Neuroprotective Role of DPP-4 Inhibitor Linagliptin Against Neurodegeneration, Neuronal Insulin Resistance and Neuroinflammation Induced by Intracerebroventricular Streptozotocin in Rat Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is an age-related, multifactorial progressive neurodegenerative disorder manifested by cognitive impairment and neuronal death in the brain areas like hippocampus, yet the precise neuropathology of AD is still unclear. Continuous failure of various clinical trial studies demands the utmost need to explore more therapeutic targets against AD. Type 2 Diabetes Mellitus and neuronal insulin resistance due to serine phosphorylation of Insulin Receptor Substrate-1 at 307 exhibits correlation with AD. Dipeptidyl Peptidase-4 inhibitors (DPP-4i) have also indicated therapeutic effects in AD by increasing the level of Glucagon-like peptide-1 in the brain after crossing Blood Brain Barrier. The present study is hypothesized to examine Linagliptin, a DPP-4i in intracerebroventricular streptozotocin induced neurodegeneration, and neuroinflammation and hippocampal insulin resistance in rat model of AD. Following infusion on 1st and 3rd day, animals were treated orally with Linagliptin (0.513 mg/kg, 3 mg/kg, and 5 mg/kg) and donepezil (5 mg/kg) as a standard for 8 weeks. Neurobehavioral, biochemical and histopathological analysis was done at the end of treatment. Dose-dependently Linagliptin significantly reversed behavioral alterations done through locomotor activity (LA) and morris water maze (MWM) test. Moreover, Linagliptin augmented hippocampal GLP-1 and Akt-ser473 level and mitigated soluble Aβ (1–42), IRS-1 (s307), GSK-3β, TNF-α, IL-1β, IL-6, AchE and oxidative/nitrosative stress level. Histopathological analysis also exhibited neuroprotective and anti-amylodogenic effect in Hematoxylin and eosin and Congo red staining respectively. The findings of our study concludes remarkable dose-dependent therapeutic potential of Linagliptin against neuronal insulin resistance via IRS-1 and AD-related complication. Thus, demonstrates unique molecular mechanism that underlie AD.


Introduction
Alzheimer's disease is an irreversible multifactorial neurodegenerative disease that leads to the deterioration of brain functions [26] and accounts for around 80% of dementia globally in elder patients [20]. There are several molecular mechanisms underlie AD yet the ideal neuropathology of AD is still unknown [3]. It is manifested by neuropathological hallmarks that include extracellular deposition of amyloid-beta (Aβ) senile plaques, intracellular deposition of hyper phosphorylated tau protein neurofibrillary tangles, neuronal and synaptic death in the hippocampus [62]. CA1/ CA3 regions of hippocampus is more vulnerable to neuronal death in the brain [43]. Currently approved medications including acetylcholinesterase (AchE) inhibitors like donepezil, rivastigmine and N-methyl-D-aspartate (NMDA) receptor antagonist like memantine are supposed as the golden standard for the symptomatic relief of AD [62] and unfortunately failed to counteract the disease progression. Therefore, it is an important need to develop more effective, therapeutic and safer drugs which may reverse the disease condition.
Numerous research evidence supports the ameliorative effect of DPP-4 inhibitors (DPP-4i) against neurodegeneration in different experimental models of brain disease [5,35,36,41] by increasing the level of Glucagon-like peptide-1 (GLP-1) in the circulation that crosses Blood Brain Barrier (BBB) [31] where binds with GLP-1 receptor (GLP-1R) and mitigates neurodegeneration and neuronal resistance [21,23,32,51]. Therefore, pharmaceuticals for the treatment of T2DM could also be effective in ameliorating hippocampal insulin resistance and AD-related complications.
Moreover, Rohnert et al. first reported that DPP-4i showed neuroprotection against stroke via intracerebral administration of sitagliptin in the rat [48]. 4 weeks peroral pre-treatment followed by 3 weeks post-stroke treatment with Linagliptin has also indicated neuroprotection by reducing brain damage after stroke in both normal and T2D/obese mice [15]. However, Darsalia et al. have also reported that chronic administration of Linagliptin against stroke mitigates neurodegeneration which could be due to the augmented proliferation of neural stem cells [17] and was not mediated by the GLP-1R [16]. Nonetheless, more research is required to understand the exact cellular and molecular mechanisms of DPP-4i against neurodegeneration in the brain.
During the Insulin Signalling pathway (ISP), the binding of insulin to its receptor instigates downstream IRS-1/PI3K/ AKT/GSK-3β pathway which results in a neuroprotective effect in the brain [6]. Downregulation of this pathway due to the deposition of Aβ oligomers/plaques in the hippocampus results in serine phosphorylation of IRS-1 at position 307 (IRS-1 ser307) leading to neuronal insulin resistance and neurodegeneration [51]. The high density of IRS-1 ser307 level in the hippocampus has also been identified during an autopsy of the brain of AD patients [10].
Streptozotocin (STZ) is a beta cytotoxic glucosaminenitrosourea drug that has been cited to induce neurodegeneration, insulin resistance in the brain and many pathological aspects of AD in subdiabetogenic doses [46]. Impaired cholinergic neurotransmission, Aβ  senile plaques, intracellular deposition of neurofibrillary tangles, cognitive dysfunctions, neuroinflammation and oxidative stress are the altered cellular conditions that have been triggered by intracerebroventricular streptozotocin (ICV-STZ) [29,40]. Considering all this evidence, the pathologies resembling AD were recapitulated in rats by administration of a subdiabetogenic ICV dose of STZ. This is our first study to examine the therapeutic effect of Linagliptin on neuronal insulin resistance via IRS-1 s307 and AD-related complications in ICV-STZ induced AD model in Albino Wistar rats (Fig. 1).
Linagliptin, a DPP-4 inhibitor exhibits therapeutic effects against peripheral insulin resistance [42] and thus holds a potential curative approach for mitigating neuronal insulin resistance and Alzheimer's disease-like complications. However, it does not cross the BBB but it inhibits DPP-4 peripherally that augments GLP-1 level in the circulation, which does cross BBB to provide consolidation of memory and synaptic plasticity [50,51]. This is our first study to evaluate the effect of Linagliptin on AD-related pathologies by modulating neuronal insulin resistance via IRS-1 s307 and to determine whether the neuroprotective effect is associated with brain insulin resistance in ICV-STZ induced AD model in Albino Wistar rats.

Drugs and Chemicals
All the drug solutions were freshly prepared before conducting the experiment. Linagliptin was procured from Cayman Chemicals Company, USA. Donepezil was purchased from Cipla Pharmaceutical Company, India. STZ for rats was purchased from Sigma Aldrich, USA. Aβ (1-42) Elisa kit was procured from Elabsciences Pvt. Ltd. India. GLP-1 Elisa kit, IRS1-s307 Elisa kit, Akt-ser473 and Gsk-3β Elisa kit were obtained from Genxbio, India. TNF-α, IL-1β and IL-6 Elisa kits were purchased from Krishgen Biosystems, India.
Three different treatment groups rats received three different doses of Linagliptin (0.513 mg/kg, 3 mg/kg, 5 mg/ kg) respectively. The dose of Linagliptin (0.513 mg/kg) was calculated from the corresponding human dose (5 mg) as recommended by FDA for the treatment of diabetes. 3 mg/kg and 5 mg/kg doses were based on the prior evidence available [30,36]. Linagliptin and reference standard donepezil (5 mg/kg) [62] were dissolved in water according to their respective doses prior to oral administration to treatment rats using oral gavage. STZ (3 mg/kg) in 5 µl citrate buffer will be administered bilaterally as the ICV route. 2.5 µl into the left ventricle and 2.5 µl into the right ventricle of the brain on days 1 and 3 in rats [62].

Treatment Schedule
Rats were randomly and equally divided into 9 groups, comprising 7 rats in each group. Group I (Control): Oral administration of water for 8 weeks; Group II (Sham): Intracerebroventricular (ICV) infusion of citrate buffer 5 μl bilaterally on 1st and 3rd day; Group III (ICV-STZ): ICV infusion of STZ bilaterally in the hippocampus on 1st and 3rd day to induce AD; Group IV (ICV-STZ + Linagliptin): ICV-STZ was infused on 1st and 3rd day and then dose of Linagliptin (0.5 mg/kg) was administered for the period of 8 weeks; Group V (ICV-STZ + Linagliptin): ICV-STZ was infused on 1st and 3rd and then 3 mg/kg dose of Linagliptin was administered for 8 weeks; Group VI (ICV-STZ + Linagliptin): ICV-STZ was infused on 1st and 3rd and then 5 mg/ kg dose of Linagliptin was administered for 8 weeks; Group VII (Linagliptin per se): Oral administration of Linagliptin (5 mg/kg) for 8 weeks. Group VIII (ICV-STZ + Donepezil): ICV-STZ was administered on the 1st and 3rd day then Donepezil (5 mg/kg) was administered for the period of 8 weeks; Group IX (Donepezil per se): rats with oral administration of Donepezil for 8 weeks. At the end of the treatment, all the rats were assessed for neurobehavioral, histopathological and biochemical parameters. (Fig. 2).

Surgical Procedure
Animals were anaesthetized with the admixture of ketamine hydrochloride (100 mg/kg) and xylazine (5 mg/kg) i.p. on the first day of our experiment. Following anaesthesia, rats were placed on stereotaxic apparatus with proper fitting of the head skull. A sagittal cut was made in the skull to recognize Bregma and lambda. Taking Bregma as the centre point, reciprocal openings were bored into the rat skull bilaterally at the directions;-3.5 mm anterior-posterior to the Bregma, 2.0 mm two-sided to sagittal stitch and 2.7 mm dorsoventral [47]. Using 10 μl of Hamilton syringe (26-gauge), a prepared solution of 3 mg/kg of STZ was gradually infused into each opening of parallel ventricles. The syringe was left in the opening for 120 s (post-infusion period) to permit legitimate implantation of the toxicity into the hippocampus [60]. Following this, the etched skull was stitched properly and antiseptic powder was sprinkled. Rats were then housed in the cages for induction of AD. The sham group went through the same procedure yet was infused with only citrate buffer (5 µl) bilaterally.

Locomotor Activity
The locomotor activity (LA) of all experimental rats was performed to determine motor impairment in the video path analyzer. Responses were recorded between 8:30 a.m. and 1:30 p.m. under controlled laboratory conditions in the open field comprised of an acrylic box (40.6 × 40.6 × 40.6 cm3), connected with the two photo beams (16 beams/dimensions; 2.5 cm between beams) of a computer interface (TrueScan 2.0 version, Coulbourn Instruments, Allentown, Pennsylvania, USA) that counted beam breaks (100 ms sampling rate). LA of each rat in each group was recorded digitally, through disruptions of IR beams caused by movements of rats placed in the activity meter and counted for 20 min after 30 min acclimatization. The box was cleaned with 70% alcohol after each recording to prevent olfactory clues from rat odours. Five responses of LA like move time (s), rest time (s), horizontal activity (cm), mean velocity (cm/s), and total movement (#) were recorded [1].

Morris Water Maze Test
The Morris Water Maze (MWM) test was conducted to survey the memory impairment in rats [33]. Time in both acquisition and probe trials was recorded using Any-maze software (ANY MAZE, Video tracking Software, US). A dark-colored water tank of stature (50 cm) and width (130 cm), topped off with water up to 30 cm in height at a temperature of 26 ± 2 °C. The water tank was virtually divided into 4 quadrants named north, east, south and west. During the acquisition trial, a black rectangular escape platform of 8 × 6 cm 2 area was submerged underwater in one quadrant (south-west) and stayed fixed. Rats were trained for 4 sequential trials for 4 consecutive days from each quadrant to explore the hidden platform (target) within the maximum time of the 60 s and were allowed to sit onto the platform for 30 s. If failed to find the platform, were physically coordinated to the platform. The escape latency period (time taken by each rat to allocate the hidden platform) was noted for 4 days. A probe trial was conducted on the 5 th day to assess the reference memory. The platform was removed from the quadrant (south-west). Rats were allowed to assign the hidden platform quadrant for the 60 s. The % dwell time (time spent by each rat to explore the hidden platform quadrant) and platform crossings (frequency to cross the target quadrant) was recorded in all the experimental groups.

Brain Homogenate Preparation
At the end of the experiment, each rat was euthanized with overdose of sodium pentothal, forfeited and perfused with phosphate buffer saline (PBS). Brains were removed and rinsed with chilled saline. Hippocampus was homogenized with chilled phosphate buffer (0.1 M; pH 7.4) at 800 × g at 4 °C for 5 min, 10 times of tissue volume. Supernatants obtained were centrifuged at 10,000 g at 4 °C for 10 min. thus, the supernatant was collected and stored at − 80 °C to estimate biochemical parameters.

Determination of Acetylcholinesterase (AChE) Level
AChE level was measured utilising Ellman's method. 0.2 ml of acetylthiocholine iodide (75 mM), 0.1 ml of buffered Ellman's reagent DTNB (5,5-dithio-bis-(2-nitrobenzoic acid, 10 mM in 15 mM NaHCO3) and 3 ml of PBS (25 mM, pH 7.4) admixture was incubated for 10 min at room temperature. Following this, 0.2 ml of supernatant prepared was added. The optical density was recorded at 412 nm within 5 min. Change in absorbance was recorded at 30 s interval using Perkin Elmer Lambda 20 spectrophotometers. Results of all groups were expressed in nanomoles of acetylthiocholine iodide hydrolyzed per min per mg of protein [19].

Determination of Lipid Peroxidation
Free radicle damage due to ICV-STZ in the hippocampus was measured as an index of lipid peroxidation, expressed in terms of TBARS level. MDA level was evaluated by using Will's method. A reaction admixture cocktail containing 0.5 ml of tris-HCL and 0.5 ml supernatant was incubated for 2 h at 37 °C. After this, 1 ml of trichloroacetic acid (TCA, 10%) was added and centrifuged at 1000 × g for 10 min. 1 ml of the supernatant obtained was mixed with 1 ml of thiobarbituric acid (TBA 0.67%). Mixture tubes were then placed in boiling water for 10 min. After cooling, distilled water (1 ml) was added to it. Absorbance was recorded at 532 nm. TBARS level in all experimental groups was expressed as nmol MDA/mg protein [49,61].

Estimation of Catalase (CAT)
CAT level was measured by using the Calibrne method (1985). Briefly, a reaction cocktail [0.05 ml supernatant + 1 ml of hydrogen peroxide (H 2 O 2 0.019 M)], 1.95 ml phosphate buffer (0.5 M, pH 7.0) were mixed to make up the final volume of 3 ml. Absorbance was recorded at 240 nm. Change in absorbance was measured at the 30 s interval using Perkin Elmer Lambda 20 spectrophotometers. CAT activity was expressed in terms of nmol H 2 O 2 /min/ mg protein in all groups [4,49].

Estimation of Nitrite Level
The level of nitrite was determined by using the Griess reagent. Accordingly, a reaction mixture containing an equal volume of supernatant and Griess reagent (0.1% N-(-naphthyl ethylenediamine dihydrochloride, 1% sulfanilamide and 5% phosphoric acid) was incubated in dark at room temperature for 10 min. Absorbance was measured at 540 nm with a Perkin Elmer Lambda 20 spectrophotometer. Nitrite level in all groups was expressed in micromoles per mg protein using the sodium nitrite standard curve [27].

Estimation of Pro-Inflammatory Cytokines TNF-α, IL-6 and IL-β
Hippocampal neuroinflammatory cytokines (TNF-α, IL-6 and IL-1β) levels in all the experimental groups were measured by using their commercially available Elisa kits in accordance to the instructions provided by the respective manufacturer.

Protein Estimation
The total concentration of protein was estimated by Lowry's method using bovine serum albumin as a standard [37].

Hematoxylin and Eosin (H and E) Staining
Whole brain was stored in 10% formalin for 48 h after which they were embedded in paraffin for four hours to create paraffin blocks. Using microtome, coronal section of thickness 5 µm were cut and silane-coated slides were prepared. Following this the slides were washed with xylene for deparaffinization and graded ethanol for rehydration. Finally slides were stained with Hematoxylin and eosin (H&E). Photomicrographs were taken using Meiji fluorescent microscope and morphology of neurons was observed in each group [57,63].

Congo Red Staining
Whole brain was stored in 10% formalin for 48 h. Later on Paraffin blocks were prepared. Brain sections were immersed in Congo red solution (0.2%) for 1 h. Following this, counter-stained with Hematoxylin. Photomicrographs were captured under a Moticam light microscope and deposition of Aβ plaques were observed in each experimental group [24].

Statistical Analysis
Data obtained from the behavioral and biochemical assessments in all the study groups were statistically analysed by using GraphPad Prism software version 5.01. Data was represented as mean ± SEM. The level of significance was taken into consideration and significant difference between each experimental groups was determined by using two-way ANOVA (escape latency in MWM) and one-way ANOVA in all other parameters followed by post-hoc Tukey-Multiple Comparison test.

Acquisition trials
In MWM test, during acquisition trials of 4 days, ICV-STZ infused rats resulted in a significant increase in escape latency on successive days (except on Day 1, df = 8, 36 F = 0.3325, ***p < 0.001) as compared to control and sham groups which showed the memory impairment in rats. On the contrary, ICV-STZ induced increase in escape latency was found to be remarkably reduced by oral administration of Linagliptin at 3 and 5 mg/kg doses (but not by 0.513 mg/kg) and positive control DNP which is indicating the memory improving effect of Linagliptin (**p < 0.01, ***p < 0.001) (Fig. 3A).

Reference memory test or probe trial
During probe trial on day 5th, reference memory was assessed in rats of all treatment groups. It was found that the rats infused with ICV-STZ spent significantly less time Table 1 Effect of Linagliptin on locomotor activity (LA) in ICV-STZ infused rat STZ infused toxic group showed significant changes in the locomotor activities as compared to control and sham group respectively. Linagliptin   3B) and platform crossing frequency (df = 8, 54 F = 23.12 *p < 0.05, **p < 0.01, ***p < 0.001) (Fig. 3C) as compared to toxic rats. These outcomes depicts that Linagliptin at 3 and 5 mg/kg dose but not at 0.513 mg/kg dose has a potential to improve learning and memory in rats with AD-like pathological changes.

Linagliptin Increases Hippocampal GLP-1 Level in STZ Induced Rats
DPP-4 inhibitors increases the level of GLP-1 in the circulation that crosses BBB to produce neuroprotective effect in brain. After depicting improvement in the neurobehavioral parameters, the effect of Linagliptin at different doses on GLP-1 level was examined. Results showed a significant decrease (df = 8, 45 F = 11.61 ***p < 0.001) in hippocampal GLP-1 level in ICV-STZ infused rats when compared with control and sham group respectively. Doses of Linagliptin (3 and 5 mg/kg) and DNP (5 mg/kg) significantly augments the level of GLP-1 in brain of STZ infused rats (*p < 0.05, ***p < 0.001). Whereas, Linagliptin at the lower dose 0.513 mg/kg did not produced any effect (p > 0.05) on GLP-1 level when compared to diseased rats (Fig. 4).

Linagliptin Ameliorates the Deposition of Soluble Aβ (1-42) Senile Plaques in Brain Hippocampus
Deposition of Aβ  peptides results in the formation of senile plaque which is the pathological hallmark of AD. Level of soluble Aβ  in the rat hippocampus using the rat specific ELISA kit was examined. ICV-STZ rats indicated prominent elevation in the level of Aβ (1-42) in hippocampus (df = 8, 45 F = 52.45 ***p < 0.001). This elevation in Aβ (1-42) level was significant when compared to other groups. 3 and 5 mg/kg dose of Linagliptin and 5 mg/kg dose of DNP remarkably reversed the STZ induced augmentation in Aβ  in rat hippocampus (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent to the trend, 0.513 mg/kg dose of Linagliptin did not show any change in Aβ (1-42) level in rat hippocampus (p > 0.05) (Fig. 5).

Linagliptin Augments AKT (ser473) Phosphorylation in Hippocampus in ICV-STZ Induced Rats
Aβ deposition in AD leads to down regulation of PI3K/ Akt signalling pathway and up regulation of GSK-3β that results in hyper phosphorylation of Tau protein which sequentially results in the intracellular deposition of neurofibrillary tangles. Results showed a significant decrease (df = 8, 45 F = 35.48 ***p < 0.001) in hippocampal AKT (ser473) level in ICV-STZ infused rats when compared with control and sham group. Linagliptin (3 and 5 mg/kg) and DNP (5 mg/kg) treatment significantly augments the level of AKT (ser473) level in brain of ICV-STZ infused rats (*p < 0.05, ***p < 0.001). Linagliptin at the lower dose 0.513 mg/kg did not produced any effect on AKT (ser473) level when compared to diseased group (Fig. 7).

Linagliptin Dose-Dependently Attenuated Acetylcholinesterase (AChE) Activity in Hippocampus
It is well known that Acetylcholinesterase (AChE) enzyme is mainly liable for the degradation of ACh (Acetylcholine), a neurotransmitter in the brain, into acetate/choline resulting into cholinergic decline responsible for cognitive deficit in AD [52]. A significant (df = 8, 45 F = 48.43 ***p < 0.001) increase in the AChE level in the ICV-STZ infused toxic group as compared to the control and sham groups. A trend was observed in the Linagliptin 0.513 mg/kg group when compared with the ICV-STZ infused toxic group. Whereas,  Table 2).

Dose-Dependent Effect of Linagliptin on Hippocampal Oxidative/Nitrosative Stress Markers (TBARS, Nitrite, and CAT)
ICV-STZ infused rats showed dose-dependent significant difference on the level of oxidative and nitrosative stress markers. A significant (***p < 0.001) increase in the level of TBARS and Nitrite and decrease in CAT enzyme (antioxidant) was observed in the ICV-STZ infused toxic rats when compared with control and sham groups. These alterations in TBARS, Nitrite and CAT enzyme level was significantly reversed by Linagliptin

Histopathological Analysis
Histological analysis of rat's hippocampus was carried out by H&E and Congo red staining specific for morphology of neurons and deposition of Aβ respectively. In control and sham groups, H&E and Congo red staining exhibited intact However, ICV-STZ infused rat's exhibits toxic effect in both the staining. In H&E staining, a significant increase in the eosinophilic cytoplasm, pyknotic nuclei and extensive vacuolization was observed in rat hippocampus. Whereas, in Congo red staining, a significant augmentation in the Aβ (1-42) plaques deposition can be observed in STZ infused rats. Moreover, treatment with Linagliptin at 0.513 mg/kg does not indicate any significant (p > 0.05) change in any of the histopathological staining when compared with toxic rats. In contrast to this notion, oral treatment with Linagliptin at 3 mg/kg and 5 mg/kg and DNP at 5 mg/kg in H and E staining and Congo red staining showed significant reduction in eosinophilic stained neurons and mild neuronal toxicity and reduction in Aβ (1-42) plaques deposition respectively when compared to toxic infused group (Figs. 10, 11).

Discussion
Alzheimer's disease (AD) is the most devastating agelinked progressive neurodegenerative disorder, manifested by several behavioral, cognitive and neuronal loss. This neurodegenerative disorder causes severe dementia over a long period of time and failure of various clinical trial studies demands the utmost need to develop the therapeutic agent that should reverse the disease progression [62]. STZ administered Intracerebroventrically in subdiabetogenic dose in rats is a well-known non-transgenic model of AD.
In our present study, in-vivo experiment was conducted in which subdiabetogenic ICV dose of STZ was administered in rats to cause neurodegeneration due to development of pathologies including cognitive deficit, Aβ (1-42) deposition, GSK-3β phosphorylation, neuroinflammation, oxidative and nitrosative stress markers in hippocampus as compared to control and sham groups [62]. Linagliptin (0.513 mg/kg, 3 mg/kg and 5 mg/kg) was investigated in the ICV-STZ induced AD in Albino Wistar rats to evaluate its neuroprotective role against hippocampal insulin resistance and AD-related complications. Moreover, an in vitro study reported that phosphorylation of IRS-1 at serine307 results in brain insulin resistance [8,9,51]. ICV-STZ infused rats exhibited an impaired motor and memory skills as assessed by behavioral paradigms viz Locomotor Activity (LA) and Morris Water Maze (MWM) test respectively. The effect of Linagliptin at different doses on motor and memory responses exhibited a dose-dependent significant changes in the photocell beam crossings in LA, acquisition and probe trial in MWM test when compared with toxic group. The observed outcomes are in line with the previous findings that acknowledged motor and memory impairment due to impairment in locomotor responses and MWM [34].
GLP-1 level in hippocampus by peripherally blocking the action of DPP-4 (enzyme that degrades GLP-1) have already proved its therapeutic benefits against various neurodegenerative disorders [5,35,36,41]. ICV infusion of STZ have reduced the level of GLP-1 in rats as compared to control and sham. Treatment with Linagliptin for period of 8 weeks significantly and dose-dependently increases the hippocampal level of GLP-1 which mitigates the pathological hallmarks of AD. Prior evidence have reported that brain insulin resistance leads to neurodegeneration [9] due to deposition of Aβ plaques [51]. Augmented GLP-1 level in hippocampus upregulates AKT/PI3K pathway whereas, downregulates GSK-3β and hyperphosphorylation of tau [11].
Deposition of Aβ in hippocampus is a key pathological hallmark of AD. Aβ  and Aβ  are the two major forms of Aβ senile plaques in the hippocampus. Aβ  is much more prone to aggregate and is toxic to neurons as compared to Aβ (1-40) [18,28]. Therefore, in our present study treatment of ICV-STZ treated rats with Linagliptin for 8 weeks resulted in a dose-dependent and significant reduction of Aβ  level as compared to control and sham rats. Our finding is consistent with a prior evidence reported by Kosaraju et al. where Linagliptin was used for the treatment of AD in 3XTg-AD mouse model of the disease [36]. The proteolytic cleavage of APP by β and γ secretases in amyloidogenic pathway is the limiting step in neurons for the generation of Aβ plaques [12]. The significant reduction in BACE 1 and γ secretase enzyme may indicate decreased deposition of plaques but no such literature is available depicting role of Linagliptin on these enzymes.
Numerous studies have reported the connection between AD and brain insulin resistance [8,9,51]. This is the first study to evaluate that Linagliptin significantly and dosedependently decreases IRS-1 S307 phosphorylation induced by i.c.v administration of STZ. Prior evidence have demonstrated the profound action of Linagliptin in increasing insulin sensitivity in ICV-Aβ (1-42) infused rat model of AD [51]. The effect of Linagliptin for 8 weeks on the level of IRS-1 (s307) followed by the ICV-STZ in hippocampus exhibited dose-dependent and significant reduction in the neuronal resistance by decreasing the phosphorylation of IRS-1 (s307). It is well reported that deposition of Aβ oligomers results in Jun amino-terminal kinases -1 (JNK-1) activation leading to neuronal insulin resistance [56]. Therefore, decrease in Aβ (1-42) and pro-inflammatory cytokines level can be the probable reason behind the neuroprotection. Moreover, effect of Linagliptin on IRS-1 (s612) and (s632/ s635) through activation of IKK (NF-κB) and Extracellular signal-regulated kinase (ERK) respectively is not yet available.
PI3K/Akt and Glycogen Synthase Kinase-3β (GSK-3β) enzyme shows connection with Aβ [39]. Zhang et al. have reported that Vildagliptin treatment against cognitive deficits in a STZ-induced type 2 diabetic rat model, is mediated via enhancing Akt/BDNF/nerve growth factor expression levels [65]. Accumulation of Aβ leads to downregulation of PI3K/Akt and upregulation of GSK-3β that which eventually results in the intracellular deposition of neurofibrillary tangles [22]. Experimental data exist revealed that augmentation in Akt protein or PI3K/Akt pathway results in the mitigation of GSK-3β level [38]. Indeed even in our study, ICV-STZ rats exhibited significant decrease in Akt protein and increase GSK-3β level which was dose-dependently and significantly attenuated by Linagliptin for the 8 weeks period.
The increased level of acetylcholinesterase (AChE) enzyme has been cited to decrease the level of acetylcholine (Ach), a neurotransmitter level in the hippocampus of AD brain by enhancing its hydrolysis [55]. Deposition of Aβ (1-42) plaques exhibited impaired cholinergic neurotransmission by augmenting AChE level [14]. Present research, ICV-STZ infusion depicted significant increase in hippocampal AChE level. Contrastly, Linagliptin showed dose-dependent and significant effect on AChE activity after 8 weeks of treatment. Our results were in support of finding of Kosaraju and co-workers summarizing the dosedependent inhibitory effect of Linagliptin on AChE activity in hippocampus [36].
Neuroinflammation responses have been evidenced to be linked to free radical formation and mitochondrial dysfunction in the development of AD [58]. Corresponding to the findings of Wang et al. that STZ injection in brain increases oxidative stress we detected significant increase in level of TBARS, NO, and decrease in the level of CAT in hippocampus as compared to control and sham [59]. However, Linagliptin significantly and dose-dependently attenuated nitrite and TBARS level while, increases CAT level. Relevant to our finding it was additionally reported in previous studies that deposition of Aβ (1-42) plaques leads to the increase in oxidative/nitrosative stress markers [44]. Therefore, plausible reason behind Linagliptin mediated attenuation in oxidative/nitrosative stress can be due to decrease in Aβ  level.
Earlier evidences depicts that imbalance in the formation and clearance of Aβ peptides plaques in neurons releases pro-inflammatory cytokines [7,54]. Results of our present study showed significant increase in TNF-α, IL-6 and IL-1β cytokines level after ICV-STZ infusion in rats. Nonetheless, dose-dependent treatment with Linagliptin exhibited significant attenuation in the level of neurotoxic cytokines in hippocampus. Accumulation of Aβ (1-42) plaques incites over activation of microglial cells which leads to detrimental effect on release of pro-inflammatory cytokines causing brain damage [45].
Brain damage is correlated with infusion of ICV-STZ in rat's hippocampus. Histological examination by H and E and Congo red indicate neuronal injury characterised by high density of eosinophilic stained cytoplasm, pyknotic nuclei and neuronal shrinking [2,64] and deposition of Aβ , dipicting significant high density of red stains in STZ infused rat hippocampus respectively [13,53]. We have also analysed the histology of hippocampal sections by both H & E and Congo red staining photomicrographs. Linagliptin showed dose-dependent and significant protective effect against neuronal injury and plaque formation in ICV-STZ induced rats when compared with toxic group. These histological results were correspondence to the behavioural and biochemical assessments of our research.

Conclusion
From the outcomes of the present research, it could be concluded that Linagliptin for the period of 8 weeks mitigates cognitive impairment, hippocampal insulin resistance and AD-like complications in hippocampal neurons of rats which is attributable to the behavioural, biochemical and histological assessment in ICV-STZ rat model of AD. This research also present a perspective on the interaction between Aβ (1-42), Type 3 Diabetes Mellitus due to serine phosphorylation of IRS-1, neuroinflammation, AD-related complications and potential therapeutic role of Linagliptin [51]. To the best of our knowledge, our study is the first to evaluate the ameliorative effect of Linagliptin at three different doses against subdiabetogenic dose of ICV-STZ induced neurodegeneration and AD related complications in rats and to determine whether the neuroprotective role is associated with neuronal insulin resistance, rendering Linagliptin a promising pharmacological intervention to prevent symptoms and slow down progression of AD. The ensemble of our research findings discussed gives new insight to the mode of action of Linagliptin on insulin signalling pathway that requires the need of further investigation in order to understand the exact cellular and molecular mechanism of action of DPP-4i against pathological events similar to AD. The limitations of this research work are also highlighted which provides information for future research. Further research is necessary to determine the precise mechanisms by which linagliptin affects the insulin signalling pathway including IRS-1/PI3K/ AKT, ERK, and IKK (NF-κB).