L-Leucine Upregulates Lysosomal Biogenesis and Autophagy to Lower Plaques in 5XFAD Mouse Model of Alzheimer's Disease.

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Published in final edited form as: J Neurochem. 2026 Apr;170(4):e70432. doi: 10.1111/jnc.70432 Search in PMC Search in PubMed View in NLM Catalog Add to search L-Leucine Upregulates Lysosomal Biogenesis and Autophagy to Lower Plaques in 5XFAD Mouse Model of Alzheimer’s Disease Ramesh K Paidi Ramesh K Paidi 1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA 2 Division of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA Find articles by Ramesh K Paidi 1, 2 , Sukhamoy Gorai Sukhamoy Gorai 1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA Find articles by Sukhamoy Gorai 1 , Susanta Mondal Susanta Mondal 1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA 2 Division of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA Find articles by Susanta Mondal 1, 2 , Kalipada Pahan Kalipada Pahan 1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA 2 Division of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA Find articles by Kalipada Pahan 1, 2 Author information Copyright and License information 1 Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA 2 Division of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois, USA Author Contributions Ramesh K. Paidi: investigation, writing – original draft, data curation, visualization, validation, formal analysis, software. Sukhamoy Gorai: investigation, formal analysis, software, visualization, validation, data curation. Susanta Mondal: investigation, formal analysis, validation, data curation, visualization. Kalipada Pahan: conceptualization, funding acquisition, writing – review and editing, writing – original draft, resources, supervision, project administration, software. ✉ Correspondence: Kalipada Pahan ( [email protected] ) PMC Copyright notice PMCID: PMC13070289  NIHMSID: NIHMS2164106  PMID: 41940752 The publisher's version of this article is available at J Neurochem Abstract Twenty different amino acids are required for the human body for proper functioning as amino acids serve as building blocks for proteins. We screened different essential and non-essential amino acids for the ability to stimulate lysosomal biogenesis and, interestingly, found an essential amino acid L-leucine as the most potent one in stimulating lysosomal biogenesis in astrocytes. However, D-leucine remained weaker than L-leucine in terms of stimulation of lysosomal biogenesis. Accordingly, L-leucine increased autophagy in cultured brain cells and in vivo in the brain of 5XFAD mice, one of the animal models of Alzheimer’s disease (AD). L-Leucine also stimulated the uptake and degradation of amyloid-β in astrocytes and reduced the plaque load and improved cognitive functions in 5XFAD mice. Although L-leucine was discovered about 200 years back, until now, no receptor has been identified for L-leucine. Here, we noticed that L-leucine binds to the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) to activate this nuclear hormone receptor. Accordingly, L-leucine remained ineffective in increasing lysosomal biogenesis and autophagy in PPARα −/− brain cells. Lentiviral establishment of full-length PPARα , but not Y314D-PPARα , reinstated the autophagy-stimulating effect of L-leucine in PPARα −/− astrocytes, emphasizing the importance of leucine’s interaction with the Y314 residue. Moreover, oral L-leucine decreased the plaque load and improved spatial learning and memory in 5XFAD mice, but not in 5XFAD ΔPPARα mice ( 5XFAD lacking PPARα ), highlighting the involvement of PPARα in the neuroprotective effects of L-leucine. These results may be beneficial for AD patients. Keywords: Alzheimer’s disease, autophagy, cognitive function, L-leucine, lysosomal biogenesis, plaque 1 |. Introduction Alzheimer’s disease (AD), an age-related neurodegenerative disease, is the most common cause of human dementia ( Reitz et al. 2011 ). It is estimated that nearly 7 million individuals are living with AD in the USA and that this number may go up to 13 million by 2050. The extracellular senile plaques and intracellular neurofibrillary tangles are two most notable pathological features of AD ( Korczyn and Grinberg 2024 ; Rangasamy et al. 2018 ; Congdon et al. 2023 ; Dutta et al. 2023 ). While plaques are basically formed by short amyloid-beta (Aβ) peptides, tangles consist of hyperphosphorylated microtubule associated protein tau ( Congdon et al. 2023 ; Dutta et al. 2023 ). Clinically, AD is characterized and monitored by progressive deficiency in memory, judgment, decision making, and language usage ( Nussbaum and Ellis 2003 ). Although the etiology of AD is poorly understood, it is believed that multiple risk factors including age, stress, sleep deprivation, brain injury, genetic background, etc. may be responsible for AD ( Reitz et al. 2011 ; Biessels and Despa 2018 ). Despite intense investigations, no effective treatment is currently available for AD. Several studies have demonstrated that dysfunctional autophagy is closely associated with the pathogenesis of AD. Accordingly, autophagosomes are accumulated in neuronal dendrites rather than soma in mouse models of AD ( Yu et al. 2005 ). Interestingly, the abnormal accumulation of autophagosome occurs before the formation of amyloid plaques ( Yu et al. 2005 ). The level of different autophagy-related proteins was reported to be down-regulated in AD brains ( Heckmann et al. 2020 ; Lachance et al. 2019 ). Cathepsin B is an important lysosomal protease that is involved in the degradation of autophagic substrates. It has been shown that in a mouse model of AD, knockdown of cathepsin B aggravates plaque pathology and lentiviral overexpression of the same molecule leads to the reduction of amyloid plaques ( Mueller-Steiner et al. 2006 ). Similarly, studies have also demonstrated that up-regulation of transcription factor EB (TFEB) in tau mouse models markedly decreases the levels of both soluble phosphorylated tau and insoluble tau aggregates, resulting in improvement of cognitive functions ( Polito et al. 2014 ). Therefore, identification of nontoxic molecules for the upregulation of lysosomal biogenesis and autophagy is a promising approach for the treatment and prevention of AD. Amino acids readily available through daily diets are usually considered nontoxic. We screened different amino acids based on the ability to promote lysosomal biogenesis and found L-leucine, an essential amino acid, as a potent one to stimulate lysosomal biogenesis and autophagy in brain cells. Accordingly, orally administered L-leucine upregulated autophagy in the hippocampus, reduced hippocampal plaque load, and improved cognitive functions in 5XFAD mice. We also found that L-leucine interacted with the ligand-binding domain of PPARα and that L-leucine remained unable to increase autophagy, lower plaques, and enhance memory and learning in 5XFAD mice lacking PPARα. These results suggest that supplementation of L-leucine may be neuroprotective for AD patients. 2 |. Materials and Methods 2.1 |. Reagents Cell culture materials (DMEM/F-12, L-Glutamine, Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic) were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals. L-Leucine and other amino acids were obtained from Sigma. Primary antibodies, their sources and concentrations used are listed in Table S1 . Alexa-fluor antibodies used in immunostaining were obtained from Jackson ImmunoResearch, and IR-dye-labeled reagents used for immunoblotting were from Li-Cor Biosciences. Sequence of primers used in the present study are mentioned in Table S2 . 2.2 |. Animals PPARα −/− mice, 5XFAD [(APPwFILon,PSEN1*M146L*L286V) 6799Vas/J ; RRID:MMRRC_034840-JAX] mice ( Oakley et al. 2006 ), and C57BL/6J mice (inbred of 5XFAD mice) were obtained from Jackson Laboratory, Bar Harbor, ME, USA. Unless otherwise indicated, PPARα −/− mice ( Peters et al. 2000 ) were maintained as homozygous on the C57BL/6J background. 5XFAD ΔPPARα mice, developed earlier by us ( Corbett et al. 2015 ) were maintained transgenic for the 5XFAD mutations and homozygous for the PPARα −/− allele through genotyping as described by us ( Chandra et al. 2018 ; Corbett et al. 2015 ; Patel et al. 2020 ). Six-month-old 5XFAD and 5XFAD ΔPPARα mice (body weight 28 ± 2 g) of both sexes in equal ratio were used for experiments. Animal maintenance and experiments were performed in accordance with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use committee of the Rush University Medical Center (IACUC protocol # 20–007). Animals were housed in the state-of-the-art animal care facility of the Cohn Research Building of the Rush University Medical Center in an environmentally controlled vivarium (standard autoclaving-safe cage; 7:00 a.m./7:00 p.m. light cycle; temperature maintained at 21°C–23°C; humidity 35%–55%). Animals were provided standard mouse chow and water ad libitum and closely monitored for health and overall well-being daily by veterinary staff and the investigator. This study was not preregistered. Daily veterinary care were provided under the supervision of the attending veterinarian, Dr. Jeffrey P. Oswald, DVM, Diplomate, ACLAM. 2.3 |. Treatment of Mice With L-Leucine L-Leucine was solubilized in warm water upon gentle shaking at room temperature for 10 min. Six-month-old 5XFAD mice (body weight 28 ± 2 g) were treated orally with different doses (10, 25 or 50 mg/kg body weight/day) of L-leucine solubilized in 100 μL water via a gavage needle. Therefore, control 5XFAD mice received 100 μL water via gavage. Genotyped non-transgenic animals were also treated with L-leucine (50 mg/kg body weight/day) via a gavage needle. Typically, any animal experiment ( Modi et al. 2014 ; Modi et al. 2015 ; Rangasamy et al. 2015 ) is justifeid with 99% confidence interval that generates p = 0.99 and (1 − p ) = (1–0.99) = 0.01; ε is the margin of error = 0.05. Based on these values, the resultant sample size is: N = 1.28 2 × 0.99 ( 1 − 0.99 ) 0.05 2 = 1.28 2 × 0.99 × 0.01 0.05 2 = 0.016 0.0025 = 6.48 ~ 6 . Therefore, six mice ( n = 6) were used in each group. We used mice from both sexes (equal distribution). Therefore, 3 males and 3 females were used in each group. No randomization was performed to allocate subjects in the study. No blinding was performed as well. Treatment of 6-month-old 5XFAD mice started daily at 10 a.m. (central time) and continued for 60 days. During treatment, no mouse reached the moribund stage or died. Conditions for moribund were as follows: Central nervous system disturbance (Head tilt, Seizures, Tremors, Circling, Spasticity, and Paresis); Inability to remain upright; Evidence of muscle atrophy; Chronic diarrhea or constipation; Rough coat and distended abdomen; Spreading area of alopecia caused by disease; Coughing, rales, wheezing and nasal discharge; Distinct jaundice and/or paleness (anemia); Markedly discolored urine, polyuria or anuria; Frank bleeding from any orifice; Persistent self-induced trauma. Therefore, no mouse was excluded from the study during the treatment. 2.4 |. Checking the Entry of L-Leucine Into the Brain L-Leucine (100 μg in 100 μL saline) was tagged with 100 μL near-infrared fluorescent dye Alexa 680 (ThermoFisher) and each mouse was treated with 200 μL volume of Alexa-680-labeled L-leucine through the tail vein as described ( Brahmachari and Pahan 2007 ; Dasgupta et al. 2004 ). Similarly, different groups of control mice were also treated with only Alexa-680 and un-tagged L-leucine via tail vein. Following 2 h of injection, animals were sacrificed, and different parts of the brain taken from cortex, midbrain, and cerebellum regions were scanned under Licor Odyssey infrared scanner as described before ( Dutta, Jana, et al. 2021 ). 2.5 |. DNA Constructs and Lentiviral Transductions Generation of the pCMV6-AC-GFP lentiviral backbone expressing TurboGFP ( OriGene # PS100010 ) and FL-PPARα or Y314D-PPARα was described before ( Roy et al. 2013 , 2015 ). We bought mouse PPARα ORF in pCMV6-AC-GFP vector (cat # MG 227641) from OriGene and mutation at Tyr314 with aspartate (Y314D) was carried out by site-directed mutagenesis. For generating pLenti6.3/V5-TOPO constructs of FL-PPARα or Y314D-PPARα , each construct was amplified by PCR followed by TOPO cloning reaction using Invitrogen kit (K5315–20) with pLenti6.3/V5-TOPO vector. For transformation, one-Shot Stbl3 competent cells were used, and sequencing of the clones was performed at ACGT Inc. The ViraPower Packaging Mix and pLenti expression plasmid DNA containing either FL-PPARα or Y314D-PPARα were used for lentiviral production in 293FT cells, which are not listed as commonly misidentified by the International Cell Line Authentication Committee (ICLAC). Viral particles were concentrated with lenti-concentrator solution followed by calculation of MOI. Astrocytes were transduced with lentiviral particles at MOI 10 for 48 h at 37°C. Live GFP imaging was used to monitor viral integration. 2.6 |. Isolation of Primary Mouse Astrocytes Astrocytes were isolated from 2 to 3 days old mouse pups as described earlier ( Ghosh and Pahan 2012 ; Khasnavis et al. 2012 ; Brahmachari et al. 2006 ). In brief, mouse pups were placed on a sterile 100 mm petri dish containing ice-cold dissection solution (HBSS). When pedal reflex was lost, pups reached the proper plane of anesthesia. Then pups were beheaded by scissors, brains isolated and placed together in the DMEM/F-12 media supplemented with 10% heat-inactivated FBS followed by isolation of mixed glial cells. Therefore, brains isolated from multiple pups were pooled together. On day 9, the mixed glial cultures were subjected to shaking at 240 rpm for 2 h at 37°C on a rotary shaker for removing microglia. Flasks were allowed another round of shaking on 11 days at 190 rpm for 18 h for taking off oligodendrocytes and residual microglia. The attached cells were washed and seeded onto new plates for further studies. About 98% of this preparation was found to be positive for GFAP, a marker of astrocytes. Briefly, the brain tissues from the pups were pooled together and placed together in the DMEM/F-12 media supplemented with 10% heat-inactivated fetal bovine serum. 2.7 |. Isolation of Mouse Hippocampal Neurons Primary hippocampal neurons were prepared from fetuses (E18) of pregnant PPARα −/− mice and strain-matched C57BL/6J littermates using methods as described earlier with few modifications ( Roy et al. 2013 , 2014 , 2015 ; Jana et al. 2007 ). Briefly, the pregnant mouse was euthanized by carbon dioxide inhalation followed by cervical dislocation as a secondary physical method to confirm the death of the animal that was ascertained by the lack of pedal reflex to a toe pinch. Then the euthanized dam was pinned on its back on an absorbent pad, and the abdomen was sterilized with 70% ethanol to reduce the contamination from the fur. By abdominal incision, the abdominal cavity was opened, visible embryos separated from the uterus by cutting the tissue connecting it to the body and placed in a sterile petri dish containing ice-cold dissection solution (HBSS). When pedal reflex was lost due to hypothermia, fetal brains were separated by scissors, hippocampi isolated as a thin slice of tissue near the cortical edge of the medial temporal lobe, and placed together in the Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) media supplemented with 10% heat-inactivated FBS. Therefore, hippocampi isolated from multiple fetal brains were pooled together. Cells were dissociated by trituration and single cell suspension was plated in poly-D-lysine pre-coated 6 wells plates containing complete DMEM/F12 media. After cell attachment (5 min after plating), the DMEM/F12 media was replaced with the Neurobasal Medium containing the B27 supplements (Life Technologies). Finally, 10 μM AraC was added to remove any glial contamination from the neuronal culture. Experiments were carried out in 9–10-days-old pure hippocampal neuronal cultures. Immediately before experimental treatments, the medium was replaced with Neurobasal Medium without B27 supplements. 2.8 |. LysoTracker Staining It was performed as described earlier ( Chandra, Kundu, et al. 2017 ; Ghosh et al. 2015 ). Briefly, after treatments, cells were incubated with 75 nM LysoTracker Red DND99 (Life Technologies, Grand Island, NY) for 45 min. Cells were then washed thoroughly with filtered PBS and mounted on glass slides for viewing under a BX41 fluorescence microscope. 2.9 |. In Silico Molecular Docking Study It was performed as described earlier ( Paidi, Jana, et al. 2023 ; Roy et al. 2015 , 2016 ) with modifications. In brief, to examine whether and how L-leucine interacts with different PPARs, we first generated in silico model structure of unavailable crystal structures of PPARα, PPARβ, and PPARγ. The homology models of PPARα, PPARβ, and PPARγ was built using the SWISS-MODEL, an automated web-based server using the sequences (NCBI ID NP_001106889.1 [aa 196–468], NP_035275.1 [aa 172–440], and NP_001295283.1 [aa 205–471], respectively) available at the NCBI website. The target template was uploaded and searched for the most identical homologous sequence. The sequence identity between the two sequence NP_001106889.1 and 3sp6.1.A was 92%, NP_035275.1 and 5u42.2 and NP_001295283.1 and 3e00.1.B was 98%. Finally, the model was obtained by blasting the target sequence with the most identical homologous sequence. The quality of the model structures was further verified. Autodock4 (The Scripps Research Institute, La Jolla, USA) tool was used to dock the crystal structure of PPARβ (PDB ID: 2j14), PPARα and PPARγ homology models. Finally, the minimum energy dock structure was chosen as the best docking pose. For creating the final representable image, we used the PyMOL software (The PyMol Molecular Graphics System, Version 2.0, Schrödinger LLC). 2.10 |. Thermal Shift Assay (TSA) TSA was performed in a QuantStudio 3 real-time thermal cycler using a thermal shift dye kit ( Thermo Fisher ) as described by us ( Roy et al. 2015 , 2016 ; Paidi, Jana, Raha, et al. 2021 ). Briefly, 0.5–1 μg of purified protein was added to 18 μL of thermal shift buffer together with 1–2 μL of dye for each reaction. The reaction was fixed in a 96-well PCR plate in the dark and then placed in the thermal cycler using the following two-stage program ([25°C for 2 min] 1 cycle; [27°C for 15 s, 26°C for 1 min] 70 cycles; auto increment 1°C for both stages). The filter was set at ROX with no passive filter and no quencher filter. 2.11 |. Time-Resolved Fluorescence Energy Transfer (TR-FRET) Analysis For TR-FRET assay, the Lanthascreen TR-FRET PPARα coactivator assay kit was used as described earlier ( Roy et al. 2015 , 2016 ). Briefly, L-leucine was added to GST-tagged recombinant PPARα LBD, terbium (Tb)-tagged anti-GST antibody, and fluorescein (FL)-tagged PGC-1α as mentioned in the manufacturer’s protocol. The plate was centrifuged followed by incubation in the dark for 30 min and analysis in a Perkin–Elmer Victor X5 Luminescence spectrometer. The excitation and emission were set at 340 and 540 nm, respectively. 2.12 |. Immunoblotting Western blotting was conducted as described earlier ( Corbett et al. 2013 ; Raha et al. 2023 ). Briefly, RIPA buffer (50 mM Tris–HCl, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, protease inhibitor) was used for the lysis of brain tissue samples. After 10 min of incubation in ice, samples were centrifuged at 14 000× g at 4°C for 15 min. The Bio-Rad Bradford protein assay was used to measure the protein concentration in the supernatant. The SDS sample buffer was added to 40–60 μg of total protein and boiled for 5 min. Proteins were then transferred onto a nitrocellulose membrane (Bio-Rad), which was blocked with TBST containing BSA (5%) for 1 h and probed with the respective primary antibodies overnight at 4°C on a shaker. The next day, membranes were washed in TBST for 1 h, incubated in secondary antibodies for 1 h at room temperature, washed again for 1 more h before visualizing under the Odyssey Infrared Imaging System (Li-COR, Lincoln, NE). 2.13 |. Real-Time PCR Mice were euthanized by carbon dioxide inhalation followed by cervical dislocation as a secondary physical method. Death of mice was determined by the lack of pedal reflex to a toe pinch. Mice brains were separated by a scissor for the isolation of hippocampi, which were kept in Trizol solution (Sigma-Aldrich, St. Louis, MO) and stored at −20°C. Next day, total RNA was isolated following methods as described before ( Dutta et al. 2022 ; Paidi, Jana, Mishra, Dutta, and Pahan 2021 ; Paidi, Jana, Mishra, Dutta, Raha, and Pahan 2021 ). The isolated RNA was reverse transcribed into cDNA and real-time PCR was performed using the primers ( Table S2 ). We employed the SYBR green real-time kit obtained from QuantaBio (Beverly, MA) to carry out the PCR reaction in ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) as described before ( Dutta et al. 2022 ). Using the level of GAPDH mRNA, the mRNA expressions of respective genes were normalized. Relative expression of genes was compared among experimental animals. 2.14 |. Densitometric Analysis The ImageJ software (NIH, Bethesda, MD) was used to analyze protein blots, and bands were normalized to their respective β-actin loading controls. 2.15 |. Assay of Transcriptional Activities For assaying transcriptional activities, at first, cells were plated at 70%–80% confluence in 12-well plates for 18 h followed by co-transfection with 0.25 μg of PPRE-Luc (a PPAR-dependent reporter construct) and 12.5 ng of pRL-TK using LipofectAMINE Plus ( Corbett et al. 2012 ; Ghosh and Pahan 2012 ). After 24 h of transfection, cells were treated with L-leucine and other amino acids for 4 h followed by measuring activities of firefly and Renilla luciferases. 2.16 |. Tri-Peptidyl-Peptidase 1 (TPP1) and Cathepsin B and D Assays Cultured mouse primary astrocytes were treated with different doses (50, 100, and 200 μM) of L-Leucine, and after 24 h of treatment, the cells were pelleted down. Cell pellet was homogenized in a buffer containing 0.15 M NaCl and Triton X-100 followed by incubating the supernatant in a reaction volume of 100 μL at pH 4.0 in the presence of 200 μM of the substrate Ala-Ala-Phe-7-amido-4-methyl coumarin (Sigma-Aldrich). Victor X2 micro-plate reader (Perkin-Elmer) was employed for measuring the activity of TPP1 at excitation/emission of 360/460 nm. Data are shown as the relative fluorescence unit (RFU) fold change of the treated groups with respect to the control at 20 min. For assaying cathepsin B and D, the cells were lysed in a homogenization buffer (pH 5.5; 2.5 mM EDTA, Triton X-100, 2.5 mM DTT). For monitoring the activity of cathepsin B, the supernatant was incubated with 100 μM of the substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride at pH 6.0 followed by measuring the plate at excitation/emission of 355/460 nm. On the other hand, for cathepsin D, the supernatant was incubated at pH 4.0 with the 10 μM of the substrate 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D-Arg-amide (Enzo Life Sciences). The plate was examined every 10 min for 90 min at excitation/emission of 320/420 nm as described by us ( Chandra et al. 2018 ; Ghosh et al. 2012 ). Data are shown as the RFU fold change of the treated groups compared with the control at 30 min. 2.17 |. Amyloid β Uptake and Degradation Assay Mouse primary astrocytes were cultured in 96-well plates (Thermo Fisher Scientific) for treatment with 100 μM of L-leucine and D-leucine. After 24 h, the cells were incubated in medium containing 500 nM oligomeric FAM-tagged Aβ (1–42) (Anaspec) for 2 or 4 h. For assaying the degradation, after 4 h of incubation with FAM-Aβ, the cells were allowed to grow in Aβ-free medium for an additional 6 h before measuring the fluorescence. Next, the cells were washed in regular medium, and for quenching extracellular signal, Trypan blue solution was added. After another media wash, the fluorescence was recorded at excitation/emission of 485/535 in Perkin-Elmer Victor X2. Next, the cells were incubated with Hoechst and the fluorescence was measured at excitation/emission of 360/465 nm. FAM-Aβ fluorescence was normalized with Hoechst ( Chandra et al. 2018 ; Raha et al. 2021 ). 2.18 |. Monitoring Aβ Uptake and Degradation by Immunocytochemistry Following a 24-h treatment with 100 μM L-Leucine and D-Leucine, mouse primary astrocytes cultured on coverslips were incubated with 500 nm Hilyte Fluor 647-tagged Aβ (1–42) (Anaspec) for 4 h. The cells were washed in regular Aβ-free media for an extra 6 h for tracking the degradation. After that, chilled methanol was used to fix the cells. Nuclei were stained with Hoechst ( Chandra et al. 2018 ). Before visualizing under a BX41 fluorescence microscope, coverslips were mounted using Fluoromount (Sigma-Aldrich). 2.19 |. Immunofluorescence Analysis It was performed as described earlier ( Brahmachari et al. 2009 ; Chakrabarti et al. 2023 ; Saha et al. 2009 ). Briefly, cover slips containing 100–200 cells/mm 2 were fixed with 4% paraformaldehyde followed by treatment with cold ethanol and two rinses in phosphate-buffered saline (PBS). Samples were blocked with 3% bovine serum albumin (BSA) in PBS-Tween-20 (PBST) for 30 min followed by incubation in PBST containing 1% BSA and primary antibodies. After three washes in PBST (15 min each), slides were further incubated with Cy2 (Jackson ImmunoResearch Laboratories Inc.). For negative controls, a set of culture slides was incubated under similar conditions without the primary antibodies. The samples were mounted and observed under an Olympus BX-41 fluorescence microscope. 2.20 |. Immunohistochemistry (IHC) It was performed as described before ( Dutta, Jana, et al. 2021 ; Dutta et al. 2022 ; Dutta, Majumder, et al. 2021 ; Paidi et al. 2022 ). In brief, mice were anesthetized using a mixture of xylazine (10 mg/kg i.p.) and ketamine (100 mg/kg i.p.) in 0.9% normal saline. The absence of a pedal reaction to a pinch on the toe guaranteed the depth of anesthesia. After the heart was revealed and the right ventricle cut, a bore needle was then inserted into the left ventricle. Mice were first perfused with PBS (pH 7.4) and then 4% (w/v) paraformaldehyde solution in PBS using a peristaltic pump. After perfusion, brains were removed for IHC. Samples were incubated for 3 h in PBS with 10% sucrose and 0.05% Tween 20 (PBST), followed by an overnight incubation at 4°C with 30% sucrose. After the procedure, the brain was prepared for traditional cryosectioning and embedded in OCT (Tissue Tech) at −80°C. Following two rinses in PBS, blocking with 3% BSA in PBST, and double labeling with two antibodies, frozen sections (30 μm) were treated with cold ethanol (−20°C) ( Table S1 ). Sections were incubated with Cy2 or Cy5 conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc.) following three PBST washes. The samples were mounted and observed under the Olympus BX41 fluorescent microscope equipped with a Hamamatsu ORCA-03G camera. 2.21 |. Thio-S Staining Thioflavin S (Thio-S) solution was prepared in TBS in the dark and filtered to prevent aggregation on the sections. After washing and incubation with secondary antibodies for 1 h, free-floating sections were incubated in 1% Thio-S for 5 min. Sections were washed twice in 50% ethanol for 1 min and twice in TBS for 5 min before drying and mounting in Fluoromount (Sigma). Thio-S quantification was performed on two sections (one image per section) of each of six mice per group as described before ( Chandra et al. 2018 ; Corbett et al. 2015 ; Paidi, Raha, et al. 2023 ). First, grayscale images were converted to binary using Fiji and uniformly thresholded. Using the analyze particles function, several parameters such as Thio-S-positive area %, Thio-S puncta count, and Thio-S puncta size were calculated. 2.22 |. Measurement of Mean Fluorescence Intensity (MFI) We employed ImageJ (NIH) for measuring MFI and counting target proteins or cells as described before ( Chandra, Roy, et al. 2017 ; Dutta et al. 2022 ). A contour was drawn around the target’s area in the corresponding region of interest in order to quantify the MFI. The MFI was then measured using the software’s Analyze-Measure option. Each MFI value was subtracted from the background fluorescence according to the equation given in the ImageJ manual. 2.23 |. ELISA for Aβ40 and Aβ42 For measuring the levels of insoluble Aβ ( Chandra et al. 2018 ; Rangasamy et al. 2015 , 2018 ), hippocampal tissues were homogenized in TBS containing protease inhibitors for centrifugation at 150 000 g for 30 min. The resulting pellet was suspended in three volumes (wt/vol original tissue weight) of TBS + 1% Triton X-100 with same homogenizing procedure and centrifuged again at 150 000 g for 30 min. Protein concentrations were measured using Bio-Rad Bradford protein assay and samples diluted in accordance with the manufacturer’s instructions followed by running ELISA for Aβ1–40 and Aβ1–42 using Invitrogen ELISA kits (KMB 3481, Aβ1–40; KMB3441, Aβ1–42) from ThermoFisher. 2.24 |. Barnes Maze and T Maze These experiments were performed as described before ( Corbett et al. 2013 ; Roy et al. 2013 ; Dutta et al. 2023 ; McKay et al. 2024 ). Briefly, all behavioral experiments were conducted during the light phase (within 10 a.m. and 4 p.m.). We trained mice for 2 days followed by taking the examination on day 3 on Barnes maze. For increasing motivation and performance on Barnes maze, mice were deprived of food overnight. During training, food-deprived mice were placed in the center of the maze in a 10 cm high cylinder-shaped start chamber. After 10 s, the start chamber was removed and mice were allowed to move around the maze to search for the color food chips in the enticed tunnel. When the mouse entered the baited tunnel, the session was considered over. On day 3, a video camera ( Basler Gen I Cam—Basler acA 1300–60 ) connected to a Noldus computer system was placed above the maze. The maze was brightened with high wattage light in order to motivate mice for entering into the escape tunnel. The performance was monitored by the EthoVision XT video tracking system ( Noldus ). Cognitive parameters were evaluated by measuring latency (duration before all four paws were on the floor of the escape box) and errors (incorrect responses before all four paws were on the floor of the escape box). Similarly, for T-maze as well, food-deprived mice were also adapted to the T-maze for 2 days so that during a 10-min training period, a mouse could eat food rewards at least five times. Since the right arm was always baited with colored food chips, each trial required a mouse to turn into the right arm after being held at the starting point for 30 s. The mouse was allowed to enter the right arm, stay there for 30–40 s, return to the starting position, hold it for 30 s, and then turn right once more. The mouse was tested on day 3 by assessing the capacity of each mouse to make both positive and negative turns. The reward side is typically accompanied by a visual cue. The number of times the animal eats the food incentive would determine a favorable turn. After every training session, both the Barnes maze and the T-maze were thoroughly cleaned with a mild detergent to make sure that any cues left behind by previous mice are removed. 2.25 |. Novel-Object Recognition (NOR) Test For monitoring the short-term memory, we employed the NOR test as described before ( Rangasamy et al. 2018 , 2020 ; Guha et al. 2022 ; Saha et al. 2020 ). A wooden floor square arena measuring 40 × 40 cm with walls 30 cm high was used as an open field arena during the training period. Two plastic objects that varied in color, shape, and texture were placed in specific locations in the environment 18 inches away from each other. A video camera ( Basler Gen I Cam—Basler acA 1300–60 ) connected to a Noldus computed system was fixed facing down on the center of the open field arena. The mouse was allowed to survey the environment and objects freely for 5 min followed by keeping the animal back in its individual home cage. After 15 min, the mouse was again placed back into the environment with two objects in the same locations, replacing one of the familiar objects with a third novel object. The mouse was allowed to explore again for 5 min. The time spent by the mice towards the novel object (TN) and familiar object (TF) was measured. The discrimination index (DI) and preference index ( Rega et al. 2016 ) were derived according to the formulae: DI = (TN − TF)/(TN + TF), PI = TN/(TN + TF) × 100. 2.26 |. Statistical Analysis Results were statistically analyzed using GraphPad Prism version 10.6.1 (892). We used the Shapiro–Wilk test for normal distribution validation. The normality of data was verified using Quantile-Quantile (Q-Q) plots generated from the Shapiro–Wilk test. No test for outliers was conducted. Values are expressed as either mean ± SD or mean ± SEM. Statistical comparisons between two different samples were conducted by two-sample un-paired t -test. On the other hand, one-way and two-way ANOVAs followed by Tukey’s multiple comparisons were performed for statistical analyses among multiple groups. The criterion for statistical significance was p < 0.05. Statistical details of experiments including sample distribution, degree of freedom, etc. are mentioned under figure legends. 3 |. Results 3.1 |. Effect of Different Essential and Non-Essential Amino Acids on Lysosomal Biogenesis in Mouse Primary Astrocytes Since astrocytes are the major cell type in the brain, employing astrocytes for the clearance of depositions from the brain is considered as an important area of research. However, such molecules are poorly described. Therefore, we tested the efficacy of different essential and non-essential amino acids on lysosomal biogenesis in astrocytes. Although according to Ghislat et al. (2012) , taking away of essential amino acids could increase autophagy, we screened different amino acids for the efficacy in inducing lysosomal biogenesis in mouse primary astrocytes. Since LysoTracker Red selectively stains the acidic lysosomal organelles, to determine the lysosome content of astrocytes, cells were treated with 100 μM concentration of different essential and non-essential amino acids followed by staining with Lysotracker Red and counting red puncta. Among different essential amino acids tested, L-leucine was found to be very efficient in stimulating lysosomal biogenesis ( Figure 1A , C ). When tested at different doses (50, 100, and 200 μM), maximum efficacy of L-leucine in upregulating lysosomal biogenesis was observed at a dose of 100 μM ( Figure 1D , E ). Accordingly, L-leucine treatment also increased the level of cathepsin D ( Figure S1A ) and cathepsin B ( Figure S1B ), lysosomal markers of autophagy, in primary astrocytes. We also tested whether L-leucine could increase the activity of lysosomal enzyme tri-peptidyl-peptidase 1 (TPP1), dysfunction of which causes a lysosomal disorder known as late-infantile neuronal ceroid lipofuscinosis ( Johnson et al. 2019 ; Ghosh et al. 2012 ). We found that different doses of L-leucine markedly upregulated TPP1 activity in primary astrocytes with maximum increase seen at 100 μM ( Figure S1C ). FIGURE 1 |. Open in a new tab Effect of different amino acids on lysosomal biogenesis. Mouse primary astrocytes from C57BL/6 mouse pups were incubated with 100 μM of different essential and non-essential amino acids separately under serum-free condition for 24 h, followed by staining with Lysotracker red, which specifically labels lysosomes and acidic organelles in live cells (A, B). Quantification of the number of the LysoTracker-positive puncta per cell for at least 15 images per group from three independent set of experiments calculated by using Image J (C). Dose dependent effect of L-Leucine in mouse primary astrocytes (D) and mouse primary hippocampal neurons (F). Primary astrocytes and hippocampal neurons were isolated from C57BL/6 mice and treated with different doses of Leucine (50, 100, 200 μM) for 24 h under serum free condition followed by Lysotracker red staining to detect the lysosomes and acidic organelles in live cells. Hoechst was used for nuclei stain. Scale bar 2 μm. For checking specificity, hippocampal neurons were also treated with L-alanine (100 μM). Number of LysoTracker-positive puncta per cell were from 15 different images per group from three independent experiments using Image J software (E, G). Data are shown as mean ± SEM form lysosomal count per cell for at least 15 images per group from three independent set of experiments analyzed by using Image J. One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 10,140 = 84.03, p < 0.0001 (C); F 3,60 = 30.77, p < 0.0001 (E); F 3,64 = 50.21, p < 0.0001 (G)]. NS, not significant; *** p ≤ 0.001. For detailed statistical results, please see Table S3 . It has been reported that unlike L-leucine, D-leucine potently ended seizures even after the onset of seizure activity ( Hartman et al. 2015 ). Therefore, we also examined the effect of D-leucine on lysosomal biogenesis. Although D-leucine also significantly increased lysotracker staining in astrocytes, it was much less efficient than L-leucine in stimulating lysosomal biogenesis ( Figure 1A , C ). On the other hand, other essential amino acids such as L-lysine, L-histidine, and L-tryptophan remained ineffective in increasing lysosomal biogenesis in astrocytes ( Figure 1A , C ). Among different non-essential amino acids tested, L-alanine, but not L-glycine, L-glutamate, L-aspartate, and L-arginine, showed some efficacy in upregulating lysosomal biogenesis ( Figure 1B , C ). However, L-leucine was stronger than L-alanine in increasing lysosomal biogenesis in astrocytes ( Figure 1A – C ). Next, we examined whether in addition to astrocytes, L-leucine was also capable of stimulating lysosomal biogenesis in other brain cells. Since hippocampal neurons are primarily affected in AD, we investigated the effect of L-leucine in these cells. Mouse primary hippocampal neurons were treated with L-leucine for 24 h followed by lysotracker staining. For comparison purposes, neurons were also treated with L-alanine. Similar to astrocytes, L-leucine also strongly increased lysosomal biogenesis in hippocampal neurons ( Figure 1F , G ). In contrast, very little stimulation of lysosomal biogenesis was observed in L-alanine-treated hippocampal neurons ( Figure 1F , G ). Treatment with L-leucine also increased the level of cathepsin D ( Figure S2A , B ) in primary hippocampal neurons. Together, these results suggest that L-leucine could stimulate lysosomal biogenesis and augment lysosomal functionality in brain cells. 3.2 |. L-Leucine Augments Uptake and Degradation of Aβ in Primary Astrocytes We further investigated whether L-leucine-mediated lysosomal biogenesis could elevate the cellular degradative capability of Aβ in astrocytes. We performed a quantitative in vitro assay to study the effect of L-leucine on uptake and degradation of FAM-tagged Aβ1–42 in astrocytes. After 4 h of incubation with (Aβ1–42)-containing medium, L-Leucine treatment significantly enhanced the intracellular amount of Aβ 42 compared with control, indicating that L-leucine increases the uptake of Aβ by astrocytes ( Figure S3A ). After the 4 h Aβ1–42 incubation, when cells were grown for an additional 6 h in Aβ-free medium (6 h wash), Leucine-treated astrocytes demonstrated a significantly lower level of intracellular Aβ than the control, suggesting that the internalized Aβ was effectively degraded ( Figure S3A ). In contrast, such internalization and degradation of Aβ was not seen in D-Leucine-treated astrocytes ( Figure S3A ). We further confirmed these observations by labeling astrocytes with HiLyte Fluor (HF)-tagged Aβ1–42. Similar to the in vitro kinetic assay, immunocytochemistry showed that L-leucine enhanced the uptake of Aβ and the degradation of internalized Aβ compared with the control after 4 h of incubation with HF-Aβ1–42 ( Figure S3B , D ), and an additional 6 h wash, respectively ( Figure S3C , D ). On contrary, D-Leucine did not show any effect on uptake and degradation of Aβ ( Figure S3B , D ). Collectively, these results illustrate that L-leucine is capable of enhancing astrocytic clearance of Aβ via increasing the uptake and lysosomal degradation of Aβ by astrocytes. 3.3 |. Oral L-Leucine Stimulates Lysosomal Biogenesis In Vivo in the Brain of 5XFAD Mice Next, we investigated whether L-leucine treatment could upregulate lysosomal biogenesis in vivo in the hippocampus of the 5XFAD mouse model of AD. However, before this preclinical study, it is important to understand whether L-leucine enters the brain. Therefore, L-leucine was conjugated with a near-infrared fluorescent dye Alexa-Flour 680 and 2-month-old C57/BL6 mice were treated with Alexa-Flour 680-conjugated L-leucine via tail vein injection. After 1 h of injection, infrared signals were seen in different parts of the brain (frontal cortex, midbrain, and cerebellum) of mice receiving Alexa-Flour 680-conjugated L-leucine, but neither leucine-treated nor Alexa 680-treated, mice ( Figure S4A , B ), indicating that L-leucine can enter into the brain. Then 6-month-old 5XFAD transgenics were treated with L-leucine (50 mg/kg body weight/day) or vehicle orally for 2 months followed by double-labeling of hippocampal sections for MAP2 & TFEB ( Figure 2A ), MAP2 & LAMP2 ( Figure 2B ) and MAP2 & TPP1 ( Figure 2C ). As expected, MAP2-positive neurons in hippocampal sections of non-Tg mice expressed TFEB ( Figure 2A ), LAMP2 ( Figure 2B ) and TPP1 ( Figure 2C ). However, levels of TFEB ( Figure 2A , D ), LAMP2 ( Figure 2B , E ) and TPP1 ( Figure 2C , F ) decreased in the hippocampus of 5XFAD mice as compared to non-Tg mice. On the other hand, consistent with that found in cultured brain cells, oral L-leucine treatment increased the levels of TFEB ( Figure 2A , D ), LAMP2 ( Figure 2B , E ) and TPP1 ( Figure 2C , F ) in vivo in the hippocampus of 5XFAD mice. To confirm these findings further, hippocampal extracts were immunoblotted with antibodies against TFEB, LAMP2 and TPP1. Again, we found a decrease in TFEB ( Figure 2G , H ), LAMP2 ( Figure 2G , I ) and TPP1 ( Figure 2G , J ) in the hippocampus of 5XFAD mice as compared to non-Tg mice. However, similar to immunostaining results, L-leucine treatment restored and/or increased the levels of TFEB ( Figure 2G , H ), LAMP2 ( Figure 2G , I ) and TPP1 ( Figure 2G , J ) in the hippocampus of 5XFAD mice. The mRNA expression analysis also indicates a decrease in TFEB ( Figure S5A ), LAMP1 ( Figure S5B ), LAMP2 ( Figure S5C ), Sqstm1 ( Figure S5D ), CatB ( Figure S5E ), CatD ( Figure S5F ), Cln1 ( Figure S5G ), Cln2 ( Figure S5H ), and Cln3 ( Figure S5I ) in the hippocampus of 5XFAD mice as compared to non-Tg mice, which increased after L-leucine treatment. Accordingly, we found the presence of cathepsin D in the hippocampus of non-Tg mice, which decreased drastically in 5XFAD mice ( Figure S6A , B ). However, significant restoration and/or increase in cathepsin D was observed in the hippocampus of 5XFAD mice after L-leucine treatment ( Figure S6A , B ). Therefore, several molecules involved in autophagy-lysosomal functioning could be upregulated in the hippocampus of 5XFAD mice by L-leucine treatment. FIGURE 2 |. Open in a new tab Oral administration of L-leucine enhances lysosomal biogenesis in vivo in the hippocampus of 5XFAD mice. Six-month-old 5XFAD mice ( n = 6 per group) were treated with L-leucine (50 mg/kg body wt/day) via gavage once daily for 60 days. Since L-leucine was dissolved in 100 μL warm water, control 5XFAD mice were also treated with same volume of water as vehicle. Hippocampal sections were double labeled for MAP2 and TFEB (A), MAP2 and LAMP2 (B), MAP2 and TPP1 (C) followed by quantification of TFEB (D), LAMP2 (E), and TPP1 (F) MFI on 10 images from a total of 6 mice per group. Hippocampal extracts were immunoblotted for TFEB, LAMP2, and TPP1 (G). For raw blots, please see Figure S18 . Bands were scanned and values (H, TFEB; I, LAMP2; J, TPP1) normalized with β actin. Values are mean ± SEM of six mice per group. One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 2,33 = 254.5, p < 0.0001 (D); F 2,22 = 431.77, p < 0.0001 (E); F 2,33 = 154.6, p < 0.0001 (F); F 2,10 = 33.50, p < 0.0001 (H); F 2,10 = 26.41, p = 0.0001, Non-Tg vs. 5XFAD + Leu (50 mg/kg) p = 0.2523, 5XFAD vs. 5XFAD + Leu (50 mg/kg) p = 0.0010 (I); F 2,10 = 33.80, p < 0.0001 (J)]. *** p < 0.001. For detailed statistical results, please see Table S3 . There appears to be a loss of MAP2 staining in 5XFAD mice as compared to non-Tg mice. To investigate the basis of this loss of MAP2 staining, hippocampal extracts of non-Tg, 5XFAD and L-leucine-treated 5XFAD mice were immunoblotted for MAP2. It was evident from Figure S7A , B that the protein level of MAP2 decreased in the hippocampus of 5XFAD mice as compared to non-Tg mice and that L-leucine treatment could restore the level of MAP2 partially. 3.4 |. Oral L-Leucine Lowers the Amyloid Burden in the Hippocampus of 5XFAD Mice Since L-leucine enhances lysosomal biogenesis in cultured brain cells and in vivo in the hippocampus of 5XFAD mice, we intended to evaluate the functional implications of this induction of the cellular clearance pathways. We examined whether oral L-leucine could reduce the amyloid load in the hippocampus of 5XFAD mice. The main components of plaques are Aβ peptides consisting of common isoforms Aβ 40 and Aβ 42 . Since these isoforms are recognized by the 6E10 monoclonal antibodies (mAb), brain sections are immunostained with this mAb. We found a remarkable increase in the Aβ in the hippocampus ( Figure 3A ) and cortex ( Figure 3B ) of 5XFAD mice in comparison to non-Tg mice. It was corroborated by quantification of plaque count ( Figure 3C , G ), amyloid plaque burden ( Figure 3D , H ), plaque density ( Figure 3E , I ), and amyloid plaque area ( Figure 3F , J ) in the hippocampus ( Figure 3C – F ) and cortex ( Figure 3G – J ) of 5XFAD mice as well as non-Tg mice. However, oral administration of L-leucine significantly reduced the level of Aβ in the hippocampus and cortex of 5XFAD mice ( Figure 3A – J ). FIGURE 3 |. Open in a new tab Oral L-leucine decreases plaque burden in the hippocampus and cortex of 5XFAD mice. Six-month-old 5XFAD mice ( n = 6 per group) were administered L-leucine (50 mg/kg body wt) via gavage once daily for 60 days. Since water was used as solvent for L-leucine, control 5XFAD mice also received the same volume of water as vehicle via gavage. After 60 days of treatment, hippocampal sections were DAB immunostained with 6E10 antibody (A, hippocampus of different magnifications; B, cortex of different magnifications; C, number of plaques in the hippocampus; D, amyloid plaque burden; E, density of plaques in the hippocampus; F, area of plaques in the hippocampus; G, number of plaques in the cortex; H, amyloid plaque burden in cortex; I, density of plaques in the cortex; J, area of plaques in the cortex). For quantification, two sections (one image per section) of each of six mice per group were considered. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. *** p < 0.001. Hippocampal isolates were immunoblotted for Aβ plaques using 6E10 antibody (K). For raw blots, please see Figure S19 . The bands were scanned, and intensity ratio values of Aβ/actin are represented (L). Results are mean ± SEM of six mice per group. One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 2,15 = 237.5, p < 0.0001 (C); F 2,15 = 41.84, p < 0.0001 (D); F 2,15 = 441.6, p < 0.0001 (E); F 2,10 = 78.02, p < 0.0001 (F); F 2,10 = 285.5, p < 0.0001 (G); F 2,10 = 66.28, p < 0.0001 (H); F 2,10 = 1022, p < 0.0001 (I); F 2,10 = 160.7, p < 0.0001 (J); F 2,15 = 126.4, p < 0.0001 (L)]. *** p < 0.001. For detailed statistical results, please see Table S3 . To confirm it further, we performed Western blot analysis of hippocampal homogenates with 6E10 mAb and found markedly higher level of Aβ peptides in the hippocampus of 5XFAD mice as compared to non-Tg mice ( Figure 3K , L ). However, similar to immunostaining results, a significant decrease in Aβ was seen in 5XFAD mice upon L-leucine treatment ( Figure 3K , L ). To further strengthen our findings, we quantified levels of Aβ1–40 and Aβ1–42 in the hippocampus by ELISA, which showed that levels of Triton-X-insoluble Aβ1–40 ( Figure S8A ) and Aβ1–42 ( Figure S8B ) were markedly higher in the CNS of 5XFAD mice as compared to non-Tg mice. Interestingly, treatment of 5XFAD mice with L-leucine led to a significant decrease in both Triton-X-insoluble Aβ1–40 ( Figure S8A ) and Aβ1–42 ( Figure S8B ), indicating that oral L-leucine can reduce the burden of Aβ in the hippocampus of 5XFAD mice. Together, these results demonstrate that L-leucine treatment is capable of decreasing the plaque burden in 5XFAD mice. 3.5 |. Oral L-Leucine Enhances Autophagic Clearance of Aβ in 5XFAD Mouse Brains Further, we explored whether L-leucine oral administration could enhance autophagic clearance of Aβ in the hippocampus of 5XFAD mice. Therefore, we performed colocalization immunohistology studies using autophagy markers with Amyloid β 6E10 mono clonal antibody. Mice were treated with L-leucine (50 mg/kg body weight/day) or vehicle orally for 1 month followed by double-labeling of hippocampal sections with antibodies against either LC3 & Aβ 6E10 ( Figure S9A ) or p62 & Aβ 6E10 ( Figure S9C ). While levels of LC3 ( Figure S9A , B ) and p62 ( Figure S9C , D ) decreased, the level of amyloid plaques ( Figure S9 ) increased in the hippocampus of 5XFAD mice as compared to non-Tg mice. On the other hand, L-leucine treatment increased the levels of LC3 ( Figure S9A , B ) and p62 ( Figure S9C , D ), while decreasing the level of amyloid plaques ( Figure S9 ) in the hippocampus of 5XFAD mice. 3.6 |. Activation of PPARα by L-Leucine Next, we investigated the mechanism by which L-leucine up-regulates lysosomal biogenesis and lowers plaque. Peroxisome proliferator-activated receptor α (PPARα) is a lipid-lowering transcription factor ( Roy and Pahan 2015 ; Kersten et al. 2000 ; Majumder et al. 2026 ) and recently, we have demonstrated that activation of PPARα plays an important role in lysosomal biogenesis via direct transcriptional regulation of TFEB ( Chandra et al. 2018 ; Ghosh et al. 2015 ; Ghosh and Pahan 2016 ). Therefore, we examined whether L-leucine was capable of activating PPARα. As evident from Figure 4A , L-leucine dose-dependently induced PPRE-dependent luciferase activity with the highest induction shown at 100 μM concentration. On the other hand, D-leucine at a concentration of 100 μM remained unable to induce the activation of PPRE ( Figure 4B ). Other essential and non-essential amino acids such as L-lysine, L-histidine, L-tryptophan, L-alanine, L-glycine, L-glutamate, and L-aspartate also did not induce the activation of PPRE ( Figure 4B ), indicating the specificity of the effect. FIGURE 4 |. Open in a new tab Characterization of interaction of L-leucine with PPARα. Wild type (A), PPARα−/− (C), and PPARβ−/− (D) astrocytes were transfected with pPPRE-luciferase construct for 24 h, followed by treatment with different doses of Leucine (50, 100, and 200 μM) for 6 h and were examined by luciferase assay. (B) Wild type astrocytes were transfected with pPPRE-luciferase construct for 24 h, followed by treatment with different amino acids under serum free condition for 6 h and then luciferase assay. (E) A rigid body in silico docked pose of the PPARα LBD with L-leucine was derived using AutoDock 4.2. Three-D view of the docked protein showing the binding site and (F) key residues (Ser280, Y314, Y464 and H440) involved in leucine and PPARα interaction. (G) Thermal shift assay of PPARα was conducted with 10 μM Leucine. The melting of PPARα was monitored using an SYBR Green real-time melting strategy. (H) Thermal shift assay of PPARα was conducted with 10 μM L-alanine. The melting of PPARα was evaluated using an SYBR Green real-time melting strategy. The results were analyzed and confirmed after three independent experiments. (I) A time-resolved fluorescence resonance energy transfer (TR-FRET) analysis was performed to examine whether Leucine is a ligand of PPARα. The curve was plotted as a 520/490 nm ratio of response with increasing doses of Leucine. Curve fit was done in GraphPad Prism software. The analysis generated an EC50 of 2.3 μM and a Hill slope of 0.5245. (J) Astrocytes isolated from PPARα−/− mice were transduced with either Lenti-FL-PPARα, Y314D-PPARα or Naked Lenti virus for 48 h. Then groups were transfected with pPPRE-luciferase construct for 24 h, followed by treatment with different doses of Leucine (50, 100, and 200 μM) for 6 h and then luciferase assay. Results are represented as mean ± SEM of three independent experiments of Arbitrary fluorescence unit of luciferase activity monitored by TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 3,44 = 145.7, p < 0.0001 (A); F 8,72 = 2.193, p = 0.1155 (B); F 3,19 = 1.350, p = 0.2879 (C); F 3,25 = 40.90, p < 0.0001 (D); F 10,65 = 76.95, p < 0.0001 (J)]. NS, not significant, * p ≤ 0.05; *** p ≤ 0.001. For detailed statistical results, please see Table S3 . To understand whether L-leucine-mediated activation of PPRE is due to PPARα or PPARβ, PPRE activation was monitored in astrocytes isolated from PPARα −/− or PPARβ −/− mice. Induction of PPRE activation by L-leucine in PPARβ −/− ( Figure 4D ), but not PPARα −/− ( Figure 4C ), astrocytes indicates that L-leucine-mediated activation of PPRE in astrocytes is due to PPARα, but not PPARβ. Next, we investigated mechanisms by which L-leucine could activate PPARα. While short- and medium-chain fatty acids are ligands of PPARα, it is not known whether any amino acid could be a ligand of PPARα. Until now, it is also not known whether L-leucine binds to a receptor. Therefore, in the beginning, we employed a rigid body protein-ligand docking tool (SwissDock) for searching the interaction between L-leucine and different PPARs. We mainly focused on the ligand-binding domain (LBD) because a well-defined LBD capable of binding to different ligands is present in all three PPARs. Three key residues in the ligand-binding pocket of PPARα are S280, Y314, and Y464 ( Patel et al. 2018 ; Roy et al. 2015 ). While aspirin binds to the Y314 residue, gemfibrozil interacts with the Y464 residue ( Patel et al. 2018 ; Roy et al. 2015 ). Here, we noticed that L-leucine fitted nicely in the LBD of PPARα ( Figure 4E ) to display H-bonding with Y314 (1.9 Å), Y464 (2.0 Å), H440 (2.4 Å), and S280 (1.6 Å) ( Figure 4F ). On the other hand, L-leucine did not dock in the LBD of either PPARβ ( Figure S10A , B ) or PPARγ ( Figure S10C , D ). In the case of both PPARβ ( Figure S10A , B ) and PPARγ ( Figure S10C , D ), L-leucine interacted nonspecifically on the surface of the protein. It is always necessary to strengthen in silico results with experimental evidences. Therefore, to confirm the interaction between L-leucine and PPARα, we performed thermal shift assay (TSA). PPARα protein was conformationally stable as evident from the typical sigmoidal melting curve ( Figure 4G ). However, 10 μM L-leucine strongly shifted the melting curve of PPARα by 7.36 (49.36–42)°C ( Figure 4G ), indicating strong binding of L-leucine with PPARα. On the other hand, a very weak shift of PPARα melting curve by only 1.92°C was seen for L-alanine ( Figure 4H ), suggesting the specificity of the effect. To further confirm, we performed time-resolved fluorescence resonance energy transfer (TR-FRET) assay ( Roy et al. 2016 ). As evident from Figure 4I , L-leucine displayed a strong contact with PPARα. The binding curve ensued a Hill slope of 0.5245 with an EC 50 value of 2.3 μM ( Figure 4I ). To confirm the functional significance of this biophysical interaction between PPARα and L-leucine, primary astrocytes isolated from PPARα −/− mice were transduced with lenti-full-length PPARα and lenti-Y314DPPARα followed by treatment with L-leucine. Although L-leucine remained unable to stimulate PPRE-driven luciferase activity in PPARα −/− astrocytes ( Figure 4C ), L-leucine upregulated PPRE luciferase activity in PPARα −/− astrocytes that were transduced with lenti-full-length PPARα , but not lenti-Y314DPPARα ( Figure 4J ). These results indicate the importance of binding of L-leucine with the Y314 residue of PPARα in the activation of PPARα. 3.7 |. L-Leucine Upregulates Lysosomal Biogenesis in Brain Cells via Its Interaction With PPARα Next, we examined whether L-leucine increased lysosomal biogenesis via PPARα. As evident from lysotracker staining, L-leucine increased lysosomal biogenesis in WT ( Figure 5A , D ) and PPARβ −/− ( Figure 5C , D ), but not PPARα −/− ( Figure 5B , D ) astrocytes, indicating that L-leucine stimulates lysosomal biogenesis in astrocytes via PPARβ, but not PPARα. Accordingly, in contrast to that found in WT astrocytes ( Figure S1 ), L-leucine remained unable to increase the activities of cathepsin B ( Figure S11A ), cathepsin D ( Figure S11B ), and TPP1 ( Figure S11C ) in PPARα −/− astrocytes. Similarly, L-leucine treatment upregulated the levels of TFEB ( Figure 6A , B ), LAMP2 ( Figure 6C , D ), and TPP1 ( Figure 6E , F ) in primary hippocampal neurons isolated from WT , but not PPARα −/− , mice. FIGURE 5 |. Open in a new tab L-leucine induces lysosomal biogenesis in astrocytes via PPARα, not PPARβ. Primary astrocytes were isolated from C57BL/6 (A), PPAR α null (B), and PPAR β (C) null mice and were incubated with Leucine (100 μM) under serum free condition for 24 h, followed by staining with LysoTracker red to selectively stain the lysosomes and acidic organelles in live cells. Hoechst was used for nuclei stain. Number of LysoTracker-positive puncta per cell were from 15 different images per group from three independent experiments using Image J software (D). Data are shown as mean ± SEM. (E) PPARα−/− astrocytes were transduced with either lenti-FL-PPARα, Y314D-PPARα or naked lenti virus for 48 h. Cells were incubated with 100 μM Leucine for 16 h followed by staining with Lysotracker red. (F) Results were shown as Lysotracker puncta/cell. Two-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 2,90 = 19.69, p < 0.0001 (D)]. One-way mixed model ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 5,101 = 72.38, p < 0.0001 (D); F 4,84 = 115.0, p = 09947 (F)]. NS, not significant; *** p < 0.001. For detailed statistical results, please see Table S3 . FIGURE 6 |. Open in a new tab L-Leucine enhances lysosomal biogenesis in mouse primary hippocampal neurons via PPARα. Hippocampal neurons isolated from WT and PPARα null mice were treated with leucine in serum free conditions for 24 h., followed by monitoring TFEB (A), LAMP2 (C) and, TPP1 (E), which were double-labeled with MAP2. Scale bar, 20 μM. Data are shown as mean ± SEM of mean fluorescence intensity (MFI) of TFEB (B), LAMP2 (D), and TPP1 (F). One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 3,20 = 88.12, p < 0.0001 (B); F 3,20 = 133.9, p < 0.0001 (D); F 3,20 = 82.87, p < 0.0001 (F)]. NS, not significant; *** p < 0.001. For detailed statistical results, please see Table S3 . Next, PPARα −/− astrocytes were transduced with lenti-full-length (FL)-PPARα for 2 days followed by overnight treatment with L-leucine. Interestingly, the introduction of FL-PPARα significantly increased lysosomal biogenesis in L-leucine-stimulated PPARα −/− astrocytes ( Figure 5E , F ), confirming an important role of PPARα in L-leucine-mediated upregulation of lysosomal biogenesis. Next, in order to delineate a direct role of the Y314 residue of PPARα in L-leucine-mediated lysosomal biogenesis, PPARα −/− astrocytes were transduced with lenti-Y314DPPARα for 2 days followed by stimulation with 100 μM of L-leucine. In contrast to lenti-FL-PPARα , L-leucine remained unable to increase lysosomal biogenesis in lenti-Y314DPPARα -transduced PPARα −/− astrocytes ( Figure 5E , F ), suggesting that the binding of L-leucine with the Y314 residue of PPARα is important for L-leucine-mediated upregulation of lysosomal biogenesis. In addition to the upregulation of TFEB and lysosomal biogenesis, PPARα activation is also known to augment CREB ( Roy et al. 2013 ), SOCS3 ( Chakrabarti et al. 2019 ), IL-1Ra ( Chakrabarti et al. 2021 ), and Nurr1 ( Gottschalk et al. 2019 ) in brain cells as well as in vivo in the brain. Since L-leucine induced the activation of PPARα, we examined the effect of L-leucine treatment on the expression of these genes in vivo in the hippocampus of 5XFAD mice. While the mRNA expression of CREB ( Figure S12A ), SOCS3 ( Figure S12B ), IL-1Ra ( Figure S12C ), and Nurr1 ( Figure S12D ) decreased in the hippocampus of 5XFAD mice as compared to non-Tg mice, a marked increase in mRNA expression of these genes was observed in 5XFAD mice after L-leucine treatment ( Figure S12A , D ), indicating that in addition to TFEB, many other PPARα-dependent genes also could be upregulated in the brain of 5XFAD mice by L-leucine. 3.8 |. Oral L-Leucine Increases Lysosomal Biogenesis and Decreases Plaque in the Hippocampus of 5XFAD Mice via PPARα Next, we investigated whether L-leucine required PPARα to stimulate lysosomal biogenesis in vivo in the brain of 5XFAD mice. Double-labeling of hippocampal sections with NeuN and PPARα showed significant loss in PPARα in the hippocampus of 5XFAD mice as compared to non-Tg mice ( Figure S13A , C ). However, L-leucine treatment markedly restored and/or increased the level of PPARα in the hippocampus of 5XFAD mice ( Figure S13A , C ). On the other hand, decrease in PPARβ was not observed in the hippocampus of 5XFAD mice as compared to non-Tg mice ( Figure S13B , D ). Accordingly, L-leucine treatment also did not modulate the level of PPARβ in the hippocampus of 5XFAD mice ( Figure S13B , D ), indicating the specificity of the effect. Therefore, to delineate the role of PPARα in vivo in 5XFAD mice, we used 5XFAD ΔPPARα mice ( 5XFAD mice lacking PPARα) ( Corbett et al. 2015 ; Roy et al. 2015 ). Six-month-old 5XFAD ΔPPARα mice were fed with L-leucine orally for 60 days followed by monitoring lysosomal biogenesis in the hippocampus. In contrast to the upregulation of TFEB, LAMP2 and TPP1 in the hippocampus of 5XFAD mice by L-leucine ( Figure 2 ), this essential amino acid remained unable to increase the levels of TFEB ( Figure S14A , D ), LAMP2 ( Figure S14B , E ) and TPP1 ( Figure S14C , F ) in the hippocampus of 5XFAD ΔPPARα mice. To confirm the finding further, we performed Western blot of hippocampal extracts. Again, similar to immunofluorescence results, L-leucine treatment remained unable to increase the protein levels of TFEB ( Figure S14G , H ), LAMP2 ( Figure S14G , I ) and TPP1 ( Figure S14G , J ) in the hippocampus of 5XFAD ΔPPARα mice. These results demonstrate that L-leucine upregulates lysosomal biogenesis in vivo in the hippocampus of 5XFAD mice via PPARα. Next, we investigated the role of PPARα in L-leucine-mediated reduction of plaques using 5XFAD ΔPPARα mice. Although L-leucine treatment decreased plaques from the hippocampus and cortex of 5XFAD mice ( Figure 3 ), this amino acid could not lower plaque load from the brain of 5XFAD ΔPPARα mice as evident from Thio-S & 6E10 double-labeling ( Figure 7A & Figure S15 ), Thio-S puncta ( Figure 7B ), Thio-S-positive and 6E10-positive puncta ( Figure 7C ), Thio-S-positive and 6E10-positive plaque size ( Figure 7D ), percent of Thio-S-positive and 6E10-positive plaque burden ( Figure 7E ), and Thio-S-positive and 6E10-positive plaque area ( Figure 7F ). These results were also confirmed by DAB staining of hippocampal ( Figure S16A , C – F ) and cortical sections ( Figure S16B , G – J ) as well as Western blot of hippocampal extract ( Figure S16K , L ). These results suggest that L-leucine requires PPARα to decrease amyloid plaques in 5XFAD mice. FIGURE 7 |. Open in a new tab Oral L-leucine requires PPARα to decrease Aβ plaque in the brain of 5XFAD mice. Six-month-old 5XFAD and 5XFAD ΔPPARα ( 5XFAD lacking PPARα ) mice ( n = 6 per group) were treated with leucine (50 mg/kg body wt/day) via gavage once daily for 60 days. Since leucine was solubilized in 100 μL water, control 5XFAD and 5XFAD ΔPPARα mice also received the same volume of water as vehicle via gavage. After 60 days of treatment, hippocampal sections were double labeled with thioflavin-S ( Heckmann et al. 2020 ) and 6E10 antibody (red) (A, double-labeled image; B, Thio-S puncta count; C, Thio-S+ Aβ 610 puncta count; D, Thio-S+ Aβ 610 puncta size; E, Thio-S+ positive area percentage; F, Thio-S+ Aβ 6e10 plaque area). For quantification, two sections (one image per section) of each of six mice per group were considered. One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 4,20 = 140.6, p < 0.0001 (B); F 4,25 = 69.89, p < 0.0001 (C); F 4,20 = 116.8, p < 0.0001 (D); F 4,20 = 52.64, p < 0.0001 (E); F 4,25 = 19.52, p < 0.0001 (F)]. *** p < 0.001; ** p < 0.01; ns, not significant. For detailed statistical results, please see Table S3 . 3.9 |. L-Leucine Improves Cognitive Functions in 5XFAD Mice via PPARα Since cognitive dysfunction is the most important clinical symptom of AD and such symptoms are seen in 5XFAD mice ( Patel et al. 2018 , 2020 ), we examined whether oral administration of L-leucine could improve cognitive functions in 5XFAD mice. After 60 days of treatment with 10, 25, and 50 mg/kg/day L-leucine, 5XFAD mice were tested by Barnes maze, hippocampus-dependent spatial learning and behavior ( Roy et al. 2013 , 2015 ; Sarkar et al. 2025 ). Six-month-old non-Tg animals were treated with L-leucine (50 mg/kg/day) to validate the effect of leucine in normal animals. Consistent with earlier reports ( Roy et al. 2015 ; Rangasamy et al. 2015 ), 5XFAD mice exhibited impairment in spatial behaviors as evident by heat map ( Figure 8A ), latency ( Figure 8B ), and errors ( Figure 8C ) in comparison to age-matched non-transgenic mice. On the T maze, 5XFAD mice also exhibited poor performance in contrast to non-Tg mice as demonstrated by positive turn ( Figure 8D ) and negative turn ( Figure 8E ). On the other hand, at both the doses (25 and 50 mg/kg/day) tested, L-leucine significantly improved the performance of 5XFAD mice on the Barnes maze ( Figure 8A , heat map; Figure 8B , latency; Figure 8C , errors) and T maze ( Figure 8D , positive turn; Figure 8E , negative turn). However, L-leucine was more effective at 50 mg/kg/day than 25 mg/kg/day in improving spatial learning and memory of 5XFAD mice ( Figure 8A – E ). In contrast, at a dose of 10 mg/kg/day, L-leucine remained ineffective in improving the performance of 5XFAD mice on the Barnes maze ( Figure 8A – C ) and T maze ( Figure 8D , E ). We also monitored short-term memory by novel object recognition (NOR) and found impairment in 5XFAD mice as compared to non-Tg mice and improvement in NOR performance of 5XFAD mice by L-leucine treatment. However, L-leucine treated non-Tg animals did not exhibit any statistically significant difference compared to untreated non-Tg animals. This is evident from heat map ( Figure S17A ), time spent ( Figure S17B ), discrimination index ( Figure S17C ), and preferential index ( Figure S17D ). FIGURE 8 |. Open in a new tab Oral L-leucine requires PPARα to improve spatial learning and memory in 5XFAD mice. Six-month-old 5XFAD mice ( n = 6 per group) were treated with L-leucine (10, 25 and 50 mg/kg body wt/day) via gavage once daily for 60 days. Six-month-old non-transgenic animals were also treated with 50 mg/kg/day of L-leucine. Since Leucine was solubilized in 100 μL water, control 5XFAD mice also received the same volume of water as vehicle via gavage. After 60 days of treatment, mice were tested for Barnes maze (A, heatmap; B, latency; C, errors) and T maze (D, positive turn; E, negative turn). Six-month-old 5XFAD ΔPPARα ( 5XFAD lacking PPARα ) mice ( n = 6 per group) were treated with Leucine (50 mg/kg body wt/day) via gavage once daily for 60 days. After 60 days of Leucine treatment, mice were tested for Barnes maze (F, heatmap; G, latency; H, errors) and T maze (I, positive turn; J, negative turn). For statistical analyses, results are mean ± SEM of six mice per group. One-way ANOVA followed by Tukey’s multiple comparison post hoc test indicates [ F 5,24 = 18.81, p < 0.0001 (B); F 5,29 = 18.49, p < 0.0001 (C); F 5,28 = 40.84, p < 0.0001 (D); F 5,23 = 43.57, p < 0.0001 (E); F 2,15 = 110.2, p < 0.0001 (G); F 2,15 = 40.87, p < 0.0001 (H); F 2,15 = 53.39, p < 0.0001 (I); F 2,15 = 60.55, p < 0.0001 (J)]. NS, not significant. For detailed statistical results, please see Table S4 . * p < 0.05; *** p < 0.001. Next, we inspected whether L-leucine required PPARα to protect cognitive functions in 5XFAD mice. Therefore, we used 5XFAD ΔPPARα mice ( 5XFAD mice lacking PPARα) ( Corbett et al. 2015 ; Roy et al. 2015 ). Six-month-old 5XFAD ΔPPARα mice were fed with 50 mg/kg/day L-leucine for 60 days followed by monitoring memory and learning. Although L-leucine treatment improved the performance of 5XFAD mice on Barnes maze ( Figure 8A – C ) and T maze ( Figure 8D , E ), this amino acid remained unable to increase spatial learning and memory in 5XFAD ΔPPARα mice as evident from heat map ( Figure 8F ), latency ( Figure 8G ) and error ( Figure 8H ) from Barnes maze and positive turn ( Figure 8I ) and negative turn ( Figure 8J ) from T maze. Similarly, L-leucine was also ineffective in improving NOR task as shown by heat-map ( Figure S17E ), time spent ( Figure S17F ), discrimination index ( Figure S17G ), and preferential index ( Figure S17H ). These results suggest that L-leucine improves cognitive functions in 5XFAD mice via PPARα. 4 |. Discussion Accumulated evidence suggests that the impaired Aβ removal process is one of the underlying mechanisms in sporadic AD patients. Unquestionably, the available drugs target symptoms, and so-called novel treatments have failed miserably in therapeutic clinical interventions. Therefore, promoting the cellular degradative mechanism for the efficient clearance of insoluble amyloid deposition is considered an attractive therapeutic strategy in AD. Earlier studies showed that TFEB decreases Aβ levels by promoting lysosomal biogenesis and increasing Aβ clearance ( Xiao et al. 2014 ). It has also been shown that neuron-specific expression of TFEB by AAV vectors leads to a decrease in APP production, total Aβ levels, and plaque burden by enhancing flux via the endosome-lysosome pathway ( Xiao et al. 2015 ). Here, we delineate that L-leucine, one of the essential amino acids to be consumed through diets, upregulates lysosomal biogenesis and autophagy and reduces amyloid plaque pathology in a mouse model of AD. In cultured brain cells, treatment with L-leucine led to an increase in different lysosome-specific molecules and upregulated lysosomal biogenesis. Similarly, upon oral administration, L-leucine led to an increase in lysosomal proteins in the hippocampus and a decrease in plaque load in 5XFAD mice. Since L-leucine is usually nontoxic, our study suggests that L-leucine supplementation may have a therapeutic value for the treatment of AD. Earlier studies have shown that withdrawal of essential amino acids increases autophagy via involving Ca 2+ /Calmodulin-dependent Kinase Kinase-β ( Ghislat et al. 2012 ). They have also mentioned that autophagy induced by amino acid starvation requires adenosine monophosphate-activated protein kinase ( Ghislat et al. 2012 ). In contrast, in the present study, we delineated that treatment of astrocytes and neurons with a particular essential amino acid (L-leucine) led to an increase in lysosomal biogenesis and autophagy. Supplementation of L-leucine also increased autophagy in the brain of 5XFAD mouse model of AD. Therefore, the function of L-leucine in autophagy is just the opposite of what has been reported by Ghislat et al. (2012) for the withdrawal of essential amino acids. How does L-leucine couple lysosomal biogenesis and autophagy in brain cells? TFEB is a basic helix loop helix transcription activator belonging to the MiT family of transcription factors ( Sardiello et al. 2009 ; Martini-Stoica et al. 2016 ). Promoter of various lysosome associated genes harbor coordinated lysosomal expression and regulation (CLEAR) element and through binding to this element, TFEB positively regulates multiple cellular processes such as lysosomal biogenesis, autophagy, exocytosis and endocytosis. Here, we have demonstrated that L-leucine upregulates TFEB in cultured brain cells and in vivo in the hippocampus of 5XFAD mice. Accordingly, oral L-leucine also upregulated other lysosomal proteins and autophagy markers in the hippocampus of 5XFAD mice. It has been shown that enhancing astrocytic lysosomal biogenesis by targeted TFEB expression through viral gene transfer reduces amyloid pathogenesis via facilitating the uptake and the degradation of Aβ in lysosomes ( Xiao et al. 2014 ). Similarly, upregulating lysosomal function with TFEB accelerates lysosomal degradation of holo-APP in neurons to attenuate Aβ generation and reduce plaque pathogenesis ( Xiao et al. 2015 ). Therefore, L-leucine supplementation may exhibit beneficial effects in AD via upregulation of TFEB and enhancement of lysosomal biogenesis. Now, how does L-leucine upregulate TFEB? Although L-leucine was discovered from cheese in 1819 as the first branched chain amino acid, until now, it is not known whether there is any receptor for L-leucine. By using structural, functional, mutagenesis, and biochemical methodologies, here, we have demonstrated that L-leucine binds and activates PPARα, a transcription factor known to be involved in fatty acid metabolism. The strong interaction between L-leucine and PPARα was confirmed by TR-FRET analysis and thermal shift assay. In silico docking study using Autodock4 followed by site-directed mutagenesis uncovered the association between L-leucine and a tyrosine residue (Y314) of the PPARα ligand-binding domain. Reporter assay revealed an increase in luciferase activity by L-leucine in WT and PPARβ −/− , but not PPARα −/− , astrocytes, indicating the involvement of PPARα, but not PPARβ, in L-leucine-mediated activation of PPRE. Furthermore, restoration of PPRE reporter activation in PPARα −/− astrocytes by L-leucine upon transduction with lenti-full-length-PPARα , but not lenti-Y314D-PPARα , indicated the importance of the Y314 residue in L-leucine-mediated activation of PPARα. Interestingly, PPRE reporter activation was not seen in astrocytes by other amino acids such as L-lysine, L-histidine, L-tryptophan, L-alanine, L-glycine, L-glutamate, and L-aspartate, indicating the specificity of the effect. PPARs belonging to the class of ligand-inducible transcription factors are nuclear hormone receptors. Prototypically, being activated by fatty acids and their derivatives, this group of molecules acts as lipid sensors. However, here, we have described that PPARα could be activated by an amino acid as well. Earlier we demonstrated that activation of PPARα by gemfibrozil ( Ghosh et al. 2015 ; Majumder et al. 2026 ) and aspirin ( Chandra et al. 2018 ) leads to increased transcription of TFEB and upregulation of lysosomal biogenesis in brain cells. Consistently, in 2020, Luo et al. have also shown that activation of PPARα-mediated autophagy reduces AD-like pathology and cognitive decline in PSEN1ΔE9 mice ( Luo et al. 2020 ). Here, we have seen that L-leucine is capable of increasing the level of TFEB in astrocytes isolated from WT , but not PPARα −/− , mice. Similarly, L-leucine also could not stimulate the level of LAMP2 and TPP1 and upregulate lysosomal biogenesis in brain cells isolated from PPARα −/− mice. Our findings may have therapeutic potential and applicability in the modulation of lysosomal proteostasis and rescuing enzyme homeostasis in various lysosomal storage disorders (LSDs) because TFEB has been established as a specific modulator of lysosomal proteostasis in LSDs ( Song et al. 2013 ). Overexpression of TFEB has been shown to reduce lysosomal size and improve overall autophagy in Pompe disease, which is characterized by dysfunctional autophagy and lysosomal abnormality ( Spampanato et al. 2013 ). According to Rega et al. (2016) , genistein treatment stimulates TFEB to lower cystine levels and rescue the abnormalities of lysosomal compartments in cystinosis. In addition to LSDs, genetic activation of TFEB is reported to exhibit robust neuroprotection via reducing the load of α-synuclein in dopaminergic neurons, establishing an inverse connection between TFEB and α-synuclein toxicity ( Decressac et al. 2013 ). Therefore, L-leucine supplement may be beneficial for several LSDs and neurodegenerative disorders via up-regulation of TFEB and induction of lysosomal biogenesis and autophagy. At present, no effective therapy is available for preventing or halting the progression of AD. Pharmacological molecules directed towards TFEB as a therapeutic option in AD are still understudied. L-leucine is an essential amino acid that we regularly consume through diets. Therefore, L-leucine is considered nontoxic. Although at a dose of 25 mg/kg body wt/day, L-leucine showed some efficacy, at a dose of 50 mg/kg body wt/day, L-leucine was very effective in protecting memory and learning and reducing plaques in the 5XFAD mouse model of AD. If our mouse dose of L-leucine (50 mg/kg body weight/day) is translated to human, L-leucine supplement at a dose of 3 to 4 g per adult per day may be helpful for controlling AD-related symptoms and pathology in patients. In summary, here, we describe that L-leucine, an essential amino acid, binds to the LBD of PPARα, activates this nuclear hormone receptor, and stimulates lysosomal biogenesis and autophagy via PPARα. Moreover, orally administered L-leucine clears amyloid plaques from the brain, stimulates hippocampal function, and defends spatial learning and memory in an animal model of AD via PPARα. Thus L-leucine supplement may be beneficial for AD. Supplementary Material Supplement NIHMS2164106-supplement-Supplement.pdf (2MB, pdf) Additional supporting information can be found online in the Supporting Information section. Data S1: jnc70432-sup-0001-DataS1.zip. Funding This study was supported by a grant from NIH (AT10980) and merit awards (1I01BX005002 and I01BX005613) from US Department of Veterans Affairs. Moreover, Dr. Pahan is the recipient of a Research Career Scientist Award (1IK6 BX004982) from the Department of Veterans Affairs. However, the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the Department of Veterans Affairs or the United States government. Abbreviations: AD Alzheimer’s disease Aβ Amyloid beta FAD Familial AD FAM 6-carboxyfluorescein GAPDH Glyceraldehyde 3-phosphate dehydrogenase GFAP Glial fibrillary acidic protein GFP Green fluorescent protein HBSS Hanks’ balanced salt solution MFI Mean fluorescence intensity MOI Multiplicity of infection OCT Optimal cutting temperature PBS Phosphate-buffered saline PBST PBS-Tween-20 PPAR Peroxisome proliferator-activated receptor PPRE Peroxisome proliferator-responsive element RRID Research resource identifier TBST TBS plus Tween 20 TFEB Transcription factor EB TPP1 Tri-peptidyl-peptidase 1 TR-FRET Time-resolved fluorescence energy transfer TSA Thermal shift assay Y314 Tyrosine 314 Footnotes Ethics Statement Animal maintenance and experiments were in accordance with National Institute of Health guidelines and were approved (protocol ID: 20-007) by the Institutional Animal Care and Use committee of the Rush University of Medical Center. 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