Immunoperoxidase staining of NF, as a marker of axonal integrity, demonstrated clear degeneration of AD optic nerves compared to control tissues (Fig. 5). This example is representative of the study population. The majority of the AD optic nerves from the Alzheimer Disease Research Center came from patients with severe AD (Braak stage V-VI; Table 1) and were all severely affected as evidenced by greatly decreased NF staining.
Double-immunfluorescence labeling revealed colocalization of LRP within GFAP-positive astrocytes of optic nerves in both age-matched controls (Fig. 2 A, C) and AD nerves (Fig. 2 B, D) but to a greater degree in the AD tissues.
We demonstrated a decrease in the expression of LRP in AD optic nerves compared to that in the age-matched controls; this corroborates with other studies showing decreased LRP in AD patients (24,30). We presented histochemical evidence of AD optic nerve degeneration by showing a large decrease in NF staining, which is interpreted as a large loss of axons. This is consistent with the previous reports identifying optic neuropathy in AD patients (3-7).
In humans and animal models, LRP has been shown to be responsible for clearing Aβ out of the CNS via transport across the BBB (19,29,30). Reduced LRP has been identified in the cerebral vasculature of AD patients and appears to be associated with the accumulation of Aβ in the brain, which is believed to initiate pathogenic cascades seen in AD (17). Furthermore, the accumulation of Aβ within cerebral blood vessels in AD, known as cerebral amyloid angiopathy (CAA), is associated with the cognitive decline of this disease. Decreased clearance of Aβ across the BBB may contribute to CAA and parenchymal Aβ deposits (17). This is supported by studies that have demonstrated that impairment in the clearance of CNS beta-amyloid may be fundamental to the pathophysiology of AD (41). These findings suggest that the decreased expression of LRP found in our study could, by reducing the efflux of Aβ out of the optic nerve, play an important role in the pathogenesis of AD optic neuropathy.
sLRP under normal conditions is the major endogenous Aβ chaperone protein in plasma, acting as a peripheral “sink” pulling Aβ from the brain to the blood, preventing Aβ entrance into the CNS and facilitating its clearance in the liver (29,34). However, there is decreased sLRP in AD, most of which may become impaired by oxidation initiated by reactive oxygen species generated by the receptor for advanced glycation end products (RAGE) proinflammatory cascade (17). RAGE receptors in the BBB are responsible for the influx of Aβ into the CNS and have been shown to be upregulated in AD optic neuropathy (42). This suggests that the sLRP “peripheral sink” for Aβ is compromised in AD, leading to elevated free Aβ levels in plasma that exacerbate the increased Aβ levels in the CNS via RAGE-mediated transport across the BBB (29).
Astrocytes are normally neuroprotective, functioning to monitor the surrounding tissue environment, supply nutrients, support BBB functions, and repair by way of gliosis (48). When chronically activated, they release neurotoxic cytokines and activate destructive pathways (49,50). Cellular LRP not only clears Aβ but also has the potential to produce Aβ. LRP can internalize amyloid precursor protein (APP) and deliver it to the endosomal compartment where it can undergo amyloidogenic processing by β-secretase. This is followed by γ-secretase action to produce Aβ. As a consequence of increased APP internalization, LRP can enhance Aβ secretion (31,51). However, LRP has been shown to modulate APP trafficking between the cell surface and the compartments of the endocytic pathway by interacting with APP in the endoplasmic reticulum (ER) (52). By doing so, LRP has been shown to retain APP in the ER, reducing the levels of APP that reach the plasma membrane (53). Thus, LRP is intricately involved with cellular pathways that suppress Aβ generation but that can be altered to facilitate production (54). While we were able to show colocalization of LRP within astrocytes, future studies examining LRP immunolabeling with microglial markers and NF protein may further uncover the complicated role of LRP in the pathogenesis of AD optic neuropathy.
It should be noted that while the AD subjects were slightly older than the controls, this was not statistically significant (P = 0.13). LRP has been shown to decrease with normal aging in rodents, non-human primates, and AD patients, but it has not been shown to decrease with age in normal humans (29).
A potential therapeutic approach to AD optic neuropathy might be to target the interruption of LRP-mediated internalization of APP, a necessary step for LRP-facilitated production of Aβ. LRP and APP must form a complex with the adaptor protein FE65 to internalize APP from the plasma membrane in order to process it into Aβ (55). Interrupting this process could harness the protective effects of LRP effluxing Aβ from the CNS without the downside of producing more Aβ. This is supported by studies that suggest that LRP can be protective against AD (56). Another potential avenue of treatment would be to develop a synthetic sLRP, which could be administered intravenously that would bind to Aβ with high affinity and minimal toxicity. This could shift the Aβ transport equilibrium toward the plasma (21). Furthermore, other studies have shown that administering recombinant LRP clusters can effectively sequester plasma Aβ in human AD plasma and in AD mice (18). In mice, sequestration resulted in reductions of Aβ accumulation in the brain parenchyma and vasculature. This resulted in improvements in memory, learning, and cerebral blood flow responses (57).
In conclusion, our findings demonstrate a significantly reduced expression of LRP in AD optic nerves as compared to that in age-matched controls. This result, along with the LRP expression in the AD microvasculature, is concordant with other investigators' work and supports the hypothesis that decreased LRP may play a role in the underlying pathophysiology of AD optic neuropathy by reduced efflux of Aβ out of the optic nerve into the systemic circulation.
The authors express their gratitude to the USC Alzheimer's Disease Research Center and Lions Eye Bank of Oregon for supplying the tissues. Additionally, the authors thank Laurie Dustin for her assistance with the statistics and Ernesto and Eric Barron for facilitating the use of the confocal microscope and formatting the figures.
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