The forebrain synaptic transcriptome is organized by clocks but its proteome is driven by sleep

Neurons have adapted mechanisms to traffic RNA and protein into distant dendritic and axonal arbors. Taking a biochemical approach, we reveal that forebrain synaptic transcript accumulation shows overwhelmingly daily rhythms, with two-thirds of synaptic transcripts showing time-of-day-dependent abundance independent of oscillations in the soma. These transcripts formed two sharp temporal and functional clusters, with transcripts preceding dawn related to metabolism and translation and those anticipating dusk related to synaptic transmission. Characterization of the synaptic proteome around the clock demonstrates the functional relevance of temporal gating for synaptic processes and energy homeostasis. Unexpectedly, sleep deprivation completely abolished proteome but not transcript oscillations. Altogether, the emerging picture is one of a circadian anticipation of messenger RNA needs in the synapse followed by translation as demanded by sleep-wake cycles

Sleep-wake cycles drive daily dynamics of synaptic phosphorylation

The circadian clock drives daily changes of physiology, including sleep-wake cycles, through regulation of transcription, protein abundance, and function. Circadian phosphorylation controls cellular processes in peripheral organs, but little is known about its role in brain function and synaptic activity. We applied advanced quantitative phosphoproteomics to mouse forebrain synaptoneurosomes isolated across 24 hours, accurately quantifying almost 8000 phosphopeptides. Half of the synaptic phosphoproteins, including numerous kinases, had large-amplitude rhythms peaking at rest-activity and activity-rest transitions. Bioinformatic analyses revealed global temporal control of synaptic function through phosphorylation, including synaptic transmission, cytoskeleton reorganization, and excitatory/inhibitory balance. Sleep deprivation abolished 98% of all phosphorylation cycles in synaptoneurosomes, indicating that sleep-wake cycles rather than circadian signals are main drivers of synaptic phosphorylation, responding to both sleep and wake pressures

Lsd1 ablation triggers metabolic reprogramming of brown adipose tissue

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT- selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1- deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT

An RNAi screen identifies genes associated with the nucleo-cytoplasmic translocation machinery important for circadian dynamics.

<p>U-2 OS <i>Bmal1-</i>luciferase reporter cells were transduced with RNAi constructs targeting 62 genes associated with nucleo-cytoplasmic translocation and bioluminescence rhythms were recorded for several days. Significant period alteration compared to non-silencing controls (ns, blue) was determined by one-way ANOVA of all constructs targeting one gene; a Dunnetts posttest assessed the effects of individual constructs. Multiple testing was corrected for by a Bonferroni-Holm correction. Horizontal dotted lines indicate two standard deviations of the ns control period. Targeting <i>Fbxl3</i> was used as positive control (green). Genes for which at least two shRNA constructs led to a significant period alteration in the same direction are labeled (red dots); non-significant values are depicted in grey; dot size indicates the significance of period deviation: smallest: 0.05 > q > 0.005, medium: 0.005 > q > 0.001, largest: q < 0.001). When only one RNAi construct was available for gene knockdown, period deviation is depicted as empty circle (error bars = SD, n = 3).</p

Knockdown and knockout of TNPO1 shortens the circadian period.

<p>(<b>A)</b> Left: Representative time series for the effect of three different sh<i>Tnpo1</i> constructs compared to a non-silencing (ns) control shRNA. Right: Mean period deviation and relative <i>Tnpo1</i> mRNA and protein levels. Bottom: representative western blot of TNPO1 protein levels in ns control and <i>Tnpo1</i> depleted cells; lanes are from the same blot with identical exposure time. Error bars = SD, n_{period deviation} = 7 to 14 individual recordings, n_{mRNA and protein} = 4–10 individual cell lysates; one-sample t-test to test whether period deviations are different from zero, *** p < 0.001. (<b>B)</b> Top: Schematic illustration of the CRISPR/Cas9-mediated genome editing of the <i>Tnpo1</i> gene. Bottom left: Representative time series of U-2 OS reporter cells transduced with Cas9 expression vector and indicated guide RNA (gRNA). Middle: Mean periods of independent bioluminescence recordings (error bars = SD, n = 3, one-way ANOVA with Bonferroni posthoc test comparing sgRNAs to wild-type: *** p < 0.001, n.s. = non- significant. Right: Representative western blot of residual TNPO1 protein levels in genome-edited cells.</p

TNPO1 interacts with PER1.

<p><b>(A)</b> Scatterplot of the results of two different anti-CLOCK immunoprecipitations (IPs) from unsynchronized U-2 OS cell nuclei followed by mass-spectrometry. Depicted is the enrichment of identified proteins relative to the starting cell lysate for two CLOCK-antibodies against each other after normalization to an IgG control IP (n = 3 independent IPs, Welch’s t-test). <b>(B)</b> Representative MYC- or V5-CoIPs (empty bars) normalized to IgG control IPs (filled bars) of HEK293 lysates (mean ± SD, n = 3 independent IPs, Student’s t-test: n.s. = non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001). To control for efficient IP, western blots of either MYC-TNPO1 or PER1/2-V5 were performed using the specific anti-MYC or anti-V5 IPs as well as the unspecific control IgG IPs. Experiments were repeated two to seven times. (<b>C</b>) Co-immunoprecipitation from U-2 OS cells stably expressing a PER1-LUCIFERASE fusion protein with either an antibody targeting endogenous TNPO1 (IP_TNPO1) or an IgG control (IP_C). Shown are luciferase intensities of αTNPO1 IPs (empty bar) normalized to counts from IgG control IPs (filled bar). Given are means ± SD, n = 3 independent IPs (** p < 0.01, Student’s t-test). Representative western blots to control for efficient IPs are shown below. (<b>D</b>) TNPO1 interaction region is located in the C-terminal part of PER1. Top: Schematic illustration of the primary structure of human PER1. Shown are nuclear export sequences (NES, green), the classical nuclear localization sequence (cNLS, blue), the three PY-motifs (i.e. putative non-classical nuclear localization motifs, red), the CRY interaction domain (brown) as well as 25 cysteine residues that are conserved within human and mouse PER1 (yellow and orange). Different grey/black shades indicate the PER1 fragments used. Bottom left: Co-immunoprecipitation from HEK293 cells expressing MYC-TNPO1 and indicated fragments of PER1-LUCIFERASE with anti-MYC (empty bars) or control IgG (filled bars). For comparison, data for full-length PER1 are re-plotted from panel B. Given are means ± SD from nine independent IPs performed at three experimental days. One-way ANOVA with Bonferroni corrected posthoc test revealed indicated significance; ** p < 0.01; n.s. = non-significant). Controls for efficient IP are shown below. Bottom right: Co-immunoprecipitation from HEK293 cells expressing MYC-TNPO1 and a mutant version of PER1-LUCIFERASE (PER1_{PY/Cys_mut}), in which both C-terminal PY-motifs (PY_883/834 and PY_935/936) as well as seven C-terminal cysteine residues (orange) are exchanged to alanine residues, with anti-MYC (empty bars) or control IgG (filled bars). Given are means ± SD of five independent immunoprecipitations. Representative western blots to control for efficient IPs are shown below.</p