s2fieldsofstudy: Biology Psychology

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Acute Down-regulation of BDNF Signaling Does Not Replicate Exacerbated Amyloid-β Levels and Cognitive Impairment Induced by Cholinergic Basal Forebrain Lesion

Degeneration of basal forebrain cholinergic neurons (BFCNs) precedes hippocampal degeneration and pathological amyloid-beta (Aβ) accumulation, and underpins the development of cognitive dysfunction in sporadic Alzheimer’s disease (AD). We hypothesized that degeneration of BFCNs causes a decrease in neurotrophin levels in innervated brain areas, which in turn promotes the development of Aβ pathology and cognitive impairment. Here we show that lesion of septo-hippocampal BFCNs in a pre-symptomatic transgenic amyloid AD mouse model (APP/PS1 mice) increases soluble Aβ levels in the hippocampus, and induces cognitive deficits in a spatial memory task that are not seen in either unlesioned APP/PS1 or non-transgenic littermate control mice. Furthermore, the BFCN lesion results in decreased levels of brain-derived neurotrophic factor (BDNF). However, viral knockdown of neuronal BDNF in the hippocampus of APP/PS1 mice (in the absence of BFCN loss) neither increased the level of Aβ nor caused cognitive deficits. These results suggest that the cognitive decline and Aβ pathology induced by BFCN loss occur independent of dysfunctional neuronal BDNF signaling, and may therefore be directly underpinned by reduced cholinergic neurotransmission.

INTRODUCTION
Alzheimer's disease (AD) is histopathologically characterized by the accumulation of two proteins: extracellular deposits of amyloid-β (Aβ) that form amyloid plaques, and intracellular inclusions of the microtubule-associated protein tau that give rise to neurofibrillary tangles (NFTs). In conjunction with this pathology, post-mortem assessment of the brains of AD patients has shown that selective degeneration of the cholinergic neurons in the basal forebrain is a significant pathological feature of the sporadic form of the disease and likely underpins aspects of cognitive decline (Mesulam, 2004;Contestabile, 2011;Schliebs and Arendt, 2011). Consistent with this, modern structural magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging of amyloid have demonstrated that significant basal forebrain volume loss occurs in prodromal stages of the disease (Hall et al., 2008;Grothe et al., 2013;Kerbler et al., 2015), and that this degeneration correlates with and predicts subsequent cognitive decline, cortical Aβ burden and hippocampal degeneration (Grothe et al., 2010;Kerbler et al., 2015;Schmitz and Nathan Spreng, 2016). Transgenic mice that model specific aspects of AD have been widely used to understand the etiology of amyloidogenesis and the development of NFTs. As early degeneration of basal forebrain cholinergic neurons (BFCNs) does not occur in AD mouse models (Perez et al., 2007), an immunotoxin that selectively targets cholinergic neurons through their expression of the p75 neurotrophin receptor (p75 NTR ), the anti-p75 NTR antibody-conjugated ribosome-inactivating toxin (p75-saporin), has been used to induce BFCN lesions and investigate the subsequent downstream effects. BFCN lesioning in mice that are genetically programmed to overproduce Aβ exacerbates amyloid and tau pathology and increases the associated cognitive deficits (Gil-Bea et al., 2012;Laursen et al., 2013;Ramos-Rodriguez et al., 2013;Hartig et al., 2014). However, an exacerbation in tau pathology is not seen following BFCN lesioning in tau-only transgenic mice (Turnbull and Coulson, 2017), indicating that this pathology is independent of cholinergic degeneration, and is likely driven by the induced Aβ accumulation. However, it remains unclear how reduced cholinergic innervation results in an increase in Aβ in the cortex and/or hippocampus.
We and others have reported that reduced cholinergic innervation results in decreased hippocampal brain-derived neurotrophic factor (BDNF) expression (Gil-Bea et al., 2011;Turnbull and Coulson, 2017). Furthermore, in human AD post-mortem tissue, BDNF mRNA and protein, as well as its precursor form, pro-BDNF, its receptor tropomyosin receptor kinase B (TrkB) and activation of downstream signaling molecules such as cAMP response element binding (CREB) protein, are all decreased (Phillips et al., 1991;Yamamoto-Sasaki et al., 1999;Holsinger et al., 2000;Siegel and Chauhan, 2000;Peng et al., 2005). A reduction in the serum BDNF level also correlates with an increase in the severity of dementia (Laske et al., 2006(Laske et al., , 2011Yasutake et al., 2006). These findings implicate reduced trophic signaling in AD pathogenesis; however whether this effect is causative or a consequence of the disease is unclear.
Reduced BDNF and nerve growth factor (NGF) signaling can induce amyloidogenic processing of amyloid precursor protein (APP), which results in the apoptotic cell death of cultured hippocampal neurons (Matrone et al., 2008). Furthermore, enhancing BDNF signaling in vitro can reduce Aβ production or enhance the clearance  and dephosphorylation of tau protein (Elliott et al., 2005). However, genetic down-regulation of BDNF (heterozygous gene knockout) in AD mouse models does not result in exacerbated AD-like pathology (Castello et al., 2012), although these mice harbor BDNF deficits throughout their entire lives and may develop compensatory mechanisms that overcome the effect of reduced BDNF (Rantamäki et al., 2013). Therefore the effect of acute neurotrophic dysfunction, as might occur in conjunction with BFCN denervation in an AD mouse model, is yet to be studied.
In order to test the role of impaired cholinergic and neurotrophic signaling in the development of AD, we compared the effect of lesioning BFCNs with that of an acute reduction in BDNF expression in the hippocampus.

Animals
All experiments were approved by the University of Queensland Animal Ethics Committee, in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Mice were housed on a 12 h dark/light cycle with water and standard chow available ad libitum.

APP/PS1 Transgenic Mice
The APP/PS1 mice (Jax Mice Database strain B6C3 Tg (APPswe, PSEN1dE9) 85Dbo/J; stock number #004462) express human APP695 with Swedish mutations (K595N and M596L) and mutant human presenilin 1 carrying the exon-9-deleted variant (Jankowsky et al., 2004). Hemizygous APP/PS1 transgenic males aged 5 months were used, together with age-matched non-transgenic littermate controls. Only male mice were used in these studies as gender differences in plaque deposition for this transgenic line have been reported (Burgess et al., 2006).

Tau Knockout Transgenic Mice
A single homozygotic male tau knockout mouse (Tau KO; Jax Mice Database strain Mapttm1 (EGFP) Klt; stock number #004779) was used as a negative control for tau expression in western blotting experiments.
Two groups of mice underwent stereotaxic injection of AAV-Synapsin-Cre-GFP into the hippocampus, one group at 5 months of age and another at 7 months of age. All mice were analyzed 2 months post-viral infusion for BDNF protein and total Aβ levels, hippocampal amyloid plaque load, and behavioral parameters in a Y maze test. As no differences in any parameter were found between the mice of different ages, all data presented in the results are from pooled groups.

Tissue Preparation
Mice were sacrificed either by sodium pentobarbital (100 mg/kg i.p.) overdose followed by transcardial perfusion of 4% paraformaldehyde (PFA) or by cervical dislocation. Tissue that was to be assessed by biochemical methods was dissected out from the freshly isolated brain and snap frozen in liquid nitrogen. The entire hippocampus of BFCN-lesioned mice was used for biochemical analysis, whereas for BDNF fl/wt mice, only the anterior hippocampus containing the AAV-infected GFP-positive tissue was used. Brain areas to be assessed histologically were post-fixed in 4% PFA overnight at 4 • C, and then left for 24 h in phosphate-buffered saline (PBS) containing 30% sucrose. The following day, the tissue was embedded in Frozen Section Compound (FSC22; Leica) and basal forebrain and hippocampal sections were cut in the coronal plane (40 µm) using a sliding microtome (SM2000r, Leica). All sections used for comparative analysis were processed, stained and analyzed together.
Alternatively, hippocampal and cortical Aβ plaques were identified by Thioflavin-S staining. The sections were incubated in PBS-Triton X-100 with DAPI (1:4000; Thermo Scientific) and then washed in PBS-Triton X-100 before incubation in the dark for 5 min in twice-filtered 0.1% Thioflavin-S solution. The slides were then washed in 70% ethanol followed by distilled water to remove excess stain, after which they were allowed to dry before being coverslipped with fluorescence mounting medium (Dako).
Fluorescence and bright-field microscopy were performed using a fluorescence slide scanner (Axio, Zeiss) and a bright-field slide scanner (Axio, Zeiss), respectively.

Quantification of Immunohistochemistry
The numbers of ChAT-and parvalbumin-positive neurons in the basal forebrain were counted for each animal, as per Boskovic et al. (2014). Briefly, anatomically matched sections (10 coronal sections per animal) containing the medial septum and diagonal bands of Broca were analyzed (beginning at 1.18 mm anterior to Bregma). Bilateral ChAT-and parvalbuminlabeled cells were counted manually within Imaris 7.2.3 software (Bitplane), and statistics were performed on the average of the total cells counted for each group using Graphpad Prism 6.
Quantification and measurements of amyloid plaques were obtained using a custom pipeline in CellProfiler, and the hippocampal area was calculated in Fiji (ImageJ). For each animal, every third hippocampal section (10 sections per animal) was counted, starting from the beginning of the hippocampus (1.06 mm posterior to Bregma).

Measurement of Aβ and BDNF by ELISA
The levels of human Aβ 42 (Invitrogen) and mouse BDNF (Biosensis) in supernatant prepared from hippocampal homogenates were measured using commercial ELISA kits.
Human Aβ 42 levels were measured following extraction from hippocampal lysate. Briefly, hippocampal homogenate was homogenized in 5 M guanidine-HCl (diluted in 50 mM Tris, pH 8.0) as per the manufacturer's instructions (Invitrogen). A bicinchoninic acid (BCA) assay (Thermo Scientific) to measure the total protein content was performed to ensure that the soluble lysate concentrations were within the assay range and could be standardized. The solid phase sandwich Aβ 42 ELISA was performed according to the manufacturer's instructions (Invitrogen) using a monoclonal antibody specific for the NH2-terminus of human Aβ and a rabbit antibody specific for the COOH-terminus of the 1-42 Aβ sequence, with the resultant measurements (pg) being normalized per mg of total soluble protein.
To measure BDNF levels, soluble proteins were extracted using an acid extraction protocol as per the manufacturer's instructions. Briefly, hippocampi were suspended in 20 volume/weight extraction buffer (0.05 M sodium acetate, 1 M sodium chloride, 1% Triton-X100, Roche complete inhibitor cocktail tablet) and homogenized. A BCA assay (Thermo Scientific) was performed, followed by an ELISA for BDNF according to the manufacturer's instructions (Biosensis). The resulting measurements (pg) were normalized per mg of total soluble protein.

Sequential Extraction of Tau Protein
To measure the levels of tau and hyperphosphorylated tau epitopes, soluble (in reassembly buffer (RAB)) and insoluble (RIPA) fractions of hippocampal homogenate were sequentially extracted as previously described (Probst et al., 2000). Briefly, hippocampi were suspended in 10 volumes/weight ice-cold RAB buffer (0.01 M MES, 1 mM EGTA, 0.5 mM MgSO 4 , 0.75 M NaCl, 0.02 M NaF, 1 mM Na 3 VO 4 , 1 mM PMSF) containing Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche), and homogenized. Samples were centrifuged at 21,000 g for 90 min at 4 • C and the supernatant was extracted as the RAB fraction. The remaining pellet was resuspended and homogenized in the same volume of ice-cold RIPA buffer (Cell Signaling) with 0.02 M NaF, 1 mM Na 3 VO 4 , 1 mM PMSF, Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP Phosphatase Inhibitor Cocktail (Roche). Samples were centrifuged at 21,000 g for 90 min at 4 • C and the supernatant was extracted as the RIPA fraction. A BCA assay (Thermo Scientific) was performed in order to measure the total protein content for each sample from each fraction to ensure that changes in tau were comparable across samples.
Levels of ChAT, and phosphorylated and total levels of the TrkB receptor were quantified from hippocampal homogenates by western blot analysis as described above. The following primary antibodies were used: goat anti-ChAT (1:1000; Invitrogen), rabbit anti-phosphoTrkB (phospho S478; 1:1000; Biosensis), goat anti-TrkB (1:1000; R&D Systems) and the loading control mouse anti-GAPDH (1:4000; Cell Signaling). Imaging and analysis were performed as described above.

Y Maze
To measure short-term working memory, mice were tested in a two trial forced Y maze paradigm. The Y maze consisted of an elevated Y-shaped maze in clear plexi-glass with arms that were 40 cm long, 9 cm wide and 21 cm high. A removable barrier was used to shut off a maze arm of choice (randomized across genotype) during habituation. Extra-maze visual cues were placed around the room and the maze was wiped with ethanol after every trial to remove odor cues. The test was divided into two phases: a 5 min habituation in which mice could freely explore two arms of the maze, a 20 min interphase interval in the home cage, and a 5 min test phase in which all three arms were accessible.
In these tests, no group of mice showed a significant preference for the novel arm over the previously explored arms in terms of total time spent in the arm or their first entry choice (data not shown). A subgroup of 10 (five per condition) APP/PS1 transgenic mice were tested 1 month post-BFCN lesion in the Y maze (another group of 16 mice were tested in the Morris water maze, see below). As one mouse spent a considerable portion of the habituation trial immobile, its data were not included in the subsequent analysis. The APP/PS1 × BDNF fl/wt mice were tested at 7 or 9 months of age, 2 months after AAV injection. Data were collected and analyzed using Ethovision XT (Noldus).

Morris Water Maze
Spatial memory was assessed using the Morris water maze as previously described (Turnbull and Coulson, 2017). Briefly, a subgroup of APP/PS1 mice were placed in a circular pool filled with opaque water with four virtual quadrants indicated by cues mounted on the surrounding walls. The mice underwent a 5 day acquisition phase in which they were released from three different start positions per day, with 20 min intervals between each training run. On the 6th day, the mice were subjected to a probe trial (in which the platform was removed) to test spatial memory retention. To investigate BFCN-mediated spatial learning, the platform was moved to a new location and the mouse's ability to ''update'' its memory of the platform location was assessed over the 7th and 8th days (reversal task). A probe trial was again performed on the 9th day.
Latency was measured as the time from when the mouse was placed in the water until it had remained in the arena for a maximum of 60 s or until it had remained on the platform for a total of 10 s. Data were collected and analyzed using Ethovision XT (Noldus).

Statistics
All data are expressed as mean ± SEM. Statistical tests, including appropriate post hoc analysis, were performed for each result and are reported in the figure legends. The significance threshold was set at p < 0.05 and analyses were performed using Graphpad Prism 6.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

p75-Saporin Toxin Injection into the Basal Forebrain Selectively Lesions Cholinergic Neurons
In order to selectively ablate the cholinergic septo-hippocampal pathway, a single injection of p75-saporin, or its non-specific IgG-saporin control, was stereotaxically injected into the medial septum of 5 month old APP/PS1 transgenic mice (Figures 1A,B). As p75 NTR expression is restricted to cholinergic neurons in the basal forebrain, endocytosis of the toxin induces the selective death of cholinergic neurons. One month post-injection, the basal forebrain was assessed for cholinergic neuron loss by counting the number of ChAT-positive neurons in histologic sections ( Figure 1C). p75-saporin injection resulted in a 40% loss of BFCNs (Figure 1D). The selectivity of the toxin was verified by counting the number of parvalbumin-positive neurons. As expected, no change in the number of these neurons, which do not express p75 NTR , was observed ( Figure 1E). The 40% loss of cholinergic neurons caused by the lesion was sufficient to reduce cholinergic innervation to the hippocampus, as western blot analysis of hippocampal homogenates from lesioned animals revealed a significant decrease in the level of ChAT protein ( Figure 1F).

Memory Retention in a Morris Water Maze Task Is Impaired Following Basal Forebrain Cholinergic Neuron Lesion in APP/PS1 Mice
To determine if BFCN lesions in APP/PS1 transgenic mice accelerated cognitive decline, we first tested 6 month old mice for working memory in the Y maze 1 month post-injection of IgG-or p75-saporin into the basal forebrain. Although there was no significant difference in the distance each group of mice traveled (Figure 2A), BFCN lesioned mice displayed a significant reduction in spontaneous entries into alternate arms of the maze compared to non-lesioned mice (Figure 2B), indicating a degree of hippocampal dysfunction. Frontiers in Molecular Neuroscience | www.frontiersin.org FIGURE 2 | Memory retention is impaired in a Morris water maze task following BFCN lesion in APP/PS1 mice. Graphs of total distance traveled (A) and number of maze arm alternations (B) of basal forebrain-lesioned (p75-saporin) APP/PS1 mice and their non-lesioned (IgG-saporin) transgenic controls (mean ± SEM, * * * p < 0.001, unpaired t-test). Graph of the escape latency (time to find the hidden platform) averaged per day in an 8 day Morris water maze task of spatial learning of (C) basal forebrain-lesioned (p75-saporin) APP/PS1 mice and their non-lesioned (IgG-saporin) transgenic controls, and (D) basal forebrain-lesioned (p75-saporin) non-lesioned (IgG-saporin) and naïve non-transgenic littermates of the same age. A probe trial in which the platform was removed was performed on day 6. The platform location was then changed for testing on days 7 and 8. Two-way analysis of variance (ANOVA) with Tukey's post hoc test, * p < 0.05. (E) Quantification of time spent in the target quadrant, (F) latency to the platform, and (G) platform crossing frequency during the probe trial on day 6, between basal forebrain-lesioned and non-lesioned APP/PS1 transgenic mice and basal forebrain-lesioned, non-lesioned, and naïve non-transgenic littermate controls. (H) Quantification of velocity during the acquisition stage. Data are presented as mean ± SEM with sample size indicated in the bars. One-way ANOVA with Tukey's post hoc, * p < 0.05, ns, not significant.
Although APP/PS1 transgenic mice display obvious cognitive impairments by 12 months of age, at 6 months of age their performance is comparable to that of wild-type mice in the hippocampal-dependent spatial learning task, the Morris water maze (Lalonde et al., 2005;Volianskis et al., 2010). To test for changes in long-term memory in the Morris water maze paradigm, we compared the task performance of IgGand p75-saporin-injected APP/PS1 transgenic mice with age-, gender-, and treatment-matched non-transgenic littermates, together with naïve (no stereotaxic surgery) non-transgenic mice. During the 5 day acquisition phase of the task, all experimental groups demonstrated learning as measured by a significant decrease in the time taken to reach the hidden escape platform. This indicates that BFCN lesioning in both non-transgenic and APP/PS1 transgenic mice does not impact learning acquisition at this age (Figures 2C,D). However, in the probe trial, BFCN-lesioned APP/PS1 mice spent less time in the target quadrant, and made fewer crosses over the platform location than non-lesioned APP/PS1 mice or their non-transgenic littermates, regardless of BFCN lesion status (Figures 2E-G).
On days 7 and 8, all experimental groups underwent a reversal test where the platform location was changed. Non-transgenic littermates and APP/PS1 transgenic mice that had BFCN lesions took longer to locate the platform than unlesioned and naïve mice (Figures 2C,D). This is consistent with previous reports in which ''updating'' of memory is linked to basal forebrain function (Tait and Brown, 2008;Al-Onaizi et al., 2017). Thus, a deficit in reversal task learning serves as an internal control for an effective BFCN lesion. All experimental mice traveled equivalent distances in the maze and there were no obvious differences in velocity (Figure 2H), ruling out altered motor function as an explanation for the above results. Taken together, these results indicate that the combination of the APP/PS1 genotype and the BFCN lesion resulted in an impairment of the cognitive processes required for optimal memory performance in the Morris water maze and Y maze tasks.

Total Aβ 42 Levels in the Hippocampus Increase Following Basal Forebrain Cholinergic Neuron Lesion
The APP/PS1 transgenic mouse line carries human APP and PS1 (presenilin 1) transgenes that harbor mutations associated with early onset AD, as a result of which Aβ (particularly the aggregation-prone longer form, Aβ 42 ) is overproduced and accumulates as plaques in the brains of these animals (Jankowsky et al., 2004). To determine if cholinergic denervation exacerbated this accumulation of Aβ, we lesioned BFCNs in APP/PS1 transgenic mice and measured the Aβ level in FIGURE 3 | Total Aβ 42 levels increase in the hippocampus following BFCN lesion. (A) Quantification of total Aβ 42 levels by ELISA from hippocampal lysates of control and basal forebrain-lesioned APP/PS1 mice. Quantification of (B) Aβ plaque number with Thioflavin S staining per µm 2 and (C) plaque size across the complete hippocampus of basal forebrain-lesioned and control mice. Loss of BFCNs had no effect on plaque number or size in the hippocampus but resulted in an increase in total Aβ 42 levels in the hippocampus. Data are presented as mean ± SEM, with sample size indicated in the bars. Unpaired t-test, * p < 0.05. (D) Representative fluorescence images of amyloid plaques (visualized with Thioflavin S staining) in the hippocampus of APP/PS1 transgenic mice with IgG-saporin injection (control) or p75-saporin injection (lesion) into the basal forebrain. Plaques are indicated by white arrows and a magnified view of a plaque is visible in the top right corner. FIGURE 4 | Tau phosphorylation does not change in the hippocampus following BFCN lesion. (A) Representative western blots of the soluble tau fraction of hippocampal homogenates from naïve tau knockout mice, and naïve, control (IgG-saporin) and basal forebrain-lesioned (p75-saporin) APP/PS1 mice probed for the pan-tau marker Tau5 and the tau hyperphosphorylation epitopes AT8, AT180, AT270, pSer235, pSer262 and pSer422. Each tau band is compared to its respective GAPDH loading control band. (B) Quantification of tau protein bands from the soluble tau fraction. No significant differences (two-way ANOVA) in hyperphosphorylated tau epitopes were observed between naïve, control and basal forebrain-lesioned APP/PS1 mice. Bands were normalized to the GAPDH loading control levels and the levels are plotted relative to those of control APP/PS1 mice. Data are presented as the mean ± SEM, with the sample size indicated in the bars.
hippocampal homogenates by ELISAs, as well as determining the size and number of Aβ plaques in histological sections. Our results revealed an increase in total Aβ 42 levels in the hippocampal lysates following BFCN lesion that was not observed in IgG-saporin-injected mice ( Figure 3A). However, this increase in total Aβ 42 was not reflected in hippocampal amyloid plaque number or size (Figures 3B-D). Indeed there were only rare amyloid deposits in the hippocampus of these 6 month old mice ( Figure 3D). The size and number of cortical plaques was also determined, but again, no changes were observed following BFCN lesion (data not shown). These results suggest that cholinergic degeneration exacerbates the production of soluble Aβ 42 , which has not yet accumulated and been deposited into plaques. Ramos-Rodriguez et al. (2013) have previously reported an increase in a single hyperphosphorylated tau epitope in the cortex following BFCN lesion of APP/PS1 transgenic mice. To assess whether tau pathology was also exacerbated in APP/PS1 transgenic mice following loss of BFCNs, we performed sequential extraction of tau protein and measured the levels by western blot analysis of hippocampal homogenates. The levels of total tau and a range of tau phosphorylation epitopes that are considered to be markers of pathological hyperphosphorylation were assessed. However, no changes in the levels of pan-tau or a variety of phospho-tau epitopes in the soluble (Figures 4A,B) and insoluble (data not shown) fractions were observed following BFCN lesion of the transgenic mice.
FIGURE 5 | Brain-derived neurotrophic factor (BDNF) protein and TrkB receptor signaling are reduced following BFCN lesion. (A) Quantification of BDNF protein levels by ELISA from hippocampal lysates of naïve wild-type mice and control and basal forebrain-lesioned APP/PS1 mice. Loss of BFCNs resulted in a decrease in the BDNF protein level. (B) Representative western blots of hippocampal homogenates from control (IgG-saporin) and basal forebrain-lesioned (p75-saporin) APP/PS1 mice probed for phosphorylated TrkB (pTrkB) and total TrkB. Each band is compared to its respective GAPDH loading control band. (C) Quantification of pTrkB and TrkB bands expressed as a percentage of the levels in control mice. Loss of the cholinergic projection to the hippocampus resulted in a reduction in phosphorylated/active TrkB receptor. Data are presented as mean ± SEM, with the sample size indicated in the bars. One-way ANOVA, * * p < 0.01, * * * p < 0.001.

A Reduction in BDNF Protein and TrkB Receptor Signaling Are Observed Following Basal Forebrain Cholinergic Neuron Lesion
To test whether reduced cholinergic innervation to the hippocampus following BFCN lesion affected neurotrophic signaling, the level of BDNF protein was measured by ELISA, while TrkB receptor activation was measured by western blot analysis. BFCN lesion in APP/PS1 transgenic mice resulted in a reduction in the level of BDNF protein in the hippocampus ( Figure 5A). This decrease was confirmed by a reduction in the level of phosphorylated TrkB, reflecting reduced activation of the receptor (Figures 5B,C).

A Reduction in the Anterior Hippocampal BDNF Level Does Not Affect Working Memory
To test whether the effects seen following BFCN lesioning were due to the reduction in hippocampal BDNF, we reduced BDNF expression to a level equivalent to that observed in BFCN-lesioned mice by injecting a neuron-specific AAV-Synapsin-Cre-GFP bilaterally into the anterior hippocampus of 5 or 7 month old APP/PS1 × BDNF fl/wt mice (Figures 6A,B). Six weeks after injection of the virus, we assessed its spread by staining histological sections with an anti-GFP antibody, revealing labeling throughout the CA1/CA2 subregions of the anterior hippocampus, but not in the dentate gyrus ( Figure 6C). The BDNF protein levels in homogenates of the anterior hippocampus of virus-injected animals were measured by ELISA, revealing a significant decrease compared to saline-injected controls and naïve animals ( Figure 6D). To assess whether this had an effect on memory, mice were assessed in the Y maze 2 months after their surgery. No difference in the total distance traveled or alternation was observed between saline-and virusinjected APP/PS1 × BDNF fl/wt mice (Figures 6E,F).

Reduction of Anterior Hippocampal BDNF Levels Do Not Alter Aβ Levels or Affect Amyloid Pathology
We next determined if the reduced hippocampal BDNF protein levels (in the absence of coincident cholinergic dysfunction) induced an increase in hippocampal Aβ 42 levels in APP/PS1 × BDNF fl/wt mice injected with AAV-Synapsin-Cre-GFP. There was no change in plaque number or size in either the area of the hippocampus infected with virus, as indicated by immunostaining for GFP, or in non-infected areas (Figures 7A-C). The total Aβ 42 levels were also unchanged in lysates of the anterior hippocampus between mice injected with AAV-Synapsin-Cre-GFP or saline ( Figure 7D). These results suggest that, although BDNF signaling is reduced following BFCN lesion in APP/PS1 mice, reduced BDNF levels alone do not exacerbate the accumulation of Aβ.

DISCUSSION
Degeneration of BFCNs is an early and core feature of sporadic AD that underpins aspects of cognitive deterioration and contributes to disease progression (Grothe et al., 2010;Kerbler et al., 2015;Schmitz and Nathan Spreng, 2016). In genetically susceptible mouse models, inducing cholinergic degeneration can exacerbate the production and/or accumulation of Aβ, and cause cognitive impairment (Gil-Bea et al., 2012;Laursen et al., 2013;Ramos-Rodriguez et al., 2013;Hartig et al., 2014), as well as resulting in reduced levels of BDNF, another feature of AD (Egan et al., 2003;Tapia-Arancibia et al., 2008;Fahnestock, 2011). However, the etiological relationship between lowered BDNF levels and (C) Representative fluorescence image of a hippocampal cross-section demonstrating the spatial distribution of the virus carrying a GFP tag (in green) after stereotaxic injection into an APP/PS1 × BDNF fl/wt mouse. (D) Quantification of the BDNF protein levels (pg/mg total protein) by ELISA from anterior hippocampal lysates of naïve (gray bars), saline-injected and virus-injected APP/PS1 × BDNF fl/wt mice. Stereotaxic injection of AAV-Synapsin-Cre-GFP into the hippocampus of mice carrying a floxed BDNF allele resulted in a decrease in BDNF protein. One-way ANOVA, * * p < 0.01. Graphs of total distance traveled (E) and the number of maze arm alternations (F) of APP/PS1 × BDNF fl/wt mice injected with a saline control or AAV Synapsin-Cre virus (unpaired t-test), ns, not significant.
Aβ accumulation are unclear. Here we show that although BFCN lesions in adult mice induce reduced neurotrophin signaling, this loss of neuronal BDNF is unlikely to be directly responsible for the coincident increase in Aβ accumulation.
We report that loss of cholinergic neurons in young presymptomatic Aβ-overproducing mice triggers an increase in the level of soluble Aβ 42 in the hippocampus, a post-synaptic target of BFCNs, causing cognitive dysfunction in two hippocampal-dependent spatial navigation tasks. This finding is consistent with previous reports which demonstrated exacerbation of Aβ pathology and cognitive impairment in AD model mice (Gil-Bea et al., 2012;Laursen et al., 2013;Ramos-Rodriguez et al., 2013;Hartig et al., 2014). However, the memory deficits we observed in the probe trial of the Morris water maze 1 month after BFCN lesioning of 5 month old APP/PS1 mice occurred at an earlier age than previously reported, with Ramos-Rodriguez et al. (2013) describing deficits in 7 month old mice that had harbored BFCN lesions for 4 months. In both studies, BFCN lesions in wild-type mice did not cause equivalent memory deficits, indicating that cholinergic denervation (including BDNF down-regulation) alone is insufficient to impair memory retention. The most likely explanation for the observed memory deficits in the APP/PS1 BFCN-lesioned mice is the increase in soluble Aβ levels observed in our study (this was not measured in the Ramos-Rodriguez et al.'s (2013) study). This is also a possible explanation for the Y maze alternation deficits which were observed in lesioned but not APP/PS1 × BDNF fl/wt mice. However, similar phenotypes have been reported not only for AD model mice but in situations of cholinergic neurotransmission impairment and reduced BDNF levels (Kokkinidis and Anisman, 1976;Tempier et al., 2013;Chen et al., 2016). As the reduced BDNF may have contributed to the Aβ-induced cognitive impairment in the Morris water maze (Rantamäki et al., 2013), we asked whether the decrease in BDNF was required for the rise in Aβ levels.
Dysfunctional neurotrophic signaling is a feature of AD and a risk factor for cognitive decline (Peng et al., 2005;Fahnestock, 2011). In particular, the BDNF polymorphism resulting in a Val66Met substitution, which reduces the activity-dependent neuronal secretion of BDNF (Egan et al., 2003), accounts for up to 30% of the variability in cognitive decline in AD patients (Lim et al., 2013(Lim et al., , 2015. In our experiments, the levels of hippocampal BDNF protein and TrkB receptor signaling were reduced following BFCN lesion, consistent with previous studies showing that a complete lesion of BFCNs in rats results in reduced BDNF mRNA in the hippocampus (Kokaia et al., 1996;Ferencz et al., 1997) and that cholinergic basal forebrain innervation of the hippocampus is involved in the regulation of BDNF protein and mRNA expression (Lapchak et al., 1993;Gil-Bea et al., 2011). However, despite achieving a significant decrease in hippocampal BDNF level in APP/PS1 × BDNF fl/wt mice injected with a synapsin Cre recombinase-carrying AAV, equivalent to that of a BDNF heterozygous animal, we did not observe an exacerbation in Aβ pathology or an increase in the level of Aβ.
Although in our study only neuronal BDNF was reduced, previous in vivo studies have reported that heterozygous BDNF;APP/PS1 mice display no alteration in amyloid plaque formation at 9 months of age (Rantamäki et al., 2013). Similarly, the levels of Aβ and tau pathology in both heterozygous BDNF;3XTg-AD mice and TrkB;3XTg-AD mice are reported to be comparable to those of transgenic controls (Castello et al., 2012). Why might we expect a different result in our experiments when BDNF is lost only from neurons? Loss of the BDNF gene from conception could lead to compensation; despite the 43% reduction in cortical BDNF protein reported by Castello et al. (2012) and Rantamäki et al. (2013), the authors also described an age-dependent increase in mature BDNF protein, with BDNF mRNA and phospho-TrkB levels being unaltered in the cortical tissue of the APP/PS1 mice. Acute loss of BDNF, such as occurs following BFCN lesion or damage, may therefore trigger different effects. In vitro, Matrone et al. (2008) demonstrated that complete withdrawal of BDNF from hippocampal neurons using a blocking antibody resulted in increased Aβ production. Therefore the acute change in BDNF levels and TrkB signaling in our study, equivalent to the BDNF loss that occurs in human AD patients (Tapia- Arancibia et al., 2008), could have induced increased Aβ production; however, we did not observe this. As there is now a considerable body of research, including work in humans (Lim et al., 2013(Lim et al., , 2015, which indicates that neither a chronic nor an acute reduction of neuronal BDNF is sufficient to cause Aβ accumulation in vivo, it is reasonable to conclude that reduced BDNF levels do not directly cause AD. What then is responsible for the increased Aβ levels that we and others have reported? Recent reports indicate that cholinergic deficiency, induced by knockout of the cholinergic vesicle transporter and mediated by reduced M1 muscarinic receptor signaling, could result in changes in BFCN-derived microRNAs and the splicing of the β-site APP cleaving enzyme 1 (BACE1) mRNA and BACE1 overexpression (Fisher, 2012;Kolisnyk et al., 2016Kolisnyk et al., , 2017. In turn, increased BACE activity would favor the amyloidogenic cleavage pathway, raising the level of soluble Aβ 42 . Furthermore, Aβ could be released by degenerating BFCNs, and neurotrophin signaling within BFCNs could control Aβ production. Therefore, there are a number of possible mechanisms by which cholinergic degeneration could lead directly to Aβ generation. We surmise that increased Aβ in combination with reduced BDNF levels results in cognitive dysfunction. In support of this idea, BDNF is known to be beneficial for synaptic and cognitive function, including in the presence of Aβ pathology. For example, Blurton-Jones et al. (2009) demonstrated that transplanted neural stem cell-derived BDNF increased the density of synapses in the hippocampus of 3XTg-AD mice and reversed cognitive deficits without alterations in Aβ or tau pathology. Therefore, although dysfunctional neurotrophin signaling does not appear to directly affect the level of Aβ in vivo, the therapeutic potential of enhancing neurotrophic signaling remains a viable option (Nagahara and Tuszynski, 2011).


RHO Gene Polymorphisms in Patients with Pterygium †

: Pterygium is one of the most common ocular surface diseases, and characterized by inflammatory infiltrates, proliferation, fibrosis, angiogenesis, and extracellular matrix breakdown. We investigated the association of polymorphisms in the RHO genes RHOA , RHOB , RHOC , RHOD , and RND3 ( RHOE ). The results of this study demonstrate for the first time the association of RHO genes with the pterygium. We displayed that the RHO gene polymorphisms were significantly associated with pterygium in the Turkish population.

Introduction
Pterygium, one of the most common eye disorders, is the growth of subepithelial fibrovascular tissue starting from the bulbar conjunctiva onto the cornea [1]. It is characterized by an altered basal epithelial cell proliferation, vascularization, and invasion of the adjacent corneal epithelium. Independent risk factors include age, male sex, rural life, and exposure to sun or ultraviolet (UV) light [2]. The pathogenesis of this disease remains unclear, but evidences suggest that UV light is a major contributor in the formation of pterygia [3]. Genetic and environmental factors are important in the etiology of pterygium. Genetic factors are thought to have a role in the development of pterygium, but the most of the molecular mechanisms leading to pterygium are still unknown. Pterygium formation impairs vision, limits eye movements, and can cause eye irritation, foreign body sensation, and dryness. Currently, surgery is the only effective treatment for pterygium, but recurrences are common [4]. Rho proteins are members of the Ras superfamily of small GTPases that play critical roles in many cellular processes including actin dynamics, vesicular trafficking, gene transcription, cytoskeletal reorganization, cell-cycle progression, differentiation, adhesion, and migration [5]. These proteins also contribute to pathological processes such as cancer cell migration, invasion, metastasis, fibrosis, inflammation, angiogenesis, and wound repair. Rho proteins consist of a family of 20 intracellular signaling molecules including RhoA, RhoB, RhoC, RhoD, and RhoE [6].
Involvement of the genetic variants of RHO genes in pterygium development has not been examined yet. Therefore, the purpose of the present study was to investigate possible associations between RHO gene polymorphisms and pterygium in a Turkish population.

Experimental Procedure
A total of 379 patients, diagnosed as pterygium at the Ophthalmology Clinic of the Gaziantep University Hospital and 324 unrelated healthy controls, were included to this study. Routine ophthalmologic evaluations were performed on all subjects. A control group of age-and sex-matched individuals was chosen randomly from a sample of patients admitted to the ophthalmology outpatient department for routine ophthalmic examinations, refractive errors, blepharitis, conjunctivitis, burning, itching, presbyopia, or senile cataracts. This study was approved by the local Ethics Committee. Peripheral venous blood samples (5 mL) were obtained and collected in sterile siliconized vacutainer tubes with 2 mg/mL disodium ethylenediaminetetraacetic acid. Genomic DNA was extracted from whole peripheral blood samples with salting-out method and stored at −20 °C until analysis [7]. Genomic DNAs were analyzed in all patients and controls using the dynamic array system (Fluidigm, South San Francisco, CA, USA) as previously described [8]. A 96.96 dynamic array on the BioMark HD system (Fluidigm, South San Francisco, CA, USA) was used for detection of polymorphism. The Digital PCR Analysis software (Fluidigm, South San Francisco, CA, USA) was used to process the data after the reaction. In the present study, 15 single nucleotide polymorphisms (SNP) were assayed. For calculation of the significance of differences in allele frequencies, the Chi-square test (with Yate's correction) or Fisher's exact test was used. A statistical comparison of two groups was performed with unpaired Student t-test. The original significance level was set at a p value of 0.05. Bonferroni correction for multiple testing was used for polymorphism studies, and a P value of <0.0033 (0.05/15) was considered statistically significant.

Results
The mean age and gender of the patients (50.9 ± 12.4 years, 51.5% female) and control (52.1 ± 14.7 years, 51.9% female) groups were similar (p > 0.05). Table 1 shows the distributions of genotypes and alleles between the case and control groups. There were marked associations for the genotype frequencies of the RHOA gene rs6784820 polymorphism between the patients and the control group. Both genotype and allele frequencies of RHOB gene rs2602160, rs62121967, rs11541350 SNPs, RHOC gene rs2306937 and rs11102522 SNPs, RHOD gene rs61891303 and rs7112925 SNPs, and RND3 (RHOE) gene rs13418763 and rs1441982 SNPs were significantly associated with pterygium. There were no marked associations with the other 5 RHO polymorphisms (RhoA gene rs2177268, rs2878298 SNPs, RhoC gene rs2999156 SNP, RhoD gene rs3923203 SNP, RhoE gene rs1528428 SNP) studied (Table 1, p > 0.0033).

Discussion
This is the first study to examine the association of the RHO gene polymorphisms with the risk of developing pterygium. In this case-control study, we showed that the RHO gene polymorphisms were significantly associated with pterygium in the Turkish population. Therefore, RHO gene variants could be a risk factor for developing pterygium, and may increase susceptibility to pterygium in Turkish patients.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Medicine

text:
1,25-Dihydroxyvitamin D3 protects obese rats from metabolic syndrome via promoting regulatory T cell-mediated resolution of inflammation

Vitamin D3 has been found to produce therapeutic effects on obesity-associated insulin resistance and dyslipidemia through its potent anti-inflammatory activity, but the precise immunomodulatory mechanism remains poorly understood. In the present study we found that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D3, significantly attenuated monosodium glutamate (MSG)-induced obesity and insulin resistance as indicated by body weight reduction, oral glucose tolerance improvement, and a glucose infusion rate increase as detected with hyperinsulinemic-euglycemic clamp. Moreover, 1,25(OH)2D3 not only restored pancreatic islet functions but also improved lipid metabolism in insulin-targeted tissues. The protective effects of 1,25(OH)2D3 on glycolipid metabolism were attributed to its ability to inhibit an obesity-activated inflammatory response in insulin secretory and targeted tissues, as indicated by reduced infiltration of macrophages in pancreas islets and adipose tissue while enhancing the expression of Tgf-β1 in liver tissue, which was accompanied by increased infiltration of Treg cells in immune organs such as spleen and lymph node as well as in insulin-targeted tissues such as liver, adipose, and muscle. Together, our findings suggest that 1,25(OH)2D3 serves as a beneficial immunomodulator for the prevention and treatment of obesity or metabolic syndrome through its anti-inflammatory effects.

Introduction
Obesity is associated with multiple adverse health outcomes collectively summarized as the "metabolic syndrome", consisting of insulin resistance, dyslipidemia, and cardiovascular diseases 1 . A linking role of inflammation between obesity and metabolic syndrome has been established which mostly refers to the release of various adipocyte products, such as cytokines, fatty acids, and oxygen free radicals from the white adipose tissue in obese individuals 2 . Many of these products function as molecules of damage-associated molecular patterns (DAMPs) to initiate chronic low-degree inflammation to promote the infiltration of adipose tissue with activated macrophages through the activation of pattern reorganization receptors (PRRs) such as Toll-like receptors (TLRs) expressed on immune cells and residual cells 3,4 . Indeed, accumulated evidence has indicated that both obesity and metabolic syndrome are inflammatory disorders and inflammatory responses are causally involved in insulin resistance by ultimately suppressing the insulin signaling pathway 5 .
Epidemiological studies indicate that insufficient vitamin D status is a potential contributor to insulin resistance and obesity [6][7][8] . Data from a pilot study examining vitamin D deficiency in type 1 and type 2 diabetes suggests that vitamin D insufficiency is more common in type 2 diabetes than in type 1 diabetes, unrelated to age, sex, or insulin treatment 9 . However, studies on the administration of vitamin D supplements to vitamin D-sufficient patients with IGT or type 2 diabetes have yielded conflicting results. Some have reported an improvement, others no effect 10 . One study even showed a worsening of type 2 diabetes: supplementation in three British Asians with vitamin D deficiency and type 2 diabetes led to increased insulin resistance and the deterioration of glycemic control 11 . Interestingly, Wortsman et al. 12 reported that obesity-associated vitamin D deficiency is largely because of the decreased bioavailability of vitamin D 3 from cutaneous and dietary sources due to its deposition in body fat compartments. Furthermore, chronic feeding of mice with 1,25(OH) 2 D 3 , the biologically active form, suppressed the inflammatory responses in a variety of animal models including experimental asthma, encephalomyelitis, and rheumatoid arthritis [13][14][15] . The potential immune modulating effects of 1,25(OH) 2 D 3 on the immune system have been initially derived from in vitro observations following the treatment of dendritic cells (DC) with 1,25(OH) 2 D 3 which could inhibit the maturation of myeloid DC via reducing secretion of IL-12 as well as the expression of co-stimulatory molecules. 1,25(OH) 2 D 3 also enhances the secretion of CCL22 by DC in vitro, which is a chemokine that attracts T cells into the skin 16 . IL-10-producing regulatory T cells can be induced in vitro by 1,25(OH) 2 D 3 in the presence of dexamethasone 17 . In addition, 1,25(OH) 2 D 3 can hamper the secretion of IFN-α from T cells and induce Th2 cell development with increased production of IL-4, IL-5, and IL-10 18 . Nonetheless, the immunoregulatory mechanism of 1,25(OH) 2 D 3 on obesity-associated insulin resistance and dyslipidemia is not fully understood.
In this study we have investigated the effects and mechanism of 1,25(OH) 2 D 3 in the regulation of insulin sensitivity and glucolipid metabolism in MSG-obese rats. We found that a short course of treatment with 1,25(OH) 2 D 3 improved insulin resistance and glucolipid metabolism disorder of MSG-obese rats by inhibiting the inflammatory responses in primary insulin-targeted tissues including adipose, liver, and muscle, which is largely due to increasing the infiltration of CD4 þ CD25 þ FoxP3 þ regulatory T-cells in these tissues. Our studies indicate that administration of 1,25(OH) 2 D 3 protects MSG-obese rats from the development of obesity and its related metabolic risks.

Animal model
Wistar rats (newborn) were obtained from Vital River Laboratory Animal Technology (Beijing, China). All animal protocols conformed to the Guidelines for the Care and Use of Laboratory Animals prepared and approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. Newborn Wistar rats were s.c. injected with monosodium L-glutamate (MSG) at 4 g/kg/day for seven successive days as described previously 19 . In contrast to the normal rats, the MSG rats developed obesity with increased plasma TG, cholesterol, and free fatty acid contents as well as impaired insulin sensitivity in their adulthood 20 . Experimental protocols are outlined in Fig. 1. In protocol A (Fig. 1A), the four-week-aged MSG rats were given s.c. injections of 1 μg/kg 1,25(OH) 2 D 3 twice a week for 16 weeks, and insulin tolerance test (ITT) and the euglycemic hyperinsulinemic clamp were assayed at the end of the experiment to evaluate the incidence of insulin resistance. In protocol B (Fig. 1B), obese MSG rats were sorted into three groups according to body weights and fasting plasma glucose values, and then were treated with 1 μg/kg 1,25(OH) 2 D 3 (twice a week), 4 mg/kg/day rosiglitazone or vehicle for 8 weeks.

Oral glucose tolerance test
The OGTT and ITT were carried out as previously described 21 . In brief, after fasting for 6 h on the last treatment day, the animals were administrated an oral dose of 2 g/kg glucose. Blood samples were collected at 0, 30, 60, and 120 min after glucose loading. Blood glucose was analyzed by the glucose-oxidase method, and the area under the curve (AUC) was generated from the data of OGTT.

Biochemical analysis
On the final day blood samples were collected. Serum TG (triglyceride), T-CHO (total cholesterol), and LDL-C (low-density lipoproteins cholesterol) were assayed using enzymatic colorimetric methods provided by commercial kits (Biosino Bio-Technology and Science Inc., Beijing, China). Serum insulin was measured using radio-immunoassay kit (Chinese Institute of Atomic Energy, Beijing, China) according to the manufacturer's instructions.

Clamp experiments in MSG-obese rats
The insulin sensitivity was analyzed using euglycemic hyperinsulinemic clamp technique. Ten rats per group were subjected to this test. The clamp experiments were carried out as previously described 22 . Briefly, rats fasted for 6 h were anesthetized with 45 mg/kg i.p. of pentobarbital. After tracheotomy, the right jugular vein (infusion of insulin and glucose) and left carotid artery (collecting blood sample) were catheterized. Thirty minutes after catheterization, the basal glucose level was tested, and followed by insulin infusion (8 mU/kg/min). At the same time, a variable rate of glucose (10%) was infused and blood glucose levels were measured at 5-min intervals. The steady state (blood glucose levels at 5-6 mmol/L) was reached about 2 h later and sustained for 30 min with constant glucose infusion. Glucose disappearance was represented by average glucose infusion rate (GIR, mM/kg/min) during steady state (the final 30 min of the clamp).

Western blot analysis
Briefly, the homogenized tissue extract (40 mg protein) from the liver was fractionated on a 10% SDS-PAGE gel and the proteins were transferred to PMSF membranes. After blocking with 5% nonfat milk, membranes were incubated with antibodies to pNF-κB, NF-κB, STAT3 and pSTAT3 (Cell Signaling Technology, USA) at 4°C overnight. The membranes were washed and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Enhanced chemiluminescence reagents (Amersham, USA) were employed to visualize the protein bands on the membranes. The densities of specific bands were normalized with the β-actin signal and were quantitated by densitometry using gel-pro software.

Immunolabelling and confocal microscopy
At the end of experiment the liver, pancreas and infrarenal adipose tissues were removed from MSG rats, fixed with 4% polyformalin in phosphate-buffered saline and embedded in paraffin. Liver and adipose tissue sections were stained with hematoxylin-eosin (H&E) and fat cell sizes were measured. The pancreas was fixed in bourin for paraffin embedding and stained with gomori and H&E for analysis of pancreatic β cell mass and islet morphology. Immunostaining was performed using GLUT2 (Bioss, China) and CD68 (BD, USA) antibody. An ABC kit and 3,3-diaminobenzidine (DAB) were used for amplification and staining, respectively. Quantitative analysis of the positive area was performed by image analysis using a computer with Image analyzer (Image Pro-Plus 5.1). Twenty fields were randomly selected from the each section, and the positively stained area was measured and expressed as IOD for each field. Liver, fat and muscle sections were subjected to immunofluorescence for identifying CD4 þ CD25 þ FoxP3 þ Tregs. Tissue sections were incubated with CD4 þ , CD25 þ and FoxP3 þ primary antibodies (Cell Signaling Technology) at 4°C overnight. The sections were washed thrice and then incubated with Alexa488-, Alexa647-or Alexa350-conjugated secondary antibodies (Invitrogen) for 30 min. The colocalization of CD4 (green), CD25 (red) and FoxP3 (blue) were acquired using a confocal microscope FV1000 (Olympus Microsystems) and the Tregs were identified by the coexpression of CD4, CD25 and FoxP3 (white dots).

Figure 1
Experimental protocol for studying the effects of 1,25(OH) 2 D 3 on the development of insulin resistance in MSGobese rats. MSG-obese rats were rendered by s.c. injection of MSG (4 g/kg/day) to newborn Wistar rats for seven successive days. (A) In protocol A, four-week-aged MSG rats were given s.c. injections of 1,25(OH) 2 D 3 (1 g/kg, twice a week) for 16 weeks and an insulin tolerance test (ITT) and the euglycemic hyperinsulinemic clamp were conducted at the end of experiment to measure the incidence of insulin resistance. (B) In protocol B, the obese MSG rats were divided into three groups according to body weights and fasting plasma glucose values and were respectively treated with 1,25(OH) 2 D 3 (1 g/kg, twice a week), rosiglitazone (4 mg/kg/day), or vehicle for 8 weeks.

Flow cytometry analysis
CD4 þ CD25 þ FoxP3 þ Tregs were identified by methods described previously 23 . Briefly, single-cell suspensions from spleens and mesentery lymph nodes were stained with antibodies that recognize the following antigens: CD4, CD25 and FoxP3, which were labeled with FITC, APC or PE (eBioscience, USA), respectively. More than 10,000 events were recorded through a CD4 þ cell gate, and then CD5 þ FoxP3 þ Tregs were determined using a PARTEC CyFlow (Munich, Germany) and data were analyzed using FCS EXPRESS.

Statistical analyses
All values are presented as mean7standard error of mean (SEM). Student's t-test was used for two-group comparisons. Multiple comparisons among three or more groups were performed by oneway ANOVA. Po0.05 or Po0.01was considered statistically significant.

1,25(OH) 2 D 3 protected mice from developing MSG-induced insulin resistance
As obesity is the most important determinant of metabolic syndrome which is characterized by insulin resistance, hypertriglyceridaemia, hypo-HDL-cholesterolaemia and hypertension, we are firstly interested in analyzing whether 1,25(OH) 2 D 3 protected MSG-induced obese rats from insulin resistance. The male and female MSG rats were given an early-life treatment with 1 μg/kg of 1,25(OH) 2 D 3 twice a week from 4 to 20 weeks of age, corresponding to early childhood in humans (Fig. 1A). Using the oral glucose tolerance test, we found that the incidence of insulin resistance in vehicle-treated controls was 61% in male rats and 78% in females. However, only 16% of male rats and 27% of female rats treated with 1,25(OH) 2 D 3 developed insulin resistance ( Fig. 2A). Moreover, 1,25(OH) 2 D 3 increased the glucose infusion rate (GIR) of MSG rats with early-life treatment (Fig. 2B). These data suggest that 1,25(OH) 2 D 3 protects against the development of MSG-induced obesity.

1,25(OH) 2 D 3 improved insulin sensitivity of MSG-obese rats
To determine the potential therapeutic significance of 1,25(OH) 2 D 3 on established obesity, the body weight, glucose/ lipid metabolism, and insulin sensitivity were then chosen to evaluate the efficacy of 1,25(OH) 2 D 3 in the insulin resistant adult MSG rats (Fig. 1B). After an eight-week treatment, the body weight of the 1,25(OH) 2 D 3 -treated MSG rats was significantly lower than that of the vehicle-or rosiglitazone-(a positive control) treated rats (Fig. 3A), and there was no change in serum calcium and serum phosphorus levels (data not shown). As expected, the blood glucose (Fig. 3B) and plasma insulin levels (Fig. 3C) of 1,25(OH) 2 D 3 -treated MSG rats were dramatically decreased compared to the vehicle-treated MSG rats. We further examined the effects of 1,25(OH) 2 D 3 on the improvement of insulin resistance in the MSG-obese rats by conducting OGTT and euglycemichyperinsulinemic clamp tests. In the OGTT assay, the integrated blood glucose (AUC) levels of both 1,25(OH) 2 D 3 and rosiglitazone-treated MSG rats significantly decreased at 2 h after glucose loading (Fig. 3D). Using the euglycemic-hyperinsulinemic clamp, we observed that there was significant insulin resistance in MSG-obese rats, characterized by decreased GIR. However, both 1,25(OH) 2 D 3 and rosiglitazone treatment increased the GIR of MSG rats by 107% and 49%, respectively, compared with the vehicle-treated MSG rats (Fig. 3E). Corresponding to decreased insulin sensitivity, the mRNA expression of Irs-1, which plays an important biological function for glucose uptake and augmenting insulin signaling, was dramatically up-regulated in the 1,25(OH) 2 D 3 -treated MSG rat livers (Fig. 3F). As accumulated evidence has shown that obesity is characterized by both peripheral insulin resistance and impaired insulin secretion, we are interested to know whether 1,25(OH) 2 D 3 could be beneficial to improve MSG-induced dysfunction of pancreatic islets. In the MSG rats the islets showed substantial fibrosis, whereas there was little fibrosis in the 1,25(OH) 2 D 3 -treated MSG rats and the overall morphology of the islets was more regular and rounded (Fig. 3G). GLUT2 is the most important glucose transporter ensuring glucose uptake for most tissues. The suppression of GLUT2 in β-cells is associated with glucose-sensitive insulin secretion and the loss of high-K m glucose transport. We found GLUT2 loss in MSG rats and 1,25(OH) 2 D 3 normalized the expression of GLUT2 (Fig. 3H). All together, these results indicate that 1,25(OH) 2 D 3 administration leads to an improvement of glucose metabolism and insulin sensitivity in MSG-induced obese rats.

1,25(OH) 2 D 3 ameliorated lipid metabolism of MSG-obese rats
As abnormal lipid metabolism is an important consequence and contributing factor in insulin resistance in the pathogenesis of obesity, we investigated the effect of 1,25(OH) 2 D 3 on the regulation of lipid metabolism. We found that 1,25(OH) 2 D 3 treatment significantly reduced the serum TG level of MSG rats (Fig. 4A), but did not change the level of the serum T-CHO and LDL-C ( Fig. 4B and C). We further examined the effect of 1,25(OH) 2 D 3 in liver and adipose tissues, both of which are the most important insulin-targeted tissues. PPAR-α is believed to participate in the regulation of fatty acid uptake and β-oxidation in the liver tissue. The mRNA expression of Ppar-α was significantly down-regulated in the vehicle or rosiglitazone-treated MSG rat 1,25-Dihydroxyvitamin D3 protects obese rats from metabolic syndrome liver, which was significantly up-regulated by 1,25(OH) 2 D 3 (Fig. 4D). Histomorphological analysis of liver tissues using H&E staining revealed severe hepatic steatosis in the MSG rats, characterized by excessive accumulation of fat in hepatic intracellular vesicles. Treatment of the MSG rats with 1,25(OH) 2 D 3 completely eliminated the steatosis. Liver slices from the 1,25(OH) 2 D 3 treated MSG rats were found to be histologically comparable to those from normal rats (Fig. 4E). Additionally, it has been proved that the accumulation of "dysfunctional" adipose tissue characterized by the presence of "large" lipid-laden adipocytes contributes to the insulin resistance of obese individuals. For adipose tissue, MSG induced a dramatic increase in the fat mass and adipocyte size, while this adipocyte hypertrophy was attenuated by 1,25(OH) 2 D 3 treatment ( Fig. 4F and G). All of these data suggest that 1,25(OH) 2 D 3 has a critical role in the regulation of lipid metabolism in liver and adipose tissues, both of which are the primary insulin-targeted tissues involved in the regulation of lipid metabolism.

1,25(OH) 2 D 3 exerted anti-inflammatory effects in MSGobese rats
The macrophage is a major cell-type involved in chronic inflammation, and studies in obese humans and rodents have shown a dramatic activation of macrophages that produce inflammatory factors in pancreatic islets and adipose tissues. MSG-induced dysregulation of glucose and lipid led to high-level expression of CD68, a marker of activated macrophages, in pancreatic islets (Fig. 5A) and adipose tissues (Fig. 5B), indicating more pronounced macrophage infiltration in the insulin-secretory organ and insulin-targeted tissues. In comparison to the MSG rat, treatment with 1,25(OH) 2 D 3 resulted in significant suppression of macrophage infiltration in both tissues ( Fig. 5A and B). Interleukin-6 (IL-6), a typical inflammatory cytokine that is secreted by macrophages and Th2 cells, can alter insulin sensitivity by mediating different steps in the insulin signaling pathway. We found that serum IL-6 was increased in MSG rats (Fig. 5C), and a corresponding increase was also observed on the mRNA expression of Il-6 in MSG rat livers (Fig. 5D). Furthermore, we found that phosphorylation of NF-κB, a nuclear factor playing a central role in inflammatory disease, was increased in MSG rat livers (Fig. 5E). 1,25(OH) 2 D 3 elicited a distinct anti-inflammatory role by decreasing the IL-6 serum level, as well as the infiltration of IL-6 and activation of NF-κB in MSG rat livers (Fig. 5C-E). Additionally, we tested the activity of STAT3, a transcription factor which can suppress the production of pro-inflammatory cytokines. In the livers of MSG rats the phosphorylation of STAT3 was lower than normal rats, which was partly recovered in the livers of MSG rats treated with 1,25(OH) 2 D 3 , but not rosiglitazone (Fig. 5F). Transforming growth factor-β1 (TGF-β1) is an inhibitory inflammatory factor secreted by Treg cells. We found a reduction of Tgf-β1 in MSG rat livers and 1,25(OH) 2 D 3 normalized its expression (Fig. 5G). These results indicate that treatment with 1,25(OH) 2 D 3 inhibits the MSG-enhanced pro-inflammatory response, but restores the inhibitory inflammatory response in adipose and liver tissues of obese rats.

1,25(OH) 2 D 3 increased the number of Tregs in spleen and lymph node
Accumulated evidence suggests that regulatory T cells play a key role in controlling the inflammation status. To confirm our above findings that the decreased TGF-β1 in MSG rat livers was markedly restored by treatment of 1,25(OH) 2 D 3 , we performed further studies using flow cytometry to detect the number of Treg cells in secondary immune organs such as spleen and lymph node. We found that the number of CD4 þ CD25 þ FoxP3 þ Tregs did not change in spleen (Fig. 6A) but significantly decreased in the lymph node (Fig. 6B) of MSG rats. However, 1,25(OH) 2 D 3 -treated MSG rats revealed a significant increase of Tregs in both spleen and lymph node, compared to vehicle-treated MSG rats ( Fig. 6A and B). Because nonresolving inflammation is known to drive insulin resistant in insulin-targeted tissues, we further examined whether the treatment of 1,25(OH) 2 D 3 could impact the infiltration of Treg in these tissues. Using confocal microscopy, we observed that 1,25(OH) 2 D 3 could dramatically enhance the frequency of CD4 þ CD25 þ FoxP3 þ Tregs in primary insulin-targeted tissues such as liver (Fig. 6C), adipose (Fig. 6D) and muscle (Fig. 6E). These findings indicate that Tregs have a protective role in the pathogenesis of obesity as well as insulin resistant and that 1,25(OH) 2 D 3 may have general therapeutic efficacy for obesity by increasing the secretion or infiltration of Tregs in immune-or insulin-dependent tissues.

Discussion
It has become clear that obesity is one of the major risk factors for the onset of metabolic syndrome and type 2 diabetes mellitus. Recent studies have shown that obesity patients and animals appear to be in a chronic low-grade inflammation status characterized by increased plasma level of TNF-α, IL-6, IL-1, IL-8, MCP-1, and PAI-1 [24][25][26] . The accumulation of lipids and the expansion of the fat mass initiate the production of cytokines and chemokines. The proinflammatory factors inducing and maintaining the chronic inflammatory response are the critical inhibitors of insulin action on target organs, especially liver and adipose tissue. It is attributed to their architectural organization in which metabolic cells (hepatocytes or adipocytes) are in close proximity to immune cells (Kupffer cells or macrophages). For instance, co-cultured 3T3-L1 adipocytes and macrophages alter the expression of GLUT4 and insulin receptor substrate-1 27 . Studies in MCP-1 receptor-deficient mice suggest that these mice developed obesity after high-fat feeding but had less macrophage infiltration and weaker insulin resistance than control mice 28 . Macrophages in adipose tissue are suspected to be the major source of inflammatory factors such as TNF-α and IL-6. Corresponding with the Th1/Th2 concept of T-cell activation, a concept of M1/M2 polarization has recently been advanced for macrophages. In adipose tissue macrophages normally are of M2-like phenotype, characterized by expression cytokines involved anti-inflammation including IL-10, TGF-β, IL-4 and IL-13 29 . Consumption of high-fat diet shifts cytokine expression of adipose tissue macrophages from M2-to M1-patterns by decreasing expression of IL-10 and increasing TNF-α and IL-6 30 . Islets isolated from high-fat-fed mice produce much more inflammatory factors, including IL-6, IL-8, chemokine KC, granulocyte colony-stimulating factor, and MIP-1α 31 . In our studies, newborn rats were subjected to monosodium L-glutamate (MSG) s.c. injection which caused chemical ablation of the hypothalamic arcuate nucleus, and as a result induced progressive obesity, insulin resistance, and dyslipidemia. In MSG rats, several cellular lesions have been associated with insulin resistance, including decreased expression of insulin signal protein such as IRS-1 in liver and GLUT2 in pancreas, adipocyte hypertrophy and ECM deposition in pancreas islets. These lesions are concomitant with a chronic inflammatory status involving enhanced macrophage infiltration in adipose tissue and pancreas, an up-regulated IL-6 level and down-regulated TGF-β expression.
Numerous evidence has suggested that inflammation promotes energy expenditure in liver, adipose and skeletal muscle tissues to fight against energy surplus. Over-expression of NF-κB p65, the most crucial pro-inflammatory transcription factor, protects mice from high fat diet-induced obesity and insulin resistance through up-regulation of the expression of TNF-α and IL-6 in adipose tissue and macrophages 32 . In addition, inflammatory cytokines, such as TNF-α, IL-1 and IL-6, can also serve as anti-obesity signals by modifying both energy intake and energy expenditure [33][34][35] . However, both obesity and inflammation could be reciprocal causative factors, and breaking the obesity-inflammation cycle with anti-inflammation reagents seems to be a more effective strategy against obesity from metabolic syndrome and type 2 diabetes. Indeed, IL-1R antagonist and salicylates, a treatment that inhibits the activity of NF-κB, have been used in animal models to improve insulin sensitivity 36 . Agents such as thiazolidinedione (TZD) have been proven to have anti-inflammatory activities that partially contributes to their insulin-sensitizing effects 37 . In this work, we found that rosiglitazone exhibited antiinflammatory properties by reducing macrophage infiltration in adipose tissue and pancreas. In recent years, 1,25(OH) 2 D 3 has been shown not only to be important for insulin secretion from the pancreatic beta-cell but also for apoptosis of adipose cells, both of which are closely associated with 1,25(OH) 2 D 3 -triggered intracellular uptake of calcium [38][39][40][41] . Furthermore, most of the known biological effects of 1,25(OH) 2 D 3 are mediated through the vitamin D 3 receptor (VDR), which can be found in a wide range of immune cells, including lymphocytes, dentritic cells and macrophages. Accumulating research suggest that the immune properties of 1,25(OH) 2 D 3 include inhibition of DC differentiation and maturation, suppression of T cell activation, and enhancement of phagocytosis in monocytes or macrophages.
It has been shown that 1,25(OH) 2 D 3 could reshape immune homeostasis involving a shift of production of T-cell cytokines from predominantly Th1 (IL-2, IFNγ) to Th2 (IL-4, IL-10). This shift probably is due to a direct interference of 1,25(OH) 2 D 3 with the antigen-presenting dendritic cells. Indeed, 1,25(OH) 2 D 3 induces a transformation of dendritic cells towards a tolerogenic phenotype 42 . In vitro treatment with 1,25(OH) 2 D 3 inhibited IFNγ-induced phosphorylation of STAT1 and IL-12-triggered phosphorylation of TYK2, JAK2, STAT3, and STAT4 in association with a decrease in T cell proliferation 43 . In another study Gregori et al. 44 found that the analog of 1,25(OH) 2 D 3 inhibited IL-12 production, blocked pancreatic infiltration of Th1 cells, and enhanced CD4 þ CD25 þ Tregs. In our studies, we found exogenous 1,25(OH) 2 D 3 induced the infiltration of Treg cells not only in secondary immune organ such as spleen and lymph node, but also in primary insulin-targeted tissues such as liver, adipose and muscle, which provide straightforward in vivo evidence that 1,25(OH) 2 D 3 can be used as an immunomodulator for the therapy of obesity via enhanced Tregs-mediated inhibition of the inflammatory response in both immune organs and insulin-dependent tissues.

Conclusions
In summary, our studies demonstrate that 1,25(OH) 2 D 3 treatment can reduce body weight, improve glucose and lipid metabolism, decrease the incidence of obesity-induced insulin resistance and improve established insulin resistance in MSG-obese rats. Moreover, the chronic inflammatory status of insulin target tissues was ameliorated and was clearly linked with enhanced CD4 þ CD25 þ FoxP3 þ regulatory T cells. These findings add substance to the inflammatory theory behind the pathogenesis of obesity and insulin resistance. Furthermore, our study indicates that 1,25(OH) 2 D 3 has potential immunoregulatory efficacy on obesity and insulin resistance, both of which are becoming the leading risk factors for morbidity and mortality in metabolic syndrome and diabetic patients.


extfieldsofstudy: Biology Medicine

text:
Genome of tiny predator with big appetite

The capture and enslavement of eukaryotic algae by unicellular predators to acquire photosynthesis was a major driving force in early eukaryotic diversification. A genome presented in BMC Biology provides a glimpse of how such a tiny predator might have preyed on red algae and detained them to create new lineages of photosynthetic organisms.

plastids in these photosynthesizers have two bounding membranes, now clearly understood to derive from the pair of membranes that enclose your typical, Gram-negative cyanobacterium.
But primary endosymbiosis was merely the first chapter in the acquisition of photosynthesis by eukaryotes. Once the cyanobacterium was tamed, it was transplanted horizontally into several disjunct eukaryotic lineages by multiple secondary endosymbioses. These secondary endosymbioses-sometimes involving a red alga and sometimes involving a green alga, but curiously never a glaucophyte-resulted in the eukaryotic alga becoming the detainee in one or another phagotroph to create a metaorganism comprising a relic cyanobacterium inside a eukaryote inside yet another eukaryote. How do we know this with certainty? Cryptomonads. These little algae are a classic Darwinian transition form in this shell game, retaining all the shells of the two sequential engulfment processes, plus a few tell-tale bits and pieces of the swallowed ones.
The crucial piece in cryptomonads is the vestigial nucleus of the engulfed red alga, which we now refer to as the nucleomorph in recognition of Dennis Greenwood's seminal observation that it had the morphology of a little nucleus sequestered between the shells (membranes) of the metaorganism (references in [2]). Nucleomorph genomes confirm that this little structure is indeed the relic nucleus of the red algal endosymbiont and prove unequivocally that secondary endosymbiosis did happen, and that cryptomonads are one result [3]. Nucleomorphs give credence to all the other putative secondary endosymbioses where the nucleomorph, and even some of the membrane shells, have apparently disappeared with reduction of the endosymbiont through increased integration into the metaorganisms.
Goniomonas genomes and a window into the origins of photosynthesis presence of ejectisomes (enigmatic ejectile structures that burst like party streamers from the cell under duress), and the two unequal flagella emerging from a deep, complicated anterior invagination [4] (Fig. 1). Where Goniomonas differs to most cryptomonads-which are either blueish-greenish or reddish-brownish depending on which of the endosymbiont's phycobilin pigments they retain-is it lacks any color, and apparently any plastid [5]. This raises the question of whether it had a plastid and carelessly lost it, or whether it is a representative of the cryptomonads before they entered into their complex secondary endosymbiotic relationship with a red alga? A first step would be to infer how Goniomonas relates to photosynthetic cryptomonads, and DNA sequences were the obvious approach.
To get DNA sequences from organisms one typically needs a goodly amount of them. Goniomonas is a predator, so when Hill and I wanted to grow it up in large numbers, we opted to provide it with loads of bacteria to engulf and digest-this was as simple as me popping a grain of wheat into several flasks of culture media to support bacterial growth, whilst Hill arduously plucked out individual Goniomonas cells with a micropipette and placed them in solitary confinement with only food for company. Hill and I got a clonal culture and a ribosomal RNA sequence from Goniomonas truncata, and could show it was sister to photosynthetic cryptomonads [6].
In their recent article in BMC Biology, Cenci et al. report the entire genome of a related species, Goniomonas avonlea [7], which hails from the setting of Lucy Montgomery's novel Anne of Green Gables. The G. avonlea genome paints the first complete picture of how cryptomonads might have functioned before they crossed the tracks and made a pact with an alga to become autotrophic. It is a fascinating window into how things probably were with cryptomonads before they switched lifestyles.
As genomes go, the G. avonlea blueprint is still a bit of a roughie, remaining in~32,000 unjoined pieces. That's because the genome is huge for such a tiny organism, with a final size approaching 100 megabases; the effort to get it fully assembled and polished would be formidable. Gene count, at~18,000 non-redundant proteins, is also impressive, and these genes are, on average, interrupted by about five introns each. But numbers aside, what does the genome tell us about how G. avonlea makes its living, now and in the past? Quite a bit as it happens. A concerted search turned up no convincing evidence for G. avonlea now having, or ever having had, a plastid. Thus, we can be pretty certain this type of cryptomonad is ancestrally heterotrophic. Metabolic pathways typically taken care of by the plastid in plants and algae-namely fatty acid synthesis, isoprenoid precursor synthesis, iron sulfur cluster generation, and heme synthesis-are apparently done in the cytosol of G. avonlea using canonical eukaryotic machinery not related to cyanobacteria. Thus, there are no traces of plastid-type metabolisms lurking in the genome, and Goniomonas appears to be a living representative of the pre-secondary-endosymbiosis cryptomonads.

An ancestral phagotroph?
G. avonlea also seems well equipped gene-wise to digest its prey, having a panoply of lysozymes to chew through the cell walls of those bacteria unfortunate enough to end up going down the gullet of the predator and into its food vacuoles. For me though, the jewel-in-the-crown of the G. avonlea genome are glycan hydrolases belonging to the GH50 family, which cleave β-1,4 glycosidic bonds of agarose, a principal component of red algal cell walls. G. avonlea thus seems equipped to be algivorous, capable of digesting the walls of red algal prey cells. In today's oceans and streams, most red algae are multicellular and too large to be preyed upon by a miniscule flagellate like G. avonlea, but unicellular red algae small enough for a Goniomonas cell to engulf are not uncommon and were perhaps more so in earlier times. Thus, if G. avonlea does indeed engulf and digest red algae, it is not too much of a stretch to imagine a scenario where the prey cell is detained and not digested-exactly the kind of event predicted to have occurred at the outset of a secondary endosymbiosis creating the photosynthetic cryptomonads [2,3].
Goniomonas is thus a nice fit for the ancestral phagotroph that was routinely capturing and digesting red algae and could have commenced a longer and more sustained relationship with its prey to embark on the acquisition of photosynthesis through a secondary endosymbiosis. But what does Goniomonas tell us about the other, non-cryptomonad eukaryotes that also have reduced red algal endosymbionts for plastids?
Major eukaryote groups including the heterokonts/stramenopiles (algae like brown kelps, diatoms, and golden flagellates), haptophytes (abundant limestone-armoured phytoplankton whose dead ancestors comprise most of the white cliffs of Dover), dinoflagellates (including the symbionts of corals crucial for reef building and the toxic basis of certain red tides), and alveolates (parasites of animals and protists that cause diseases such as malaria and toxoplasmosis and possess relic, non-photosynthetic plastids) all harbour secondary endosymbionts of red algal origin. A long-standing debate about whether all these different types of organisms gained their red algal endosymbiont in one event-perhaps akin to the one discussed here in which a Goniomonas-like phagotroph captured and retained a red alga-or whether each of them descends from a separate capture of a red alga by different ancestors remains wide open, despite 40 years of investigation [8][9][10].
The genome of Goniomonas doesn't yet resolve this debate, but it gives us an extant model with which to explore what the phagotrophic partner in the extraordinary amalgam that led to at least one group of complex algae, the cryptomonads, was like. We might just have extant descendants of the two partners-predator and preywith which to better understand how a great swathe of eukaryotic diversity originated through secondary endosymbiosis. Maybe we should feed our Goniomonas on red algae rather than bacteria.