Neuroprotection by WldS depends on retinal ganglion cell type and age in glaucoma

Background Early challenges to axonal physiology, active transport, and ultrastructure are endemic to age-related neurodegenerative disorders, including those affecting the optic nerve. Chief among these, glaucoma causes irreversible vision loss through sensitivity to intraocular pressure (IOP) that challenges retinal ganglion cell (RGC) axons, which comprise the optic nerve. Early RGC axonopathy includes distal to proximal progression that implicates a slow form of Wallerian degeneration. In multiple disease models, including inducible glaucoma, expression of the slow Wallerian degeneration (WldS) allele slows axon degeneration and confers protection to cell bodies. Methods Using an inducible model of glaucoma along with whole-cell patch clamp electrophysiology and morphological analysis, we tested if WldS also protects RGC light responses and dendrites and, if so, whether this protection depends upon RGC type. We induced glaucoma in young and aged mice to determine if neuroprotection by WldS on anterograde axonal transport and spatial contrast acuity depends on age. Results We found WldS protects dendritic morphology and light-evoked responses of RGCs that signal light onset (αON-Sustained) during IOP elevation. However, IOP elevation significantly reduces dendritic complexity and light responses of RGCs that respond to light offset (αOFF-Sustained) regardless of WldS. As expected, WldS preserves anterograde axon transport and spatial acuity in young adult mice, but its protection is significantly limited in aged mice. Conclusion The efficacy of WldS in conferring protection to neurons and their axons varies by cell type and diminishes with age.


Background
Age-related neurodegenerative disorders of the brain differ in etiology but often share similar important features [1,2]. Among these are early challenges to axonal physiology, active transport, and ultrastructure [1,3]. These pathological features are also endemic in diseases that affect the optic nerve, which conveys visual signals from the retina to central projection sites in the brain. Glaucomatous optic neuropathy (or glaucoma) is characterized by early axonopathy, including deficits in axonal anterograde transport from retina to central brain targets [4,5]. The disease causes vision loss through progressive degeneration of retinal ganglion cells (RGCs) and their axons, which comprise the optic nerve. The disease remains a prevalent neurodegenerative disorder and is the leading cause of irreversible blindness worldwide, with an estimated 100 million people afflicted by 2040 [6]. Sensitivity to intraocular pressure (IOP) is an associated risk factor for glaucoma, and higher IOP is linked to accelerated pathology [5,7,8]. Thus, current treatments focus on lowering IOP. However, regardless of hypotensive therapeutic intervention, many patients continue to lose vision [9]. Thus, like age-related diseases of the brain itself, therapeutics are needed that address neurodegeneration directly [10].
Abating axon dysfunction early in neurodegeneration can prevent subsequent stages of glaucomatous progression [2,3,11,12]. This is so as well in experimental models of glaucoma, which generally rely upon inducible or transgenic-related elevations in IOP [13,14]. In rodents, experimental interventions that slow or prevent loss of anterograde transport to RGC projection sites in the brain also impede axon degeneration in the optic nerve and cell body loss in the retina [5,[15][16][17]. Without intact transport, axons undergo Wallerian or Wallerian-like degeneration [1]. Expression of the Wld S (slow Wallerian degeneration) allele significantly delays axon degeneration in numerous disease models [18], though protection is dose-and possibly age-dependent [19,20]. In glaucoma models, Wld S preserves RGC axon structure, delays cell body loss, and preserves measures of gross retinal function [21][22][23][24][25]. The Wld S mutation creates a chimeric fusion protein containing 70 N-terminal amino acids of ubiquitination factor Ube4b linked to full-length nicotinamide mononucleotide adenylyltransferase 1 or Nmnat1, the enzyme responsible for synthesis of nicotinamide adenine dinucleotide or NAD, a cofactor involved in oxidative phosphorylation [26][27][28]. Glaucoma compromises RGC axon metabolism long before outright degeneration primarily through elevated oxidative stress and reduced mitochondrial efficiency [4,5,29]. Supplementation with nicotinamide, a NAD precursor, preserves RGC metabolism, anterograde axon transport and optic nerve integrity in the DBA/2 J mouse model of glaucoma [24,30].
Here we asked whether Wld S also confers protection to RGC dendritic arbors in the retina and, if so, whether this protection depends upon RGC type. In glaucoma, RGCs that depolarize to light onset (ON cells) or light offset (OFF cells) may show different susceptibility to IOP elevations [31][32][33]. Using our microbead occlusion paradigm [14,29,34], we elevated IOP (+ 33%) in Wld S mice and recorded light responses from a major class of ON and OFF RGCs, the α-Sustained type [32,33,35,36]. By comparing wild-type (WT) and Wld S RGC responses and morphology, we find that Wld S protects αON-Sustained light responses and dendritic morphology following IOP elevation for 1 month. However, Wld S αOFF-Sustained RGCs showed reduced light responses and dendritic complexity, similar to WT RGCs. Interestingly, while Wld S preserved anterograde axonal transport to the brain and spatial contrast acuity in young mice, its protection diminished with age.

Animals
The Vanderbilt University Institutional Animal Care and Use Committee approved all experimental procedures described below. We purchased 6 to 8-week-old and 1-year old male wild type C57BL/6 mice (WT) from the Charles River Laboratory (Wilmington, MA). Two breeding pairs of homozygous slow Wallerian degeneration mutant mice (Wld S ) on C57BL/6 background were kindly donated by Dr. Shu-Wei Sun from Loma Linda University [37]. We bred Wld S mice in the Vanderbilt University Medical Center Division of Animal Care. For our experiments we used 6 to 10-week-old and 1-year old male and female homozygous Wld S mice (see Genotype Confirmation). Mice were maintained on a 12-h light-dark cycle with standard rodent chow available ad libitum.

Genotype confirmation
We genotyped Wld S mice following the published protocol [38]. Briefly, we extracted genomic DNA from tails (DNeasy Blood & Tissue, Qiagen, Hilden, Germany). Because the Wld S mutation contains an in tandem triplication of a region intrinsic to the mouse genome, we determined the copy number of mutant Wld S alleles (QuantStudio 3 Real Time PCR, Applied Biosystems, Waltham, MA) using the following primers: Wld S forward: 5′-GGC AGT GAC GCT CAG AAA TTC-3′ and Wld S reverse: 5′-GTT CAC CAG GTG GAT GTT GCT-3′. β-tubulin forward: 5′-GCC AGA GTG GTG CAG GAA ATA-3′ β-tubulin reverse: 5′-TCA CCA CGT CCA GGA CAG AGT-3′. For each mouse, Wld S allele copy number was measured in triplicate, normalized to β-tubulin as endogenous control, and we quantified the copy number using the comparative ΔCT method [39]. We then normalized Wld S copy number relative to Wld S copy number measured in WT C57 mice obtained from Charles River Laboratory. As previously observed [38], Wld S copies formed a continuum. We defined animal genotype by Wld S copies using criteria established by [38]. Wld S null mice copy number ranged from 0 to 1.4 (Wld S−/− ), Wld S heterozygotes 1.4-2.5 (Wld S+/− ), and Wld S homozygotes ≥2.5 (Wld S+/+ , Fig. 1A).

Retinal ganglion cell dendritic morphology analysis
After imaging, micrographs of RGCs dendritic arbors were montaged and manually traced using Adobe Illustrator and Adobe Photoshop, respectively. We analyzed the skeletonized arbors by measuring the following parameters: dendritic field area, total dendritic length, dendritic intersections, and branch points. Dendritic field area was defined by outlining the distal dendritic tips and calculating the area of the resultant polygon. Total dendritic length was defined as the summation of all dendrites. The number of dendritic intersections was determined by Sholl analysis (ImageJ, 1.53c). Dendritic branch points were analyzed manually by counting the number of dendritic bifurcations [34].

Anterograde axonal transport
We anesthetized mice with 2.5% isoflurane and injected 1.5 μL of 1 μg/ μL solution of cholera toxin subunit B (CTB) conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR) into the vitreous of both eyes. Forty-eight hours later we perfused mice transcardially with PBS followed by 4% paraformaldehyde. Brains were removed and cryoprotected in 20% sucrose and coronal midbrain sections (50 μm) cut on a freezing sliding microtome.
We imaged alternating sections of the superior colliculus (SC) using a Nikon Ti Eclipse microscope (Nikon Instruments Inc., Melville, NY) and quantified the intensity of CTB signal (intact transport) using a custom ImagePro macro (Media Cybernetics, Bethesda, MD) as previously described [4]. We confirmed CTB uptake by RGCs in the retinas using an Olympus FV-1000 inverted confocal microscope.

Behavioral spatial acuity
We placed unrestrained mice on an elevated platform located in the center of an arena surrounded by four adjoining computer monitors (OptoMotry; Cerebral Mechanics Inc., Canada [45]). We measured spatial frequency thresholds by assessing the optomotor response to drifting sinusoidal gratings at 100% contrast. Grating spatial frequency was systemically adjusted based on the optomotor response noted by naïve experimenters. Mice were tested 3 times before microbead injection (baseline) and twice a week for 4 weeks after injection [29,43]. We analyzed spatial acuity data by calculating the difference in spatial acuity (Δ spatial acuity) of microbead and saline injected eyes.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistics were performed using Graphpad 8.0 (Graphpad Software, San Diego, CA). We first assessed data for outliers using Grubb's test. Then, we determined whether data best fit normal or lognormal distributions. We used the Shapiro-Wilk test for normality. If data best fit a lognormal distribution, datasets were transformed by computing the logarithm (base 10) [34,46]. For normal or transformed data, we performed parametric statistics. If datasets fit undetermined distributions or mix distributions (normal and lognormal), we performed non-parametric statistics. Statistical significance was defined as p ≤ 0.05.

Neuroprotection by Wld S is dependent on RGC type during glaucoma
We verified the genotype of each Wld S mouse by measuring the number of transgene copies relative to C57Bl/ 6 (WT) mice. These formed a continuum ranging from null (Wld S−/− , corresponding to WT) to 2.5 and above (Fig. 1A), which we defined as Wld S+/+ following previous studies [27,38]. For a subset of mice, we compared Wld S+/+ , Wld S+/− , and WT designation in whole retina by probing for both Wld S transcripts (318 bp and 478 bp) compared to GAPDH (71 bp; Fig. 1B). While naïve intraocular pressure (IOP) did not differ between WT left and right eyes nor between Wld S+/+ left and right eyes (p ≥ 0.17), we detected a modest difference in IOP between WT and Wld S+/+ naïve eyes (14.64 ± 0.13 vs. 14.1 ± 0.13 mmHg, respectively, p = 0.006). This small difference persisted in eyes receiving a saline injection as internal control (Fig. 1C) but was not significant when averaged over the one-month experimental period (p = 0.79, Fig. 1D). In contrast, an injection of microbeads into the anterior chamber following our established protocol elevated IOP in both WT (+ 32.6%, 19.64 ± 1.82 vs. 14.81 ± 1.5 mmHg) and Wld S+/+ (+ 36.8%, 19.48 ± 1.97 vs. 14.23 ± 1.61 mmHg) compared to saline-injected eyes (p < 0.001, Fig. 1D). Next, we determined if genotype or IOP elevation influenced RGC density by immunolabeling whole-mount retinas against RNA-binding protein with multiple splicing (RBPMS, [47], Fig. 1E). When quantified, we did not detect a significant difference in RBPMS+ RGCs regardless of genotype or IOP condition (WT saline: 3662. 15 Fig. 1F).
With a month of elevated IOP, αOFF-S RGCs in WT retina demonstrated less overall activity before, during and after light presentation (Fig. 7A), consistent with our previous work [29]. As before, the integrated response to light offset reduced significantly (− 20%, p = 0.011), while the mean and peak response to offset did not change (p ≥ 0.12, Fig. 7B). The RMP did not change with elevated IOP, in contrast to the more depolarized RMP of WT αON-S RGCs (p = 0.55, Fig. 4C). In contrast to αON-S RGCs, elevated IOP decreased the light response of αOFF-S RGCs from Wld S+/+ retinas (Fig.  7D), reducing the peak firing rate by 42% (p = 0.01, Fig.  7E). Thus, Wld S+/+ is insufficient for the protection of mechanisms driving light-evoked responses of αOFF-S RGCs following a month of IOP elevation Table 1.
Age-dependent influence of Wld S+/+ on axon transport and spatial acuity The protective capacity of Wld S depends on age, even though Wld S expression does not change with age [20,49,50]. Here, we tested how aging impacts Wld S+/+ protection of anterograde axon transport to the superior colliculus (SC) and spatial frequency threshold (i.e., spatial acuity) in the context of glaucoma. We first established a basis for testing the influence of aging on Wld S+/+ protective capacity in young mice (aged 6-8 weeks). A month of IOP elevation significantly degraded anterograde transport of cholera toxin subunit B (CTB) to the SC in WT mice (Fig. 8A), while transport from Wld S+/+ eyes remained largely intact (Fig. 8B). When quantified, elevated IOP reduced the percentage of intact transport to the SC by 36% compared to control in WT animals (p < 0.001, Fig. 8C), a significant difference compared to Wld S+/+ (p = 0.007, Fig. 8C). For WT mice, spatial acuity significantly diminished over the experimental period (R 2 = 0.80, p = 0.002), while acuity for Wld S+/+ did not change (R 2 = 0.18, p = 0.28) and remained better than WT over the course of IOP elevation (p ≤ 0.05, Fig. 8D). Our results indicate Wld S+/+ prevents significant loss of axon transport and spatial acuity following 1 month of IOP elevation in young mice.
Next, we tested the influence of age on the protective capacity of Wld S on anterograde axon transport and spatial acuity following IOP elevation. We again elevated IOP by unilateral microbead injection but using aged (1 year old) WT and Wld S+/+ animals (Fig. 9A). A single injection of microbeads similarly increased IOP in both WT (+ 29.6%, 19.45 ± 1.52 vs. 15.01 ± 1.28 mmHg) and Wld S eyes (+ 29.76, 19.23 ± 1.67 vs. 14.82 ± 1.37 mmHg). Four weeks of IOP elevation reduced intact anterograde axon transport in aged WT mice slightly more than it did for young, 41% vs. 36% (p = 0.74; Fig. 9B, D). Interestingly, aged Wld S+/+ animals also demonstrated degraded transport with elevated IOP compared to control 82.8 ± 9 spikes, p = 0.011), C but RMP of αOFF-S RGCs from Ctrl and microbead-injected eyes is similar (p = 0.55). D Mean light-evoked spike rate histograms of Wld S+/+ αOFF-S RGCs from Ctrl and microbead eyes. E IOP elevation significantly decreased the peak response to light offset in Wld S+/+ αOFF-S RGCs (− 42%, 40 ± 5.7 vs. 69 ± 7.5 spikes/s, p = 0.01), and modestly reduced the mean (p = 0.12) and integrated (p = 0.09) response to light offset. F RMP is similar for Wld S+/+ αOFF-S RGCs from Ctrl and microbead-injected eyes (p = 0.13). Statistics: Student's t-test (B, C, E, F); Mann-Whitney test (E, peak spike rate). Data = mean ± SEM Table 1 Descriptive Statistics for Dendritic Morphological and Physiological Measurements of αONand αOFF-S RGCs from Control and Microbead Eyes of WT and Wld S+/+ Animals. Values are expressed as mean ± SEM eyes (− 15%; p ≤ 0.03, Fig. 9C,D), though still retaining significantly more intact transport compared to either young or aged WT eyes challenged by elevated IOP (p ≤ 0.002). Aging also influenced spatial acuity. Elevated IOP induced a decrease not only in WT (p = 0.0064), but also in Wld S+/+ animals (p = 0.0058, Fig. 9E), which significantly exceeded the rate of reduction in younger Wld S+/+ animals (p < 0.001). The rate of degradation in acuity was similar between aged WT and Wld S+/+ mice (p = 0.35). Thus, aging reduces the neuroprotective benefit of Wld S+/+ not only on axonal transport but also throughout the optic projection.
Although Wld S+/+ provides extensive protection during glaucoma for dendritic arbors and mechanisms that drive RMP and the light-evoked response of αON-S RGCs, neuroprotection by Wld S+/+ is not robustly afforded to αOFF-S RGCs. We find IOP elevation significantly reduces the number of dendritic branch points of αOFF-S RGCs from WT and Wld S+/+ eyes (Fig. 6C,  F). Moreover, we find 1 month of IOP elevation decreases the integrated light-evoked response of WT αOFF-S RGCs (Fig. 7B) and the peak response of Wld S+/+ αOFF-S RGCs (Fig. 7E).
Based on these data, Wld S+/+ appears to protect αON-S RGCs but not αOFF-S cells. In support for this argument, evidence suggests OFF RGCs are more vulnerable to stress caused by IOP elevation [31][32][33]. Although this is a tantalizing conclusion, qualitative analysis reveals Wld S protein is expressed in the nuclei of most cells in the RGC layer of Wld S+/+ mice, indicating RGCs should Fig. 9 Aging Reduces the Neuroprotective Capacity of Wld S+/+ . A IOP of aged WT (circles) and Wld S+/+ (squares) mice before (day 0) and after (days ≥1) a single unilateral injection of microbeads (blue symbols) or equivalent volume of saline (beige symbols). Microbeads elevated IOP by 29.5% in aged WT (19.45 ± 1.5 vs. 15.01 ± 1.3 mmHg, p < 0.001) and 29.7% in Wld S+/+ eyes compared to saline-injected eyes (19.23 ± 1.6 vs. 14.82 ± 1.3 mmHg, p < 0.001). B, C Example coronal sections (top) of the SC after intravitreal injection of CTB-488 (green) into saline-and microbead eyes of aged WT (B) and Wld S+/+ mice (C). Transport deficits are indicated by dashed lines. Retinotopic maps (bottom) recreated from SC sections with optic disc indicated by white filled circles. Scale bar = 500 μm. D Quantification of anterograde axon transport to the SC. Data from young (1.5-2.5 months old) animals are replotted from Fig. 8C for comparison. We did not detect a difference in the percent of intact transport to the SCs corresponding to saline eyes of young vs. aged WT and Wld S+/+ animals (p = 0.60). For aged animals, IOP elevation decreased transport in WT (p < 0.001) and Wld S+/+ mice (p = 0.03) when compared to respective saline eyes. Axon transport of aged Wld S+/+ microbead-injected eyes was greater than that observed in SC of aged WT microbead eyes (p = 0.001). E Quantification of Δ spatial acuity. Data for young animals replotted from Fig. 8D for comparison. Spatial acuity linearly diminishes over the duration of IOP elevation in aged WT (R 2 = 0.74, p = 0.0064) and Wld S+/+ mice (R 2 = 0.81, p = 0.0058). Statistics: Student's t-test (A), Kruskal-Wallis test, Dunn's post hoc (D) Linear mixed model (E), Linear regression (E). Multiple comparison significance indicators: * Young WT vs. Young Wld S+/+ ; # Aged WT vs. Young Wld S+/+ ;Ŷ oung Wld S+/+ vs. Aged Wld S+/+ (E). Data presented = mean ± SEM be equally protected from stress by Wld S [52]. However, the magnitude of neuroprotection following acute axonal injury is correlated with Wld S expression levels in αand γ-motor neuron neurons [53]. Thus, the possibility exists that Wld S expression is dependent on RGC type.
The discrepancy between the extent of neuroprotection provided by Wld S to αONand αOFF-S RGCs is further highlighted by the fact the duration of Wld S neuroprotection is dependent on glaucoma model. Wld S neuroprotection lasts months in chronic DBA2/J glaucoma but is restricted to 2-4 weeks following IOP elevation by photocoagulation of the trabecular meshwork [22,52]. Therefore, it might be the case that both αONand αOFF-S RGC respond to Wld S during stress, but the duration of protection by Wld S is more transient for αOFF-S RGCs.
The duration of neuroprotection by Wld S may be shorter for αOFF-S RGCs due to their intrinsic physiological properties. αOFF-S RGCs intrinsically generate greater spontaneous activity compared to αON-S RGCs (Figs. 2 and 5B). Thus, αOFF-S RGCs may be more sensitive to stress due to the metabolic burden of persistent action potential generation and maintaining RMP. Cell excitability determines the magnitude of neuroprotection by Wld S after nerve sectioning. "Enhancing axon excitability by high-frequency stimulation or blunting excitability by blocking voltage-gated sodium channels with tetrodotoxin both accelerate axon degeneration following axotomy of neuromuscular junctions in Wld S mice" [54]. Considering these findings, αOFF-S RGCs may be more vulnerable to stress due to their intrinsically high spontaneous activity, which is further enhanced during early progression of glaucoma [29,34].
Neuroprotective capacity of Wld S is dependent on age during glaucoma Our second key finding is neuroprotection by Wld S on anterograde axonal transport and spatial acuity during glaucoma is dependent on age. As we have previously shown [29,43], IOP elevation significantly reduces anterograde axonal transport and spatial acuity in young adult WT mice (Fig. 8). Both anterograde axon transport and spatial acuity are protected during glaucoma in young adult Wld S+/+ mice (Fig. 8). Our data provide additional confirmatory evidence that Wld S+/+ provides neuroprotection for axon transport during glaucoma [22][23][24]. Furthermore, our results indicate if axonopathy caused by IOP elevation is reduced, as is the case for young adult Wld S+/+ animals (Fig. 8C), degradation of spatial acuity slows (Fig. 8D).
Although we find anterograde axonal transport and spatial acuity are preserved in young adult Wld S+/+ animals, neuroprotection by Wld S is reduced in aged, oneyear old, animals following IOP elevation (Fig. 9). Even so, Wld S still offers some protection on anterograde axon transport in aged mice relative to age-matched WTs (Fig. 9B-D). However, IOP elevation significantly reduces spatial acuity similarly for aged WT and Wld S+/+ mice (Fig. 9E).
Our finding that neuroprotection by Wld S+/+ depends on age is surprising because axon integrity and RGC pattern electroretinogram responses are preserved in 10 to 12 month old DBA2/J.Wld S mice [22]. However, when the age range is narrowed to include only 12 month old DBA2/J.Wld S mice, optic nerves possess more degenerating axons compared to age-matched DBA2/J.Wld S mice supplemented with nicotinamide, indicating neuroprotection by Wld S on axonopathy is reduced by advanced age in chronic DBA2/J.Wld S glaucoma [24]. These data suggest neuroprotection by Wld S falls off precipitously between 10 to 12 months of age in DBA2/J glaucoma. Following an inducible injury, the neuroprotective capacity of Wld S is also age dependent, even though Wld S expression levels do not change with age [20,49,50]. Taken together, the degree of neuroprotection by Wld S appears to be highly sensitive to age, limiting the utility of targeting only Wld S to block the progression of age-related neurodegenerative diseases [24,55,56].

Conclusions
We have demonstrated that Wld S+/+ does not equally protect all RGC types even in young adult animals during glaucoma and neuroprotection by Wld S+/+ on axonopathy is dependent on age. We find αOFF-S RGCs from Wld S+/+ retinas are similarly susceptible to dendritic degeneration and degradation of RGC signaling during glaucoma as their WT counterparts. While Wld S+/+ protects αON-S RGC dendrites and light-evoked activity. The influence of Wld S+/+ on anterograde axonal transport and spatial acuity is significantly blunted in aged animals. Although Wld S+/+ confers significant neuroprotection of anterograde axon transport and spatial acuity during axonopathy in otherwise uncompromised systems, this protection is limited at the level of different RGC types and is age dependent. Funding Support provided by a departmental unrestricted award by the Research to Prevent Blindness Inc., Research to Prevent Blindness Inc. Stein Innovation Award, the Stanley Cohen Innovation Fund, and National Institutes of Health grants EY017427, EY024997, and EY008126 to DJC. Imaging supported through the Vanderbilt University Medical Center Cell Imaging Shared Resource core facility and NIH grants CA68485, DK20593, DK58404, and DK59637.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate The Vanderbilt University Institutional Animal Care and Use Committee approved all experimental procedures described in this manuscript.