DRGs
Prepared from Sprague Dawley pups (P5-P14) and finally incubated in complete medium (Neurobasal A (ThermoFisher), B27 supplement (ThermoFisher), Gentamicin and glutaMAX (Thermofisher)). 
After 24hrs, the medium was replaced with fresh medium containing 1.5µM cytarabine (Ara-c; SigmaAldrich) to reduce proliferation of fibroblasts. 

DRG treatments
Cells were habituated to hypoxia at approximately DIV 7 (hypoxia: 3% O2, 5% CO2 and 92% N2). 
At approximately DIV10, cells were treated with rotenone (SigmaAldrich, 0, 1nM, 10nM, 100nM, 500nM) and/or levodopa methyl ester (0, 3µM, 30µM, 300µM) for 24 hours or 7 days. 
Vehicle controls for rotenone and levodopa were DMSO (Sigma Aldrich) and distilled water, respectively. 

Measurement of mitochondrial membrane potential
Membrane potential was measured using TMRM (Thermofisher).
Medium was removed and replaced with medium containing 10 nM TMRM and treatments for 30 min.
Cells were then imaged using a confocal microscope (LSM780, ex 561nm, em 566-669nm; 0.05 x 0.05µm per pixel using Plan-Apochromat 40x/1.3 oil DIC M27 objective).
Photomicrographs were analysed using ImageJ (doi:10.1038/nmeth.2019) software. Briefly, each soma was outlined to create an ROI and the mean pixel intensity of TMRM-stained mitochondria measured.

Oxidative stress assay
Dihydroethidium (DHE; Santa Cruz Biotechnology) was added to cells (final concentration 10µM DHE) and were incubated for 30 minutes.
Cells were then imaged using a confocal microscope (LSM780, ex 561nm, em 585-733nm; 0.83 x 0.83µm per pixel, 1024 x 1024 pixels, taken using Plan-Apochromat 10x/0.45 M27 objective).
Images were analysed with ImageJ (doi:10.1038/nmeth.2019) using particle analysis. Briefly, images were thresholded using manual intensity threshold boundaries of 120-255. Particle sizes 200µm2-infinity in size and with a circularity of 0.10-1.00 were measured, providing an outcome of percent area above threshold per image.

Immunostaining
Cells were fixed for 10 minutes using 4% paraformaldehyde (Sigma Aldrich) containing 250mM sucrose (Fisher Bioreagent) and stored at -4° C.
Cells were washed (3 x 5mins in 0.01M PBS), then permeabilised with 0.1% Triton X-100 for 10 minutes and then blocked in 5% goat serum in 0.01M PBS (Jackson ImmunoResearch) for 1 hr.
Cells were then incubated in primary antibodies, diluted in block, overnight with gentle rotation. 
Antibodies:
anti-MAP2, Abcam cat# ab5392 RRID:AB_2138153, 1 : 10 000
anti-beta III tubulin antibody, Abcam Cat# ab18207, RRID:AB_444319, 1 : 2500
anti-ATP5b, Millipore Cat# MAB3494, RRID:AB_177597, 1 : 500
Cells were then washed (3 x 5mins in 0.01M PBS) and then incubated with secondary antibody for 2 hr (Jackson ImmunoResearch, 1:200 in block).
Following washing (3 x 5mins in 0.01M PBS), cells were counterstained with 0.5 μg/ml Hoechst solution (Hoechst-34580 dye, Sigma Aldrich) for 10 minutes, then washed in PBS.
  
Imaging of neurites based upon MAP2 staining
LSM780 AxioObserver, plan-apochromat 10x/0.45 M27 objective, image resolution 1.38x1.38µm/pixel, 1024x1024 pixels/image.
Excitations and emissions were 405nm, 410-585nm (Hoechst), 488nm, 490-594nm (MAP2). 
Percent area positive for MAP2 staining was determined following auto-thresholding in ImageJ (doi:10.1038/nmeth.2019).

Imaging of neurites based upon beta III tubulin staining was completed using an LSM780 AxioObserver, plan-apochromat 10x/0.45 M27 objective, image resolution 1.66x1.66µm/pixel, 512x512 pixels/image.
Excitations and emissions were 405nm, 410-499nm (Hoechst), 561nm, 585-733nm (beta III tubulin).
Channels were separated, the red channel was auto-thresholded and then percent area above threshold per image was quantified in ImageJ (doi:10.1038/nmeth.2019).
Using the thresholded images to “IdentifyPrimaryObjects”, fluorescence intensity per neurite per image was quantified from the red channel (beta III tubulin) in CellProfiler (doi:10.1186/s12859-021-04344-9).

Cell bodies were imaged using LSM780 AxioObserver, plan-apochromat 40/1.3 oil DIC M27 objective, image resolution 0.05x0.05µm/pixel, 1024x1024 pixels/image.
Excitations and emissions were 405nm, 415-572nm (Hoechst), 488nm, 490-594nm (ATP5b), 561nm, 585-733nm (beta III tubulin).
The images were then analyzed in ImageJ (doi:10.1038/nmeth.2019). Channels were split and each soma was outlined based upon beta III tubulin staining, avoiding the nucleus.
Staining for ATP5b was auto-thresholded, and the percent area above threshold, within the soma ROIs, was quantified and expressed relative to control-treated cells.

Lysosome content and acidity measurements
Medium was removed and replaced with medium containing treatments and Lysotracker red (100nM, L7528 Thermofisher) and CellTracker green (6µM, C7025, Thermofisher).
Cells were incubated in the dark, at 37°C, for 1 hour, washed gently and imaged in warmed Krebs.
A 10-µm stack through the middle of each DRG soma, identified based upon CellTracker, was taken using an LSM780 confocal Plan-Apochromat 63x/1.4 Oil DIC M27 (pixel size 0.7µm x 0.7µm x 0.34µm).
Using a beam splitter, a line scan was taken, ex405nm em410-556nm (Lysosensor), ex561nm em 566-691nm (LysoTracker).
Channels were split, auto-thresholded and despeckled. A 5µm block of soma was outlined and surrounding area cleared.
For total lysosomal content, area stained by Lysotracker per optical slice was quantified, expressed as a proportion of cell area and a mean per soma generated.
For acidity, the proportion of Lysosensor contained with Lysotracker-stained ROIs per slice was quantified and a mean per soma generated. 

50B11 (doi:10.1177/1744806920970368) cell culturing
Cells were cultured in Neurobasal medium supplemented with 10% fetal bovine serum, 2% B-27, 20mM D-glucose and 0.2 M L-glutamine (GlutaMAX). All media and supplements were from Thermo Fisher Scientific, USA. Cells were used at passages 5-9.

50B11 treatments
Cells were habituated to hypoxia (3% O2, 5% CO2 and 92% N2) for 24hrs and then treated with combinations of levodopa methyl ester, entacapone (1µM [37]) and/or homocysteine (20µM [38,39]) or vehicle for 24hrs.

Lysosome analysis
Medium was replaced with medium containing treatments and Lysotracker red (100nM, L7528 Thermofisher Scientific) and Hoechst-34580 dye (500nM; Sigma Aldrich) for 30 minutes.
Cells were then imaged using StereoInvestigator (V5.00, MBF Bioscience) on a Zeiss Z1 microscope at ×63 magnification.
Images of nuclei and lysosomes were taken separately through the depth of each cell. 
The image stacks were then rendered using Helicon Focus (V8.2.2, Kharkiv, Ukraine) and cropped to 1550 x 1180 pixels.
Resultant images were then processed in CellProfiler (doi:10.1186/s12859-021-04344-9).
The size of all lysosomes was quantified.
In addition, Hoechst-stained nuclei were co-localised within these photo-micrographs in CellProfiler (doi:10.1186/s12859-021-04344-9) and then expanded to create masks using boundaries of 10, 20, 30, 40, 50 and 60 pixels from original size. Lysosomes within each boundary were identified and quantified for size and fluorescence intensity in CellProfiler (doi:10.1186/s12859-021-04344-9).


Datafile
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Each individual worksheet is labelled according to the appropriate figure/figure part.