Abstract

   Nicotinamide adenine dinucleotide (NAD^+) is a critical metabolic
   co-enzyme implicated in brain aging, and augmenting NAD^+ levels in the
   aging brain is an attractive therapeutic strategy for
   neurodegeneration. However, the molecular mechanisms of brain NAD^+
   regulation are incompletely understood. In cardiac tissue, the
   circadian nuclear receptor REV-ERBα has been shown to regulate NAD^+
   via control of the NAD^+-producing enzyme NAMPT. Here we show that
   REV-ERBα controls brain NAD^+ levels through a distinct pathway
   involving NFIL3-dependent suppression of the NAD^+-consuming enzyme
   CD38, particularly in astrocytes. REV-ERBα deletion does not affect
   NAMPT expression in the brain and has an opposite effect on NAD^+
   levels as in the heart. Astrocytic REV-ERBα deletion augments brain
   NAD^+ and prevents tauopathy in P301S mice. Our data reveal that
   REV-ERBα regulates NAD^+ in a tissue-specific manner via opposing
   regulation of NAMPT versus CD38 and define an astrocyte
   REV-ERBα–NFIL3–CD38 pathway controlling brain NAD^+ metabolism and
   neurodegeneration.

   Subject terms: Alzheimer's disease, Circadian mechanisms, Astrocyte,
   Ageing
     __________________________________________________________________

   Lee et al. show that the circadian clock protein REV-ERBα controls
   brain NAD^+ levels by regulating the NAD^+-consuming enzyme CD38.
   Global or astrocytic REV-ERBα deletion or pharmacologic REV-ERB
   inhibition protects against tau pathology in mice.

Main

   NAD^+ is an abundant metabolite required for multiple cellular
   functions, including redox homeostasis, DNA repair, metabolism and
   histone/protein deacetylation via control of sirtuins
   (SIRTs)^[54]1–[55]3. NAD^+ homeostasis is dependent on a balance
   between production and consumption, with NAMPT being the rate-limiting
   enzyme for production, whereas several proteins, including CD38,
   poly-ADP-ribose polymerases (PARPs) and SIRTs, are
   consumptive^[56]1–[57]3. Interestingly, NAD^+ is intertwined with
   circadian clock function, as NAMPT expression is under circadian
   control in the liver, whereas both NAD^+ itself and NAD^+-dependent
   deacetylase SIRT1 regulate core clock function directly^[58]4,[59]5.
   Because NAD^+ levels decline with aging in most tissues, including the
   brain^[60]3, augmentation of NAD^+ levels has been proposed as a
   therapeutic strategy to counteract aging and prevent neurodegenerative
   diseases^[61]2,[62]3,[63]6.

   REV-ERBα (encoded by Nr1d1) is a circadian clock protein, nuclear
   receptor and transcriptional repressor, which serves critical functions
   in the control of metabolism and inflammation^[64]7–[65]11. REV-ERBα
   expression is dampened in patients with Alzheimer’s disease (AD), who
   often suffer from sleep fluctuations and disrupted circadian
   function^[66]12, and global REV-ERBα deletion can prevent amyloid
   plaque pathology in an AD mouse model by enhancing microglial
   phagocytic activity^[67]13. Conversely, microglia-specific REV-ERBα
   deletion can exacerbate tau pathology in male mice^[68]12, suggesting
   that REV-ERBα may exert complex cell-type-specific effects on AD
   pathology. In cardiac tissue, REV-ERBα is required to maintain NAD^+
   levels via rhythmic expression of NAMPT^[69]14. In that setting,
   REV-ERBα deletion de-represses NFIL3/E4BP4, leading to suppression of
   NAMPT, loss of NAD^+ levels and cardiomyopathy. However, it is unknown
   if a similar pathway is present in the brain or how such a
   REV-ERBα–NAD^+ axis might influence neurodegeneration. As
   REV-ERBα-targeted therapeutics are being developed, understanding the
   overall and cell-type-specific effects of this pathway on brain NAD^+
   and neurodegeneration has clear translational importance^[70]15,[71]16.
   Thus, we investigated REV-ERBα effects on brain NAD^+ levels and
   neurodegeneration under basal conditions and in the setting of tau
   pathology, which plays a critical role in AD and other age-related
   neurodegenerative conditions. We observed that REV-ERBα regulates NAD^+
   levels in the brain through an NFIL3–CD38 pathway in astrocytes and
   that global or astrocyte-specific REV-ERBα deletion augments brain
   NAD^+ levels and protects mice from tauopathy. Here we provide initial
   findings that pharmacological antagonism of REV-ERBα can mitigate tau
   pathology in PS19 mice, suggesting that the protective effects of
   REV-ERBα inhibition predominate in the brain and that REV-ERBα
   inhibitors may hold promise for AD therapy.

Results

REV-ERBα deletion suppresses CD38 expression and induces NAD^+ levels in the
brain

   To investigate the effects of REV-ERBα on gene expression in the brain,
   we generated global, postnatal REV-ERBα knockout (RKO) mice using the
   tamoxifen-sensitive CAG::CreER^T2 line and Nr1d1^fl/fl mice, which
   produce full loss of REV-ERBα expression^[72]17. REV-ERBα deletion was
   induced by tamoxifen at 2 months of age, and hippocampal transcriptomes
   were analyzed by bulk RNA sequencing (RNA-seq) at 10 months of age
   (Fig. [73]1a). We observed abundant expression of Cre in the
   hippocampus of the Cre^+ group and found more than approximately 50%
   Nr1d1 reduction by tamoxifen treatment (Fig. [74]1b). We performed bulk
   RNA-seq and identified 470 differentially expressed genes (DEGs) in
   Cre^+ versus Cre^− hippocampus (P < 0.05, |fold change (FC)| > 50%; 262
   higher, 208 lower). Elevated genes in Cre^+ mice included Tex11, Plvap,
   Bmal1(Arntl), C4b, Bag3, Plin4 and Cxcl5, and downregulated transcripts
   included Apoa1, Apoa2, Lcat and Ccr7 (Fig. [75]1c). Gene Ontology term
   pathway analysis using these DEGs identified ‘Cation transport’ and
   ‘Steroid metabolic process’ as top processes altered by postnatal
   REV-ERBα deletion in the brain (Fig. [76]1d,e). Interestingly, we
   observed that the transcription factor Nfil3, a known target of
   REV-ERB-mediated transcriptional repression^[77]14, was upregulated in
   Cre^+ mice, whereas the critical NAD^+-consuming enzyme Cd38 was
   strongly downregulated after REV-ERBα deletion (Fig.
   [78]1e,f)^[79]18,[80]19. As NAD^+ levels can vary with time of day in
   liver^[81]4,[82]5, we prepared hippocampal lysates from Cre^− and Cre^+
   mice euthanized every 6 hours across the day under standard
   12-hour:12-hour lighting, to exclude time-of-day effects. We saw
   upregulation of the REV-ERB target Fabp7 at all timepoints, indicating
   strong REV-ERBα deletion. Cd38 transcript did not vary by time of day
   in Cre^− mice and was consistently suppressed across timepoints in
   Cre^+ mice (although the 12:00 timepoint did not meet significance),
   suggesting that REV-ERBα regulation of Cd38 is not time-of-day
   dependent (Extended Data Fig. [83]1a). Moreover, CD38 protein level was
   also downregulated in RKO hippocampus (Extended Data Fig. [84]1b). We
   next measured the NAD^+ concentration using brain cortex samples and
   found that it was significantly increased in RKO brain (Fig. [85]1g)
   compared to wild-type (WT) (Cre^−) controls, in sharp contrast to a
   previous report in cardiac tissue^[86]14. NAD^+ metabolism is generally
   driven by NAD^+-consuming enzymes such as CD38, SIRTs and
   PARPs^[87]1,[88]20. Among these, only Cd38 was significantly
   downregulated by REV-ERBα deletion in hippocampus (Fig. [89]1h),
   suggesting that REV-ERBα could affect brain NAD^+ level through CD38
   modulation.

Fig. 1. REV-ERBα deletion suppresses the NAD^+-consuming enzyme CD38 and
enhances brain NAD^+ levels.

   [90]Fig. 1
   [91]Open in a new tab

   a, Schematic showing experiments with inducible global REV-ERBα KO mice
   (CAG::Cre^ERT2;Nr1d1^fl/fl mice, termed RKO). TAM, tamoxifen. b, Cre
   expression (WT, n = 13 mice; RKO, n = 17 mice) and Nr1d1 (REV-ERBα)
   deletion efficiency (WT, n = 14 mice; RKO, n = 18 mice) in RKO (Cre^+)
   compared to WT (Cre^−) mouse brain. c, Volcano plot showing
   differential gene expression from WT and RKO hippocampus (log[2]FC
   cutoff = 0.5, −log[10] (P value) cutoff = 1.3, corresponds to P value
   of 0.05 using limma-voom). d, Top five upregulated or downregulated
   biological processes identified for DEGs in REV-ERBα KO brain from bulk
   RNA-seq. e, Heatmap representing genes from ‘Cation transport;
   upregulation’ and ‘Steroid metabolic process; downregulation’. Nfil3
   and Cd38 are noted with red asterisks. f, Expression of Nfil3 and Cd38
   from WT and RKO hippocampus by qPCR (WT, n = 12 mice; RKO, n = 9 mice).
   g, Increased NAD^+ levels in RKO cerebral cortex compared to WT (WT,
   n = 12; RKO, n = 13). h, Transcripts of three major NAD^+-consuming
   enzymes, Sirt1, Parp1 and Cd38, in WT and RKO hippocampus (from RNA-seq
   data in c). i, Nampt expression in hippocampal tissue is unchanged in
   RKO group compared to WT group (WT, n = 12 mice; RKO, n = 9 mice,
   qPCR). j, Expression of Nfil3 transcript in Arc nucleus from control
   (n = 4 mice) and hypothalamic REV-ERB α/β KO brain (H-DKO, n = 5 mice)
   and cardiomyocyte-specific REV-ERBα/β KO heart (CM-DKO, n = 3 mice per
   group). k, Cd38 and Nampt transcript expression in Arc nucleus tissue
   from control (n = 4 mice) and hypothalamic REV-ERBα/β KO (H-DKO, n = 5
   mice). l, Cd38 and Nampt transcript expression in heart tissue from
   control and cardiomyocyte REV-ERBα/β KO (CM-DKO) mice (n = 3 mice per
   group). m, Knockdown of Nfil3 with siNfil3 siRNA causes increased
   expression of Cd38 in primary mouse astrocyte cultures. Control siRNA
   is labeled as ‘siCon’. n, Diagram depicting proposed indirect
   regulation of CD38 and NAD^+ by REV-ERBα via NFIL3 inhibition. Note
   that the NFIL3–NAMPT interaction is minimal in brain but predominates
   in heart. **P < 0.01, ***P < 0.005, ****P < 0.001; ‘NS’ is
   non-significant by two-tailed t-test. Error bars represent
   mean ± s.e.m. ptn, protein.

   [92]Source data

Extended Data Fig. 1. Dampened Cd38 mRNA and protein expression on Rev-erbα
knock-out (KO) mouse hippocampus across the day.

   [93]Extended Data Fig. 1
   [94]Open in a new tab

   (a) Tamoxifen treated global REV-ERBα KO mice (CAG::CreER^T2;
   Nr1d1^fl/fl; RKO) and Cre- controls (WT) were sacrificed every 6 hours
   over a 24-hour period under standard 12 h:12 h light:dark conditions
   and gene expression was assayed using hippocampal tissue. Cd38
   transcript did not vary by time of day, but was decreased at all
   timepoints (except 12 pm). A known target of REV-ERBα mediated
   repression, Fabp7, was highly induced compared to WT (Cre-) at all
   timepoints. N = 3 mice/genotype/ in each time point. (b) Lower level of
   CD38 protein in RKO hippocampus than WT at 12 pm (WT, n = 5; RKO, n = 4
   mice). *p < 0.05, **p < 0.01, and ****p < 0.001 by 2-tailed T-test.
   Error bars represent mean ± SEM.

   [95]Source data

REV-ERBα regulates NAD^+ level via NFIL3–CD38 axis not through Nampt in the
brain

   In cardiac tissue, REV-ERBα directly represses Nfil3, which is required
   for expression of Nampt, an enzyme that catalyzes the first reversible
   step in NAD^+ biosynthesis and nicotinamide (NAM) salvage^[96]14.
   REV-ERBα deletion in heart, therefore, reduces Nampt expression and
   causes NAD^+ depletion^[97]14, the opposite of what we observed in
   brain (Fig. [98]1g). Moreover, we found that REV-ERBα KO had no effect
   on Nampt expression in hippocampus (Fig. [99]1i), suggesting divergent
   regulatory pathways in heart versus brain. To address this further, we
   examined transcriptomic data from previous studies of hypothalamic and
   heart-specific REV-ERBα/β deletion^[100]14,[101]21. As expected, Nfil3
   expression was increased in both the arcuate (Arc) nucleus of the brain
   and heart tissue after REV-ERB deletion (Fig. [102]1j). REV-ERB
   deletion significantly reduced Arc Cd38 levels without any effect on
   Nampt, similar to our data in hippocampus (Fig. [103]1k). However,
   myocardial REV-ERB deletion had no effect on Cd38 expression but
   strongly suppressed Nampt, which was previously shown to cause NAD^+
   depletion^[104]14 (Fig. [105]1l). As Cd38 is primarily expressed in
   astrocytes in mouse brain according to a brain transcriptomic atlas
   ([106]https://brainrnaseq.org/)^[107]22, we suspected that astrocytes
   may be the main site of the REV-ERBα effects on NAD^+. We hypothesized
   that astrocyte REV-ERBα regulates Cd38 expression via NFIL3. To address
   this, we knocked down Nfil3 using small interfering RNA (siRNA) in
   primary cultured astrocytes and observed a decrease in Cd38 levels
   (Fig. [108]1m). Although REV-ERBβ (Nr1d2) can have overlapping
   functions with REV-ERBα (refs. ^[109]8,[110]23), we observed only weak
   induction of Nfil3 and no significant Cd38 reduction after
   siRNA-mediated REV-ERBβ knockdown in cultured astrocytes, suggesting
   that REV-ERBβ has a minimal impact (Extended Data Fig. [111]2).

Extended Data Fig. 2. REV-ERBβ (Nr1d2) knockdown does not alter Cd38
expression in primary astrocytes.

   [112]Extended Data Fig. 2
   [113]Open in a new tab

   Knockdown of Nr1d2 (REV-ERBβ) by siRNA transfection in cultured primary
   astrocytes induces Bmal1, has a small effect on Nfil3 transcript
   levels, but Cd38 does not significantly change (siControl, n = 7;
   siNr1d2, n = 7 plates from two mice). **p < 0.01, ****p < 0.001, and ns
   is non-significant by 2-tailed T-test. Error bars represent mean ± SEM.

   [114]Source data

   To further support our hypothesis that NFIL3 regulates Cd38
   transcription directly, we analyzed an existing mouse single-cell assay
   for transposase-accessible chromatin using sequencing (ATAC–seq)
   dataset, which probes accessible chromatin in more than 800,000
   individual nuclei from 45 regions that span multiple brain regions and
   maps the state of 491,818 candidate cis-regulatory DNA elements in 160
   distinct cell types, for NFIL3 binding motifs in the Cd38
   promoter^[115]24. We retrieved astrocyte-specific candidate
   cis-regulatory elements (cCREs) and identified a peak (cCREs353987)
   that was specific to astrocytes from the mouse brain white and gray
   matter and located at the 5′ transcription start site (TSS) of the Cd38
   genomic region (Extended Data Fig. [116]3). Furthermore, multiple lines
   of evidence from three additional databases support the functionality
   of this genomic region, including the Mouse ATAC–seq Atlas database,
   DNase-seq data from the Cistrome database as well as the ENCODE cCREs
   database from the ENCODE project^[117]25–[118]27. Using the position
   weight matrix (PWM) of NFIL3 (motif M01819 from the Catalog of Inferred
   Sequence Binding Preferences (CIS-BP) database), the Patser