Novel therapeutic potential of angiotensin receptor 1 blockade in a rat model of diabetes-associated depression parallels altered BDNF signalling

Abstract

Aims/hypothesis: Diabetes is a worldwide epidemic linked with diverse diseases of the nervous system, including depression. A few studies suggested a connection between renin-angiotensin-aldosterone system blockers and reduced depressive symptoms, although underlying mechanisms are unclear. Here we investigated the antidepressant effect and the mechanisms of action of the angiotensin receptor 1 blocker (ARB) losartan in an experiential model of diabetes-associated depression.

Methods: Experimental diabetes was induced by streptozotocin in adult male Wistar rats. After 5 weeks of diabetes, rats were treated for 2 weeks with a non-pressor oral dose of losartan (20 mg/kg). In protocol 1, cerebrovascular perfusion and glial activation were evaluated by single-photon emission computed tomography-MRI and immunohistochemistry. In protocol 2, behaviour studies were performed (forced swim test and open field test). Hippocampal proinflammatory response and brain-derived neurotrophic factor (BDNF) signalling were also assessed.

Results: Here, we show that diabetic rats exhibit depression-like behaviour, which can be therapeutically reversed by losartan. This action of losartan occurs via changes in diabetes-induced neuroinflammatory responses rather than altered cerebral perfusion. We also show that as a part of its protective effect losartan restores BDNF production in astrocytes and facilitates BDNF-tropomyosin receptor kinase B-cAMP response element-binding protein signalling in the diabetic brain.

Conclusions/interpretation: We identified a novel effect of losartan in the nervous system that may be implemented to alleviate symptoms of diabetes-associated depression. These findings explore a new therapeutic horizon for ARBs as possible antidepressants and suggest that BDNF could be a target of future drug development in diabetes-induced complications.

Keywords: Angiotensin receptor 1 blocker; Brain-derived neurotrophic factor; Depression; Neuroinflammation; Renin–angiotensin–aldosterone system.

Figures

Fig. 1

Losartan attenuates depressive behaviour of diabetic rats. OFT and FST were performed on control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). (a) In OFT, locomotor activity is represented by number of grid crossings. (b–f) In FST, floating reflects immobility (b), while time spent in active mobility (c) is represented by struggling (d), swimming (e) and diving (f). Each moving pattern is represented as percentage of time. Data are presented as mean ± SD (n = 6–8/group). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control rats; †p < 0.05 vs DM

Fig. 2

Decreased cerebral perfusion of diabetic rats is unaltered by losartan. (a) In control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS), mean arterial pressure was calculated. (b, c) Cerebral blood perfusion was measured by SPECT–MRI based on [99mTc]HMPAO uptake. Representative images from different sections of the whole brain. Colours represent the magnitude of [99mTc]HMPAO uptake (green, purple and yellow represent low, middle and high uptake, respectively) (b). Quantified data revealed significantly decreased perfusion in all investigated brain regions in DM and DM+LOS rats vs controls (c). (d, e) Cerebrovascular activation was assessed by ICAM-1 immunostaining in coronal brain sections of the dentate gyrus. Images are representative. Scale bar, 50 μm. (d) Integrated density of fluorescent images was determined by using Image J and expressed as arbitrary units (e). (f, g) Protein levels of p-eNOS (f) and endothelin-1 (g) in rat hippocampus were also evaluated (fold change vs control, which was set as 1). Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3a). Data are presented as mean ± SD (n = 4–5/group for imaging and n = 6–8/group for inflammatory markers and histology). *p < 0.05 and **p < 0.01 vs control rats. AU, arbitrary units; B. olfactorius, bulbus olfactorius

Fig. 3

Losartan mitigates proinflammatory cytokine responses in diabetic rats. Neuroinflammation was assessed by various methods in control (non-diabetic), vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). (a, b) Microglia activation was measured indirectly by SPECT–MRI based on [125I]CLINME uptake. (a) Representative images from different sections of the whole brain. Colours represent the magnitude of [125I]CLINME uptake (green, purple and yellow represent low, middle and high uptake, respectively) (b) Quantified data revealed significantly increased CLINME uptake in all investigated brain regions in DM and DM+LOS rats. (c) Microglial activation was assessed by P2Y12 immunostaining. Arrowheads showing P2Y12-positive microglia with thicker cell bodies and a partial withdrawal of processes (indicative of higher level of microglial activity). Scale bar, 50 μm. Quantification was based on unbiased densitometric analysis of P2Y12 signals in coronal brain sections of the dentate gyrus. (d) GFAP immunostaining showing astrocyte responses to diabetes and losartan and unbiased densitometric analysis showing reduced GFAP levels in coronal brain sections of the dentate gyrus in the DM+LOS group compared with the DM group (and control group). Scale bar, 100 μm. Due to technical problems (inappropriate immunostaining), 5/7 rats were analysed in the DM group. In (c) and (d), images are representative; integrated density of fluorescent images was determined by using Image J software. (e–h) Elevated protein level of NF-κB and mRNA expression of Il1a, Il6 and Tnf were measured in the vehicle-treated diabetic rats; these effect was abolished by losartan treatment. Data are presented as fold change vs control, which was set as 1. Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3a.). Il1a, Il6 and Tnf mRNA expression levels were normalised to Rn18s expression. For Il6 and Tnf, due to technical problems (inappropriate RNA isolation and PCR), 7/8 rats were analysed in the vehicle-treated diabetic group. Data are presented as mean ± SD (n = 4–6/group for imaging and n = 6–8/group elsewhere). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control rats; †p < 0.05 and †††p < 0.001 vs DM. AU, arbitrary units; B. olfactorius, bulbus olfactorius

Fig. 4

BDNF production and localisation is normalised by losartan in diabetic rats. (a–f) Representative blot (a) and quantification of protein levels of proBDNF (b), mBDNF (c) and cleavage enzymes (d–f) in the hippocampus of control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). Data are presented as fold change vs control, which was set as 1. Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3b). (g) Triple immunofluorescence staining showing BDNF, GFAP-positive astrocytes and Iba1-positive microglia in the brain. Arrowheads in inserts indicate astrocytes with BDNF immunopositivity. Representative images of coronal brain sections of the dentate gyrus from three rats (representing each group) are shown. Scale bar, 200 μm. Data are presented as mean ± SD (n = 6–8/group). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control rats; †p < 0.05 and †††p < 0.001 vs DM

Fig. 5

Losartan alters neuronal responses via mBDNF–TrkB signalling. (a–f) Representative blot and protein levels of p75NTR (b), p-JNK (c), TrkB (d), p-ERK (e) and p-CREB (f), in the hippocampus of control, diabetic (DM) and losartan-treated diabetic (DM+LOS) rats. Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3b). (g, h) mRNA expression levels of Bax (g) and Bcl2 (h) in the hippocampus. mRNA expression of Bax and Bcl2 was normalised to Rn18s mRNA expression. In (a–h), Data are expressed as fold change vs control, which was set as 1. (i) Immunofluorescence images showing a marked upregulation of p-CREB in the hippocampal dentate gyrus in response to treatment with losartan. Integrated density of fluorescent images was determined using Image J software. Densitometric analysis showed a significant difference in p-CREB levels in DM+LOS compared with control and DM. No colocalisation of p-CREB with P2Y12-positive microglia (green) or blood vessels identified by tomato lectin (blue) can be seen. Scale bar, 100 μm. (j) p-CREB staining shows colocalisation with neurons identified by the pan-neuronal marker protein gene product 9.5 (PGP 9.5). Scale bar, 50 μm. In (i) and (j), representative images of the dentate gyrus are shown as captured in coronal brain sections. . Data are presented as means ± SD (n = 6–8/group). *p < 0.05 and **p < 0.01 vs control rats; †p < 0.05 and ††p < 0.01 vs DM. AU, arbitrary units