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Extracts of total protein were prepared by lysis of neurons on the cell culture dish in 2 x Roti-Load sample buffer (Carl Roth, Germany). Western blotting was performed as described previously (Loy et al., 2011). Membranes were blocked with 5% milk powder in TBST (0.1% Tween 20 in TBS) for 1 hr and then incubated overnight at 4°C with the corresponding primary antibodies, diluted in blocking solution. After incubation with HRP-coupled secondary antibodies, the blots were developed using ECL reagent (GE Healthcare, Chicago, IL). Blots were either developed using CP100 photo-developer (Agfa, Belgium) or FUSION-FX7 chemiluminescence detection system (Vilber Lourmat, France). Quantification of protein expression was carried out either by using ImageJ (RRID:SCR_003070) or FUSION-CAPT image analysis software in the linear range of detection.

other

Mice were anesthetized and transcardially perfused with 4% PFA (w/v) in PBS (pH 7.4). Brains were removed, post-fixed for 2 hr in 4% PFA, cryo-protected in 30% (w/v) sucrose at 4°C and embedded in Tissue-Tek medium (Sakura Finetek Europe, The Netherlands). Free-floating cryosections (40 µm) were washed three times in Tris-Buffered Saline (TBS), permeabilized in 0.3% (v/v) Triton-X-100 in TBS for 5 min and incubated with blocking solution (10% (v/v) normal serum, 1% (w/v) BSA, 0.3% Triton-X-100 in TBS) for 2 hr. Sections were incubated with primary antibodies overnight at 4°C. After three washes in 0.025% Triton-X-100 in TBS sections were incubated with corresponding secondary antibodies. Sections were counterstained with Hoechst 33258 (H-3569, Molecular Probes, Grand Island, NY) for 5 min or with RedDot2 (40061, Biotium, Fremont, CA) for 20 min. After three washes in TBS sections were mounted with Roti-Mount FluorCare mounting medium (Carl Roth, Germany). Images were acquired using a LSM 510/Axiovert200M microscope (Carl Zeiss, Germany).

other

AAV8-hSyn-eGFP and AAV8-hSyn-Satb2-V5 were delivered by stereotaxic injection into the dorsal hippocampus of 12 week old male mice (Satb2 cKO and control littermates). A burr hole was drilled into the skull, and 1 μl of viral stock (1012–1013 vg/ml) was injected bilaterally at a rate of 100 nl/min. The following coordinates (relative to Bregma) were used: antero-posterior, −2.1 mm; medio-lateral, ± 1.5 mm; dorso-ventral, −1.5 mm from the skull surface. The needle was left in place for 5 min after the injection. The mice were allowed to recover for four weeks after stereotaxic injections. Infection efficiencies of AAVs were determined by immunohistochemistry using antibodies to Satb2 or V5-tag or by analyzing the fluorescence of eGFP.

other

Total RNA was isolated from primary hippocampal cultures or dissected CA1 hippocampal tissue using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized following the High-Capacity cDNA Reverse Transcription Kit protocol (Thermo Fisher Scientific). qPCR was performed using Fast SYBRGreen Master Mix (Thermo Fisher Scientific). All reactions were run in duplicates. The relative expression values were determined by normalization to Gapdh transcript levels and calculated using the ∆∆CT method (Pfaffl, 2001). Primers used for RT-qPCR analysis are listed in Supplementary file 1. Real-time PCR analysis of miRNAs was carried out using the miScript PCR System (Qiagen, Germany) following the manufacturer’s instructions.

other

RNA was isolated from dissected CA1 hippocampal tissue using TRIzol (Thermo Fisher Scientific). Library preparation and cluster generation for mRNA and small RNA sequencing was performed according to Illumina standard protocols (TruSeq, Illumina, San Diego, CA). Libraries were quality-controlled and quantified using a Nanodrop 2000 (Thermo Fisher Scientific), Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and Qubit (Thermo Fisher Scientific).

other

Base calling from raw images and file conversion to fastq-files was achieved by Illumina pipeline scripts. Subsequent steps included quality control (FastQC, www.bioinformatics.babraham.ac.uk/projects/fastqc/), mapping to reference genome (mm10, STAR aligner v2.3.0 (Djebali et al., 2012), non-default parameters), read counting on genes or exons (HTSeq, http://www-huber.embl.de/users/anders/HTSeq, mode: intersection-non-empty) and differential gene expression (DESeq2_1.4.5 [Love et al., 2014]) bio-statistical analysis.

other

AAV8-hSyn-Satb2-V5-transduced DIV11 primary hippocampal neurons were cross-linked with formaldehyde at 1% final concentration for 10 min at room temperature, and chromatin was prepared using the Zymo-Spin ChIP kit (Zymo Research, Irvine, CA) following manufacturer’s instructions. Sonication was performed at high power setting for 40 cycles (30 s ON, 30 s OFF) using a Bioruptor Plus (Diagenode Inc., Denville, NJ), yielding fragment size range of 200–700 bp. ChIP assays were performed in triplicates using 20 μg of chromatin and 10 μg of anti-V5 tag antibody (ab15828). IgG (12–370, Millipore, Billerica, MA) was included as a negative control. ChIP DNA was purified using ChIP DNA Clean and Concentrator (Zymo Research) and the relative abundance of a control region in V5-immunoprecipitated DNA was quantified by qPCR with sequence-specific primers. DNA libraries (Satb2-ChIP and Input control DNA) were prepared and sequenced on a HiSeq 2000 sequencer (Illumina). Sequencing resulted in 39,309,191 and 88,731,003 high quality filtered 20–50 bp single end reads for ChIP- and Input DNA respectively. These reads were aligned to the mm10 mouse genome using the BWA short read aligner (Li and Durbin, 2009) (version 0.7.12). The aligned reads were filtered to keep only uniquely mapping reads (ChIP: 15,507,953 Input: 51,700,265). A cross-correlation analysis was performed to assess quality metrics (Phantompeakqualtools package, RRID:SCR_005331). The resulting NSC (=1.24) and RCS (=0.82) values met the ENCODE quality thresholds (NSC ≤ 1.05, RCS ≤ 0.8) (Landt et al., 2012). Peaks of enriched Satb2 binding were called using MACS2 (RRID:SCR_013291) (Zhang et al., 2008) (version 2.1.0), by allowing only one tag at the same location and setting the false discovery rate to 0.01 and the fold-enrichment cutoff to 2. This resulted in 8414 high confidence peaks and a fraction of reads in peaks (FriP) of 6.2% which is well above the ENCODE requirements for good quality data. Peak annotation and comparisons with other public data sets were performed with the R/Bioconductor packages ChIPseeker (Yu et al., 2015) and ngs.plot (RRID:SCR_011795) (Shen et al., 2014). Testing of ChIP-seq peak data for the enrichment of biological pathways and Gene Ontology terms was performed by using ChIP-Enrich tool (Welch et al., 2014). Public datasets (GSE63271, GSE66701, GSE21161, GSE65159) (Gjoneska et al., 2015; Kim et al., 2010; Telese et al., 2015; Wang et al., 2015) were downloaded from GEO and genomic coordinates were lifted over to mouse mm10 for data sets which were only available in a differing genome assembly version. Datasets that did not contain peak data (BED files) were reanalyzed by mapping quality filtered raw reads to mm10 and calling peaks using MACS2.

other

ChIP was performed on microdissected adult mouse CA1 tissue (a pool of 8–10 mice) by using the Zymo-Spin ChIP kit (Zymo Research) following manufacturer’s instructions. Briefly, 100 mg of CA1 tissue were cross-linked with 1% formaldehyde (Sigma) for 10 min at RT and neutralized with 0.125M glycine. Chromatin was fragmented using a Bioruptor Plus sonicator (Diagenode Inc., Denville, NJ), yielding fragment size range of 200–500 bp. Sonicated chromatin (10–15 µg) was incubated at 4°C overnight with 5 µg antibodies (ab34735, AB_2301417, Abcam or normal rabbit IgG 12–370, AB_145841, Millipore). After washing, ChIP-ed DNA was eluted; reverse cross-linked at 65°C for 2 hr and purified using Zymo-Spin ChIP kit (Zymo Research).

other

Contextual fear conditioning was performed in a 25 × 25 × 35 cm chamber with transparent walls and a metal rod floor, cleaned with water and illuminated to 300 lux (TSE, Bad Homburg, Germany) as previously described (Busquet et al., 2008; Dobi et al., 2013; Sartori et al., 2011). After a 120 s acclimation period, mice were conditioned with three presentations of a 0.60 mA scrambled foot shock, with a 120 s inter-shock interval. The mice were allowed to remain in the chamber for an additional 120 s following the last stimulus presentation. Short-term and long-term fear memories were tested 1 hr and 24 hr later respectively in the conditioning chamber. Freezing was measured as an index of fear (Blanchard and Blanchard, 1969) manually scored based on DVD recordings, defined as no visible movement except that required for respiration, and converted to a percentage [(duration of freezing /total time) × 100] by a trained observer blind to the animals’ group/genotype.

other

Prior to training, mice were handled 1–2 min for five days and then habituated to the experimental apparatus (a 41 × 41 × 41 cm open field arena containing home-cage floor bedding and illuminated to 150 Lux; Tru Scan, Coulbourn Instruments, Holliston, MA) for 5 min a day for three consecutive days in the absence of objects. During the training period, mice were placed into the experimental apparatus containing two identical objects (blue colored Lego Duplo blocks 2.5 × 2.5 × 5 cm) and allowed to explore for 10 min. During the short-term (1 hr) or long-term (24 hr) retention tests, mice were placed in the experimental apparatus for 5 min. For assessment of spatial object location-dependent memory, one copy of the familiar object was placed in the same location as during the training trial, and one copy of the familiar object was moved and placed in the middle of the box. For the novel object recognition test (Antunes and Biala, 2012), one copy of the familiar object and a new object (100 ml glass beaker) were placed in the same location as during the training trial. Exploration was scored when the mouse’s nose touched the object. All training and testing trials were videotaped and analyzed by individuals blind to the genotype of subjects. The relative exploration time (t) was recorded and expressed as a percent discrimination index (D.I. = (tnovel − tfamiliar) / (tnovel + tfamiliar) × 100%). Mean exploration times were then calculated and the discrimination indexes between treatment groups compared. Animals that explored less than 3 s total for both objects during either training or testing were removed from the analysis.

other

Reactivity to the foot shock was evaluated in the same apparatus used for contextual fear conditioning as previously described (Sartori et al., 2011). After a 120 s acclimation period, mice were subjected to a series of 1 s shocks of gradually increasing amperage (0.1 mA every 30 s) starting from 0.1 mA. Mice were scored for their first visible response to the shock (flinch), their first pronounced motor response (run or jump), and their first vocalized distress, as previously described (Wittmann et al., 2009).

other

Acute hippocampal slices were prepared from 2–3 month old mice. In brief, mice were anesthetized and decapitated; the brain was quickly transferred into ice-cold carbogenated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) for 3 min. The ACSF used for electrophysiological recordings contained 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 26 mM NaHCO3, 2 mM CaCl2, and 25 mM glucose. Hippocampi were cut with a vibratome (VT 101200S; Leica, Germany) into 400 µm thick transversal slices. Recordings were performed in a submerged recording chamber at 32°C. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region with a glass micropipette (resistance: 3–15 MΩ) filled with 3 M NaCl at a depth of ca. 150–200 µm. Monopolar tungsten electrodes were used for stimulating the Schaffer collaterals at a frequency of 0.1 Hz. Stimulation was set to elicit a fEPSP with a slope of ca. 40–50% of maximum for LTP recordings. After 40 min of baseline stimulation, LTP was induced by applying theta-burst stimulation (TBS), in which a burst consisted of 4 pulses at 100 Hz which were repeated 10 times in a 200 ms interval (5 Hz). Three of such trains were used to induce LTP at 0.1 Hz. Basic synaptic transmission and presynaptic properties were analyzed via input-output (IO) measurements and paired-pulse facilitation (PPF). The IO-measurements were performed by application of a defined value of current (25–250 µA in steps of 25 µA). PPF was performed by applying a pair of two stimuli by different inter-stimulus intervals (ISI) ranging from 10, 20, 40, 80 to 160 ms. Data were collected, stored, and analyzed with LABVIEW software (National Instruments, Austin, TX). The initial slope of fEPSPs elicited by stimulation of the Schaffer collaterals was measured over time, normalized to baseline, and plotted as average ± SEM. Statistical analyses were performed using Student's t-test.

other

Statistical analysis was conducted as indicated in the figure legends using SPSS software (SPSS Inc). Statistical significance was determined by Student’s t-test, one-way ANOVA or two-way ANOVA followed by appropriate post hoc test (Tukey, Hochberg, Bonferroni or Fisher’s LSD). Data represent mean ± SEM of at least three independent biological replicates. No statistical methods were used to predetermine sample sizes; however our sample sizes were similar to those reported in previous studies. The data distribution was assumed to be normal, but it was not formally tested, except for datasets with n > 10.

other

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

clinical case

Thank you for submitting your article "Satb2 determines miRNA Expression and Long-Term Memory in Adult CNS" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.

clinical case

The chromatin-associated protein Satb2 is known to play important roles in neuronal fate determination in the developing brain, however its functions in mature neurons have not been explored. In this manuscript the authors use a Camk2-Cre to conditionally knockout Satb2 in mature forebrain neurons revealing impairments in hippocampal synapse plasticity and memory consolidation. in vivo RNAseq experiments revealed that a few dozen mRNAs and several hundred miRNAs were differentially expressed in hippocampus of cKO mice. They choose Arc as a potential microRNA target, found that its protein expression was reduced in Satb2 cKO hippocampus, and saw improved memory performance when Arc expression was rescued. Intermingled with these experiments, in cultured neurons they found that expression of Satb2 was induced by exogenous BDNF or bicuculline and showed that overexpressed Satb2 binds the promoters of both protein coding and non-coding genes, including a number of microRNA genes.

study

All three reviewers were convinced of the high quality of the data and the novelty of the behavioral findings. However, all three raised concerns about the links between the mechanistic data and the behavioral data. Some of these concerns can be addressed in the text or through additional data analysis, but a few new experiments are essential to make the mechanistic data on Satb2 chromatin binding a compelling story to explain the behavioral results.

study

1) A major weakness of the ChIP experiments is that they are performed in cultured neurons with overexpressed Satb2 whereas the RNAseq data are derived from hippocampus of the cKO mice. To confirm that the culture/overexpression ChIP experiments are relevant for the in vivo gene expression data the authors need to confirm by ChIP-PCR that endogenous Satb2 is bound to these same regions in hippocampus of the cKO mice. Furthermore, as a control for the specificity of the V5 antibody in the overexpression ChIP, the minimum control is ChIP from uninfected neurons – this could be ChIP-PCR as well.

study

The authors overemphasize the miRNA findings at the expense of discussing the mRNAs that are changed. In principle, there is nothing from the ChIP data selectively linking Satb2 with miRNA expression that justifies the emphasis on miRNAs when interpreting the behavioral results.

other

The authors report that nearly 50% of all microRNAs are dysregulated in Satb2 cKO mice, whereas only a very small number of mRNAs show differences. This is not clearly explained by the ChIP data (which show Satb2-V5 bound to both sets of promoters). Some explanation should be offered.

study

3) As presented, the BDNF-dependent regulation of Satb2 doesn't help to build a cogent model to explain the functions of Satb2 in learning and memory or LTP. Do the authors think that regulation of expression contributes to the requirement for Satb2 in learning and memory? If so it would be interesting to see if activity-dependent expression of Arc is dysregulated in Satb2 knockouts. Do physiologically relevant stimuli induce the expression of Satb2 in vivo? If these data cannot be more tightly tied to the behavior they would be better off moved to the supplementary data.

study

Thank you for resubmitting your work entitled "Satb2 determines miRNA expression and long‐term memory in adult central nervous system" for further consideration at eLife. Your revised article has been favorably evaluated by a Senior Editor, and a Reviewing Editor.

study

In this revision the authors have provided essential additional data needed to support the findings of this study. In particular, the chromatin immunoprecipitation controls (e.g. the V5 ChIP from nontransduced neurons and the endogenous Satb2 ChIP from hippocampus) provide crucial support for the model of direct transcriptional regulation by Satb2 in hippocampal neurons of the mRNA and miR targets identified as dysregulated in the sequencing studies. Further the validation of the mIR findings by RT-PCR and ChIP-PCR strengthen the focus of the manuscript on these targets.

study

The major finding of this study – that Satb2 has an important gene regulatory function in mature hippocampal neurons relevant to synaptic plasticity and learning and memory – is a novel and useful addition to the literature that will be of interest to a broad range of neurobiologists.

other

1) The BDNF data remain partially disconnected from the rest of the story and the "synapse to nucleus feedback loop" idea that is repeated several times in the manuscript is not supported by the data. The experiments demonstrating BDNF-dependent regulation of Satb2 expression in cultured neurons are well done, and the authors make a reasonable argument to keep these data in the main figures. However, they do not have relevant evidence for BDNF-dependent regulation of Satb2 in vivo or any evidence that induction of Satb2 expression is required for its role in hippocampal function. (Dark rearing is not a relevant experiment to demonstrate activity- or BDNF-dependent gene regulation in vivo. The more usual experiment would be dark adaption after eye opening followed by light exposure, since dark rearing induces developmental delays.) I do not think it is required that the authors demonstrate BDNF-dependent regulation of Satb2 expression in vivo. However in lieu of these data or other data showing that BDNF- or activity-dependent regulation of Satb2 levels matter for the functions of Satb2 identified in this study, then the authors cannot conclude in the Discussion that they have identified a synapse to nucleus feedback circuit. This language needs to be removed from the last line of the Abstract, the last line of the Introduction, and the first line of the Discussion. It is reasonable based on the BDNF data provided that the authors can speculate in the Discussion that BDNF-dependent regulation of Satb2 might regulate synapse plasticity as they propose. Finally, the Discussion section on the relationship between BDNF and Satb2 in synapse plasticity (third paragraph) remains beyond the data. Lots of things disrupt LTP in the hippocampus in addition to BDNF and Satb2, so just because those two have LTP disruption in common does not bind them tightly together.

study

2) The authors have improved their commentary on the possibility that Satb2 is acting to regulate mature hippocampal neuronal function via chromatin looping, but several sentences in the Discussion still remain that are overstatements from the data presented.

study

"Our findings suggest that some of the key functions of the hippocampus depend on changes in the higher-order chromatin architecture." The authors' findings suggest these functions "may" depend on chromatin looping. Yes, the literature shows that Satb2 regulates chromatin architecture but it very well is likely to have additional functions. Unless the authors were to study chromatin architecture in these neurons of this mouse, it cannot be concluded that is the mechanism of action in this study.

study

"Our results in primary hippocampal cultures also suggest that this type of higher-order chromatin rearrangement is an activity- and BDNF dependent process that involves changes in Satb2 expression levels." This is speculation since this manuscript provides no evidence for changes in chromatin looping associated with changes in BDNF-induced Satb2 expression. This can be proposed, but the data do not "suggest" this conclusion.

study

Subsection “Satb2 occupies active gene promoters”, first paragraph: "suggesting that Satb2 predominantly binds to active promoters of transcription factors and synaptic plasticity genes." It is not clear the authors have the data to support the word "predominantly" which would be quantified as well more than half of the targets are these genes. Unless this fact can be provided the phrase should be cut from the sentence.

study

Subsection “Satb2 occupies active gene promoters”, third paragraph: "our analysis revealed strong association of Satb2 with mIR promoters" The word "strong" is not quantitative. Do the authors have a statistical way to show this is "significant"? Otherwise the observation should just be stated with no qualifier.

study

Subsection “Satb2 determines the expression of protein-coding genes and miRNAs linked to learning and memory in the CA1 hippocampal field”, last paragraph: The word "pivotal" for the role of Satb2 in microRNA regulation is very strong and would suggest it is more important than other mIR regulators. "Important" would be more accurate for the data presented here.

study

1) A major weakness of the ChIP experiments is that they are performed in cultured neurons with overexpressed Satb2 whereas the RNAseq data are derived from hippocampus of the cKO mice. To confirm that the culture/overexpression ChIP experiments are relevant for the in vivo gene expression data the authors need to confirm by ChIP-PCR that endogenous Satb2 is bound to these same regions in hippocampus of the cKO mice. Furthermore, as a control for the specificity of the V5 antibody in the overexpression ChIP, the minimum control is ChIP from uninfected neurons – this could be ChIP-PCR as well.

study

Despite these substantial technical hurdles we were able to confirm Satb2 binding sites initially revealed by in vitro ChIP-seq experiments in CA1 tissue. In these newly added experiments we used a rabbit polyclonal antibody against Satb2 (offered by Abcam, ad34735) that has been previously used in EMSA assays and in a single study describing ChIPseq of chromatin derived from embryonic cortex (Mutual regulation between Satb2 and Fezf2 promotes subcerebral projection neuron identity in the developing cerebral cortex, McKenna et al., 2015). As a negative control, we used chromatin from Satb2 cKO CA1 tissue. With this Satb2-specific antibody we succeeded in demonstrating Satb2 enrichment at various Satb2 target regions that we had previously identified in our in vitro ChIPseq in chromatin ex vivo samples from control but not Satb2 cKO mice. The results are included in the revised version of the manuscript (Figure 5—figure supplement 4).

other

We also carried out ChIP qPCR assays using AAV-Satb2-V5 transduced and non-transduced primary hippocampal neurons. We performed one ChIP assay using AAV-Satb2-V5 transduced primary neurons (50 million cells) and at least two independent ChIP assays using non- transduced cultures (50 million cells per assay). The experiments in non-transduced cells showed minimal interaction with Satb2 for all tested target regions. The results are included in the revised version (Figure 5—figure supplement 1). Furthermore, we complemented the ChIP qPCR data derived from AAV-Satb2-V5 transduced neurons with a second biological replicate of the ChIP seq experiment (in which libraries for input and pooled ChIP DNA from three independent ChIP assays were used). The results from the second biological replicate completely confirmed the mapped Satb2 genomic binding sites and will be deposited at the GEO.

other

The authors overemphasize the miRNA findings at the expense of discussing the mRNAs that are changed. In principle, there is nothing from the ChIP data selectively linking Satb2 with miRNA expression that justifies the emphasis on miRNAs when interpreting the behavioral results.

other

Pre-miRNAs were not detected in our RNAseq and sRNAseq experiments. We used the TruSeq RNA v2 kit which relies on polyA selection and on essence precludes the analysis of pre-miRNAs. The sRNAseq protocol we applied also does not allow pre-miRNAs quantification because of size selection during library prep (only fragments with 147-157 bp length including adaptors which correspond to miRNAs (22 bp) and piwi-interacting RNAs or some other regulatory small RNA molecules (about 35 bp) are selected).

other

The authors report that nearly 50% of all microRNAs are dysregulated in Satb2 cKO mice, whereas only a very small number of mRNAs show differences. This is not clearly explained by the ChIP data (which show Satb2-V5 bound to both sets of promoters). Some explanation should be offered.

study

3) As presented, the BDNF-dependent regulation of Satb2 doesn't help to build a cogent model to explain the functions of Satb2 in learning and memory or LTP. Do the authors think that regulation of expression contributes to the requirement for Satb2 in learning and memory? If so it would be interesting to see if activity-dependent expression of Arc is dysregulated in Satb2 knockouts. Do physiologically relevant stimuli induce the expression of Satb2 in vivo? If these data cannot be more tightly tied to the behavior they would be better off moved to the supplementary data.

study

Our data in primary cultures reveal the potential of the Satb2 promoter to respond to BDNF-trkB signaling in hippocampal neurons. In our opinion, this is a novel finding deserving description in the main text since to our knowledge a similar mode of regulation has not so far been demonstrated for any other transcription factor required for learning and memory apart from immediate early genes. These data give us grounds to hypothesize that Satb2 is subjected to the same type of regulation also in vivo, e.g. in CA1 following behavioral learning or in the visual cortex after modulation of sensory input. Our behavior results showing that some of the best investigated functions of BDNF in the hippocampus, i.e. modulation of late-LTP and memory formation, depend on Satb2 are consistent with this hypothesis. However, it is very challenging to reveal regulation of Satb2 in vivo in the CA1 hippocampal area, following associative learning for the following reasons:

study

The use of whole CA1 hippocampal homogenates (e.g. in Western blotting experiments) will dilute the signal when only a small proportion of the cells in the sample are responsible for the changes. Tagging of activated neurons, e.g. in H2B-GFP TetTag mice, has shown that only 10-15% of CA1 pyramidal neurons become activated and labeled during fear conditioning (Reactivation of Neural Ensembles during the Retrieval of Recent and Remote Memory, Taylor et al., 2013). Thus, a special transgenic line should be used (such as H2B-GFP TetTag mice) to permanently tag neurons that are active during contextual fear conditioning and then examine Satb2 levels exclusively in the labeled neurons.

other

To circumvent this issue we used the dark-rearing paradigm to manipulate activity in the visual cortex. Unlike learning paradigms where only a small proportion of neurons are engaged, visual deprivation upon dark rearing causes a more general and robust decrease in activity levels in the visual cortex making it a more suitable model. Dark-rearing of mice for 4 days lead to a significant decrease in Egr1 levels in all layers of visual cortex (Author response image 1A) as previously shown (Differential induction and decay curves of c-fos and zif268 revealed through dual activity maps; Zangenehpour and Chaudhuri, 2002). Similarly, we found a small, but significant reduction in Satb2 levels by dark-rearing (Author response image 1B). This finding demonstrates the possibility of Satb2 regulation by neuronal activity. The small effect size that we observed points to the necessity of more specific measurements in learning paradigms such as determining Satb2 in activity-tagged cells or after mosaic DREADD silencing. In addition, a circumstantial evidence for Satb2 regulating or being regulated by ensemble dynamics is an observation of variability of Satb2 nuclear intensities between cells. It is currently under investigation. We did not include these data in the current manuscript because we consider them beyond the scope of the current investigations focusing on Satb2 function in hippocampus.10.7554/eLife.17361.028Author response image 1.(A) Mean intensity of Egr1 in each layer was normalized to corresponding layer 4 for each mouse to obtain relative Egr1 expression. Dark-rearing leads to a significant reduction in relative Egr1 levels in layer 2/3 and 6. (B) Dark-rearing leads to a reduction in relative Satb2 levels in layer 2/3 and 6.DOI: http://dx.doi.org/10.7554/eLife.17361.028

study

(A) Mean intensity of Egr1 in each layer was normalized to corresponding layer 4 for each mouse to obtain relative Egr1 expression. Dark-rearing leads to a significant reduction in relative Egr1 levels in layer 2/3 and 6. (B) Dark-rearing leads to a reduction in relative Satb2 levels in layer 2/3 and 6.

other

We also tested the idea of Satb2 regulation being necessary for learning-induced Arc expression by using IHC with tyramide signal amplification after contextual fear conditioning. The experiment compared three groups, naïve animals, 2h and 12h after training for both genotypes with 6 mice per group. Arc intensity was measured in a region of interest within the dorsal CA1-stratum pyramidale. The analysis did not reveal any effect of Satb2 loss on either early or late wave of Arc induction. Here, we would like to point out that intensity measurements by IHC cannot be directly compared to Western blot quantification of Arc protein in the CA1 area since in IHC experiment Arc expression in the cell soma is only considered whereas Western blot measures also Arc in dendrites where Arc mRNA is translated.

other

In this revision the authors have provided essential additional data needed to support the findings of this study. In particular, the chromatin immunoprecipitation controls (e.g. the V5 ChIP from nontransduced neurons and the endogenous Satb2 ChIP from hippocampus) provide crucial support for the model of direct transcriptional regulation by Satb2 in hippocampal neurons of the mRNA and miR targets identified as dysregulated in the sequencing studies. Further the validation of the mIR findings by RT-PCR and ChIP-PCR strengthen the focus of the manuscript on these targets.

study

The major finding of this study – that Satb2 has an important gene regulatory function in mature hippocampal neurons relevant to synaptic plasticity and learning and memory – is a novel and useful addition to the literature that will be of interest to a broad range of neurobiologists.

other

1) The BDNF data remain partially disconnected from the rest of the story and the "synapse to nucleus feedback loop" idea that is repeated several times in the manuscript is not supported by the data. The experiments demonstrating BDNF-dependent regulation of Satb2 expression in cultured neurons are well done, and the authors make a reasonable argument to keep these data in the main figures. However, they do not have relevant evidence for BDNF-dependent regulation of Satb2 in vivo or any evidence that induction of Satb2 expression is required for its role in hippocampal function. (Dark rearing is not a relevant experiment to demonstrate activity- or BDNF-dependent gene regulation in vivo. The more usual experiment would be dark adaption after eye opening followed by light exposure, since dark rearing induces developmental delays.) I do not think it is required that the authors demonstrate BDNF-dependent regulation of Satb2 expression in vivo. However in lieu of these data or other data showing that BDNF- or activity-dependent regulation of Satb2 levels matter for the functions of Satb2 identified in this study, then the authors cannot conclude in the Discussion that they have identified a synapse to nucleus feedback circuit. This language needs to be removed from the last line of the Abstract, the last line of the Introduction, and the first line of the Discussion. It is reasonable based on the BDNF data provided that the authors can speculate in the Discussion that BDNF-dependent regulation of Satb2 might regulate synapse plasticity as they propose. Finally, the Discussion section on the relationship between BDNF and Satb2 in synapse plasticity (third paragraph) remains beyond the data. Lots of things disrupt LTP in the hippocampus in addition to BDNF and Satb2, so just because those two have LTP disruption in common does not bind them tightly together.

study

2) The authors have improved their commentary on the possibility that Satb2 is acting to regulate mature hippocampal neuronal function via chromatin looping, but several sentences in the Discussion still remain that are overstatements from the data presented.

study

The discussion of functional interactions between BDNF and Satb2 has been slashed. Although we are convinced of the proposed BDNF and activity-dependent regulation of Satb2 in vivo, we agree that the in vitro data in the present manuscript, although providing strong indications, have been over-interpreted by us. Work concerning Satb2 regulation by dark adaptation/re-exposure to light in V1 (which in fact was what we were referring to in our previous letter) is in progress in our laboratory and we will discuss mechanism of in vivo-Satb2 regulation and its functional connex with BDNF in due time.

study

Sclerosing mesenteritis is a rare non-neoplastic disorder of unknown etiology that primarily affects the mesentery of the small intestine with chronic fibrosing inflammation during late adult life [1–3]. Only about twenty pediatric cases have been reported to date , but none has been reported in Chinese children. It can be an acute or insidious onset and lacks specific clinical manifestations. Abdominal pain and mass are the main symptoms. Here we report the first case of 5-year-old Chinese boy with sclerosing mesenteritis who presented with intractable abdominal pain, bloating, intestinal obstruction and massive ascites.

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A 5-year-old Chinese boy presented with a 4-week history of recurrent bloating, abdominal pain, anorexia and vomiting. There was no past or family history of any IgG4 and/or auto-immune related diseases. On admission, physical examination showed a moderately distressed patient with a temperature of 36.2 °C, a respiratory rate of 23 breaths/min, a blood pressure of 92/63 mmHg and a pulse rate of 102 beats/min. Abdominal examination revealed a severely distended abdomen with massive ascites. There was positive shifting dullness and diminished bowel sounds, but no abdominal wall rigidity. A complete blood count revealed a white blood cell count of 10.2 × 109/L with 64% neutrophils, a hemoglobin level of 108 g/L and hematocrit of 35%. Biochemical investigations showed significantly increased serum levels of IgE (280 IU/ml; normal reference 0 IU/ml to 30 IU/ml) and C-reactive protein (11 mg/L; normal reference < 0.5 mg/L). Liver transaminases, albumin, total bilirubin, alkaline phosphatase, glucose, erythrocyte sedimentation rate, electrolytes and renal function tests were all within the normal ranges. Both stool culture and parasite tests were negative. An abdominal CT scan revealed bowel wall thickening on the right side and massive ascites. A positron emission tomography was performed to exclude any malignancies and showed a large soft tissue density around the pancreas, with inflammatory lesions and mild metabolic activity. There was widespread bowel edema. A diagnostic paracentesis revealed slightly yellow-colored fluid containing white blood cells (1000 × 109/L). Rivalta test of the ascites was positive for exudate and there was no tubercle bacillus or malignancy. An abdominal film showed intestinal obstruction (Fig. 1a). An initial barium study suggested an incomplete jejunal obstruction with delayed transit and only a small amount of barium reaching the distal jejunum (Fig. 1b).Fig. 1Imaging studies. a The abdominal film showed ileus and massive ascites; b The initial barium study showed partial jejunum obstruction and slow transit with only a small amount of barium reaching to the distal jejunum; c The 2nd barium study showed slow bowel peristalsis, and delayed transit in the duodenum and jejunum; d The 3rd barium study showed mildly delayed gastric emptying and delayed transit in jejunum

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Imaging studies. a The abdominal film showed ileus and massive ascites; b The initial barium study showed partial jejunum obstruction and slow transit with only a small amount of barium reaching to the distal jejunum; c The 2nd barium study showed slow bowel peristalsis, and delayed transit in the duodenum and jejunum; d The 3rd barium study showed mildly delayed gastric emptying and delayed transit in jejunum

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An exploratory laparotomy was performed. There were widespread dilatation, edema and thickening of the small bowel, with massive clear slightly-yellowish ascites of about 1300 ml. Small intestine mesentery and ascending colon were also thickened, with multiple yellowish nodules. There were extensive intraperitoneal adhesions. No evidence of obstructive pathology was identified. The disease was centered more on the mesentery. With concern for malignancy, nearly 60 cm of bowel including the terminal ileum, ileocecal valve, part of ascending colon and their mesentery, and part of omentum, were resected. An end ileostomy was created.

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Histopathological examination showed significant thickened serosa and eosinophilic infiltration (5-30/high power field) in the lamina propria. Mesentery and nodules had inflammatory cell infiltration, fat necrosis, fibroblast proliferation and fibrosis. There was omental fibrous connective tissue hyperplasia with infiltration of lymphocytes and adipose tissue (Fig. 2 a-c).Fig. 2First histopathological examination showed (1) eosinophilic infiltration (5-30 /HPF) in the lamina propria (2A); (2) fibrous connective tissue hyperplasia in omentum, lymphocytic infiltration and adipose tissue (2B); and (3) mesentery tissue with inflammatory cell infiltration, fat necrosis, fibroblastic proliferation and fibrosis (1C). The 2nd histopathological examination (4 months after initial surgery) showed inflammatory cell infiltration and fibrinoid necrosis in serosal surface (2D; mesentery tissue with inflammatory cell infiltration, fibroblasts proliferation (2E) and fat necrosis (2F)

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First histopathological examination showed (1) eosinophilic infiltration (5-30 /HPF) in the lamina propria (2A); (2) fibrous connective tissue hyperplasia in omentum, lymphocytic infiltration and adipose tissue (2B); and (3) mesentery tissue with inflammatory cell infiltration, fat necrosis, fibroblastic proliferation and fibrosis (1C). The 2nd histopathological examination (4 months after initial surgery) showed inflammatory cell infiltration and fibrinoid necrosis in serosal surface (2D; mesentery tissue with inflammatory cell infiltration, fibroblasts proliferation (2E) and fat necrosis (2F)

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Post-surgically, the patient was treated with parenteral nutrition, intravenous fluids, gastrointestinal decompression, and pro-motility agents such as mosapride and erythromycin. However, severe bloating persisted and more than 1000 ml of green-yellowish fluids was drained from the decompression tube and 200 ml from the ileostomy daily. With suspicion of eosinophilic enteritis based on pathology report, he was treated with intravenous dexamethasone at 0.2 mg/kg/d, which was gradually decreased to 0.1 mg/kg/day over a 2-week period and stopped after 4 weeks. His abdominal pain decreased and ascites resolved, with a gradual improvement in enteral nutrition intake. While the decompression tube drainage was decreased, the ileostomy drainage increased to more than 1000 ml daily. The second and third barium studies showed some degree of gastrointestinal motility improvement (Fig. 1c and d). Subsequently at 4 months after the initial exploratory laparotomy, a 2nd surgery was performed in order to close the ileostomy and revealed that the bowel was still severely edematous, with very tight adhesions between the bowel and the abdominal wall. Ileostomy closure couldn’t be performed. Tissues near the ileostomy, including bowel and mesentery, were resected and sent for pathological examination, which showed chronic inflammatory cell infiltration, fat necrosis and fibrosis (Fig. 2 d-f). Based on pathological features and clinical presentations, a diagnosis of sclerosing mesenteritis was finally established. Prednisolone at 2 mg/kg was started and he experienced rapid clinical improvement in 4 weeks and was discharged from the hospital. After 8 weeks of treatment, prednisolone was slowly weaned and stopped after 24 weeks. At 8-month after hospital discharge, he was asymptomatic with adequate weight gain. A final surgery (15 months after the initial exploratory laparotomy) was performed to close the ileoostomy and there was no edema but he was noted to have diffusely enterocolonic adhesions. The boy remained asymptomatic at a 6-month post-operation clinic visit.

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Sclerosing mesenteritis is a rare chronic fibrosing inflammatory disease of ambiguous pathogenesis primarily diagnosed in late adult life while pediatric cases are very uncommon because children have mesenteric fat than adults [1–3]. It is a benign disorder mainly involving the small-bowel mesentery; however, mesocolon, peripancreatic region, omentum, pelvis or retroperitoneum may be also involved. Both mesenteric panniculitis and mesenteric lipodystrophy are considered to be histological variants of sclerosing mesenteritis . Viswanathan and Murray reported one case and summarized another 16 pediatric cases from the literature and found that the average age at diagnosis in children was 6.5 years . In our case, the lesion was extensive and involved small intestine, ascending mesocolon, omentum and peritoneum, leading to intractable bowel obstruction. Clinical symptoms are usually non-specific, including abdominal pain, anorexia, fatigue, weight loss, fever, abdominal mass, ascites, pericardial effusion, diarrhea and constipation [1–3]. Laboratory examinations including C-reactive protein, erythrocyte sedimentation rate, complete blood count, and biochemistry are also non-specific, which renders a diagnostic challenge for clinicians. Abdominal CT or ultrasonography may play an important role in diagnostic evaluation and the most common finding is a soft tissue mass at the root of small bowel mesentery . In the present case, massive peritoneal cavity effusion resulted in a negative finding on CT scan. A definite diagnosis is dependent on histopathology, which usually reveals fat necrosis, chronic inflammation and fibrosis but can be variable from case to case. These histological variants may reflect the state of a chronic inflammatory process; necrosis in mesenteric fat may represent disease progression and eventually progress to fibrosis which leads to retractile mesenteritis . In the present case, infiltration of chronic inflammatory cells, fat necrosis and fibrosis were all observed in the histological findings from initial and subsequent surgical specimens, but due to lack of awareness of the disease, a diagnosis of sclerosing mesenteritis was not recognized at the first operation. Therefore, due to the nonspecific signs and symptoms of sclerosing mesenteritis, diagnosis is mainly made through a combination of histopathological and imaging (preferably abdominal CT) findings .

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A recently defined subtype, IgG4-related sclerosing mesenteritis, has been reported to be closely related to IgG4-related disease and one such case was reported in children [8–10]. In our case, IgG4 serum subclass levels or tissue immunohistochemistry were not performed.

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The optimal therapeutic strategy for sclerosing mesenteritis remains unclear. Some asymptomatic or mild clinical cases may resolve spontaneously without therapy. In some cases with intensive fibrosis leading to intractable bowel obstruction or perforation, surgical resection or ileostomy might be required [1–3]. Medications including corticosteroids, thalidomide, cyclophosphamide, colchicine, azathioprine and tamoxifen have been reported to induce remission of sclerosing mesenteritis [11–13]. Most of the previous reported pediatric cases were successfully treated with corticosteroids. Our patient was started on corticosteroids at 2 mg/kg after definitive diagnosis was made and had a clinical remission in 4 weeks. Due to the severity of intestinal inflammation, we decided to keep him on corticosteroids at 2 mg/kg for another 4 weeks and then slowly weaned off it over 6 months.

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We describe a very rare case of sclerosing mesenteritis in a Chinese boy. We failed to make a diagnosis at the first operation due to lack of awareness of the disease. Sclerosing mesenteritis should be considered as a differential diagnosis in patients with intractable bowel obstuction and serous cavity effusion. A definite diagnosis is dependent on histopathology. Judicious use of corticosteriods therapy can lead to clinical remission.

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A 52-year-old right-handed man presented for the evaluation of progressive emotional flattening, social withdrawal, and loss of empathy. Prior to symptom onset, his wife of 21 years described him as “easy going, loving, kind, and generous.” As symptoms progressed, the subject began to alienate coworkers with unwelcome and persistent practical jokes. These disinhibited behaviors were accompanied by new, compulsive Internet usage. No language, visuospatial, memory, or motor symptoms were reported.

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Past medical history included chronic headaches and multiple concussions but no seizures, psychiatric disorders, or drug use. He denied cutaneous, dental, or renal abnormalities. His childhood development was unremarkable. He completed 13 years of formal education and worked in the upper echelons of management at a professional firm.

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The subject’s family history (Supplementary Fig. S1a) was notable for a father with multiple concussions and late-life seizures, alcoholism, impulsiveness, and poor decision-making. An older sibling with left arm hemihypertrophy (Parkes-Weber Syndrome) was deceased after post-operative stroke. A younger sibling developed epilepsy at age 19 and underwent frontal lobe resection surgery at age 43; this individual experienced progressive difficulty with concentration and multitasking, qualifying for a diagnosis of mild cognitive impairment (MCI) in the executive domain.

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On exam, the proband was 72 inches tall and weighed 220 lb (BMI of 29.8). Skin exam revealed ungual fibromas and a shagreen patch. His neurological exam was normal except for an expansive mood, intrusive commentary, and jocularity verging on the inappropriate. Neuropsychological testing revealed impaired confrontational naming with mild deficits in semantic knowledge. Visual episodic memory, executive functioning, and information processing speed were also impaired. Other cognitive domains were within normal limits for age. Deficits localized to the right greater than left temporal and frontal lobes. Magnetic resonance imaging (MRI) revealed corresponding areas of atrophy and white matter lesions (Fig. 1a). Electroencephalogram (EEG) was negative for epileptiform activity.Fig. 1Proband clinical MRI, novel TSC1 mutation, cell model of TSC1/hamartin haploinsufficiency, and neuropathology of a TSC1 mutation carrier. a Magnetic resonance imaging (MRI) FLAIR sequences revealed severe atrophy of the right anterior temporal lobe, particularly the entorhinal/perirhinal cortices and inferior temporal gyrus, as well as the hippocampus and amygdala. There was also involvement of the left anterior/mesial temporal lobe and the bilateral frontal and right parietal lobes. T2/FLAIR hyperintensities are seen within the subcortical white matter, some of which extended to the superolateral aspects of the lateral ventricles (arrowheads). These lesions can be seen in tuberous sclerosis or focal cortical dysplasias as a transmantle sign. b Sanger sequencing confirmed a novel frameshift variant in the TSC1 gene (NM_000368.4: c.62_63insTG: p. Arg22CysfsTer5) that segregated with the proband and affected sibling. c Representative micrographs of the enlarged cell body in the undifferentiated TSC1 heterozygous mutant SH-SY5Y lines. Scale bars represent 50 microns. d Representative western blots showing decreased TSC1/hamartin and increased phopho-P70S6Kthr389, total tau, and phospho-tau levels in retinoic acid differentiated TSC1 +/− SH-SY5Y cells. TDP-43 levels are unchanged. e–g Fragment of frontal cortex resection from the proband’s sibling was received and stained for hyperphosphorylated tau, revealing patchy staining in neurons, glia, and surrounding neuropil. Scale bars represent 1000 (e), 250 (f), and 25 (g) microns

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Proband clinical MRI, novel TSC1 mutation, cell model of TSC1/hamartin haploinsufficiency, and neuropathology of a TSC1 mutation carrier. a Magnetic resonance imaging (MRI) FLAIR sequences revealed severe atrophy of the right anterior temporal lobe, particularly the entorhinal/perirhinal cortices and inferior temporal gyrus, as well as the hippocampus and amygdala. There was also involvement of the left anterior/mesial temporal lobe and the bilateral frontal and right parietal lobes. T2/FLAIR hyperintensities are seen within the subcortical white matter, some of which extended to the superolateral aspects of the lateral ventricles (arrowheads). These lesions can be seen in tuberous sclerosis or focal cortical dysplasias as a transmantle sign. b Sanger sequencing confirmed a novel frameshift variant in the TSC1 gene (NM_000368.4: c.62_63insTG: p. Arg22CysfsTer5) that segregated with the proband and affected sibling. c Representative micrographs of the enlarged cell body in the undifferentiated TSC1 heterozygous mutant SH-SY5Y lines. Scale bars represent 50 microns. d Representative western blots showing decreased TSC1/hamartin and increased phopho-P70S6Kthr389, total tau, and phospho-tau levels in retinoic acid differentiated TSC1 +/− SH-SY5Y cells. TDP-43 levels are unchanged. e–g Fragment of frontal cortex resection from the proband’s sibling was received and stained for hyperphosphorylated tau, revealing patchy staining in neurons, glia, and surrounding neuropil. Scale bars represent 1000 (e), 250 (f), and 25 (g) microns

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The proband met criteria for probable behavioral variant frontotemporal dementia (bvFTD), a sub-type of frontotemporal lobar degeneration (FTLD), based on early behavioral disinhibition, loss of empathy, perseverative behaviors, neuropsychological profile, and MRI findings . Given his family history, he was screened for known genetic causes of FTLD with no observed pathogenic variants. Therefore, the proband and his affected sibling underwent whole-exome sequencing. Among variants identified, a novel frameshift mutation in one allele of the TSC1 gene, predicted to result in early termination of the TSC1/hamartin protein, was shared by the proband and his younger sibling (Fig. 1b, Supplementary Fig. S1b, c; Table S1).

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TSC1/hamartin is an upstream inhibitor of mTOR, which regulates cell growth and protein degradation . To determine the significance of the TSC1 loss of function variant, CRISPR/Cas9 genome editing was used to generate a cellular model of TSC1/hamartin deficiency in SH-SY5Y cells, a human neuroblastoma cell line that can be differentiated into neuronal-like cells (Supplementary Fig. S2a). We confirmed that these lines exhibited decreased TSC1/hamartin, increased cell size, and elevated P70S6K phosphorylation, consistent with mTOR over activity (Fig. 1c, Supplementary Fig. S2b, c). After retinoic acid (RA) differentiation, TSC1 +/− cells maintained lower levels of TSC1/hamartin and increased mTOR activity. Interestingly, the RA-differentiated TSC1 +/− lines accumulated phospho-tau and total tau. However, levels of TDP-43, another protein that aggregates in FTLD , were unchanged compared to control (Fig. 1d, Supplementary Fig. S2d–h).

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Histological samples were obtained from the partial frontal lobe resection of the affected sibling, also a TSC1 mutation carrier. Similar to SH-SY5Y model of TSC1 heterozygosity, immunostaining of the resected tissue for phosphorylated tau revealed patchy neuropil staining as well as diffuse cytoplasmic staining of scattered neurons and glia, with variable intensity (Fig. 1e–g). No neurofibrillary tangles, Pick bodies, glial inclusions, or other features of an inherited or sporadic tauopathy were observed. Sections were also stained for TDP-43, phospho-TDP-43, amyloid beta (Aβ), α-synuclein, and ubiquitin. Surprisingly, they did not demonstrate elevated immunoreactivity for any other neurodegenerative disease proteins (data not shown).

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In this case report, we describe the first case of bvFTD in an adult with sub-clinical tuberous sclerosis complex (TSC) due to a novel TSC1 frameshift variant. Although TSC has been called an “infantile tauopathy” , TSC1 mutations have not previously been implicated in age-associated neurodegenerative disease. This report, therefore, potentially adds TSC1 to the list of genes that are implicated in both a juvenile-onset lysosomal storage disease and adult-onset neurodegeneration. Others include GBA and ATP13A2 in Parkinson’s disease, CTSD in Alzheimer’s disease, and PGRN in FTLD [10, 15]. The TSC1/TSC2 complex normally inhibits mTOR activity, with loss of function mutations resulting in overactive mTOR and consequential increased protein synthesis and decreased protein degradation [3, 8, 9]. Strikingly, the decreased protein degradation seems selective for tau, as evidenced from both our cell-based and neuropathological data. This case now suggests that with age, accumulated tau can precipitate neuron loss and neurodegeneration. It also raises the intriguing possibility that tau metabolism is selectively regulated by the mTOR pathway. Our data suggest that TSC1 loss of heterozygosity, which is necessary for TSC-related tumors , is not required for TSC1-related tau accumulation and neurodegeneration.

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Our findings could be highly significant to the care of TSC patients. Many features of tuberous sclerosis associated neuropsychiatric disorders (TAND) [4, 5], including obsessive behavior, attention deficits, altered eating, impulsivity, memory loss, and language dysfunction, overlap with bvFTD criteria. Thus, these two clinical entities may represent overlapping disorders precipitated by progressive tau pathology. Additional sequencing of the TSC1/2 genes in FTLD cohorts, use of new tau-based PET imaging, and review of neuropathological specimens from older TSC patients may further support the association between TSC, TAND, and FTLD.

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This study extends the relationship between mTOR and tau metabolism [1, 16] to include a potential age-related component. How does aging alter the TSC1/mTOR axis to potentially contribute to the development of neurodegeneration? Why do tau and phospho-tau accumulate, while related proteins that undergo chaperone mediated autophagy, such as TDP-43, do not? Do TSC2 mutations also link to age-related tauopathies? These questions and others are stimulated by our report. In the meantime, this study supports trials of rapamycin analogs which are already underway for neurodegeneration , and moves us towards a personalized medicine future in which sub-populations of tauopathy patients may be selectively responsive to this therapeutic approach.

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The formation and spread of small aggregates of proteins such as α‐synuclein, β‐amyloid or tau is reported in a wide range of neurodegenerative diseases.1, 2 Of these, Parkinson's disease (PD) is characterised by the accumulation of a misfolded and aggregated protein called α‐synuclein within neurons to form Lewy neurites and Lewy bodies.3 Genetic and pathological evidence suggests that the protein α‐synuclein is central to neurodegeneration in PD.4 Specifically, the transition from an intrinsically disordered α‐synuclein monomer through a series of oligomeric intermediates (with varying structures and size) to a highly structured filament5, 6 is recognised to drive pathogenesis in α‐synucleinopathies. Furthermore, aggregates of α‐synuclein exhibit cell–cell transfer, leading to seeding and recruitment of more protein molecules to form additional aggregates that can generate new seeds in an exponential way,7 leading to the region–region spread of disease. The distinct structure of α‐synuclein aggregates has a role in its pathogenic properties, in particular, the toxicity of the aggregate, the cell type affected, seed competency, and the regional transfer of pathology.8, 9 This dramatic effect of the structure of the ordered assembly on the pathogenic pathway in the brain underpins the importance of understanding the order/structure of α‐synuclein aggregates.

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Previous studies have shown aggregates to be very diverse in terms of their mechanisms of formation, size and structure.10, 11, 12, 13 Bulk measurements obtained with conventional, ensemble‐based, biophysical techniques are able to characterise many features of these heterogeneous aggregates,14, 15 but new quantitative tools are needed to specifically characterise in greater detail the structural features of individual aggregates, particularly in human tissue and biological fluids.

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By means of single‐molecule fluorescence resonance energy transfer (smFRET) experiments, a subpopulation of aggregates formed with fluorescently labelled α‐synuclein has previously been shown to undergo a slow structural rearrangement before growing into fibrils.12 This conversion can generate more cross‐β structure and the resulting aggregates were reported to be both, more resistant to proteinase‐K and more toxic to cells. Unlabelled fibrils of amyloid‐β or α‐synuclein can be imaged with total internal fluorescence (TIRF) microscopy and structurally specific dyes such as thioflavin T (ThT),16, 17 opening up the possibilities of studying aggregates in human biofluids.18 At a single fibril level, dyes such as ThT or Congo Red have been shown to bind insulin fibrils in an ordered way, and by monitoring the intensity as a function of the polarisation angle, these dye classes can be provide information on the structure of fibrils.19

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In this work, we have characterised structural features of aggregates formed by an amyloidogenic protein, by implementing a highly sensitive fluorescence anisotropy setup. Fluorophores absorb light with a probability proportional to the square of the dot product of the local optical electric field and the molecular transition dipole moment. Thus when a dye binds in a defined orientation it emits highly polarised anisotropic fluorescence. We therefore built a bespoke anisotropy instrument to study the structure of spatially isolated amyloid aggregates by placing a polariser in the detection path which rotates continuously during image acquisition (Figure 1 a). ThT is a widely‐used benzothiazole dye that increases its fluorescence quantum yield by several orders of magnitude upon binding extended cross‐β structures. Experiments suggest that the dye preferentially binds with its long axis parallel to the long axis of fibrils,20, 21, 22 but depending on the protein under study and the structure of the fibril other binding sites may exist. When single α‐synuclein fibrils are imaged as a function of the angle of the axis of polarisation, their fluorescence modulates sinusoidally between a maximum when the axis is aligned with the fibril and a minimum if the axis is perpendicular to the fibril (Figure 1 b–d). This further confirms that the dominant binding site (or possibly sites) of ThT is aligned with the axis of the α‐synuclein fibrils.

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Procedure to monitor anisotropy: a) Fluorescence anisotropy was measured on a total internal fluorescence microscopy (TIRF) arrangement in which a rotating polariser is mounted between the dichroic and the tube lens of the microscope. b) ThT binds specifically to the cross‐β architecture of amyloid fibrils in a well‐defined manner. c) The detected fluorescence of ThT for α‐synuclein fibrils is maximum when the polariser is aligned with the fibril, and minimum when it is orthogonal to the fibril (scale bar=5 μm). d) Montage of the modulating fluorescence of an α‐synuclein fibril as the polariser rotates (each frame corresponds to the intensity averaged during a rotation of approximately 3.6°). e) This intensity can be fitted to a sinusoidal curve to quantify the degree of modulation and hence structural order.

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A measure of the degree of extended ordered cross‐β structure in an aggregate is the amplitude of the fluorescence signal of ThT as the polariser rotates. The normalised fluorescence intensity as a function of polariser angular displacement of each individual protein aggregate was fitted to a sinusoid (see Methods section and Figure S1 in the Supporting Information (SI)). The fluorescence was fitted to y=acosbx+c+d , where a represents the amplitude of the signal, b the constant angular frequency (user‐defined rotation velocity of the polariser), c the phase and d an offset (Figure 1 e). The response to the anisotropy measurement is defined by: modulation=2a/(a+d) . A small modulation value implies disordered β‐sheet content in the aggregate, whereas a larger modulation value implies spatially aligned β‐sheet content.

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We used a single‐molecule sensitive TIRF imaging mode to measure the structural arrangement of individual spatially isolated diffraction‐limited aggregates which we have previously characterised using super‐resolution techniques.23 We performed an aggregation reaction for recombinant α‐synuclein and focused on the kinetics of the lag phase of the aggregation.24 The reaction was done at a concentration of 70 μm under agitation at 200 rpm in 25 mm Tris buffer (pH 7.4) supplemented with 0.1 m NaCl and 0.01 % NaN3 at 37 °C. The aggregation reaction was performed in low binding polypropylene tubes to minimise protein adhering to the tubes. More specifically we analysed samples taken from the aggregation reaction at times between 1 h and 4 h. At longer times aggregates larger than the diffraction limit of optical light (i.e. ≈170 nm) start to form. A histogram of modulation values for each time point revealed that oligomers present at 1 h and 2 h have low modulation values (typically lower than 0.5), while at 3 h we found a distinct population of oligomers that respond with high modulation values (Figure 2). These data are indicative of the structural rearrangement from a relatively amorphous oligomer into a “fibril‐like” periodic structure. The modulation measurement does not correlate with the fluorescence intensity of the aggregate (SI, Figure S2 and S3), suggesting that the number of ThT binding sites can remain constant during a structural re‐arrangement. Although there is a variability associated with the stochasticity of the nucleation process during the lag phase of the aggregation, independent experiments show the presence of the two populations of aggregates and the same trend for the evolution of the species (SI, Figure S4).

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Structural analysis of the species observed during an α‐synuclein aggregation reaction: a) Modulation measurements of detected aggregates during an aggregation reaction at 70 μm show an evolution from relatively disordered aggregates (1 h and 2 h) to a mixture of relatively disordered and “fibril‐like” aggregates (3 h and 4 h). Each histogram pools data from 3 independent aggregations. Representative fluorescence images are shown in (b) (scale bar=5 μm) with magnified sub‐images (2.5x); number of aggregates analysed in each time point: 1 h n=121, 2 h n=385, 3 h n=581, 4 h n=719.

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Combining all the detected aggregates, the overall distribution of the degree of modulation can be fitted to two Gaussian distributions (Figure 3 a). When compared to the modulation of long fibrils (for example formed after 24 h of aggregation, yielding fibrils that are several μm long), we observe that the fibrils typically have higher modulation values (Figure 3 b). The histogram of oligomers and fibrils can be therefore fitted to three Gaussian distributions (Figure 3 c). The population corresponding to low modulation values have some cross‐β content (as ThT binds to them), and behaves in a similar way to fluorescent beads (SI, Figure S5), meaning that these aggregates are not structurally aligned. The scatter plot of modulation vs. the mean intensity of all measured aggregates suggested once again that disordered aggregates convert to fibril‐like aggregates without an increase in integrated fluorescence intensity (Figure 3 d). We observed that fibrils are characterised by high modulation and intensity values (Figure 3 d). This strong fluorescence anisotropy response of fibrils can be achieved by labelling with other fluorescent dyes such as pentameric formyl thiophene acetic acid (pFTAA) which has also been shown to bind to cross‐β structures25 (SI, Figure S6).

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Modulation and toxicity of α‐synuclein during aggregation: a) Number of modulating and non‐modulating α‐synuclein aggregates obtained from the time evolution of the two populations shown in Figure 2 a. (error bars correspond to error in population fits multiplied by the bin size). b) Ca2+ influx kinetics obtained with α‐synuclein aggregates (average of the three independent aggregation reactions, error bars are standard deviation between three replicates). c) Example of liposome assay, showing liposomes with disrupted membranes as fluorescent puncta. A higher number of fluorescent liposomes can be seen when incubating with 4 h aggregates as compared with 1 h aggregates (scale bars=5 μm).

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To correlate the structural information of the aggregated species with the ability of these aggregates to generate toxic effects in cells we evaluated their capability to disrupt membranes with a technique that uses vesicles filled with a Ca2+ sensitive dye.26 Upon the interaction of a protein aggregate with the vesicle's membrane, Ca2+ ions enter the vesicle from the surrounding solution and hence becomes fluorescent. This change in fluorescence can be detected using TIRF microscopy. We imaged individual liposomes in the presence of Ca2+ buffer (blank), followed by the addition of an aliquot of α‐synuclein aggregates at a concentration of 50 nm and subsequent addition of ionomycin. In the presence of only Ca2+ buffer, the fluorescence intensity of the vesicles was low and comparable to that of background noise due to minimal Ca2+ presence within the vesicle.26 After incubation (for 10 minutes) with α‐synuclein samples, we detected an increase in the localised fluorescence intensity of the vesicles showing that Ca2+ ions could enter the vesicles as a consequence of the aggregates’ induced membrane permeability. Subsequent addition of ionomycin, an ionophore enabling Ca2+ to enter the vesicles, results in the saturation of all vesicles with Ca2+ ions, allowing us to quantify the extent of membrane disruption (Figure 4 c).

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Modulation landscape of small aggregates and fibrils of α‐synuclein: a) Modulation measurements of all species detected in three independent aggregation reactions fitted to two Gaussian functions (1149 species in total). b) Long fibrils (formed after 24 h aggregation, typically several μm in length) respond with high modulation values, which can be fitted adding a third Gaussian (511 species considered). c) The complete landscape of aggregates and fibrils can be fitted with three Gaussian functions. d) Scatter plot of mean intensity of each aggregate (green dots) and fibril (magenta dots) and its corresponding modulation values.

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In order to understand further the evolution of aggregates we globally fitted two Gaussian functions to each time point, pooling data obtained in three independent aggregation reactions to describe better the landscape of aggregates (Figure 3 a). The integrated areas of the Gaussians give an estimate of the number of aggregates in each population (Figure 4 a). The results show that non‐modulating aggregates appear before modulating ones, and at a slower rate, suggesting that there is a conversion of non‐modulating to modulating aggregates. In contrast to these well‐defined populations, aggregates formed during an aggregation reaction of α‐synuclein at low monomer concentration (1 μm for 1 month) display a broader distribution of modulation responses, suggesting that a wider variety of species are formed over long periods of time (SI, Figure S7).

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The permeabilisation assay showed a linear increase of the Ca2+ influx after incubation with aggregates (previously aggregated for 1 h to 4 h), suggesting that both disordered and fibril‐like aggregates induce calcium influx in the liposomes (Figure 4 b). Given that the trend of modulating species dominates the later time points while the abundance of non‐modulating aggregates increases at a slower rate (Figure 4 a), our results suggest that modulating species have a higher ability to disrupt membranes.

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To demonstrate the broad applicability of our technique, we also applied the method to samples of human cerebrospinal fluid (CSF), comparing aged‐matched healthy controls (HC) to Parkinson's disease (PD) patients. By analysing the modulation of individual species in each group (obtained from 4 HC and 4 PD samples), we found that the large majority of species show non‐modulating behaviour, (Figure 5 a,b) meaning that they are disordered. Only a small fraction (≈1 %) of species showed modulating behaviour in both HC and PD groups (Figure 5 c). Species with higher modulation values (modulation over 0.45) in PD patients have a mean of 0.57 compared to HC with a mean of 0.48 (inset Figure 5 c). The abundance of these modulating species in CSF is very low and therefore prevents us from a robust statistical analysis. Further studies need to be done to characterise these ordered species, as they are candidates to be involved in toxicity and spreading mechanisms. However, this does demonstrate the technique is capable of making structural measurements in human CSF.

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Anisotropy of species observed in human cerebrospinal fluid: Fluorescence anisotropy histogram of species in a) HC CSF and b) PD CSF obtained by pooling aggregates from four different individuals in each case (HC: n=1461 species, mean=0.3112, standard deviation=0.1015; PD: n=1069 species, mean=0.2929, standard deviation=0.1136). c) Cumulative distribution of species from the same data set showing higher number of modulating species (modulation>0.45) in PD.

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In summary, we have demonstrated that by combining sensitive TIRF microscopy with anisotropy measurements, one can directly characterise the structural features of individual oligomers. This method is highly flexible as it does not require protein labelling, but rather a dye that recognises cross‐β motifs. We have shown the conversion from disordered aggregates of α‐synuclein to fibrillar aggregates, in agreement with previously reported smFRET measurements. Furthermore, our experiments suggest that modulating aggregates have a higher capacity to disrupt lipid membranes. Our results provide clear evidence that most ThT active species in CSF are disordered, but do, however, contain cross‐β sheet structure. Our ability to analyse single aggregates individually allowed us to detect an ultra‐low abundance of fibril‐like species in human CSF. This methodology is compatible with other proteins whose aggregation has been associated with human disorders such as amyloid‐β, tau, lysozyme or insulin. Overall this approach provides a new method to characterise the degree of fibrillation in individual protein aggregates, contributing to the set of biophysical methods needed to understand some of the most fundamental mechanisms in neurodegeneration.

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Ultraviolet-B (UVB) radiation of the skin is considered to be the major determinant of vitD levels [3, 8, 14]. However, due to latitude, cutaneous synthesis of vitD occurs less than six months of the year in the Nordic countries, and dietary content is also limited [9, 14–17]. 25-hydroxyvitamin D (25(OH)D) is metabolized in the kidneys by 1-alpha-hydroxylase to the active form, 1,25-dihydroxyvitamin D (1,25(OH)2D) [3, 4, 18]. Through the vitamin D receptor, regulates 1,25(OH)2D hundreds of genes in a variety of body tissues [6, 14, 19, 20]. A major proportion of 25(OH)D and 1,25(OH)2D binds to vitamin D-binding protein (DBP) or albumin (>99%) [21, 22]. Free and bioavailable fractions seem to be more strongly correlated to the biological activity [14, 22–24]. Serum levels of 25(OH)D are used for evaluation of vitD status [3, 14, 15, 21]. The optimal levels of 25(OH)D and 1,25(OH)2D during pregnancy are, however, not settled, and recommendations concerning vitD intake are diverging [4–6, 14, 17, 25].

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In pregnancy, the calcium requirement of the fetus results in profound changes in maternal calcium homeostasis [4, 26]. Whereas parathyroid hormone (PTH) plays a major role in calcium and bone metabolism in the non-pregnant state, vitD appears to be a prominent regulator during pregnancy [8, 27]. This is reflected in a two- to threefold rise in 1,25(OH)2D levels to increase intestinal calcium absorption, and ensure mineralization of the fetal skeleton [4, 26, 27]. This rise is dependent on sufficient 25(OH)D . The relationship between 25(OH)D and 1,25(OH)2D during pregnancy remains, however, unclear [18, 23, 26]. There are many determinants of 25(OH)D contributing to the diverse prevalence rates reported during pregnancy [3, 5, 9, 16, 29, 30]. A large variation in 1,25(OH)2D levels in third trimester has also been observed . Larger longitudinal studies concerning 1,25(OH)2D, free and bio-available vitamin D in pregnancy are scarce [18, 21, 23].

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clinical case

Vitamin D (vitD) inadequacy among pregnant women is prevalent worldwide, and has been associated with adverse pregnancy outcomes [1–10]. Developmental origins of disease have gained increasing attention, and maternal hypovitaminosis D during fetal life is one of the factors suggested to be of significance for future disease, including osteoporosis and cardiovascular disease [11–13].

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We aimed to assess 25(OH)D, 1,25(OH)2D and PTH levels and the relationship between these parameters among Caucasian women in the two final trimesters. DBP was analyzed for calculation of free and bioavailable 25(OH)D and free 1,25(OH)2D. Furthermore, we aimed to investigate determinants of vitD, including seasonal and latitudinal variation, and adherence to the recommendations on vitD and calcium intake. Finally, we wanted to explore the association of total and free 25(OH)D, 1,25(OH)2D and PTH levels with pregnancy outcomes (gestational diabetes mellitus (GDM) and birthweight).

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The current study included 855 pregnant Norwegian women from the cities of Trondheim (n = 660), latitude 63°N and Stavanger (n = 195), latitude 58°N (Fig 1). They participated originally in a randomized controlled trial (RCT), conducted between 2007 and 2009, aiming to investigate the antenatal health effects of an exercise program, and the primary outcome was gestational diabetes mellitus . The present study was a secondary analysis of the RCT. Healthy Caucasian women, 18 years and older, with a singleton live fetus were included. Exclusion criteria were high-risk pregnancies and diseases that could hinder participation . The two groups were homogenous at inclusion and after the intervention, and were merged in the current study.

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Serum 25(OH)D levels <50 nmol/L were classified as insufficiency and 25(OH)D levels <30 nmol/L as deficiency (VDD) according to the US Institute of Medicine (IOM) and Nordic Nutrition recommendations [17, 25, 32]. The seasons were divided as follows: winter (December-February), spring (March-May), summer (June-August) and autumn (September-November). Gestational hypertension was defined as systolic blood pressure (BP) >140 mm Hg, diastolic BP >90 mm Hg, or both in women with no pregestational hypertension. The criteria for GDM were fasting glucose level in whole blood ≥6.1 mmol/L, or plasma glucose ≥7.0 mmol/L, or 2-hour glucose level ≥7.8 mmol/L after oral glucose tolerance test in women with no pregestational diabetes mellitus .

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The study was conducted in accordance with the ethical principles in the declaration of Helsinki, approved by the Regional Committee for Medical and Health Research Ethics (REK 4.2007.81) and registered in the ClinicalTrials.gov (NCT 00476567). Written informed consent was obtained from all participants.

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Blood samples were collected after fasting, and the sera were stored at -80°C. The following analyses were conducted at Trondheim University Hospital: 25(OH)D and PTH by electrochemiluminescence immunoassay (ECLIA), calcium by a colorimetric method, and magnesium, phosphate, albumin and creatinine by photometric methods. All assays were delivered by Roche Diagnostics Ltd., Switzerland. Total calcium was corrected for the albumin concentration . DBP and 1,25(OH)2D were analyzed at the Hormone Laboratory, Oslo University Hospital; DBP by an in-house competitive radioimmunoassay with GC-globulin (Sigma-Aldrich Corp, St. Louis, MO, USA) and polyclonal anti-GC-globulin antibodies (DakoCytomation, Glostrup, Denmark), and 1,25(OH)2D by an enzyme immunoassay (IDS Nordic A/S immunodiagnosticsystems) [39, 40]. Reference range, limit of detection and coefficient of analytical variation (CV) for the different analyses are presented in S1 Table.

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We compared two methods reported by Vermeulen et al. and Bikle et al. for calculation of free 25(OH)D, and the estimates were similar (difference of ~1%) [41–43]. Free 25(OH)D and 1,25(OH)2D are presented according to Bikle et al. [41, 42, 44]: Dfree=Dtotal(1+([bindingconstantalbumin]×albumin)+([bindingconstantDBP]×DBP))

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The participants were recruited consecutively, and clinical data and blood samples were collected in second and third trimester (pregnancy week 18–22 and 32–36) . Questionnaires regarding sociodemographic variables, diet and supplements, childbirths, medical history, smoking behavior and physical activity were completed. A sub-analysis of circulating 1,25(OH)2D was performed in 250 women from Trondheim. To ensure that participants with both low and high levels of 25(OH)D were included in the subgroup analysis of 1,25(OH)2D, the serum levels of 25(OH)D in third trimester were divided into five categories: ≤30 nmol/L, 25(OH)D >30 to ≤50 nmol/L, 25(OH)D >50 to ≤75 nmol/L, 25(OH)D >75 to ≤100 nmol/L and 25(OH)D >100 nmol/L. From each category of 25(OH)D, 50 women were sampled for analysis of 1,25(OH)2D in second and third trimester. We have applied probability weights (the inverse of the probability of an observation being selected into the sample) in the statistical analyses of 1,25(OH)2D to produce estimates representative of the total Trondheim population [33, 34]

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A self-administered optical mark readable Food Frequency Questionnaire (FFQ) containing around 180 food items was used to collect information about vitD and calcium intake [35, 36]. The serving size alternatives were specified in household units, and calculated in grams using a software developed at the Institute for Nutrition Research, University of Oslo . The FFQ was established for dietary surveys in the general Norwegian population age 16 to 67 years, and has been validated [35, 37].

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The binding constant was 6 x105 M-1 between 25(OH)D and albumin, and 5.4 x 104 M-1 between 1,25(OH)2D and albumin. The binding constant was 7 x 108 M-1 between 25(OH)D and DBP, and 3.7 x 107 M-1 between 1,25(OH)2D and DBP [41, 42, 44]. Bioavailable 25(OH)D was calculated as the sum of albumin-bound and free 25(OH)D . The percentage of free 25(OH)D was estimated as : (free25(OH)Dtotal25(OH)D)x100

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