PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins.

Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.

Haemolysin Ahh1 secreted from Aeromonas dhakensis activates the NLRP3 inflammasome in macrophages and mediates severe soft tissue infection.

Severe soft tissue infections caused by Aeromonas dhakensis, such as necrotizing fasciitis or cellulitis, are prevalent in southern Taiwan and around the world. However, the mechanism by which A. dhakensis causes tissue damage remains unclear. Here, we found that the haemolysin Ahh1, which is the major virulence factor of A. dhakensis, causes cellular damage and activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome signalling pathway. Deletion of ahh1 significantly downregulated caspase-1, the proinflammatory cytokine interleukin 1ß (IL-1ß) and gasdermin D (GSDMD) and further decreased the damage caused by A. dhakensis in THP-1 cells. In addition, we found that knockdown of the NLRP3 inflammasome confers resistance to A. dhakensis infection in both THP-1 NLRP3-/- cells and C57BL/6 NLRP3-/- mice. In addition, we demonstrated that severe soft-tissue infections treated with antibiotics combined with a neutralizing antibody targeting IL-1ß significantly increased the survival rate and alleviated the degree of tissue damage in model mice compared control mice. These findings show that antibiotics combined with therapies targeting IL-1ß are potential strategies to treat severe tissue infections caused by toxin-producing bacteria.

Natural nanogels crosslinked with S-benzyl-L-cysteine exhibit potent antibacterial activity.

Biofilm-forming bacteria E. coli and P. aeruginosa have both exhibited resistance against multiple antibiotics in clinical settings. To find a solution, researchers have turned to antibacterial structurally modified from natural materials that are harmless to the human body. Among these is DNA, a natural polymer composed of deoxyribose that when treated with HCl exposes its aldehyde groups and produces DNA-HCl. Here, we crosslinked these aldehyde groups with the primary amines in S-benzyl-L-cysteine (SBLC) using a Schiff reaction to obtain DNA-HCl-SBLC. We additionally treated alginate acid (AA) with EDAC, obtaining AA-EDAC, and substituting it with SBLC to produce AA-SBLC. We incorporated the above reactions with an emulsification process to produce nanogels (NGs) that were verified to be spherical and possessing benzene rings successfully grafted onto DNA-HCl and AA-EDAC. These natural NGs were proven to be negatively charged through zeta potential analysis and presented low cytotoxicity toward normal cells in cell organoid viability assays. These SBLC-modified polymers provided better inhibition of bacterial growth than those without modification. Moreover, after incubation with SBLC-modified NGs, bacteria expressed intracellular recA or pvdA in a dose-dependent manner, which was consistent with SEM data from damaged bacteria. Out of four tested NGs, DNA-HCl-SBLC NGs suppressed P. aeruginosa-induced sepsis most effectively and extended the lifespan of C. elegans. This study provides an alternative clinical solution to antibiotics-resistant biofilm strains.

Isocitrate Dehydrogenase Alpha-1 Modulates Lifespan and Oxidative Stress Tolerance in Caenorhabditis elegans.

Altered metabolism is a hallmark of aging. The tricarboxylic acid cycle (TCA cycle) is an essential metabolic pathway and plays an important role in lifespan regulation. Supplementation of α-ketoglutarate, a metabolite converted by isocitrate dehydrogenase alpha-1 (idha-1) in the TCA cycle, increases lifespan in C. elegans. However, whether idha-1 can regulate lifespan in C. elegans remains unknown. Here, we reported that the expression of idha-1 modulates lifespan and oxidative stress tolerance in C. elegans. Transgenic overexpression of idha-1 extends lifespan, increases the levels of NADPH/NADP+ ratio, and elevates the tolerance to oxidative stress. Conversely, RNAi knockdown of idha-1 exhibits the opposite effects. In addition, the longevity of eat-2 (ad1116) mutant via dietary restriction (DR) was reduced by idha-1 knockdown, indicating that idha-1 may play a role in DR-mediated longevity. Furthermore, idha-1 mediated lifespan may depend on the target of rapamycin (TOR) signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Together, our data provide new insights into the understanding of idha-1 new function in lifespan regulation probably via DR and TOR signaling and in oxidative stress tolerance in C. elegans.

The conserved regulator of autophagy and innate immunity hlh-30/TFEB mediates tolerance of enterohemorrhagic Escherichia coli in Caenorhabditis elegans.

Infection with antibiotic-resistant bacteria is an emerging life-threatening issue worldwide. Enterohemorrhagic Escherichia coli O157: H7 (EHEC) causes hemorrhagic colitis and hemolytic uremic syndrome via contaminated food. Treatment of EHEC infection with antibiotics is contraindicated because of the risk of worsening the syndrome through the secreted toxins. Identifying the host factors involved in bacterial infection provides information about how to combat this pathogen. In our previous study, we showed that EHEC colonizes in the intestine of Caenorhabditis elegans. However, the host factors involved in EHEC colonization remain elusive. Thus, in this study, we aimed to identify the host factors involved in EHEC colonization. We conducted forward genetic screens to isolate mutants that enhanced EHEC colonization and named this phenotype enhanced intestinal colonization (Inc). Intriguingly, four mutants with the Inc phenotype showed significantly increased EHEC-resistant survival, which contrasts with our current knowledge. Genetic mapping and whole-genome sequencing (WGS) revealed that these mutants have loss-of-function mutations in unc-89. Furthermore, we showed that the tolerance of unc-89(wf132) to EHEC relied on HLH-30/TFEB activation. These findings suggest that hlh-30 plays a key role in pathogen tolerance in C. elegans.

Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans.

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

OmpR coordinates the expression of virulence factors of Enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans.

Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.

Effects of electrotactic exercise and antioxidant EUK-134 on oxidative stress relief in Caenorhabditis elegans.

Antioxidant uptake and regular exercise are two well-acknowledged measures used for rejuvenation and oxidative stress elimination. Previous studies have revealed that moderate exercise mildly increases intracellular signaling oxidant levels and strengthens the ability of an organism to deal with escalating oxidative stress by upregulating antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase. Antioxidant supplementation directly scavenges intracellular reactive oxygen species (ROS) to reduce oxidative stress. However, research to understand the impacts of these enzymes on mitigating oxidative stress from the perspective of simple animals is limited. Herein, we show that exercise combined with antioxidant supplementation ameliorates the physiological phenotypes and markers of aging in wild-type and SOD/CAT-deficient Caenorhabditis elegans. We discovered that treated wild-type and gene-deficient worms show better survivorship, reproduction, and motility compared with their control counterparts. Assays of biochemical indices revealed that variations in sod-3 expression under different stress levels imply an inducible enzyme response resulting from exercise training and antioxidant supplementation. In addition, induced ROS resistance obtained from any type of treatment could persist for several days even after treatment cessation, thus suggesting a potential long-term antioxidative stress effect. Our findings confirm that exercise, antioxidant supplementation, and their combination could significantly improve the ability of C. elegans to withstand adverse stress. Our observations provide promising insights into future therapies of anti-oxidative stress in higher animals.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

IGLR-2, a Leucine-Rich Repeat Domain Containing Protein, Is Required for the Host Defense in Caenorhabditis elegans.

Enterohemorrhagic Escherichia coli (EHEC), a human pathogen, also infects Caenorhabditis elegans. We demonstrated previously that C. elegans activates the p38 MAPK innate immune pathway to defend against EHEC infection. However, whether a C. elegans pattern recognition receptor (PRR) exists to regulate the immune pathway remains unknown. PRRs identified in other metazoans contain several conserved domains, including the leucine-rich repeat (LRR). By screening a focused RNAi library, we identified the IGLR-2, a transmembrane protein containing the LRR domain, as a potential immune regulator in C. elegans. Our data showed that iglr-2 regulates the host susceptibility to EHEC infection. Moreover, iglr-2 is required for pathogen avoidance to EHEC. The iglr-2 overexpressed strain, which was more resistant to EHEC originally, showed hypersusceptibility to EHEC upon knockdown of the p38 MAPK pathway. Together, our data suggested that iglr-2 plays an important role in C. elegans to defend EHEC by regulating pathogen-avoidance behavior and the p38 MAPK pathway.

UvrY is required for the full virulence of Aeromonas dhakensis.

Aeromonas dhakensis is an emerging human pathogen which causes fast and severe infections worldwide. Under the gradual pressure of lacking useful antibiotics, finding a new strategy against A. dhakensis infection is urgent. To understand its pathogenesis, we created an A. dhakensis AAK1 mini-Tn10 transposon library to study the mechanism of A. dhakensis infection. By using a Caenorhabditis elegans model, we established a screening platform for the purpose of identifying attenuated mutants. The uvrY mutant, which conferred the most attenuated toxicity toward C. elegans, was identified. The uvrY mutant was also less virulent in C2C12 fibroblast and mice models, in line with in vitro results. To further elucidate the mechanism of UvrY in controlling the toxicity in A. dhakensis, we conducted a transcriptomic analysis. The RNAseq results showed that the expression of a unique hemolysin ahh1 and other virulence factors were regulated by UvrY. Complementation of Ahh1, one of the most important virulence factors, rescued the pore-formation phenotype of uvrY mutant in C. elegans; however, complementation of ahh1 endogenous promoter-driven ahh1 could not produce Ahh1 and rescue the virulence in the uvrY mutant. These findings suggest that UvrY is required for the expression of Ahh1 in A. dhakensis. Taken together, our results suggested that UvrY controls several different virulence factors and is required for the full virulence of A. dhakensis. The two-component regulator UvrY therefore a potential therapeutic target which is worthy of further study.

The role of the bacterial protease Prc in the uropathogenesis of extraintestinal pathogenic Escherichia coli.

BACKGROUND: Extraintestinal pathogenic E. coli (ExPEC) remains one of the most prevalent bacterial pathogens that cause extraintestinal infections, including neonatal meningitis, septicemia, and urinary tract (UT) infections (UTIs). Antibiotic therapy has been the conventional treatment for such infections, but its efficacy has decreased due to the emergence of antibiotic-resistant bacteria. Identification and characterization of bacterial factors that contribute to the severity of infection would facilitate the development of novel therapeutic strategies. The ExPEC periplasmic protease Prc contributes to the pathogen's ability to evade complement-mediated killing in the serum. Here, we further investigated the role of the Prc protease in ExPEC-induced UTIs and the underlying mechanism. METHODS: The uropathogenic role of Prc was determined in a mouse model of UTIs. Using global quantitative proteomic analyses, we revealed that the expression of FliC and other outer membrane-associated proteins was altered by Prc deficiency. Comparative transcriptome analyses identified that Prc deficiency affected expression of the flagellar regulon and genes that are regulated by five extracytoplasmic signaling systems. RESULTS: A mutant ExPEC with a prc deletion was attenuated in bladder and kidney colonization. Global quantitative proteomic analyses of the prc mutant and wild-type ExPEC strains revealed significantly reduced flagellum expression in the absence of Prc, consequently impairing bacterial motility. The prc deletion triggered downregulation of the flhDC operon encoding the master transcriptional regulator of flagellum biogenesis. Overexpressing flhDC restored the prc mutant's motility and ability to colonize the UT, suggesting that the impaired motility is responsible for attenuated UT colonization of the mutant. Further comparative transcriptome analyses revealed that Prc deficiency activated the σE and RcsCDB signaling pathways. These pathways were responsible for the diminished flhDC expression. Finally, the activation of the RcsCDB system was attributed to the intracellular accumulation of a known Prc substrate Spr in the prc mutant. Spr is a peptidoglycan hydrolase and its accumulation destabilizes the bacterial envelope. CONCLUSIONS: We demonstrated for the first time that Prc is essential for full ExPEC virulence in UTIs. Our results collectively support the idea that Prc is essential for bacterial envelope integrity, thus explaining how Prc deficiency results in an attenuated ExPEC.

Rationally designed divalent caffeic amides inhibit amyloid-ß fibrillization, induce fibril dissociation, and ameliorate cytotoxicity.

One of the pathologic hallmarks in Alzheimer's disease (AD) is extracellular senile plaques composed of amyloid-ß (Aß) fibrils. Blocking Aß self-assembly or disassembling Aß aggregates by small molecules would be potential therapeutic strategies to treat AD. In this study, we synthesized a series of rationally designed divalent compounds and examined their effects on Aß fibrillization. A divalent amide (2) derived from two molecules of caffeic acid with a propylenediamine linker of ∼5.0 Šin length, which is close to the distance of adjacent ß sheets in Aß fibrils, showed good potency to inhibit Aß(1-42) fibrillization. Furthermore, compound 2 effectively dissociated the Aß(1-42) preformed fibrils. The cytotoxicity induced by Aß(1-42) aggregates in human neuroblastoma was reduced in the presence of 2, and feeding 2 to Aß transgenic C. elegans rescued the paralysis phenotype. In addition, the binding and stoichiometry of 2 to Aß(1-40) were demonstrated by using electrospray ionization-traveling wave ion mobility-mass spectrometry, while molecular dynamic simulation was conducted to gain structural insights into the Aß(1-40)-2 complex.

RIOK-1 Is a Suppressor of the p38 MAPK Innate Immune Pathway in Caenorhabditis elegans.

Innate immunity is the primary defense mechanism against infection in metazoans. However, aberrant upregulation of innate immune-signaling pathways can also be detrimental to the host. The p38 MAPK/PMK-1 innate immune-signaling pathway has been demonstrated to play essential roles in cellular defenses against numerous infections in metazoans, including Caenorhabditis elegans. However, the negative regulators that maintain the homeostasis of this important innate immune pathway remain largely understudied. By screening a focused RNAi library against the kinome of C. elegans, we identified RIOK-1, a human RIO kinase homolog, as a novel suppressor of the p38 MAPK/PMK-1 signal pathway. We demonstrated that the suppression of riok-1 confers resistance to Aeromonas dhakensis infection in C. elegans. Using quantitative real time-PCR and riok-1 reporter worms, we found the expression levels of riok-1 to be significantly upregulated in worms infected with A. dhakensis. Our genetic epistasis analysis suggested that riok-1 acts on the upstream of the p38 MAPK/pmk-1 genetic pathway. Moreover, the suppression of riok-1 enhanced the p38 MAPK signal, suggesting that riok-1 is a negative regulator of this innate pathway in C. elegans. Our epistatic results put riok-1 downstream of skn-1, which encodes a p38 MAPK downstream transcription factor and serves as a feedback loop to the p38 MAPK pathway during an A. dhakensis infection. In conclusion, riok-1 is proposed as a novel innate immune suppressor and as a negative feedback loop model involving p38 MAPK, SKN-1, and RIOK-1 in C. elegans.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

A multi-omic analysis reveals the role of fumarate in regulating the virulence of enterohemorrhagic Escherichia coli.

The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Several molecular mechanisms have been described for the pathogenicity of EHEC; however, the role of bacterial metabolism in the virulence of EHEC during infection in vivo remains unclear. Here we show that aerobic metabolism plays an important role in the regulation of EHEC virulence in Caenorhabditis elegans. Our functional genomic analyses showed that disruption of the genes encoding the succinate dehydrogenase complex (Sdh) of EHEC, including the sdhA gene, attenuated its toxicity toward C. elegans animals. Sdh converts succinate to fumarate and links the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) simultaneously. Succinate accumulation and fumarate depletion in the EHEC sdhA mutant cells were also demonstrated to be concomitant by metabolomic analyses. Moreover, fumarate replenishment to the sdhA mutant significantly increased its virulence toward C. elegans. These results suggest that the TCA cycle, ETC, and alteration in metabolome all account for the attenuated toxicity of the sdhA mutant, and Sdh catabolite fumarate in particular plays a critical role in the regulation of EHEC virulence. In addition, we identified the tryptophanase (TnaA) as a downstream virulence determinant of SdhA using a label-free proteomic method. We demonstrated that expression of tnaA is regulated by fumarate in EHEC. Taken together, our multi-omic analyses demonstrate that sdhA is required for the virulence of EHEC, and aerobic metabolism plays important roles in the pathogenicity of EHEC infection in C. elegans. Moreover, our study highlights the potential targeting of SdhA, if druggable, as alternative preventive or therapeutic strategies by which to combat EHEC infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Design and Synthesis of Malonamide Derivatives as Antibiotics against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious threat to humans. Most existing antimicrobial drugs, including the ß-lactam and quinoxiline classes, are not effective against MRSA. In this study, we synthesized 24 derivatives of malonamide, a new class of antibacterial agents and potentiators of classic antimicrobials. A derivative that increases bacterial killing and biofilm eradication with low cell toxicity was created.

Neuroprotective Effects of Betulin in Pharmacological and Transgenic Caenorhabditis elegans Models of Parkinson's Disease.

Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system in the elderly. It is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta, as well as by motor dysfunction. Although the causes of PD are not well understood, aggregation of α-synuclein (α-syn) in neurons contributes to this disease. Current therapeutics for PD provides satisfactory symptom relief but not a cure. Treatment strategies include attempts to identify new drugs that will prevent or arrest the progressive course of PD by correcting disease-specific pathogenic process. Betulin is derived from the bark of birch trees and possesses anticancer, antimicrobial, and anti-inflammatory properties. The aim of the present study was to evaluate the potential for betulin to ameliorate PD features in Caenorhabditis elegans ( C. elegans) models. We demonstrated that betulin diminished α-syn accumulation in the transgenic C. elegans model. Betulin also reduced 6-hydroxydopamine-induced dopaminergic neuron degeneration, reduced food-sensing behavioral abnormalities, and reversed life-span decreases in a pharmacological C. elegans model. Moreover, we found that the enhancement of proteasomes activity by promoting rpn1 expression and downregulation of the apoptosis pathway gene, egl-1, may be the molecular mechanism for betulin-mediated protection against PD pathology. Together, these findings support betulin as a possible treatment for PD and encourage further investigations of betulin as an antineurodegenerative agent.