Neurobiology of Aging 34 (2013) 1581e1588
`
`Contents lists available at SciVerse ScienceDirect
`
`Neurobiology of Aging
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / n e u a g i n g
`
`Nicotinamide riboside restores cognition through an upregulation of
`proliferator-activated receptor-g coactivator 1a regulated b-secretase 1
`degradation and mitochondrial gene expression in Alzheimer’s mouse models
`Bing Gong a, Yong Pan a, Prashant Vempati a, Wei Zhao a, Lindsay Knable a, Lap Ho a, Jun Wang a,
`Magdalena Sastre c, Kenjiro Ono a,1, Anthony A. Sauve d, Giulio M. Pasinetti a,b, *
`a Department of Neurology, Center of Excellence for Novel Approaches to Neurotherapeutics, Mount Sinai School of Medicine, New York, NY, USA
`b Geriatric Research, Education and Clinical Center, James J. Peters Veteran Affairs Medical Center, Bronx, NY, USA
`c Division of Brain Sciences, Imperial College London, London, UK
`d Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA
`
`a r t i c l e i n f o
`
`a b s t r a c t
`
`Article history:
`Received 5 November 2012
`Received in revised form 5 December 2012
`Accepted 9 December 2012
`Available online 9 January 2013
`
`Keywords:
`Nicotinamide riboside
`Alzheimer’s disease
`b-secretase (BACE1)
`Promotes peroxisome proliferator-activated
`receptor (PPAR)-g coactivator 1 (PGC)-1a
`Ubiquitineproteasome system
`Mitochondrial metabolism
`Synaptic plasticity
`Long-term potentiation
`
`þ
`
`, a coenzyme involved in redox activities in the mitochondrial
`Nicotinamide adenine dinucleotide (NAD)
`electron transport chain, has been identified as a key regulator of the lifespan-extending effects, and the
`þ
`expression has been linked with a decrease in beta-amyloid (Ab) toxicity in Alzheimer’s
`activation of NAD
`þ
`precursor, it promotes peroxisome proliferator-
`disease (AD). Nicotinamide riboside (NR) is a NAD
`activated receptor-g coactivator 1 (PGC)-1a expression in the brain. Evidence has shown that PGC-1a is
`a crucial regulator of Ab generation because it affects b-secretase (BACE1) degradation. In this study we
`tested the hypothesis that NR treatment in an AD mouse model could attenuate Ab toxicity through the
`activation of PGC-1a-mediated BACE1 degradation. Using the Tg2576 AD mouse model, using in vivo
`behavioral analyses, biochemistry assays, small hairpin RNA (shRNA) gene silencing and electrophysio-
`logical recording, we found (1) dietary treatment of Tg2576 mice with 250 mg/kg/day of NR for 3 months
`significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-
`þ
`in the cerebral cortex; (2) application of NR to hippocampal slices (10 mM) for 4 hours
`state levels of NAD
`abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treat-
`ment promotes PGC-1a expression in the brain coinciding with enhanced degradation of BACE1 and the
`reduction of Ab production in Tg2576 mice. Further in vitro studies confirmed that BACE1 protein content is
`decreased by NR treatment in primary neuronal cultures derived from Tg2576 embryos, in which BACE1
`degradation was prevented by PGC-1a-shRNA gene silencing; and (4) NR treatment and PGC-1a
`overexpression enhance BACE1 ubiquitination and proteasomal degradation. Our studies suggest that
`dietary treatment with NR might benefit AD cognitive function and synaptic plasticity, in part by promoting
`PGC-1a-mediated BACE1 ubiquitination and degradation, thus preventing Ab production in the brain.
`Ó 2013 Elsevier Inc. All rights reserved.
`
`1. Introduction
`
`þ
`
`has been identified
`Nicotinamide adenine dinucleotide (NAD)
`as a key regulator in the lifespan-extending effects of calorie
`restriction in a number of species. Numerous studies have sug-
`þ
`mediates multiple major biological processes,
`gested that NAD
`including calcium homeostasis, energy metabolism, mitochondrial
`functions, cell death, and aging in various tissues including brain.
`
`* Corresponding author at: Mount Sinai School of Medicine, Department of
`Neurology, 1468 Madison Avenue, New York, NY 10029, USA. Tel.: þ1 212 241 7938
`or þ1 212 241 5563; fax: þ1 212 876 9042.
`E-mail address: giulio.pasinetti@mssm.edu (G.M. Pasinetti).
`1 Current address: Department of Neurology and Neurobiology and Aging,
`Kanazawa University Graduate School of Medical Science, Kanazawa, Japan.
`
`0197-4580/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved.
`http://dx.doi.org/10.1016/j.neurobiolaging.2012.12.005
`
`þ
`might play important
`Increasing evidence has suggested that NAD
`roles in metabolic processes in the brain, and has effects on brain
`functioning such as neurotransmission,
`learning, and memory.
`Recent studies have shown that the activation of NAD expression
`has been linked with a decrease in the amyloid toxicity in
`Alzheimer’s disease (AD) animal models (Kim et al., 2007; Qin et al.,
`2006),
`in which it might relate to the interactions with the
`expression of peroxisome proliferator-activated receptor-g coac-
`tivator 1 (PGC)-1a (Nemoto et al., 2005) and through the activation
`of neuronal NAD-dependent deacetylase sirtuin-1 (SIRT1) activa-
`tion (Qin et al., 2006; Rodgers et al., 2005). It has been shown that
`during metabolic stress conditions such as the fasting state,
`hypoxia, NADþ levels, and SIRT1 protein levels are increased,
`leading to deacetylation of PGC-1a, subsequently increasing the
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`expression of PGC-1a, promoting gluconeogenic transcriptional
`program (Rodgers et al., 2005), consequently protecting the mito-
`þ
`chondrial energy metabolism. Thus, the benefits of the NAD
`stimulating cell survival have raised the hope that using pharma-
`þ
`concentrations might provide
`cological agents to increase NAD
`therapeutic benefits in delaying the onset and slowing the
`progression of AD dementia.
`Nicotinamide riboside (NR) is a NAD precursor, which is con-
`verted to NAD through action of human NrK1 and NrK2 genes in the
`de novo fashion (Bieganowski and Brenner, 2004; Bieganowski et al.,
`2003). Evidence shows that NR treatment increases intracellular
`þ
`þ
`concentration and improves NAD
`-dependent activities in the
`NAD
`cell by increasing silent mating-type information regulation 2 (Sir2)-
`dependent gene silencing and longevity via nicotinamide riboside
`þ
`synthesis (Belenky et al., 2007).
`kinase (NRK) 1-dependent NAD
`Thus it is possible that the exogenous application of NR is capable of
`promoting the biosynthesis of NAD, thus promoting the beneficial
`effects of NAD (Braidy et al., 2008). Excitingly, it has been reported
`that treatment with nicotinamide prevents cognition in AD trans-
`genic mice via a mechanism involving sirtuin inhibition and reduc-
`tion of tau phosphorylation (Green et al., 2008). However, the role of
`NR in the beta-amyloid (Ab) deposition in AD brain is still not clear.
`It has been shown that PGC-1a also plays an important role in
`energy metabolism by regulating mitochondrial
`function in
`different tissues. The expression of PGC-1 has been found signifi-
`cantly decreased in Alzheimer’s brains, and it is involved in the
`Ab pathological generation by affecting the processing of amyloid
`precursor protein (APP), at least partially through enhancing the
`a-secretase activity (Qin et al., 2009; Wu et al., 2006). Recently, our
`group and others reported that 1 of the mechanisms in which the
`PGC-1 decreases the Ab burden is also involved in the regulation of
`the F-Box (FbX)2-E3-ligase-mediated b-secretase (BACE1) degra-
`dation (Gong et al., 2010; Katsouri et al., 2011) as it does in other E3
`ligases in the ubiquitin system in other tissues. Encouraged by the
`effects of NAD on promoting the PGC-1 expression, in this study, we
`tested the hypothesis that exogenous treatment of NR might reduce
`the Ab burden in AD brain via enhancing PGC-1a expression, which
`increases BACE1 ubiquitination, degradation, and improves mito-
`chondrial metabolism. Our study provides a novel therapeutic
`strategy for the treatment of AD.
`
`2. Methods
`
`2.1. Animals
`
`/
`mice (Qin et al.,
`Tg2576 mice were crossed with PGC-1a
`/
`/Tg2576 mice. Animals were back-
`2009) and generated PGC-1a
`crossed at least 10 generations onto normal C57BL/6 mice (Jackson
`Laboratories, Bar Harbor, ME, USA). All experiments were approved
`by the Mount Sinai School of Medicine Animal Care committees.
`
`2.2. Primary neuronal cell culture
`
`Tg2576 mouse primary neuronal cell cultures were prepared
`from the brains of 14.5-day-old embryos bred from wild type
`C57BL/6 females crossed with Tg2576 males, as described previ-
`ously (Gong et al., 2010). Briefly, after isolation, cerebral hemi-
`spheres were placed into Dulbecco’s modified Eagle’s medium
`supplemented with 10% fetal bovine serum (FBS) and 1 penicillin-
`streptomycin. Brain tissue was dissociated and the single cells were
`suspended in Dulbecco’s modified Eagle’s medium supplemented
`with 10% FBS and 1 penicillin-streptomycin. Cells were seeded
`into precoated 12-well plates (BD Biosciences) at a concentration of
`8  105 cells per well. After 30 minutes of incubation in a tissue
`culture incubator, the cells were changed to neural basal medium
`
`(Invitrogen) supplemented with 0.5 mM L-glutamine (Cellgro), 1 
`B-27 (Invitrogen), and 1 penicillin-streptomycin (Invitrogen). The
`cells were kept in an atmosphere of 95% air and 5% CO2. After 7
`more days of incubation, cells were used for the treatment.
`
`2.3. BACE1 activity measurements and quantification of amyloid
`peptides by enzyme-linked immunosorbent assay
`
`The measurement of the Ab levels has been described previously
`(Gong et al., 2004). Briefly, levels of Ab1e40 and Ab1e42 in primary
`cultured Tg2576 neurons infected with various adenoviral vectors
`were determined using sandwich type enzyme-linked immuno-
`sorbent assay (ELISA) (Biosource International, Camarillo, CA, USA).
`The background from control medium (transfected with the adeno-
`green fluorescent protein [GFP] vector) was subtracted from the
`sample values.
`
`2.4. NR treatment and behavioral assessment
`
`to 8-month-old Tg2576 mice were treated with
`Seven-
`250 mg/kg/day NR; control Tg2576 mice were treated with saline.
`Treatments started at approximately 5e6 months of age, and lasted
`until 10e11 months of age. Mice had their cognitive functions
`assessed by the object recognition protocol. Mice were first placed
`in an apparatus and allowed to explore an object. After a certain
`interval, the mouse was returned to the apparatus, which contained
`the familiar object and a novel object (Bevins and Besheer, 2006).
`The time which the mouse spent on the novel object was calculated
`and was compared between treated and control groups. After
`behavioral assessment, mice were sacrificed, and their brains were
`dissected. One hemisphere was snap frozen for subsequent
`assessment of Ab-specific ELISA as discussed above. The other
`hemisphere was fixed in formaldehyde for subsequent stereological
`quantitative assessments of neuritic plaque pathology, as previ-
`ously described (Green et al., 2008; Wang et al., 2008). In parallel,
`control studies using age-, sex-, and strain-matched wild type (WT)
`mice, were conducted to evaluate the potential effect of NR treat-
`ment on cognitive function in the absence of Ab neuropathology.
`
`2.5. Western blot
`
`Cells and tissues were lysed in either radioimmunoprecipitation
`assay (RIPA) buffer lysis buffer or Cell Signaling Lysis Buffer sup-
`plemented with protease inhibitors. Fifty to 100 mg of protein lysate
`was then run on sodium dodecyl sulfate polyacrylamide gel elec-
`trophoresis. Proteins were transferred to a nitrocellulose transfer
`membrane (Whatman). The membranes were blocked in 5% fat-
`free milk for 1 hour, then incubated in primary antibody for 1
`hour, horseradish peroxidase (HRP)-conjugated secondary anti-
`body for 1 hour, and developed in enhanced chemiluminescence
`(ECL) substrate.
`
`2.6. Hippocampal slice preparation and electrophysiology recording
`
`We cut four hundred mm brain slices from Tg2576 mice, and WT
`littermates, and maintained them in an interface chamber at 29 C
`for 90 minutes before recording, as previously reported (Gong et al.,
`2006). The bath solution consisted of 124.0 mM NaCl, 4.4 mM KCl,
`1.0 mM Na2HPO4, 25.0 mM NaHCO3, 2.0 mM CaCl2, 2.0 mM MgSO4,
`and 10.0 mM glucose. The stimulating electrode, a bipolar tungsten
`electrode, was placed at the level of the Schaeffer collateral fibers,
`whereas the recording electrode, a glass electrode filled with bath
`solution, was placed at the level of the CA1 stratum radiatum. Basal
`synaptic transmission was assayed by plotting the stimulus voltages
`against slopes of field excitatory postsynaptic potentials. For the
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`long-term potentiation (LTP) experiments, a 15-minute baseline
`was recorded every minute at an intensity that evoked a response
`of approximately 35% of the maximum evoked response. LTP was
`induced using ø-burst stimulation (4 pulses at 100 Hz, with the
`bursts repeated at 5 Hz and each tetanus, including three 10-burst
`trains, separated by 15 seconds). Two-way analysis of variance
`followed by Bonferroni’s test was used for statistical analysis. Data
`are mean  standard error of the mean (error bar) of the results
`from 2 independent experiments.
`
`2.7. Reverse transcription (RT)-polymerase chain reaction on
`mitochondrial gene expression in the brains of Tg2576 mice
`
`Five-month-old Tg2576 mice were treated with 250 mg/kg/day
`of NR for approximately 3 months, and total RNA from the cerebral
`cortex was extracted using RNeasy Mini Kit (Qiagen) 24 hours after
`behavioral testing. Complementary DNA was synthesized using
`Superscript III First-Strand Synthesis SuperMix for qRT-polymerase
`chain reaction (PCR) (Invitrogen) with 1 mg of total RNA. Quanti-
`tative RT-PCR was performed using Maxima SYBR Green Master
`Mix (Fermentas).
`
`3. Results
`
`3.1. NR treatment promotes cognition coincided with induction of
`PGC-1a
`
`To assess if NR has any protective effects on cognitive function as
`we proposed, we first treated Tg2576 AD transgenic (APP) mice
`(Hsiao et al., 1996) (7e8-month-old) with 250 mg/kg/day of NR
`(equivalent to 1300 mg/kg/day in the human) for 3 months via
`
`drinking water. We found that the NR treatment significantly
`improved cognitive performance of these mice in an object recog-
`nition test, which is a cognitive task to examine hippocampal- and
`cortical-dependent learning. This cognition function is compro-
`mised by AD pathology in this mouse model (Oddo et al., 2003). In
`particular, nontreated control Tg2576 mice performed at the chance
`level (42.0  9.2%), and the chance of NR-treated Tg2576 mice
`recognizing a novel level object was significantly better (63.2 
`1.7%; p < 0.05) (Fig. 1A). To confirm the improvement of the
`behavior in Tg2576 mice is because of the effects of the NR treat-
`ment and its link with NAD, next we tested the bioavailability of NR
`in the brain in this treatment protocol. We found that this NR
`þ
`in
`treatment significantly increased the steady-state levels of NAD
`the cerebral cortex (Fig. 1B). Our data implicates NR as a potential
`feasible therapy for treating energy metabolism defects in the AD
`þ
`levels in brain.
`brain through enhancing NAD
`
`þ
`3.2. NR treatment enhances NAD
`
`and PGC-1a expression
`
`þ
`has been associated with the
`Because the increase in NAD
`promotion of PGC-1a expression in various tissues, we further
`explored whether the treatment with NR could have any effects on
`PGC-1a expression and further affect the Ab levels. Indeed, our
`quantitative RT-PCR analysis in the brain samples from the mice of
`NR-treated and control groups showed NR significantly increased
`the PGC-1a gene expression compared with the untreated group,
`p < 0.05 (Fig. 1C). One of the important roles of PGC-1a has been
`reported to be reduction of Ab burden, thus we further tested the
`Ab levels in these mice. Consistent with the increase in the PGC-1a
`expression, the Ab levels are significantly decreased in the groups
`treated with NR (Fig. 1D).
`
`þ
`Fig. 1. NR improves cognitive function in Tg2576 mice via a promotion of NAD
`and PGC-1a levels. (A) Treatment with NR 250 mg/kg/day in Tg2576 mice for 3 months improves
`cognitive function. Object recognition memory test, performed as described by Bevins and Besheer (2006). Values are expressed as mean  standard error of the mean, n ¼ 10 mice per
`þ
`levels measured by NAD/NADH Assay Kit (Abcam). (C) PGC-1a mRNA levels in
`group. * p < 0.05, 2-tailed Student t test. (B) The NR treatment significantly increased the levels of NAD
`brain. Values are expressed as mean  standard error of the mean, n ¼ 8 mice per group. * p < 0.05, n ¼ 5 mice per group, 2-tailed Student t test. (D) Enzyme-linked immunosorbent
`assay showed the levels of Ab1e42 levels in brains treated with NR comparing with placebo-treated brains in Tg2576 mice. n ¼ 8 mice. Abbreviations: Ab, beta-amyloid; CTL, control;
`mRNA, messenger RNA; NAD, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; PGC-1a, peroxisome proliferator-activated receptor-g coactivator 1a.
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`3.3. NR improves synaptic plasticity in Tg2576 mice
`
`Because NR treatment improves cognitive function, we next
`used electrophysiological recording to study if NR has effects on
`synaptic plasticity in hippocampal CA1 region, reflected by LTP. We
`found there was no difference in the input-output curves generated
`using the field excitatory postsynaptic potential versus stimulus
`intensity between the NR treatment and control Tg2576 mice
`(Fig. 2A). For the LTP, after a 60-minute NR treatment (20 mM), the
`responses were significantly increased, and were 224  15% at 120
`minutes of the theta-burst stimuli, and in the control mice, the
`response were 164  12% (Fig. 2B) (n ¼ 8 slices; p < 0.05). The NR
`perfusion in this condition does not affect the input-output curve,
`nor the LTP levels in WT mice (Fig. 2C and D); p > 0.05.
`
`3.4. PGC-1a deficiency promotes the generation of Ab peptide in AD
`models
`
`It has been suggested that reduced PGC-1a expression in the AD
`brain strongly correlates with the progression of clinical dementia
`and neuropathology (Qin et al., 2009), based on the data showing
`that NR treatment increases PGC-1a expression. Thus it is inter-
`esting to know if PGC-1a deficiency might causally promote AD
`type b-amyloidosis in in vitro and in vivo experimental models.
`/
`) mice
`First, we crossed Tg2576 mice with PGC-1a null (PGC-1a
`þ/
`/APP double transgenic
`(Leone et al., 2005) to generate PGC-1a
`þ/
`/APP F1 mice with PGC-1a
`mice, followed by crossing PGC-1a
`/
`null mice to generate PGC-1a
`/APP (PGC-1a null/APP) mice.
`Assessed by ELISA, we found that the homozygous knockout of
`/
`PGC-1a in PGC-1a
`/APP mice significantly promoted Ab neuro-
`pathology (reflected by Ab1e40 and Ab1e42 levels in the cerebral
`cortex and hippocampus) by approximately 40%, in comparison
`with age- and sex-matched APP (Tg2576) mice (Fig. 3A) (n ¼ 3;
`p < 0.05). Further, in in vitro feasibility studies using primary
`
`cortical-hippocampal neuron cultures derived from Tg2576 mice,
`we found that reducing the cellular levels of PGC-1a by approxi-
`mately 1.5-fold (data not shown) by infecting cultured cells with an
`adenoviral PGC-1a silencing shRNA significantly increased the
`accumulation of b-amyloid Ab1e40 and Ab1e42 peptides in the
`conditioned medium by 1.2-fold or more (Fig. 3B). Collectively,
`these studies support our working hypothesis that downregulation
`of PGC-1a in the brain might causally promote amyloid neuropa-
`thology, and NR treatment improves the PGC-1a expression, thus
`reducing the Ab burden in AD mouse models.
`
`3.5. NR reduces BACE1 levels through promotion of PGC-1a
`expression
`
`Because NR treatment reducing Ab levels is coincident with the
`enhanced expression of PGC-1a, which has been associated with
`degradation of BACE1 (Gong et al., 2010), next we tested if the
`effects of NR on the Ab levels is also via a regulation on BACE1
`degradation.
`We tested the protein levels of BACE1 and PGC-1a in primary
`cultured cortical neurons from brains of Tg2576 mice treated with
`NR. We first confirmed the effects of NR on the promotion of
`PGC-1a expression by Western blot analysis, though this increase
`was abolished by the infection of adenoviral PGC-1a shRNA
`(Fig. 4A). Next, we probed the levels of BACE1 protein and found
`that BACE1 protein levels were significantly decreased by NR tre-
`atment, and this decrease was largely abolished by PGC-1a sile-
`ncing by adenoviral PGC-1a shRNA (Fig. 4B), suggesting that NR
`affects the Ab levels through regulation of PGC-1a expression.
`To further mechanistically explore the association between PGC-
`1a and BACE1, we overexpressed PGC-1a in cortical-hippocampal
`neuron cultures derived from Tg2576 mice. We found that the
`BACE1 levels were decreased by the PGC-1a expression, and this
`decrease was abolished by the PGC-1a gene silencing assessed by
`
`Fig. 2. NR-treated slices from Tg2576 mice improves synaptic function but has no effect on WT mice. (A) Application of NR at 20 mM for 4 hours in hippocampal slices from Tg2576
`mice (12e14-month-old) has no significantly effects on basal synaptic transmission recorded in CA1 region (A) (p > 0.05), however, the deficits of LTP in the Tg2576 mice was greatly
`rescued, n ¼ 8 slices, 2-way analysis of variance; p < 0.01 (B). Perfusion of NR has no effects on the basal synaptic transmission (C) nor on the LTP (D) in hippocampal slices from WT
`littermates (n ¼ 8, 2-way analysis of variance; p > 0.05). Abbreviations: fEPSP, field excitatory postsynaptic potentials; LTP, long-term potentiation; NR, nicotinamide riboside; WT,
`wild type.
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`Fig. 3. PGC-1a deficiency promotes the generation of Ab peptides in vivo and in vitro. (A) Increased Ab1e40 or Ab1e42 concentrations in approximately 4-month-old PGC-1a
`/APP
`mice relative to age- and sex-matched control APP (Tg2576) mice as assessed by enzyme-linked immunosorbent assay. (B) Silencing PGC-1a in primary corticohippocampal neurons
`derived from Tg2576 embryos resulted in elevation of Alzheimer’s disease-type Ab levels in the cultures with conditioned medium relative to control neuron cultures. PGC-1a
`silencing was achieved by infecting neurons with a PGC-1a shRNA adenovirus (a gift from Dr Puigserver) at 10 MOI. Control cells were infected with a GFP adenovirus. Ab
`accumulation in cultured media was assessed 24 hours after adenoviral infection. Values are expressed as mean  standard error of the mean from 2e3 independent experiments,
`with n ¼ 3e4 per group; * p < 0.05; ** p < 0.01; 2-tailed Student t test. Abbreviations: Ab, beta-amyloid; GFP, green fluorescent protein; MOI, multiplicity of infection; PGC-1a,
`peroxisome proliferator-activated receptor-g coactivator 1a; shRNA, small hairpin RNA.
`
`Western blot analysis (Fig. 5A). Because the ubiquitin-proteasome
`system (UPS) has been reported to be linked with BACE1 degra-
`dation, we then treated the adenoviral PGC-1a-infected neurons
`with lactacystin (5 mM), a specific proteasome inhibitor, for 4 hours.
`Consistent with our previous report,
`lactacystin significantly
`inhibited BACE1 degradation, suggesting that PGC-1a might exert
`influence on BACE1 via UPS-mediated degradation (Fig. 5B).
`
`control cells, suggesting that PGC-1a promotes BACE1 degradation
`in UPS through BACE1 ubiquitination (Fig. 6, upper panel).
`Consistent with the increase in ubiquitinated BACE1 levels, the
`levels of monoubiquitin levels were decreased in the cells with NR
`treatment or PGC-1a expression (Fig. 6, lower panel).
`
`3.7. NR induces PGC-1a-associated energy metabolism genes
`
`3.6. NR promotes BACE1 degradation through an increase in the
`BACE1 ubiquitination
`
`Because protein ubiquitination is an essential step for most
`proteins degraded by UPS, we further explored whether NR treat-
`ment increased the BACE1 ubiquitination. HEK293 cells stably
`transfected with BACE1 vector with Myc-tag (Myc-BACE1) were
`treated with NR, and infected with adenoviral PGC-1a, and
`adenoviral PGC-1a shRNA. The protein extracts were probed by
`Western blot with anti-ubiquitin antibody. We found that the
`ubiquitinated BACE1 protein levels markedly increased in NR-
`treated and PGC-1a overexpressing HEK293 cells relative to
`
`Because the treatment of NR promotes PGC-1a expression and
`þ
`levels,
`it is interesting to know
`increases intracellular NAD
`whether the PGC-1a associated energy metabolism genes were
`affected by the NR treatments. Because it has been reported that
`some of these genes were downregulated in the AD brain, we tested
`the messenger RNA (mRNA) levels of these genes in the brain
`extract from Tg2576 mice. We found that treating Tg2576 mice with
`NR for 3 months significantly promoted the expression of gene
`products involved in a wide range of mitochondrial metabolism,
`such as citrate synthase (Fig. 7A) and aconitase genes (Fig. 7B) in the
`tricarboxylic acid cycle; pyruvate dehydrogenase kinase (Fig. 7E) in
`pyruvate metabolism, cytochrome c subunit Vic in mitochondrial
`
`Fig. 4. NR promotes PGC-1a responses and BACE1 degradation in the brain. (A) NR-treated primary neuron cultures derived from Tg2576 mice showed increased PGC-1a protein
`levels probed by Western blot analysis with anti-PGC-1a antibody (Sigma). Values are expressed as mean  standard error of the mean; n ¼ 3; * p < 0.05, Student t test. (B) BACE1
`protein levels are decreased by NR treatment and the decrease was attenuated by PGC-1a-shRNA probed by Western blot analysis with anti-BACE1 antibody (Sigma). Values are
`expressed as mean  standard error of the mean; n ¼ 3; * p < 0.05, Student t test. Inset shows the protein of adeno-PGC-1a and PGC-1a-shRNA after infected in neurons.
`Abbreviations: BACE1, b-secretase; CTR, control; NR, nicotinamide riboside; PGC-1a, peroxisome proliferator-activated receptor-g coactivator 1a; shRNA, small hairpin RNA.
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`Fig. 5. PGC-1a expression promotes BACE1 degradation. (A) Primary hippocampal-cortical neurons derived from Tg2576 embryos at 14 days in vitro were infected by adenoviral-GFP
`PGC-1a, scramble PGC-1a-shRNA, or PGC-1a-shRNA, respectively, 72 hours after infection, cell lysates were collected and analyzed via Western blot using anti-BACE1 antibodies.
`BACE1 levels were quantified and normalized against the level of ß-tubulin and plotted as percentage of CTL. Data are expressed as mean  standard error of the mean (n ¼ 5). * p < 0.05
`compared with CTL group. Inset represents PGC-1a immunoreactive signals. (B) The degradation of BACE1 caused by PGC-1a expression on was blocked by lactacystin treatment (5
`mM). Data are expressed as mean  standard error of the mean (n ¼ 5). * p < 0.05 compared with CTL group. Inset represents BACE1 immunoreactive signals. Abbreviations: BACE1,
`b-secretase; CTL, control; GFP, green fluorescent protein; PGC-1a, peroxisome proliferator-activated receptor-g coactivator 1a; shRNA, small hairpin RNA.
`
`oxidative-phosphorylation (Fig. 7F); the human phosphoglycerate
`kinase and glucose phosphate isomerase 1 genes in glycolysis
`(Fig. 7G), assessed by quantitative RT-PCR suggesting that NR might
`promote cognitive performance, in part, by inducing expression of
`gene products involved in mitochondrial energy metabolism and
`linked with PGC-1a expression.
`
`4. Discussion
`
`This study for the first time mechanistically explored the effects
`of NR on the attenuation of amyloid toxicity, improves cognitive
`function, and synaptic plasticity in AD mouse models. We for the
`first time demonstrated that the effects of NR are associated with the
`promotion of PGC-1a function and the ubiquitin proteasome system.
`
`The latter 2 have been indicated to play important roles in AD
`pathogenesis, and PGC-1a has been especially indicated in diabetes-
`related metabolism deterioration and aging-related dementia.
`It has been shown that the effects of NAD on increasing life span
`have been linked with the activation of Sir1, Sir2, and further
`activation of PGC-1a expression, consequently affecting mitochon-
`drial metabolism. NR is a NAD precursor. Evidence shows that extra-
`cellular NR application could increase intracellular NAD levels
`through glutamine-dependent NAD(þ) synthetase (Qns) 1-indepen-
`dent and Nrk1-dependent pathways (Bieganowski and Brenner,
`2004). Our data shows that treatment with NR in Tg2576 mice
`improves synaptic plasticity and behavioral function and is coincident
`þ
`and PGC-1a levels, suggesting that the
`with the increase in the NAD
`NR protective effects might be linked to the PGC-1a-regulated
`
`Fig. 6. NR promotes BACE1 ubiquitination and degradation is linked with PGC-1a. (A) BACE1 stable HEK293 cells were infected with adeno-PGC-1a or adeno-PGC-1a-shRNA and
`scramble shRNA (B). The adeno-GFP viral constructs were used as control. Cells were treated with NR 200 mg and 5 mM lactacystin. After 48 hours of transfection, cell lysate was
`collected and BACE1 immunoprecipitated using anti-BACE1 antibody, and probed with anti-ubiquitin antibodies (Sigma). (C) Quantification of the square area in the left panel by
`ImageJ and represented in the graph. This area represents BACE1 proteins at different ubiquitinated levels. Data are mean  SEM (error bar) of the results from 2 independent
`experiments. Student t test was performed; * p < 0.05 compared with GFP-lactacystin treated cells. Abbreviations: BACE1, b-secretase; CTL, control; GFP, green fluorescent protein;
`NR, nicotinamide riboside; PGC-1a, peroxisome proliferator-activated receptor-g coactivator 1a; SEM, standard error of the mean; WB, Western blot analysis.
`
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`B. Gong et al. / Neurobiology of Aging 34 (2013) 1581e1588
`
`1587
`
`Control
`
`NR
`
`OXPHOS
`
`*
`
`*
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`200
`
`175
`
`150
`
`125
`
`C
`
`α
`
`PGC-1
`
`*
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`B
`
`Aconitase
`
`TCA
`
`*
`
`Pyruvate metabolism
`
`E
`
`150
`
`125
`
`100
`
`*
`
`F
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`A
`
`CS
`
`D
`
`140
`
`120
`
`100
`
`*
`
`100
`
`75
`
`50
`
`25
`
`0
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`COX6C
`
`I
`
`GPI1
`
`75
`
`50
`
`25
`
`0
`
`PDK3
`
`Glycolysis
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`H
`
`M-PFK
`
`*
`
`80
`
`60
`
`40
`
`20
`
`0
`
`175
`
`150
`
`125
`
`100
`
`75
`
`50
`
`25
`
`0
`
`PDHA
`
`G
`
`PGK1
`
`Fig. 7. Energy metabolism gene products are induced in response to short-term NR treatment in the brains of Tg2576 mice. In this study, 5-month-old Tg2576 mice were treated
`with 250 mg/kg/day of NR for approximately 3 months, and total RNA from the cerebral cortex was extracted using RNeasy Mini Kit (Qiagen) 24 hours after behavioral testing.
`Complementary DNA was synthesized using Superscript III First-Strand Synthesis SuperMix for quantitative RT-PCR (Invitrogen) with 1 mg of total RNA. Quantitative RT-PCR was
`performed using Maxima SYBR Green Master Mix (Fermentas). Gene targets examined here included (A) CS, (B) aconitase, (C) PGC-1a, (D) PDHA, (E) PDK3, (F) COX6C, (G) PGK1, (H)
`M-PFK, and (I) GPI1. Fold changes between groups were calculated using the DCt method. Statistical analysis was done in GraphPad Prism 4. Abbreviations: COX6C, cytochrome c
`subunit Vic; CS, citrate synthase; GPI1, glucose phosphate isomerase 1; M-PFK, muscle phosphofructokinase; NR, nicotinamide riboside; OXPHOS, oxidative phosphorylation
`system; PCR, polymerase chain reaction; PDHA, pyruvate dehydrogenase; PDK3, pyruvate dehydrogenase kinase 3; PGC-1a, peroxisome proliferator-activated receptor-g coactivator
`1a; PGK1, phosphoglycerate kinase; RT, reverse transcription; TCA, tricarboxylic acid cycle.
`
`reduction of Ab. Silencing PGC-1a abolished the effects of NR on the
`BACE1 degradation, further supporting this hypothesis.
`Recently, evidence showed that BACE1 posttranslational degra-
`dation is a potential novel link in PGC-1a mediated protection
`against AD amyloid neuropathology (Gong et al., 2010; Katsouri
`et al., 2011; Kwak et al., 2011). BACE1 is a key secretase involved
`in the processing of APP, ultimately resulting in generation of
`amyloidogenic Ab peptides. Here, we found that PGC-1a plays
`a important role in enhancing BACE1 degradation through mecha-
`nisms influencing UPS-mediated responses as NR does, suggesting
`that NR might also affect the UPS system through the regulation of
`PGC-1a. Thus, our study for the first time demonstrates this novel
`feature that NR promotes PGC-1a expression and BACE1 degrada-
`tion in mechanisms associated with APP processing and AD
`b-amyloidosis in the brain.
`Cerebral glucose hypometabolism