The Involvement of Progranulin for α-Synuclein Reduction through Autolysosome Formation

Abstract

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by motor symptoms and neuropathological features, such as loss of dopaminergic neurons in the substantia nigra pars compacta and accumulation of alpha-synuclein (α-Syn). Progranulin (PGRN) is a secreted growth factor that exhibits anti-inflammatory properties and regulates lysosomal function. Although autophagy-lysosome pathway is the main degradative pathway for α-Syn, the molecular mechanistic relationship between PD and PGRN remains unclear. In this study, we investigated the role of PGRN in PD pathology. PGRN protein expression in striatum was increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. Intracerebroventricular (i.c.v.) administration of PGRN ameliorated the decrease in expression of tyrosine hydroxylase, a dopaminergic neuron marker, in MPTP-treated mice. Furthermore, i.c.v. administration of PGRN ameliorated 6-hydroxydopamine-induced motor deficits. In SH-SY5Y human neuroblastoma cells, 1-methyl-4-phenylpyridinium ion (MPP⁺), an active metabolite of MPTP, increased α-Syn expression. In contrast, PGRN ameliorated MPP⁺-induced increase in α-Syn expression. Although PGRN decreased the levels of autophagy-related proteins Sequestosome-1 (p62) and MAP1LC3 (LC3)-II, PGRN did not influence the phosphorylation of AMP-activated protein kinase and mechanistic target of rapamycin, which are also proteins that regulate autophagy. Immunostaining analysis showed that PGRN ameliorated MPP⁺-induced increase of LC3 puncta, indicator of autophagosome, and co-localization of LC3 and α-Syn. The DALGreen assay showed that PGRN ameliorated MPP⁺-induced decreasing trend of autolysosomes. These results suggest that PGRN participates in α-Syn degradation via acceleration of the autophagy-lysosome pathway and is a potential therapeutic target for PD.

INTRODUCTION

Parkinson’s disease (PD) is the secondary most common neurodegeneration in the world.¹⁾ PD causes motor impairments, bradykinesia, tremors, gait disturbance, and balance dysfunction.²⁾ PD is characterized by the accumulation of alpha-synuclein (α-Syn) forming Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta. Current therapies for PD mainly target dopamine receptor activation.^3,4) Although there are many drugs available in the market that help relieve the symptoms of PD and improve QOL, there is no cure yet and the cause of PD remains unclear. Therefore, further studies must be conducted to elucidate the etiology of PD and establish fundamental treatments.

Intracellular proteins are degraded via the ubiquitin-proteasome system (UPS) and autophagy. While the UPS degrades small molecular proteins, autophagy is responsible for the degradation of large molecular proteins, such as aggregated proteins and cytoplasmic organelles.⁵⁾ There are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy. Unwanted proteins and organelles in the cytoplasm are sequestered by autophagosomes composed of Sequestosome-1 (p62) and MAP1LC3 (LC3), and then subsequently degraded upon fusion with lysosomes.⁶⁾ There is growing evidence to suggest that autophagy is dysregulated in the brains of PD patients. The following observations have been reported in patients with PD: i) decreased chaperone-mediated autophagy in the substantia nigra,⁷⁾ ii) Lewy bodies containing LC3,⁸⁾ and iii) LC3-II accumulation in melanized dopaminergic neurons of the substantia nigra.⁹⁾ Proteins are degraded by lysosomal enzymes during autophagy, thus lysosomal inhibition leads to autophagosome accumulation.^10,11) Therefore, lysosomes play a significant role in autophagic proteolysis.

Progranulin (PGRN) is an acrosomal glycoprotein that is cleaved into smaller peptides called granulins, which display pro-inflammatory properties.¹²⁾ In the brain, PGRN, a neurotrophic factor is mainly expressed in the neurons and microglia, and exhibits various neuroprotective effects, such as anti-inflammatory effects, promotion of neuronal survival and neurite growth, and participation in lysosomal biogenesis and function.¹³⁾ Heterozygous and homozygous mutations in the GRN gene encoding PGRN are associated with frontotemporal dementia (FTD) and lysosomal storage diseases.^14,15) A meta-analysis of genome-wide association studies revealed that GRN is a novel risk locus for PD.¹⁶⁾ In addition, patients with PD had decreased serum PGRN levels¹⁷⁾ and some FTD patients with a mutation that reduced PGRN mRNA expression had synucleinopathy.¹⁸⁾

In a previous study, PGRN-deficient mice displayed aggravated dopaminergic neuron loss and increased microgliosis compared to that in wild-type mice in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model.¹⁹⁾ Viral vector delivery of the GRN ameliorated MPTP-induced behavioral abnormalities and dopaminergic neuron loss.²⁰⁾ However, the molecular mechanisms through which PGRN contributes to α-Syn pathogenesis remains unclear. In this study, we elucidated the role of PGRN in α-Syn reduction via autolysosome formation, using in vivo and in vitro PD models.

MATERIALS AND METHODS

Animals

Eight-week-old male C57BL6/J mice (15–20 g) were obtained from Japan SLC (Hamamatsu, Japan). The mice were kept at 24 ± 2 °C with exposure to a 12 h light/dark cycle and allowed ad libitum access to food and water. Every procedure was carried out in accordance with the animal care guidelines issued by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University (Gifu, Japan).

MPTP-Induced PD Model Mice

MPTP (Sigma-Aldrich, MO, U.S.A.) at 20 mg/kg was intraperitoneally administered to mice four times a day every 2 h, as previously reported.²¹⁾ The control group was treated with saline. PGRN (R&D Systems, MN, U.S.A.) dissolved in saline was administered intracerebroventricularly (i.c.v.; 1 ng) post-MPTP administration. On day 1 and day 3, post-MPTP administration, mouse brains were removed with or without perfusion of the whole body. Perfused mouse brains were used for immunofluorescence analysis and the brains collected without perfusion were used for Western blot analysis.

Tissue Preparation

Mice were anaesthetized with a mixture of three different drugs dexmedetomidine (Tokyo Chemical Industry, Tokyo, Japan) 0.3 mg/kg, midazolam (Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) 4.0 mg/kg, and butorphanol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) 5.0 mg/kg. After perfusion with saline for 3 min, the mice were perfusion-fixed with 4% paraformaldehyde for 3 min. Brains were prepared by removing the brain and postfixed at 4 °C for 24 h using the same fixative. Brain sections were equilibrated in 25% sucrose buffer and flash frozen with Tissue-Tek O.C.T (Leica, Wetzlar, Germany). At last, 30 µm thick slices of brain were obtained using a cryostat.

Immunohistochemistry

Frozen slices were washed with phosphate-buffered saline (PBS); after blocking using 10% horse serum (Vector Labs, CA, U.S.A.) for 1 h at room temperature, the slides were incubated with rabbit anti-tyrosine hydroxylase (TH) antibody (1 : 100 Santa Cruz Biotechnology, TX, U.S.A.) overnight at 4 °C. The sections were then washed with PBS and incubated with Alexa Fluor^® 546 goat anti-mouse immunoglobulin G (IgG) (1 : 1000, Thermo Fisher Scientific Inc., MA, U.S.A.) secondary antibody for 1 h at room temperature. The sections were sealed using Fluoromount (Diagnostic BioSystems, Pleasanton, CA, U.S.A.). An All-in-One fluorescence microscope (BZ-X710: Keyence, Osaka, Japan) was used to capture representative data. The number of TH-positive neurons in two sections of the magnification of ×400, and the mean number of TH-positive cells was calculated. The results were then expressed as a % of the control.

Western Blot Analysis

Mouse brains and human neuroblastoma SH-SY5Y cells obtained from the European Collection of Cell Culture (Wiltshire, U.K.) were lysed in radio immunoprecipitation assay buffer [150 mM NaCl, 50 mM Tris–HCl (pH 8.0), 1% Igepal CA-630, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS)] with protease (Sigma-Aldrich) and phosphatase inhibitor cocktails (Sigma-Aldrich). Lysates were subjected to centrifugation at 12000× g for 20 min at 4 °C. Protein density was determined comparing the BCA protein assay kit (Thermo Fisher Scientific) to known concentrations of bovine serum albumin. SDS sample buffer (FUJIFILM Wako) was added and the mixture was boiled for 5 min. Prepared samples were applied to 5–20% SDS-polyacrylamide gel electrophoresis. Isolated proteins were transfer to polyvinylidene difluoride membranes (Immobilon-P: Millipore Corporation, Bedford, MA, U.S.A.). The membrane was also the subject of incubation with the follow primary antibodies: goat polyclonal anti-PGRN antibody (1 : 500, R&D Systems), TH mouse monoclonal antibody (1 : 1000, Santa Cruz) β-actin mouse monoclonal antibody (1 : 5000, Sigma-Aldrich) α-Syn antibody (1 : 1000, Proteintech Group, Inc, IL, U.S.A.), lysosomal-associated membrane protein 1 (Lamp1) rat monoclonal antibodies (1 : 1000, Abcam PLC, Cambridge, U.K.) and phosphorylated-mechanistic target of rapamycin (p-mTOR), mechanistic target of rapamycin (mTOR), phosphorylated-AMP-activated protein kinase (p-AMPK), AMPK, LC3, p62, Beclin e-1 rabbit monoclonal antibodies (1 : 1000, Cell Signaling Technology Inc., MA, U.S.A.). After incubation with the primary antibody, the membrane was incubated with the secondary antibodies described below: horseradish peroxidase-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG (1 : 1000, Pierce Biotechnology Inc., MA, U.S.A.) secondary antibodies. Visualization of immunoreactive bands was performed using a LAS-4000 luminescent image analyzer (FUJIFILM Corp., Tokyo, Japan) and Amersham™ Imager 680 (Cytiva, Tokyo, Japan).

6-Hydroxydopamine (6-OHDA)-Induced PD Model Mice

The 6-OHDA (Sigma-Aldrich) hemi-injection was performed as previously reported.²²⁾ Briefly, mice were anesthetized using ketamine 43.8 mg/kg (Daiichi-Sankyo, Tokyo, Japan) and xylazine 2.5 mg/kg (Bayer Health Care, Tokyo, Japan) administered intraperitoneally. The skull of the mouse was then exposed, and 6-OHDA (2 µL) was injected in stratum at the following points: anterior-posterior (AP, +0.5 mm), lateral (L, −2.0 mm, left), and dorsal/ventral (DV, −3.0 mm) from the bregma. The 6-OHDA solution (5 µg/µL in 0.9% NaCl with 0.02% ascorbic acid) was injected over 3 min. Control and vehicle animals were administered 2 µL of 0.9% NaCl with 0.02% ascorbic acid in the left striatum. PGRN (1 ng) or saline was administered i.c.v. post-6-OHDA administration. After 7 d, apomorphine 0.5 mg/kg (Sigma-Aldrich), a dopamine agonist, was administered subcutaneously and a rotation test was performed.

Rotation Test

Mice were administered apomorphine (0.5 mg/kg) subcutaneously and then placed in a circular apparatus. Their behavior was observed for 10 min. Rotation to the right was scored +1, rotation to the left was scored −1, and the rotation score per minute was calculated.

Cell Culture

SH-SY5Y human neuroblastoma cells were cultured with Dulbecco’s modified Eagle’s medium (D-MEM: Nacalai Tesque, Kyoto, Japan) including 10% fetal bovine serum (FBS: Valeant, CA, U.S.A.), penicillin (Meiji Seika Co., Ltd., Tokyo, Japan) 100 U/mL, and streptomycin (Meiji Seika Co., Ltd.) 100 µg/mL in a controlled atmosphere humidified with 95% air and 5% CO₂ at 37 °C, as previously described.²³⁾ Cells were passaged by trypsinization every three or four days and maintained in 10 cm dishes (BD Biosciences, NJ, U.S.A.).

1-Methyl-4-phenylpyridinium Ion (MPP⁺) Damage in SHSY5Y Cells

When cells were 70% confluent, they were seeded in 12-well plates or 8-well chambers (100000 cells/mL). Twenty-four hours later, medium was replaced with DMEM containing 1% FBS and PGRN (200 ng/mL) was added. One hour later, MPP⁺ (500 µM) was added and incubated for 72 h. Western blotting or fluorescent immunostaining was then performed.

Immunocytochemistry

The cells were cultured on 8-well chamber slides non-coat (Matsunami, Osaka, Japan). Cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then washed three times for 5 min each with PBS. After fixing, the cells were permeabilized with 50 µg/mL saponin (FUJIFILM Wako) in PBS for 5 min and washed three times for 5 min each with PBS. Slides were blocked with 3% goat serum in PBS for 30 min at room temperature. Subsequently, the slides were incubated with rabbit anti-LC3 (1 : 100, Cosmobio, Tokyo, Japan) and α-Syn (1 : 100, Proteintech) antibody diluted in 3% Goat serum in PBS solution overnight at 4 °C. The cells were then washed three times for 5 min with PBS and incubated with the following secondary antibodies: Alexa Fluor^® 546 goat anti-rabbit IgG and Alexa Fluor^® 488 donkey anti-mouse IgG (1 : 1000, Thermo Fisher Scientific) diluted in 3% goat serum in PBS solution for 1 h at room temperature. Finally, the nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) and coverslips were mounted on glass slides. Confocal images were acquired by FLUOVIEW FV3000 (Olympus Corporation, Tokyo, Japan).

DALGreen Assay

SH-SY5Y cells were planted in 96-well plates at 1 × 10⁴ cells/well and cultured for 24 h at 37 °C in moistened air with 5% CO₂. For staining with DALGreen as described,²⁴⁾ seeded cells were preincubated with the dyes in the culture medium for 30 min at 37 °C. The medium was exchanged for fresh medium supplemented with 1% FBS, and then PGRN and MPP⁺ were added simultaneously. After 72 h, the absorption at 540 nm (reference wavelength, 360 nm) was measured using SkanIt Re for Varioskan Flash version 2.4 software (Thermo Fisher Scientific). Then Hoechst 33342 (Molecular Probes, OR, U.S.A.) was added and All-in-One fluorescence microscope (BZ-X710: Keyence) was used to capture representative data. The ratio of absorbance of DALGreen to cell number was used as an indicator of autolysosome formation.

Statistical Analysis

Data are shown as mean ± standard error of the mean (S.E.M.). Any statistical comparisons were performed using one-way ANOVA followed by Tukey–Kramer’s test. Statistically significant was set at p < 0.05. All statistical evaluations were conducted using SPSS Statistics software (IBM, NY, U.S.A.).

RESULTS

PGRN Expression Levels in the Striatum of MPTP-Treated Mice

Mice were injected with MPTP (20 mg/kg, i.p.) four times a day every 2 h. PGRN expression in the striatum was evaluated on day 1 and day 3 post-administration. Different molecular weight bands of progranulin were detected, resulting from different glycosylation. It is known that progranulin has two isoforms, a mature secreted form of 88 kDa and an immature form of 58–68 kDa, depending on the state of glycosylation.²⁵⁾ Brain damage, such as cerebral ischemia, increases immature isoforms.²⁶⁾ An increase in immature form of PGRN expression was seen in Figs. 1A and B.

Fig. 1. The Expression Level of PGRN in Striatum of MPTP-Treated Mice

Different molecular weight bands of PGRN were seen, resulting from different glycosylation. Immature forms of 58–68 kDa were detected. (A) The expression level of total double band density of PGRN and actin were evaluated by Western blot analysis in the striatum of the mice one or three days after administration of MPTP (20 mg/kg, i.p.). (B) The expression levels of PGRN were expressed as the ratio to actin. Data presented as mean ± S.E.M. (n = 5). #, p < 0.05 ##, p < 0.01, versus Control (Dunnett’s T3 test).

Effect of PGRN on Dopaminergic Neurons in the Substantia Nigra and Striatum of MPTP-Treated Mice

To investigate the effect of external administration of PGRN on MPTP-treated mice, PGRN (i.c.v.) was administered to the mice post-MPTP administration. MPTP-treated mice decrease dopaminergic innervation but do not stably cause motor deficits.²⁷⁾ Therefore, in this study, we evaluated a different model to examine the effects on dopamine nerves using the MPTP model and on behavioral abnormalities using the 6-OHDA model, which allows for simple behavioral assessment. MPTP decreased the expression of TH, a dopaminergic neuron marker, in substantia nigra, as determined by Western blotting and fluorescence immunostaining (Figs. 2A–D). In contrast, PGRN tended to ameliorate the MPTP-induced decrease the number of TH positive cell in the substantia nigra and improve TH expression and the striatum (Figs. 2A–D).

Fig. 2. The Effect of PGRN on MPTP-Induced Dopaminergic Neuron Injury

(A) The expression level of TH and actin were evaluated by Western blot analysis in striatum of MPTP-treated mice with or without PGRN. (B) The expression levels of tyrosine hydroxylase (TH) were expressed as the ratio to actin. Data presented as mean ± S.E.M. (n = 5). #, p < 0.05 versus Control. *, p < 0.05 versus Vehicle (Tukey–Kramer’s test). (C) TH immunofluorescence staining in substantia nigra of the mice three days after administration of MPTP (20 mg/kg, i.p.) with or without PGRN (1 ng, i.c.v.). (D) TH positive cells in substantia nigra were counted from TH immunofluorescence images and Data presented as mean ± S.E.M. (n = 3–6). #, p < 0.05 versus Control (Tukey–Kramer’s test).

Effect of PGRN on 6-OHDA-Induced Motor Dysfunction

Rotation tests were performed to assess the effect of PGRN on motor functions in unilaterally lesioned 6-OHDA-induced PD mice. 6-OHDA was administered to the mice through the left striatum under anesthesia. PGRN or saline (vehicle) was administered i.c.v. after 6-OHDA administration. Seven days later, apomorphine was administered subcutaneously. Post-apomorphine administration, rotation tests were performed. PGRN improved the 6-OHDA-induced increase in rotational behavior (Fig. 3). 6-OHDA decreased the expression of TH in the ipsilateral and contralateral striatum, as determined by Western blotting (Supplementary Figs. 2A–D). PGRN tended to ameliorate the 6-OHDA-induced decrease TH expression in the substantia nigra and striatum (Figs. 2A, B).

Fig. 3. The Effect of PGRN on 6-OHDA-Treated Mice in Behavior Test

Right rotation per minutes in mice seven days after administration of 6-OHDA (10 µg, intrastriatal administration) with or without PGRN (1 ng, i.c.v.). Left rotation was represented by a minus sign. Data presented as mean ± S.E.M. (n = 5–8). ##, p < 0.01 versus Control. *, p < 0.05 versus Vehicle (Tukey–Kramer’s test).

Effect of PGRN on MPP⁺-Induced Increase in α-Syn in SH-SY5Y Cells

MPP⁺, an active metabolite of MPTP was used to develop an in vitro PD model.²⁸⁾ The effect of PGRN on MPP⁺ treatment for 72 h was examined in SH-SY5Y cells. While MPP⁺ treatment increased α-Syn expression, PGRN ameliorated this increase in α-Syn expression (Figs. 4A, B).

Fig. 4. The Effect of PGRN on the Expression Levels of α-Syn in SH-SY5Y Cells Treated with MPP⁺

(A) The expression level of α-Syn and actin were evaluated by Western blot analysis on SH-SY5Y cells treated with MPP⁺ (500 µM) for 72 h with or without PGRN (100, 200 ng/mL). (B) The expression levels of α-Syn were expressed as the ratio to actin. Data presented as mean ± S.E.M. (n = 6). #, p < 0.05 versus Control. *, p < 0.05 versus Vehicle (Tukey–Kramer’s test).

Effect of PGRN on Autophagy-Related Proteins in SH-SY5Y Cells

The initiation of autophagy is regulated by mTOR and AMPK in response to dynamic changes in cellular nutrients and energy levels.²⁹⁾ MPP⁺ treatment for 72 h increased phosphorylated AMPK and decreased phosphorylated mTOR; however, PGRN did not affect the levels of phosphorylated AMPK and mTOR (Figs. 5A, B). Unwanted intracellular proteins are first surrounded by a membrane composed of LC3-II and p62 to form autophagosomes, which then fuse with lysosomes (forming autolysosomes) and are subsequently degraded.³⁰⁾ While MPP⁺ treatment increased LC3-II and p62 expression, PGRN inhibited LC3-II and p62 expression (Figs. 5C, D). Results from immunostaining showed that MPP⁺ treatment increased LC3 puncta and co-localization of α-Syn and LC3 (Supplementary Fig. 3). In contrast, PGRN suppressed the upregulation of LC3 puncta and co-localization of α-Syn and LC3 (Supplementary Fig. 3).

Fig. 5. The Effect of PGRN on MPP⁺-Induced Changing of Autophagic Pathway in SH-SY5Y Cell

(A) The expression level of, p-AMPK AMPK, p-mTOR, and mTOR were evaluated by Western blot analysis in SH-SY5Y cells treated with MPP⁺ (500 µM) for 72 h with or without PGRN (200 ng/mL). (B) The expression levels of p-AMPK were normalized by AMPK and p-mTOR were normalized by mTOR. (C) The expression level of LC3, p62, and actin were evaluated by Western blot analysis in SH-SY5Y cells treated with MPP⁺ (500 µM) with or without PGRN (200 ng/mL). (D) The expression levels of p62 were normalized by actin and LC3-II were by normalized by LC3-I. Data presented as mean ± S.E.M. (n = 6). #, p < 0.05, ##, p < 0.01 versus Control. **, p < 0.05, p = 0.11 versus Vehicle (Tukey–Kramer’s test).

Effect of PGRN on Autolysosome Formation in SH-SY5Y Cells

DALGreen permeates the plasma membrane and is incorporated into autophagosomes formed upon induction of autophagy. Subsequently, DALGreen fuses with lysosomes (forming autolysosomes) and emits fluorescence within the acidic environment of autolysosomes.²⁴⁾ Treatment with MPP⁺ tended to decrease DALGreen fluorescence intensity and percentage of autolysosome formation. In contrast, PGRN improved DALGreen fluorescence intensity and autolysosome formation (Figs. 6A, B).

Fig. 6. The Effect of PGRN on Autolysosome Formation in SH-SY5Y Cell

(A) The pictures were represented as autolysosome formation using Lysotracker in SH-SY5Y cells. The green dots indicated autolysosome and blue indicated nuclei. Scale bar = 30 µm. (B) The level of autolysosome formation treated with MPP⁺ (500 µM) for 72 h, with or without PGRN (200 ng/mL). Data presented as mean ± S.E.M. (n = 7). p = 0.211 versus control. *, p < 0.05 versus Vehicle (Tukey–Kramer’s test).

DISCUSSION

In this study, we evaluated the effects of PGRN on MPTP-induced dopaminergic neuron damage, 6-OHDA-induced motor dysfunction in vivo, and MPP⁺-induced autophagic abnormalities in vitro.

PGRN exhibits various neuroprotective effects, such as anti-inflammatory effects, promotion of neuronal survival and neurite growth, and participation in lysosomal function.¹³⁾ PGRN expression in the striatum of mice was increased on day 1 and day 3 post-MPTP administration (Fig. 1). PGRN is reportedly enhanced at the site of acute inflammation to suppress local inflammation, such as in the heart after myocardial infarction in mice³¹⁾ and retinal cells injured by needle puncture in zebrafish.³²⁾ Acute MPTP administration provokes inflammatory responses, such as activation of microglia.^33,34) Thus, an increase in PGRN may have suppressed inflammation in the striatum of MPTP-treated mice (Fig. 1).

PGRN has anti-inflammatory properties, but when converted to GRN by neutrophil elastase, it has pro-inflammatory properties.³⁵⁾ In a mouse MCAO model, cerebral ischemia caused increased PGRN expression and neutrophil elastase activity, resulting in increased GRN.³⁶⁾ Furthermore, Horinokita et al. also showed that elastase inhibition suppressed cerebral ischemic injury, suggesting that increased GRN promotes inflammation during brain injury. In addition, intracerebroventricular administration of low-dose PGRN (1 ng) was neuroprotective in the MCAO mouse model, while high-dose PGRN (5 ng) was not.³⁷⁾ In the present study, PGRN (1 ng) but not PGRN (5 ng) inhibited MPTP-induced dopamine neuronal loss (data not shown). Therefore, it is possible that low-dose PGRN (1 ng) was neuroprotective, but high-dose PGRN (5 ng) increased GRN expression by neutrophil elastase and failed to suppress inflammation.

MPTP increased α-Syn expression and impaired autophagy in the striatum of rodents,^38,39) but no results showing increased α-Syn in the substantia nigra of MPTP-treated mice were obtained (data not shown). Fourteen-day chronic MPTP administration model increases α-Syn in TH-positive cells, but a subacute administration model (20 mg/kg every 2 h for 4 doses; similar protocol to the present study) reported to produce no increase in α-Syn.⁴⁰⁾ Therefore, as a future work, the MPTP chronic administration model, in which accumulation of α-Syn occurs, will be used for evaluation against α-Syn in vivo.

Accumulation of α-Syn in the substantia nigra and striatum is believed to promote neurodegeneration, subsequently leading to PD.⁴¹⁾ Degradation by chaperone-mediated autophagy inhibits α-Syn.⁴²⁾ Subsequently, macroautophagy is induced and autophagosomes accumulate in dopaminergic neurons.⁴³⁾ Autophagosomes then fuse with lysosomes (forming autolysosomes) which are degraded by lysosomal enzymes. Both chaperone-mediated autophagy and macroautophagy involve degradation by lysosomal enzymes; therefore, lysosomes are essential in α-Syn degradation by autophagy. In the MPTP subacute administration model, we also observed a trend of increase in Lamp1 and LC3-I, p62, and Becline-1 (Supplementary Fig. 1). Lamp1 is a lysosomal membrane protein and is involved in the fusion of lysosomes and autophagosomes.⁴⁴⁾ Deletion of Lamp1 in Drosophila increased susceptibility to oxidative stress and exacerbated α-Syn-induced motor deficits,⁴⁵⁾ suggesting that PGRN may improve MPTP-induced neuropathy via increasing Lamp1 and reducing oxidative stress MPTP-induced neuropathy may have been ameliorated. It has also been reported that subacute MPTP administration reduced LC3 and Becline-1 expression,⁴⁶⁾ and a similar trend was observed, although the difference was not significant (Supplementary Figs. 1B, C). Thus, subacute MPTP administration may have caused autophagy impairment, and PGRN may have exhibited neuroprotective effects through improved autophagy.

Several autosomal dominant and recessive genes encoding for lysosomal, autophagic, and endosomal proteins and genetic risk factors are associated with the development of PD.⁴⁷⁾ PGRN regulates lysosomal function and biogenesis via acidification of lysosomes.⁴⁸⁾ PGRN is modulated by nuclear localization of the transcription factor EB, a key regulator of lysosomal proteins.⁴⁸⁾ In addition, pathway analysis on neuronal cells showed that silencing of autophagy-related genes enhanced PGRN expression,⁴⁹⁾ suggesting that increased PGRN expression (Fig. 1) may be a compensatory mechanism for impaired autophagy. PGRN administration (i.c.v.) improved TH expression in the substantia nigra of MPTP-treated mice (Fig. 2A). These results are in agreement with earlier studies showing that viral vector delivery of the GRN gene improved the PD-like state,²⁰⁾ while PGRN-deficient mice displayed worsening symptoms in the MPTP-induced PD model.¹⁹⁾ Therefore, these results suggest that PGRN exerts neuroprotective effects by ameliorating autophagy and improving lysosomal function.

6-OHDA selectively injures monoaminergic neurons but cannot cross the blood-brain barrier; therefore, 6-OHDA hemi-injection into the striatum can injure dopaminergic neurons in the substantia nigra and striatum.⁵⁰⁾ Apomorphine administration to 6-OHDA-treated rodents induced asymmetric rotational behavior; therefore, 6-OHDA-treated rodent models have been widely used to study PD-like behavioral disorders.⁵¹⁾ PGRN exhibits anti-inflammatory properties, such as inhibition of neutrophil degranulation, inhibition of tumor necrosis factor α (TNF-α) and cyclooxygenase-2 (COX-2) transmission,^52,53) and suppression of macrophage M1 polarization.⁵⁴⁾ 6-OHDA induces the release of inflammatory cytokines, such as TNF-α, COX-2, and inducible nitric oxide synthase.⁵⁵⁾ Therefore, PGRN may exert protective effects on dopaminergic neurons and improve motor function by blocking the release of these inflammatory cytokines. In addition, rotational asymmetry correlates well with dopaminergic neuronal loss in the 6-OHDA intrastriatal model,⁵⁶⁾ and PGRN improved rotational movements. In the ipsilateral striatum of 6-OHDA-treated mice, PGRN had a weak tendency to improve TH expression (Supplementary Figs. 2A, C), no significant difference was found. This could be due to the smaller injury caused by 6-OHDA compared to MPTP damage or the fact that it could not be detected by Western blotting. Further studies are needed to examine the counting of TH-positive cells and the direct effect of PGRN.

MPTP is converted in the body to its active metabolite MPP⁺, which is transported via the dopamine transporter and selectively damages dopaminergic neurons.²⁸⁾ Accumulation of α-Syn has been reported in MPP⁺-treated neuroblastoma cells.⁵⁷⁾ Sortilin plays a major role in the uptake of extracellular PGRN.⁵⁸⁾ SHSY5Y cells express sortilin, suggesting that PGRN is taken up and acts intracellularly.⁵⁹⁾ PGRN tended to have ameliorated the accumulation of α-Syn (Fig. 5), decreased p62 and LC3 expression (Fig. 5), and enhanced lysosomal function (Fig. 6). PGRN is known to regulate lysosomal function.^12,14) PGRN-deficient mice displayed autophagic impairments, such as TDP-43 accumulation in neurons⁶⁰⁾ and podocyte injury in diabetic nephropathy.⁶¹⁾ MPP⁺ treatment significantly increased LC3 puncta, the colocalization of α-Syn and LC-3 (Supplementary Fig. 3) and tended to decrease autolysosome formation (Fig. 6). These results suggest that MPP⁺-induced accumulation of α-Syn is due to autophagy dysfunction associated with suppression of autolysosome formation. In contrast, PGRN suppressed α-Syn expression (Fig. 4), improved LC-3 and p62 accumulation (Figs. 5C–E), and promoted autolysosome formation (Fig. 6). Therefore, PGRN decreased α-Syn expression through improved autophagy via enhancement of lysosomal function.

In conclusion, the present study revealed that PGRN exerts neuroprotective effects against MPP⁺- and 6-OHDA-induced dopaminergic cell damage in vivo and in vitro, respectively. Additionally, the results from the study showed that neuroprotective effects of PGRN may be attributed to its ability to improve autophagy by promoting autolysosome formation. These findings suggest that PGRN may be a potential therapeutic target for PD.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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