Francesco Bellia

Cellular stress response: a novel target for chemoprevention and nutritional neuroprotection in aging, neurodegenerative disorders and longevity

The predominant molecular symptom of aging is the accumulation of altered gene products. Moreover, several conditions including protein, lipid or glucose oxidation disrupt redox homeostasis and lead to accumulation of unfolded or misfolded proteins in the aging brain. Alzheimer’s and Parkinson’s diseases or Friedreich ataxia are neurological diseases sharing, as a common denominator, production of abnormal proteins, mitochondrial dysfunction and oxidative stress, which contribute to the pathogenesis of these so called “protein conformational diseases”. The central nervous system has evolved the conserved mechanism of unfolded protein response to cope with the accumulation of misfolded proteins. As one of the main intracellular redox systems involved in neuroprotection, the vitagene system is emerging as a neurohormetic potential target for novel cytoprotective interventions. Vitagenes encode for cytoprotective heat shock proteins (Hsp) Hsp70 and heme oxygenase-1, as well as thioredoxin reductase and sirtuins. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Thus, the impact of dietary factors on health and longevity is an increasingly appreciated area of research. Reducing energy intake by controlled caloric restriction or intermittent fasting increases lifespan and protects various tissues against disease. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin, a group of proteins linked to aging, metabolism and stress tolerance in several organisms. Recent findings suggest that several phytochemicals exhibit biphasic dose responses on cells with low doses activating signaling pathways that result in increased expression of vitagenes encoding survival proteins, as in the case of the Keap1/Nrf2/ARE pathway activated by curcumin and NAD/NADH-sirtuin-1 activated by resveratrol. Consistently, the neuroprotective roles of dietary antioxidants including curcumin, acetyl-l-carnitine and carnosine have been demonstrated through the activation of these redox-sensitive intracellular pathways. Although the notion that stress proteins are neuroprotective is broadly accepted, still much work needs to be done in order to associate neuroprotection with specific pattern of stress responses. In this review the importance of vitagenes in the cellular stress response and the potential use of dietary antioxidants in the prevention and treatment of neurodegenerative disorders is discussed.

Neuroprotective features of carnosine in oxidative driven diseases.

Carnosine (β-alanyl-L-histidine) is a natural dipeptide widely and abundantly distributed in excitable tissues of several animal tissues. Although its physiological role has not been completely understood yet, many beneficial actions have been attributed to carnosine, such as being an antioxidant, antiglycating and ion-chelating agent, a wound healing promoter and a free-radical scavenger. The role of carnosine in the neuroprotection of oxidative stress-driven disorders has been reviewed. The effects of carnosine have been extensively studied both in vivo and in vitro models of cerebral damages, such as neurodegenerative disorders and hypoxia-ischemia injuries. Beside the classical sacrificial agent, carnosine has been reevaluated as a molecular chaperon and an inducer of antioxidant systems in oxidative stress conditions. Thus, beneficial effects on most of the common biochemical events that characterize neurological disorders make carnosine a very promising molecule among all the endogenous compounds in the treatment and/or prevention of oxidative driven diseases.

Extracellular N-acetylaspartate depletion in traumatic brain injury

N‐Acetylaspartate (NAA) is almost exclusively localized in neurons in the adult brain and is present in high concentration in the CNS. It can be measured by proton magnetic resonance spectroscopy and is seen as a marker of neuronal damage and death. NMR spectroscopy and animal models have shown NAA depletion to occur in various types of chronic and acute brain injury. We investigated 19 patients with traumatic brain injury (TBI). Microdialysis was utilized to recover NAA, lactate, pyruvate, glycerol and glutamate, at 12‐h intervals. These markers were correlated with survival and a 6‐month Glasgow Outcome Score. Eleven patients died and eight survived. A linear mixed model analysis showed a significant effect of outcome and of the interaction between time of injury and outcome on NAA levels (p = 0.009 and p = 0.004, respectively). Overall, extracellular NAA was 34% lower in non‐survivors. A significant non‐recoverable fall was observed in this group from day 4 onwards, with a concomitant rise in lactate–pyruvate ratio and glycerol. These results suggest that mitochondrial dysfunction is a significant contributor to poor outcome following TBI and propose extracellular NAA as a potential marker for monitoring interventions aimed at preserving mitochondrial function.

Administration of carnosine in the treatment of acute spinal cord injury.

L-Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. L-Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha in different experimental settings. In the present study, we investigated the efficacy of L and D-carnosine on the animal model of spinal cord injury (SCI). The spinal cord was exposed via a four-level T5-T8 laminectomy and SCI was produced by extradural compression of the spinal cord at level T6-T7 using an aneurysm clip with a closing force of 24 g. Treatment with D-carnosine (150 mg/kg administered i.p., 1 h and 6h, after SCI), but not L-carnosine significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation, inducible NO synthase (iNOS) and Hsp70 expression, (d) proinflammatory cytokines, and (e) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Furthermore, D-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI) significantly ameliorated the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate the strong difference between L-carnosine and D-carnosine. The result strongly suggests that D-carnosine treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Carnosine derivatives: new multifunctional drug-like molecules

Carnosine (β-alanyl-l-histidine) is an endogenous dipeptide widely and abundantly distributed in the muscle and nervous tissues of several animal species. Many functions have been proposed for this compound because of its antioxidant and metal ion-chelator properties. Many potential therapeutic properties have been recognized especially related to the antioxidant activity, but the therapeutic uses are strongly limited by the mechanism governing its homeostasis. This fact has been the main reason for developing the synthesis of carnosine derivatives with interesting potentiality, but until now there have been very few applications. These derivatives could represent the future drugs for many pathologies related to oxidative stress and metal ion dyshomeostasis.

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: synthesis and characterization of their copper(II) complexes.

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of several animal species. Many functions have been proposed for this compound, such as antioxidant and metal ion-chelator properties. However, the main limitation on therapeutic use of carnosine on pathologies related to increased oxidative stress and/or metal ion dishomeostasis is associated with the hydrolysis by the specific dipeptidase carnosinase. Several attempts have been made to overcome this limitation. On this basis, we functionalized carnosine and its amide derivative with small sugars such as glucose and lactose. The resistance of these derivatives to the carnosinase hydrolysis was tested and compared with that of carnosine. We found that the glycoconjugation protects the dipeptide moiety from carnosinase hydrolysis, thus potentially improving the availability of carnosine. The copper(II) binding properties of all the new synthesized compounds were investigated by spectroscopic (UV-Visible and circular dichroism) and ESI-MS studies. Particularly, the new family of amide derivatives that are not significantly hydrolyzed by carnosinase is a very promising class of carnosine derivatives. The sugar moiety can act as a recognition element. These new derivatives are potentially able to act as chelating agents in the development of clinical approaches for the regulation of metal homeostasis in the field of medicinal inorganic chemistry.

The role of copper(II) and zinc(II) in the degradation of human and murine IAPP by insulin-degrading enzyme

Amylin or islet amyloid polypeptide (IAPP) is a 37-residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β-cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin-degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper- and zinc-induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion-induced changes in substrate accessibility.

Soluble Sugar-Based Quinoline Derivatives as New Antioxidant Modulators of Metal-Induced Amyloid Aggregation

Oxidative stress and protein aggregation have been demonstrated to be the major factors involved in neurodegenerative diseases. Metal ions play a pivotal role, acting as mediators of neurotoxicity either by favoring or redox cycling. Thus, they represent a promising and suitable therapeutic target for the treatment of neurodegenerative disorders. In particular, the development of bifunctional or multifunctional molecules, which have antiaggregant and metal-chelating/antioxidant properties, may be considered as a valuable strategy for the treatment of neurodegeneration considering its multifactorial nature. Herein, we report the design and the characterization of four new multifunctional sugar-appended 8-hydroxyquinolines focusing on the effects of the conjugation with trehalose, a nonreducing disaccharide involved in the protection of proteins and cells against environmental stresses. These glycoconjugates do not exhibit any antiproliferative activity against three human cell lines of different histological origin, unlike 8-hydroxyquinolines. The multiple properties of the new derivatives are highlighted, reporting their Cu(2+) and Zn(2+) binding ability, and antioxidant and antiaggregant capacities. In particular, these latter were determined by different assays, including the evaluation of their ability to modulate or even suppress the aggregation of Aβ1-40 and Aβ1-42 peptides induced by copper or zinc ions.

Carnosinases, Their Substrates and Diseases

Carnosinases are Xaa-His dipeptidases that play diverse functions throughout all kingdoms of life. Human isoforms of carnosinase (CN1 and CN2) under appropriate conditions catalyze the hydrolysis of the dipeptides carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyryl-l-histidine). Alterations of serum carnosinase (CN1) activity has been associated with several pathological conditions, such as neurological disorders, chronic diseases and cancer. For this reason the use of carnosinase levels as a biomarker in cerebrospinal fluid (CSF) has been questioned. The hydrolysis of imidazole-related dipeptides in prokaryotes and eukaryotes is also catalyzed by aminoacyl-histidine dipeptidases like PepD (EC 3.4.13.3), PepV (EC 3.4.13.19) and anserinase (EC 3.4.13.5). The review deals with the structure and function of this class of enzymes in physiological and pathological conditions. The main substrates of these enzymes, i.e., carnosine, homocarnosine and anserine (β-alanyl-3-methyl-l-histidine) will also be described.

Unusual Cyclodextrin Derivatives as a New Avenue to Modulate Self‐ and Metal‐Induced Aβ Aggregation

Mounting evidence suggests an important role of cyclodextrins in providing protection in neurodegenerative disorders. Metal dyshomeostasis is reported to be a pathogenic factor in neurodegeneration because it could be responsible for damage involving oxidative stress and protein aggregation. As such, metal ions represent an effective target. To improve the metal-binding ability of cyclodextrin, we synthesized three new 8-hydroxyquinoline-cyclodextrin conjugates with difunctionalized cyclodextrins. In particular, the 3-difunctionalized regioisomer represents the first example of cyclodextrin with two pendants at the secondary rim, resulting in a promising compound. The derivatives have significant antioxidant capacity and the powerful activity in inhibiting self-induced amyloid-β aggregation seems to be led by synergistic effects of both cyclodextrin and hydroxyquinoline. Moreover, the derivatives are also able to complex metal ions and to inhibit metal-induced protein aggregation. Therefore, these compounds could have potential as therapeutic agents in diseases related to protein aggregation and metal dyshomeostasis.