Chronic AdipoRon Treatment Mimics the Effects of Physical Exercise on Restoring Hippocampal Neuroplasticity in Diabetic Mice

Administration of exercise mimetic drugs could be a novel therapeutic approach to combat comorbid neurodegeneration and metabolic syndromes. Adiponectin is an adipocyte-secreted hormone. In addition to its antidiabetic effect, adiponectin mediates the antidepressant effect of physical exercise associated with adult hippocampal neurogenesis. The antidiabetic effect of the adiponectin receptor agonist AdipoRon has been demonstrated, but its potential pro-cognitive and neurotrophic effects in the hippocampus under diabetic condition are still unclear. This study reported that chronic AdipoRon treatment for 2 weeks improved hippocampal-dependent spatial recognition memory in streptozotocin-induced diabetic mice. Besides, AdipoRon treatment increased progenitor cell proliferation and neuronal differentiation in the hippocampal dentate gyrus (DG) of diabetic mice. Furthermore, AdipoRon treatment significantly increased dendritic complexity, spine density, and N-methyl-D-aspartate receptor-dependent long-term potentiation (LTP) in the dentate region, and increased BDNF levels in the DG of diabetic mice. AdipoRon treatment activated AMPK/PGC-1α signalling in the DG, whereas increases in cell proliferation and LTP were not observed when PGC-1α signalling was pharmacologically inhibited. In sum, chronic AdipoRon treatment partially mimics the benefits of physical exercise for learning and memory and hippocampal neuroplasticity in the diabetic brain. The results suggested that AdipoRon could be a potential physical exercise mimetic to improve hippocampal plasticity and hence rescue learning and memory impairment typically associated with diabetes. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-021-02441-7.

Activating a common molecular target that mediates the eumetabolic and pro-cognitive effects of physical exercise can be a novel therapeutic approach to combat diabetes and its associated neurodegeneration. The adipocyte-secreted hormone adiponectin is deemed to be a worthy candidate since it possesses antidiabetic property by promoting insulin sensitivity (27). Hypoadiponectinemia is restored in obese and diabetic individuals undergoing physical exercise interventions (28,29). The central adiponectin level could be linked to neurodegeneration and cognitive decline as suggested by reduced adiponectin levels in the hippocampus of diabetic mice (30) and cerebrospinal fluid of AD patients (31). Importantly, further studies have unravelled the pivotal role of adiponectin in cognitive function and hippocampal plasticity. Learning and memory deficits are presented in adiponectin-deficient mice (32). Also, adiponectin regulates dendritic and spine formation (33) as well as synaptic plasticity (32) in the hippocampus, where adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) are expressed in the hippocampal neural progenitor cells (34), neurons (35), and synapses (32). Our team has previously shown that adiponectin mediates exercise-induced adult neurogenesis, dendritic arborisation, and AMP-activated protein kinase (AMPK) activation in the hippocampus (30,34,36). BDNF is a crucial downstream effector to regulate adult neurogenesis, synaptic plasticity, and cognitive function (37). The concomitant increases in brain-derived neurotrophic factor (BDNF) and adult hippocampus neurogenesis mimic the beneficial effects of voluntary running on cognitive enhancement in mice (38). Notably, activating PGC-1α signalling is required for physical exercise to increase BDNF levels (39) and dendritic spine formation (40) in the hippocampus. In addition, an early study has demonstrated that adiponectin promotes PGC-1α activity through AdipoR1/AMPK signalling (41). These studies collectively suggest that activating adiponectin signalling cascade may contribute to raising BDNF level.
Administration of AdipoRon, an agonist of AdipoR1 and AdipoR2, resembles the effects of adiponectin administration and physical exercise on improving insulin sensitivity and oxidative metabolism in diabetic mice (42). Since AdipoRon can cross the blood-brain barrier (31,43), this study aimed to investigate whether chronic treatment with AdipoRon could act as a pro-cognitive exercise mimetic to restore hippocampal neuroplasticity in animals experiencing diabetes-associated hippocampal dysfunction.

Animals and Experimental Design
All experimental procedures were approved and followed the Animal Subjects Ethics Sub-Committee's guidelines at The Hong Kong Polytechnic University. Five-week-old C57BL/ 6J male mice were fed with standard chow and water ad libitum and kept under a 12-h light-dark cycle. A singledose streptozotocin (STZ; 195 mg/kg i.p.) (Santa Cruz Biotechnology Ltd., USA) was delivered intraperitoneally to induce diabetes-associated hippocampal impairment as previously described (30). Tail blood samples were measured by glucometer (Accu-Chek® Performa, Australia) 7 days after STZ administration, and only animals that exhibited hyperglycaemia (≥ 20 mmol/L) were included in the experiments (Fig. S1).
AdipoRon (20 mg/kg in 0.5% carboxymethylcellulose salt solution, i.p. injection) (Sigma-Aldrich, MO, USA) or vehicle was delivered continuously for 14 days (44). In the voluntary exercise regime, mice were group housed in the holding cage, with shared running wheels as previously described (30,34). Mice were injected with bromodeoxyuridine i.p. (50 mg/kg in 0.9% saline) before AdipoRon treatment to label surviving new-born cell in the DG. The day after the last treatment, mice were subjected to the open field test and Y-maze task (Fig.  1A).

Y-Maze Task
Three identical arms (L × W × H: 10 × 6 × 8 cm) in Y-maze were pre-designated as the starting arm, the familiar arm, and the novel arm, respectively (44). Three arms were counterbalanced to avoid recognition bias. Tests were carried out in the dim light setting. In the 10-min habituation session, a mouse was released in the designated starting arm and was allowed to explore the maze with a blocked novel arm. After a 4-h intermission, the mouse was allowed to explore the maze with the unblocked novel arm for 5 min. Time spent in each arm was hand-scored by the trained researcher in a sample-blinded manner. Exploration ratios were calculated as (time spent in the novel arm (N)/(time spent in novel + familiar arms) and (time spent in the familiar arm (F))/(time spent in novel + familiar arms), and an exploration index (in a −1 to 1 scale) was calculated as (N−F)/(N+F).

Tissue Preparation for Immunostaining
Mice were deeply anaesthetised with isoflurane (Zoetis, UK). Blood samples were collected transcardially by using a 1-mL syringe with a 25-G needle. Blood samples were clotted for 30 min at room temperature, and blood serum samples were Fig. 1 AdipoRon treatment ameliorated diabetes-associated spatial recognition memory. A Animal treatment timeline (n = 7 animals per group). Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study and are available at doi: 10.3390/ijms22042068 (44). B Exploration ratios in Y-maze. Neither AdipoRon treatment nor exercise altered novelty preference in the Y-maze in healthy control mice. Diabetic mice showed impairment in Y-maze as shown with a greater preference towards the familiar arm (paired t-test: *P < 0.005 vs. Novel arm), while both AdipoRon treatment and exercise restored the memory deficit in diabetic mice (**P < 0.005 vs. Novel arm). C Exploration indices in Y-maze. Diabetic mice presented spatial memory deficit (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle), whereas AdipoRon treatment (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle) and exercise (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle) ameliorated memory deficit. D Diabetic mice showed anxietylike behaviour (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle), which could be restored by exercise (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle). E Diabetic mice had reduced locomotor activity (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle). AdipoRon treatment and exercise (Tukey's post hoc test: P > 0.05 vs. STZ-Vehicle) did not improve locomotor activity in diabetic mice collected by centrifugation at 1,000g for 20 min at 4°C, and then stored at −80°C until measurements using enzymelinked immunosorbent assay (ELISA). Animals were then perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA) in 0.01 M phosphate-buffered saline (PBS). The isolated brains were post-fixed with 4% PFA at 4°C overnight. The brains were then stored in 30% sucrose until they sank. Coronal sections (1-in-6 series, 30-μm thickness) were prepared using a vibratome (Leica Biosystems, Germany). The brain slices were stored in cryoprotectant (30% glycerol and 30% ethylene glycol) at 4°C until immunostaining.

Immunohistochemistry
Immunostaining was performed by the free-floating method as previously performed (44). For BrdU staining, the antigens were retrieved in the citric acid buffer (pH 6.0) at 95°C for 15 min, then denatured in 2 N HCl for 30 min and neutralised by 0.1 M borate buffer (pH 8.5) for 15 min at room temperature. The sections were incubated overnight with mouse anti-BrdU antibody (1:1,000; Roche Life Science, Germany) and then incubated with the biotinylated goat anti-mouse IgG (1:200; Vector Laboratories, CA, USA) for 2 h at room temperature. For doublecortin (DCX) and Ki-67 staining, sections were incubated with mouse anti-DCX (1:200; Santa Cruz Biotechnology, TX, USA) or rabbit anti-Ki67 (1:1,000; Abcam, UK) antibodies, then secondary antibodies: biotinylated goat anti-mouse or goat anti-rabbit IgG (1:200; Vector Laboratories, CA, USA). Positive staining was visualised with the peroxidase method using the VECTASTAIN® ABC kit (HRP) (Vector Laboratories, CA, USA) and the DAB peroxidase substrate kit (Vector Laboratories, CA, USA).

Immunofluorescent Co-labelling
Immunofluorescent co-labelling of BrdU and DCX was performed as previously described (34,44). After antigen retrieval, sections were incubated with primary antibodies overnight and secondary antibodies: goat anti-rabbit IgG Alexa Fluor-488 and goat anti-mouse IgG Alexa Fluor-568 for 2 h at room temperature. After washes with PBS, the sections were counterstained by DAPI and coverslipped with the fluorescent mounting medium (Dako, CA, USA).

Quantifications of BrdU+, Ki67+, and DCX+ cells
Cells were counted in the 1-in-6 series of nine sections (from bregma −1.34 to −3.80 mm), using optical fractionators system (grid size: 55 μm × 55 μm; counting frame: 35 μm × 35 μm) of Stereo Investigator (MicroBrightField Inc., VT, USA) as previously described (44). Cells located in the dentate subgranular zone and granular cell layer were counted, whereas those located in the uppermost focal plane were excluded. Quantification was performed in a sample-blinded manner.

Quantifications of DCX+/BrdU+ co-labelled cells
Images of six sections from each animal were captured using LSM 800 confocal laser scanning microscope (Carl Zeiss Microscopy, NY, USA). Fifty BrdU positive cells were randomly selected, followed by confirming the DCX co-labelled ratio to indicate neuronal differentiation (34,45). Quantification of the co-labelling was performed in a sample-blinded manner (30,44).

Golgi Staining
Golgi staining was performed using the commercially available FD Rapid GolgiStain™ Kit (FD Neurotechnologies, Inc., MD, USA). Solutions A and B in FD Rapid GolgiStain™ Kit were pre-mixed in a 1:1 ratio for a week. Mice were deeply anaesthetised by isoflurane (Zoetis, UK) and rapidly decapitated to obtain the whole brain. The hemispheres were halved and kept in solution A-B mixture in the dark at room temperature for 6 days. Then, the halved brains were immersed in cryoprotectant at 4°C in the dark until they sank. Cryoprotectant was replaced after the first 24 h. Coronal brain slices with 200-μm thickness were obtained from rostral to caudal (from bregma −1.34 to −3.80 mm) using a vibratome (Leica Biosystems, Germany) filled with ice-cold, 6% sucrose solution. Sections were immediately mounted onto a chrome alum-coated slide with solution C and dried in a dark chamber overnight at room temperature. The staining solution was freshly prepared by mixing solution D, solution E, and Milli-Q water in 1:1:2 ratio. Samples were stained for 10 min and were briefly rinsed by Milli-Q water. At last, dried samples were dehydrated through 70%, 90%, and 100% ethanol (5 min each, 10 min for absolute ethanol) and xylene for 15 min. Samples were then coverslipped.

Dendritic Branch Analyses
Granule cells were selected based on the following criteria (45)(46)(47)(48): (1) Neuronal cell bodies located in the middle part of the section thickness to minimise the truncated branch segments; (2) consistent impregnation along with the entire extent of all dendrites; (3) located in the appropriate subregion of the dorsal hippocampi. Specifically, granule cells in the hippocampal DG were selected based on their soma position because granule cells with different soma position in the dentate gyrus could have different morphologies. Granule cells located closer to the subgranular zone show a simpler dendritic architecture with typically one primary dendrite extension. The granule cells located in the outer granule cell layer display multiple primary dendritic extensions and have more complex dendritic branching. Single-branched granule cells were selected from the inner cell layer, and multiple-branched granule cells were selected from the outer cell layer as previously performed (45)(46)(47)(48). Five single-branched and five multiplebranched granule cells were selected from each sample to measure the total dendritic length, and Sholl analysis was performed using Neurolucida (MicroBrightField Bioscience, VT, USA).

Dendritic Spine Analyses
Five single-branched and five multiple-branched granule cells were selected from each sample. Five segments of tertiary or quaternary dendritic branches (longer than 15 μm) were selected from a granule cell using ×400 magnification under a light microscope (Axioplan, Zeiss, Oberkochen, Germany) (45,47). Quantification of dendritic spines was performed using ×630 magnification.

Protein Extraction
Mice were rapidly decapacitated. The hippocampal DG was isolated from the Ammon's horn as previously described (49). The dentate gyrus samples were homogenised from a pool of seven mice from the same treatment group in ice-cold radioimmunoprecipitation assay buffer (Abcam, Cambridge, UK) containing Halt© phosphatase/protease cocktail (Thermo F i s h e r S c i e n t i f i c , W a l t h a m , M A , U S A ) a n d phenylmethanesulfonyl fluoride (Thermo Fisher Scientific, Waltham, MA, USA). Samples were then sonicated for 20 s with a 50% pulse. Supernatants were collected by centrifugation (14,000g) at 4°C for 30 min. Protein concentrations of supernatant were quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and stored at −80°C.

Long-term Potentiation Field Recording
Hippocampal DG was positioned on the 64 extracellular electrodes in a recording probe (P515A, Alpha MED Scientific Inc., Osaka, Japan) to stimulate granule neurons in the middle molecular layer (medial perforant pathway) of the suprapyramidal blade. Recordings of field excitatory postsynaptic potentials (fEPSPs) were made on adjacent channels located midway between the crest and the distal end of the limb. Slices were perfused at a rate of approximately 2 mL/ min. fEPSPs were acquired using the recording amplifiers (MED-A64MD1 and MED-A64HE1S, Alpha MED Scientific Inc., Osaka, Japan) connected to a Windows computer running Mobius software. For each slice, stimulus intensity (20-35 μA) was adjusted to yield 40-50% of the maximal response slope without population spikes. Baselines fEPSP measurements were obtained by delivering single-pulse stimulation at 15-s interstimulus intervals. After a steady baseline of at least 20 min, a conditioning protocol was used to induce synaptic plasticity, followed by baseline measurement for 1 h as previously performed (50). High-frequency stimulation (HFS) protocol (4 trains of 50 pulses, delivered at 100 Hz with 30-s intertrain interval) was applied in the presence of bicuculline methiodide (5 μM) to inhibit the γ-aminobutyric acid-A (GABA A ) receptors and facilitate LTP induction in the DG. In pharmacological intervention, brain slices were acutely perfused in PEAQX tetrasodium hydrate (0.1 μM; NVP-AAM077) (Sigma-Aldrich, MO, USA) to inhibit GluN2A subunit (51) or in Ro 25-6981 maleate (0.5 μM) (Alomone Labs, Israel) to inhibit GluN2B subunit (52) for 30 min before HFS. Similarly, SR-18292 maleate (1 nM; Sigma-Aldrich, MO, USA), a PGC-1α blocker, was perfused 20 min before HFS. AdipoRon (500 nM dissolved in aACSF) was bath perfused in the last 10 min in the presence of inhibitors.

Immunoassays for Adiponectin and BDNF Levels
Levels of adiponectin and BDNF in the collected sera and dentate gyri were determined by using the commercial ELISA kits, including mouse adiponectin ELISA kit for measuring serum adiponectin levels (Immunodiagnostics, University of Hong Kong, Hong Kong), mouse adiponectin ELISA kit for measuring adiponectin levels in the hippocampal DG (AdipoGen Life Sciences, Switzerland), and the total BDNF Quantikine ELISA kit (R&D system, MN, USA) according to the manufacturer's instructions.

N2a Mouse Neuroblastoma Cell Line Maintenance
Murine neuroblastoma cell line N2a (ATCC, VA, USA) was maintained in the Gibco® Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, MA, USA) containing 10% foetal bovine serum (FBS), 2 mM L-glutamine, and 50 μg/mL penicillin-streptomycin. After reaching 90% confluence, cell cultures were washed with Gibco® Dulbecco's PBS (DPBS, Thermo Fisher Scientific, MA, USA) and incubated with the mixture of 0.25% trypsin/0.02% EDTA at 37°C for 10 min. Trypsinization was halted by FBS-containing DMEM. The cell suspension was pelleted at 300g for 7 min and split in four. Passages 3-7 (the initial cell stock as passage 0) were used for the following assays.

Cell Proliferation Assay
CyQUANT TM NF Cell Proliferation Assay Kit (Invitrogen, MA, USA) was used to examine the proliferative effect of AdipoRon in vitro. CyQuant® dye is a fluorochrome that intercalates with the nuclear DNA content in living cells (53). The emission intensity is correlated linearly with cell number over the range of 10 to 50,000 cells (53). One hundred microliters trypsinised subculture was seeded at a density of 0.02 million per well in the 96-well flat-bottom microplates (Falcon, Fisher Scientific, NH, USA) coated with 0.1% poly-L-lysine (Sigma, MO, USA). The subculture was maintained in the drug-free culture medium for 24 h. The next day, the medium was decanted and replaced by a new medium containing AdipoRon alone or with the inhibitors. Specifically, AdipoRon (40 mg/mL), GW9662 (20 mg/mL), and SR-18292 maleate (20 mg/mL) were dissolved in DMSO as stocks and then diluted with the culture medium. To examine the proliferative effect of AdipoRon, subculture was independently treated by AdipoRon (0.5, 1, 5, or 25 μM) for 4 h or 24 h. To examine the involvement of PPAR-γ and PGC-1α in AdipoRon-induced cell proliferation, subculture was first pretreated by inhibitors PPAR-γ blocker: GW9662 (10 μM; APE×BIO, TX, USA) (54) and PGC-1α blocker: SR-18292 (20 μM; Sigma-Aldrich, MO, USA) (55) for 1 h, followed by AdipoRon and inhibitor co-treatment for 24 h. All control samples were treated with 2% DMSO (v/v) as vehicle treatment. After treatments, culture media were decanted. One hundred microliters 1× CyQuant® NF dye was added to each well. One hour after, sample fluorescence (excitation/emission λ = 480/520 nm) was measured using the Varioskan TM Flash Plate Reader (Thermo Fisher Scientific, MA, USA). Cell proliferation rate was determined by comparing emission intensity for a drug-treated subculture to DMSO-treated controls. The relative cell proliferation was calculated as follows: where readings of the cell-free well and untreated wells were the blank and the positive control, respectively.

Statistical Analyses
Data were shown as mean ± SEM. A paired t-test was performed to compare the novel/familiar arm visit in the Y-maze task. To evaluate the effects of AdipoRon treatment and STZdiabetes, two-way ANOVA was employed in behavioural assessments, histological analyses, fEPSP analyses, and ELISA by Tukey post hoc test. Wilcoxon matched-pairs signed-rank test between AdipoRon-and vehicle-treated animals in the control and diabetic animals separately to address dendritic complexity. Two-way ANOVA with LSD post hoc test was applied in the densitometric analyses of immunoblotting. One-way ANOVA with LSD post hoc test was used to evaluate the AdipoRon dosage effect in in vitro proliferation assay. Statistical analyses were performed in Prism 8.0 software (GraphPad Software, USA). P < 0.05 was considered statistically significant.

Chronic Treatment with AdipoRon Mimicked Voluntary Running to Ameliorate Diabetes-Impaired Spatial Memory
Hippocampal-dependent spatial recognition memory was assessed by a Y-maze test (56). Control mice with an intact spatial recognition memory showed a significantly higher exploration ratio in the novel arm ( Fig. 1B and Appendix Table S1; P < 0.0005 vs. Novel arm). The STZ-diabetic mice showed a greater preference towards the familiar arm (

AdipoRon Treatment Ameliorated Impairments in Dendritic Arborisation and Spine Loss in Diabetic Mice
Structural changes in dendritic arborization could also affect hippocampal function; and hence hippocampal-dependent learning and memory (45,62,63). Voluntary running exercise significantly promotes dendritic enrichment in the hippocampus (18,45,64). The Golgi-stained neurons in diabetic mice showed significant reductions in total dendritic length in both younger (

AdipoRon-Restored LTP Deficits in the Hippocampal DG of Diabetic Mice
Previous studies have shown that long-term voluntary wheel running exercise (2 to 3 months) promotes hippocampal synaptic plasticity (62,65). To determine whether AdipoRon promotes synaptic plasticity as exercise does, we examined its effect on LTP formation in the hippocampal DG of diabetic mice (Fig. 4A). Diabetes significantly impaired LTP formation ( Fig. 4C; P < 0.0001 vs. Control-Vehicle), which was restored by bath application with AdipoRon (10 min; 0.5 Control-Vehicle; *P < 0.05 vs. STZ-Vehicle) on increasing proliferating cell in both control and diabetic mice. n = 5 per group. Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). B STZ-diabetes reduced the density of DCX + immature neuron (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle). AdipoRon treatment (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle) restored the density of immature neurons as running did in diabetic mice (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle), but not increasing the immature neuron density in control mice (Tukey's post hoc test: *P > 0.05 vs. Control-Vehicle). Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). C STZ reduced cell survival in the DG (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle) marked by BrdU. Voluntary running increased cell survival in both control (Tukey's post hoc test: *P < 0.05 vs. Control-Vehicle) and diabetic animals (Tukey's post hoc test: *P < 0.05 vs. STZ-Vehicle). However, AdipoRon treatment showed no significant effects (Tukey's post hoc test: *P > 0.05 vs. STZ-Vehicle). Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). D STZ impaired neuronal differentiation (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle) with reduced percentages of BrdU/DCX co-labelling. AdipoRon treatment (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle) restored neuronal differentiation of new-born cells in diabetic mice (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle), as runners did. However, AdipoRon did not show effects on neuronal differentiation in healthy control mice (n = 5 animals per group). Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). AdipoRon exerted proliferating effect in a time-and dose-dependent manner (6 replicates per independent experiment; n = 3 independent experiments/treatment group) in CyQuant assay (LSD post hoc test: *P < 0.05 vs. Control). J In vitro examination of the involvement of PPARγ/PGC-1α signalling in AdipoRon-induced cell proliferation in culture N2a cells. AdipoRon-induced cell proliferation was mediated by PPARγ/PGC-1α signalling. Blocking either PPAR-γ by SR-18292 or PGC-1α by GW9662 or co-treatment diminished AdipoRon-induced cell proliferation (9 replicates per independent experiment; n = 4 independent experiments/group) (LSD post hoc test: *P < 0.05 vs. untreated control) μM) ( Fig. 4C; P < 0.0001 vs. STZ). Two-way ANOVA revealed significant effects of AdipoRon and diabetic on LTP formation ( Fig. 4B and C: Effect of STZ-diabetic: F 1,40 = 26.25, P < 0.0001; effect of AdipoRon: F 1,40 = 23.08, P < 0.0001; effect of interaction: F 1,40 = 12.69, P = 0.001). PGC-1α is a master regulator of mitochondrial biogenesis (66,67) which is necessary for LTP formation (68). As expected, pharmacological inhibition of PGC-1α by SR-18292 (Fig. 4D) diminished the promoting effect AdipoRon on LTP formation in diabetic mice (Fig. 4E & 4F; P = 0.514 vs. STZ-AdipoRon). Bath application with NMDA receptor subunit blockers, including NVP-AAM077 (GluN2A inhibitor) or Ro 25-6981 (GluN2B inhibitor) (Fig. 4G), showed similar effects ( Fig. 4H and I; GluN2A: P < 0.0188 vs. STZ-AdipoRon; GluN2B: P < 0.011 vs. STZ-AdipoRon). Oneway ANOVA revealed significant effects of pharmacological inhibitions of NMDAR subunits on LTP formation (F 2,27 = 6.133, P = 0.0064). Altogether, the findings demonstrated the Fig. 3 AdipoRon restored dendritic arborisation, soma size, and spine density of dentate granule cells in diabetic mice. A, B In granule cells with single primary dendrite, STZ reduced A total dendritic length (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle) and B dendritic arborisation in diabetic mice (Tukey's post hoc test: # P < 0.05 vs. STZ-Vehicle), which could be restored by AdipoRon treatment (Tukey's post hoc test: *P < 0.05 vs. STZ-Vehicle). C Diabetic condition and AdipoRon treatment did not alter soma size in single-branched neurons. D, E: D In granule cells with multiple primary dendrites, STZ reduced total dendritic length (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle). Meanwhile, AdipoRon treatment restored total dendritic length (Tukey's post hoc test: *P < 0.05 vs. STZ-Vehicle) and E promoted dendritic arborisation in diabetic mice (Tukey's post hoc test: # P < 0.05 vs. STZ-Vehicle), whereas AdipoRon restored the dendritic length (Tukey's post hoc test: *P < 0.05 vs. STZ-Vehicle). F AdipoRon treatment restored reduction in soma size in diabetic mice (Tukey's post hoc test: # P < 0.05 vs. Control-Vehicle, *P < 0.05), but AdipoRon did not affect soma size in multiple-branched neurons of control mice. G AdipoRon treatment increased spine density in the granule cells with single primary dendrite in diabetic mice (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle). H Diabetes reduced spine density in granule neurons with multiple primary dendrites (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle), which could be restored by AdipoRon treatment in both control and diabetic mice (Tukey's post hoc test: **P < 0.005). n = 5 animals per group. I Representative Golgi staining of the hippocampal dentate gyrus, with green arrow marking the multiplebranched (more mature) neuron at the outer granule cell layer, and the red arrow marking a single-branched (late-born) neuron at the inner granule cell layer involvement of PGC-1α and NMDA receptor subunits GluN2A and GluN2B in the action of AdipoRon on restoring synaptic plasticity in diabetic mice.

AdipoRon Treatment Promoted BDNF Levels and Activated AMPK/PGC-1α Signalling in the Hippocampal DG
Voluntary running exercise elevates adiponectin levels in the whole hippocampus but not in the serum of healthy mice (69). Our results showed that AdipoRon treatment restored serum adiponectin levels in diabetic mice ( Fig. 5A; STZ-AdipoRon: P = 0.0434 vs. STZ-Vehicle) resembling the effect of voluntary wheel running ( Fig. 5A; STZ-Exercise: P = 0.011 vs. STZ-Vehicle). However, AdipoRon treatment did not restore the decrease in adiponectin level in the DG of diabetic mice ( Fig. 5B; P = 0.994 vs. STZ-Vehicle) and reduced adiponectin levels in the control mice ( Fig. 5B; P < 0.0001 vs. Control-Vehicle).
BDNF is a critical neurotrophin required for physical exercise to induce hippocampal neurogenesis. We examined whether AdipoRon enhances BDNF levels and AMPK/ PGC-1α signalling that is also be activated by physical exercise (34,39,70). AdipoRon and exercise treatment elevated BDNF levels in the control mice ( Fig. 5C; Control-AdipoRon: P < .0001 and Control-Exercise: P < 0.0001 vs. Control-Vehicle). Diabetic condition reduced serum BDNF levels ( Fig. 5C; STZ-Vehicle P < 0.0001 vs. Control-Vehicle). AdipoRon and voluntary running restored serum BDNF levels in diabetic mice ( Fig. 5C; STZ-AdipoRon: P = 0.0039 and STZ-Exercise: P = 0.0098 vs. STZ-Vehicle). Apart from the elevated serum BDNF levels following AdipoRon treatment, BDNF levels in the hippocampal DG were increased, though diabetes did not alter BDNF levels in the dentate region ( Fig. 5D; P = 0.743 vs. Control-Vehicle). Conversely AdipoRon treatment promoted BDNF levels in the DG of diabetic ( Fig. 5D; P = 0.0039 vs. STZ-Vehicle) and control mice ( Fig. 5D; P < 0.0001 vs. Control-Vehicle). These findings implicated that increased BDNF levels could be linked to the promoting effect of AdipoRon on hippocampal neuroplasticity.
We have previously shown that adiponectin mediates physical exercise-induced adult hippocampal neurogenesis through AdipoR1/AMP-activated protein kinase (AMPK). Besides, AMPK and PPAR-γ coactivator 1-α (PGC-1α) are potential upstream transducers of BDNF; therefore, we further tested whether AdipoRon elicited its effect via the AMPK/ PGC-1α pathway (Fig. 5E). Western blot analysis showed that AdipoRon treatment enhanced the phosphorylation of AMPK Thr172 (Fig. 5F) in both diabetic and control mice. AdipoRon treatment also activated PGC-1 α in diabetic and control mice, as indicated by reducing the inhibitory form of PGC-1α Ser571 (Fig. 5G). In sum, the results suggested that AdipoRon treatment significantly increased AMPK and PGC-1α activity and BDNF levels in the DG.

Discussion
This study demonstrates that AdipoRon treatment elicits procognitive and neurotrophic effects indicating its potential in acting as an exercise mimetic. Here we demonstrated that AdipoRon treatment rescued spatial memory deficits in diabetic mice in a manner that was reminiscent of the procognitive effects observed with voluntary running exercise (see also Appendix Tables S1 and S2). AdipoRon also rescued adult hippocampal neurogenesis, increased dendritic arborisation and spine density, enhanced hippocampal LTP, and increased serum and hippocampal DG levels of BDNF in diabetic mice.
The antidiabetic effects of AdipoRon were first demonstrated in high-fat diet-fed mice and leptin-deficient (db/db) mice (42). In this study, we used a diabetic mouse model presenting more pronounced cognitive impairments to examine the therapeutic effects of AdipoRon for cognition. We have previously reported that streptozotocin-induced diabetes impairs adult hippocampal neurogenesis (30). Our results . n = 7 per group. Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). B Diabetes significantly reduced adiponectin level in DG (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle), whereas AdipoRon treatment reduced adiponectin levels in control (Tukey's post hoc test: **P < 0.005 vs. Control-Vehicle). AdipoRon treatment did not restore adiponectin levels in DG from diabetic mice. n = 5 replicates. C Diabetes reduced serum BDNF levels (Tukey's post hoc test: ## P < 0.005 vs. Control-Vehicle). AdipoRon and exercise treatment increased serum BDNF levels in control animals (Tukey's post hoc test: **P < 0.005 vs. Control-Vehicle), while both treatments restored serum BNDF levels in diabetic mice (Tukey's post hoc test: **P < 0.005 vs. STZ-Vehicle). n = 7 per group. Previously reported Control-Vehicle and Control-AdipoRon data were used to support this study (44). D Diabetes did not alter BDNF level in DG, whereas AdipoRon treatment increased BDNF levels in control (Tukey's post hoc test: **P < 0.005 vs. Control-Vehicle) and diabetic mice (Tukey's post hoc test: *P < 0.05 vs. STZ-Vehicle). n = 5 replicates. E Representative images of western blot analysis. (n = 4 technical replicates from 7 pairs of DG pooled-sample/group). F AdipoRon treatment also increased p-AMPKα Thr172 expression in the hippocampal DG (LSD post hoc test: *P < 0.05 vs. Control-Vehicle). G AdipoRon treatment significantly suppressed the expressions of p-PGC-1α Ser571 , a phosphorylated inhibitory form of PGC-1α, in the DG of control (LSD post hoc test: *P < 0.05 vs. Control-Vehicle) and diabetic mice (LSD post hoc test: ** P < 0.05 vs. STZ-Vehicle) support a prior study that a single injection of streptozotocin (65 mg/kg i.v.) can induce hippocampal atrophy, spine loss, and hippocampal-dependent spatial learning and memory impairment (71).
Physical exercise facilitates learning and memory formation, enhances adult hippocampal neurogenesis, increases dendritic arborisation, and improves hippocampal synaptic plasticity (72)(73)(74)(75)(76). In addition to promoting adult hippocampal neurogenesis (33) and learning and memory performance (32), adiponectin can mediate the effects of physical exercise on reducing depression and promoting hippocampal cell proliferation (30,34). In the current study, we showed that the adiponectin receptor agonist, AdipoRon, could similarly restore hippocampal cell proliferation, BDNF levels, and memory performance in diabetic mice (34,62). Furthermore, AdipoRon treatment restored dendritic atrophy and spine density in DG granule cells of the diabetic mice. Adiponectin has shown to be indispensable for dendritic and spine formation in the DG granule neurons (33). Our results showed that AdipoRon treatment promoted dendritic complexities and spine densities in the dentate granule neurons associated with the increased serum and dentate BDNF levels, whereas diabetic mice showed a significant reduction in serum and dentate adiponectin levels. AD mice show decreased adiponectin levels and spine density in the cortex and hippocampus, which could be restored by AdipoRon treatment (31). Our results collectively suggest that AdipoRon treatment resembles the pro-cognitive and neurotrophic effects of voluntary running and could be an effective treatment for restoring structural plasticity and improving learning and memory associated with diabetes.
Both adiponectin and AdipoRon can cross the blood-brain barrier (31,34). To examine the direct effect of AdipoRon, we showed that AdipoRon, like adiponectin, can elicit direct proliferative effects in the N2a cultured cell, where adiponectin receptors are expressed (34). The current results have demonstrated the direct effect of AdipoRon on restoring LTP formation in the dentate region in diabetic mice. It is known that adiponectin modulates LTP formation in the hippocampal CA1 (8) and CA3 (32) regions, whereas AdipoRon can restore LTP deficits induced by adiponectin deficiency (32). Our findings indicated the significant effect of AdipoRon on restoring synaptic deficits in the hippocampus, a brain region with AdipoR1 and AdipoR2 being expressed (32). NMDA receptor (NMDAR) subunits, such as GluN2A and GluN2B, mediate the LTP induction in the hippocampus (77,78), while adiponectin deficiency reduces the expressions of the αamino-hydroxy-methyl-isoxazolepropionic acid receptor (AMPAR) and NMDAR subunits in the hippocampus (79). The expressions of AMPAR and NMDAR subunits and adiponectin receptors are increased in post-recording 5×FAD hippocampal slices (80). The present study also addressed the critical roles of GluN2A/B in AdipoRon-induced LTP formation in hippocampal slices from diabetic mice, suggesting the involvement of NMDA receptors in AdipoRoninduced synaptic plasticity in the hippocampus.
AdipoRon might exert indirect action on promoting hippocampal plasticity because serum adiponectin levels in diabetes were increased by AdipoRon treatment. Adiponectin levels in the DG were markedly reduced in diabetic mice, and AdipoRon treatment failed to restore the decrease. Besides, AdipoRon treatment reduced adiponectin levels in the DG in physiological condition. A feedback mechanism may be involved in downregulating the adiponectin levels in the brain in response to the AdipoRon action on activating central adiponectin signalling. In contrast, excessive adiponectin in the brain or hyperactivation of the adiponectin signalling pathway may impair neural plasticity. Another possibility is that AdipoRon may increase the expression levels of adiponectin receptors in the hippocampal DG. Therefore, the reduction in adiponectin levels serves as a negative feedback regulation to avoid over-activation of adiponectin signalling. Lastly, adiponectin receptors are expressed on endothelial cells of the blood-brain barrier (BBB) (81), and adiponectin signalling maintains the BBB integrity (82). AdipoRon administration and elevated circulating adiponectin may alter the BBB permeability, limiting the entry of adiponectin to the brain from the circulation.
Elevated BDNF levels promote adult neurogenesis (83) and synaptic plasticity (84), which in turn facilitate learning and memory formation (85). Our findings demonstrated that enhancement in structural and synaptic plasticity is associated with elevated dentate and serum BDNF levels following AdipoRon treatment. The central BDNF levels are known to be positively correlated with peripheral BDNF levels (86). Physical exercise promotes hippocampal BDNF levels through PGC-1α signalling (39). Over-expression of PGC-1α restores BDNF levels in Aβ-treated N2a cells (87). We have previously shown that adiponectin is required for exercise to induce AMPK activation in the hippocampus (34); however, administration of AdipoRon activated AMPK/ PGC-1α signalling without increasing adiponectin levels in diabetic mice, suggesting a direct action of AdipoRon on activating adiponectin receptors and thus its downstream signalling cascade. The beneficial effects of AdipoRon on restoring hippocampal neuroplasticity could be linked to the activation of AMPK/PGC-1α signalling pathway (Fig. 6). Our study reported that AdipoRon treatment enhances PGC-1α activity in the DG. The in vitro proliferative assay has suggested the involvement of PGC-1α/PPAR-γ in the AdipoRon-induced cell proliferation and the involvement of PGC-1α in the AdipoRon-induced LTP formation in diabetic mice. PGC-1α mediates the AMPK-induced neuronal growth and maturation (88). PGC-1α is also essential for spine formation (40) and is a master regulator of mitochondrial biogenesis (66). Mitochondrial Ca 2+ uptake is involved in LTP formation in the hippocampus (68). It is possible that AdipoRon could activate PGC-1α activity to promote mitochondrial biogenesis, which in turn promotes Ca 2+ influx; and facilitates LTP formation.
Furthermore, AMPK is a master regulator of metabolism (89) and is necessary for maintaining neural stem cell proliferation (90). Voluntary running promotes hippocampal AMPK activation (40), which is absent in adiponectin knockout mice (34). Conversely, adiponectin deficiency reduces hippocampal AMPK activation (32). Activating AMPK activity leads to increased BDNF levels in a dose-dependent manner (91). We reported that AdipoRon treatment increased the serum adiponectin levels, but not in the dentate gyrus in diabetic mice, suggesting the activation of AMPK in the dentate region could be a direct effect of AdipoRon. Taken together, our results have suggested the involvement of AMPK/PGC-1α/BDNF signalling in the action of AdipoRon on rescuing hippocampal atrophy in diabetic mice.

Conclusion
Our results demonstrated that chronic treatment with AdipoRon mimicked the beneficial effects of physical exercise on promoting hippocampal-dependent learning and memory function and restoring adult neurogenesis in diabetic condition. In addition, AdipoRon treatment promoted dendritic complexity and synaptic plasticity as well as serum and dentate BDNF levels in the hippocampus of diabetic mice. The beneficial effects could be dependent on downstream AMPK/ PGC-1α signalling. An adiponectin-based therapeutic which can enhance hippocampal BDNF levels could serve as a pharmacological intervention for restoring diabetesassociated learning and memory impairment.

Declarations
Ethics Approval This study was approved by the Animal Subjects Ethics Sub-Committee (ASESC Code: 16-17/10-RS-R-GRF), The Hong Kong Polytechnic University. Fig. 6 Summary of the effects of AdipoRon on promoting hippocampal neuroplasticity. Two weeks of AdipoRon treatment significantly promoted cell proliferation and neuronal differentiation in the dentate region. AdipoRon restored diabetes-induced impairment in adult hippocampal neurogenesis, dendritic complexity, and spine density, as well as NMDA receptor-dependent synaptic plasticity, through activating AMPK/PGC-1α/BDNF signalling in the hippocampus. Graphical images are created in BioRender© Consent to Participate N/A, Consent for Publication N/A.

Conflict of Interest The authors declare no competing interests.
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