Protective Role of AMPK against PINK1B9 Flies' Neurodegeneration with Improved Mitochondrial Function

Adenosine 5′-monophosphate-activated protein kinase (AMPK)'s effect in PTEN-induced kinase 1 (PINK1) mutant Parkinson's disease (PD) transgenic flies and the related mechanism is seldom studied. The classic MHC-Gal4/UAS PD transgenic flies was utilized to generate the disease characteristics specifically expressed in flies' muscles, and Western blot (WB) was used to measure the expression of the activated form of AMPK to investigate whether activated AMPK alters in PINK1B9 PD flies. MHC-Gal4 was used to drive AMPK overexpression in PINK1B9 flies to demonstrate the crucial role of AMPK in PD pathogenesis. The abnormal wing posture and climbing ability of PINK1B9 PD transgenic flies were recorded. Mitochondrial morphology via transmission electron microscopy (TEM) and ATP and NADH: ubiquinone oxidoreductase core subunit S3 (NDUFS3) protein levels were tested to evaluate the alteration of the mitochondrial function in PINK1B9 PD flies. Phosphorylated AMPKα dropped significantly in PINK1B9 flies compared to controls, and AMPK overexpression rescued PINKB9 flies' abnormal wing posture rate. The elevated dopaminergic neuron number in PPL1 via immunofluorescent staining was observed. Mitochondrial dysfunction in PINK1B9 flies has been ameliorated with increased ATP level, restored mitochondrial morphology in muscle, and increased NDUFS3 protein expression. Conclusively, AMPK overexpression could partially rescue the PD flies via improving PINK1B9 flies' mitochondrial function.


Introduction
PD symptoms are the result derived from dopamine, dopaminergic, DA selectivity in the two areas of the brain induced by dopamine neurons loss in the substantia nigra compacta (substantia nigra pars compacta, SNc) and striatum degeneration [1,2].Te other feature of PD exists in surviving neurons with a synapse nucleoprotein (α-synuclein) as the main composition of the formation of Lewy bodies (LB) [3,4].Sporadic forms of PD are usually due to oxidative stress caused by environmental toxins, such as rotenone, N, N′-dimethyl-4,4′-bipyridinium dichloride (paraquat), and 1-methyl-4-phenyl-1, 2, 3, 6tetrahydropyridine (MPTP), and these substances target mitochondrial electron transport complexes from which they impair mitochondrial function [5,6].Although the low incidence of sporadic PD is extremely low, recent studies found that many of the genes in familial PD plays an essential role in mitochondrial function, such as α-synuclein (PARK1), UCHL1 (PARK5), LRRK2 (PARK8) dominant mutation, PINK1 (PARK6), DJ-1 (PARK7) mutations, and Parkin (PARK2) [7].PD can be classifed into familial and sporadic types according to genetic pathogenesis, and gene mutations are more common in familial PD [8][9][10].PINK1 genes are considered one of the common pathogenic genes that lead to autosomal recessive PD [11][12][13].PINK1 gene encodes mitochondrial serine/threonine kinase associated with mitochondrial autophagy and quality control [14].Terefore, PINK1 mutations related to PD models that present with the obvious mitochondrial dysfunctioninduced neurodegenerative disease have become a hot research issue.Te vital role of mitochondria is to generate ATP as the energy core station as it is the most critical organelles in eukaryotes to meet the need for energy metabolism [15,16].Te morphology of mitochondria is in a state of constant changes in response to the diferent metabolic situations [17,18].Under normal conditions, the fssion and fusion of mitochondria are in a state of constant conversion, and PINK1 gene mutation can disrupt the balance of such conversion [19,20].Te disorder of mitochondrial quality control (MQC) is closely related to neurodegenerative diseases [21].
Numerous studies have indicated the fight muscle degeneration of PINK1 B9 in the PD transgenic fruit fy model caused by the PINK1 gene mutation resulting in fying capacity impairment, abnormal wing posture, signifcantly shortened life span, and signifcantly lowered dopamine neuron content than the normal control group [22,23].
Mitochondrial dysfunction is the most obvious pathogenic characteristic in PINK1 B9 fies, with hyperinfated and degenerated plaques of mitochondria in the fight muscle [24,25].Terefore, direct energy regulation of PINK1 B9 fies might improve their mitochondrial function.
AMPK is a highly conserved energy sensor in eukaryotes that is capable of sensing metabolic stress [26,27].When energy balance is disturbed in cellular metabolism, AMPK activation induces alterations in metabolism of cells and regulates gene expression to decrease anabolic metabolism, promote catabolism, and recover ATP level [27].AMPK is rapidly activated after nearly all mitochondrial stresses, even those that do not disrupt the mitochondrial membrane potential.AMPK regulates autophagy and mitophagy through the activation of the kinase ULK1, the mammalian homologue of ATG1 [28].AMPK phosphorylates mitochondrial fssion factor and promotes mitochondrial fssion upon energetic stress.By simultaneously regulating mitochondrial fssion, mitophagy, and transcriptional control of mitochondrial biogenesis, AMPK acts as a signal integration platform to maintain mitochondrial health [29].
It has been found that deactivated AMPK exacerbates loss of neurons and associated phenotypes in LRRK and parkin mutant fies, while activated AMPK may lead a potential therapeutic clue for familial PD [30].However, its role in PINK1-mutated PD models has not been studied.
Neurons mainly use glucose as the main energy source.While the brain is one of the most crucial organs in the body to absorb glucose, more than 50% of the total glucose is used; as a result, the neurons have extremely high energy demand, and however, they cannot store too much energy [31,32].Hence, the energy storage capacity and demand are crucial since neurons are sensitive to cellular energy fuctuations [33].Terefore, AMPK keeps an essential function in the central nervous system (CNS) in maintaining the neurons' function and in maintaining homeostasis of cellular energy [34].AMPK is the crucial kinase in regulating energy metabolism, which might be an important clue in PINK1-mutated PD pathogenesis.However, the function or regulation of AMPK in the PINK1-mutated PD model and its mechanism have not been studied.
Tis study is to investigate AMPK's function in PINK1 B9 PD transgenic fies, interfering AMPK expression for PD transgenic fies.Intervention was conducted to observe whether AMPK overexpression (AMPK OE) or RNA interference with AMPK (AMPK RNAi) had protective efects on PD transgenic fies and further explore the mechanism of such protective efects.

Materials and Methods
2.1.Drosophila Lines and Husbandry.All fies were treated in a 12-hour light-dark cycle with the standard cornmeal, agar medium, yeast at 50% relative humidity, 25 °C.MHC-Gal4 (muscle) and TH-Gal4 (tyrosine hydroxylase) were used to construct the corresponding systems in this study for the tissue-specifc genes' knockdowns and upregulation.Te isogenic W 1118 and UAS-AMPK OE (32108) were obtained from Bloomington Drosophila Stock Center.PINK1 B9 fy line was a gift given by Dr. J Chung from KAIST, Korea.Separately, we used MHC-Gal4 and TH-Gal4 driver to cross with W 1118 to generate the control fies.Only male fies were chosen from the day they hatched and were transferred to the new tubes within ten days.

Abnormal Wing Postures
Quantifcation.One hundred male fies were chosen randomly from each experimental group and were put in the transparent tubes (25 cm in length and 1.6 cm in diameter) with putting 5 fies in each after anesthesia.Ten, it was incubated at room temperature for 20 min to acclimate to the environment.Data obtained from more than three independent experiments are presented as mean ± S.D. of scores.At room temperature and under standard light conditions, all behavioral tests were performed.

Climbing Assay.
Climbing assay was applied to evaluate locomotor function.One hundred fies from each indicated group were applied to in this assay for more than three independent experiments.Each fy was placed in a vertically placed tube (1.6 cm in diameter and 25 cm in length).Te fies were tapped to the bottom of the tube.Te total climbing time phase was collected by crossing over the 6 cm line from the tube bottom.Fly numbers were noted that could go over the 6 cm line within 10 seconds.Te data were shown as the percentage of total fies in this experiment and were shown as average +SEM by more than three independent experiments.Te statistical analysis was processed via two-way ANOVA (p < 0.05).

Transmission Electron Microscopy (TEM).
Ten-day-old fy thoraxes were prepared following fxing for 12 hours in 2.5% glutaraldehyde and 2% paraformaldehyde.A transmission electron microscope is used to examine the ultrathin sections.Ten thoraxes were selected from each experimental group.At Guangxi Medical University, Pathology Department, we collected the TEM images.At 10,000 x magnifcation, the slices were recorded by using the electron microscopy (H-7650, HITACHI, Japan).Mitochondrial perimeter was determined using ImageJ software.

Dopaminergic Neurons
Immunostaining.PPL1 TH-positive neurons quantifcation was evaluated using whole-mounted brain samples immune-stained from each experimental group.Ten-day-old fy brain samples were dissected in cold phosphate-bufered saline (PBS).Te brain samples were put in 0.3% Triton X-100 (Sigma) and 4% paraformaldehyde in PBS and set at 25 °C for 1 hour.Ten, the brains were kept incubated at 4 °C overnight by antityrosine hydroxylase (TH) (1 : 500 diluted for use, Millipore, Boston, MA).Te following day, the brains were washed in 0.3% Triton X-100 in PBS three times and each for 20 minutes at room temperature.Afterwards, brain samples were then incubated using fuorescent secondary antibody at 25 °C for 1 hour.After mounting the samples, they were imaged via the confocal microscope (Wetzlar, Germany).PPL1 TH-positive neuron quantifcation was analyzed from the brain hemisphere.

ATP Content Measurement.
Five thoraxes from each group were harvested and the tissue was washed in cold PBS.Te tissue was homogenized in 100 μL ice-cold 2N PCA with a homogenizer sitting on ice.Samples were centrifuged at 13,000 g for 2 minutes at 4 °C in a cold centrifuge and the supernatant was transferred to a fresh tube.After homogenizing the tissue in 100 μL of PCA, the volume was diluted to 500 μL with the Assay Bufer XXIII/ATP Assay Bufer included in the ATP kit (ab83355).Te ATP content of each group was extracted and was detected following the protocol of manufacturer and was detected by Ex/ Em � 535/587 nm fuorometric to show mitochondrial functions for more than three independent experiments.Protein amount was standardized by protein assay (Bradford), while the abundance of ATP was analyzed according to the protein amount.

Reactive Oxygen Species (ROS) Level
Detection.ROS was measured using the CellROX Orange reagent (cat.no.BB-470512; Shanghai Bio-Tech Co., Ltd.).Te thoraxes of 30 Drosophila were obtained, homogenized, and centrifuged at 1,000 x g for 10 min at 4 °C, and the supernatant was collected.Te supernatant and 20 μM CellROX Orange Reagent were mixed and incubated at 37 °C in the dark for 30 min, and the fuorescence intensity at 510 and 610 nm (maximum excitation light and maximum emission wavelength) was measured using a multifunction microplate reader.

Respirometry Measurement.
Using the high-resolution respirometer, Oxygraph-2K equipment, mitochondrial oxygen consumption was collected.Torax tissues were isolated freshly, homogenized on ice, and placed in the reaction bufer (80 mmol/l KCl, 3 mmol/l magnesium chloride, 1 mmol/l EDTA, 10 mmol/l Tris/HCl, and 5 mmol/l potassium phosphate, pH 7.4).Ten, inhibitors, substrates, and uncouplers were used as described below: substrates including 2M glutamate, 0.8M malate, and 2M pyruvate (P + M + G), 4 mM cytochrome C (Cyt-c), and 0.5M ADP + Mg 2+ ; uncoupler including rotenone and antimycin A (AMA); and 1.0 mM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for measuring mitochondrial respiratory chain complexes.Using malate, pyruvate, and glutamate as the substrates, complex I respiration was measured.Complex II was assessed via respiration bufer mixed with 10 mM succinate and 1 mM rotenone.Oxygen fux and oxygen concentration indicating CI and CII capacity were collected by Oroboros Instruments DatLab software for more than three independent experiments.
Endogenous control used in this study was 18S.

Pathogenic Features in PINK1 B9
Flies.As previous studies presented, PINK1 B9 fies presented the severely abnormal wing posture and climbing ability (Figures S1A-S1C) accompanied with the severely damaged mitochondrial morphology and the decreased mitochondrial number per observation feld in fight muscle tested by TEM (Figures S1D-S1F).ATP level decreased dramatically in PINK1 B9 compared to the W 1118 fies (Figure S1G), and ROS (reactive oxygen species) increased signifcantly in PINK1 B9 compared to the W 1118 fies (Figure S1H) indicating the severely imbalanced energy metabolism.MHC-Gal4 drove AMPK OE fies did not show signifcant diference compared to W 1118 control fies (Figures S1A-S1H).

Discussion
PD is one of the most common seen neurodegenerative diseases with motor dysfunction.At present, PD afects about 5∼6 million people around the world.With the rapid growth of the world population, the number is expected to reach 10 million by 2030.Te pathogenesis of PD is mainly due to aging and environmental factors, while the specifc pathogenesis is still unclear.Dysfunction of mitochondria is increasingly regarded as a threatening factor in the susceptibility of PD dopaminergic neurons and has become the pathogenesis characteristic of familial PD.Recently, researchers have shown that PINK1 gene mutation is one of the primary pathogenic genes causing familial PD, and PINK1 protein has been proved to be a vital element for MQC [36].Terefore, we concluded that in the mutant PD drosophila model, PINK1 B9 fies, the indirect fight muscles were not orderly with the decreased dopaminergic neuron number in PPL1, and the number of mitochondrial number per observation feld was reduced accompanied by the decreased ATP level, which directly refecting mitochondrial function was signifcantly decreased consistently to previous studies [37].
AMPK is an energy sensor of cells, capable of sensing metabolic stress and integrating many physiological signaling pathways restore energy balance [38].Some studies have shown that abnormal energy metabolism is a risk factor associated with neurodegenerative diseases [39].Furthermore, recent studies suggest that AMPK activation has a neuroprotective function [40].AMPK's activator, 5-amimidazole 4-formamide ribonucleotide (AICAR), protects hippocampal neurons by resisting glucose defciency damage and glutamate toxicity and inhibits the apoptotic level of astrocytes induced by ceramide.At the same time, some studies have found that gallic acid can signifcantly inhibit dopamine and mitochondrial dysfunction with its efects depending on the activity of AMPK, as the protective efect of gallic acid is eliminated when AMPK activity is inhibited.Since numerous dietary supplements and pharmaceuticals (e.g., AICAR or metformin) that increase AMPK activity are available for use in humans which might slow down the disease progression of PD, the clinical studies of their efects in PD patients are limited.Recent studies indicated that when treated with AMPK activator, parkin and LRRK2 mutants signifcantly improve the DA neuronal and climbing phenotypes in Ddc-GAL4-LRRK2 G2019S fies [30].
So that in our study, regulate AMPK expression in PD transgenic fies, especially the overexpression of AMPK might rescue its neurodegeneration phenotypes.Te experimental results showed that in the PD transgenic fies, the main AMPK activation form p-AMPK-α was signifcantly lower than normal controls.Tus, we speculated that the overexpression of AMPK in PD transgenic fies might rescue its phenotype and other disease features.As our expectation, AMPK OE can indeed improve PD transgenic fies' abnormal wing posture and the climbing ability.Consistently, dopaminergic neuron numbers increased signifcantly in AMPK OE interfered with PINK1 B9 fies so that the upregulation of AMPK rescues PD transgenic fies' muscle mitochondrial morphology and improves the mitochondrial content per unit area.Also, AMPK overexpression shown that the substantia nigra striatum in PD disease models, mitochondrial respiratory chain CI, and the activity of the coenzyme Q10 declined [41].Here, we found the increased CI subunit, NDUFS3 protein expression tested by WB, and the increased CI function in AMPK OE interfered PD.Tis study used a classical model of PD fies verifed increased activation of AMPK expression-protected PINK1 mutant PD transgenic fies.Te activity of AMPK enables the energy metabolism to reach a balanced state, thus fnding a possible therapeutic target for neurodegenerative diseases treatment like PD.
Our study is the frst to show that the expression level of p-AMPK-α protein, the main activated form of AMPK, was signifcantly decreased in PINK1 B9 mutant PD Drosophila model compared with the normal control.After interfering by AMPK OE with the PINK1 B9 , the abnormal wing posture of the PD transgenic drosophila model could be restored signifcantly, while the expression level of the activated form of AMPK protein in PINK1 B9 fies' muscle tissue increased dramatically.
When detecting AMPK OE interfered PINK1 B9 phenotypes, we found that the abnormal wing posture rate decreased signifcantly and improved climbing ability with the increased dopaminergic neuron numbers in PPL1, indicating the protective role of AMPK on neurodegeneration of PINK1 B9 fies.We also found the severely impaired mitochondrial function in the PD transgenic fies fight muscle was restored accompanied by the restored mitochondrial morphology, increased ATP levels, and the increased expression of the key marker of mitochondrial respiratory chain Complex I subunits and NDUFS3 protein.Furthermore, understanding overexpressed AMPK protects PINK1mutated PD fies which might indicate a therapeutic target on PINK1 mutant form of PD.

Figure 6 :
Figure 6: AMPK overexpression improves PINK1 B9 fies' CI function of ETC.(a-c) Quantifcation of mitochondrial respiratory CI and CII function of ETC tested in thorax tissue (n ≥ 5 group).(d,e) Equal amounts of protein (80 μg), isolated from fy thorax muscles of the indicated phenotype (n ≥ 10/group), were separated by SDS-PAGE and immunoblotted using the NDUFS3 antibody.Relative NDUFS3 protein expression in the thorax muscles of the indicated genotypes of fies (mean ± S.D.; * P < 0.05, # P < 0.0001).
Values represented as means ± SEM.Data were calculated by GraphPad Prism 8 via either unpaired two-tailed Student's t-test or one-way ANOVA test to determine signifcance.* p < 0.05, * * p < 0.01, * * * p < 0.005, and # p < 0.001 were expressed in all fgures to show the signifcant diference between other groups (NS, not signifcantly diferent).
Interfered PINK1B9Flies.AMPK OE and AMPK RNAi transgenic fies driven by the classical MHC-Gal4 muscle promoter were verifed by RT-PCR to confrm the correct AMPK expression alterations in the mRNA level compared with the normal control group (FigureS2A).Te expression level of AMPK mRNA and p-AMPK-α was signifcantly higher in AMPK OE fies and lower in AMPK RNAi compared with the normal controls (Figures2(a)-2(c) and S2A).Te construction strategy of AMPK overexpressed PINK1 B9 fies is shown in Figure2(d).More importantly, as the increased expression of AMPK mRNA was examined in AMPK OE PINK1 B9 fies, we confrmed the main activated form of AMPK protein ex-we observed the increased p-ULK1 expression in AMPK OE PINK1 B9 fies (Figures 2(i) and 2(j)).