Fig 2: Cytosolic localization studies between cis-FITC and endosomes of cisplatin delivery with or without milk-exosomes. (A) Intracellular trafficking of internalized cis-FITC monitored by co-localization with the endosome marker Rab5 (red fluorescence) and lysosome marker LysoTracker Red (red fluorescence) by confocal (red fluorescence microscopy. Green represents cis-FITC, and blue represents DAPI stained nuclei. (B) Intracellular trafficking of internalized milk-exosome/cis-FITC monitored by co-localization with the endosome marker Rab5 (red fluorescence) and lysosome marker LysoTracker Red (red fluorescence) by confocal (red fluorescence microscopy. Green represents cis-FITC, and blue represents DAPI stained nuclei.

Fig 3: Rotenone causes accumulation of early endosomes prior to deficits in macroautophagy in rats. The number of early endosomes in dopaminergic neurons were assessed by immunohistochemistry using the early endosomal specific marker Rab5 and the dopaminergic specific marker TH in aged rats. Representative photomicrographs of Rab5 (Red) in nigral TH+ (Green) neurons in rats dosed with vehicle, acute or endpoint rotenone (A). The quantification for the average number of Rab5 puncta per dopaminergic neuron; n = 4/grp (B). The number of autophagosomes in dopaminergic neurons were assessed by immunohistochemistry using the p62/SQSTM1 and TH (C). The quantification for the average number of p62/SQSTM1 puncta per dopaminergic neuron; n = 4/grp (D). Data are analyzed using unpaired t-test * p < .05, ** p < .001, *** p < .001 graphs are expressed as mean ± SEM. Symbols represent individual brains. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig 4: Endolysosomal deficits in human iPD postmortem brain tissue. Paraffin embedded tissue sections of the substantia nigra from age-matched control human brains and individuals with iPD. Immunoreactivity of the early endosomal protein Rab5 in surviving nigral dopaminergic neurons of iPD patients in comparison to age-matched controls (A). Quantification of the average number and size of each Rab5 puncta per dopaminergic neuron in the substantia nigra; n = 8/grp (B). Representative photomicrographs of transferrin in surviving nigral dopaminergic neurons of iPD patients in comparison to age-matched controls (C). Quantification of the average number transferrin puncta per dopaminergic neuron in the substantia nigra; n = 4/grp (D). The number of late endosomes and lysosomes are assessed using specific markers: lysosomal-associated membrane protein 1 (Lamp1) and mannose-6-phosphate receptor (M6PR). The number of late endosomes (Lamp1+ and M6PR+) and lysosomes (Lamp+ and M6PR−) in iPD patient dopaminergic neurons compared with age-matched controls (E). Quantification for average number of late endosomes and lysosomes per dopaminergic neuron in the substantia nigra; n = 4/grp (F). As shown, representative photomicrographs of lysosomal (Lamp+ and cathepsin D +) and non-lysosomal (Lamp− and cathepsin D+) cathepsin D immunoreactivity in surviving nigral dopaminergic neurons of iPD patients in comparison to age-matched controls (G). Quantification for the average number of cathepsin D puncta per dopaminergic neuron in the substantia nigra of iPD and age-matched controls; n = 5/grp (H). Quantification for the average size of each cathepsin D puncta per dopaminergic neuron in the substantia nigra of iPD and age-matched controls; n = 5/grp (I). Representative photomicrographs of lysosomal GCase (GCase+ and Lamp1+) immunoreactivity in surviving nigral dopaminergic neurons of iPD patients in comparison to age-matched controls (J). Quantification of the average number and size of each lysosomal GCase puncta per dopaminergic neuron in the substantia nigra; n = 4/grp (K). Data are analyzed using unpaired t-test ** p < .01, *** p < .001 graphs are expressed as mean ± SEM. Symbols represent individual brains.

Fig 5: Left panel: Balanced autophagic flux. Rab5-positive early endosomes are the initial sorting station of the endocytic pathway. Normally, endocytic cargoes are either recycled to the cell surface or degraded via lysosomes. Rab5-positive early endosomes mature into late endosomes through a process called ‘Rab conversion’. Late endosomes, containing cargo from early endosomes, acquire newly synthesized lysosomal hydrolases and continue to mature and may eventually fuse with lysosomes. The resultant endolysosome matures into a classical highly acidic lysosome containing Lamp1 and the hydrolases and proteases necessary for degradation. Autophagosomes, the major cargo vesicle for macroautophagy, also traffic cargo to the lysosome for degradation, thereby forming an autolysosome. Under normal cellular homeostasis, endolysosomal function and autophagic flux are ‘balanced’.Middle panel: Acute rotenone dosing causes selective endolysosomal defects. Rotenone causes oxidative stress and activates LRRK2, and the resultant increase in LRRK2 kinase activity leads to phosphorylation of Rab10 and accumulation of swollen, Rab5-positive early endosomes. The apparent failure of Rab5-positive early endosomes to mature properly decreases the number of late endosomes and lysosomes, which likely impairs degradation and likely explains the accumulation of α-synuclein. At this stage, autophagosomes, labeled with p62 are unaffected.Right panel: Combined endolysosomal and autophagic deficits in endpoint rotenone and iPD. With continued cellular stress (mitochondrial impairment and LRRK2 activation), the endolysosomal deficits persist and worsen, and there are eventually fewer lysosomes with which autophagosome can fuse. As a result, p62-positive autophagosomes accumulate. Additionally, the finding of extra-lysosomal cathepsin D suggests that lysosomal integrity may be compromised. At this stage, there is ongoing neurodegeneration and depletion of dopamine neurons.