Intranasal treatment of lixisenatide attenuated emotional and olfactory symptoms via CREB-mediated adult neurogenesis in mouse depression model

Intranasal treatment of LXT reserved CUMS induced emotional and olfactory dysfunctions

We first created CUMS model for mimicking depression symptoms. LXT with two dosages (10 nmol/kg/d and 50 nmol/kg/d) were administrated by intranasal treatment from day-15 to day-40 (Figure 1A). Depression and anxiety behaviors were then detected. FST and TST demonstrated that 50 nmol/kg/d treatment of LXT significantly reduced the immobility time, indicating the effects of LXT intranasal treatment to depressive symptoms. Anxiety behaviors were assessed with open field test (OFT) and elevated plus maze (Figure 1B, 1C, one-way ANOVA, p<0.01 LXT-50 vs. CUMS). In open field test, treatment of LXT intranasally resulted the remarkable prolonged movement in center area (Figure 1D, one-way ANOVA, p<0.001 LXT-50 vs. CUMS). Consistently, LXT treatment also increased the movement duration of the mice in open arm area in EPM (Figure 1F, one-way ANOVA, p<0.001 LXT-50 vs. CUMS). Olfactory dysfunctions also related with the emotional symptoms in depression [6]. Herein, we tested the olfactory discrimination behavior to evaluate the olfactory functions. Mice were repeatedly exposed to butanol (Buta) and the sniffing duration was gradually decreased. As expected, sniffing duration was dramatically prolonged when the mice was exposed to pentanol (Penta), indicating the olfactory discrimination ability. The CUMS model showed dramatic decreased of the separation index (sniffing duration of Pento/sniffing duration of Pento and last Buto*100%) in compared with control (Figure 1H, one-way ANOVA, p<0.01, CUMS vs. Control). While administration of LXT by intranasal treatment recovered the olfactory function, which reflected by the significant increased separate index to CUMS (Figure 1H, one-way ANOVA, p<0.01, CUMS vs. LXT-50). Therefore, LXT intranasal treatment could improve the emotional and olfactory functions in depression animal model.

Figure 1. Intranasal treatment of LXT reserved CUMS induced emotional and olfactory dysfunctions. (A) Schematic picture to show the experimental process. Mice were performed with CUMS protocol and conducted with the intranasal administration of the LXT. (B, C) Immobility time of the mice to show the antidepressant behaviors in FST and TST. (D) Time of the mice to spend in center area of the region in open field test. (E) Time of mouse to spend in open arm in elevated plus maze test. (F, G) Duration of the sniff and the separation index (sniff duration of pentanol) of mice in olfactory discrimination test.

LXT intranasal treatment enhanced adult neurogenesis in hippocampus and olfactory bulbs

Adult neurogenesis at olfactory bulbs (OB) and hippocampus plays the critical roles in regulating olfactory functions and emotional behaviors [4]. To further explore whether adult neurogenesis is correlated with the effects of LXT to improve the behavioral dysfunctions in CUMS model, we labelled the new generated cells with BrdU and co-stained the BrdU with immature neuronal marker doublecortin (DCX) at both OB and hippocampal DG region. As the result shown, BrdU+ cells as well as BrdU and DCX dual positive cells were considerably decreased at OB region in CUMS mice (Figure 2A–2C, one-way ANVOA, p<-0.001 CUMS vs. Control). LXT treatment increased the number of both BrdU+ cells and BrdU+/DCX+ cells (Figure 2A–2C, one-way ANVOA, p<-0.001 CUMS vs. LXT-50). The same condition was observed in hippocampal region. Intranasal treatment of LXT with 50mg/kg/d resulted in the significant increased BrdU+ cells and BrdU+/DCX+ cells in hippocampus, when comparing with CUMS model (Figure 2E, 2F, one-way ANOVA, p<0.001 CUMS vs. LXT-50). The results indicate that LXT treatment could improve the adult neurogenesis in both OB and hippocampal region. Such biological effects might involve with the behavioral improvement of LXT to CUMS animal model.

Figure 2. LXT intranasal treatment enhanced adult neurogenesis in hippocampus and olfactory bulbs. (A, D) Confocal image to show the distribution and cell number of DCX+ immature neurons (Red) and BrdU+ (Green) newborn cells in olfactory bulb and hippocampal DG region. (B, C) DCX+ immature neuron cell density and the newborn DCX+/BrdU+ cell density in olfactory bulb was recovered by LXT treatment. (E, F) DCX+ immature neuron cell density and the newborn DCX+/BrdU+ cell density in hippocampal DG region was recovered by LXT treatment.

Hippocampal neurogenesis is required in effects of LXT to CUMS model

The antimitotic drug temozolomide (TMZ) has previously been used successfully as an experimental tool in animals to deplete adult neurogenesis and is used regularly as a standard chemotherapy for brain cancer [11]. Here we treated the mice with TMZ to inhibit the adult neurogenesis (Figure 3A). Treatment of TMZ remarkably decreased the adult neurogenesis. It was shown that density of DCX+ cells in hippocampus was dampened (Figure 3B, 3C, student t-test, p<0.001). Moreover, the dendritic fibers labelled with DCX were also dramatically decreased after TMZ treatment (Figure 3B, 3D, student t-test, p<0.001). Those results indicate that TMZ exerted the inhibitory effects to adult neurogenesis in hippocampus. As the consequence, inhibition of hippocampal neurogenesis compromised the behavioral improvement of the LXT. Injection of TMZ increased the immobility time in FST and simultaneously decreased the movement duration in center region in OFT (Figure 3E, 3F, student t-test, p<0.001). Therefore, LXT intranasal treatment ameliorated the depression and anxiety like behaviors depending on the enhancement of neurogenesis.

Figure 3. Hippocampal neurogenesis is required in effects of LXT to CUMS model. (A) Schematic picture to show the experimental process. Mice with CUMS protocol was conducted with TMZ treatment and combined with the intranasal administration of the LXT. (B) Confocal image to show the distribution and cell number of DCX+ immature neurons (Red). (C, D) Statistical analysis of DCX+ immature neurons and DCX+ neural fibers. (E, F) Statistical analysis of immobility time in FST as well as the time spend in center region in open field test.

CREB mediates the effects of LXT to improve adult neurogenesis and related behaviors

CREB pathway is the critical signaling to regulate adult neurogenesis. In hippocampal tissue, we observed that LXT intranasal treatment significantly elevated the CREB phosphorylation level at Ser133 in comparing with CUMS mode, which suggesting LXT recovered the activity of CREB (Figure 4A, 4B, one-way ANOVA, p<0.05, LXT50 vs. CUMS). This result indicates that CREB might be one of the important regulators involving with the effects of LXT to improve behaviors via promoting neurogenesis. To further confirm the role of CREB signaling in therapeutic effects of LXT intranasal treatment, we injected the treatment mice with 666-15 to inhibit the activation of CREB (Figure 4C, 4E). By labelling the immature neurons in hippocampal DG region with DCX, we detected that 666-15 administration to LXT treatment group remarkably decreased the density of DCX+ immature neuronal density (Figure 4D, 4E, one-way ANOVA, p<0.05 CUMS+LXT50 vs. CUSM+LXT50+666-15). Behavioral test showed that 666-15 treatment prolonged the immobility time in FST and inhibited the duration of sniffing to Penta in olfactory discrimination test (Figure 4F, one-way ANOVA, p<0.05 CUMS+LXT50 vs. CUSM+LXT50+666-15; G, two-way ANVOA, p<0.001, CUMS+TLX50 trail 4 vs. Penta; in significance, p=0.9937, CUMS+TLX50+666-15 trail 4 vs. Penta). These two behavioral tests indicate that inhibition of CREB signaling blocked the behavioral effects of LXT with intranasal treatment. Thus, CREB mediated adult neurogenesis the critical mechanism underlying the effects of LXT intranasal treatment to depression.

Figure 4. CREB mediates the effects of LXT to improve adult neurogenesis and related behaviors. (A, B) Western blot band and statistical analysis to show the phosphorylation of CREB at pSer333 and the total protein level. (C) Schematic picture to show the experimental process. Mice with CUMS protocol was conducted with 666-15 treatment and combined with the intranasal administration of the LXT. (D) Confocal image to show the distribution and cell number of DCX+ immature neurons (Red) in hippocampal DG region. (E) Statistical analysis of DCX+ immature neuronal density. (F) Statistical analysis of immobility time in FST. (G) Statistical analysis of the duration of the sniff and the separation index (sniff duration of pentanol) of mice in olfactory discrimination test.

Animals

C57BL/6N mice (age of 8 weeks, male) were obtained from laboratory animal center in Shanxi Medical University. Mice were maintained in the standard environment (12 h light/dark cycle, with lights on at 8:00 A.M., ad libitum access to dry food pellets and water). All animal performance were followed the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Shanxi Medical University Research Ethic Committee. After a 1-week adaptation period, mice were processed for the chronic unpredictable mild stress (Figure 1A). Briefly, the CUMS paradigm consisted in exposure, once daily, to one of the following aversive stressors: restraint-mice were placed in a 50 ml plastic tube (Falcon) with openings in both sides for breathing, for 1 h; shaking-groups of five mice were placed in a plastic box container and placed in an orbital shaker for 1 h at 150 rpm; social defeat-mice were introduced in a cage of an aggressive mice and after being defeated, they were placed in a transparent and perforated plastic container, to avoid further physical contact, inside the resident homecage for 30 min; hot air stream-mice were exposed to a hot air stream from a hairdryer for 10 min; overnight illumination-mice were exposed to regular room light during the night period; inverted light cycle-regular room light was off during daytime and on during nighttime for 2 days; tilted cage-homecages were tilted in a 45° angle during 1 h [25]. The process was repeated for 40 days, and the LXT treatment were conducting from the 15^th day of modelling process. LXT were treated based on clinical dosage and separated with dosages as 10 nmol/kg and 50 nmol/kg, dissolved in PBS and performed intranasal treatment to mice with total volume of 50μl daily. The CREB inhibitor 666-15 (10 mg/kg) was injected to the mice with i.p. every two days. Administration was conducted using a gentle pipette of 2.5 μL per nostril with no anesthetics. Temozolomide (TMZ) was treated to the mice with i.p. on a daily dosage of 20mg/kg.

Behavioral tests

Forced swim test

FST was conducted in a water tank (cylinder 30 cm height x 20 cm diameters). Mice were gently placed into the water and allowed for total 6 min free swimming. First 2 min was considered as the environmental habituation, and mobility time including swim and struggle in last 4 min was recorded to analyze the learned helplessness.

Tail suspension test

Mice were suspended on the tap-top of their tails for 6 min. Distance from mice nose to the floor was 25cm. The mobility time (excluding the swim-like oscillations and pendulum) was recorded as the indicator of depressive mood.

Open field test

OFT was processed in the 40cm×40cm×40cm white box. Mice were put into the box with allowance of free movement for 10min. The time percentage of the mice in central regions (20cm×20cm in center) was analyzed for evaluating the anxiety mood.

Elevated plus maze

Mice were performed with EPM to further confirm the anxiety mood. Mice were placed on the platform comprises two open arms (25 x 5 x 0.5 cm) across from each other and perpendicular to two closed arms (25 x 5 x 16 cm) with a center platform (5 x 5 x 0.5 cm), allowing for move freely for 10 min. The time of the mice staying on open arm were recorded for analyzing the anxiety-mood.

Immunofluorescence

Mice were sacrificed with cardiac perfusion by 4% paraformaldehyde (PFA). Before cardiac perfusion, mice were anaesthetized with 100-200 mg/kg body weight for Ketamine and 5-16 mg/kg body weight for Xylazine injected intraperitoneally. Brain was collected and post-fixed in 4% PFA for 24 h. Tissue was then embedded after dehydration with 30% of sucrose. Hippocampal and olfactory bulb cryosections were prepared with 20μm thickness. Briefly, sections after antigen retrieve (pH 6.0 citrate acid) and washing with 0.3% TritionX-100 PBS were incubated with 5% goat serum (0.3 TritionX-100). For BrdU staining, sections were incubated with 2M HCl for 1 h at room temperature (RT) and wash with PBST for 3 times (15min each time). Primary antibodies (Mouse-anti-BrdU, CST, 1:400; Rabbit-anti-DCX, CST, 1:400) were incubated with tissue sections at 4° C overnight. Secondary antibodies (Goat-anti-Rabbit-Alexa fluor 488, ThermoFisher, 1:800; Goat-anti-Mouse-Alexa fluor 568, ThermoFisher, 1:800) were incubated with sections for 2 h RT. DAPI (Sigma) was incubated with sections for 10min. Tissue IF images were obtained with confocal microscopy (Carl Zeiss, LSM 700) with Z-stack for 20μm. Image was shown as maximum projection of Z-stack. Required positive cell number was calculated and displayed as the cell density per mm².

Western blot

Hippocampal tissue was collected, and the protein lysate was prepared with RIPA buffer. Briefly, primary antibodies (rabbit anti-pCREB (Ser133), CST, 1:1000; rabbit anti-CREB, CST, 1:1000) were incubated with membrane at 4° C overnight. Secondary antibody (Goat-anti-rabbit HRP, CST, 1:2000) was incubated with membrane at room temperature for 1 hour. Protein bands were visualized with enhanced chemiluminescent (ECL, Pierce, Rockford, IL) and analyzed using the image system of Quantity One (Bio-Rad, Quantity One 1-D Analysis Software).

Statistics

All values are expressed as mean ± SEM. For analyzing across multiple groups, comparisons were performed by one-way or two-way analysis of variance (ANOVA) followed by multiple comparison test via GraphPad Prism 5 (GraphPad software Inc, CA, USA). Two group comparing was performed with student t-test. A p value of 0.05 was cut off as statistically significance.