Mitofusin 1 silencing decreases the senescent associated secretory phenotype, promotes immune cell recruitment and delays melanoma tumor growth after chemotherapy.

Cellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment.

Hepatic metabolism of grazing cows of two Holstein strains under two feeding strategies with different levels of pasture inclusion.

The objective of the study was to characterize adaptations of hepatic metabolism of dairy cows of two Holstein strains with varying proportions of grazing in the feeding strategy. Multiparous autumn calving Holstein cows of New Zealand (NZH) and North American (NAH) strains were assigned to a randomized complete block design with a 2 x 2 factorial arrangement with two feeding strategies that varied in the proportions of pasture and supplementation: maximum pasture and supplementation with a pelleted concentrate (MaxP) or fixed pasture and supplementation with a total mixed ration (FixP) from May through November of 2018. Hepatic biopsies were taken at - 45 ± 17, 21 ± 7, 100 ± 23 and 180 ± 23 days in milk (DIM), representing prepartum, early lactation, early mid-lactation and late mid-lactation. The effects of DIM, feeding strategy (FS), strain and their interactions were analyzed with mixed models using repeated measures. Cows of both strains had similar triglyceride levels, mitochondrial function and carnitine palmitoyltransferase activity in liver during lactation. However, there was an effect of DIM and FS as liver triglyceride was higher for the MaxP strategy at 21 DIM and both mitochondrial function and carnitine palmitoyltransferase activity in liver were lower for the MaxP strategy at 21 DIM. Hepatic mitochondrial function and acetylation levels were affected by the interaction between strain and feeding strategy as both variables were higher for NAH cows in the MaxP strategy. Mid-lactation hepatic gene expression of enzymes related to fatty acid metabolism and nuclear receptors was higher for NZH than NAH cows. This work confirms the association between liver triglyceride, decreased hepatic mitochondrial function and greater mitochondrial acetylation levels in cows with a higher inclusion of pasture and suggests differential adaptative mechanisms between NAH and NZH cows to strategies with varying proportions of grazing in the feeding strategy.

CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline.

Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f-/- and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.

A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Computational and mitochondrial functional studies of novel compound heterozygous variants in SPATA5 gene support a causal link with epileptogenic encephalopathy.

The SPATA5 gene encodes a 892 amino-acids long protein that has a putative mitochondrial targeting sequence and has been proposed to function in maintenance of mitochondrial function and integrity during mouse spermatogenesis. Several studies have associated homozygous or compound heterozygous mutations in SPATA5 gene to microcephaly, intellectual disability, seizures and hearing loss. This suggests a role of the SPATA5 gene also in neuronal development. Recently, our group presented results validating the use of blood cells for the assessment of mitochondrial function for diagnosis and follow-up of mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsy. In this study, we were able to diagnose a patient with epileptogenic encephalopathy using next generation sequencing. We found two novel compound heterozygous variants in SPATA5 that are most likely causative. To analyze the impact of SPATA5 mutations on mitochondrial functional studies directly on the patients' mononuclear cells and platelets were undertaken. Oxygen consumption rates in platelets and PBMCs were impaired in the patient when compared to a healthy control. Also, a decrease in mitochondrial mass was observed in the patient monocytes with respect to the control. This suggests a true pathogenic effect of the mutations in mitochondrial function, especially in energy production and possibly biogenesis, leading to the observed phenotype.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Differential hepatic mitochondrial function and gluconeogenic gene expression in 2 Holstein strains in a pasture-based system.

The objective of this study was to assess hepatic ATP synthesis in Holstein cows of North American and New Zealand origins and the gluconeogenic pathway, one of the pathways with the highest ATP demands in the ruminant liver. Autumn-calving Holstein cows of New Zealand and North American origins were managed in a pasture-based system with supplementation of concentrate that represented approximately 33% of the predicted dry matter intake during 2017, 2018, and 2019, and hepatic biopsies were taken during mid-lactation at 174 ± 23 days in milk. Cows of both strains produced similar levels of solids-corrected milk, and no differences in body condition score were found. Plasma glucose concentrations were higher for cows of New Zealand versus North American origin. Hepatic mitochondrial function evaluated measuring oxygen consumption rates showed that mitochondrial parameters related to ATP synthesis and maximum respiratory rate were increased for cows of New Zealand compared with North American origin. However, hepatic gene expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase was increased in North American compared with New Zealand cows. These results altogether suggest an increased activity of the tricarboxylic cycle in New Zealand cows, leading to increased ATP synthesis, whereas North American cows pull tricarboxylic cycle intermediates toward gluconeogenesis. The fact that this occurs during mid-lactation could account for the increased persistency of North American cows, especially in a pasture-based system. In addition, we observed an augmented mitochondrial density in New Zealand cows, which could be related to feed efficiency mechanisms. In sum, our results contribute to the elucidation of hepatic molecular mechanisms in dairy cows in production systems with higher inclusion of pastures.

Blood cell respiration rates and mtDNA copy number: A promising tool for the diagnosis of mitochondrial disease.

Human mitochondrial diseases are a group of heterogeneous diseases caused by defects in oxidative phosphorylation, due to mutations in mitochondrial (mtDNA) or nuclear DNA. The diagnosis of mitochondrial disease is challenging since mutations in multiple genes can affect mitochondrial function, there is considerable clinical variability and a poor correlation between genotype and phenotype. Herein we assessed mitochondrial function in peripheral blood mononuclear cells (PBMCs) and platelets from volunteers without known metabolic pathology and patients with mitochondrial disease. Oxygen consumption rates were evaluated and respiratory parameters indicative of mitochondrial function were obtained. A negative correlation between age and respiratory parameters of PBMCs from control individuals was observed. Surprisingly, respiratory parameters of PBMCs normalized by cell number were similar in patients and young controls. Considering possible compensatory mechanisms, mtDNA copy number in PBMCs was quantified and an increase was found in patients with respect to controls. Hence, respiratory parameters normalized by mtDNA copy number were determined, and in these conditions a decrease in maximum respiration rate and spare respiratory capacity was observed in patients relative to control individuals. In platelets no decay was seen in mitochondrial function with age, while a reduction in basal, ATP-independent and ATP-dependent respiration normalized by cell number was detected in patients compared to control subjects. In summary, our results offer promising perspectives regarding the assessment of mitochondrial function in blood cells for the diagnosis of mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsies, and for following disease progression and response to treatments.

Mitochondrial Bioenergetics and Dynamics in Secretion Processes.

Secretion is an energy consuming process that plays a relevant role in cell communication and adaptation to the environment. Among others, endocrine cells producing hormones, immune cells producing cytokines or antibodies, neurons releasing neurotransmitters at synapsis, and more recently acknowledged, senescent cells synthesizing and secreting multiple cytokines, growth factors and proteases, require energy to successfully accomplish the different stages of the secretion process. Calcium ions (Ca2+) act as second messengers regulating secretion in many of these cases. In this setting, mitochondria appear as key players providing ATP by oxidative phosphorylation, buffering Ca2+ concentrations and acting as structural platforms. These tasks also require the concerted actions of the mitochondrial dynamics machinery. These proteins mediate mitochondrial fusion and fission, and are also required for transport and tethering of mitochondria to cellular organelles where the different steps of the secretion process take place. Herein we present a brief overview of mitochondrial energy metabolism, mitochondrial dynamics, and the different steps of the secretion processes, along with evidence of the interaction between these pathways. We also analyze the role of mitochondria in secretion by different cell types in physiological and pathological settings.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo.

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

Particles from the Echinococcus granulosus Laminated Layer Inhibit CD40 Upregulation in Dendritic Cells by Interfering with Akt Activation.

The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS). We also reported that the presence of pLL inhibited the activating phosphorylation of the phosphatidylinositol 3-kinase (PI3K) effector Akt induced by granulocyte-macrophage colony-stimulating factor or interleukin-4. We now show that the inhibitory effect of pLL extends to LPS as a PI3K activator, and results in diminished phosphorylation of GSK3 downstream from Akt. Functionally, the inhibition of Akt and GSK3 phosphorylation are linked to the blunted upregulation of CD40, a major feature of the unconventional maturation phenotype. Paradoxically, all aspects of unconventional maturation induced by pLL depend on PI3K class I. Additional components of the phagocytic machinery are needed, but phagocytosis of pLL particles is not required. These observations hint at a DC response mechanism related to receptor-independent mechanisms proposed for certain crystalline and synthetic polymer-based particles; this would fit the previously reported lack of detection of molecular-level motifs necessary of the effects of pLL on DCs. Finally, we report that DCs exposed to pLL are able to condition DCs not exposed to the material so that these cannot upregulate CD40 in full in response to LPS.

Mitofusins modulate the increase in mitochondrial length, bioenergetics and secretory phenotype in therapy-induced senescent melanoma cells.

Cellular senescence is an endpoint of chemotherapy, and targeted therapies in melanoma and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and an enhanced mitochondrial energy metabolism supports resistance to therapy in melanoma cells. Herein, we assessed the mitochondrial function of therapy-induced senescent melanoma cells obtained after exposing the cells to temozolomide (TMZ), a methylating chemotherapeutic agent. Senescence induction in melanoma was accompanied by a substantial increase in mitochondrial basal, ATP-linked, and maximum respiration rates and in coupling efficiency, spare respiratory capacity, and respiratory control ratio. Further examinations revealed an increase in mitochondrial mass and length. Alterations in mitochondrial function and morphology were confirmed in isolated senescent cells, obtained by cell-size sorting. An increase in mitofusin 1 and 2 (MFN1 and 2) expression and levels was observed in senescent cells, pointing to alterations in mitochondrial fusion. Silencing mitofusin expression with short hairpin RNA (shRNA) prevented the increase in mitochondrial length, oxygen consumption rate and secretion of interleukin 6 (IL-6), a component of the SASP, in melanoma senescent cells. Our results represent the first in-depth study of mitochondrial function in therapy-induced senescence in melanoma. They indicate that senescence increases mitochondrial mass, length and energy metabolism; and highlight mitochondria as potential pharmacological targets to modulate senescence and the SASP.

Impaired hepatic mitochondrial function during early lactation in dairy cows: Association with protein lysine acetylation.

Early lactation is an energy-deming period for dairy cows which may lead to negative energy balance, threatening animal health and consequently productivity. Herein we studied hepatic mitochondrial function in Holstein-Friesian multiparous dairy cows during lactation, under two different feeding strategies. During the first 180 days postpartum the cows were fed a total mixed ration (70% forage: 30% concentrate) ad libitum (non-grazing group, G0) or grazed Festuca arundinacea or Mendicago sativa plus supplementation (grazing group, G1). From 180 to 250 days postpartum, all cows grazed Festuca arundinacea were supplemented with total mixed ration. Mitochondrial function was assessed measuring oxygen consumption rate in liver biopsies revealed that maximum respiratory rate decreased significantly in grazing cows during early lactation, yet was unchanged in non-grazing cows during the lactation curve. While no differences could be found in mitochondrial content or oxidative stress markers, a significant increase in protein lysine acetylation was found in grazing cows during early lactation but not in cows from the non-grazing group. Mitochondrial acetylation positively correlated with liver triglycerides ß-hydroxybutyrate plasma levels, well-known markers of negative energy balance, while a negative correlation was found with the maximum respiratory rate sirtuin 3 levels. To our knowledge this is the first report of mitochondrial function in liver biopsies of dairy cows during lactation. On the whole our results indicate that mitochondrial function is impaired during early lactation in grazing cows that acetylation may account for changes in mitochondrial function in this period. Additionally, our results suggest that feeding total mixed ration during early lactation may be an efficient protective strategy.

Deep sequencing discovery of causal mtDNA mutations in a patient with unspecific neurological disease.

Mitochondrial diseases (MD) are a group of diseases that can be caused by either mutations in the mitochondrial genome or nuclear DNA. MD may be difficult to diagnose since very often they are highly heterogeneous and with overlapping phenotypes. Molecular genomics approaches, especially NGS have helped in this sense. In this study we have sequenced the mitochondrial genome of a girl with an unspecific neurological disorder and her mother. The later, while neurologically unaffected, suffers from a myopathy without clear cause. We were able to detect two non-synonymous mutations in the MT-ATP6 gene, which we propose are strong candidates for causative agents. 9017C as the main candidate present at high heteroplasmy frequency in the patient (83,2%) and moderate in the mother (45,4%) while it has a low frequency in the general population. It might act alone or in conjunction with 9010A as an accessory mutation. Evolutionary analysis showed that both mutations were located in a critical position in the F0 a subunit, from F0-F1 ATPase. Functional studies showed that carriers of those mutations in comparison to an unaffected individual (father) presented a decrease in the basal and ATP-dependent oxygen consumption rate and a decrease in the maximum respiration rate.

Impact of monomeric, oligomeric and fibrillar alpha-synuclein on astrocyte reactivity and toxicity to neurons.

Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of aggregated and fibrillar forms of alpha-synuclein (α-syn). Here, we analyze the effect of different species of α-syn, including monomeric, oligomeric and fibrillar forms of the protein, on rat astrocytes. Astrocytes treated with these distinct forms of α-syn showed an increase in long and thin processes and glial fibrillary acidic protein expression, indicating cell activation, high levels of intracellular oxidants and increased expression of cytokines. Moreover, astrocytes incubated with the different species induced hippocampal neuronal death in co-culture, and cytotoxicity was particularly enhanced by exposure to fibrillar α-syn. Further exploration of the mechanisms behind astrocyte activation and cytotoxicity revealed differences between the assessed α-syn species. Only oligomers induced mitochondrial dysfunction in astrocytes and significantly increased extracellular hydrogen peroxide production by these cells. Besides, TNF-α and IL-1ß (interleukin 1ß) expression presented different kinetics and levels depending on which species induced the response. Our data suggest that α-syn species (monomeric, oligomeric and fibrillar) induce astrocyte activation that can lead to neuronal death. Nevertheless, the tested α-syn species act through different preferential mechanisms and potency. All together these results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of Parkinson's disease and related α-synucleinopathies.

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies.

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Worsening of memory deficit induced by energy-dense diet in a rat model of early-Alzheimer's disease is associated to neurotoxic Aß species and independent of neuroinflammation.

Diet is a modifiable risk factor for Alzheimer's disease (AD), but the mechanisms linking alterations in peripheral metabolism and cognition remain unclear. Since it is especially difficult to study long-term effects of high-energy diet in individuals at risk for AD, we addressed this question by using the McGill-R-Thy1-APP transgenic rat model (Tg(+/-)) that mimics presymptomatic AD. Wild-type and Tg(+/-) rats were exposed during 6months to a standard diet or a Western diet (WD), high in saturated fat and sugar. Results from peripheral and hippocampal biochemical analysis and in situ respirometry showed that WD induced a metabolic syndrome and decreased presynaptic bioenergetic parameters without alterations in hippocampal insulin signaling or lipid composition. Cognitive tests, ELISA multiplex, Western blot, immunohistochemistry and RT-qPCR indicated that WD worsened cognition in Tg(+/-) rats, increased hippocampal levels of monomeric Aß isoforms and oligomeric species, promoted deposits of N-Terminal pyroglutamate-Aß (AßN3(pE)) in CA1 pyramidal neurons and interneurons, decreased transcript levels of genes involved in neuroprotective pathways such as Sirtuin-1 and increased nitrated proteins. Our results support the concept that in the presence of early Aß pathology, diet-induced metabolic dysfunctions may contribute as a "second hit" to impair cognition. Noteworthy, such effect is not mediated by higher microglia activation or disruption of blood brain barrier. However, it may be attributed to increased amyloidogenic processing of amyloid precursor protein, generation of AßN3(pE) and dysregulation of pathways governed by Sirtuin-1. This evidence reinforces the implementation of prophylactic interventions in individuals at risk for AD.

Synaptosomal bioenergetic defects are associated with cognitive impairment in a transgenic rat model of early Alzheimer's disease.

Synaptic bioenergetic deficiencies may be associated with early Alzheimer's disease (AD). To explore this concept, we assessed pre-synaptic mitochondrial function in hemizygous (+/-)TgMcGill-R-Thy1-APP rats. The low burden of Aß and the wide array of behavioral and cognitive impairments described in 6-month-old hemizygous TgMcGill-R-Thy1-APP rats (Tg(+/-)) support their use to investigate synaptic bioenergetics deficiencies described in subjects with early Alzheimer's disease (AD). In this report, we show that pre-synaptic mitochondria from Tg(+/-) rats evidence a decreased respiratory control ratio and spare respiratory capacity associated with deficits in complex I enzymatic activity. Cognitive impairments were prevented and bioenergetic deficits partially reversed when Tg(+/-) rats were fed a nutritionally complete diet from weaning to 6-month-old supplemented with pyrroloquinoline quinone, a mitochondrial biogenesis stimulator with antioxidant and neuroprotective effects. These results provide evidence that, as described in AD brain and not proven in Tg mice models with AD-like phenotype, the mitochondrial bioenergetic capacity of synaptosomes is not conserved in the Tg(+/-) rats. This animal model may be suitable for understanding the basic biochemical mechanisms involved in early AD.