Epigenetic Regulation of Mammalian Target of Rapamycin Debilitates Insulin Resistance Associated Alzheimer Disease Condition in Rats


 Insulin resistance (IR) and accumulation of amyloid beta (Aβ) oligomers are potential causative factor for Alzheimer Disease (AD). Simultaneously, enhanced clearance level of these oligomers through autophagy activation bring novel insights into their therapeutic paradigm. Autophagy activation is negatively correlated with mammalian target of rapamycin (mTOR) and dysregulated mTOR level due to epigenetic alterations can further culminate towards AD pathogenesis. Therefore, in the current study we explored the neuroprotective efficacy of rapamycin and vorinostat in-vitro and in-vivo. Aβ1−42 treated SH-SY5Y cells were exposed to rapamycin (20µM) and vorinostat (4µM) to analyse mRNA expression of amyloid precursor protein (APP), brain derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), neuronal growth factor (NGF), beclin-1, microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate, lysosome-associated membrane protein 2 and microtubule associated protein 2. In order to develop IR condition, rats were fed a high fat diet (HFD) for 8weeks and then subjected to intracerebroventricular Aβ1−42 administration. Subsequently, their treatment was initiated with rapa (1mg/kg, i.p.) and vori (50mg/kg, i.p.) once daily for 28days. Morris water maze was performed to govern cognitive impairment followed by sacrification for subsequent biochemical and histological estimations. For all the measured parameters, a significant improvement was observed amongst the combination treatment group in contrast to that of the HFD + Aβ1−42 group and that of the groups treated with the drugs alone. Outcomes of the present study thus suggest that combination therapy with rapa and vori provide a prospective therapeutic approach to ameliorate AD symptoms exacerbated by IR.


Introduction
Given the fact that the brain is an insulin-sensitive organ, numerous mechanism(s) converge towards the possibility that insulin resistance (IR) condition is one of the major factor for aggravating Alzheimer disease (AD) (Boles et al., 2017), although the exact known mechanism is far from being established. The presence of insulin can be seen in various parts of the brain, including hippocampus where AD patients are mostly affected. Insulin also plays vital roles in the brain like maintenance of synaptic plasticity (Ferreira et al., 2018), regulation of pro-in ammatory cytokine secretion in microglia and astrocytes in-vitro etc (J Spielman et al., 2015;Labandeira-Garcia et al., 2017). However, given allied functional roles played by insulin in the brain, the exact pathological mechanism(s) that leads to aggravation of AD due to IR remains enigmatic.
AD is a progressive memory related neurodegenerative disorder evoking devastating cognitive impairments in affected individuals. Symptoms usually start with forgetting names/dates of important persons or events that eventually lead up to a complete loss of memory (Jahn, 2013). In many cases, the predominant pathological hallmark of AD typically embodies misfolding of amyloid beta (Aβ) and hyperphosphorylation of tau paving way for formation of neuro brillary tangles (NFTs) (M Ashraf et al., 2014), although in some cases the cause can also be genetic.
In addition to the classical pathological hallmark of AD, epidemiological studies also suggest a direct correlation between IR and AD mainly through the formation of Aβ oligomers (Mullins et al., 2017). A recently performed preclinical study in our lab further con rmed this hypothesis where nanoformulated form of rosiglitazone, a well-known drug for treatment of type 2 diabetes mellitus (T2DM) was used for the treatment of IR complicated streptozotocin (STZ) induced AD in mice (Sarathlal et al., 2020). Such kind of metabolic complication induced AD are further associated with typical epigenetic modi cations that might deleteriously affect the disease condition, majorly being histone modi cations. In continuation to the above study we have also examined the bene ciary effect of rosiglitazone in combination with a potent histone deacetylase inhibitor (HDACi), vorinostat (vori) in exerting neuroprotection against IR induced AD in mice (K C et al., 2021).
We have already explored the therapeutic e cacy of anti-diabetic drugs and HDACis alone and in combination in decreasing the IR associated AD pathology, however, recently our interest extended towards autophagy, a mechanism responsible for ushing away misfolded proteins to maintain homeostasis in the body. It is believed that during neurodegeneration autophagy has a crucial role to play, however its combination with epigenetics to unravel their therapeutic e cacy during IR associated AD is not yet explored. In this regard, we selected a highly potent autophagy activator, rapamycin (rapa) and vori to explore its potential in the aforementioned disease pathogenesis.
In AD, increase in the aggregated/mutant forms of Aβ renders failure in the process of autophagy resulting in a toxic build-up and neuronal degeneration over time. Recent studies have con rmed the role of rapa as an autophagy inducer and promotor of delayed aging extending lifespan in mice neurodegenerative models (Selman et al., 2009). Rapa mainly orchestrates its signalling through target of rapa (TOR) pathway divided into two complexes, mTORC1 and mTORC2 (Guertin et al., 2006) that senses nutrient/growth factor availability with cell metabolism (Sarbassov et al., 2005). mTORC1 inhibits autophagy through Unc51-like kinase 1 (ULK1) phosphorylation and mAtg13, the mammalian homologs of the yeast kinase Atg1 and Atg13 respectively, which are essential for the formation of preautophagosomal structures (Mizushima, 2010) in response to the presence of abundant nutrients and energy. On the other hand, mTORC2 also regulates autophagy through mTOR, phosphorylating and activating Akt and PKC (Zeng et al., 2007). Positive modulation of mTORC1 by Akt through its phosphorylation by mTORC2 activates mTORC1 function, thereby inhibiting autophagy. Long term treatment with rapa in transgenic mouse models (hAPP (J20) or PDAPP) have prevented AD-like cognitive de cits with decreased levels of Aβ 42 (Spilman et al., 2010). The results of the study con rmed that inhibition of mammalian target of rapa (mTOR) activated autophagy in the hippocampus of rapa treated mice, however, similar result was not being observed in non-transgenic PDAPP mice delineating the fact that Aβ reduction with cognitive ability improvement may be due to increased neuronal autophagy as a result of response to high Aβ levels in transgenic mice.
Further, in a study conducted by Madeo, Kroemer, and coworkers, it was established for the rst time that autophagy induction could su ce upon histone posttranslational modi cations where the cytoprotective autophagy activation in aging yeast was con rmed upon treatment with spermidine majorly due to inhibition of histone acetylation, particularly, Iki3 and Sas3, causing global histone H3 hypoacetylation, thereby repressing gene expression. Additionally, acetylation of histone H4 lysine 16 (H4K16ac) is reduced upon autophagy induction by numerous stimuli. Decreased levels of autophagy-related genes, such as, ATG9A, GABARAPL2, MAP1LC3, ULK1, ULK3, and VMP1 is correlated with H4K16 deacetylation (Füllgrabe et al., 2013). Experimental evidence strongly suggests that inhibition of the autophagic H4K16 deacetylation does not necessarily inhibit autophagy but indeed increases autophagic ux which is consistent with genome-wide investigations. Similarly, histone methylation, particularly, di and trimethylation of histone H3 at lysine 4 (Wend et al., 2013) and H4K20 (Schotta et al., 2004) are critically associated with controlling the expression of major autophagy related proteins/genes modulating transcriptional activation and repression. Previous results from our lab have already demonstrated that Sodium Butyrate (SB), a pan HDACi can elicit major neuroprotective potency against 6-hydroxydopamine induced PD in rats owing to its rigorous histone acetylation action and great blood brain barrier permeability (Sharma, Taliyan, & Singh, 2015). Moreover, several models of AD evidenced lower histone acetylation levels (Marques & Outeiro, 2013), for instance, vori in combination with curcumin elicits synergistic neuroprotective e cacy in Aβ toxicity induced in PC12 cells (Meng et al., 2014).
Hence, in the current study, initially, SH-SY5Y cells were used to determine the neuroprotective potential of a potent combination of rapa and vori. High fat diet (HFD) fed animals were induced with Aβ 1−42 peptide to mimic IR induced AD condition in wistar rats. Post induction of AD, rats were administered with rapa and vori for a period of 28days. Both the drugs were administered alone as well as in combination to investigate the possible effects on the behavioural outcome, mRNA expression of relevant marker proteins, biochemical estimation of growth factors and pyknotic neuronal count.
Other reagents of analytical grade were obtained from CDH and SRL, India, and all the biological solvents for the study were freshly prepared just before use.

In-vitro experimental procedures
Cell culture SH-SY5Y cell lines were obtained from National Centre for Cell Science, Pune, India and were maintained in DMEM/F12 and 10% FBS supplemented with 0.584g/L of glutamine and 1000µl/L of antibiotics (penicillin-streptomycin). After the cells were 70% con uent they were sub-cultured and maintained in an incubator with 5% CO 2 at 37˚C.

MTT assay
Brie y, con uent cells were trypsinized and transferred to a 2ml media containing 15ml centrifuge tube which was then centrifuged at 2000rpm for 5min in 24˚C (Eppendorf, 5430R). After centrifugation the supernatant was discarded and the formed pellet was re-suspended in 1ml media. 10 4 cells/ml was diluted and into each well of 96 well plate was added 200µl of cell suspension. A culture condition of 37ºC and 5% CO 2 was maintained for incubation of the 96 well plate. To evaluate the percentage growth inhibition at 12, 24 and 36h, three 96 well plates were prepared. Further, rapa at a concentration of 0, 5, 10, 20 and 40µM and vori at a concentration of 0, 1, 2, 4, 8µM was added separately as well as in combination, after the cells were adhered to the culture plate. Cells were washed with 1X phosphate buffered saline (PBS) and 200µl of MTT (0.5mg/ml) was added and incubated for 3 hours. MTT was then washed away with 1X PBS after 3h followed by addition of dimethyl sulfoxide (DMSO, 100µl). The mixture was then kept at room temperature for 15min until the cells were lysed and forms a violet colour.
The optical density (OD) was recorded at 573nm with 670nm as reference wavelength. The formula used for calculating the percentage growth inhibition of cells treated with rapa and vori was: % Inhibition = 100 -(Test OD/Non-treated OD)*100. Each sample was analysed in triplicate.

Drug treatment
For treatment of the respective drugs, SH-SY5Y cells were seeded at a concentration of 2.5 × 10 4 cells/ well in a 6 well plate provided with DMEM/F12 supplemented with 10% FBS and maintained at 37˚C with 5% CO 2 . After gaining 50-60% con uency, the cells were treated with 25 mM of glucose except for control (Khan et al., 2019;Yang et al., 2013). Further, 4 wells were exposed to 10µM of Aβ 1−42 alone (Ozansoy et al., 2020) as well as in the presence of 20µM rapa, 4µM vori and their combination for 24 h (rapa, 10µM and vori, 2µM) ( Table 1). Ribonucleic acid (RNA) isolation and cDNA synthesis The total RNA was isolated from the cell lysate using TRIzol™ Reagent (Invitrogen™, 15596018) according to manufacturer's protocol. Complementary deoxyribonucleic acid (cDNA) was further synthesized using RevertAid Reverse Transcriptase (Thermo Fisher, EP0441) and Random hexamer (Thermo Fisher, SO142).

Real-time quantitative polymerase chain reaction (qRT-PCR)
The qRT-PCR assays were performed using the CFX Connect Optics Module (Bio-Rad). For ampli cation 2X Maxima SYBR Green/ROX qRT-PCR Master Mix (Thermo Fisher, K0221) was used according to manufacturer's instructions with speci c primers (Table 2). PCR conditions were 95°C for 10min followed by 40cycles of 95°C for 15s, 60°C for 30s and 72°C for 30s. The relative expression levels of various mRNA were calculated using the 2 −ΔΔCT method (Sarathlal et al., 2020). Each sample was analysed in triplicate.

Induction of insulin resistance
Animals were fed with a HFD for a period of 8weeks and the HFD composition was similar to as described by Srinivasan et al (Srinivasan et al., 2005). The mice subjected to HFD exhibited characteristic features of IR as con rmed after assessment of serum TC, TG and LDL using commercially available colorimetric tests.

Induction of Alzheimer disease by Aβ 1−42
IR induced Alzheimer type of dementia animal model was developed by feeding mice with HFD for 8weeks, followed by injecting freshly prepared Aβ 1−42 peptide solution as discussed previously (Krishna et al., 2020) at a nominal dose of 5µM/kg into the lateral cerebral ventricles using the previously reported coordinates . Brie y, midline sagittal incision was made over the scalp to open the rat skull by placing the rat head in the exact location of the stereotaxic apparatus (Inco Ambala, India). Prior to the stereotaxic surgery, rats were anesthetized using an intraperitoneal injection of ketamine-xylazine mixture (80mg/kg and 5mg/kg, respectively). Antiseptic powder (Neosporin) and a single dose of intraperitoneal injection of gentamicin (40mg/kg) were administered after surgery to prevent sepsis.

Experimental protocol
Brie y, animals were divided into 6 groups and each group comprised of 6 animals as described in Table  3. The treatment was continued for 28 days. The drugs (rapa and vori) were prepared freshly just before use every day. Dose for vori was selected based on study conducted previously in our lab (K C et al., 2021;Sharma & Taliyan, 2016) whereas dose for rapa for neuroprotection was selected based on available literature (Lu et al., 2015). Rapa was initially dissolved in 100% ethanol, stored at 20°C, and diluted immediately before use in a vehicle solution containing 5% Tween 80, 5% PEG 400, and 4% ethanol. Rats were injected i.p. once daily for 28 days. Vori was prepared by dissolving in 12.5% v/v DMSO in saline and injected i.p once daily for 28 days. The behavioural parameters were assessed during the last week. Afterwards the animals were sacri ced and various biochemical parameters, mRNA analysis and histological assessments were performed. The experimental regimen and treatment groups are described in Fig. 1 and Table 3 respectively. Animals were subjected to feeding with NPD.
2 HFD Animals were fed with HFD and infused with buffer into the lateral cerebral ventricles.
Behavioural assessment: Morris water maze test Morris water maze (MWM) test is the most widely used behavioural test to analyse spatial learning and memory. Brie y, the latency to escape towards the submerged platform was recorded. The animal attains a memory for the submerged position of platform using spatial information (Morris, 1984). A circular tank of 120cm diameter and 60cm height with water lled upto 40cm divided into four equal quadrants as south-west (SW), north-west (NW), north-east (NE) and south-east (SE) was used. The platform was kept 2cm below the level of water on a randomly chosen quadrant of the pool and the position was kept Page 9/30 constant throughout the experiment. Initially animals were allowed to swim freely for 60 seconds without platform. For the training session each animal received 4 trials once from each starting point. The ceiling time was kept to 60 seconds. The animals are allowed to remain on the platform for 30 seconds before initiating the next trial. The escape latency (the time taken to locate hidden platform) was recorded using ANY-Maze video tracking system (Stoelting, USA) (Sharma, Taliyan, & Ramagiri, 2015;Sharma & Taliyan, 2014).

Probe trial
The reference memory was assessed using probe trial after 24 hours of MWM trial. In brief, the platform was removed and animals were released from one of the quadrant and allowed to explore the location of platform for 60 seconds. To evaluate the reference memory, the time it spent on target quadrant was recorded using ANY-Maze video tracking system (Stoelting, USA) (Sharma, Taliyan, & Ramagiri, 2015;Sharma, Taliyan, & Singh, 2015).

Blood Collection and serum isolation
Blood was collected using retro-orbital method. Each animal was hand restrained, the neck was gently scuffed and the eye was made to bulge. A glass capillary was inserted. Blood was allowed to ow by capillary action into the 2ml centrifuge tube. The tubes were then centrifuged at 4000 rpm, 4°C for 20min and the serum was separated in another centrifuge tube.

Serum metabolic parameters
Serum TG, TC and LDL cholesterol levels were measured using commercially available kits as per manufacturer's instructions. Each sample was analysed in triplicate.

Hippocampus homogenate preparation
Animals were sacri ced by decapitation, brains were removed and rinsed with ice-cold (0.9% w/v NaCl) isotonic saline. The whole hippocampal region was isolated and the CA1 and DG region was also separated (Chen et al., 2005) which was homogenized with ice cold 0.1 M phosphate buffer (pH 7.4) in a volume 10 times (w/v) the weight of tissue for ELISA estimations and with TRIzol™ Reagent for mRNA estimation. The homogenate was centrifuged at 12000g for 15min (4°C) and aliquots of supernatant were separated .

Protein determination
Protein content in brain hippocampus samples was measured by the method of Lowry using BSA (1 mg/ml) as a standard (Classics Lowry et al., 1951). Each sample was analysed in triplicate.
Real-time quantitative polymerase chain reaction (qRT-PCR) The total RNA was isolated from hippocampal homogenates and qRT-PCR assay was performed with protocol described under in-vitro experiments. The primers used for in vivo mRNA expression level is listed in Table 4. Estimation of global histone H3 acetylation level EpiQuik™ Global Histone H3 Acetylation Assay Kit was used to determine the level of Global Histone H3 acetylation according to manufacturer's instruction. The protocol consists of nucleic extraction whereby the hippocampus was homogenized using lysis buffer, followed by histone extraction where the histone proteins are extracted using the extraction buffer and then protein content was measured using Lowry's method and lastly histone H3 acetylation detection where the histone proteins can be stably spotted on the strip wells. A high a nity antibody was used to detect the acetylated histone H3. HRP conjugated secondary antibody-colour development system quanti ed the amount of acetylated histone H3 and was proportional to the intensity of the developed colour. Each sample was analysed in triplicate.

Histopathological analysis
The brains were rapidly removed cut into 5µm thick sections, xed with formalin (10% v/v) and embedded into para n wax followed by standard rehydration steps. Further, the sections were stained with haematoxylin and eosin stain (H & E) followed by standard dehydration steps (Sharma & Taliyan, 2014). The slides were coverslipped and hippocampal DG and CA1 region were examined under bright eld illumination using Optika TCB5 microscope (Optika Research Microscope, Italy) at 100X and 400X magni cations. Each sample was analysed in triplicate.

Statistical Analysis
The results were expressed as mean ± SD. The behavioural and biochemical parameters were analysed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using statistical graph pad prism software (version 6.01 and 9.0.0). The percentage degenerative area for the H&E staining was analysed using ImageJ software.
Results And Discussion 1) In vitro experiments a. MTT assay: MTT is a colorimetric assay that measures the reduction of yellow MTT by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dark purple) formazan product which is measured photometrically. Here, the percentage inhibition of the cells treated with rapa and vori increased in a time and concentration dependent manner.
On 12h treatment with rapa at 10μM, only 10% cells were inhibited, but 2μM of vori inhibited cells more than 20% and their combination treatment inhibited cells near to 40%. At 20μM of rapa and 4μM of vori the cell growth was inhibited by less than 40%, however their combination inhibited cell growth more than 50% ( Fig. 2A). On 24h treatment with rapa at a dose of 10μM, 20% inhibition was observed and for vori at 2μM it was nearly 35%, however their combination inhibited cell growth near to 40%. Further, 20μM rapa and 4μM vori alone inhibited 35% and 45% of cell growth respectively, however their combination drastically inhibited cell growth at about 70%. Inhibition rate was increased to 90% upon increasing the dose of rapa to 40μM and vori 8μM (Fig. 2B). On 36h treatment with rapa (20μM) and vori (4μM) inhibited cell growth more than 50% and their combination drastically inhibited growth by 80% (Fig. 2C). Based on these results we have selected rapa, 20µM; vori, 4µM for treatment in alone and rapa, 10µM; vori, 2µM for combination treatment.
b. Effect of rapa and vori on mRNA expression level of APP, BDNF, GDNF and NGF The mRNA expression of APP was increased in SH-SY5Y cells treated with high glucose and Aβ 1-42 .
c. Effect of rapa and vori on mRNA expression level of beclin-1, LC3, LAMP2 and MAP2 During AD pathogenesis, the levels of beclin-1, LC3, LAMP-2 are found to impair with disease progression leading to disruption in the autophagy machinery. Hence, in this regard we wanted to check the levels of beclin-1, LC3 and LAMP-2 in high glucose+Aβ 1-42 group and if these disrupted levels can be reversed after treatment with the combination of rapa and vori. Beclin-1 is a 60kDa protein playing a critical role in autophagy induction. It is known to control the initiating step in autophagosome assembly from preautophagic structures. LC3 is a reliable marker for autophagosomes while LAMP-2 is critically involved in lysosomal stability and autophagy. MAP-2 is a cytoskeletal protein known to stabilize the microtubule assembly.
2. In-vivo experiments a. Effect of HFD feeding on overall lipid pro le for con rmation of IR condition Before proceeding with the estimations for AD progression, we rst con rmed that the rats fed with HFD have become insulin resistant. For this purpose, we had performed the serum estimations of TC, TG and LDL levels and it was observed that a consecutive increase of TC, TG and LDL cholesterol was attained starting from week 4 up to week 8. The TC ( **** P≤0.0001), TG ( ** P≤0.01) and LDL cholesterol ( ** P≤0.01) levels of HFD fed rats considerably increased as compared with the normal pellet diet (NPD) fed group ( Supplementary Fig. 1A-1C) con rming the presence of IR condition.

b. Morris water maze
MWM is an internationally acclaimed and a well-accepted behavioural model to assess any cognitive abnormality and has been recognised as a gold standard model to evaluate AD pathology progression in rats. In the present study, we tested rats on this task and as per the anymaze tracking software, on the nal day of training we found out that the IR rats and the IR induced AD rats displayed similar delay in locating the platform. However, the animals treated with rapa and vori alone faintly decreased the time duration to locate the hidden platform when compared with HFD+Aβ 1-42 . Interestingly the rats treated with a combination of rapa and vori exhibited considerable improvement in locating the platform as compared to the HFD+Aβ 1-42 group with a signi cance of **** P≤0.0001. Similar effect was observed during the probe trial where the combination group animals spent greater time near the target quadrant when compared to the HFD+Aβ 1-42 group with a signi cance of **** P≤0.0001, but the groups treated with rapa and vori in alone spent considerably less time as compared to the HFD+Aβ 1-42 group in target quadrant with a signi cance of * P≤0.05 and ** P≤0.01 respectively.
Further, the combination treatment demonstrates signi cant improvement in the spatial memory of HFD+Aβ 1-42 group on comparison with the treatment in alone in MWM task ( ** P≤0.01, rapa vs combination; ** P≤0.01 vori vs combination) and in probe trial ** P≤0.01, rapa vs combination; * P≤0.05, vori vs combination). Figure 5 shows the track plot for the 4 th day of MWM trial (Panel A) and probe trial (Panel B). In Figure 6A and 6B, the quanti ed bar diagram for the tracking of rat head for the nal day of the MWM training (Panel A) and for probe trial (Panel B) is depicted.
To further con rm the neuroprotective e cacy of rapa and vori on the levels of Aβ 1-42 , pTau, BDNF, GDNF, and NGF levels we performed ELISA in which the elevated level of Aβ 1-42 and pTau in HFD+Aβ 1-42 group was attenuated with rapa and vori treatment alone and in combination with marked signi cant change (Supplementary Fig. 2A-2B). Further the BDNF, GDNF, and NGF levels was decreased in HFD+Aβ 1-42 group with respect to NPD group, however that could be reversed with the combination treatment with rapa and vori. Hence, the results of the ELISA based assay signi cantly correlated with the RT-PCR assay ( Supplementary Fig. 3A-3C).
IR induced AD complications continue to emerge as a global threat as the exact pathological cause is still unknown and hence therapies to halt the disease progression has failed to reach the market. The only available treatments till now are based on a symptomatic approach. Pathologically characterized as Aβ accumulation and hyperphosphorylation of tau protein particularly in the hippocampal region of the brain, AD is primarily known to affect cognitive functions of an individual.
In addition to the pathological connection between IR and AD, what drives misfolding of Aβ remains enigmatic, but irrespective of the cause removing these misfolded proteins with time is crucial for maintaining a healthy neuronal environment. Autophagy is a self-conserved mechanism popular for its role as a usher of damaged and misfolded proteins. Recent research has focused on enhancement of neuronal autophagy as an effective therapeutic approach to remove aggregated proteins proving to be a boon for neurodegenerative disorders. Autophagy is an oriented mechanism that demands the aid of numerous autophagy related proteins in completing the overall process. In turn, these autophagy related proteins are believed to be under the control of post translational histone modi cations. Studies suggest that histone deacetylase inhibition elicits neuroprotection in several models of AD and Parkinson disease (PD). Hence, in the current study we mainly focused on investigating the combined e cacy of vori, a widely used pan HDACi, and a potent autophagy inducer, rapa in ameliorating IR induced AD pathology both in-vitro and in-vivo.
Initially, MTT assay on SH-SY5Y cell line was performed and a 20 and 35 % of cell growth was inhibited with rapa and vori at a dose of 10µM and 2µM respectively and, 40% with their combination dose. Again, 20µM of rapa and 4µM of vori inhibited cell growth by 35% and 45% respectively and their combination inhibited 70% of cell growth. mRNA analysis of relevant autophagy related proteins along with proteins known to affect AD pathogenesis was conducted to explore the neuroprotective e cacy of rapa and vori alone as well as in combination in high glucose and Aβ 1-42 treated SH-SY5Y cells. mRNA analysis revealed that the expression level of BDNF, NGF, GDNF, beclin-1, LC3, LAMP-2 and MAP2 were upregulated and the expression of APP was downregulated for the combination group (rapa+vori) when compared to the high glucose+Aβ 1-42 treated group as well as upon comparison with the drugs treated alone.
Later we proceeded with a battery of behavioural, biochemical and histological analysis to prove our hypothesis that rapa and vori in combination has the capacity to mitigate IR induced AD symptoms in wistar rats. Firstly, we wanted to check the TC, TG and LDL levels to con rm the IR state of the rodents.
Our lab have already successfully reported and validated the HFD model giving rise to IR condition in rats (Sharma & Taliyan, 2018). However, before proceeding with the administration of Aβ 1-42 peptide into the rodent brain we wanted to make sure that the rats have successfully developed IR condition. What we observed was that the levels of TC, TG and LDL were found to be up regulated in the HFD group when compared to the NPD group. All these estimations were performed on a weekly basis for 8 weeks and a steep and continuous increase was observed in the serum TC, TG and LDL levels for the rats fed with the HFD which was not the case observed in the NPD group. These estimations were only performed for the NPD and HFD group with the intention to con rm the IR state of the rodents.
After con rming the IR state the animals were administered with Aβ 1-42 peptide directly into the lateral cerebral ventricles. Post-surgical care was provided and treatment was commenced with rapa and vori from the third day post Aβ 1-42 administration, i.p. once daily for 28days. On completion of the treatment, MWM was performed to assess any sort of cognitive anomaly where we found out that the latency to locate the target platform for the HFD group induced with Aβ 1-42 was much higher as compared to the group treated with a combination of rapa and vori. Favourable results for the combination group were also being observed during the probe trial.
Further, in vivo mRNA analysis for the levels of APP, BDNF, GDNF, NGF, and beclin-1, LC3, LAMP-2 and MAP2 revealed that except for APP, the levels of BDNF, GDNF, NGF and MAP2 up regulated in the combination treatment group when compared to the HFD+Aβ 1-42 group. Similarly, the levels of APP signi cantly down regulated in the treated group with both rapa and vori when compared to the HFD+Aβ 1-42 group. Moreover, the impaired levels of beclin-1, LC3 and LAMP-2 in the disease group seem to considerably improve after treatment with the combination of rapa and vori.
We also performed ELISA estimations of Aβ 42 , ptau, BDNF, GDNF and NGF and observed that the HFD+Aβ 1-42 group had elevated levels of Aβ and ptau and decreased levels of BDNF, GDNF and NGF.
These effects were found to reverse after treatment with a combination of rapa and vori.
Lastly, histological analysis of both the DG and CA1 region using H&E stain exposed signi cant pyknotic neuronal count, considerable vacuolization, cell in ltration etc with resultant degenerated neurones for both the groups of HFD alone and HFD with the Aβ 1-42 group. However, after treatment with combination of rapa and vori, the percentage of neurodegenerative area as analysed by imageJ software declined, eliciting possible neuroprotection.

Conclusion
Page 17/30 IR associated AD poses a great threat to the economy worldwide as cases continue to rise with no available therapeutic cure except symptomatic management. Autophagy inducers have recently evolved as a potent neuroprotective target delivering considerable e cacy in preclinical AD models but its usage is limited by factors such as over-accumulation of misfolded proteins leading to failure in its e cacy.
However, when combined with HDACi, this limitation is overcome which can effectively emerge as a favourable therapeutic option for IR induced AD. Hence in the current study, we used a leading autophagy inducer, namely rapa alone as well as in combination with vori for the treatment of HFD induced AD which is the rst study performed to the best of our knowledge and its preliminary outcomes are satisfactory enough to further explore possible therapeutic outcome in this area.  Figure 1 In-vivo experimental regimen.