Early age changes in mRNA expression related to NAD-dependent enzymes and mitochondrial proteins in the brain cortex of an Alzheimer’s disease mouse model

Early age changes in mRNA expression related to NAD-dependent enzymes and mitochondrial proteins in the brain cortex of an Alzheimer’s disease mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Early age changes in mRNA expression related to NAD-dependent enzymes and mitochondrial proteins in the brain cortex of an Alzheimer’s disease mouse model S Żulińska, I Wieczorek, PL Wencel, RP Strosznajder, GY Sun, JB Strosznajder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1481945/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oxidative stress and disturbances of mitochondria function in the brain have been recognized to play a crucial role in the pathophysiological mechanism of Alzheimer’s Disease (AD). However, little is known about these changes at an early age of AD, which could be crucial for therapeutic strategy for the disease. In this study, we used biochemical, and quantitative polymerase chain reaction for determination of expression of genes encoding enzymes related to the antioxidative defence including Sirtuins (Sirts) and DNA-bound poly (ADP-ribose) polymerases (PARPs). Moreover, expression of genes related to mitochondrial dynamic, biogenesis and function in the brain cortex of 3- and 6-month-old FVB mice with London mutation (V7171) was analysed and compared with mice without transgene. Results indicated significant decreases in mRNA expression encoding SOD2, Sirt1 and PARP1 in the 3-month-old AD Tg mice and an increase in expression of PARP-1 in the 6-month-old AD Tg. Although levels of mRNA encoding subunits of mitochondrial respiratory complexes (I-III) were negligible altered, there was upregulation of gene encoding subunit of complex IV and proteins related to mitochondria biogenesis and dynamic, such as the Neuronal Respiratory Factors (NRF1) and NRF2, Opa1, Fis1, and Drp1 in the AD mice. Our data indicate downregulation of genes related to antioxidation and activation of genes encoding mitochondrial biogenesis and fission / fusion at early age of the AD mice. The ability to identify changes in gene expression for Sirt1, SOD2, Fis1 and Drp1 at an early age suggest potential therapeutic targets for retarding the pathological progression in AD. Alzheimer’s disease gene expression antioxidative defense mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction It is widely suggested that activation of free radicals, oxidative stress and alterations of mitochondrial functions are crucial pathophysiological mechanisms of neurodegenerative disorders including Alzheimer’s disease (AD) (Wang et al., 2020 ; Schmitt et al., 2012 ; Oliver and Reddy, 2019 ). Mitochondria are responsible not only for ATP biosynthesis but also for nicotine adenine dinucleotide (NAD) synthesis, and NAD is the substrate for an important family of enzymes such as Sirtuins (histone deacetylase type III, ) and poly(ADP-ribose)polymerases (PARPs). Mitochondria dysfunctions could lead to activation of free radical cascade which may evoke changes in several transcription factors including peroxisomes proliferator activated receptors (PPARs) and alterations of genes expression involved in amyloid beta precursor protein (APP) metabolism and release of amyloid beta (Aβ) peptides. Environmental factors and systemic inflammation could lead to alteration of redox homeostasis, free radical liberation, and molecular changes evoked also by modulation of NAD-dependent enzymes such as histone deacetylases type III, Sirtuins and DNA bound poly (ADP-ribose) polymerases (PARP1, PARP2, PARP3) (Cantó et al., 2013 ). These changes may induce mitochondria failure and suppress activity of electron transport chain (ETC). Consequently, free radicals may activate cascades of events leading to oxidative stress, synaptic dysfunctions, cells degeneration and death (Schmitt et al 2012 ; Czapski et al., 2017). Mitochondrial dysfunction has been shown to occur early in neurodegenerative disorders, including the initial stage of AD (Reddy et al., 2012). In AD brain, oxidative damage leading to alteration of mitochondria membrane permeability can lead to alterations of Ca 2+ channels and stimulate Ca 2+ dependent processes which may further activate the free radical cascade and resulting in generation of pro-apoptotic signals and mitochondria fission (Moneim, 2015 ; Manczak et al., 2016 ; Yang et al., 2021 ). Fission and fusion of mitochondria are highly modulate by free radical production, oxidative stress, and alteration of ion homeostasis within the cell. Despite of an abundance of literature about mitochondria failure in neurodegeneration, our understanding of the processes underlying their dysfunction in different part of the brain in AD and other neurodegenerative disorders is still limited. Substantial studies have revealed that alterations of mitochondria in neuronal cells and in synaptic endings are early pathophysiological events in AD (Schmitt et al., 2012 ; Du et al., 2012 ). Nevertheless, questions on this subject including sequence of molecular events and their localization in particular brain regions have not yet been fully resolved. Moreover, the role of protective processes activated simultaneously with pathological changes is not well recognized. There is indication that energy production not only is related to complexes/super complexes of the electron transport chain (ETC) but also is dependent on mitochondrial dynamic and their ability to undergo cycles of fission and fusion (Manczak et al., 2016 ; Oliver and Reddy, 2019 ). Mitochondrial dynamic machinery depends mainly on dynamin related protein 1 (Drp1), mitochondria fission protein 1 (Fis1), mitochondria fission factor (Mff), and proteins regulating mitochondrial fusion, e.g., mitofusin-1, mitofusin-2 (Mfn1, Mnf2) and optical atrophy protein 1 (Opa1). These proteins are known to participate in the assembly and stability of ETC super complexes, in remodeling of mitochondrial cristae, and in shaping mitochondrial morphology in response to environmental conditions. However, only 13 proteins involved in mitochondria function are encoding by mtDNA. The nuclear respiratory factors, NRF1 and NRF2, are implicated in the transcription of genes encoding respiratory subunits, including 10 subunits of cytochrome c oxidase of complex IV, and transcription factor A mitochondria (TFAM) and both TFB (Transcription factor B) isoforms. Moreover, members of the nuclear receptors (NRs) superfamily including PPAR-α can also play a significant role in the transcriptional control and in mitochondrial lipids and fatty acids metabolism (Wójtowicz et al., 2020 , Strosznajder et al., 2021 ). Recent studies demonstrated excessive mitochondria division in AD patients and in experimental animal models of AD (Hu et al., 2017 ; Wang et al., 2020 ; Oliver and Reddy, 2019 ). Our data from Cieślik et al. ( 2020 ) showed significant downregulation of genes such as Sirt 1, mt-Nd1, Mfn1 and concomitant upregulation of Dnm1 in the 12-month-old AD Tg mice model (with London mutation). In the human AD brain, changes in microRNA patterns (miRNA-9, miRNA-34a, miRNA-146 and miRNA-155) were found, and these changes are probably responsible for the downregulation of Sirt1 expression (Cieslik et al., 2020). Alterations of transcription of genes encoding proteins related to mitochondria biogenesis can also affect the progression of AD. Through interaction with NRF1, PGC-1α (Peroxisome Proliferator-Activated Receptor Gamma Coactivator alpha), the key protein of mitochondria biogenesis, can lead to activation of gene coding TFAM and then mitochondrial DNA (mtDNA). This transcription factor TFAM is crucial not only for mtDNA transcription but also in mtDNA maintenance and mtDNA nucleoid formation (Kang and Hamasaki, 2005 ). Therapeutic modulations of Sirt1 (Silent information regulator 1) and SOD2 have been suggested to offer potential for treatment of aged related neurodegenerative disorders connected with mitochondria alteration. Until now, neuroprotective strategies for AD are largely unsuccessful mainly because they are introduced too late at rather advance stage of the disease. The effect of several pharmacological compounds acting on Sirtuins and DNA-bound PARPs, both NAD-dependent families of enzymes, and on their role in different stages of AD, has not been fully elucidated. Little is known on the effect of these compounds at early stage of AD because diagnosis is too late. Therefore, more studies are needed to understand the dynamic and time-dependent molecular alterations of genes encoding proteins related to anti-oxidative defense, such as Sirts /PARPs /SODs, and mitochondria dynamics and function in animal models of AD at early stage of the disease. This subject was recently highlighted by Wang et al. ( 2020 ) and Yang et al. ( 2021 ). The mitochondria changes were also observed in lymphocytes of sporadic AD patients and the question arise if they could be an early marker for the diagnosis and prognosis of disease (Jörg et al. 2021 ). Recently, several pharmacological and natural compounds for treatment of AD also modulating expression and activity of Sirt1 have been tested in pre-clinical/clinical studies of human disorders (Manjula et al., 2020; Eckert et al 2020 ; Cummings et al., 2021 ). Considering these deficiencies, this study used a transgenic AD mouse model to determine gene expression encoding proteins of anti-oxidative defense machinery, including SOD1 and mitochondrial SOD2, transcription profiles of NAD-dependent enzymes, and mRNA expression related to mitochondria dynamics, biogenesis and function in brain cortex of 3- and 6-month-old AD Tg mice as compared with corresponding age-matched controls (without transgene). The major goal here is to explore early target(s) for cytoprotection and promising therapeutic strategy in AD. Materials And Methods Animal model of AD Female FVB-Tg (Thy1; APP LD2/B6) mice, aged 3 and 6 months, were used. These mice overexpressed human AβPP with the “London” V717I mutation under control of a fragment of Thy1 promoter with specificity towards brain and spinal cord neurons. Mice without the transgene were used as controls. This animal model had successfully recreated a relatively broad spectrum of behavioral, electrophysiological and biochemical features of AD (Moechars et al., 1999; Van Drope et al., 2000). These mice display behavioral abnormalities starting at 8 weeks of age (Moechars et al., 1999). Starting from the age of approximately 3 months, these mice gradually develop agitation and cognitive disturbances and cognitive impairment. These changes were accompanied by altered reactivity to neurotransmitters (observed at 3–4 months of age) and electrophysiological alterations (between 5 and 7 months) (Moechars et al., 1999). Animals were bred under specific pathogen-free (SPF) conditions in controlled temperature and humidity conditions and 12-h light/dark cycle in the Animal House of the Mossakowski Medical Research Centre PAS, Warsaw, Poland. At 3 or 6 months, mice were decapitated. Cerebral cortices were quickly isolated on ice and frozen in liquid nitrogen. The protocol was approved by the Warsaw Local Ethics Committee for Animal Experimentation and performed in accordance with guidelines of Polish National Ethics Committee and the EU Directive 2010/63/EU. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All efforts were made to minimize suffering and to reduce the number of animals used. The experiments were performed in accordance with good laboratory practice protocols and quality assurance methods. Analysis of Gene Expression RNA was isolated using the TRI reagent (Sigma –Aldrich/Merck) as described in the manufacturer’s protocols and purified by using DNase I according to the manufacturer’s protocols (Sigma-Aldrich/Merck). The concentration and purity of obtained RNA was determined spectrophotometrically (A260/A280). Reverse transcription was performed by using the High-capacity cDNA Reverse Transcription Kit according to the manufacturer’s instruction (Applied Biosystems). The level of mRNA for studied genes was analyzed by using TaqMan Gene Expression Assays (Applied Biosystems). Cat (Mm00437992_m1), Dnm1l (Mm01 3 42 9 03 _ m 1), Fis1 (Mm004815 8 0 _ m 1), Gpx4 (Mm0051 5041_m1), Mfn1 (Mm00612599 _ m 1), Mfn2 (Mm00500120_m1), mt-Co1 (Mm04225243_g1), mt-Cytb (Mm04225271_g1 ), mt-Nd1 (Mm042 2 5 2 7 4- s 1), Opa 1 (Mm01 3 4 9 70 7 g1), Sdha (Mm01352366_m1), Sirt1 (Mm00490762_m1), Sirt3 (Mm00452131_m1), Sirt4 (Mm01201915_m1), Sirt5 (Mm0135 15 76_m1), SOD 1 (Mm0134423_g1), SOD2 (Mm01313000_m1), Sdhc (Mm00481172_m1) PARP1 (Mm01321084) PARP2 (Mm00456462_m1) PARP3 (Mm00467486_m1) Quantitative polymerase chain reaction (PCR) was performed on an Applied Biosystems 7500 Real-Time PCR System using TaqMan Gene Expression Master Mix according to the manufacturer’s instructions. The relative levels of mRNA were calculated using the ΔΔCt Method and were normalized against beta actin ( ACTB, Mm4352341E) . Determination of enzymatic activity of cytochrome c oxidase and Complex IV of mitochondria respiratory chain . Activity of cytochrome c oxidase (COX) in brain cortex homogenate lysate was determined as described by Spinazii et al. (2012). To prepare lysates, brain cortex previously frozen in liquid nitrogen and stored in -80C were thawed, dissected, and then homogenized in 20 mM phosphate buffer pH 7.5 (0.5 ml per 1mg tissue) using 1ml syringe and passing through the needle until homogenous solution appeared. Then the homogenate and lysate were frozen and thawed three times. Cytochrome c was reduced by incubation in the presence of 0.5 mM DTT for 20 min at room temperature in dark. The efficacy of cytochrome c reduction was checked by calculating the ratio of the absorbance 550 nm/565 nm, ratio > 6 indicates effective reduction. To determined Cox activity, 40 ul of lysate ± 100 µg protein was incubated with 25uM reduced cytochrome c in 25 mM phosphate buffer pH 7.0 at room temperature. The decrease of absorbance at 550 nm was measured during 3 min. The extinction coefficient for reduced cytochrome c was 18.5mM -1 x cm -1 . Statistical Analysis The results were expressed as mean values ± SEM from 4 to 8 animals, independent experiments carried out in triplicate. Differences between the means were analyzed using a Student's t-test for two groups, * p <0.05, ** p <0.01 *** p <0.001 were considered significant. The statistical analyses were performed using Graph Pad Prism version 8.0 (Graph Pad Software, San Diego, CA, USA). Results In this study, we determined mRNA profiles encoding anti-oxidative enzymes in the brain cortex of AD Tg and aged-matched control mice at 3- and 6-month of age. Results indicated a small but significant decrease in the level of mRNA encoding the Mn 2+ -dependent SOD2 in the 3-month Tg AD mice (Fig. 1). Under this condition, mRNA levels for SOD1, glutathione peroxidases GPX1, GPX4 and catalase were not changed (Fig. 1). NAD-dependent enzymes such as Sirtuins and PARPs are sensitive in oxidative/genotoxic stress conditions. Among them, Sirt1 and PARP1 are known to play a crucial role in regulating transcription factors for gene expression and cells survival. These enzymes as well as other members of the Sirtuin’s family, such as the mitochondrial Sirt3, Sirt4 and Sirt5 and other DNA-bound PARPs (PARP2and PARP3), may also play a role in the pathophysiology of neurodegenerative diseases including AD. In this study, analysis of mRNA levels demonstrated a significant decrease in expression encoding Sirt1 in the brain cortex of the 3-month-old AD Tg mice but mRNA levels for the mitochondrial Sirts (Sirt 3, 4, 5) were not significantly altered (Fig. 2a). Although mRNA encoding PARP-1 was decreased in the 3-month-old AD Tg mice and, there was a significant increase in the 6-month-old AD Tg mice (Fig. 2b). Nevertheless, there were no significant differences in mRNA levels for the DNA-bound PARP2, and PARP3 (Fig. 2b). Subsequently, we examined the mRNA levels in selected sub-units of the mitochondrial respiratory complex in the brain cortex of AD Tg mice and compared with the non Tg controls. In this study, mRNA expressions encoding the subunit of mitochondria respiratory complex I, mt-Nd1, and subunits of complex II, Sdha and Sdhc in the brain cortex of 3- and 6-month-old AD Tg mice were not altered (Fig. 3). However, mRNA levels for Cytb, encoding the subunits of complex III was decreased slightly (not-significant), whereas mRNA encoding mt-Co 1, subunit of complex IV, was increased in brain cortex of the 6-month-old AD Tg mice as compared with age-matched controls (Fig. 3). In this study, we also measured cytochrome C activity, which was not significantly altered in the 3-month-old AD Tg mice as compared with age-match controls (Fig. 3). In a subsequent study, we analyzed expression of genes encoding proteins involved in mitochondria biogenesis. In this study, mRNA levels for PPAR-α and Ppargc1 were not altered (Fig. 4). However, there were significant increases in levels of mRNA encoding NRF1 and, NRF2 in the 3-month-old AD Tg mice as compared with controls but transcription of NRF2 was downregulated in 6 months old mice instead (Fig. 4). In addition, mRNA for TFAM, another mitochondrial transcription factor, was significantly increased in 6-month-old AD Tg mice as compared with control. Lastly, we examined mRNA expression encoding proteins involved in mitochondria dynamic. Although there were no changes in expression of genes encoding Mnf1 and Mnf2, a pronounce increase in mRNA expression encoding OPA1, a key regulator of mitochondria fusion, in the 3 and 6-month-old AD Tg mice was observed (Fig. 5). Concomitantly, there were significant increases in mRNA levels encoding Fis1 and Drp1 proteins in the 6-month-old AD Tg mice (Fig. 5). Discussion As summarized in Fig. 6 , this study identified significant alterations of mRNA expression encoding enzymes involved in antioxidative defense and in dynamic of mitochondria in the cerebral cortex of 3- and 6-month-old AD Tg mice as compared with age-matched controls. The most significant changes are related to alterations of genes encoding Sirt1 and PARP1, both NAD-dependent enzymes present mainly in the nucleus (but are also located in mitochondria). These enzymes are sensitive sensors for maintenance of free radical homeostasis (Strosznajder et al., 2012 ; Felici et al., 2014 ; Lapucci et al., 2011 ; Dawson and Dawson, 2017 ). Downregulation of transcription of genes encoding SOD2 and Sirt1 could be a crucial early event in AD, and these changes may create conditions for enhancement of the free radical cascade and oxidative stress, and further leading to changes in transcription of genes encoding proteins related to mitochondria function. The functional crosstalk between Sirt1 and PARP1 is crucial for genome integrity, chromatin structure and response to free radicals DNA damage (El Ramy et al., 2009 ). Although not fully understood, there is an apparent close relationship between Sirt1, PARP1 and transcription processes in mitochondria. There is indication that PARP-1 inhibitors are best activators of Sirts (Bai et al., 2011 ) and both enzymes are molecular regulators of cells survival and death (Bai et al., 2011 ; Strosznajder et al., 2012 ; Jęśko et al., 2017 ). Sirt1 may play a significant role in APP metabolism and in antioxidative defense. Sirt1 downregulation may lead to disturbances of homeostasis between non-amyloidogenic pathway of APP degradation by α secretase and amyloidogenic pathway of APP metabolism by β secretase, subsequently leading to excessive release of Aβ peptides (Jęśko et al., 2017 ). Our previous study with the 12-month-old AD Tg mice indicated significant lower expression of genes encoding Sirt1, subunit of complex I, mt-Nd1 and concomitant increase in genes encoding subunit of complex IV, mtCo1, as well as upregulation of Drp 1, the gene encoding the crucial protein of mitochondria fission (Cieślik et al., 2020 ). In our present study, mRNA encoding Sirt1 was significantly decreased in the 3-month-old AD Tg mice. Since Sirt1 is the most studied member of the Sirtuin family, the decrease of transcription of gene coding this enzyme at the early stage of AD may play an important role in the progression of pathology of AD (Jęśko et al., 2017 ; Jęśko and Strosznajder, 2016 ). The brain function is critically relying on ATP synthesis through the ETC respiratory complexes in the mitochondria. In the present study, subunits of respiratory complexes engaged in ETC were slightly decreased, albeit not significantly. In contrast, there was a significant upregulation of expression of gene encoding subunit of complex IV which probably served as a compensatory mechanism for the small changes in other complexes and lower cytochrome c oxidase activity (albeit not significant) observed in the 3-month-old AD Tg brain cortex as compared with controls. A selective defect of cytochrome c oxidase was observed in the brain of AD patients (Mauer et al., 2000). An important by-product of electron transport processes is superoxide anion which is converted by superoxide dismutase SOD2 to the less reactive H 2 O 2 (Flyn and Melov, 2013). Analysis of post-mortem samples from Alzheimer’s patients demonstrated elevated lipid peroxidation and upregulation of SOD2 (De Leo et al., 1998 ). However, other studies using transgenic AD mice model indicated a reduction of SOD2 instead (Esposito et al., 2006 ). Our previous study demonstrated a pronounced enhancement of gene transcription encoding SOD2 in microglia cells (BV2) upon subjecting the cells to the toxic Aβ oligomers but not in neuronal (SH-SY5Y) cells, which showed a opposite effect (Cieślik et al., 2020 ). In the present study, levels of SOD2 mRNA were decreased in the 3- and slightly in 6-month-old AD Tg mouse brain (Fig. 1 ). In the AD Tg mouse model, lower transcription of genes encoding Sirt1 and SOD2 may create conditions for alterations of transcription of other genes encoding Drp1 and Fis1, which are crucial proteins of mitochondria fission. The lower transcription of genes encoding enzymes of antioxidative defense could be a prerequisite event in molecular cascade leading to mitochondria degeneration and cell death. Among the proteins related to mitochondria, those engaged in regulation of fission and fusion are probably most susceptible to alterations at the early stage of the disease (Wang et al., 2020 ; Yang et al., 2021 ). Results in the present study showed significant alterations of transcription of genes related to mitochondria dynamic, upregulation of genes encoding Opa1 protein involved in mitochondrial fusion, suggesting activation of some compensatory or protective processes evoked by activation of transcription of genes coding Fis1 and Drp1 engaged in mitochondria fission. In another study, significant increase in Fis1, gene involved in regulation of mitochondria fission was also observed in AD brains compared with age-matched controls (Wang et al., 2009 ). In AD patients and in AD animal models, different parts of the brain and different cell populations may exert different susceptibility to pathological insults which may lead to changes in expression of genes involved in mitochondria dynamic, function and mitochondria biogenesis. TFAM is a crucial transcription factor engaged in mitochondria biogenesis. The ability of TEAM transcription to increase the level of mtDNA suggests that this factor is linked the nuclear transcription response to mtDNA and mitochondrial biogenesis (Picca et al., 2015). In addition, studies have demonstrated that TFAM upregulation may protect against oxidative stress and mitochondrial failure (Campbell et al., 2012 ). In this study, expression of gene encoding TFAM was enhanced significantly in brain cortex of 6 months old AD Tg mice. Early pharmacological intervention to enhance transcription of TFAM and mtDNA stability and mitochondrial biogenesis could be a promising approach to retard the progression of AD. Moreover, our data demonstrated significant enhancement of genes expression coding NRF1 and NRF2 in brain cortex of 3 months old AD Tg mice which may suggest activation of some protective /adaptive pathway(s). Transcription of NRF2 observe in 3 months old AD Tg mice was subsequently decreased in the brain cortex of 6 months old AD Tg mice. NRFs are crucial transcription factors engaged in mitochondria biogenesis and in the regulation of expression of genes encoding several mitochondrial proteins involved in bioenergetics function and several other genes with diverse functions including cell growth, autophagy, apoptosis and antioxidant defense (Brandes and Gray, 2020 ; Urfer-Buchwalder and Urfer, 2017 ). NRF1 and NRF2 could be very promising targets in early therapeutic approaches in AD or in strategy of prevention against oxidative stress and neurodegeneration during brain aging. NRF2 through activation of genes expression, coding proteins engaged not only in antioxidative but also anti-inflammatory defense could exert significant neuroprotective effect. NRF 2 activating pharmacological compounds are recently considered by FDA for treatment of Multiple Sclerosis (MS) and other neurodegenerative disorders (Brandes and Gray 2020 ). Our data should be helpful in better understanding of early changes in transcription of genes in AD Tg mice and could contribute to a better understanding of molecular processes involved in mitochondria dysfunction in AD. Moreover, based on our findings in this study, we suggest that Sirt1, SOD2, Fis1 and Drp1 are the important targets for therapeutic approaches in the early stage of AD. The therapeutic strategy should support endogenously activated neuroprotective processes occur concomitantly with pathological changes. Declarations Conflict of interest: The authors have declared that there are no competing interests. Acknowledgements - Supported by National Science Centre (PL) Grant no 2019/35/N/NZ4/03706 and by MMRC, PAS statutory budget theme no 7. References Bai, P., Cantó, C., Oudart, H., Brunyánszki, A., Cen, Y., Thomas, C., Yamamoto, H., Huber, A., Kiss, B., Houtkooper, R. H., Schoonjans, K., Schreiber, V., Sauve, A. A., Menissier-de Murcia, J., & Auwerx, J. (2011). 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Antioxidants & redox signaling, 16(12), 1456–1466. https://doi.org/10.1089/ars.2011.4400 Spinazzi, M, Casarin, A., Pertegato, V., Salviati, L., Angelini, C. (2012). Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 7:1235–1246. https://doi.org/10.1038/nprot.2012.058 Strosznajder, J.B., Czapski, G.A., Adamczyk, A., Strosznajder, R.P. (2012). Poly(ADP-ribose) polymerase-1 in amyloid beta toxicity and Alzheimer's disease. Mol Neurobiol. 46(1):78–84. doi: 10.1007/s12035-012-8258-9 . Epub 2012 Mar 20. PMID: 22430645. Strosznajder, A. K., Wójtowicz, S., Jeżyna, M. J., Sun, G. Y., & Strosznajder, J. B. (2021). Recent Insights on the Role of PPAR-β/δ in Neuroinflammation and Neurodegeneration, and Its Potential Target for Therapy. Neuromolecular medicine, 23(1), 86–98. https://doi.org/10.1007/s12017-020-08629-9 Urfer-Buchwalder, A., & Urfer, R. (2017). Identification of a Nuclear Respiratory Factor 1 Recognition Motif in the Apolipoprotein E Variant APOE4 linked to Alzheimer's Disease. Scientific reports, 7, 40668. https://doi.org/10.1038/srep40668 Van Dorpe, J., Smeijers, L., Dewachter, I., Nuyens, D., Spittaels, K., Van Den Haute. C., Mercken, M., Moechars, D., Laenen, I., Kuiperi, C., Bruynseels, K., Tesseur, I., Loos, R., Vanderstichele, H., Checler, F., Sciot, R., Van Leuven, F. (2000). Prominent cerebral amyloid angiopathy in transgenic mice overexpressing the London mutant of human APP in neurons. Am J Pathol, 157(4):1283–98. doi: 10.1016/S0002-9440(10)64644-5 . PMID: 11021833; PMCID: PMC1850171. Wang, W., Zhao, F., Ma, X., Perry, G., & Zhu, X. (2020). Mitochondria dysfunction in the pathogenesis of Alzheimer's disease: recent advances. Molecular neurodegeneration, 15(1), 30. https://doi.org/10.1186/s13024-020-00376-6 Wang, X., Su, B., Lee, H.G., Li, X., Perry, G., Smith, M.A., Zhu, X.. (2009). Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci , 15;29(28):9090 – 103. doi: 10.1523/JNEUROSCI.1357-09.2009 . Wójtowicz, S., Strosznajder, A. K., Jeżyna, M., & Strosznajder, J. B. (2020). The novel role of PPAR alpha in the brain: Promising target in therapy of Alzheimer’s disease and other neurodegenerative disorders. Neurochemical Research, 45(5):972–988. doi: 10.1007/s11064-020-02993-5. Epub 2020 Mar 13. PMID: 32170673; PMCID: PMC7162839. Yang, D., Ying, J., Wang, X., Zhao, T., Yoon, S., Fang, Y., Zheng, Q., Liu, X., Yu, W., & Hua, F. (2021). Mitochondrial Dynamics: A Key Role in Neurodegeneration and a Potential Target for Neurodegenerative Disease. Frontiers in neuroscience, 15, 654785. ://doi.org/10.3389/fnins.2021.654785 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1481945","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":93280206,"identity":"8dc2ceee-829e-43fa-8654-f0ca0dde0e5b","order_by":0,"name":"S Żulińska","email":"","orcid":"","institution":"Mossakowski Medical Research Centre","correspondingAuthor":false,"prefix":"","firstName":"S","middleName":"","lastName":"Żulińska","suffix":""},{"id":93280207,"identity":"d31bf204-3b91-4c59-b375-0499080c8d7f","order_by":1,"name":"I Wieczorek","email":"","orcid":"","institution":"Mossakowski Medical Research Centre","correspondingAuthor":false,"prefix":"","firstName":"I","middleName":"","lastName":"Wieczorek","suffix":""},{"id":93280208,"identity":"e1ade014-c4ad-4adf-9b63-32031929de08","order_by":2,"name":"PL Wencel","email":"","orcid":"","institution":"Mossakowski Medical Research Centre","correspondingAuthor":false,"prefix":"","firstName":"PL","middleName":"","lastName":"Wencel","suffix":""},{"id":93280209,"identity":"a83d2cb7-4137-42a6-9c48-b9768e583904","order_by":3,"name":"RP Strosznajder","email":"","orcid":"","institution":"Mossakowski Medical Research Centre","correspondingAuthor":false,"prefix":"","firstName":"RP","middleName":"","lastName":"Strosznajder","suffix":""},{"id":93280210,"identity":"39377f7c-72aa-4b15-b175-dce1af2dce37","order_by":4,"name":"GY Sun","email":"","orcid":"","institution":"University of Missouri","correspondingAuthor":false,"prefix":"","firstName":"GY","middleName":"","lastName":"Sun","suffix":""},{"id":93280211,"identity":"060b1e93-a75b-4392-8755-fc364586649d","order_by":5,"name":"JB Strosznajder","email":"data:image/png;base64,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","orcid":"","institution":"Mossakowski Medical Research Centre","correspondingAuthor":true,"prefix":"","firstName":"JB","middleName":"","lastName":"Strosznajder","suffix":""}],"badges":[],"createdAt":"2022-03-23 13:59:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1481945/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1481945/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19706018,"identity":"974b3fca-81c0-4997-a681-8bc05e53a213","added_by":"auto","created_at":"2022-03-28 20:36:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":153056,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative RT-PCR to determine levels of mRNA expression of antioxidant genes in brain cortex of 3- and 6-month-old mice: APP- (control without transgene) and APP+ (AD Tg). Results were normalized to beta Actin gene expression. Data are mean ± S.E.M. for 3- 7 animals in each age (3- and 6- month-old) group. Statistical analysis was performed using Student’s t test *p\u0026lt;0.05, **p\u0026lt;0.01 as compared to the respective control group\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/ebe929de06bf796a59f17016.png"},{"id":19705640,"identity":"31f06e1b-940c-4aac-a44f-7586c12e1658","added_by":"auto","created_at":"2022-03-28 20:31:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":188417,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of mRNA levels encoding selected NAD dependent enzymes in AD Tg mice brain cortex. \u003cstrong\u003ea\u003c/strong\u003e The levels of mRNA for Sirt1 and mitochondria Sirt3, Sirt4 and Sirt5 in AD Tg (APP+) brain cortex of 3- and 6-month-old mice. Data are mean ± SEM for 4-7 animals in each age group. Statistical evaluation was carried out using Student’s t test *p\u0026lt; 0.05 as compared to the respective control group (APP-) mice\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e The levels of mRNA for DNA-bound PARP1, PARP2 and PARP3 in brain cortex of 3- and 6-month-old AD Tg (APP+) mice. mRNA expression was analyzed using qRT-PCR and results were normalized to beta Actin as described in Material and Methods. Data are mean ± SEM for 3-4 animals in each age group. Statistical evaluation was carried out using Student’s t test *p\u0026lt; 0.05 as compared to the respective control group (APP-) mice\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/561343ae4ec3f0ab2aa572e0.png"},{"id":19705638,"identity":"069c5334-8be5-4bf3-b38a-5b3007bdf055","added_by":"auto","created_at":"2022-03-28 20:31:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":197889,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression encoding subunits of respiratory complexes of ETC and activity of cytochrome c oxidase in brain cortex of 3- and 6-month-old AD Tg mice (APP+). mRNA expression was analyzed using qRT-PCR and results were normalized to expression of beta Actin. Values are means ±SEM for 3-8 animals. Statistical analysis was performed using Student’s t test *p\u0026lt;0.05 as compared to respective control (APP-) group\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/75d519e791337bc9a9d0158c.png"},{"id":19705641,"identity":"0929ebcd-9525-4865-84bd-52c298cb8e9d","added_by":"auto","created_at":"2022-03-28 20:31:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161315,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression encoding PPAR-α and proteins engaged in mitochondria biogenesis in AD Tg (APP+) brain cortex of 3- and 6-month-old mice. Expression of mRNA was analyzed by qRT-PCR and results were normalized to expression of beta Actin. Values are means ± SEM for 3-8 animals in each age group. Statistical analysis was performed using Student’s t test *p\u0026lt;0.05 as compared to respective control (APP-) group\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/80dfeb51cc971767ef446cce.png"},{"id":19706017,"identity":"c19a3761-236a-4429-a77f-92744c899c00","added_by":"auto","created_at":"2022-03-28 20:36:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":140658,"visible":true,"origin":"","legend":"\u003cp\u003emRNA expression encoding proteins engaged in fission and fusion of mitochondria in AD Tg (APP+) brain cortex of 3- and 6-month-old mice. Expression of mRNA was analyzed by qRT-PCR and results were normalized to beta Actin. Values are means ± SEM for 3-8 animals in each age group. Statistical analysis was performed using Student’s t test *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 as compared to the respective control groups\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/532a8c13e0774dac38f22583.png"},{"id":19705639,"identity":"1a3baa7c-f91c-496c-9058-0ffeea1c660f","added_by":"auto","created_at":"2022-03-28 20:31:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69239,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic representation depicting the main findings of this study. Arrows\u003cstrong\u003e ↑\u003c/strong\u003e and \u003cstrong\u003e↓\u003c/strong\u003e indicate an increase and decrease in mRNA expression encoding proteins related to anti-oxidative defense and mitochondria function respectively\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/6f7f6fdf4406bb1dc52dac8e.png"},{"id":30818939,"identity":"dbb3993b-41ae-4b8b-bbc2-cae410e5b634","added_by":"auto","created_at":"2022-12-28 01:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1198986,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1481945/v1/6e3bdca3-8b4a-4a80-af89-1603a59fe9b0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Early age changes in mRNA expression related to NAD-dependent enzymes and mitochondrial proteins in the brain cortex of an Alzheimer’s disease mouse model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIt is widely suggested that activation of free radicals, oxidative stress and alterations of mitochondrial functions are crucial pathophysiological mechanisms of neurodegenerative disorders including Alzheimer\u0026rsquo;s disease (AD) (Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schmitt et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Oliver and Reddy, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mitochondria are responsible not only for ATP biosynthesis but also for nicotine adenine dinucleotide (NAD) synthesis, and NAD is the substrate for an important family of enzymes such as Sirtuins (histone deacetylase type III, ) and poly(ADP-ribose)polymerases (PARPs). Mitochondria dysfunctions could lead to activation of free radical cascade which may evoke changes in several transcription factors including peroxisomes proliferator activated receptors (PPARs) and alterations of genes expression involved in amyloid beta precursor protein (APP) metabolism and release of amyloid beta (Aβ) peptides. Environmental factors and systemic inflammation could lead to alteration of redox homeostasis, free radical liberation, and molecular changes evoked also by modulation of NAD-dependent enzymes such as histone deacetylases type III, Sirtuins and DNA bound poly (ADP-ribose) polymerases (PARP1, PARP2, PARP3) (Cant\u0026oacute; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These changes may induce mitochondria failure and suppress activity of electron transport chain (ETC). Consequently, free radicals may activate cascades of events leading to oxidative stress, synaptic dysfunctions, cells degeneration and death (Schmitt et al \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Czapski et al., 2017).\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction has been shown to occur early in neurodegenerative disorders, including the initial stage of AD (Reddy et al., 2012). In AD brain, oxidative damage leading to alteration of mitochondria membrane permeability can lead to alterations of Ca\u003csup\u003e2+\u003c/sup\u003e channels and stimulate Ca\u003csup\u003e2+\u003c/sup\u003e dependent processes which may further activate the free radical cascade and resulting in generation of pro-apoptotic signals and mitochondria fission (Moneim, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Manczak et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Fission and fusion of mitochondria are highly modulate by free radical production, oxidative stress, and alteration of ion homeostasis within the cell.\u003c/p\u003e \u003cp\u003eDespite of an abundance of literature about mitochondria failure in neurodegeneration, our understanding of the processes underlying their dysfunction in different part of the brain in AD and other neurodegenerative disorders is still limited. Substantial studies have revealed that alterations of mitochondria in neuronal cells and in synaptic endings are early pathophysiological events in AD (Schmitt et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Du et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Nevertheless, questions on this subject including sequence of molecular events and their localization in particular brain regions have not yet been fully resolved. Moreover, the role of protective processes activated simultaneously with pathological changes is not well recognized. There is indication that energy production not only is related to complexes/super complexes of the electron transport chain (ETC) but also is dependent on mitochondrial dynamic and their ability to undergo cycles of fission and fusion (Manczak et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Oliver and Reddy, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mitochondrial dynamic machinery depends mainly on dynamin related protein 1 (Drp1), mitochondria fission protein 1 (Fis1), mitochondria fission factor (Mff), and proteins regulating mitochondrial fusion, e.g., mitofusin-1, mitofusin-2 (Mfn1, Mnf2) and optical atrophy protein 1 (Opa1). These proteins are known to participate in the assembly and stability of ETC super complexes, in remodeling of mitochondrial cristae, and in shaping mitochondrial morphology in response to environmental conditions. However, only 13 proteins involved in mitochondria function are encoding by mtDNA. The nuclear respiratory factors, NRF1 and NRF2, are implicated in the transcription of genes encoding respiratory subunits, including 10 subunits of cytochrome c oxidase of complex IV, and transcription factor A mitochondria (TFAM) and both TFB (Transcription factor B) isoforms. Moreover, members of the nuclear receptors (NRs) superfamily including PPAR-α can also play a significant role in the transcriptional control and in mitochondrial lipids and fatty acids metabolism (W\u0026oacute;jtowicz et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Strosznajder et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies demonstrated excessive mitochondria division in AD patients and in experimental animal models of AD (Hu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Oliver and Reddy, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our data from Cieślik et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) showed significant downregulation of genes such as Sirt 1, mt-Nd1, Mfn1 and concomitant upregulation of \u003cem\u003eDnm1\u003c/em\u003e in the 12-month-old AD Tg mice model (with London mutation). In the human AD brain, changes in microRNA patterns (miRNA-9, miRNA-34a, miRNA-146 and miRNA-155) were found, and these changes are probably responsible for the downregulation of Sirt1 expression (Cieslik et al., 2020). Alterations of transcription of genes encoding proteins related to mitochondria biogenesis can also affect the progression of AD. Through interaction with NRF1, PGC-1α (Peroxisome Proliferator-Activated Receptor Gamma Coactivator alpha), the key protein of mitochondria biogenesis, can lead to activation of gene coding TFAM and then mitochondrial DNA (mtDNA). This transcription factor TFAM is crucial not only for mtDNA transcription but also in mtDNA maintenance and mtDNA nucleoid formation (Kang and Hamasaki, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherapeutic modulations of Sirt1 (Silent information regulator 1) and SOD2 have been suggested to offer potential for treatment of aged related neurodegenerative disorders connected with mitochondria alteration. Until now, neuroprotective strategies for AD are largely unsuccessful mainly because they are introduced too late at rather advance stage of the disease. The effect of several pharmacological compounds acting on Sirtuins and DNA-bound PARPs, both NAD-dependent families of enzymes, and on their role in different stages of AD, has not been fully elucidated. Little is known on the effect of these compounds at early stage of AD because diagnosis is too late. Therefore, more studies are needed to understand the dynamic and time-dependent molecular alterations of genes encoding proteins related to anti-oxidative defense, such as Sirts /PARPs /SODs, and mitochondria dynamics and function in animal models of AD at early stage of the disease. This subject was recently highlighted by Wang et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Yang et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The mitochondria changes were also observed in lymphocytes of sporadic AD patients and the question arise if they could be an early marker for the diagnosis and prognosis of disease (J\u0026ouml;rg et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recently, several pharmacological and natural compounds for treatment of AD also modulating expression and activity of Sirt1 have been tested in pre-clinical/clinical studies of human disorders (Manjula et al., 2020; Eckert et al \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cummings et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering these deficiencies, this study used a transgenic AD mouse model to determine gene expression encoding proteins of anti-oxidative defense machinery, including SOD1 and mitochondrial SOD2, transcription profiles of NAD-dependent enzymes, and mRNA expression related to mitochondria dynamics, biogenesis and function in brain cortex of 3- and 6-month-old AD Tg mice as compared with corresponding age-matched controls (without transgene). The major goal here is to explore early target(s) for cytoprotection and promising therapeutic strategy in AD.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal model of AD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale FVB-Tg (Thy1; APP LD2/B6) mice, aged 3 and 6 months, were used. These mice overexpressed human AβPP with the “London” V717I mutation under control of a fragment of Thy1 promoter with specificity towards brain and spinal cord neurons. Mice without the transgene were used as controls. This animal model had successfully recreated a relatively broad spectrum of behavioral, electrophysiological and biochemical features of AD (Moechars et al., 1999; Van Drope et al., 2000). These mice display behavioral abnormalities starting at 8 weeks of age (Moechars et al., 1999). Starting from the age of approximately 3 months, these mice gradually develop agitation and cognitive disturbances and cognitive impairment. These changes were accompanied by altered reactivity to neurotransmitters (observed at 3–4 months of age) and electrophysiological alterations (between 5 and 7 months) (Moechars et al., 1999). Animals were bred under specific pathogen-free (SPF) conditions in controlled temperature and humidity conditions and 12-h light/dark cycle in the Animal House of the Mossakowski Medical Research Centre PAS, Warsaw, Poland. At 3 or 6 months, mice were decapitated. Cerebral cortices were quickly isolated on ice and frozen in liquid nitrogen. The protocol was approved by the Warsaw Local Ethics Committee for Animal Experimentation and performed in accordance with guidelines of Polish National Ethics Committee and the EU Directive 2010/63/EU. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. All efforts were made to minimize suffering and to reduce the number of animals used. The experiments were performed in accordance with good laboratory practice protocols and quality assurance methods. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of Gene Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated using the TRI reagent (Sigma –Aldrich/Merck) as described in the manufacturer’s protocols and purified by using DNase I according to the manufacturer’s protocols (Sigma-Aldrich/Merck). The concentration and purity of obtained RNA was determined spectrophotometrically (A260/A280). Reverse transcription was performed by using the High-capacity cDNA Reverse Transcription Kit according to the manufacturer’s instruction (Applied Biosystems). The level of mRNA for studied genes was analyzed by using TaqMan Gene Expression Assays (Applied Biosystems). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCat (Mm00437992_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dnm1l (Mm01 3 42 9 03 _ m 1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Fis1 (Mm004815 8 0 _ m 1),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGpx4 (Mm0051 5041_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp;Mfn1 (Mm00612599 _ m 1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Mfn2 (Mm00500120_m1),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003emt-Co1 (Mm04225243_g1), \u0026nbsp; \u0026nbsp; mt-Cytb (Mm04225271_g1 ), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; mt-Nd1 (Mm042 2 5 2 7 4- s 1),\u003c/p\u003e\n\u003cp\u003eOpa 1 (Mm01 3 4 9 70 7 g1), \u0026nbsp; Sdha (Mm01352366_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sirt1 (Mm00490762_m1),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSirt3 (Mm00452131_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sirt4 (Mm01201915_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Sirt5 (Mm0135 15 76_m1),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSOD 1 (Mm0134423_g1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;SOD2 (Mm01313000_m1), \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sdhc (Mm00481172_m1)\u003c/p\u003e\n\u003cp\u003ePARP1 (Mm01321084) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; PARP2 (Mm00456462_m1) \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; PARP3 (Mm00467486_m1) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantitative polymerase chain reaction (PCR) was performed on an Applied Biosystems 7500 Real-Time PCR System using TaqMan Gene Expression Master Mix according to the manufacturer’s instructions. The relative levels of mRNA were calculated using the\u0026nbsp;ΔΔCt Method and were normalized against beta actin (\u003cem\u003eACTB,\u0026nbsp;\u003c/em\u003eMm4352341E)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of enzymatic activity of cytochrome c oxidase and Complex IV of mitochondria respiratory chain\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eActivity of cytochrome c oxidase (COX) in brain cortex homogenate lysate was determined as described by Spinazii et al. (2012). To prepare lysates, brain cortex previously frozen in liquid nitrogen and stored in -80C were thawed, dissected, and then homogenized in 20 mM phosphate buffer pH 7.5 (0.5 ml per 1mg tissue) using 1ml syringe and passing through the needle until homogenous solution appeared. Then the homogenate and lysate were frozen and thawed three times. Cytochrome c was reduced by incubation in the presence of 0.5 mM DTT for 20 min at room temperature in dark. The efficacy of cytochrome c reduction was checked by calculating the ratio of the absorbance 550 nm/565 nm, ratio \u0026gt; 6 indicates effective reduction. To determined Cox activity, 40 ul of lysate ± 100 µg protein was incubated with 25uM reduced cytochrome c in 25 mM phosphate buffer pH 7.0 at room temperature. The decrease of absorbance at 550 nm was measured during 3 min. The extinction coefficient for reduced cytochrome c was 18.5mM\u003csup\u003e-1\u003c/sup\u003e x cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results were expressed as mean values ± SEM from 4 to 8 animals, independent experiments carried out in triplicate. Differences between the means were analyzed using a Student's t-test for two groups, * p \u0026lt;0.05, ** p \u0026lt;0.01 *** p \u0026lt;0.001 were considered significant. The statistical analyses were performed using Graph Pad Prism version 8.0 (Graph Pad Software, San Diego, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, we determined mRNA profiles encoding anti-oxidative enzymes in the brain cortex of AD Tg and aged-matched control mice at 3- and 6-month of age. Results indicated a small but significant decrease in the level of mRNA encoding the Mn\u003csup\u003e2+\u003c/sup\u003e-dependent SOD2 in the 3-month Tg AD mice (Fig.\u0026nbsp;1). Under this condition, mRNA levels for SOD1, glutathione peroxidases GPX1, GPX4 and catalase were not changed (Fig.\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eNAD-dependent enzymes such as Sirtuins and PARPs are sensitive in oxidative/genotoxic stress conditions. Among them, Sirt1 and PARP1 are known to play a crucial role in regulating transcription factors for gene expression and cells survival. These enzymes as well as other members of the Sirtuin’s family, such as the mitochondrial Sirt3, Sirt4 and Sirt5 and other DNA-bound PARPs (PARP2and PARP3), may also play a role in the pathophysiology of neurodegenerative diseases including AD. In this study, analysis of mRNA levels demonstrated a significant decrease in expression encoding Sirt1 in the brain cortex of the 3-month-old AD Tg mice but mRNA levels for the mitochondrial Sirts (Sirt 3, 4, 5) were not significantly altered (Fig.\u0026nbsp;2a). Although mRNA encoding PARP-1 was decreased in the 3-month-old AD Tg mice and, there was a significant increase in the 6-month-old AD Tg mice (Fig.\u0026nbsp;2b). Nevertheless, there were no significant differences in mRNA levels for the DNA-bound PARP2, and PARP3 (Fig.\u0026nbsp;2b).\u003c/p\u003e\n\u003cp\u003eSubsequently, we examined the mRNA levels in selected sub-units of the mitochondrial respiratory complex in the brain cortex of AD Tg mice and compared with the non Tg controls. In this study, mRNA expressions encoding the subunit of mitochondria respiratory complex I, mt-Nd1, and subunits of complex II, Sdha and Sdhc in the brain cortex of 3- and 6-month-old AD Tg mice were not altered (Fig.\u0026nbsp;3). However, mRNA levels for Cytb, encoding the subunits of complex III was decreased slightly (not-significant), whereas mRNA encoding mt-Co 1, subunit of complex IV, was increased in brain cortex of the 6-month-old AD Tg mice as compared with age-matched controls (Fig.\u0026nbsp;3). In this study, we also measured cytochrome C activity, which was not significantly altered in the 3-month-old AD Tg mice as compared with age-match controls (Fig.\u0026nbsp;3).\u003c/p\u003e\n\u003cp\u003eIn a subsequent study, we analyzed expression of genes encoding proteins involved in mitochondria biogenesis. In this study, mRNA levels for PPAR-α and Ppargc1 were not altered (Fig.\u0026nbsp;4). However, there were significant increases in levels of mRNA encoding NRF1 and, NRF2 in the 3-month-old AD Tg mice as compared with controls but transcription of NRF2 was downregulated in 6 months old mice instead (Fig.\u0026nbsp;4). In addition, mRNA for TFAM, another mitochondrial transcription factor, was significantly increased in 6-month-old AD Tg mice as compared with control.\u003c/p\u003e\n\u003cp\u003eLastly, we examined mRNA expression encoding proteins involved in mitochondria dynamic. Although there were no changes in expression of genes encoding Mnf1 and Mnf2, a pronounce increase in mRNA expression encoding OPA1, a key regulator of mitochondria fusion, in the 3 and 6-month-old AD Tg mice was observed (Fig.\u0026nbsp;5). Concomitantly, there were significant increases in mRNA levels encoding Fis1 and Drp1 proteins in the 6-month-old AD Tg mice (Fig.\u0026nbsp;5).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, this study identified significant alterations of mRNA expression encoding enzymes involved in antioxidative defense and in dynamic of mitochondria in the cerebral cortex of 3- and 6-month-old AD Tg mice as compared with age-matched controls. The most significant changes are related to alterations of genes encoding Sirt1 and PARP1, both NAD-dependent enzymes present mainly in the nucleus (but are also located in mitochondria). These enzymes are sensitive sensors for maintenance of free radical homeostasis (Strosznajder et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Felici et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lapucci et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Dawson and Dawson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Downregulation of transcription of genes encoding SOD2 and Sirt1 could be a crucial early event in AD, and these changes may create conditions for enhancement of the free radical cascade and oxidative stress, and further leading to changes in transcription of genes encoding proteins related to mitochondria function. The functional crosstalk between Sirt1 and PARP1 is crucial for genome integrity, chromatin structure and response to free radicals DNA damage (El Ramy et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although not fully understood, there is an apparent close relationship between Sirt1, PARP1 and transcription processes in mitochondria. There is indication that PARP-1 inhibitors are best activators of Sirts (Bai et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and both enzymes are molecular regulators of cells survival and death (Bai et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Strosznajder et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jęśko et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Sirt1 may play a significant role in APP metabolism and in antioxidative defense. Sirt1 downregulation may lead to disturbances of homeostasis between non-amyloidogenic pathway of APP degradation by α secretase and amyloidogenic pathway of APP metabolism by β secretase, subsequently leading to excessive release of Aβ peptides (Jęśko et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our previous study with the 12-month-old AD Tg mice indicated significant lower expression of genes encoding Sirt1, subunit of complex I, mt-Nd1 and concomitant increase in genes encoding subunit of complex IV, mtCo1, as well as upregulation of Drp 1, the gene encoding the crucial protein of mitochondria fission (Cieślik et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our present study, mRNA encoding Sirt1 was significantly decreased in the 3-month-old AD Tg mice. Since Sirt1 is the most studied member of the Sirtuin family, the decrease of transcription of gene coding this enzyme at the early stage of AD may play an important role in the progression of pathology of AD (Jęśko et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jęśko and Strosznajder, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe brain function is critically relying on ATP synthesis through the ETC respiratory complexes in the mitochondria. In the present study, subunits of respiratory complexes engaged in ETC were slightly decreased, albeit not significantly. In contrast, there was a significant upregulation of expression of gene encoding subunit of complex IV which probably served as a compensatory mechanism for the small changes in other complexes and lower cytochrome c oxidase activity (albeit not significant) observed in the 3-month-old AD Tg brain cortex as compared with controls. A selective defect of cytochrome c oxidase was observed in the brain of AD patients (Mauer et al., 2000).\u003c/p\u003e \u003cp\u003eAn important by-product of electron transport processes is superoxide anion which is converted by superoxide dismutase SOD2 to the less reactive H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Flyn and Melov, 2013). Analysis of post-mortem samples from Alzheimer\u0026rsquo;s patients demonstrated elevated lipid peroxidation and upregulation of SOD2 (De Leo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). However, other studies using transgenic AD mice model indicated a reduction of SOD2 instead (Esposito et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Our previous study demonstrated a pronounced enhancement of gene transcription encoding SOD2 in microglia cells (BV2) upon subjecting the cells to the toxic Aβ oligomers but not in neuronal (SH-SY5Y) cells, which showed a opposite effect (Cieślik et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, levels of SOD2 mRNA were decreased in the 3- and slightly in 6-month-old AD Tg mouse brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the AD Tg mouse model, lower transcription of genes encoding Sirt1 and SOD2 may create conditions for alterations of transcription of other genes encoding Drp1 and Fis1, which are crucial proteins of mitochondria fission.\u003c/p\u003e \u003cp\u003eThe lower transcription of genes encoding enzymes of antioxidative defense could be a prerequisite event in molecular cascade leading to mitochondria degeneration and cell death. Among the proteins related to mitochondria, those engaged in regulation of fission and fusion are probably most susceptible to alterations at the early stage of the disease (Wang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Results in the present study showed significant alterations of transcription of genes related to mitochondria dynamic, upregulation of genes encoding Opa1 protein involved in mitochondrial fusion, suggesting activation of some compensatory or protective processes evoked by activation of transcription of genes coding Fis1 and Drp1 engaged in mitochondria fission. In another study, significant increase in Fis1, gene involved in regulation of mitochondria fission was also observed in AD brains compared with age-matched controls (Wang et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In AD patients and in AD animal models, different parts of the brain and different cell populations may exert different susceptibility to pathological insults which may lead to changes in expression of genes involved in mitochondria dynamic, function and mitochondria biogenesis. TFAM is a crucial transcription factor engaged in mitochondria biogenesis. The ability of TEAM transcription to increase the level of mtDNA suggests that this factor is linked the nuclear transcription response to mtDNA and mitochondrial biogenesis (Picca et al., 2015). In addition, studies have demonstrated that TFAM upregulation may protect against oxidative stress and mitochondrial failure (Campbell et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, expression of gene encoding TFAM was enhanced significantly in brain cortex of 6 months old AD Tg mice. Early pharmacological intervention to enhance transcription of TFAM and mtDNA stability and mitochondrial biogenesis could be a promising approach to retard the progression of AD. Moreover, our data demonstrated significant enhancement of genes expression coding NRF1 and NRF2 in brain cortex of 3 months old AD Tg mice which may suggest activation of some protective /adaptive pathway(s). Transcription of NRF2 observe in 3 months old AD Tg mice was subsequently decreased in the brain cortex of 6 months old AD Tg mice. NRFs are crucial transcription factors engaged in mitochondria biogenesis and in the regulation of expression of genes encoding several mitochondrial proteins involved in bioenergetics function and several other genes with diverse functions including cell growth, autophagy, apoptosis and antioxidant defense (Brandes and Gray, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Urfer-Buchwalder and Urfer, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). NRF1 and NRF2 could be very promising targets in early therapeutic approaches in AD or in strategy of prevention against oxidative stress and neurodegeneration during brain aging. NRF2 through activation of genes expression, coding proteins engaged not only in antioxidative but also anti-inflammatory defense could exert significant neuroprotective effect. NRF 2 activating pharmacological compounds are recently considered by FDA for treatment of Multiple Sclerosis (MS) and other neurodegenerative disorders (Brandes and Gray \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur data should be helpful in better understanding of early changes in transcription of genes in AD Tg mice and could contribute to a better understanding of molecular processes involved in mitochondria dysfunction in AD. Moreover, based on our findings in this study, we suggest that Sirt1, SOD2, Fis1 and Drp1 are the important targets for therapeutic approaches in the early stage of AD. The therapeutic strategy should support endogenously activated neuroprotective processes occur concomitantly with pathological changes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict of interest:\u003c/strong\u003e \u003cp\u003eThe authors have declared that there are no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements -\u003c/h2\u003e \u003cp\u003eSupported by National Science Centre (PL) Grant no 2019/35/N/NZ4/03706 and by MMRC, PAS statutory budget theme no 7.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBai, P., Cant\u0026oacute;, C., Oudart, H., Bruny\u0026aacute;nszki, A., Cen, Y., Thomas, C., Yamamoto, H., Huber, A., Kiss, B., Houtkooper, R. H., Schoonjans, K., Schreiber, V., Sauve, A. A., Menissier-de Murcia, J., \u0026amp; Auwerx, J. (2011). PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation. 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Frontiers in neuroscience, 15, 654785. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e://doi.org/10.3389/fnins.2021.654785\u003c/span\u003e\u003cspan address=\"://10.3389/fnins.2021.654785\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, gene expression, antioxidative defense, mitochondria ","lastPublishedDoi":"10.21203/rs.3.rs-1481945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1481945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress and disturbances of mitochondria function in the brain have been recognized to play a crucial role in the pathophysiological mechanism of Alzheimer’s Disease (AD). However, little is known about these changes at an early age of AD, which could be crucial for therapeutic strategy for the disease. In this study, we used biochemical, and quantitative polymerase chain reaction for determination of expression of genes encoding enzymes related to the antioxidative defence including Sirtuins (Sirts) and DNA-bound poly (ADP-ribose) polymerases (PARPs). Moreover, expression of genes related to mitochondrial dynamic, biogenesis and function in the brain cortex of 3- and 6-month-old FVB mice with London mutation (V7171) was analysed and compared with mice without transgene. Results indicated significant decreases in mRNA expression encoding SOD2, Sirt1 and PARP1 in the 3-month-old AD Tg mice and an increase in expression of PARP-1 in the 6-month-old AD Tg. Although levels of mRNA encoding subunits of mitochondrial respiratory complexes (I-III) were negligible altered, there was upregulation of gene encoding subunit of complex IV and proteins related to mitochondria biogenesis and dynamic, such as the Neuronal Respiratory Factors (NRF1) and NRF2, Opa1, Fis1, and Drp1 in the AD mice. Our data indicate downregulation of genes related to antioxidation and activation of genes encoding mitochondrial biogenesis and fission / fusion at early age of the AD mice. The ability to identify changes in gene expression for Sirt1, SOD2, Fis1 and Drp1 at an early age suggest potential therapeutic targets for retarding the pathological progression in AD.\u003c/p\u003e","manuscriptTitle":"Early age changes in mRNA expression related to NAD-dependent enzymes and mitochondrial proteins in the brain cortex of an Alzheimer’s disease mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-28 20:31:24","doi":"10.21203/rs.3.rs-1481945/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f113e337-bdcf-4a47-a3b1-0f5030d471b5","owner":[],"postedDate":"March 28th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-28T01:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-28 20:31:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1481945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1481945","identity":"rs-1481945","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}