Dysfunction of axonal transport in normal-tension glaucoma: a biomarker of disease progression and a potential therapeutic target

Kazuyuki Hirooka, Tohru Yamamoto, Yoshiaki Kiuchi

Glaucoma and dysfunction of axonal transport:One of the leading causes of irreversible blindness worldwide is glaucoma, with increased intraocular pressure (IOP) being the most common risk factor. However, in some glaucoma patients it has been shown that the IOP does not differ from that of the normal population. In Japan, normal-tension glaucoma (NTG), which accounts for 92% of primary open-angle glaucoma, has been shown to be more frequent in the population. Primarily, open-angle glaucoma treatments are almost exclusively focused on lowering the IOP through the use of drugs, laser therapy or surgery. However, glaucomatous optic nerve changes are believed by many investigators to occur not only due to increases in the IOP, but also because of other factors that are unrelated to the IOP, and which can play significant roles in some of these NTG cases. Glaucoma is a disease that causes vision loss through the degeneration and eventual apoptotic death of retinal ganglion cells (RGCs). The complex and multifactorial diseases caused by glaucoma are likely the result of the convergence of several molecular pathways that then induce RGC loss. Human glaucoma studies have demonstrated the presence of impaired axonal transport along the RGCs (Knox et al., 2007). Furthermore, since axonal transport is known to have a critical role with regard to the survival of RGCs, glaucomatous optic neuropathy may be associated with a failure of this transport. It has been clearly demonstrated that impaired axonal transport is an early, reversible, sensitive change in injured RGCs that precedes cell death (Fahy et al., 2016). Brain-derived neurotrophic factor, nerve growth factor, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor all help to both mediate the activity and ensure the survival of the RGCs. Other neurotrophic factors, such as fibroblast growth factor-2, neurotrophin 3, neurotrophin 4, and interleukin-10 have all been found to be neuroprotective in RGCs (Nafissi and Foldvari, 2016). The initial pathological events that are normally observed in neurodegenerative disorders include impairment of axonal transport, as the transduction of trophic signals requires the presence of intact axonal transport. Therefore, one of the attractive treatment areas when attempting to stop the RGC loss in glaucoma is to address the changes associated with the neurotrophic factor deprivation or adjust the insufficient levels of other essential molecules in order to prevent any axonal transport blockade.

Glaucoma and Alzheimer’s diseases (AD):AD is defined as a progressive neurodegenerative disorder. This disease is associated with changes in personality, cognitive and memory deteriorations, and an impaired ability to perform routine daily activities. The loss of neurons in the hippocampus and cerebral cortex that occurs in AD patients has been reported to be caused by plaque accumulation in the brain related to abnormally folded amyloid β (Aβ) and tau protein. Results from transgenic AD animal models have reported finding that there is synaptic dysfunction and axonal pathology that occurs before the deposition of the amyloid plaques and tau aggregation. Along with these findings, the brain areas affected in AD also exhibit a significant reduction in myelination, which in conjunction with the previous findings suggest that axonal dysfunction and degeneration are important components with regard to the development of AD. One of the factors that has recently been commonly seen in AD is the impairment of axonal transport. Moreover, both glaucoma and AD have been shown to share several common features. These include the presence of slow, chronic neurodegenerative disorders in conjunction with an age-related incidence. When the optic nerves of both glaucoma and AD patients were evaluated, structural studies demonstrated that there was both degeneration and a loss of RGCs (Hinton et al., 1986). Furthermore, Lin et al. (2014) previously examined glaucoma with regard to other potential associations and reported that glaucoma appeared to be a significant predictor for the development of AD. McKinnon et al. (2002) evaluated a rat model of chronic intraocular hypertension and determined that caspase activation on a molecular level induced augmented generation of the C-terminal fragment of the amyloid precursor protein (APP), which was subsequently shown to be the key event in the pathogenesis of AD. Glaucoma has been shown to be a widespread neurodegenerative condition, as confirmed by both preclinical and clinical evidence, in addition to having common pathogenetic mechanisms with AD.

Alcadein (Alc) and retinal ganglion cell:The brain abundant type I membrane protein family, which is known as Alc, has been shown to consist of closely related family members: Alcα, Alcβ, and Alcγ (Araki et al., 2003). During the transportation of molecules that need to be preferentially delivered, Alcα plays an important role in this process. Alcα and APP form a complex through their association with the membrane scaffold protein X11L. Furthermore, they are involved in AD pathobiology and pathogenesis, with both Alcα and APP acting as cargo receptors for the kinesin-1 motor protein that plays a major role in fast anterograde axonal transport. Alcα-deficient mice were generated using a standard gene knockout method. In these mice, the coding sequence of exon 1 in the target vector was replaced with the LacZ-pA-PGK-Neo-pA cassette, thereby making it possible to evaluate the role of Alcα in endogenous APP metabolism. Evaluations of the brains in Alcα-deficient mice showed that, 1) there was significant enhancement of the amyloidogenic β-site cleavages of APP, thus leading to the augmented generation of Aβ peptides as well as Aβ-generating C-terminal fragments of APP (CTFβ), 2) there was progression of the AD pathology in the human APP-Tg mice with an Alcα-deficient background, and 3) there was significant attenuation of the association of APP with X11L that could lead to enhanced amyloidogenic cleavage of APP. In addition, we recently found significant RGC loss in Alcα-deficient adult mice (Nakano et al., 2020). We also found that elevated IOP was not associated with the observed RGC loss, being completely independent of changes (Nakano et al., 2020). Examinations of other retinal cells have shown that even though Alcα may be expressed in these cells in the retina, an Alcαdeficiency selectively affects the survival of the RGCs. In the experimental mice, starting at 3 months of age, there was a loss of RGCs observed. However, although the apparent ratio of the lost RGCs appeared to be rather constant (approximately 30%) in conjunction with the age, the numbers of eyes exhibiting ratios that were lower than the mean minus 2 standard deviations of the wild type mice appeared to increase in the older mice (Figure 1). The reason why the Alcα-deficient mice exhibited RGC loss remains unclear. The explanation for these findings could be due to the fact that some RGC populations are more susceptible to accumulated circumstantial stress and/or these changes only become evident due to changes in the age. We were able to affirm the importance of axonal transport with regard to the maintenance of RGCs in addition to also speculating that Alcα plays a role that is required for RGC survival. Thus, these findings suggest that this may be closely associated with NTG pathology. Overall, the results of this study suggest that this animal model could potentially be utilized as a tool for investigating the mechanisms of neurodegeneration in NTG, in addition to helping to develop treatments that can be used for IOP-independent RGC loss. Since Alcα is known to be involved in anterograde axonal transport, any Alcα deficiencies could potentially cause changes in the homeostatic regulation of axonal transport. This would additionally involve the retrograde transport, as both the motors and adaptors that are necessary for retrograde transport are required to be delivered to nerve terminals in order to ensure associated functions can continue. Thus, for neurons, including RGCs, to survive, target-derived neurotrophic factors are essential. As a result, in Alcα-deficient mice, any inefficient delivery of these trophic factors from the targets could very well lead to a loss of RGCs in these animals.

Figure 1 ｜The number of RGCs of knockout mice at 1.5, 3, 6 and 15 months after birth.

p3-Alcα and glaucoma:It is worth pointing out here that Alc and APP share the same function as the kinesin-1 cargo receptor and form a complex through the interaction with X11L, as previously mentioned. In addition, they are also processed with α- and γ-secretase in the same manner. Aβ has been reported to be a causative metabolic peptide of AD, and thus, is of great importance when diagnosing AD patients (Benilova et al., 2012). Since Aβ is progressively aggregation-prone, the metabolically stable nature of the peptide makes it possible to detect its quantitative or qualitative changes in the plasma. However, it turns out that the same aggregation-prone nature of the Aβ peptide along with the various aggregated soluble Aβ oligomers makes it difficult to determine the precise alterations of the Aβ levels in body fluids. Furthermore, APP generates another metabolite peptide called p3. However, p3 is metabolically labile and difficult to detect in both the cerebrospinal fluid (CSF) and in the plasma. Interestingly, as previously stated, it has been shown that Alcα is also processed by both α- and γ-secretase to generate a non-aggregation-prone p3-Alc peptide, while APP is processed by the α- and γ-secretase to generate the p3 peptide. As the p3 of APP differs, this means that the p3-Alc can be detected in CSF and plasma. In addition, it has also been shown that p3-Alc may very well reflect the pathological alterations in APP processing, which would also include dysfunction of the γ-secretase. In order to determine this γ-secretase dysfunction in sporadic AD patients, several previous studies have suggested that it might be informative and important to monitor the p3-Alcα C-terminal cleavage site alterations in the CSF, as this would directly reflect the γ-secretase functional alteration. Omori et al. (2014) used a newly established sandwich ELISA system with a C-terminal end-specific monoclonal antibody to analyze the levels of plasma p3-Alcα and reported finding there was an increase in the total plasma p3-Alcα levels in AD patients, when these patients were age-matched with subjects without dementia. When AD subjects were treated with acetylcholinesterase inhibitor (donepezil), there were significantly lower total plasma p3-Alcα levels observed as when compared to the AD patients without donepezil treatment (Omori et al., 2014). These results suggested that the increase in plasma p3-Alcα levels that were found in patients in whom AD was due to a progressing cognitive impairment in subjects with a γ-secretase malfunction, or due to a disorder of the clearance of peptides, might be due to a specific endophenotype in these individuals. The p3-Alcα level in body fluid, which includes plasma, could also reflect impaired axonal transport in the nervous system, as Alcα is efficiently cleaved en route to the cell surface (Maruta et al., 2012). Moreover, the attenuated axonal transport could also enhance the cleavage of Alcα and lead to the augmented generation of p3-Alcα. Furthermore, we recently evaluated Alcα (Nakano et al., 2020) and determined that both its expression in the mouse retina, which included RGCs, and the Alcα-deficiency, selectively affected the survival of the RGCs in the retina without any changes in the IOP elevation. Thus, it is possible that Alcα could play a very important role with regard to RGC survival, in addition to being closely associated with NTG pathology. Therefore, we hypothesize that there could be an increase in the plasma p3-Alcα levels in NTG patients. We are currently in the process of performing additional studies in order to confirm our hypothesis. In addition to the use of biomarkers such as p3-Alcα, neuroimaging techniques such as diffusion tensor imaging may also be useful for the detection and evaluation of glaucomatous damage in the optic nerve (Chang et al., 2014).In conclusion, significant evidence exists for the contribution of axonal transport to NTG, with the occurrence of axonal transport dysfunction leading to a worsening of the disease process. At the present time, however, there have been few studies that have examined the specifics of the axonal transport pathology with regard to the development of therapeutically relevant targets for drug design. It is possible that promising approaches for improving or supplementing current NTG therapeutic methodology could be developed if there was further incorporation of the axonal transport-targeted treatment into the present combinatorial therapies. It is our sincere hope that future research will more specifically examine the effects on the precise mechanism of abnormal axonal transport in NTG, as these findings could potentially serve as the basis for new treatments that would surely benefit and help to protect NTG patients.

Kazuyuki Hirooka*, Tohru Yamamoto, Yoshiaki Kiuchi

Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan (Hirooka K, Kiuchi Y)

Department of Molecular Neurobiology, Kagawa University Faculty of Medicine, Kagawa, Japan (Yamamoto T)

*Correspondence to:Kazuyuki Hirooka, MD, PhD, khirooka9@gmail.com.https://orcid.org/0000-0003-2977-124X(Kazuyuki Hirooka)

Received:July 5, 2020

Peer review started:July 8, 2020

Accepted:August 27, 2020

Published online:September 22, 2020

https://doi.org/10.4103/1673-5374.293145

How to cite this article:Hirooka K, Yamamoto T, Kiuchi Y (2021) Dysfunction of axonal transport in normal-tension glaucoma: a biomarker of disease progression and a potential therapeutic target. Neural Regen Res 16(3):506-507.

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Open peer reviewer:Xuesong Mi, Jinan University, China.

Additional file:Open peer review report 1.