Autophagic vesicle numbers increase with glaucoma pathology, with D2 numbers significantly higher than D2-
Gpnmb+ for both distal and proximal ON (
t-test,
P < 0.05 for proximal, and
P < 0.001 for distal;
Fig. 7A). The D2 ON increases in autophagic vesicles are observed primarily in the axons within optic nerves exhibiting anterograde transport deficit (
Fig. 7B), suggesting increased autophagic activity with loss of anterograde transport (paired
t-test for proximal ON,
P < 0.001; distal ON,
P < 0.01). We categorized the autophagic vesicles into six groups based on morphology, and some of these groups correspond to known autophagy profiles (
Fig. 8A). To facilitate comparisons across groups, average numbers of autophagic profiles were normalized to the total volume of axons analyzed. In axons from CTB+ optic nerves, comparing proximal and distal autophagic vesicles showed higher numbers of all autophagic profiles in distal CTB+ ON (
Figs. 8B–G). In the case of endosomes, there were significantly more in distal CTB+ ON than proximal CTB+ (Wilcoxon rank sum test,
P < 0.01). There were also significantly more lysosomes in distal over proximal CTB+ ON (Wilcoxon rank sum test,
P < 0.05). Loss of anterograde transport (CTB
− ON) altered the average number of most profiles in both proximal and distal ON. Statistically significant increases in CTB
− ON over CTB+ included the vesicle-in-vesicle clear (ViV-clear) profiles (Mann-Whitney
U test,
P < 0.05), endosomes (Mann-Whitney
U test,
P < 0.05), and lysosomes (Mann-Whitney
U test,
P < 0.001;
Figs. 8C–E). In distal ON, loss of transport led to a trend toward higher numbers of ViV-dense and clear profiles, lysosomes, and endosomes, though these were not statistically significant changes. ViV-dense profiles, with single membranes and electron dense vesicular inclusions, may be late autophagosomes. Endosomes were vesicles without obvious inclusions. These were observed most frequently in distal ON, regardless of transport status (
Fig. 8D). Transport loss significantly decreased the average number of autophagosomes (APs) in distal CTB− ON axons (Mann-Whitney
U,
P < 0.05). The average number of degradative autophagic vacuoles (dAV; also known as autolysosomes) were not different across proximal or distal axons, regardless of transport status.
Table 3 shows the percentage of the total profiles observed in each category in the D2 and D2-
Gpnmb+ (D2G) proximal and distal ONs. Interestingly, endosomes made up roughly 30% of all profiles in proximal ON, and approximately 50% of all profiles in distal ON, independent of transport status. The percentage of AP and dAV profiles decreased with transport loss in both proximal and distal axons. We do not directly compare autophagic vesicle percentages in D2 against D2-
Gpnmb+ because so few autophagic vesicles were observed in D2-
Gpnmb+ ON (
Fig. 7A;
Table 3).