular significance to the understanding of the molecular mechanisms of RDH12-induced LCA.