Investigating degeneration of the retina in young and aged tau P301L mice

Life Sciences 124 (2015) 16–23

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Investigating degeneration of the retina in young and aged tau P301L mice Wing Lau Ho a,1, Yen Leung a,1, Sally Shuk Yee Cheng a, Carmen Ka Ming Lok a, Yuen-Shan Ho a,e, Larry Baum f, Xifei Yang g, Kin Chiu a,b,⁎, Raymond Chuen-Chung Chang a,c,d,⁎⁎ a

Laboratory of Neurodegenerative Diseases, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, China Department of Ophthalmology, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China d State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong, China e School of Nursing, Faculty of Health and Social Sciences, Hong Kong Polytechnic University, Hunghom, Kowloon, Hong Kong, China f School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China g Shenzhen Center of Disease Control and Prevention, Shenzhen, China b c

a r t i c l e

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Article history: Received 3 June 2014 Accepted 20 December 2014 Available online 12 January 2015 Keywords: Aging Neurodegeneration Retina Retinal ganglion cells Tau

a b s t r a c t Aims: Tau is a microtubule-binding protein facilitating the stability of the cytoskeleton. It is important for neurons as several neurodegenerative diseases involve hyperphosphorylation and aggregation of tau. It is known that mutated tau P301L results in aggregation of tau proteins, leading to neuronal loss in the brain. The aim of this study was to investigate the effect of tau mutation on the retina using a transgenic tau P301L mouse model. Main methods: Morphometric analysis was utilized to quantify the neurodegenerative changes, including the thickness of the inner nuclear layer (INL), and the density and size of retinal ganglion cells (RGCs). Sections of retina tissue stained by hematoxylin and eosin (H&E) and immunohistochemistry were analyzed. Comparisons were made between the tau P301L mice and control mice, as well as between different age groups. Key findings: A significant decrease in the thickness of the INL in tau P301L mice was found when compared with that of control mice. The effect was more pronounced in the peripheral area, and the effect increased with age. Regarding density of RGCs, tau P301L mice showed a similar age-related decline as in control mice. Furthermore, the RGCs from tau P301L mice increased in size with age, and the RGCs from control mice decreased in size with age. Significance: Tau may be an age-independent factor of accelerated neurodegeneration, with effects differing by types of neurons and regions of the retina. © 2015 Elsevier Inc. All rights reserved.

Introduction Gradual visual loss has been documented in Alzheimer's disease (AD) patients starting at the early stages of the disease, including difficulties in reading and finding objects [17,18,20], depth perception, perceiving structure from motion [6,18,20,22], color recognition [5,18], and impairment of spatial contrast sensitivity [6,8]. Previously, all these defects have been attributed to cortical pathologies; however, new studies have revealed changes within the retina itself. A slew of clinical evidence shows that AD patients also suffer from changes in the retina. Optical coherence tomography showed a decrease ⁎ Correspondence to: K. Chiu, Department of Ophthalmology, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong. ⁎⁎ Correspondence to: R.C.-C. Chang, Rm. L1-49, Department of Anatomy, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong. Tel.: +852 39179127; fax: +852 28170857. E-mail address: [email protected] (K. Chiu), [email protected] (R.C.-C. Chang). 1 These two authors have equal contribution to the study of this work.

http://dx.doi.org/10.1016/j.lfs.2014.12.019 0024-3205/© 2015 Elsevier Inc. All rights reserved.

in thickness of peripapillary retinal fiber layer (RFL) in patients with early AD when compared with age-matched controls [2,11,16,19,25, 26,32]. Visual field loss [2,18] and reduction of macular thickness have also been reported in AD, and the total volume of the macula was inversely correlated with the severity of the disease [16]. Reduction in thickness of RFL has been revealed by confocal scanning laser ophthalmoscopy, along with reductions in neuroretinal rim volume and area, and an increased cup–disk ratio; this suggests an overall reduction in the number of optic nerve fibers passing through the optic nerve head [7]. Furthermore, laser Doppler velocimetry has demonstrated that retinal blood-flow rate is reduced in AD patients [2]. Histological analysis of postmortem AD samples has shown prominent retinal defects, including axonal degeneration in optic nerves, reduced thickness of the RFL, and a significant reduction in the number of large-diameter retinal ganglion cells (RGCs) [12,27]. In fact, many changes of RGCs have been found through structural analysis of the retina, including pale cytoplasm with swollen mitochondria and endoplasmic reticulum, pale nuclei with dispersed chromatin at early stages,

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vacuolated cytoplasm, and clumped chromatin at late stages [3]. Furthermore, apoptosis of RGCs and astrocytosis have been observed in AD animal models [10]. Taken together, recent studies show that retinal degeneration is present in AD patients as well as in animal models of AD; and these retinal changes may be linked to the visual defects reported in AD patients. The underlying cause of retinal degeneration has been previously proposed to be due to β-amyloid pathology [10,24,29]. However, recently it has been suggested that tau pathology may also play a role in retinal degeneration in AD [13]. Tau has been shown to play a pivotal role in multiple neurological pathways [13], and it interacts with various signaling pathways such as those of microtubule stability and axonal transport. A recent study using double transgenic APP/PS1 mice found hyperphosphorylated tau in the retina, which correlated with increased formation of p35 and p25, subsequent aberrant activation of Cdk5/p25, and upregulation of calpain. Zhao and others concluded that the pathogenic mechanism of AD in retinal degeneration was triggered by accelerated tau pathology via calpain-mediated tau hyperphosphorylation [34]. However, there remains a lack of direct evidence regarding the effect of tau on retinal neurodegeneration. This study addresses this question using mice expressing tau P301L mutation with morphometric analysis of histological retina specimens. The three main aims of our study are as follows: (1) to validate the morphometric protocol; (2) to establish the natural course of histological changes in wild type mice; and (3) to compare the course of retinal neurodegeneration between wild type and transgenic mice. Determining these trends will lay the foundation for targeted therapeutic studies in the future. Materials and methods Animals Twenty-one mice in total were used in this study. Eleven were homozygous tau transgenic mice of mixed gender with a mutant form (P301L) of human tau protein including four-repeats without amino terminal inserts, and driven by the mouse prion promoter 6 (MoPrP) (Strain Tg(MAPT)JNPL3Hlmc). Ten age-compatible wild type mice of mixed gender served as controls. The mice originated from Taconic Laboratories, USA, and were housed in the Laboratory Animal Unit at the Chinese University of Hong Kong under a 12 h light:dark cycle. Food and water were provided ad libitum. All laboratory procedures were conducted according to the Statement for the Use of Animals in Ophthalmic and Vision Research by the Association for Research in Vision and Ophthalmology and were approved by The University of Hong Kong Committee on the Use of Live Animals in Teaching and Research. The transgenic mice consisted of five young mice (10 months) and six aged mice (30 months). The wild type control mice consisted of four young mice (10 months) and six aged mice (24 months). The sample size has been limited by the number of available mice. Histological slide preparation The mice were euthanized at the designated age as described above. They were anesthetized with an overdose of ketamine/xylazine and subsequently perfused with 4% paraformaldehyde in phosphate buffer (pH 7.4). Both eyes were enucleated and post-fixed in the same fixative for 60 min. Retinal sections 4 μm thick were cut using a microtome (2035 Biocut, Leica). Sections were mounted and stained with hematoxylin and eosin for morphometric analysis or with immunohistochemistry for tau expression in a masked manner. Immunohistochemistry Immunohistochemistry was performed as previously described [14]. Briefly, retinal sections were treated with 0.01 M citrate buffer/0.1%

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Tween 20 (pH 6.0) at 90 °C for 15 min after dewaxing and rehydration. Sections were then blocked with 10% normal goat serum before incubation in primary antibodies K9JA for total tau (Dako, Denmark) and tau phosphorylated at serines 396 and 404 (Invitrogen, USA) overnight at 4 °C. Sections were then incubated with secondary Alexa-Fluor 488 for 1 h at room temperature prior to staining with DAPI to stain for nuclei. Sections were mounted with an Anti-fade Gold mounting medium and then imaged using fluorescence microscopy (Olympus BX51, Japan). Morphometric analysis Retinal sections were imaged at 40 × magnification by brightfield microscopy (Olympus BX51, Japan). The sections were analyzed at 2 different regions, the central retina and the peripheral retina. The central retina was defined as the region one third along the length of the retina starting from the optic nerve outwards to the periphery. The peripheral retina was defined as the region two thirds along the same length (Fig. 1A). The protocol to measure thickness of the inner nuclear layer (INL), and the size and density of the RGC layer, has been described in detail previously [13]. Images were captured and all measurements were conducted using the software Stereo Investigator (MBF Bioscience — MicroBrightField, Inc.). In brief, the thickness of the INL was measured by averaging the length of 4 tangent lines that were drawn between the outer and inner boundaries of the INL and perpendicular to the outer boundary. For RGC size and density measurements, only cells with a comparably round shape having discernable nucleus and cytoplasm were selected. Condensed cells indicated dead cells; cells with long and irregular shape were identified as non-neuronal cells, which were excluded [13]. The linear density of RGCs was measured as follows. A section of the border of the inner retina was outlined and the length measured. The number of RGCs along the section was counted. The RGC density was calculated as the number of RGCs counted divided by the length of the section. The external border of each RGC was traced as a polygon, and the area was recorded as the cell size (Fig. 1B). Statistical analysis Statistical analysis was performed using SigmaPlot 12.0. Data between experimental groups were analyzed by one-way analysis of variance, with post hoc analysis using the Holm–Sidak test. Significance level was set at p b 0.05. All the results are expressed in terms of value ± SEM. Results Expression of tau in the retina To investigate the expression of tau in wild type and P301L transgenic mice, the retina was stained for total tau (K9JA) and tau phosphorylated at serines 396 and 404. There was a prominent increase in total and phosphorylated tau expression in transgenic mice compared to control (Fig. 2). Also, tau aggregates can been seen throughout the different layers of the retina, as indicated by the arrowheads (Fig. 2). Therefore, P301L transgenic mice do express tau pathology within the retina. Mean thickness of the inner nuclear layer The effect of aging on the retina was established by comparing young and aged wild type mice (Fig. 3). It was found that the mean central thickness of the INL was 46.8 ± 4.7 μm in young control mice and 36.5 ± 2.4 μm in aged control mice (Fig. 4A), with no statistically significant (p = 0.61) difference between the two groups. However, in the peripheral retina, a significant difference was found (p b 0.001). Compared with young control mice (28.8 ± 0.9 μm), the mean

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Fig. 1. A. Photograph of a complete section of a mouse retina stained with H&E at 10 × 40 magnification showing the 4 predefined regions for further morphometric analysis: A. upper peripheral region (2/3 of the total linear length of the upper part of the retina from the optic nerve head); B. upper central region (1/3 of the total linear length of the upper part of the retina from the optic nerve head); C. lower central region (1/3 of the total linear length of the lower part of the retina from the optic nerve head); D. lower peripheral region (2/3 of the total linear length of the lower part of the retina from the optic nerve head). B. An illustrated diagram showing how the measurements were made.

thickness of the INL in the peripheral retina decreased by 19% to 23.4 ± 0.6 μm in old control mice (Fig. 4B). The effect of age in mice expressing the tau P301L mutation on retinal degeneration was examined by H&E staining and quantification (Fig. 3). In the central retinal region, the mean thickness of the INL in young tau P301L mice was 34.1 ± 3.0 μm, significantly (p = 0.046) thicker than that in aged tau P301L mice (Fig. 4A). In the peripheral retina, the mean thickness of the INL in young tau P301L mice was 21.2 ± 0.9 μm, 26.6% thinner than that in young control mice (p b 0.01) (Fig. 4B). The mean thickness of the central INL in aged tau P301L mice was 24.7 ± 1.3 μm, which was 32.4% thinner (p = 0.02) than that in control mice (Fig. 4A). Finally, the mean thickness of the INL in the peripheral retina of aged tau P301L mice was 16.7 ± 0.8 μm, which was 28.5% thinner (p b 0.001) than that in control mice

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Fig. 2. Immunohistochemical analysis of the retina in control and transgenic P301L mice for total tau, tau phosphorylated at serine 396 (pS396 Tau) and tau phosphorylated at serine 404 (pS404 Tau). Transgenic mice displayed a prominent increase in tau expression with the presence of tau aggregation (as indicated by the arrows) compared to control.

(Fig. 4B). The results suggested that overexpression of human mutant tau resulted in neuronal loss in the INL of young or aged mice.

Mean density of retinal ganglion cells Young control mice were found to have a mean density of RGCs of 129.1 ± 9.5 cells/mm in the central retina, whereas the density of RGCs for aged control mice was 87.7 ± 9.4 cells/mm. The percentage decrease was 32.1%. The difference between the two groups was statistically significant (p = 0.04) (Figs. 3 & 5A). Similarly, the mean density of RGCs in the peripheral retina of aged control mice was 55.0 ± 6.1 cells/mm compared with 80.6 ± 2.9 cells/mm in young control mice, a statistically significant (p = 0.04) decrease of 37.3% (Fig. 5B).

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Fig. 3. H&E stained cross sections of the central and peripheral retina of (A&C) young and (B&D) aged control (left) and tau P301L (right) mice, respectively.

Young transgenic and control mice were compared (Fig. 3). The density of RGCs in the central retina was 128 ± 16 cells/mm for young tau P301L mice; the difference between the tau P301L mice and control mice was not statistically significant (Fig. 5A). Similarly, the density of RGCs in the peripheral retina of young tau P301L mice was 100 ± 12 cells/mm. The difference was not statistically significant between the young tau P301L mice and the control mice (p = 0.51) (Fig. 5B). Findings in the aged mice were similar. The mean density of RGCs at the central retina (Fig. 5A) was 78.9 ± 5.3 cells/mm for aged tau P301L mice. The difference between the tau P301L mice and control mice was not statistically significant (p = 0.78). In the peripheral retina (Fig. 5B), no significant difference was found regarding the density of RGCs (p = 0.73). The RGC density was 58.6 ± 6.7 cells/mm for aged tau P301L mice. Thus, overexpression of human mutant tau did not significantly affect the mean density of RGCs in either young or aged mice.

Mean size of retinal ganglion cells The mean size of RGCs at the central retina was 80.0 ± 5.0 μm2 for young control mice, 36.3% larger than that of the RGCs of aged control mice, which had a mean size of 58.7 ± 2.8 μm2 (Figs. 3 & 6A). This difference was significant (p b 0.01). On the other hand, in the periphery, the mean size of RGCs of the young control mice was 62.6 ± 4.5 μm2, and the mean size of RGCs of old control mice was 52.2 ± 1.5 μm2 (Figs. 3 & 6B). This 20.0% difference was not significant (p = 0.09). The mean size of RGCs in the central retina of young tau P301L mice was 38.5 ± 4.8 μm2, which was significantly smaller (51.9%, p b 0.001) than in young control mice (Fig. 6A). Similarly, the mean size of RGCs in young tau P301L mice was 36.1 ± 4.3 μm2 in the peripheral retina, which was significantly smaller (42.3%, p b 0.001) than that in young control mice (Fig. 6B). In the tau transgenic mice, RGCs increased in size with age. Compared to RGCs from young tau P301L mice, RGCs from aged tau P301L

mice were larger by 54.4% in the central retina (p b 0.01), and by approximately 60.6% in the peripheral retina (p b 0.001) (Fig. 6). When the size distribution of the RGCs was plotted, it was observed that RGCs in young tau P301L mice tended to be smaller than RGCs in young control mice (Figs. 3 & 7). This phenomenon was observed in both the central retina and the peripheral retina. Thus, overexpression of human P301L tau leads to a reduction in mean size of RGCs in both the central retina and the peripheral retina of young mice. When the cell size distribution patterns were plotted to compare young and aged mice, it was observed that the distribution patterns differed with age in both the tau P301L and the control mice, and the differences could be observed in both the central and peripheral areas (Fig. 8). Regarding the control group, RGCs in the central retina tended to be smaller in aged mice than those in young mice, suggesting that RGCs tended to decrease in size with increasing age. In the peripheral retina, RGCs in aged mice had a narrower range of sizes than RGCs in young mice. For tau P301L mice, RGCs tended to be larger in aged than those in young mice. This indicated that RGCs tended to increase in size with age. This phenomenon was observed in both the central retina and the peripheral retina. Discussion This study documented morphological changes in the retina of normal mice and tau P301L mice with aging. Changes in the thickness of the INL as well as size and density of RGCs were analyzed separately in the central retina and the peripheral retina, providing new insight to the differential effects of tau P301L mutation on different regions of the retina. Changes associated with normal aging were identified by comparing young and aged control mice. Regarding the thickness of the INL, only the peripheral retina showed a statistically significant reduction, by 19%. This can be compared to the 15% decrease in overall retinal thickness found in a study by Samuel and colleagues [28]. The researchers compared C57BL/6 mice of 3–5 months with those of 24–28 months and found a decrease in retinal thickness with age, while the ratio of

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Fig. 4. A. Graph comparing the mean thickness of the INL of the central retina of each experimental group. At a young age, the INL was thinner in P301L tau mice (p = 0.03) than in control mice. When these mice aged, the P301L mice also had a significantly (p = 0.02) thinner INL in the central retina. With aging, there was a significant (p = 0.05) decrease in thickness of the INL of P301L tau mice but not in control mice. Error bars represent the standard error of the mean. B. Graph comparing the mean thickness of the INL of the peripheral retina of each experimental group. Compared to control mice, P301L mice had a significantly (p b 0.01) thinner INL in the peripheral retina in both young and aged groups. Both showed a significant decrease in thickness of INL with aging. Error bars represent the standard error of mean.

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retina with age [28]. Moreover, the total neuronal number in the retina was found to be preserved in aged mice [28]. This suggests that the decrease in RGC density found in our study is a result of the increase in retinal area rather than neuronal loss. From the same study, RGC soma size was found to decrease by about 20% with age. This result may be the average decrease in RGC soma size over the whole retina, while our results suggest a region-dependent effect of RGC soma size reduction with age. Apart from establishing the normal changes in aging, our study also examined the effect of overexpressing human P301L mutant tau on retinal neurodegeneration. This was done by comparing the transgenic tau mice with control mice at each age time point. The expression and aggregation of both tau and phosphorylated tau was confirmed in the retina of P301L transgenic mice. Regarding the changes in thickness of the INL, tau mutation elicits a significant thinning effect only in the peripheral retina at a young age, while in aged tau mice, both the central

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thickness between layers remained the same [28]. In our study, we found a similar reduction in the thickness of INL in the peripheral retina, but no significant thinning was found in the central retina. This suggests that there is a differential reduction of thickness in different regions of the retina. For age-related changes of RGCs, the average density of RGCs was found to decrease with age by 32–37%, depending on the region of the retina. The size of RGCs decreased only in the central retina by 36%, but there was no significant change in the peripheral retina. In the C57BL/6 mice study mentioned above, RGC density changes were not reported, but there was a reduction of 13% in the dendritic field area [28]. It should be pointed out that thinning of the retina was accompanied by an increase in area, indicating a preservation of volume of the

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Fig. 5. A. Graph comparing the average density of RGCs of the central retina of control and tau P301L mice. Both groups showed significant decrease in average density of RGCs when they became aged. However, there was no significant difference between the control and tau P301L mice in either young or aged groups. Error bars represent the standard error of the mean. B. Graph comparing the average density of RGCs of the peripheral retina of control and tau P301L mice. Both groups showed significant decrease in average density of RGCs when they became aged. However, there was no significant difference between the control and tau P301L mice. Error bars represent the standard error of mean.

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Fig. 6. A. Graph comparing the average size of RGCs of the central retina of control and tau P301L groups. RGCs of young tau mice were significantly smaller than those in young control mice. However RGCs of tau and control mice behaved differently as they aged, becoming significantly (p b 0.01) larger in tau P301 mice, but significantly (p b 0.01) smaller in control mice. Error bars represent the standard error of the mean. B. Graph comparing the average size of RGCs of the peripheral retina of control and tau P301L groups. RGCs of young tau mice were significantly (p b 0.01) smaller than those in young control mice. However, RGCs became significantly (p b 0.01) larger when tau mice aged. In contrast to the central retina, the size of RGCs of control mice in the peripheral retina did not show any significant reduction in size with age. Error bars represent the standard error of mean.

retina and the peripheral retina showed reduction of thickness in the INL. Combined with the effects of normal aging, it can be observed that the thinning effect has a greater and earlier onset with the tau P301L mutation. It is important to note that there is a differential effect of tau P301L overexpression related to degeneration in different regions of the retina. The peripheral retina was significantly affected at a young age, but not the central retina. Possible mechanisms for this differential effect include difference in gene expression, existence of distinct subpopulations of neurons in the central retina and the peripheral retina, or difference in the microenvironment in the central retina and the peripheral retina in terms of physical stresses or availability of survival factors such as neurotrophins. It is known that this mutation also affects other parts

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of the central nervous system (CNS). For example, in the available strains of tau mutant P301L mice, JNPL3 mice exhibit changes predominantly in the spinal cord [21], while pR5 mice exhibit changes in the cortex and the spinal cord [9], and Tg P301L mice show degeneration mainly in the temporal lobe [23]. For P301S mutants, the hippocampus may be preferentially affected [1]. Linking the patterns observed in the CNS and the retina can be useful in extending our understanding of the neurodegenerative process. Our results also demonstrate other features of tau related to neurodegeneration in the retina. In terms of cell density, tau P301L and control mice showed no significant difference, while the age related changes in these two groups were similar. This indicates that the tau P301L mutation does not have an effect on neuronal survival. For changes in RGC size, it was found that young tau P301L mice had smaller RGCs than those of control mice. At old age, there was no significant difference between tau mutant and control mice. One possible explanation is that the tau P301L mutation initially induced stress in RGCs, causing them to shrink in size, but the mutant tau protein accumulated over time in surviving RGCs, causing the soma to swell. This may be due to changes in cellular transportation or autophagy, but immunohistochemical staining of proteins like APP, caspase, or TUNEL staining would be required to further explain the neuronal size changes in follow-up analyses. Combined with functional experiments such as electrophysiological studies, these findings would increase our understanding of the underlying mechanisms. One advantage of this study is that the central and peripheral regions of the retina were analyzed separately, so that the overall effect as well as differential effect of tau P301L overexpression-related neurodegeneration could be observed. Most previous studies investigated the process of neurodegeneration in the retina as a whole, but in fact the process may occur at different rates in the central and peripheral regions. Our design was useful in identifying the differences more precisely, as well as examining all the changes as a whole. There are several limitations of this study. First, the sample size was limited. Second, the limited number of time points meant that a more detailed time course of the neurodegenerative changes could not be established. Third, the morphological analysis was two dimensional, so that the overall volumetric variations could not be detected. Lastly, to examine the independent effects of tau on retinopathy, it would be worthwhile to examine retinal changes in conditional tau knockout mice. A recent review discussed retinal abnormalities and optic nerve pathologies possibly related to AD [19]. These retinal abnormalities included drusen, tau, amyloid precursor protein, and amyloid-associated proteins at the macula. Related optic nerve pathologies included loss of retinal nerve fiber layer thickness with depletion of RGCs and optic nerve axons, optic disk pallor, cupping and neuro-retinal rim thinning, retinal microvasculature changes, and low cerebrospinal fluid pressure causing high trans-lamina cribrosa pressure [30,31,33]. However, it was pointed out that these studies had limited sample sizes and could not rule out the possibility of co-existing ocular diseases. Nevertheless, these studies highlight the potential use of retinal studies as noninvasive modalities in examining pathologies in the brain [15]. Conclusion In summary, our results showed that the over-expression of P301L mutant human tau has an effect on retinal neurodegeneration. It accelerates age-related neurodegenerative changes. The effects are more profound in the peripheral retina, in terms of changes in INL thickness and RGC size. The process of neurodegeneration is different in terms of time course and magnitude in different layers and different areas of the retina, as observed from the pattern of change in the INL and ganglion cell layer. Findings from this study may have implications in understanding diseases with differential loss of neurons. These include intraocular

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Fig. 7. Graph displaying the size distributions of RGCs of the central retina and the peripheral retina, respectively, of each experimental group, with comparison made between control versus transgenic P301L mice. In both the central and peripheral regions of young mice, the size distributions of tau P301L mice were shifted to the left of the control group, indicating that RGCs in the tau P301L group tended to be smaller. In both the central and peripheral regions, the two aged groups have nearly identical distributions, indicating that P301L mutant tau had no effect on the size of RGCs in old age.

diseases like glaucoma or cortical disease such as frontotemporal dementia. Furthermore, it is of clinical relevance to discover the time sequence of histological changes of tau-related degeneration in the brain

versus the tau-related degeneration in the retina. Understanding a clear temporal relationship between cortical and retinal changes is important, as we can use the retinal changes observed as predictive 60

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Fig. 8. Graph displaying the size distributions of RGCs of the central retina and the peripheral retina, respectively, of each experimental group, with comparison made between young and aged mice. Compared to young mice, aged mice tend to have smaller RGCs within the central retina while having a narrower range of RGC sizes. Graph displaying the size distributions of RGCs of the central retina and the peripheral retina, respectively, of each experimental group, with comparison made between control versus transgenic P301L mice. Within the central retina and the peripheral retina of tau P301L mice, the distribution of RGCs shifted toward larger sizes with aging.

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indicators of changes in the brain, since the eye is more easily accessible from outside. A similar hypothesis has been repeatedly proposed [4,30, 31,33]. Therapeutic approaches may be better developed with more accurate markers of CNS changes. Conflict of interest The authors declare that there are no conflicts of interest.

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