(R,S)-3,5-DHPG

Inhibition of mitochondrial pyruvate carrier 1 by lapatinib ditosylate mitigates Alzheimer’s-like disease in D-galactose/ovariectomized rats

Heba M. Mansour a,*, Hala M. Fawzy a, Aiman S. El-Khatib b, Mahmoud M. Khattab b
a Department of Pharmacology, Egyptian Drug Authority, EDA, formerly NODCAR, Giza, Egypt
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Cairo, Egypt

Abstract

Mitochondrial, autophagic impairment, excitotoXicity, and also neuroinflammation are implicated in Alz- heimer’s disease (AD) pathophysiology. We postulated that inhibiting the mitochondrial pyruvate carrier-1 (MPC-1), which inhibits the activation of the mammalian target of rapamycin (mTOR), may ameliorate the neurodegeneration of hippocampal neurons in the rat AD model. To assess this, we used lapatinib ditosylate (LAP), an anti-cancer drug that inhibits MPC-1 through suppression of estrogen-related receptor-alpha (ERR-α),
in D-galactose/ovariectomized rats. AD characteristics were developed in ovariectomized (OVX) rats following an 8-week injection of D-galactose (D-gal) (150 mg/kg, i.p.). The human epidermal growth factor receptor-2 (HER-2) inhibitor, LAP (100 mg/kg, p.o.) was daily administered for 3 weeks. LAP protected against D-gal/ OVX-induced changes in cortical and hippocampal neurons along with improvement in learning and memory, as affirmed using Morris water maze (MWM) and novel object recognition (NOR) tests. Furthermore, LAP suppressed the hippocampal expression of Aβ1-42, p-tau, HER-2, p-mTOR, GluR-II, TNF-α, P38-MAPK, NOX-1, ERR- α, and MPC-1. Also, LAP treatment leads to activation of the pro-survival PI3K/Akt pathway. As an epilogue, targeting MPC-1 in the D-gal-induced AD in OVX rats resulted in the enhancement of autophagy, and suppression of neuroinflammation and excitotoXicity. Our work proves that alterations in metabolic signaling as a result of inhibiting MPC-1 were anti-inflammatory and neuroprotective in the AD model, revealing that HER-2, MPC-1,and ERR-α may be promising therapeutic targets for AD.

1. Introduction

Alzheimer’s disease (AD) is an age-related disorder distinguished by cognitive decline. Histopathology, amyloid (Aβ) plaques, and neurofi- brillary tangles characterize AD. A significant component of the Aβ plaques is the aggregation of Aβ1-42, derived from the proteolytic cleavage of the amyloid precursor protein (APP) by β-and γ-secretase enzymes (Lin and Beal, 2006). Women are shown to have a higher risk of
AD than men. Menopausal-linked estrogen decline is a major risk factor for this susceptibility. Undoubtedly, estrogen has a variety of neuroprotective benefits, including the elimination of Aβ toXicity, Apo-E upregulation, mitochondrial dysfunction inhibition, and cholinergic system modulation (Ibrahim et al., 2019). Literature has established that repeated administration of D-galactose (D-gal) to rats transforms, by galactose oXidase, into aldose and hydroperoXide, attributed to the production of reactive oXygen species. Also, caspase-3 increases in the hippocampus and diminishes cholinergic neurons in the forebrain.

Likewise, ovariectomy (OVX) has been identified as an AD-inducing model, where OVX-associated estrogen depletion inhibits neurotrophic
factors, triggers neuroinflammation, and boosts the amount of Aβ1-40 (Hua et al., 2007). Therefore, OVX along with long-term D-gal admin- istration presents a good model to mimic behavioral, pathophysiolog- ical, and biochemical changes in AD (Hua et al., 2007; Ibrahim et al., 2019).

Disease-modifying treatments for AD are not available. As a conse- quence, a more effective approach is to target molecules that are downstream in signal pathways that impair cellular activity and affect neurons (Mansour et al., 2021).Glutamate is the key excitatory neurotransmitter in the CNS. As glutamate increases to toXic levels, prolonged activation of receptors induces a neurotoXic signal cascade, causes mitochondrial dysfunction, and finally AD progression. It should be mentioned that glutamate has some other brain fates, like oXidation for energy (McKenna et al., 2012). Many attempts to target excitatory receptors such as NMDA antagonists have stopped in many neurodegenerative diseases such as stroke and

Parkinson’s disease (PD) models (Ghosh et al., 2016), suggesting that TZDs could affect the molecular pathways involved in neurodegenera- tive disorders. In light of this background, we assumed that inhibiting MPC-1 could modify molecular mechanisms that are disrupted in AD and mitigate neurodegeneration in the rat AD model.

EXpression of nuclear receptor, estrogen-related receptor α (ERR-α) is crucial to MPC-1 transcription. The knockdown of ERR-α completely
blocked the stimulating effects on MPC-1 promoter activation, proving that ERR-α is necessary for MPC-1 activation (Koh et al., 2018). A recent study has demonstrated that inhibition of ERR-α by Cpd29 interferes with pyruvate entrance into mitochondria by suppressing the expression of MPC-1. This results in a massive increase in cell dependence on the oXidation of glutamine (Park et al., 2019). Lapatinib ditosylate (LAP), an anti-cancer drug, inhibits human epidermal growth factor receptor-2 (HER-2) (Wainberg et al., 2010). ERR-α is a downstream receptor of the HER-2 receptor (Ariazi et al., 2007; Ochnik and Yee, 2012). Consequently, LAP inhibits ERR-α (Deblois et al., 2016).

Previous literature evaluating the neuroprotective effect of ERR-α has shown conflicting results. In addition to the autophagy-mediated regulation by mTOR, ERR-α negatively modulates autophagy. A recent study stated that XCT-790, an inverse agonist of ERR-α, increased
autophagy, reduced toXicity of α-synuclein, and exhibited neuroprotection in a Parkinson’s disease mouse model (Suresh et al., 2018). Another study noted an age-dependent decrease in ERR-α protein expression in APP/PS1 mice (Tang et al., 2018). Another study found that the ability to learn and memorize of ERR-α knockout mice was comparable to wild-type equivalents in the Barnes maze task (Saito and toXicity. Surprisingly, oXidation of non-glucose substrates such as Cui, 2018).

Huntington’s disease due to prohibitive neurological adverse effects, highlighting the need to pursue potential approaches to target excito glutamate can be neuroprotective in some neurodegenerative diseases (Quansah et al., 2018a; Yang et al., 2014).Metabolism of all nutrients passes through different molecular pro- cesses. All of them are linked to pyruvate metabolism. A protein complex of carrying pyruvate to mitochondria is called a mitochondrial pyruvate carrier (MPC). A recent study has shown that MPC-1 suppression uti- lizing UK-5099 saved the pyramidal neurons from excitotoXic injuries by inhibiting pyruvate entrance into the mitochondria and decreasing the glutamate surplus in hippocampal culture (Divakaruni et al., 2017). Interestingly, following MPC-1 inhibition, dependence on energy sub- strates and bio-synthetic metabolism changed from glucose to amino acid and oXidation of fatty acid. The tricarboXylic acid (TCA) cycle and fatty acid synthesis are preserved by relying more on the oXidation of glutamine and fatty acids (Vacanti et al., 2014; Quansah et al., 2018; Tiulganova et al., 2018). In conclusion, MPC-1 inhibition results in a type of metabolic flexibility consistent with the use of fats and amino acids such as glutamate as anabolic fuel. Subsequently, previous findings provide a solid platform for in vivo MPC-1 inhibition evaluation to ameliorate excitotoXic injury in AD (Gim´enez-Cassina et al., 2012).

There has been an increasing understanding of the commonality of metabolic dysfunction between type 2 diabetes and AD. Common metabolic disturbances, especially mitochondrial dysfunction, and inflammation are the cornerstones of these seemingly disparate diseases. Although the possible impact of anti-diabetic medications on AD development is still controversial, possible nexuses between pathogenic pathways in type 2 diabetes and AD have encouraged clinical studies in AD utilizing approved thiazolidinediones (TZD) used in the treatment of diabetes, such as pioglitazone and MSDS-0106 (Divakaruni et al., 2013; Geldmacher et al., 2011; Shah et al., 2014).

Insulin sensitizers such as thiazolidinedione (TZD) primarily target MPC. TZD hinders the entrance of pyruvate into the mitochondria which result in a compensated rise in the use of other substrates (Divakaruni et al., 2013). Furthermore, pioglitazone is neuroprotective in a mouse model of AD (Searcy et al., 2012). MSDC-0160, another TZD, conserved cerebral 2-deoXyglucose after 3 months of oral administration in AD patients through modulation of MPC (Shah et al., 2014). Also, MSDC-0160 improves insulin sensitivity in various cell and animal Hence, the current study aimed to clarify the possible neuro- protective effects of LAP on ameliorating learning and memory decline induced by chronic administration of D-gal in OVX rats. In addition, elucidation of the molecular insights of LAP reveals the potential for
HER-2, MPC-1, and ERR-α inhibition as an efficient approach for the treatment of AD. In addition to investigating the nexuses between MPC- 1 inhibition and the pro-survival pathway, PI3K/Akt activation. Also, exploring the relationship between MPC-1 inhibition and curbing of AD characteristics; Aβ1-42, and p-tau.

2. Materials and methods
2.1. Animals

Adult female Wistar albino rats (5-month-old) weighing 160 20 gm were used in the current study. Rats were individually housed and kept in constant conditions of 22 2 ◦C, and 12/12 h light-dark cycle. They were fed a soy-free diet and water ad libitum. The experimental protocol was approved by the Ethics Committee for Animal EXperimentation at the National Organization for Drug Control and Research (permit number: NODCAR/I/16/19), following the Guide for the Care and Use of Laboratory Animals presented by the US National Institute of Health (NIH publication No. 85-23, revised 2011).

2.2. Drugs and chemicals

Tykerb® (Lapatinib ditosylate, Batch No.18061597092234), manu- factured by GlaxoSmithKline, was purchased from the market and freshly prepared in 10% tween 80 in saline. D-gal (CAS No: 59-23-4, purity: 99%) was purchased from El-Gomhoria Company, Cairo, Egypt, and was daily prepared in normal saline. All the other chemicals were of analytical grade.

2.3. Experimental design

As shown in SUPP 1. Fig. S1., 40 rats were randomly allocated into 5 groups of 8 animals each. In the first group (SO group): Sham-operated rats were administered 0.9% saline i.p. for 8 successive weeks, followed by oral administration of 1 ml of 10% tween 80 in normal saline for 3 weeks. In the second group (D-gal/OVX group), the rats underwent two- sided ovariectomy followed by treatment with 150 mg/kg/day; i.p. D- gal for 8 weeks beginning one week after healing. Then, they adminis- tered 1 ml of 10% tween 80 in 0.9% saline p.o for 3 weeks. In the third group, D-gal/OVX animals were administered 30 mg/kg LAP p.o. for 3 weeks after the D-gal administration. In the fourth group, D-gal/OVX animals were administered 50 mg/kg LAP p.o. for 3 weeks after the D- gal administration. While in the last group, D-gal/OVX animals were administered 100 mg/kg LAP p.o. for 3 weeks after the D-gal adminis- tration. The choice of D-gal dose and duration was based on previous literature (Ibrahim et al., 2019). D-gal/OVX rats treated with 100 mg/kg LAP showed the best results in behavioral examinations with histo- pathological improvement relative to D-gal/OVX rats treated with 30 and 50 mg/kg LAP, signifying that 100 mg/kg/day LAP was the most efficient dose.

2.4. Surgery

The ovariectomy was conducted under i.p. administration of 50 mg/ kg ketamine hydrochloride (Alfasan Inc., Utrecht, Holland) and 10 mg/ kg xylazine (Adwia Pharmaceutical Co., Cairo, Egypt). Bilateral abdominal incisions were made. In the SO group, the ovaries were retracted without cutting them. While in the OVX group, the ovaries and oviducts were clamped with a homeostatic clip to prevent bleeding, then they were excised. The muscle layer and skin were sutured with silk and catgut respectively. Finally, all rats were administered the analgesic diclofenac sodium at a dose of 100 mg/kg (Voltaren; Novartis Pharma, Cairo, Egypt) and the antibiotic ceftriaxone at a dose of 100 mg/kg (Ceftotax; Epico Pharmaceutical Industry, Cairo, Egypt.) subcutaneously after ovariectomy to promote the healing process for up to 72 h.

2.5. Morris water maze (MWM) test

The Morris water maze task was done to assess memory decline and hippocampal damage. It was executed to evaluate memory performance in rats during five successive days. The device consisted of a circular pool (180 cm in diameter). The apparatus was divided into four parts and contained water to a depth of 35 cm. A platform (9 cm in diameter) was fiXed in a position in one of the four parts and submerged below the water level to be hidden from the rats. The water was made opaque by the use of dry milk. The apparatus was put in a dimly lit room and supplied with fiXed visual cues that acted as navigational guides. During the acquisition phase on the first 4 days, rats were left freely swimming to find the immersed platform for 120 s. Otherwise, they were guided to the platform. Each rat was trained for two trials during the acquisition phase. On the fifth day, the platform was removed and the rat was allowed to swim for 1 min in the probe trial. The duration spent swimming was assessed as a reference memory measure (Lutas and Yellen, 2013). The swimming orbits of rats were recorded by a video camera (NIKON COOLPIX P610, Japan) and analyzed by ANY-maze tracking software (ANY-MAZE, Stoelting Co., Wood Dale, IL, USA).

2.6. Novel object recognition (NOR) test

The novel object recognition test was executed to explore the recognition memory of rats. The test consisted of three phases; habitu- ation, familiarization, and the test. During the habituation phase, rats were placed in a glass boX (40 cm 40 cm 40 cm) without any objects for 10 min. On the next habituation day, rats were put in the boX and permitted to scrutinize two indistinguishable balls for 5 min. During the testing phase on the third day, rats were exposed to two divergent ob- jects for 5 min; one was a novel cube and the other was a familiar ball. The evidence for the investigatory behavior of a rat was that it was touching or sniffing the object. The time spent exploring the objects was using the following formulas: DI= (TN-TF)/(TN + TF), and PI = TN/(TN + TF) (Ibrahim et al., 2019).

2.7. Open-field (OF) test

The open-field test was carried out as previously described (Szy- dlowska and Tymianski, 2010). This test was performed to verify that the results of the previous behavioral tests were not due to changes in spontaneous locomotor activity. The device consists of a wooden boX (80 cm 80 cm 40 cm). The test was scored by ANY-maze tracking software (ANY-MAZE, Stoelting Co., Wood Dale, IL, USA). The distance traveled, the average speed, the number of rearing, and the number of crossed lines were used as a measurement of locomotor activity (Supp. Figure.2).

2.8. Histopathology

The entire brains of three rats, randomly chosen from each group, were fiXed at 10% buffered formol saline for 24 h. The samples were washed, dehydrated by ethanol, cleared in xylene, and placed in paraffin at 56 ◦C in hot air for a further 24 h. Parts of 3-μm thickness were coated with H&E and inspected under a light microscope. Both the histopath- ological examination and evaluation of samples were carried out by an objective observer who did not know the identity of the sample being studied to eliminate any bias. A scoring system with a 4-point scale was used to evaluate the degree of severity of the observed histopathological lesions; where 0, 1, 2, and 3 indicated none, mild (changes <30%), moderate (changes 30–50%), and severe (changes >50%) alterations, respectively (Arsad, 2014).

2.9. Enzyme-linked immunosorbent assay

Utilizing a rat-specific ELISA kit and following the manufacture’s protocol, the hippocampal content of TNF-α and HER-2 (CSB-E11987r and LSBIO, Seattle, WA, USA, Cat. No. #LS-F11388), ERR-α, Aβ1-42,GluR-II, p-mTOR, p-Akt, and P38-MAPK (MyBioSource, San Diego, CA, Cat. No: MBS456430, MBS702915, MBS087279, MBS744326,MBS57254603, and MBS765087) were measured following manufac- turers’ instructions. The results were expressed as picograms per mg tissue for ERR-α, Aβ1-42, TNF-α, and mTOR. While GluR-II, HER-2, P38-MAPK, and p-Akt were expressed as ng per mg tissue. The total protein content was measured according to a previous method (Heras-Sandoval et al., 2014).

2.10. Western blotting analysis of hippocampal MPC-1, p-(Tyr458-199) PI3K, and p-(ser404) tau expression

Estimation of hippocampal MPC-1, p-(Tyr458-199) PI3K, and p-(ser404) tau protein expression was done using the Western blot technique. Succinctly, the hippocampal specimens were homogenized in a lysis buffer with a protease inhibitor miXture. Following protein quantifica-
tion using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, California, USA), a portion of 50 μg protein from each specimen was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transmitted to the nitrocellulose membrane (Amersham Bioscience, Piscat). Using a semi-transfer system (Bio-Rad, Hercules, California, USA), Pre- cision Plus Protein standards (Bio-Rad, Hercules, CA, USA) were used as molecular weight markers. To check the protein transition, the mem- branes were coated with Ponceau S solution (Sigma – Aldrich, St. Louis, MO, USA) transiently. The membranes were incubated with a blocking solution at 4 ◦C overnight. Then, the membranes were treated with TBST and incubated with the following primary antibodies; anti-MPC-1 or anti-p-(Tyr458-199) PI3 or anti-p-(ser404) tau (Thermo Fischer Scientific, Waltham, USA), or anti-β-actin (cell signaling technology, USA) at room temperature with constant trembling for 1 h. The filters were screened recorded. The discrimination and preference indices were calculated with horseradish peroXidase-conjugated goat anti-mouse immunoglobulin (Amersham, Life Science Inc., Arlington Heights, IL,USA). Finally, the detection of chemiluminescence was achieved using the Amersham detector kit following the manufacturer’s instructions and was exposed to X-ray film. The amount of MPC-1, p-(Tyr458-199) PI3K,and p-(ser404) tau were quantified using densitometric analysis by Laser Densitometer scanning (Biomed Instrument Inc., Brooklyn, NY, USA). The results of the expression of β-actin protein were expressed as arbi- trary units after normalization.

2.11. Quantitative real-time PCR

The hippocampal NADPH oXidase-1 (NOX1) assay was performed by quantitative RT-PCR. Using the RNeasy Mini kit (Qiagen, Venlo,
Netherlands), RNA was extracted according to the manufacturer’s protocol. DNase has removed the remaining DNA. The purified DNA was quantified spectrophotometrically. A reverse transcription system (Promega, Leiden, Netherlands) was used for complementary DNA synthesis. RNA was incubated at 42 ◦C for 1 h with 25 mM MgCl2, 10 X RTase buffer, 20 U RNase inhibitor, oligo d(t) primers, 10 mM dNTP miXture, and 20 U/μl AMV reverse transcriptase. SYBR Green PCR Master MiX with an ABI PRISM 7500 Fast sequence detection system and its quantification program performed the NOX-1 expression level analysis. The sequence of primers used has been described in (SUPP.Table 1). Using the 2 —ΔΔCT approach using β-actin as a house-keeping gene, the relative expression of the gene was determined (Livak and Schmittgen, 2001).

2.12. Statistical analysis

Most of the results were expressed as mean ± SD and analyzed using One-way ANOVA followed by Tukey’s post-hoc test. Two-way ANOVA
was utilized to assess the acquisition phase of the MWM test. Scoring of histopathological lesions was expressed as (median [IQR]) and analyzed using the Kruskal Wallis test followed by Dunn’s multiple comparison test. While the ambulation frequency, and rearing in the OF test were expressed as median ± range and analyzed using the Kruskal-Wallis test followed by Dunn’s multiple comparisons tests; (n 8, p < 0.05). Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA, USA). Fig. 1. Effect of LAP on behavioral functions of D-gal/OVX rats. (a) MWM acquisition phase, (b) Time spent in the target quadrant in the probe phase, (c) Representative behavior tracking plot reports of different groups showed the rapid development of a place-specific preference for the platform position where the southwest quadrant was the platform quadrant., (d) discrimination index in NOR test, and (e) Preference index in NOR test. All data were expressed as (mean ± SD) (n = 8 per group &*p ≤ 0.05 compared with SO group) and analyzed using one-way ANOVA followed by Tukey’s post-hoc test; except for the escape latency in the acquisition phase that was analyzed using two-way ANOVA followed by Tukey’s multiple comparison test. *vs SO group, # vs D-gal/OVX group, @ vs LAP (30 mg/ kg), & vs LAP (50 mg/kg), SO; sham-operation, D-gal; D-galactose, LAP; lapatinib ditosylate, MWM; Morris water maze, NOR; novel object recognition. 3. Results 3.1. Effect of different doses of lapatinib on D-galactose/ovariectomy induced-cognitive deterioration in Morris Water Maze, novel object recognition, and open-field tests The Morris water maze (MWM) and Novel object recognition (NOR) experiments evaluated the impact of lapatinib ditosylate (LAP) on cognitive impairment in D-gal/OVX rats. As seen in Figure (1a), in the acquisition phase, D-gal/OVX rats demonstrated higher escape latencies during the third, and fourth days by 5.2- and 4.8-fold respectively, relative to sham-operated (SO) rats, revealing the existence of memory deficits in those rats. On the contrary, LAP administration strengthened the cognitive capabilities of D-gal/OVX rats, as seen by shortened escape latencies. OVX rats that received 50 mg/kg LAP had reduced escape latency by 18% on the third day of the acquisition trial compared to their D-gal/OVX counterparts. On the other hand, the administration of 100 mg/kg LAP augmented the spatial cognitive capability of D-gal/OVX rats, as demonstrated by a decline in escape latencies during the third and fourth days of the acquisition phase by 82% on both days. As seen in Figures (1b), in the probe phase, D-gal/OVX rats showed a minimal retrieval index of less time spent in the platform quadrant by 43% compared to the SO rats. While treatment of D-gal/OVX rats with 30, 50, and 100 mg/kg LAP boosted the retrieval index by 1-, 1,7-, and 2-fold respectively relative to the D-gal/OVX group. In the learning retrieval phase, D-gal/OVX rats swam blindly in the maze oblivious of the pre- vious location of the platform, while D-gal/OVX rats treated with 100 mg/kg LAP recalled the quadrant wherein the platform was inserted, as demonstrated by the distance traveled in the target quadrant Figure (1c). As seen in Figures (1d, and 1e) of the NOR test, D-gal/OVX rats exhibited decreased discrimination and preference indices by 74% and, 53% respectively relative to the SO group. Nevertheless, the 100 mg/kg LAP-treated rats had higher discrimination and preference indices by 2.9- and 1.93-fold relative to the D-gal/OVX group. Worthy of note, rats treated with 30 and 50 mg/kg LAP did not display a noticeable differ- ence in discrimination and preference indices as opposed to the D-gal/ OVX group. In the open-field (OF) experiment (SUPP.2 Fig. 2), there was no substantial variation in latency time, rearing frequency, average speed, and distance traveled between different groups, confirming that the findings of other behavioral tasks were not due to differences in loco- motive performance. 3.2. Effect of lapatinib ditosylate on histopathological changes induced in D-gal/OVX rats Microscopic inspection of the brain cortex and hippocampal regions of the brain tissue of SO rats revealed the normal histological structure of the cerebral blood vessels Figure (2a) as well as the hippocampal neurons Figure (2b). While both areas of D-gal/OVX rats revealed marked tissue alterations. Some hippocampal neurons showed necrosis and shrunken appearance with congestion, perivascular edema, peri- cellular edema of some cells Figure (2c), plaque formation, neuro- nophagia, and neuronal cell degeneration proved by abnormal scores 3 (SUPP. Fig.3). CO showing necrosis, neuronophagia, astrogliosis, peri- vascular edema, and pericellular edema Figure (2d). The hippocampus of D-gal/OVX rats treated with 30 mg/kg LAP showed pyknosis and necrotic changes of some pyramidal cells, necrobiotic changes of some granular and molecular cells layers with mild pericellular edema Figure (2e). While the cerebral cortex of those rats showed vacuolar degeneration, necrosis, and basophilic shrunken neurons with peri- vascular edema Figure (2f). The hippocampus of D-gal/OVX rats treated with 50 mg/kg LAP showed a moderate degree of restoration with scattered necrotic cells, neuronophagia, and some pyknotic shrunken neurons in the molecular layer Figure (2g). Regarding the cerebral cortex of those rats showed a moderate degree of neuronal cell necro- biotic changes of some neurons with mild perivascular edema Figure (2h). The hippocampus of D-gal/OVX rats treated with 100 mg/ kg LAP revealed mild perivascular edema Figure (2i) with good resto- ration of the cerebral cortex elements, with mild degenerative and necrotic changes of some neurons, few neurons appeared necrotic and shrunken with very few neuronophagia and mild perivascular edema (H&E) Figure (2j). The histopathological grading of histopathological lesions (SUPP. Fig. 3) has proven that LAP amended the histopatho- logical changes of D-gal/OVX rats in a dose-dependent approach, and showed that 100 mg/kg/day LAP was the most effective dose. Fig. 2. Effect of lapatinib ditosylate (LAP) on his- topathological changes induced in D-gal/OVX rats. Representative photomicrographs of 3 rats from each group depict (a) hippocampus and (b) cere- bral cortex of SO rats revealing normal histological structure. (c) the hippocampus of D-gal/OVX rats showing necrosis and shrunken neurons (arrow), congestion (short arrow), and perivascular edema (ED) with pericellular edema (dashed arrows) reflective of inflammatory and pathological accel- eration by D-gal/OVX model (d) Cerebral cortex of D-gal/OVX rats showing neuronal degeneration, necrosis (arrow), neuronophagia (dashed arrow), astrogliosis, perivascular edema (short arrow) and pericellular edema (thin arrow). (e) the hippo- campus of rats treated with 30 mg/kg LAP showing pyknosis (arrow), necrotic changes of some pyra- midal cells (thin arrow), necrobiotic alterations of some granular cells (dashed arrow), and mild perivascular edema (short arrow). (f) cerebral cortex showing necrosis, vacuolar degeneration (dashed arrow), necrosis (arrow), shrinkage (short arrow) with unstained perineuronal spaces, and perivascular edema (thin arrow) which is demon- strated by enlarged perivascular space. (g) the hippocampus of rats treated with 50 mg/kg LAP moderate degree of restoration of hippocampal neurons with scattered degenerated and necrotic cells (arrow). (h) Cerebral cortex showing a mod- erate degree of neuronal cell necrobiotic changes with mild perivascular edema (arrow). (i) the hippocampus of rats treated with 100 mg/kg LAP showing very few necrotic neurons (arrow) and mild perivascular edema (dashed arrow). (j) Ce- rebral cortex showing a good restoration of the Cerebral cortex elements with mild degenerative and necrotic changes of some neurons. (H&E). SO; sham-operated, D-gal; D-galactose, OVX; ovariec- tomized, LAP; lapatinib ditosylate. Fig. 3. Effects of LAP on mechanistic insights. LAP normalized D-gal/OVX-mediated alterations in hippocampal levels of (a) Aβ, (b,c) p-tau, (d) ERR-α, (e) HER-2, (f, c) MPC-1, (g) PI3K, (h,c) p-Akt, (i) mTOR, (j) GluR-II, and (k)TNF-α. Analysis of correlation coefficient between p-tau and (l) ERR-α, (m) MPC-1, (n) P38-MAPK, and (o) NOX-1. All data are normalized to the control group and presented as the mean ± SD (n = 5). * vs SO group, # vs D-gal/OVX group, utilizing one-way ANOVA followed by Tukey’s post-hoc test; p < 0.05. SO; Sham-operated, D-gal; D-galactose, OVX; ovariectomized, LAP; lapatinib ditosylate, Aβ; amyloid-beta, ERR-α; estrogen-related receptors alpha, MPC-1; mitochondrial pyruvate carrier-1. 3.3. Effect of LAP administration on hippocampal Aβ1-42 level and p-(ser404) tau protein expression in D-gal/OVX rats D-galactose administration in OVX rats resulted in a substantial in- crease in hippocampal Aβ1-42 and p-tau by 5- and 5.3-fold respectively relative to the SO group. Conversely, administration of 100 mg/kg LAP curbed Aβ1-42 and p-tau levels by 37% Figure (3a) and 48% respectively compared to D-gal/OVX rats Figures (3b, c). 3.4. Effect of LAP on D-gal/OVX-induced alteration in hippocampal human epidermal growth factor receptor-2 (HER-2) and estrogen-related receptor-α (ERR-α) in rats D-gal/OVX rats reported an expansion of 3.2- and 4.1-fold in human epidermal growth factor receptor (HER-2) and Estrogen-related receptor α (ERR-α) levels respectively relative to the SO group. LAP treatment substantially decreased the HER-2 Figure (3e) and ERR-α Figure (3d) levels by 51% and 51% compared to D-gal/OVX rats. 3.5. Influence of LAP on D-gal/ovariectomy-induced variations in hippocampal mitochondrial pyruvate carrier-1 (MPC-1) expression in rats Estrogen restriction aligned with D-gal administration resulted in a pronounced 5.4-fold increase in hippocampal protein expression of MPC-1 relative to SO rats. In comparison, LAP-treated rats reported a decrease of 39% in this carrier’s level relative to D-gal/OVX rats Figures (3f, 3c). 3.6. Effect of LAP on D-gal/OVX-induced alterations in PI3K/Akt/mTOR signaling pathway in rats Administration of D-gal in OVX rats resulted in a significant decrease in p-PI3K and p-Akt by 24% and 40% relative to SO rats. Conversely, the administration of LAP increased the hippocampal level of p-PI3K and p- Akt by 3-, and 2-folds respectively as compared to D-gal/OVX rats. On the other contrary, D-galactose/OVX rats revealed a marked elevation in the hippocampal mTOR level (3-fold) relative to their SO counterparts. In contrast, LAP-treated rats exhibited a lower level of mTOR (54%) compared to D-gal/OVX rats Figures (3g, 3h, 3I, 3c). 3.7. Effect of LAP on hippocampal glutamate receptors and TNF-α level in D-gal/OVX rats D-galactose injection to OVX rats resulted in a pronounced increase in hippocampal glutamatergic receptors and TNF-α levels by 4- and 4.7- fold respectively as compared to the SO group. Conversely, the admin- istration of LAP decreased GluR-II and TNF by 44% and 46% respec- tively as compared to D-gal/OVX rats Figures (3j, 3k). 3.8. Effect of LAP on P38-MAPK and NADPH oxidase-1 (NOX-1) in D- gal/OVX rats Chronic administration of D-gal in OVX rats resulted in a pronounced increase in both P38-MAPK Figure (3n) and NOX-1 Figure (3◦) by 4- and 5-fold respectively as compared to SO rats. Conversely, LAP treatment attenuated these escalations by 71% and 61% respectively as compared to D-gal/OVX rats. 3.9. Correlation studies To inspect the relationship between ERR-α, MPC-1, and amyloido- genesis, we conducted correlation analyses. Aβ1-42 concentration was found to be strongly correlated with the content of ERR-α, and MPC-1 expression (r2 0.6120, r2 0.8607, p 0.0026, and p < 0.0001 respectively) Figures (3l, 3m). 4. Discussion Until now, there is no effective medical treatment for Alzheimer’s disease (AD). New approaches are required to prevent neuro- degeneration, which needs recognition of druggable targets. Away from traditional hypotheses, the current investigation demonstrated, for the first time, that the anti-cancer drug lapatinib ditosylate (LAP), could mitigate cognitive impairment and suppress AD pathological hallmarks of hippocampal Aβ1-42, and p-tau in D-gal/OVX rats. Also, LAP decreased the hippocampal expression of mitochondrial pyruvate carrier-1 (MPC-1). This effect was partly related to the inhibition of hippocampal human epidermal growth factor receptor-2 (HER-2), and estrogen-related receptors α (ERR-α) content, along with inhibition of glutamate receptor-II (GluR-II), tumor necrosis factor-alpha (TNF-α), and phosphorylated mammalian target of rapamycin (p-mTOR), P38- MAPK, and NADPH OXidase-1 (NOX-1). While activating the pro- survival pathway, PI3K/Akt. As a consequence, excitotoXic, neuro- inflammatory, and autophagic alterations were normalized. Also, the survival of cerebral and hippocampal neurons was promoted. There are many pieces of evidence that oXidative stress and estrogen depletion are two significant risk factors directly linked to the patho- logical development of AD (Hua et al., 2007). In this model, D-galactose (D-gal) administration to ovariectomized (OVX) rats caused decreased learning and memory capabilities, which was also consistent with pre- vious studies (Hua et al., 2007; Ibrahim et al., 2019(Ibrahim et al., 2021)). In the Morris water maze (MWM) and novel object recognition (NOR) tasks, sham-operated (SO) rats demonstrated positive learning performance in the training trials and on the day of testing towards the platform and novel object in the MWM and NOR tests respectively. Conversely, D-gal/OVX rats cannot generally memorize, in line with previous results (Ibrahim et al., 2019). In the current investigation, LAP restored the memory of D-gal/OVX rats in both tests. These results were not attributed to improvements in locomotive behavior as asserted by the open-field (OF) test. While most AD researches have concentrated on the involvement of the hippocampus, the cerebral cortex also seems to be implicated. In the present research, neuronal degeneration, necrosis, neuronophagia, astrogliosis, perivascular edema, and pericellular edema were observed in the cerebral cortex. Prior studies have assisted our results demon- strating amyloidogenesis and degenerative areas in the mouse cerebral cortex in various AD models (Ibrahim et al., 2019; Kamel et al., 2018). In the current study, the highest dose of 100 mg/kg LAP exhibited the most pronounced cognitive-enhancing effects and brain tissue pre- serving actions. This dose was further investigated in mechanistic studies. The high effective dosage increases the possibility of LAP to reach maximum therapeutic concentrations that permeate the blood- brain barrier (BBB). Herein, LAP restored D-gal/OVX-induced rise in HER-2 hippocampal expression, contributing to a substantial reduction in HER-2 levels. Dysfunction of autophagy has been linked with several neurodegener- ative disorders, including AD, which is associated with the accumulation of misfolded proteins such as phosphorylated tau and Aβ 42. HER-2, an oncogenic receptor, has been reported to inhibit autophagic fluX. Inhi- bition of HER-2 by CL-387,785 suppresses the production of Aβ. In compliance with the elevated levels of HER-2 in the hippocampi of AD patients. In prospective studies, various HER-2 antagonists could be useful in autophagic-related neurodegenerative disorders. Moreover, the abnormal expression or activation of HER-2 may contribute to the pathogenesis of AD (Wang et al., 2017). In the current investigation, LAP treatment curbed D-gal/OVX induced elevation in p-mTOR. Compromised autophagy is correlated with neuronal impairment of compromised Aβ clearance and cognitive deterioration in AD (Wang et al., 2017). In response to cytokines, p-mTOR has been found to actively influence microglial activation and seems to play a pivotal role in microglial reactivity. So, p-mTOR inhi- bition by LAP can therefore be a valuable tool in regulating neuro- inflammation (Dello Russo et al., 2009). In accordance with the previous findings, our results provided convincing evidence of the anti-inflammatory and autophagy-enhancing role of LAP via p-mTOR suppression. This action partly contributed to the ameliorative effect of LAP in AD. Lapatinib ditosylate amended D-gal/OVX-induced escalation in hippocampal P38-MAPK levels. Literature suggests that P38-MAPK is implicated in myriad aspects of AD pathophysiology, such as Aβ plaque formation, tau hyperphosphorylation, neuroinflammation, apoptosis, autophagy regulation, cytokine overproduction, and glutamate- mediated excitotoXicity (Kheiri et al., 2019). In agreement with the previous literature, we postulate that the neuroprotective effect of LAP is partially attributed to P38-MAPK inhibition. The current study revealed that D-gal/OVX rats showed boosted activation of the hippocampal gene expression of NOX-1. In contrast, the administration of LAP declines this increase. Elevated expression of NOX-1 in AD may be partially due to the higher levels of Aβ1-42. NOX-1 mediated neurotoXicity and neuroinflammation are directly toXic to neurons and produce multiple pro-inflammatory cytokines such as TNF- α. Therefore, suppressing NOX-1 can reduce a wide spectrum of ROS and cytokines, which results in halting neuronal damage (Block, 2008). This result substantiates another presumptive mechanistic pathway involved in the potential treatment role of LAP in AD treatment. Our study revealed that D-gal/OVX rats exhibited reduced phos- phorylation of hippocampal PI3K and Aktser473. These results are consistent with previously described reports (Ibrahim et al., 2019; Kamel et al., 2018). Conversely, inhibition of MPC-1 by LAP increased the phosphorylation of hippocampal PI3K and ser473 Akt. Noteworthy, inhibition of mTOR by rapamycin stimulates Akt phosphorylation, established as a negative feedback mechanism for PI3K/Akt signaling pathways. The stimulation of PI3K/Akt works on mTOR to improve the autophagy and lysosomal destruction of Aβ and to reduce the level of tau hyperphosphorylation. (Haruta et al., 2000; (Mueed et al., 2019)). Notably, modulation of MPC-1 had an anti-inflammatory effect in the mouse model of PD via modulation of the Akt/mTOR pathway (Ghosh et al., 2016). Therefore, the activation of the pro-survival pathway PI3K/Akt by LAP may be attributed to inhibition of both mTOR and MPC-1, and contribute to its neuroprotective effect against AD. In this investigation, LAP reversed the increase in hippocampal GluR- II content caused by D-gal/OVX. Increased GluR-II activity contributes to excitotoXicity and promotes cell death, underpinning a potential neurodegeneration mechanism established in AD. As a result, the GluR- II down-regulation by LAP treatment in the present investigation is a plausible mechanism for its possible therapeutic efficacy against AD (Divakaruni et al., 2017; Quansah et al., 2018). A previous study showed that glutamate oXidation promoted by inhibition of MPC can conse- quently be neuroprotective. The cortical neurons treated with selective MPC-1 inhibitor, UK-5099 were protected from excitotoXicity mediated by glutamate applied in the culture medium, and this could restrict excitotoXic neuronal injury (Divakaruni et al., 2017). Together, these observations lead us to propose that LAP has a neuroprotective effect by attenuating glutamate-associated excitotoXicity. Herein, chronic administration of D-gal to OVX rats resulted in a pronounced increase in TNF-α. This finding accords with previous results (Ibrahim et al., 2020; Kamel et al., 2018). On the contrary, LAP administration reduced hippocampal TNF-α levels. Autophagy impair- ment induces an inflammatory process, and neuroinflammation is an important culprit in AD pathogenesis (Mansour et al., 2021). TNF-α induces Aβ production, neuronal degradation, as well as suppresses Aβ microglia phagocytosis. The current result is in agreement with a pre- vious investigation where MPC-1 modulation reduced lipopolysaccharides-associated induction of TNF-α in mouse primary microglial cells (Ghosh et al., 2016). Hence, the anti-inflammatory effect of LAP may be contributed to TNF-α inhibition. The present study highlighted that LAP administration revealed a distinct inhibition in D-gal/OVX mediated elevation of ERR-α. In addi- tion to the negative modulatory role of both HER-2 and mTOR, the nuclear receptor ERR-α also negatively regulates autophagy, as confirmed by over-expression and knock-down studies. Autophagy was suppressed by ERR-α over-expressed but stimulated when down- regulated. A previous study reported that XCT-790, an inverse agonist of ERR-α, enhanced autophagy, ameliorated α-synuclein toXicity, and exerted neuroprotection in a mouse model of Parkinson’s disease (Suresh et al., 2018). Herein, LAP greatly inhibited the levels of hippo- campal ERR-α, ameliorating Aβ1-42 toXicity. In the present study, we demonstrated, for the first time, a strong positive correlation between Aβ1-42 and hippocampal ERR-α levels, which strengthens the association between amyloidogenesis and these receptors. Previous literature assessing the neuroprotective effect of ERR-α has demonstrated con- flicting results. Consequently, the possible role of ERR-α in learning and memory deserves to be further explored under various physiological conditions or by different behavioral learning and memory tests. In the present study, D-gal/OVX rats had an elevated hippocampal level of Aβ1-42. Amyloid plaques are the primary fundamental cause of AD. Aβ42 is the most neurotoXic type of Aβ that is formed by the cleavage of the APP by γ-secretase (Amigoni et al., 2011). Administra- tion of LAP suppressed D-gal/OVX-induced elevation in hippocampal Aβ1-42. It has been posited that this effect is partially attributed to HER-2, ERR-α, MPC-1, P38-MAPK, and mTOR suppression-mediated stimulation of autophagy that clears Aβ plaques and prohibits neuro- degeneration (Ghosh et al., 2016; Suresh et al., 2018; Wang et al., 2017). Furthermore, we demonstrated for the first time a strong positive cor- relation between ERR-α Figure (3l), MPC-1 Figure (3m), and Aβ1-42. Consistent with these findings, correlative evidence proves that increased ERR-α or MPC-1 expression may exacerbate amyloidogenesis. In the current investigation, LAP amended D-gal/OVX-induced aberration in the hippocampal p-tau levels. Abnormal tau hyper- phosphorylation inhibits microtubule binding, triggering microtubule disintegration, disrupted axonal transport, and the inevitable death of neurons. Impairment of neuronal autophagy may contribute to the inefficient removal of proteins resulting in neuronal death. Following this notion, it is also supposed that mTOR, HER-2, MPC-1, and ERR-α regulate autophagy in neurodegenerative disorders (Ghosh et al., 2016; Suresh et al., 2018; Wang et al., 2017). Plausible evidence suggests that activation of mTOR cascade signaling enhances tau pathology since mTOR stimulation contributes to autophagy dysfunction. Additionally,tau hyperphosphorylation is exacerbated by Aβ, potentially by the activation of various tau-targeting kinases, including mTOR (Plattner et al., 2006). Therefore, the noticed normalization of the hippocampal p-tau level caused by LAP administration is potentially contributed to its inhibitory effects on Aβ1-42, ERR-α, MPC-1, HER-2, and p-mTOR. In summary, our current research reveals that chronic treatment with LAP can ameliorate cognitive dysfunction and decrease Aβ1-42 and p- tau accumulation by regulating p-mTOR, ERR-α, P38-MAPK, and HER-2 in D-gal/OVX rats. Furthermore, LAP increased the pro-survival pathway PI3K/Akt. While inhibiting neuroinflammatory via inhibition of NOX-1 and TNF-α, and suppressing excitotoXicity via inhibition of MPC-1 and GluR-II. Although LAP neuroprotective effect in multiple D-gal/OVX model of AD, through multiple attractive mechanisms, several issues remain to be rectified, such as the efficacy of LAP in other AD models, such as amyloid/p-tau bearing rodents. It would also be intriguing to screen for any possible MPC1 inhibition-mediated epige- netic changes in AD models. Because such epigenetic changes could be biomarkers to target in future clinical trials. As an epilogue, targeting MPC-1 is valuable as it affects various mechanisms involved in AD pathogenesis. Such results will encourage the engagement of MPC-1 inhibitors as possible disease-modifying drugs in AD and other neuro- degenerative diseases. Funding and disclosure The authors declare that they have no conflict of interest. This work did not receive any particular grant from any funding agency. CRediT authorship contribution statement Heba M. Mansour: Investigation, Resources, Formal analysis, Writing – original draft, Data curation. Hala M. Fawzy: Writing – re- view & editing, Visualization, Supervision. Aiman S. El-Khatib: Conceptualization, Methodology, Validation, Formal analysis, Writing – review & editing, Visualization, Supervision. Mahmoud M. Khattab: Conceptualization, Methodology, Validation, Formal analysis, Writing – review & editing, Visualization, Supervision. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.neuint.2021.105178. References Amigoni, L., Ceriani, M., Belotti, F., Minopoli, G., Martegani, E., 2011. 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