Early Cytoskeletal Protein Modifications Precede Overt Structural Degeneration in the DBA/2J Mouse Model of Glaucoma

Abstract

Axonal transport deficits precede structural loss in glaucoma and other neurodegenerations. Impairments in structural support, including modified cytoskeletal proteins, and microtubule-destabilizing elements, could be initiating factors in glaucoma pathogenesis. We investigated the time course of changes in protein levels and post-translational modifications in the DBA/2J mouse model of glaucoma. Using anterograde tract tracing of the retinal projection, we assessed major cytoskeletal and transported elements as a function of transport integrity in different stages of pathological progression. Using capillary-based electrophoresis, single- and multiplex immunosorbent assays, and immunofluorescence, we quantified hyperphosphorylated neurofilament-heavy chain, phosphorylated tau (ptau), calpain-mediated spectrin breakdown product (145/150 kDa), β-tubulin, and amyloid-β₄₂ proteins based on age and transport outcome to the superior colliculus (SC; the main retinal target in mice). Phosphorylated neurofilament-heavy chain (pNF-H) was elevated within the optic nerve (ON) and SC of 8-10 month-old DBA/2J mice, but was not evident in the retina until 12-15 months, suggesting that cytoskeletal modifications first appear in the distal retinal projection. As expected, higher pNF-H levels in the SC and retina were correlated with axonal transport deficits. Elevations in hyperphosphorylated tau (ptau) occurred in ON and SC between 3 and 8 month of age while retinal ptau accumulations occurred at 12-15 months in DBA/2J mice. In vitro co-immunoprecipitation experiments suggested increased affinity of ptau for the retrograde motor complex protein dynactin. We observed a transport-related decrease of β-tubulin in ON of 10-12 month-old DBA/2J mice, suggesting destabilized microtubule array. Elevations in calpain-mediated spectrin breakdown product were seen in ON and SC at the earliest age examined, well before axonal transport loss is evident. Finally, transport-independent elevations of amyloid-β₄₂, unlike pNF-H or ptau, occurred first in the retina of DBA/2J mice, and then progressed to SC. These data demonstrate distal-to-proximal progression of cytoskeletal modifications in the progression of glaucoma, with many of these changes occurring prior to complete loss of functional transport and axon degeneration. The earliest changes, such as elevated spectrin breakdown and amyloid-β levels, may make retinal ganglion cells susceptible to future stressors. As such, targeting modification of the axonal cytoskeleton in glaucoma may provide unique opportunities to slow disease progression.

Keywords: amyloid-beta; axonal transport; cytoskeleton; glaucoma; neurofilament; phosphorylation; spectrin; tau.

Figure 1

Early elevations in pNF-H observed in distal RGC projection with effects of age and transport outcome. (A) Quantification of pNF-H by ELISA in retina, optic nerve (ON), and superior colliculus (SC). DBA/2J retinal pNF-H levels show trending increase with age peaking at 12–15 months at which point, is significantly elevated compared to the D2G controls. In the ON, pNF-H levels were highest at 8–10 months. in the DBA/2J mice but returned to levels similar to the D2G controls at 12–15 months. In the SC, pNF-H levels remained significantly elevated in the D8-10 group compared to controls. (B) (Top) Immunofluorescence of cytoskeletal markers, SMI-310 (specific for superphosphorylated, pNF-H) and the RGC marker Brn3a in the retina of an 8-mo. DBA/2J (D8) and D2G control. Micrographs show visibly increased pNF-H and somatic/axonal ptau-231 staining in the D8 retina. The D2G pNF-H labeling is typical of mature, non-pathological axons within the retina. (Bottom) Semithin (1 μm) cross-sections taken from 8-months. DBA/2J (D8), 13-months. DBA/2J (D13), and 17-months. D2G (G17) control ON immuno-stained for β-tubulin (β-TUB) and pNF-H. A significant increase in pNF-H is observed in the D8 ON compared to G17 control. In the D13, pNF-H in the ON is nearly absent corresponding to the prominent loss of axonal structure indicative by the reduction in β-TUB. Error bars depict SEM. Asterisk indicates significant statistical difference (p <0.05).

Figure 2

Transport-dependent elevations in pNF-H observed in retina and SC of DBA/2J mice. (A) (Left) Photomicrograph from the dorsal surface of a whole-mount pathological SC in which the cortex has been removed and cholera toxin-B (CTB) labeling shows sectorial loss in the left (top) collicular lobe and complete loss in the right (bottom) lobe. Abbreviations: IC, inferior colliculus; PT, pre-tectum; LGN, lateral geniculate nucleus; SC, superior colliculus. The dotted lines demarcate areas absent of CTB indicating axonal transport deficit (CTB-). (Right) ELISA quantification of pNF-H in retina, optic nerve (ON), and SC. The transport status of retina and ON were determined by whether or not the SC was CTB+ (>90% CTB-label for partially transporting projections) or CTB- (<10% CTB-label for partially transporting projections). Levels of pNF-H were significantly elevated nearly 2-fold in both retina and SC of CTB- projections compared to CTB+ projections. Asterisks indicate significant statistical differences (p <0.05). Error bars depict SEM. (B) Top panel shows immunofluorescence of the SC, comparing 7-months. (D7), and 11-months. (D11) DBA/2J with and without CTB transport. Visibly increased pNF-H is shown in CTB- D11 SC. Bottom panel illustrates an age-dependent increase in pNF-H staining in the LGN (the visual thalamus) of 6, 11, and 14-month DBA/2J mice.

Figure 3

Phosphorylated tau (ptau-231) is elevated early in the distal projection and is observed to translocate to the retina in pathologically-aged animals. (A) Multiplex quantification of ptau-231 in DBA/2J mice shows an age-dependent decrease of ptau in SC with an age-dependent increase in retinal ptau. In the SC, ptau-231 is significantly higher in the D3-5 group compared to D12-15 and D2G control group levels. In retina, however, D12-15 ptau-231 levels are significantly higher than D2G controls. Ptau levels in ON samples did not differ between groups. (B) Immunofluorescence for ptau-231 in 10-months. DBA/2J ON shows evidence of preferential accumulation of ptau-231 in the proximal portion compared to the distal portion of the nerve. (C) An example of co-immunoprecipitation data comparing native and hyperphosphorylated tau via reaction with GSK-3 suggests an increased affinity of phosphorylated tau for the retrograde motor component, dynactin-2. This is qualitatively illustrated by the increased optical density of Tau-5 (a marker for pan-tau) in the second lane representing hyperphosphorylated tau (GSK-3+Tau-412), compared to the first lane which contained only native tau. The tau band shown here corresponds to a molecular weight of approximately 43.5 kDa corresponding to the GSK-3 phosphorylation site, pS396/404. Asterisk indicates that groups are significantly different from bracketed comparison (p <0.05). Error bars depict SEM.

Figure 4

Spectrin breakdown product (SBDP) 145/150 is elevated in young DBA/2J ON and SC while β-tubulin is decreased in a transport-dependent manner in pathologically-aged ON. (A) Chemiluminescence-based quantification using ProteinSimple Wes technology illustrates significantly higher levels of SBDP(145/150) within ON of D3-5 mice compared to all other groups, with no differences seen in pathologically-aged (10–12 months.) DBA/2J mice regardless of transport outcome. Bar graph (top) shows SBDP145/150 and β-tubulin normalized to GAPDH (a housekeeping gene marker), while corresponding examples of automated western output are shown below for ON. Importantly, a significant decrease in β-tubulin was observed in pathologically-aged ON associated with zero anterograde transport to the corresponding SC compared to controls and D10-12 mice with intact transport. (B) A significant elevation in SBDP chemiluminescence was observed in D3-5 SC compared to D2G controls (B), with no differences in collicular β-tubulin elucidated. Bar graph (top) shows SBDP145/150 and β-tubulin normalized to GAPDH, while corresponding examples of automated western output are shown below for SC. (Refer back to Figure 1 to see β-tubulin loss using immunofluorescence.) Error bars depict SEM. Asterisk indicates that groups are significantly different from bracketed comparison (p <0.05)

Figure 5

Amyloid-β₄₂ elevations provide an early retinal marker of pathology and are later observed in the distal projection. (A) Retinal Aβ₄₂ was elevated in all DBA/2J age groups compared to controls (left), with no transport-dependent effects observed (right). (B) However, collicular elevations appeared later, with 8–10 and 12–15 month DBA/2J SC having significantly elevated levels compared to controls; a significant difference was also observed between 3–5 and 8–10 month DBA/2Js (left). Levels of Aβ₄₂ were significantly elevated in SC lacking intact transport (CTB-) compared to transport-intact (CTB+) SC (right). Error bars indicate SEM. Asterisk indicates that groups are significantly different from bracketed comparison (p <0.05).

Figure 6

Timeline summary of protein pathology within the DBA/2J retinal projection. A summary schematic of changes in protein levels over disease progression (pre-glaucomatous, early, and late glaucomatous ages), in the DBA/2J mouse model, for each tissue type analyzed (retina, ON, SC) is shown here. The x-axis corresponds to time while the y-axis corresponds to general protein levels. Each colored line corresponds to a specific protein: dark blue corresponds to pNF-H, red corresponds to Aβ₄₂, light blue corresponds to SBDP, green corresponds to ptau-231, dotted purple corresponds to β-tubulin. Increased/decreased slope of each line indicates increased/decreased concentrations of each specific protein over time.