Biliverdin reductase-A protein levels are reduced in type 2 diabetes and are associated with poor glycometabolic control.

AIM: Biliverdin reductase-A (BVR-A) other than its canonical role in the degradation pathway of heme as partner of heme oxygenase-1 (HO1), has recently drawn attention as a protein with pleiotropic functions involved in insulin-glucose homeostasis. However, whether BVR-A expression is altered in type 2 diabetes (T2D) has never been evaluated. MAIN METHODS: BVR-A protein levels were evaluated in T2D (n = 44) and non-T2D (n = 29) subjects, who underwent complete clinical workup and routine biochemistry. In parallel, levels HO1, whose expression is regulated by BVR-A as well as levels of tumor necrosis factor α (TNFα), which is a known repressor for BVR-A with pro-inflammatory properties, were also assessed. KEY FINDINGS: BVR-A levels were significantly lower in T2D subjects than in non-T2D subjects. Reduced BVR-A levels were associated with greater body mass, systolic blood pressure, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), triglycerides, transaminases and TNFα, and with lower high-density lipoprotein (HDL) levels. Lower BVR-A levels are associated with reduced HO1 protein levels and the multivariate analysis showed that BVR-A represented the main determinant of HO1 levels in T2D after adjustment. In addition, reduced BVR-A levels were able to predict the presence of T2D with AUROC = 0.69. for potential confounders. SIGNIFICANCE: Our results demonstrate for the first time that BVR-A protein levels are reduced in T2D individuals, and that this alteration strictly correlates with poor glycometabolic control and a pro-inflammatory state. Hence, these observations reinforce the hypothesis that reduced BVR-A protein levels may represent a key event in the dysregulation of intracellular pathways finally leading to metabolic disorders.

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer's Disease Signatures.

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer's disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.

The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome.

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer's disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

Chronic PERK induction promotes Alzheimer-like neuropathology in Down syndrome: Insights for therapeutic intervention.

A major challenge in neurobiology is the identification of the mechanisms by which protein misfolding leads to cellular toxicity. Many neurodegenerative disorders, in which aberrant protein conformers aggregate into pathological inclusions, present the chronic activation of the PERK branch of the unfolded protein response. The adaptive effects of the PERK pathway include reduction of translation by transient inhibition of eIF2α and antioxidant protein production via induction of Nrf2 transcription factor. In contrast, PERK prolonged activation leads to sustained reduction in protein synthesis and induction of cell death pathways. To further investigate the role of the PERK pathway in neurodegenerative disorders, we focused on Down syndrome (DS), in which aging confers a high risk of Alzheimer disease (AD). By investigating human DS frontal cortices, we found early and sustained PERK activation associated with the induction of eIF2α and ATF4 downstream signals. We also observed that the Nrf2 response is uncoupled from PERK and its antioxidant effects are repressed in a mechanism implicating the transcription repressor Bach1. The pharmacological inhibition of PERK in DS mice reduced eIF2α-related translational repression and promoted Nrf2 nuclear translocation, favoring the rescue of Nrf2/Bach1 imbalance. The further analysis of peripheral cells from living DS individuals provided strong support of the pathological link between PERK and trisomy 21. Our results suggest that failure to regulate the PERK pathway is a peculiar characteristic of DS pathology and it may represent an essential step to promote cellular dysfunction, which actively contributes in the brain to the early development of AD.

BVR-A Deficiency Leads to Autophagy Impairment through the Dysregulation of AMPK/mTOR Axis in the Brain-Implications for Neurodegeneration.

Biliverdin reductase-A (BVR-A) impairment is associated with increased accumulation of oxidatively-damaged proteins along with the impairment of autophagy in the brain during neurodegenerative disorders. Reduced autophagy inhibits the clearance of misfolded proteins, which then form neurotoxic aggregates promoting neuronal death. The aim of our study was to clarify the role for BVR-A in the regulation of the mTOR/autophagy axis by evaluating age-associated changes (2, 6 and 11 months) in cerebral cortex samples collected from BVR-A knock-out (BVR-A-/-) and wild-type (WT) mice. Our results show that BVR-A deficiency leads to the accumulation of oxidatively-damaged proteins along with mTOR hyper-activation in the cortex. This process starts in juvenile mice and persists with aging. mTOR hyper-activation is associated with the impairment of autophagy as highlighted by reduced levels of Beclin-1, LC3, LC3II/I ratio, Atg5-Atg12 complex and Atg7 in the cortex of BVR-A-/- mice. Furthermore, we have identified the dysregulation of AMP-activated protein kinase (AMPK) as a critical event driving mTOR hyper-activation in the absence of BVR-A. Overall, our results suggest that BVR-A is a new player in the regulation of autophagy, which may be targeted to arrive at novel therapeutics for diseases involving impaired autophagy.

The Anti-Diabetic Drug Metformin Rescues Aberrant Mitochondrial Activity and Restrains Oxidative Stress in a Female Mouse Model of Rett Syndrome.

Metformin is the first-line therapy for diabetes, even in children, and a promising attractive candidate for drug repurposing. Mitochondria are emerging as crucial targets of metformin action both in the periphery and in the brain. The present study evaluated whether treatment with metformin may rescue brain mitochondrial alterations and contrast the increased oxidative stress in a validated mouse model of Rett syndrome (RTT), a rare neurologic disorder of monogenic origin characterized by severe behavioral and physiological symptoms. No cure for RTT is available. In fully symptomatic RTT mice (12 months old MeCP2-308 heterozygous female mice), systemic treatment with metformin (100 mg/kg ip for 10 days) normalized the reduced mitochondrial ATP production and ATP levels in the whole-brain, reduced brain oxidative damage, and rescued the increased production of reactive oxidizing species in blood. A 10-day long treatment with metformin also boosted pathways related to mitochondrial biogenesis and antioxidant defense in the brain of metformin-treated RTT mice. This treatment regimen did not improve general health status and motor dysfunction in RTT mice at an advanced stage of the disease. Present results provide evidence that systemic treatment with metformin may represent a novel, repurposable therapeutic strategy for RTT.

Shining a light on defective autophagy by proteomics approaches: implications for neurodegenerative illnesses.

Introduction: Autophagy is one of the most conserved clearance systems through which eukaryotes manage to handle dysfunctional and excess organelles and macromolecules. This catabolic process has not only a role in the maintenance of basal turnover of cellular components, but it is also essential in cells adaptation to stress conditions. In the last decades, defects in autophagic machinery have been identified as a feature in neurodegenerative diseases. In this context, mass spectrometry-based proteomics has become an important tool in the comprehensive analysis of proteins involved in the autophagic flux.Area covered: In this review, we discuss recent contributions of proteomic techniques in the study of defective autophagy related to neurodegenerative illness. Particular emphasis is given to the identification of i) shared autophagic markers between different disorders, which support common pathological mechanisms; ii) unique autophagic signature, which could aid to discriminate among diseases.Expert opinion: Proteomic approaches are valuable in the identification of alterations of components to the autophagic process at different steps of the process. The investigation of autophagic defects associated with neurological disorders is crucial in order to unravel all the potential mechanism leading to neurodegeneration and propose effective therapeutic strategies targeting autophagy.

Restoration of aberrant mTOR signaling by intranasal rapamycin reduces oxidative damage: Focus on HNE-modified proteins in a mouse model of down syndrome.

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.

Intranasal rapamycin ameliorates Alzheimer-like cognitive decline in a mouse model of Down syndrome.

BACKGROUND: Down syndrome (DS) individuals, by the age of 40s, are at increased risk to develop Alzheimer-like dementia, with deposition in brain of senile plaques and neurofibrillary tangles. Our laboratory recently demonstrated the disturbance of PI3K/AKT/mTOR axis in DS brain, prior and after the development of Alzheimer Disease (AD). The aberrant modulation of the mTOR signalling in DS and AD age-related cognitive decline affects crucial neuronal pathways, including insulin signaling and autophagy, involved in pathology onset and progression. Within this context, the therapeutic use of mTOR-inhibitors may prevent/attenuate the neurodegenerative phenomena. By our work we aimed to rescue mTOR signalling in DS mice by a novel rapamycin intranasal administration protocol (InRapa) that maximizes brain delivery and reduce systemic side effects. METHODS: Ts65Dn mice were administered with InRapa for 12 weeks, starting at 6 months of age demonstrating, at the end of the treatment by radial arms maze and novel object recognition testing, rescued cognition. RESULTS: The analysis of mTOR signalling, after InRapa, demonstrated in Ts65Dn mice hippocampus the inhibition of mTOR (reduced to physiological levels), which led, through the rescue of autophagy and insulin signalling, to reduced APP levels, APP processing and APP metabolites production, as well as, to reduced tau hyperphosphorylation. In addition, a reduction of oxidative stress markers was also observed. DISCUSSION: These findings demonstrate that chronic InRapa administration is able to exert a neuroprotective effect on Ts65Dn hippocampus by reducing AD pathological hallmarks and by restoring protein homeostasis, thus ultimately resulting in improved cognition. Results are discussed in term of a potential novel targeted therapeutic approach to reduce cognitive decline and AD-like neuropathology in DS individuals.