Mouse neuroblastoma N2a cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Meilunbio, Dalian, China) supplemented with 10% fetal bovine serum (FBS, Gibco, MA, USA) and 1% penicillin/streptomycin solution (Meilunbio) in 5% CO₂ at 37°C. N2a cells were transfected with pLKO-puro-shRNA-Nurr1 (Nurr1-KD) or pLKO-puro-shRNA (Ctrl) plasmid by lipofectamine 6,000 reagents (Beyotime, Shanghai, China) according to the manufacturer’s instructions, respectively. Transfected cells were screened by cell culture medium containing 3 μg/ml puromycin (Beyotime) until no more cell died.

For exogenous LC3B detection, the Nurr1-KD and Ctrl cells were transfected with GFP-LC3B plasmids by lipofectamine 6,000 reagents for 48 h. After that, cells were digested and seeded onto glass coverslips pre-coated with poly-lysine in 24 well-plate dishes and cultured overnight. The above cells were stained with an anti-LAMP1 antibody (methods were mentioned in the immunofluorescence staining and imaging part). For GBA overexpression and transcriptional activity assay, the coding domain sequence (NM_001077411.4) or its upstream 2,000 bp promoter sequence of mouse GBA was subcloned into pEGFP-N1 (GBA-pEGFP-N1) or pGL4.18 (GBA2000-luc) plasmid, respectively. GBA-pEGFP-N1/pEGFP-N1 plasmid was transfected into Nurr1-KD or Ctrl cells for 72 h. GBA2000-luc/pGL4.18 (Ctrl-luc) and pGL4.74 plasmids were co-transfected into Nurr1-KD and Ctrl cells for 48 h.

Nurr1^floxp/floxp mice were generated by ViewSolid Biotech (Beijing, China) and bred with CAGGCre^ERTM (Cre) mice. Cre-Nurr1^floxp/wt mice were used to cross with Nurr1^floxp/floxp mice to obtain the Cre-Nurr1^floxp/floxp mice (Nurr1^cKO). The Nurr1^floxp/floxp mice (Nurr1^cWT) were used as control. The genotype of mice was identified by PCR screening using primers as follows: Nurr1-floxp-forward: 5′-gggggcgtttggaaatga-3, Nurr1-floxp-reverse: 5′-gggcaaaagaggtggaacagc-3′; cre-forward: 5′-gctaaccatgttcatgccttc-3′, cre-reverse: 5′-aggcaaattttggtgtacgg-3′; positive control-forward: 5′-gagtgccaattcgatgatgagtc-3′, positive control-reverse: 5′-gcgcttactttgtgctgtccta-3′. Eight-week-old Nurr1^cKO and Nurr1^cWT mice were injected intraperitoneally with 10 mg/ml tamoxifen twice daily for 5 consecutive days. After 1 month, Nurr1^cKO and Nurr1^cWT mice were used (n ≥ 4 for each group) for further analysis.

Total RNA was extracted from N2a cells using RNAiso Plus reagent (Takara, Japan) and reverse-transcribed to double-strand cDNA according to the manufacturer’s instructions (TransGene Biotech, Beijing, China). The relative gene expression was determined with a SYBR Green RT-qPCR kit (TransGene Biotech) by ABI Prism 7,500 Detection System (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR (RT-qPCR) assay was repeated three times with three replicates per detection and gene expression was quantified with the 2^ΔΔCt method. The primers sequences were as follows: Nurr1 (forward: 5′-attccaggttccaggcaaac-3′, reverse: 5′-agcaaagccagggatcttct-3), GAPDH (forward: 5′-gcattgtggaagggctcatg-3′, reverse: 5′-agggatgatgttctgggcag-3′), GBA (forward: 5′-catccttgctttgtccccac-3′, reverse: 5′-ctggaagtcgttaggggtgt-3′).

Cells and SN tissues were re-suspended in radio immunoprecipitation assay (RIPA) buffer (Beyotime) with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 30 min, then centrifuged at 12,000 rpm for 15 min. The concentration of the supernatant was determined by a BCA Protein Assay kit (Takara). The remaining supernatant was added 5× sodium dodecyl sulfate (SDS) loading buffer (Beyotime) and boiled at 95°C for 10 min. Samples with equal amount of protein (20–30 μg) were subjected to 10% or 12.5% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to 0.2/0.45 μm polyvinylidene difluoride (PVDF) membranes (Merck Millipore Ltd., Germany). After blocking with 5% skimmed milk for 1 h at room temperature (RT), the membranes were incubated with the primary antibodies at 4°C overnight. After incubation, the membranes were washed with 1× tris-buffered saline containing tween-20 (TBST) three times and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 h. Specific protein bands were developed with enhanced electrochemiluminescence (ECL) plus procedure (Meilunbio) and quantified by the FluorChem Q system (Protein Simple, San Jose, CA, USA), normalized to GAPDH. Cell samples were collected at least three times, and the assays were repeated three times regarding each sample. Antibodies used were as follows: anti-Nurr1/NR4A2 antibody, anti-Cathepsin D antibody (immunofluorescence staining, IF), anti-CD107a/LAMP1 (1D4B) antibody (IF), anti-CD107b/LAMP2 antibody (IF), anti-LC3 antibody (Proteintech Group Inc., Rosemont, IL, USA), anti-β-glucosidase (GBA) antibody (Santa Cruz Biotechnology, TX, USA), anti-Cathepsin D antibody (Western blotting, WB), anti-LAMP1 antibody (WB), anti-LAMP2A antibody (WB) (Abcam, Cambridge, MA, USA), anti-GAPDH antibody (Cell Signaling Technology, Danvers, IL, USA), HRP-conjugated secondary antibodies (Proteintech). The original images of Western Blotting are available in the Supplementary material.

Cells were washed with phosphate buffered saline (PBS, Meilunbio) after seeding for 24 h and fixed in 4% paraformaldehyde (PFA, Beyotime) for 30 min at RT, then permeated with 0.25% Triton X-100 (Sigma-Aldrich) for 30 min at RT. Cell samples were washed with PBS and blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min at RT. The samples were stained with appropriate primary antibodies overnight at 4°C. After staining, the samples were washed and incubated with fluorescent secondary antibodies (Cell Signaling Technology) in a dark place for 1 h at RT. Nuclei were stained with Hoechst (1:1000) for 5 min. The fluorescence images were taken under a confocal microscope (A1R MP+, Nikon, Japan). Brain tissues were fixed with 4% PFA overnight at 4°C, then dehydrated in 15% and 30% sucrose solutions for 24 h and embedded in an optimal cutting temperature compound (Tissue-Tek, Torrance, CA, USA). Coronal sections of 40 μm were cut on a freezing microtome (Leica, Wetzlar, Germany). The slides were blocked with buffer (10% normal goat serum, 1% BSA, 0.3% Triton X-100, PBS solution) for 1 h at RT and incubated with the primary antibodies overnight at 4°C. Following incubation with the primary antibody, the slides were washed with PBS and incubated with secondary antibodies. Nuclei were stained with Hoechst (1:1000) for 5 min. The fluorescence images were taken under a confocal microscope (A1R MP+, Nikon, Japan).

Cells were seeded into a 6-well plate at a density of 5 × 10⁵ per well for 16 h. Then the samples were washed with PBS and incubated with 2 μM LysoSensor Green DND-189 dye (TransGene) at 37°C for 20 min. Fresh DMEM with 10% FBS was added to the plates after washing with PBS for three times. Then the samples were photographed by a confocal microscope (Nikon) or harvested and quantified by a flow cytometer (FACS CANTO II, BD, USA) at the wavelength of 530 nm. Cells without being stained were used as negative control.

Cells were harvested in a lysis buffer after transfection for 48 h and then centrifuged at 12,000 rpm for 10 min to get the supernatant. The activities of the firefly and renilla luminescence were measured by a dual-luciferase assay kit (Beyotime) under a microplate reader (BioTek Instruments, Inc., VT, USA). The concentration of the supernatant was determined with a BCA protein kit (Takara). The relative luciferase activity was normalized to the renilla activity and protein concentration.

The transcriptome sequencing and analysis were conducted by OE biotech Co., Ltd. (Shanghai, China). In brief, total RNA of Nurr1-KD and Ctrl cells was extracted by the RNAiso Plus reagent (Takara) and evaluated. The libraries were constructed by TruSeq Stranded mRNA LT Sample Prep Kit according to the manufacturer’s instruction (Illumina, San Diego, CA, USA) and then sequenced on the Illumina sequencing platform. DAVID online platform was utilized to perform the Gene Ontology (GO) enrichment analysis to identify significant canonical pathways. The differentially expressed genes (DEGs) were screened out with the DESeq2 Software. Gene expression was estimated by basemean value and significant difference was tested by negative binomial (NB) distribution method. Fold change ≥ 1.5 and false discovery rate (q) < 0.05 were set as the cut-off criteria for identifying the DEGs.

Cells were seeded at a density of 6 × 10⁶ in a 10 cm dish and cultured at 37°C overnight. On the next day, the cells were harvested and washed with PBS. Then the cells were pre-fixed with a fixative solution containing 2.5% glutaraldehyde (Servicebio, Wuhan, China) at RT for 30 min, and post-fixed in 1% osmium acid at RT for 2 h, successively dehydrated with graded ethanol (30%–50%–70%–80%–90%–95%–100%–100%). After embedding steps, the samples were double stained with 2% uranyl acetate saturated alcohol and 2.6% lead citrate solution, placed on grid and imaged by a transmission electron microscopy (TEM) (HITACHI, HT7700).

Cells were seeded at a density of 6 × 10⁶ into a 10 cm dish and cultured at 37°C overnight. On the next day, cells were sonicated in PBS and then centrifuged at 12,000 rpm for 15 min to get the supernatant. The supernatant was mixed with the substrate of Gcase and a compatible buffer from a β-Glucosidase enzyme activity kit (YanQi Biology, Shanghai, China) at 37°C for 2 h, then added the chromogenic reagents. The protein concentration of the supernatant was determined by a BCA kit (Takara). The standard curve was established with the concentration of the standard substance and its absorbance at 405 nm on a microplate reader (BioTek Instruments). The generation of 1 nmol p-nitrophenol from every milligram of protein per minute was defined as a unit of enzyme activity (nmol/min/mg protein).

The SN was rapidly removed, weighed and kept at −80°C. The amounts of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT) and 5-hydroxytryptamine (5-HT) were determined by high-performance liquid chromatography (HPLC) (EiCOM, HTEC-500, Kyoto, Japan) as described in detail previously (Xiao et al., 2015).

GraphPad Prism 8.0 software was used for statistical analysis and all data were expressed as mean ± SEM. Unpaired Student’s t-test was used for comparisons between two groups. Statistical significance was defined as p-value less than 0.05, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

We established stable Nurr1 knockdown (Nurr1-KD) and control (Ctrl) cells. The expression of Nurr1 was evaluated by RT-qPCR, Western blotting and Immunofluorescence staining. The results revealed that the mRNA and protein levels of Nurr1 in the Nurr1-KD group were significantly decreased to 31% (p < 0.0001) and 64% (p < 0.01) of those in the Ctrl group, respectively (Figures 1A, B). Consistently, immunofluorescence staining showed markedly weaker staining of Nurr1 intensity in the Nurr1-KD group compared to the Ctrl group (p < 0.05, Figures 1C, D), in accordance with the results of RT-qPCR and Western blotting. These results confirm the successful construction of Nurr1 knockdown cells.

Transcriptome analysis identified 1,785 DEGs between Nurr1-KD and Ctrl groups, including 835 upregulated genes and 950 downregulated genes (Fold change ≥ 1.5, q < 0.05). GO identified 22 biological pathways associated with ALP (Figure 2A), containing 45 DEGs with 13 upregulated and 32 downregulated genes (Figure 2B). Further analysis showed that these DEGs were mainly enriched in the following categories: autophagy assembly, phagosome acidification, positive regulation of phagocytosis/engulfment, etc. by biological process (Figure 2A); autophagosome and cytoplasmic vesicle, etc. by cellular component (Figure 2C); protein binding and macromolecular complex binding, etc. by molecular function (MF) (Figure 2D). Collectively, all these analysis results demonstrate a strong association between Nurr1 and the ALP.

TEM observation was performed to find the ultrastructure differences between Nurr1-KD and Ctrl group. A sharply increased number of autophagic vesicles were observed in Nurr1-KD group (p < 0.01, Figures 3A, C), indicating a dysregulated autophagic activity. Immunofluorescence analysis exhibited an increased LC3B fluorescence intensity and more LC3B-positive punctate structures in Nurr1-KD cells compared to Ctrl cells (p < 0.05, Figures 3B, D). Then, we transfected GFP-LC3B plasmids into Nurr1-KD and Ctrl cells. Immunofluorescence analysis showed that knockdown of Nurr1 significantly increased the autophagic puncta colocalized by exogenous LC3B and Lamp1 (p < 0.05, Figures 3E, F). Western blotting assay suggested the enhanced conversion of LC3B-I to LC3B-II, indicating elevated number of the autophagosomes (p < 0.01, Figures 3G, H). These results indicate that knockdown of Nurr1 promotes autophagosome accumulation.

Lysosomal acidity in Nurr1-KD and Ctrl cells was measured by the LysoSensor Green DND-189, which showed a pH-dependent increase in green fluorescence intensity in an acidified environment. The fluorescence intensity in Nurr1-KD cells showed a significant decrease compared to Ctrl cells (Figure 4A), indicating that knockdown of Nurr1 induced the alkalization of lysosomes. The same results were further confirmed by flow cytometry analysis (p < 0.01, Figures 4B, C). The expression of key lysosomal proteins (Lamp1, Lamp2, and CTSD) was assessed using Western blotting and Immunofluorescence staining. The results showed that the protein levels of Lamp1, Lamp2, and CTSD were all decreased to 46.8% (p < 0.01), 70.9% (p < 0.05), and 77.6% (p < 0.01), respectively (Figure 4D). A consistent alteration pattern in the expression of Lamp1, Lamp2, and CTSD was observed in Nurr1-KD cells through Immunofluorescence staining analysis (p < 0.01, Figures 4E–G). Immunofluorescence quantification further revealed diminished lysosomal localization of CTSD in Nurr1-KD cells (p < 0.01, Figures 4E, G), indicating impaired lysosomal protease retention. The above results demonstrate that knockdown of Nurr1 disrupts normal lysosomal function.

To further verify the regulation of Nurr1 in lysosomal function, we have established an inducible Nurr1 knockout mouse model (Nurr1^cKO) (Figure 5A) and systematically assessed the PD associated parameters. Immunofluorescence and Western blotting analysis confirmed efficient Nurr1 protein reduction in the SN (p < 0.05, Figures 5B, D, E), accompanied by a corresponding decline in TH expression (p < 0.05, Figures 5C–E). HPLC revealed significant depletion of HVA in Nurr1^cKO mice (p < 0.05), with non-significant decreasing trends in DA, DOPAC, and 5-HT levels. Notably, 3-MT concentrations remained unaltered (Figure 5F). Strikingly, lysosomal marker proteins Lamp1, Lamp2, and CTSD were significantly downregulated in the SN of Nurr1^cKO mice (p < 0.05; Figures 5G–K), mirroring our in vitro findings (Figures 4D–G). These congruent results establish Nurr1 as a key transcriptional regulator of lysosomal homeostasis in dopaminergic neurons.

Western blotting analysis showed that knockdown of Nurr1 decreased the protein level of GBA to 74.0% and 74.0% compared to the control group in vivo and in vitro, respectively (p < 0.01, Figures 6A–D). As expected, lower GBA protein level led to a significant reduction of GCase activity, with 73.7% activity remaining (p < 0.05, Figure 6E). The mRNA level of GBA decreased to 65% in Nurr1-KD cells compared to Ctrl cells (p < 0.0001, Figure 6F). To investigate the molecular mechanisms underlying the regulation of GBA expression by Nurr1, we analyzed the potential binding sites of Nurr1 with the GBA promoter (−2,000 to 0) using JASPAR database. The results showed that the GBA promoter region contains 10 binding sites of Nurr1, suggesting that Nurr1 may be the transcription factor of GBA (Figure 6G). To confirm the hypothesis, we successfully constructed a luciferase plasmid containing the GBA promoter region ranged from −2,000 to 0 (p < 0.0001, Figure 6H). The dual-luciferase reporter gene assay showed that knockdown of Nurr1 significantly inhibited the transcriptional activity of GBA with an efficiency of 62.5% (p < 0.01, Figure 6I).

To further investigate the role of GBA in Nurr1-mediated regulation of lysosomal function, we overexpressed GBA in Nurr1-KD cells and detected the expression levels of lysosomal proteins. Overexpression of GBA alleviated the decreased expression of Lamp1 and CTSD proteins, and their expression increased to 1.45-fold and 1.46-fold compared to Nurr1-KD + Ctrl group, respectively (p < 0.05, Figures 7A, B). Immunofluorescence staining demonstrated that the expression level of Lamp2 was elevated to 1.17-fold by GBA overexpression (p < 0.01, Figures 7C, D). We conclude that the lower expression and enzymatic activity of GBA induce lysosomal depletion in Nurr1-KD cells (Figure 8), and the overexpression of GBA ameliorates the defects of lysosomal proteins.