Exploring Autophagy Inducing Molecules: Targeting Diverse Pathways in Alzheimer's Disease Management

Neurodegenerative disorders are a significant burden on society, characterized by the progressive deterioration of neurons in the central nervous system (CNS). These modern‐day incongruities, including Parkinson's disease (PD), Alzheimer's disease (AD), migraine, meningitis, and schizophrenia, are on the rise due to various factors such as genetic abnormalities, congenital conditions, malnutrition, stressful lifestyles, and environmental issues [1]. Among these disorders, dementia, particularly dementia due to AD, imposes a substantial economic burden, which is escalating rapidly. The World Health Organization (WHO) reported that approximately 5.8 million people in America are affected by AD‐related dementia, costing around $290 billion. Alarmingly, these numbers are projected to reach 14 million individuals and $1.1 trillion by 2050 [2]. The mortality rate due to dementia has also seen a sharp increase, with a surge of almost 145%(Year)compared to the year 2000.

The pathogenesis of AD involves the continuous progression of neuronal deterioration causing cognitive impairment and memory loss [3]. Alois Alzheimer's pioneering work in dementia defined AD as "senile dementia". However, it is now understood as age‐related neurodegeneration, characterized by the accumulation of misfolded beta‐amyloid proteins and neurofibrillary tangles within neurons [4].

Amyloid precursor protein (APP), present on the surface of the endoplasmic reticulum, undergoes proteolytic cleavage to produce Aβ proteins, which can accumulate both intracellularly and extracellularly [5]. Two pathways, namely the amyloidogenic and non‐amyloidogenic pathways, govern the cleavage of these transmembrane proteins [6]. While Aβ₄₀ and Aβ₄₂ are normal forms present in the brain, the AD brain exhibits an increased accumulation of insoluble Aβ₄₂ [7]. Additionally, the hyperphosphorylation of tau protein leads to the formation of insoluble tangle filaments and the disintegration of microtubules, further contributing to neuronal dysfunction [8, 9].

The clearance of these unwanted and damaged protein aggregates, including misfolded Aβ and tau, is mediated by autophagy, a cellular degradative‐catabolic process crucial for maintaining cellular homeostasis [10]. Autophagy plays a prominent role in removing intracellular and extracellular protein aggregates and damaged organelles. Among its various modes, macroautophagy is the most common form, followed by chaperone‐mediated autophagy and microautophagy [11]. Notably, the dysregulation of autophagy and mitochondrial dysfunction contributes to the pathogenesis of age‐related neurodegenerative diseases like Alzheimer's disease [12].

The clinical evidence suggests that autophagy holds great promise in managing Alzheimer‐related dementia. However, a better understanding of complex mechanisms underlying autophagy dysregulation in AD diseases opens avenues for developing effective treatments. This review aims to provide a comprehensive overview of autophagy in neurodegenerative diseases including AD, emphasizing the potential of therapeutics targeting autophagy especially plant‐derived compounds that hold the promise for future research and development. Additionally, we have also included some repurposed molecules used as autophagy modulators that can be used in dementia related to Alzheimer's.

The direct evidence linking autophagy to Alzheimer's disease was first presented by Nixon and group, where the pathological evaluation revealed the presence of autophagic vacuoles, multilamellar bodies, and dense residuals in affected neuronal cells [13]. Subsequently in another study, further demonstration of the relationship between impaired autophagic vesicle clearance and the accumulation of these vesicles in AD neurons was illustrated [14]. While the endoplasmic reticulum and mitochondria have important correlations with autophagy, the phagophore is considered the initiation site of this process [15].

Regulation of autophagy mainly occurs through two pathways: the mammalian target of rapamycin (mTOR)‐dependent and mTOR‐independent pathways. The mTOR‐dependent pathway involves kinases such as ULK1 and VPS34, which create a pathway for autophagy regulation [16]. Akt‐mediated phosphorylation of mTOR inhibits autophagy, while inactivation of these kinases promotes autophagy [17]. Alternatively, AMPK activation can induce autophagy by inhibiting mTOR1 and promoting phagosome formation. Therapeutic strategies targeting these pathways, particularly inhibiting mTOR complex 1, hold promise in modulating autophagy [18].

In addition to the mTOR‐dependent pathway, independent pathways such as the inositol signaling pathway, calpain signaling pathway, and cAMP pathway have been explored as therapeutic targets for autophagy induction [17]. The inositol pathway, associated with glycolysis and mitochondrial functions, promotes cell growth, but decreased inositol levels can induce autophagy and facilitate the clearance of cellular debris [19]. Similarly, the Ca²⁺/calpain pathway, influenced by cytosolic calcium levels, affects cellular growth, and increased levels inhibit autophagy [20]. Finally, the cAMP pathway, governed by adenosine 3ʹ,5ʹ‐cyclic monophosphate, plays a crucial role in cell signal transduction and can induce autophagy through specific targets like protein kinase A and Epac [18]. Both these mTOR‐dependent and independent pathways are briefly presented in Figure 1 below.

Considering the potential of autophagy as a therapeutic target, extensive research has been focused on identifying compounds from medicinal plants with potent therapeutic activities and low toxicity. Polyphenols, alkaloids, terpenes, and terpenoids from various plant sources have shown promise in treating neurodegenerative diseases [21]. For instance, polyphenols like curcumin and resveratrol have demonstrated in vivo activity in inducing autophagy. Curcumin activates autophagy via the PI3K/Akt/mTOR pathway, while resveratrol induces autophagy through the AMPK/mTORC1 pathway [22, 23]. Additionally, compounds like quercetin, berberine, and Rg2 from ginseng, have also shown the potential to induce autophagy through specific pathways [24, 25, 26].

The neuroglia is a connective tissue present in the peripheral nervous system (PNS) and central nervous system (CNS) which is important for maintaining the homeostasis of the brain via different pathways. The glial cells of the CNS are classified into microglia, macroglia and myeloid lineage cells [27]. Autophagy is an important process in astrocytic cells to clear debris such as protein aggregates and other defective organelles. Astrocytes, meanwhile, have brain region‐specific functions such as calcium signaling at synapses, regulation of manganese concentrations in the brain, and regulation of the blood‐brain barrier, thus also referred to as housekeeping cells [28]. Although autophagy is a ubiquitous process and can be cell‐specific, the ability of astrocytes along with microglial cells to clear extracellular beta‐amyloid aggregates by an APOE‐dependent internalization process is crucial in AD [29]. A study by Simonovitch et al presented that the APOE4 knock‐in mice model had less autophagic clearance compared to the APOE3 model [30]. Similarly, astrocytes' insulin‐degrading enzyme (IDE) secretion is actively triggered via an autophagy‐dependent nonconventional pathway and influences extracellular Aβ degradation [31, 32]. Another pathway of neuroinflammation in AD can trigger the microglia and astrocytes to release anti‐ and proinflammatory cytokines leading to a decrease in mitochondrial integrity and an increase in reactive species, finally causing cell death [33, 34]. Furthermore, the microglial NLRP3‐inflammasome overexpression can cause excess production of inflammatory cytokines and neuronal death. A study by Cho et al confirmed this concept, showing that ATG7 or LC3 gene knockdown in mice model increased the NLRP3 inflammasome activity significantly [35]. In conclusion, data showed that microglial autophagy can promote cell survival by decreasing inflammasome activity, which has disease‐modifying potential.

Oligodendrocytes play an important role in the formation of myelin sheath, providing nutrition to the neurons, and signal transmission [36, 37]. NG2 cells, a subtype of oligodendrocytes show affinity towards Aβ42, which they degrade with the assistance of astrocytic projections. In a similar vein, Aber et al presented the potential role of the autophagic ability of oligodendrocytes using the Atg5 or Atg7 phenotype. The findings suggested that the abnormal thickening and accumulation of myelin sheath mimicked pathological conditions similar to AD [38]. The disruption in autophagic functioning may further lead to neurodegenerative disease progression [39].

Autophagic processes play a crucial role in clearing cellular debris, such as aggregated amyloid‐beta proteins and neurofibrillary tangles, from various neuroglial pathways. Disruption of autophagy can exacerbate Alzheimer's disease pathology. Our work has presented different phytoconstituents that can enhance disease outcomes by promoting autophagy.

Memantine is an FDA‐approved drug for AD. Its mechanism of action is commonly explained via its noncompetitive NMDA receptor‐blocking activity, which prevents the influx of excess Ca²⁺ ions into the neuronal cells, and consequently prevents neuronal damage and apoptosis. It also increases Aβ₄₂ clearance, but autophagy has also been suggested as a main mechanism [85]. Hirano and colleagues observed the increase in LC3‐II levels in the HeLa cell line by memantine and also concluded that the autophagic effects of memantine did not show involvement of mTORC1 and NMDAR pathways but may alter the activity of the VPS34 complex. They harvested Induced pluripotent stem cells (iPSCs) from the dermal fibroblast of a healthy subject, a PARK2 and a PARK 6 patient, and measured differentiation efficiency as well as mitophagic flux. Where they observed autophagic flux in damaged mitochondria of both PARK2 and PARK6 patient neurons via PINK1/parkin signaling.

Another group of researchers, Wet and colleagues, observed the concentration‐based effect of memantine on mitochondrial networks, with dose‐dependent macro‐autophagy and mitophagy activation. Júlia Companys‑Alemany et al illustrated the memantine‐autophagy relation in 5XFAD mice, where it diminished the Aβ levels and improved cognitive function. The induction of autophagy was indicated by elevated levels of autophagy markers such as LC3‐II/LC3‐I ratio, and LAMP1, whereas no alteration was seen in p62 levels [86]. In similar works, it was demonstrated that memantine can exhibit autophagy‐inducing capabilities in other disease models. One such research by Wan‐Soo Yoon et al. illustrated its effect on malignant glioma cell lines (T‐98 G and U‐251 MG) and discovered autophagy‐induced cell death. An increase in LC3‐II/LC3‐I ratio and Beclin‐1 levels was observed along with enhanced formation of autophagic vacuoles via TEM imaging. Also, the role of NMDR1 receptors was confirmed in autophagy in T‐98 G cell lines [87]. Along similar lines, Albayrak et al. explored the effects of memantine on lung cancer lines (A549) and estimated via the Western Blot technique, the levels of LC3B‐I, LC3B‐II, and SQSTM1 proteins. There was an increase in the expression of LC3B‐II and SQSTM1 after treatment with memantine, indicating a prominent role in autophagy [88]. However, the studies have shown G0/G1 cell cycle arrest in cancer cells, but its autophagic role in different disease conditions needs to be explored.

In 2019, a novel H₂S‐releasing prodrug of memantine was synthesized by Sestito and group, by substitution of free NH₂ with H₂S, thereby equipping the molecule with H₂S‐releasing property. Memantine and the newly formed compound, named 'mention', upregulated LC3II expression, while reducing expression of p62, p‐mTOR proteins as well as Akt‐Ser473 phosphorylation. This indicated that both of these molecule's function via modulation of PI3K/AKT/mTOR signaling. Memantine and memit inhibited self‐aggregation of Aβ_1‐42 as well. In conclusion, the author believes that besides its disease‐modifying potential and autophagy inducer, its use is still limited as an NMDA receptor blocker in neurodegenerative conditions [89].

Extensive research has gone into drug development and delivery, which has subsequentially led to promising therapeutic APIs being approved for various pathologies by various regulatory agencies such as the Food and Drug Administration (FDA). These APIs have been approved only after much deliberation and toxicological and pharmaceutical evaluation. This availability of details has led to the exploration of the use of these drugs approved for a certain disease for other pathologies, prominent examples being drugs such as minoxidil (alopecia areata), sildenafil (erectile dysfunction), aspirin (angina), and so forth. Also, there is incessant research into medications and drugs for conditions that have unmet needs or have only symptomatic treatment options available, for example, ulcerative colitis, rheumatoid arthritis, various carcinomas, PD, AD, and so forth.

Certain FDA‐approved drugs have been found to induce autophagy in various studies. Also, some of these have good blood bioavailability, Aβ accumulation inhibition, and tau hyperphosphorylation blocking capabilities to name a few, which have also been demonstrated by various scientific groups listed in Table 2.

These preclinical studies render them suitable candidates for use as autophagy inducers in AD. Here, we will briefly review some of the FDA‐approved drugs, focusing on their role in AD and autophagy modulation.

Carbamazepine (Dibenzoazepine): Carbamazepine (CBZ) is an FDA‐approved anticonvulsant, that has been explored in the induction of autophagy in AD by Lixi Li and colleagues [91]. They observed a notable reduction in Aβ plaque load in APP (swe)/PS1(deltaE9) mice after treatment with CBZ. This was accompanied by the induction of autophagy and reduction in mTOR signaling, both of which were notably independent of each other. Further enlightenment over this was provided through a study by Zhang et al., who also found the autophagy induction of CBZ to be independent of the mTOR pathway, along with an increase in LC3‐II expression and diminished Aβ₄₂ levels. However, dose‐dependent toxicity was also observed [90].

Several clinical studies have focused on the calming effect of CBZ on AD‐related agitation. A couple of clinical studies found CBZ to be efficacious in treating AD‐linked agitation [92, 93]. Certain drug‐related side effects were also noted such as agranulocytosis, leukopenia, and allergic reaction. However, a recent trial, namely the SYMBAD trial (NCT03031184) reported that neither CBZ nor another antidepressant, mirtazapine, could alleviate or diminish agitation related to AD, while duly mentioning that the CBZ group was discontinued due to low recruitment [138]

Rapamycin, everolimus, and temsirolimus (Macrolides): Also called sirolimus, rapamycin is an mTOR inhibitor and a known autophagy inducer. It is an FDA‐approved medication having multiple FDA approvals under its belt, viz kidney transplant rejection (1999), restenosis (2003), locally advanced metastatic or unresectable malignant perivascular epithelioid tumors (2021) and Lymphangioleiomyomatosis (2022) [95]. Rapamycin has exhibited anti‐AD activity in multiple animal models, such as APP/PS1 and P301S mice, leading to augmented memory function and attenuated Aβ load and tau hyperphosphorylation [96]. Currently there are two ongoing clinical trials for evaluating the safety and efficacy of rapamycin in AD, the Evaluating Rapamycin Treatment in Alzheimer's Disease using positron emission tomography (ERAP) phase IIa trial and another phase II trial (NCT04629495) for evaluation of safety and efficacy of rapamycin in early‐stage AD [139].

Temsirolimus is an ester of rapamycin and a 2nd gen mTOR blocker that has been FDA‐approved for renal cell carcinoma. This drug in its complex form with FKBP12 binds to mTOR at its piperidine region causing mTOR to dimerize and inactivate [99]. Another mTOR inhibitor, namely everolimus has emerged as a treatment option for early stage dementia, courtesy of a study on its effects on a 3xTG‐AD mice model. It was administered via an osmotic pump into the brain to overcome its low BBB crossing capability. A higher half‐life in the brain was recorded leading to slower metabolism. A notable enhancement in cognition and a lower negative impact on the periphery were seen, suggesting that intrathecal administration of the drug may improve neurodegeneration via inhibition of mTOR [98]. Another study by Jiang and colleagues on HEK293‐APP695 cells and APP/PS1 mice validated the anti‐AD activity of temsirolimus, which marked a reduction of hippocampal apoptosis and Aβ plaques, as well as improvement in memory and cognition [140].

Bosutinib, nilotinib, dasatinib (c‐Abl inhibitors), and masitinib (Tyrosine kinase inhibitor) (Benzamides): These benzamides have been extensively used in the treatment of leukemia and are FDA‐approved. Research by Irina Lonskaya and colleagues has unraveled that nilotinib and bosutinib increase the solubility of parkin and lead to enhanced parkin‐beclin‐1 interaction, which subsequentially leads to increased Aβ clearance and reduction in plaque load [101, 141]. A clinical study by Pagan et al. explored the effects of bosutinib in dementia with Lewy bodies (DLB) patients and noted that the drug was safe and well tolerated in DLB patients, with attenuation of alpha‐synuclein and dopamine levels in CSF, at approximately the least efficacious dose of bosutinib [103]. In another study by Mahdavi et al., it was observed that bosutinib when administered in patients with probable Alzheimer's dementia or Parkinson's spectrum disorder with dementia responded positively to the therapy over 1 year, and the cognitive and behavioral symptoms worsened with discontinuation of the therapy (NCT02921477) [102]. This study was further followed up 1 year post earlier publication with findings along similar lines, further warranting the continuation of studies investigating the therapeutic role of bosutinib in dementia and AD [142].

Dasatinib is also a promising TKI explored by multiple groups for its effect on cognition [143, 144] undergoing clinical trials in combination with quercetin. Adam Krzystyniak and colleagues evaluated the dasatinib and quercetin combination for efficacy in AD in senescent Wistar rat models. They noticed a significant improvement in cognitive ability as well as spatial awareness using active allothetic place avoidance test, also they confirmed that the combination therapy restored hippocampal functions [145]. Both quercetin (as discussed above) and dasatinib have autophagy‐inducing activity. There have been multiple reports of autophagy‐inducing activity of dasatinib in a wide array of disease models, viz ovarian cancer [107], leukemia [146], and malignant pleural mesothelioma [147]. In brief, it enhanced beclin‐1 expression, p62 degradation, and LC3B‐II levels and reduced AKT expression. The clinical investigations of the combination therapy have been discussed in a later section containing drugs in clinical trials for AD with linked autophagy mechanisms.

Dapagliflozin (c‐glucoside Sodium‐glucose co‐transporter‐2) got FDA approval for disease conditions like heart failure and type‐2 diabetes, where it blocks the reabsorption of glucose from proximal convoluted tubule. Its role in preventing heart failure is under scrutiny, however, the potential mechanism is through diminishing calcium levels in cardiac myocytes and prevention of cardiac remodeling [148]. In addition to this, Furuya et al. explored the autophagic potential of dapagliflozin in the liver and found that the prominent hepatic autophagy, where the rise in LC3‐II: LC3‐I ratio along with inhibition of mTORC1 was observed through SGLT2 inhibition. However, the expression of p62 remained unaltered [109]. In a similar study by Li et al., it was observed that the inhibition in AMPK‐mTOR signaling (enhanced activation of AMPK and decreased dephosphorylation of mTOR) and thus induces autophagy in hepatic steatosis‐induced HepG2 cells and ZDF rat models [110]. The same phenomenon was further investigated in cadmium‐induced testicular dysfunction in rat animal models. The upregulation of Beclin 1 and a decrease in SQSTM‐1/p62 accumulation lead to the activation of the autophagic pathway [149]. Samman et al examined the role of dapagliflozin therapy in aluminum chloride‐induced dementia in a Swiss rat model, which was further investigated by behavioral as well as mechanistic studies. The results from the treatment group exhibited a lower mean escape latency and improved target recognition in the Morris Water Maze (MWM) test. Also, the rise in total entries as well as spontaneous alternations in the Y‐Maze test were seen. The higher p‐AMPK and p‐mTOR activation resulting in reduced oxidative stress and inflammatory markers were further supplemented through western blot analysis. In conclusion, dapagliflozin showed a neuroprotective effect in the induced AD model, but further investigation is needed to unravel the full potential [111]. A phase‐2 clinical trial (NCT03801642) exploring the safety, tolerability and efficacy of 10 mg dapagliflozin administered in probable AD patients was performed, with results not disclosed till present [150].

Liraglutide and Semaglutide (Glucagon‐like peptide (GLP‐1) analog): Liraglutide got its approval for type 2 diabetes in pediatric patients and weight management drug in children of 12 and above (fda.gov, 2019,2020). Liraglutide has been reported to increase LC3‐II, beclin‐1 levels, and Atg7 levels in high‐fat diet rats and HepG2 cell lines. It also enhanced the activation of AMPK and reduced p‐mTOR expression. The authors concluded that liraglutide induced autophagy via the AMPK/mTOR pathway [113]. Another study explored the effects of liraglutide on hepatic steatosis. It was observed that the lipopeptide increased autophagic flux via elevated nuclear translocation of Transcription factor EB (TFEB) and Increased CTSB and LAMP1 expression which are involved in downstream signaling in the TFEB pathway [114]. The autophagic induction by liraglutide was further validated by Yuan and colleagues, in an erectile dysfunction rat model using cavernous nerve electrostimulation. The study revealed that the drug alleviated erectile dysfunction via GLP‐1R expression leading to autophagy along with reduced ROS levels [115]. Moreover, its role in the treatment of AD was further assessed in the phase IIb clinical trial (NCT01843075), where it was assessed as a GLP‐1 analog and showed a notable improvement in cognitive capabilities and MRI volume [116]. Liraglutide has also been explored as a dual treatment option for type 2 diabetes and AD in APP/PS1xdb/db mice. The results indicated that treatment with liraglutide may prevent brain damage in Type 2 diabetes when AD is concurrent but clinical application is a bit debatable [151]. However, McClean and colleagues have thoroughly explored the anti‐AD effect of liraglutide in various prophylactic and AD‐induced APP/PS1 mice models and validated the therapeutic efficacy of this GLP‐1 analog. Liraglutide exhibited the potential to reduce cognitive decline in mild AD patients by up to 18% when compared to the placebo group due to a decrease in the rate of shrinkage of cognitive parts of the brain [152].

Semaglutide is a Glucagon‐Like Peptide‐1 receptor agonist approved by the FDA for weight loss and type‐2 diabetes in adults [153]. In a similar study, it also induces autophagy in SH‐SY5Y cells impaired by Aβ_25‐35. This was validated by an increase in Atg7, Beclin‐1, p62, and LC3‐II levels in semaglutide‐treated cell lines [118]. Another study reported that semaglutide may have a positive role in diminishing dementia in patients with type‐2 diabetes [154]. Currently, two phase‐3 clinical trials, EVOKE (NCT04777396) and EVOKE+ (NCT04777409) are being conducted to garner information related to the safety and efficacy of this autophagy inducer in early‐onset AD [155].

Metformin (Biguanide): Metformin is the drug of choice for type‐2 diabetes. It has a very good safety profile and therapeutic window [156]. To validate the autophagic flux enhancement by metformin in cancer pathogenesis, Mauro de Santi and colleagues performed extensive study on multiple cell lines, viz, NIH/3T3, 3T3‐619C3, 3T3‐619D3, 3T3‐621B1, 3T3‐621B6, and3T3‐SCRD3 cell lines previously treated with MNU and H₂O₂ (for induction of tumorigenesis) and observed cytostasis in all of these, indicating an autophagic effect on cancer cells. This was further confirmed by an increase in LC3‐II and p62 expression. Also, NIH/3T3 and 3T3‐SCRD3 cell lines exhibited reduced cell viability, whereas shATG7 cell lines did not show the same, indicating that metformin‐induced autophagy in impaired cells [157]. There have been multiple reports of autophagy induction due to metformin treatment through a wide spectrum of pathways [158]. Metformin has been reported to enhance AMPK activation in the cerebrum and decrease mTOR and NFκB in APP/PS1 mice [121]. Furthermore, Campbell and colleagues discovered via a meta‐analysis of clinical studies, that metformin administration leads to reduced risk of dementia in patients with type‐2 diabetes [159]. However, metformin may cause vitamin B12 deficiency which has been reported to have a significant role in memory functions and cognitive ability [160, 161]. A case‐control study by Janet et al. investigated the risk of AD in diabetes patients administered with metformin. The team concluded that metformin not only does have any AD‐related risk, but contrarily it was associated with a lower risk of AD incidences in older diabetic patients [123]. A pilot clinical study exploring the safety and tolerability of metformin in Luchsinger et al. in prevention and treatment of hyperinsulinemia in patients with amnestic mild cognitive impairment (AMCI). The study revealed that metformin moderately improved cognitive symptoms while not causing any serious adverse effects. Metformin was tolerable at different doses for different patients, where only 10% of patients were able to tolerate the maximum dose of 1000 mg twice a day [122].

Simvastatin (hexahydro naphthalene): Statins are antihyperlipidemic drugs that inhibit HMG‐CoA reductase enzyme leading to reduced cholesterol levels, and therefore find their use as an adjuvant in a plethora of cardiovascular diseases as well, such as hypertension, angina, etc [162]. Simvastatin is derived from lovastatin which was the first statin to get FDA approval. Simvastatin is FDA‐approved as an antihyperlipidemic, however recent research has explored this statin for its therapeutic efficacy in a wide array of diseases, such as atherosclerosis [163], osteoarthritis [125], alcohol‐induced liver disease [164], hepatitis C [165], hepatocellular carcinoma [166], etc. with improvement in aforementioned pathologies via induction of autophagy. Wei and colleagues studied the effects of simvastatin in coronary artery myocytes (CAMs) and discovered that the statin significantly enhanced LC3B and Beclin1 levels. Moreover, the autophagy induction was confirmed by flow cytometry by which it was found that autophagosome generation was increased, and by confocal microscopy which permitted them to visualize the elevated lysosomal fusion of the autophagosomes, which reduced with the introduction of 3‐methyladenine, an autophagy blocker, or Atg7 gene knockdown. The mTOR‐dependent autophagy induction of simvastatin‐conjugated gelatin hydrogel administration in 10‐week‐old osteoarthritis‐induced C57BL6/J mice was demonstrated by Tanaka et al. The hydrogel reduced mTOR phosphorylation and IL‐1β levels and escalated the LC3‐II/LC3‐I ratio. Along similar lines, Atef and colleagues evaluated simvastatin activity in an alcohol‐induced liver disease model in Swiss albino rats and observed that it restored autophagy, indicated by increased LC3‐II levels as well as to some extent, reversal of alcohol‐induced morphological damage and functional abnormalities and reduction in oxidative stress and endoplasmic reticulum stress‐induced apoptosis. Furthermore, simvastatin has been reported to ameliorate AD and reduce Aβ₄₀ and Aβ₄₂ load in guinea pigs [167]. In a comparative study with atorvastatin and lovastatin, simvastatin exhibited the highest reductions in Green fluorescent protein fused Aβ levels in yeast in a dose‐dependent manner [168]. However, in a phase 3 clinical trial, simvastatin did not reduce the pace of clinical progression of AD, despite reducing the body's lipid levels [169].

Gemfibrozil (aromatic ether): Rongcan Luo and colleagues were interested in whether peroxisome proliferated activated receptor‐alpha (PPARA), which has a prominent role in autophagy and metabolism of fatty acids in the liver, could be explored as a target for Aβ clearance, as in case of PP‐PSEN1/E9 mice. Gemfibrozil, an FDA‐approved antihyperlipidemic PPARA agonist, was selected for further evaluation along with another agonist Wy14643. The results from the behavioral, in‐vitro, and in‐vivo examinations revealed that the drug significantly reduced cognitive dysfunction and enhanced spatial awareness, along with a marked reduction in anxiety. The autophagosomes generated in microglia and astrocytes near the plaques were observed, which was attributed to a reduction in soluble as well as insoluble Aβ in both cortex and hippocampus tissue [128]. In another study, gemfibrozil reduced Aβ plaques and neuroinflammatory processes such as astrogliosis and microgliosis in 5XFAD mice. Improvement in spatial awareness and cognitive function was confirmed by the Barnes maze and T‐maze test [129]. Moreover, a phase‐2 clinical trial (NCT02045056) was conducted to evaluate the safety and efficacy of gemfibrozil in predementia. The double‐blind, placebo‐controlled trial reported improvement in AD‐related neuroinflammation along with a marked reduction in Aβ₄₂ levels and atrophy in the hippocampus [130].

Nicotinamide (Vitamin B3): It is water soluble Vitamin B3, which is FDA‐approved as a food additive and is listed as a GRAS agent [170]. The mechanism by which nicotinamide has been proposed via PARP‐1 inhibition, DNA repair, ROS inhibition, etc [171, 172]. In recent times, its role in the induction of autophagy has become a topic of research, consequentially leading to further exploration [131]. In a study on H9C2 chronic hypoxic myocardial cells, it was found that nicotinamide treatment improved the viability of the cells, while also augmenting Atg5 (Autophagy‐ Related Gene 5) expression and LC3‐II: LC3‐I ratio [132]. Kang et al. demonstrated the induction of autophagy in fibroblast cells. They noted a significant attenuation of mitochondrial mass in nicotinamide‐treated cells via autophagy. Nicotinamide treatment enhanced the replicative lifespan of fibroblasts and improved mitochondrial quality expressed as increased membrane potential (ΔΨm). Senescent cells exhibit a reduction in autophagosome formation and autophagy, whereas ROS generation increases. This leads to over‐accumulation of damaged and disrupted cell organelles malfunctioning cell constituents and ultimately aging‐related abnormalities. Nicotinamide treatment led to augmentation in autophagic processes and increased autophagic flux [133].

In another study by Liu and colleagues on the effect of nicotinamide in AD, notable improvement in mitochondrial intactness was seen in nicotinamide‐treated 3xTgAD mice. It attenuated cognitive decline and AD‐related pathological markers, namely Aβ levels and tau hyperphosphorylation along with improved cognitive ability and spatial awareness [134]. However, a phase 2 clinical trial to evaluate the efficacy and safety of nicotinamide in mild to moderate AD failed to demonstrate any plausible efficacy of nicotinamide in the aforementioned conditions [135]. However, another phase 2a clinical trial exploring the role of nicotinamide in the modulation of early AD (NEAT study) has also been completed, with results showcased at the 2023 Alzheimer's Association International Conference. Nicotinamide attenuated changes in Clinical Dementia Rating‐Sum of Boxes (CDR‐SB) concerning placebo, with improved CSF p‐tau181 and total tau levels (Neurology Live, 2023).

Vorinostat (carboxylate): Vorinostat is an inhibitor of histone deacetylase (HDAC), FDA‐approved for cutaneous T‐cell lymphoma (CTCL) [137]. A research study conducted by Hrzenjak and colleagues validated the autophagy‐inducing potential of vorinostat on the ESS‐1 tumor cell line. There was an augmentation of mono dansyl cadaverine (MDC) levels after treatment with vorinostat, which indicated an elevated accumulation of autophagic vacuoles in the cells concerning control. Moreover, notable concentration‐dependent protein level attenuation of autophagy signaling proteins mTOR and phosphor‐mTOR provided further validation for the same [173]. Another study by Dupéré‐Richer unraveled that initial administration of vorinostat induces autophagy in parental U937 cells, however, there is a gradual adaptation, and the cells begin to utilize this augmentation in autophagy to enhance cell survival and consequentially there is a rise in vorinostat resistance, which was reversed with chloroquine [136]. To confirm the hypothesis of HDAC inhibitors inducing autophagy, Shao and colleagues studied observed Bcl‐XL overexpressing HeLa cells previously treated with vorinostat under a transmission electron microscope (TEM), which is regarded as the gold standard for examination of cell ultrastructure. Morphological alterations characteristic of autophagy‐induced cell death were observed in these cells, such as vacuolization, mutilated nuclei, autophagosome accumulation, and so forth [174].

HDAC inhibitors have been tested as a treatment option for AD in cell lines as well as in animal models, and vorinostat has been no exception. Kilgore and colleagues evaluated the efficacy of multiple HDAC inhibitors in the APPswe/PS1dE9 mouse model of AD and noted that vorinostat recovered memory functions and spatial awareness. It was also the only HDAC inhibitor to target HDAC6 [175]. Similarly, Benito et al. revealed that along with neurological improvement, vorinostat also reduces Aβ accumulation and neuronal inflammation as well as assists restoration of genetic changes [176]. However, another study reported limited bioavailability of vorinostat in the Tg2567 mouse model of AD. This was attributed to the poor pharmacokinetic profile of the drug [177], which may be improved by formulation development and optimization processes [178].

Furthermore, various combination therapies with vorinostat have also been explored for AD. Treatment with a cocktail therapy of curcumin, vorinostat, and silibinin in Aβ_25‐35 pretreated PC12 cells attenuated ROS levels, recorded via measurement of fluorescence under confocal microscopy. Reduction in superoxide dismutase (SOD) and malondialdehyde (MDA) levels further supported this finding. The cocktail preserved Akt/MDM2/p53 signaling and also had a neuroprotective action against Aβ25‐35‐induced cellular toxicity [179]. Furthermore, to increase the therapeutic efficacy, thereby attenuating dose‐dependent toxicity of HDAC inhibitor vorinostat, a phosphodiesterase‐5 inhibitor, namely tadalafil was administered concomitantly in an APP/PS1 mice model of AD with lower doses of the former. It was observed that the drugs worked in synergism to alleviate the symptoms and improve the pathological identifiers of AD, viz cognitive dysfunction, reduced dendritic spine density, Aβ load, and tau hyperphosphorylation [180]. Augmentation of clusterin in human astrocytes by vorinostat has also been linked with its anti‐AD activity [181]. Currently, there is an ongoing phase1b trial (NCT03056495) of vorinostat in AD [144].

Due to the limited therapeutic options for AD, a wide array of drugs is being currently explored for their safety and efficacy in the aforementioned condition. Several of these are currently in clinical trials (as mentioned in Table 3) for AD therapy, some of which have linked autophagy‐inducing mechanisms as well. For example, tyrosine kinase inhibitors such as nilotinib and dasatinib are currently undergoing clinical trials for AD [106]. As discussed above, these moieties have autophagy‐inducing capabilities. Several polyphenols such as quercetin, resveratrol, and curcumin, which have autophagy‐inducing capabilities, are also currently undergoing phase II clinical trials for AD (NCT06470061) [202]. Rapamycin and niacin, which induced autophagy as discussed above, are currently undergoing phase I clinical trials for AD (NCT05723172) (NCT06582706).

Trehalose is a carbohydrate commonly used as a food additive in various food processing industries [203]. Chen et al., in their research, have unraveled that trehalose as well as other carbohydrates such as sucrose and raffinose induce autophagy by augmenting the conversion of LC3‐I to LC3‐II via an mTOR‐independent pathway [203, 204]. Mirtazapine is an antidepressant that acts via inhibition of adrenergic α₂‐autoreceptors and α₂‐heteroreceptors as well as 5‐HT₂ and 5‐HT₃ receptors [205]. It has been found to induce autophagy, however, induction of autophagy by mirtazapine was observed to be via HMGB1‐dependent Akt/mTOR pathway, and induced cardiotoxicity in a C57BL/6 J mice model, which was further augmented by alcohol [184]. Further research is required to warrant its use as a therapeutically active autophagy inducer.

A potent vasodilator hydralazine is conventionally used for its antihypertensive properties. While not much is reported about its role in autophagy, Mirzaei et al. have mentioned that it induces autophagy at therapeutically active doses such that it enhances the clearance of protein aggregates [186]. Levetiracetam is a potent antiepileptic that has been reported to upregulate autophagy‐promoting proteins such as beclin‐1 and LC3‐II/LC3‐I ratio in both SH‐SY5Y and APPswe/PS1dE9 transgenic mice amounting to reduced cognitive disability in mice in the preclinical setting [187]. Sphingosine‐1‐phosphate receptor modulators have recently been approved for various pathologies such as multiple sclerosis and ulcerative colitis [206]. Gruchot et al. observed that autophagy was one of the various mechanisms that got enriched upon treatment of microglia with siponimod for their activation with lipopolysaccharide [188]. Vortioxetine, a serotonin reuptake inhibitor, has been observed to induce autophagy in AGS gastric cancer cells via PI3K/AKT pathway, augmenting the levels of beclin1 and LC3 [189]. In another recent study by Lopez et al., CD‐3 monoclonal antibody Foralumab exhibited the potential to upregulate autophagy‐inducing genes in a 3xTg mice model of AD [191].

β2‐agonist administration has been known to induce skeletal muscle hypertrophy. Joassard et al. observed that β2‐agonist formoterol, when administered in rat skeletal muscle, led to an increase in autophagy gene LC3b and gamma‐aminobutyric acid receptor‐associated protein‐like 1 (Gabarapl1) [207]. In a cljnical setting of PD, Abdelmoaty et al. by proteomic analysis observed that in PD patients, treatment with sargramostim, a recombinant human granulocyte‐macrophage colony‐stimulating factor, augmented the levels of autophagy‐inducing proteins ATG3, ATG7, and GABARAPL2 within 6 months of therapy with sargramostim [208]. Pimavanserin is an atypical antipsychotic conventionally used to manage symptoms of psychosis such as delusions and hallucinations. However, a recent study by Ramachandran et al has demonstrated that it may also be used as an inducer of autophagy for pancreatin cancer therapy [196] since it augments the levels of autophagy in pancreatic ducal adenocarcinoma (PDAC) cells. The same was then demonstrated in‐vivo via a Subcutaneous xenograft pancreatic tumor model.

A phosphodiesterase‐4 inhibitor roflumilast was inspected preclinically for its therapeutic effects on ovariectomy‐induced depression‐like symptoms. It was observed that roflumilast attenuated depression‐like symptoms via induction of autophagy by activation of AMPK/mTOR/ULK1‐dependent autophagy pathway in Swiss albino rats. A consequent enhancement of p‐AMPK, p‐ULK1, Beclin‐1, and LC3II/I expression and reduction in p62 and p‐mTOR protein expression was also noted [197]. In another study, idazoxan, an imidazoline, was found to induce autophagy and augment levels of autophagy marker LC3‐II in RAW.264.7 cells [198].

ANAVEX2‐73 (blarcamesine) is a Sigma Receptor (SIGMAR1) agonist. Recently, SIGMAR1 activity has been explored for its possible implication in various cognitive and neurological conditions [199]. Along similar lines, in a study by Christ et al., ANAVEX2‐73 was demonstrated as an autophagy‐inducing agent in HeLa and HEK293A cells as well as in C. elegans [209]. Methylphenidate, a CNS stimulant approved for ADHD, has exhibited autophagy‐inducing capabilities in a study by Kong et al. However, the aforementioned drug has been linked with an increased risk of damage to the retina, and autophagy was observed to be a causative factor due to its negative effects on photoreceptor cells [200]. An orphan drug Bryostatin, has been approved by the FDA for the therapy of fragile X syndrome [210] has shown promise as an autophagy‐inducing agent, enhancing the autophagic flux of Aβ protein aggregates in hippocampal neurons [201].

Autophagy is an important physiological process quintessential for the normal functioning of cells, promoting clearance of senescent or unessential cellular material and synthesis of new proteins and material required for various cellular processes. This section discusses various novel and conventional therapeutic moieties as autophagy‐inducers (also mentioned in Table 4) by various research groups.

Spermidine is a known autophagy inducer with neuroprotective effects. It induces autophagy by cleavage of Beclin‐1, which is supported by the translocation of the caspase‐3 enzyme in staurosporine‐treated PC12 cells and cortical neurons [211]. Lithium is an approved medication for bipolar disorder. It has shown promising preclinical results for its therapeutic application in other neurological disorders, including AD [227]. A couple of studies have reported autophagy‐inducing effects of lithium, via inhibition of Inositol monophosphatase (IMPase) and inositol polyphosphate‐1‐phosphatase (IPPase), which further downregulates IP₃‐DAG levels via attenuation of inositol levels [212, 228].

Metixene, an anticholinergic drug used in the therapy for PD, has been observed to induce autophagy preclinically in metastatic cancer and brain metastasis in immunodeficient mice models and BT‐474Br and MDA‐MB‐231 cancer cells. The moiety was found to induce incomplete autophagy via N‐Myc downstream regulated 1 (NDRG1) phosphorylation which subsequently caused caspase‐induced apoptosis in the cells [213]. A recent study by Shaban et al. focuses on synthesizing novel pyridine derivatives with profound apoptosis and autophagy‐inducing capabilities on MCF‐7 Cells. They observed that a novel molecule 'Compound 12' exhibited profound autophagy‐induced cell death, which was analyzed through flow cytometry using acridine orange lysosomal dye. The IC₅₀ value for the same was 0.5 and 5.27 μM for MCF‐7 and HepG2 cell lines respectively [214]. Another research group Liang et al. designed and synthesized various urea derivatives 10a ∼ 10r, 11a, 11b. and 12a ∼ 12c and examined for their autophagy‐inducing properties. The results suggested that the 10p was most potent with IC₅₀ values of 1.97 ± 0.01, 6.18 ± 0.56, 4.92 ± 0.61, 7.01 ± 2.23, and 13.79 ± 3.15 against HT‐29, HeLa, K562, MCF‐7, and HEPG2 cells respectively It was also observed that 10p caused cell cycle arrest at G2/M phase [215].

Drugs with antiparasitic activity such as albendazole and mebendazole have been conventionally used for their anthelminthic activity. A recent study by Jung et al. centered around the autophagy‐inducing potential of albendazole has revealed that it induces AMPK and ULK1 phosphorylation‐mediated autophagy in human colon adenocarcinoma cells such as HCT‐15, HCT‐116, HT‐29, and SW480 cells [216]. Similarly, mebendazole was found to induce autophagy in HUVEC cells. An increase in LC3B+ puncta in HUVEC cells at 1 and 3 μM concentrations was observed via immunofluorescence, further validated using siRNAs of ATG5, ATG7, and Beclin1 to knock out the specific autophagy mediating genes [221]. In a separate study, oxibendazole and teniposide, a podophyllotoxin derivative, were observed to induce autophagy via lysosomal co‐localization augmented by TMEM55B‐ JNK‐ interacting protein 4 (JIP‐4) in SH‐SY5Y cells and extensively verified using JIP‐4 knockout cells [217]. A couple of other anticarcinogenic agents, amsacrine and etoposide have also preclinically exhibited autophagy‐inducing activity. Lee et al. in a recent study discovered that amsacrine induces autophagy in human chronic myeloid leukemia (CML) cells by augmentation of SIDT2 levels, subsequently leading to attenuation of miR‐25. This reduction in miR‐25 expression consequently led to an aggravated NOX4‐mediated ROS production [218]. Also, ABT‐263 (Navitoclax) an experimental BCL2 inhibiting anticancer molecule had autophagy‐inducing properties, mediated by increased IKKα/β‐NFκB axis expression in U937 cells, with cell viability being diminished to 50% after 24 h of 1 μM treatment with ABT‐263 [229]. The autophagic nature of ABT has been further validated through various other studies [219]. The compound has also preclinically been able to promote the clearance of senescent glial cells, warranting further research as a therapy for AD as well [230]. The response of DNA‐damaging anticancer agents on autophagy was examined by researchers at the University of Bern, where they discovered that even nonlethal microdoses of agents such as cisplatin and etoposide were able to trigger the augmentation of ATG5, a prominent mediator of autophagy, causing mitotic catastrophe, independent of induction of autophagy. However, a separate study by Karlitepe et al. and Pilevneli et al. demonstrated that etoposide markedly induces serin/threonine phosphatase (Wip1/PPM1D) mediated autophagy in MCF‐7, D283‐med and IMR32 cells, with rise in LC3I/II and p62 expression, analyzed via western blot and immunostaining assays [220, 231].

Microcolins, obtained from marine cyanobacterium Moorea producens, are structurally identical to lipopeptides and have anticancer properties. Its anticancer effects were attributed to its modulation of phosphatidylinositol transfer protein alpha/beta isoform (PITPα/β) and triggers autophagy leading to an increase in the levels of LC3II to LC3I and Beclin‐1. However to ensure its role in AD, further research is needed [222].

Certain mTORC modulators such as NR1, DL001, and NV‐5440 have also been explored as autophagy inducers and subsequently exhibited preclinical prowess as the same [232]. NV‐5440 was observed to have an IC₅₀ value of 0.07 μM in MCF7 cells [224], whereas DL001 had an IC₅₀ value of 74.9 pM in PC12 cells [223]. Specific peptides such as Exendin‐4, apelins, and myokines also have been reported as autophagy inducers. Exendin‐4 has been recently analyzed for its role in the modulation of autophagy in an AAV‐9‐A53T‐α‐synuclein‐administered animal model of PD. It was discovered that treatment with exendin‐4 diminished the pathological α‐synuclein and consequently attenuated neuronal degradation and improved cognitive ability [225]. Apelin‐13 is another such peptide that has exhibited bone protection potential in bone marrow mesenchymal stem cells and OVX rats via activation of mitophagy [226]. Gemfibrozil, a phenolic ether conventionally used as a lipid‐lowering agent, is a PPARA agonist. It has been earlier reported and validated that PPARA activation through an agonist potentiates autophagy pathways in cells [233]. Wy14643, an agonist of PPARA receptors, has been studied for its effects on autophagy by Luo et al. both in vitro in human microglia (HM) cells and U251 human glioma cells expressing mutant APP‐p.M671L and in vivo in APP‐PSEN1ΔE9 mice. They observed a marked reduction in both soluble and insoluble Aβ protein in hippocampal and cortical regions, along with increased microglial autophagosome expression [128].

Autophagy is a complex biological process that plays a dual role in health and disease. Its manipulation, either stimulation or inhibition, has proven beneficial depending on the disease context. In cancer, autophagy can diminish tumor growth by clearing senescent cells and toxic metabolites, but it can also be exploited by cancer cells for survival under stress [234]. In neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), impaired autophagy contributes to the accumulation of misfolded proteins and cellular debris, exacerbating neurodegeneration. In AD specifically, reduced autophagy hampers the clearance of amyloid‐β (Aβ) plaques and tau protein, leading to progressive cognitive decline and memory loss [235, 236]. This culminates in progressive memory loss, mental confusion, forgetfulness, and eventually, inability to perform daily tasks and follow basic instructions [17].

Current FDA‐approved AD treatments include cholinesterase inhibitors (donepezil, rivastigmine, and galantamine), and NMDA receptor antagonists (memantine) [237], and monoclonal antibodies (aducanumab and lecanemab), primarily address symptoms or reduce amyloid load. Aducanumab and Lecanemab exhibit side effects like Amyloid Imaging Abnormalities (ARIA), headache, and delirium, which can be fatal in some cases, therefore limiting their use in patients [238].

Various autophagy inducers have been investigated for their therapeutic potential in Alzheimer's disease (AD). Some candidates have progressed to clinical trials, while others have demonstrated improvements in memory and cognitive functions in animal models. However, further research is essential to achieve the ultimate therapeutic goal for AD. Since current pharmacological treatments primarily focus on symptom management, there is an urgent need for disease‐modifying therapies. Autophagy inducers hold significant promise as future treatments for AD.

Many of these promising inducers belong to BCS Class 2 or Class 4, presenting challenges such as low bioavailability, limited permeability, and off‐target adverse effects. Nanotechnology offers innovative solutions to these limitations by facilitating targeted drug delivery, improving drug solubility and stability, and enhancing the ability of drugs to cross the blood‐brain barrier (BBB). These advancements optimize therapeutic efficacy while minimizing systemic side effects, addressing the inherent drawbacks of these drug classes [239]. Nanoparticle‐based strategies have opened new avenues for realizing the full therapeutic potential of autophagy inducers. By enhancing bioavailability and enabling precise delivery to target tissues, these approaches reduce dosing frequency and significantly lower the risk of off‐target side effects, thereby improving the safety and effectiveness of autophagy‐inducing therapies [240, 241, 242]. Continued exploration of autophagy‐based therapies, combined with nanotechnology, could lead to breakthroughs in AD treatment, expanding therapeutic options and addressing unmet needs. With further research, autophagy inducers might emerge as a cornerstone of future AD pharmacotherapy, moving beyond symptomatic relief to modifying the course of the disease.

Conceptualization was carried out by Baljinder Singh, Shubham Mahajan, Rehan Khan, and Sanjay Garg; the original draft was prepared by Baljinder Singh and Shubham Mahajan; review and editing were performed by Baljinder Singh, Shubham Mahajan, Sadikalmahdi Abdella, Rehan Khan, and Sanjay Garg; supervision was provided by Sanjay Garg and Rehan Khan. All authors have read and approved the final version of the article.

The authors declare no conflict of interest.