The ultraviolet (UV) component of solar radiation is the major driving force of skin carcinogenesis. Most of studies on UV carcinogenesis actually focus on DNA damage while their proteome-damaging ability and its contribution to skin carcinogenesis have remained largely underexplored. A redox proteomic analysis of oxidized proteins in solar-induced neoplastic skin lesion and perilesional areas has been conducted showing that the protein oxidative burden mostly concerns a selected number of proteins participating to a defined set of functions, namely: chaperoning and stress response; protein folding/refolding and protein quality control; proteasomal function; DNA damage repair; protein- and vesicle-trafficking; cell architecture, adhesion/extra-cellular matrix (ECM) interaction; proliferation/oncosuppression; apoptosis/survival, all of them ultimately concurring either to structural damage repair or to damage detoxication and stress response. In peri-neoplastic areas the oxidative alterations are conducive to the persistence of genetic alterations, dysfunctional apoptosis surveillance, and a disrupted extracellular environment, thus creating the condition for transformant clones to establish, expand and progress. A comparatively lower burden of oxidative damage is observed in neoplastic areas. Such a finding can reflect an adaptive selection of best fitting clones to the sharply pro-oxidant neoplastic environment. In this context the DNA damage response appears severely perturbed, thus sustaining an increased genomic instability and an accelerated rate of neoplastic evolution. In conclusion UV radiation, in addition to being a cancer-initiating agent, can act, through protein oxidation, as a cancer-promoting agent and as an inducer of genomic instability concurring with the neoplastic progression of established lesions.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory, reasoning and other cognitive functions. Pathologically, patients with AD are characterized by deposition of senile plaques (SPs), formed by β-amyloid (Aβ), and neurofibrillary tangles (NTFs) that consist of aggregated hyperphosphorylated tau protein. The accumulation of insoluble protein aggregates in AD brain can be associated with an impairment of degradative systems. This current study investigated if the disturbance of protein polyubiquitination is associated with AD neurodegeneration. By using a novel proteomic approach, we found that 13 brain proteins are increasingly polyubiquitinated in AD human brain compared to age-matched controls. Moreover, the majority of the identified proteins were previously found to be oxidized in our prior proteomics, and these proteins are mainly involved in protein quality control and glucose metabolism. This is the first study showing alteration of the poly-ubiquitin profile in AD brain compared with healthy controls. Understanding the onset of the altered ubiquitin profile in AD brain may contribute to identification of key molecular regulators of cognitive decline. In AD, deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins that are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology and cognitive decline.

Brain insulin resistance is associated with an increased Aβ production in AD although the molecular mechanisms underlying this link are still largely unknown. Biliverdin reductase-A (BVR-A) is a unique Ser/Thr/Tyr kinase regulating insulin signalling. Studies from our group, demonstrated that BVR-A impairment is among the earliest events favoring brain insulin resistance development. Furthermore, reported a negative association between BVR-A protein levels/activation and BACE1 protein levels in the parietal cortex of aged beagles (an animal model of AD), thus suggesting a possible interaction. Therefore, we aimed to demonstrate that BVR-A impairment is a molecular bridge linking brain insulin resistance with increased Aβ production. Age-associated changes of BVR-A, BACE1, insulin signalling cascade and APP processing were evaluated in the parietal cortex of beagles and experiments to confirm the hypothesized mechanism(s) have been performed in vitro in HEK293APPswe cells. Our results show that BVR-A impairment occurs early with age and is associated with brain insulin resistance. Furthermore, we demonstrate that BVR-A impairment favors CK1-mediated Ser phosphorylation of BACE1 (known to mediate BACE1 recycling to plasma membrane) along with increased Aβ production in the parietal cortex, with age. Overall, our results suggest that the impairment of BVR-A is an early molecular event contributing to both (I) the onset of brain insulin resistance and (II) the increased Aβ production observed in AD. We, therefore, suggest that by targeting BVR-A activity it could be possible to delay the onset of brain insulin resistance along with an improved regulation of the APP processing.

Impairment of biliverdin reductase-A (BVR-A) is an early event leading to brain insulin resistance in AD. Intranasal insulin (INI) administration is under evaluation as a strategy to alleviate brain insulin resistance; however, the molecular mechanisms underlying INI beneficial effects are still unclear. We show that INI improves insulin signaling activation in the hippocampus and cortex of adult and aged 3×Tg-AD mice by ameliorating BVR-A activation. These changes were associated with a reduction of nitrosative stress, Tau phosphorylation, and Aβ oligomers in brain, along with improved cognitive functions. The role of BVR-A was strengthened by showing that cells lacking BVR-A: (i) develop insulin resistance if treated with insulin and (ii) can be recovered from insulin resistance only if treated with a BVR-A-mimetic peptide. These novel findings shed light on the mechanisms underlying INI treatment effects and suggest BVR-A as potential therapeutic target to prevent brain insulin resistance in AD.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3+ lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3+ T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.

Biliverdin reductase-A (BVR-A) is a novel direct target of the insulin receptor, which phosphorylate BVR-A activating its Ser/Thr/Tyr kinase activity. Through this activity, BVR-A negatively regulates IRS1 activation, thus allowing the correct transduction of the insulin-mediated signaling. Along the progression of AD pathology we identified two phases in which: (1) the early impairment of BVR-A is responsible for the hyper-activation of IRS1, which then (2) causes the stimulation of feedback mechanisms including mTOR, aimed to turn-off IRS1 hyper-activity (Fig.1), thus promoting brain insulin resistance (b.i.r.). Reduced BVR-A activity is therefore an early event triggering the onset b.i.r̤ Intranasal insulin (I-Ins) administration is under evaluation as therapeutic strategy to alleviate b.i.r. in AD. However, the exact molecular mechanisms underlying I-Ins beneficial effects are still unclear. The goal of our project was to clarify whether the I-Ins-associated beneficial effects were mediated by the restoration of BVR-A activity. Changes of (1) the insulin signaling machinery (IR/IRS1/ ERK1/2/AKT/mTOR levels and activation) (2) total OS markers (PC, HNE, 3-NT) and (3) Aβ and tau levels, were evaluated in the hippocampus and cortex of 3xTg-AD and WT mice undergoing an early (4 months) or late (10 moths) I-Ins treatment (1 U/day, 3 times per week, for 2 months) (Fig.2). The morris water maze (MWM) and the novel object recognition (NOR) tasks were used to test cognitive functions. Cell-based experiments to support in vivo data were performed in HEK-APPSwe cells. I-Ins administration rescues the activation of BVR-A both in young and old 3xTg-AD mice. Improved BVR-A activity is associated with (1) a restoration of the insulin signaling cascade, (2) reduced OS markers and (3) a reduction of Tau pathology. All these changes parallel an improved cognition (Fig.3). Cell-based experiments confirmed the central role of BVR-A by showing that the effects of insulin are abolished when BVR-A is knocked-down. Our data highlight that BVR-A plays a pivotal role in the regulation of the insulin signaling in the brain. Restoration of BVR-A activity first, sheds light on the molecular mechanisms underlie I-Ins-mediated beneficial effects, and then suggest the role of BVR-A as potential therapeutic target to prevent b.i.r.in AD. (A) Proposed mechanism leading to b.i.r. in AD. Under physiological condition BVR-A regulates the acitvation of IRS1, thus controlling the correct transduction of insulin signaling cascade. In 3xTg-AD mice, an early impairment of BVR-A (6 months) is responsible for the hyper-activation of IRS1, which sustains the activation of the insulin signaling cascade for a time longer than normal (6 months). This phenomenon is aggravated by the further rise of the oxidative/nitrosative stress levels (PC, HNE and 3-NT), which results in the oxidative stress-induced impairment of BVR-A, which finally contributes to maintain IRS1 hyperactive. Persistence of IRS1 hyper-activation represents a signal for the activation of feedback mechanisms including mTOR, which at the end is responsible for the inhibitory phospshorylation of IRS1 and thus for the onset of brain insulin resistance (b.i.r.) (12 months). (B) Temporal profile of the events promoting BIR in the hippocampus 3×Tg-AD mice. Arrows, promotion; dotted lines, inhibition; Y, phosphor-Tyr residues; S, phospho-Ser residues; Y-NO2, 3-NT modifications. Scheme of the treatment used in this project. Intranasal insulin (I-Ins) administration prevents the impairment of BVR-A and the hyper-activation of IRS1. Normalization of the activity of the BVR-A/IRS1 axis is associated the correct transduction of the insulin signaling cascade, which finally prevents the cognitive decline observed in the 3xTg-AD mice.

Down Syndrome (DS) individuals by the age of 40ys develop a type of dementia that has the same characteristics as Alzheimer disease (AD). Previous studies in DS and AD brain suggest common neurodegenerative pathways including mitochondrial dysfunction, oxidative stress (OS) and reduced glucose metabolism. In addition, several studies suggest a link between insulin resistance and cognitive dysfunction in AD. The present study aims to analyze the crosstalk between the onset of brain insulin resistance (BIR) and OS as possible contributing factors to the neurodegenerative process in tg mouse model of DS (Ts65Dn). We longitudinally analyze (at 1–3-9–18 months) changes of i) IR/IRS1/ERK1/2/Akt levels and activation state iii) oxidative stress markers and iii) biliverdin reductase-A (BVR-A), SIRT1 and PTEN protein levels and activation, in the cortex of Ts65Dn mice. In parallel, changes of APP/Abeta levels have been analyzed. Our results show the mutual interaction between increased OS and BIR in Ts65dn, which does not correlate with Abeta levels. We found that OS negatively impacts the activation of insulin cascade since postnatal age that also persists with age. These findings highlight the role of BIR in the onset of AD-like neurodegeneration and suggest that aberrant insulin signaling strongly contributes to cognitive decline also in DS.