Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

Yukihiro Shiga; Aline Giselle Rangel Olguin; Sana El Hajji; Nicolas Belforte; Heberto Quintero; Florence Dotigny; Luis Alarcon-Martinez; Arjun Krishnaswamy; Adriana Di Polo

doi:10.1016/j.xcrm.2024.101839

. 2024 Nov 29;5(12):101839. doi: 10.1016/j.xcrm.2024.101839

Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

Yukihiro Shiga ^1,², Aline Giselle Rangel Olguin ³, Sana El Hajji ^1,², Nicolas Belforte ^1,², Heberto Quintero ^1,², Florence Dotigny ^1,², Luis Alarcon-Martinez ^1,^2,⁴, Arjun Krishnaswamy ³, Adriana Di Polo ^1,^2,^5,^∗

PMCID: PMC11722116 PMID: 39615485

Summary

Disruption of calcium (Ca²⁺) homeostasis in neurons is a hallmark of neurodegenerative diseases. Here, we investigate the mechanisms leading to Ca²⁺ dysregulation and ask whether altered Ca²⁺ dynamics impinge on neuronal stress and circuit dysfunction. Using two-photon microscopy, we show that ocular hypertension, a major risk factor in glaucoma, and optic nerve crush injury disrupt the capacity of retinal neurons to clear cytosolic Ca²⁺ leading to impaired light-evoked responses. Gene- and protein expression analysis reveal the loss of the sarco-endoplasmic reticulum (ER) Ca²⁺-ATPase2 pump (SERCA2/ATP2A2) in injured retinal neurons from mice and patients with primary open-angle glaucoma. Pharmacological activation or neuron-specific gene delivery of SERCA2 is sufficient to rescue single-cell Ca²⁺ dynamics and promote robust survival of damaged neurons. Furthermore, SERCA2 gene supplementation reduces ER stress, reestablishes circuit balance, and restores visual behaviors. Our findings reveal that enhancing the Ca²⁺ clearance capacity of vulnerable neurons alleviates organelle stress and promotes neurorecovery.

Keywords: retinal ganglion cell, glaucoma, traumatic optic neuropathy, calcium homeostasis, endoplasmic reticulum stress, organelle stress, in vivo imaging, neurodegeneration, sarco-endoplasmic reticulum (ER) calcium-ATPase2 pump (SERCA2/ATP2A2), gene therapy

Graphical abstract

Highlights

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Calcium (Ca²⁺) clearance capacity is compromised in injured retinal neurons
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Ocular hypertension induces SERCA2 downregulation and ER stress
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Increased SERCA2 activity restores Ca²⁺ homeostasis and mitigates ER stress
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SERCA2 gene augmentation promotes neuronal survival and restores visual function

Using two-photon microscopy, Shiga et al. show that impaired calcium clearance and ER stress are hallmarks of neuronal dysfunction in preclinical models of glaucoma and traumatic optic neuropathy. SERCA2 gene therapy restores calcium homeostasis, alleviates organelles stress, and promotes neurorecovery of vulnerable neurons.

Introduction

Neurodegenerative disorders share the common feature of progressive neuronal loss in specific regions of the central nervous system. For example, the selective death of retinal ganglion cells (RGCs) results in vision loss in glaucoma, the leading cause of irreversible blindness worldwide affecting over 80 million people globally.¹ There is no cure for glaucoma and current therapies rely solely on controlling elevated intraocular pressure, a major risk factor for developing the disease, albeit with limited success.² Despite decades of research, the factors that cause RGC demise and loss of vision in patients with glaucoma are poorly understood.³ Cumulative evidence indicates that RGC dysfunction precedes neuronal loss.⁴ Indeed, clinical studies using multiple outcome measures identified RGC dysfunction as an early pathophysiological event in patients with glaucoma.⁴ Prior to death, RGCs enter periods of aberrant activity associated with cellular stress.⁴^,⁵ A major roadblock toward developing effective therapeutics for glaucoma is that little is known about the early pathological alterations that lead to RGC dysfunction prior to cell death.

Calcium (Ca²⁺) plays an essential role in the transmission of depolarizing signals as well as synaptic activity, and is a key regulator of many cellular processes.⁶ Dynamic intracellular Ca²⁺ transients reflect electrical events in neurons; hence, Ca²⁺ imaging has emerged as a powerful tool to assess neuronal function in many systems and conditions.⁷^,⁸^,⁹ Ca²⁺ enters the soma through voltage-dependent Ca²⁺ plasma membrane channels, including L-, N-, and P/Q-type channels, as well as store-operated Ca²⁺ influx mechanisms.¹⁰ Given the crucial role of Ca²⁺ as a universal messenger, its baseline concentration within neurons must be maintained at low enough levels to allow for dynamic changes without an excessive energy cost. Indeed, increased resting cytosolic Ca²⁺ and spontaneous Ca²⁺ signals are conserved features of brain diseases.¹¹^,¹²^,¹³^,¹⁴^,¹⁵ Low cytosolic Ca²⁺ is achieved by the orchestrated action of mechanisms that pump Ca²⁺ into intracellular stores, notably the endoplasmic reticulum (ER) and mitochondria.¹⁶ The ER serves as one of the most important Ca²⁺ stores within neurons, where Ca²⁺ uptake is mediated by the sarco-ER Ca²⁺-ATPase2 (SERCA2) pump, an integral ER membrane protein.¹⁷ SERCA2 exists as two alternative splicing variants: SERCA2a is present in cardiac and skeletal muscle, and subsets of neurons; SERCA2b is found in many tissues including neurons¹⁸; and both are expressed by RGCs.¹⁹^,²⁰ However, the mechanisms that regulate Ca²⁺ homeostasis in RGCs and how they influence neuronal dysfunction in glaucoma are currently unknown.

To address this, we asked the following key questions: (1) are Ca²⁺ dynamics in RGCs disrupted early in the disease process? (2) if so, what are the molecular components that underlie RGC dysfunction? and (3) what strategy can effectively restore Ca²⁺ homeostasis and promote neurorecovery? We used two-photon microscopy to longitudinally monitor light-evoked Ca²⁺ responses in mice subjected to ocular hypertension (OHT) and optic nerve crush (ONC). Our data demonstrate that injured RGCs display substantially slower Ca²⁺ clearance, detected prior to RGC death and across all RGC subtypes, and was accompanied by a shift in ON-OFF predominance. Ca²⁺ clearance defects occurred concomitantly with ER stress and downregulation of SERCA2, a response validated in retinas from mice subjected to OHT or ONC, and from patients with primary open-angle glaucoma. Remarkably, adeno-associated virus (AAV)-mediated SERCA2 supplementation rescued light-triggered Ca²⁺ responses, reduced ER stress, promoted neuronal survival, and restored visual function. Together, our work identifies defective Ca²⁺ clearance as a signature feature of early RGC dysfunction and demonstrates that SERCA2 upregulation is an effective strategy to promote neurorecovery.

Results

Ca²⁺ clearance deficits precede overt neuronal death during glaucomatous stress

To visualize light-evoked Ca²⁺ responses, we used transgenic mice encoding the Ca²⁺ sensor GCaMP6f selectively in RGCs (Thy1-GCaMP6f).²¹ Expression of GCaMP6f in RGCs was confirmed in retinal flat mounts and cross-sections labeled with the cell-specific marker RBPMS (RNA-binding protein with multiple splicing)²² (Figures 1A and 1B). Amacrine cells in the ganglion cell layer, visualized with an antibody against the transcription factor Ap2α,²³ did not express GCaMP6f (Figure S1A). To mimic glaucomatous stress, Thy1-GCaMP6f mice received an intracameral injection of magnetic microbeads, which were attracted to the iridocorneal angle with a magnet to block aqueous humor outflow (Figure 1C). This procedure leads to a gradual increase in intraocular pressure and progressive RGC death²⁴ (Figures 1D and 1E). Ca²⁺ responses were evaluated at two weeks after microbead injection when increased intraocular pressure is stable but no significant RGC loss is detected²⁴^,²⁵ (Figure 1E). To record Ca²⁺ dynamics, we used two-photon laser scanning microscopy (TPLSM), which allows minimally invasive transscleral imaging with single-cell resolution in living mice²⁶^,²⁷^,²⁸ (Figure 1F). Mice were presented with a light stimulus, and longitudinal real-time recordings of Ca²⁺ transients in RGC soma were acquired. In human retinas, midget RGCs play a fundamental role in vision and account for >80% of all RGCs.²⁹ Recent evidence, including integrated single-cell transcriptomics of retinal atlases across species, identified mouse alpha RGCs (αRGCs) as the orthologs of human midget RGCs.³⁰ Thus, our in vivo analysis focused on ON-αRGCs, characterized by strongly labeled somata and large dendritic arbors expressing non-phosphorylated neurofilament heavy-chain protein.³¹ The identity of ON-αRGCs was confirmed by light stimulation as well as post hoc analysis of dendritic stratification in the proximal sublamina b (ON sublamina) (Figures S1B and S1C).

In sham uninjured retinas, GCaMP6f-positive RGCs elicited a brief burst of light-evoked Ca²⁺ increase followed by rapid signal decay to baseline (Figures 1G and 1I, Video S1). In contrast, RGCs from eyes subjected to OHT showed a marked increase in response decay (Figure 1H). We quantified this difference by fitting single exponential curves to the decreasing portion of each response and using the value tau (τ) as a measure of decay time constant (Figures 1I and 1J and Video S2). Quantification of Ca²⁺ dynamics confirmed that RGCs from glaucomatous eyes displayed substantially longer Ca²⁺ decay (longer time for signal to decay to baseline) relative to sham controls, whereas there were no statistically significant differences between the Ca²⁺ rise rate and signal amplitude among groups (Figures 1K–1M). To further establish whether such defects arise from increased eye pressure, we longitudinally recorded Ca²⁺ responses in the same neurons before and after OHT induction using TPLSM. We found that although light-evoked Ca²⁺ transients displayed normal dynamics prior to glaucoma induction, Ca²⁺ decay was substantially slower during OHT damage relative to sham controls (Figures 1N and 1O). No statistically significant differences were found in the Ca²⁺ rise rate and signal amplitude between both conditions (Figures 1P and 1Q). Taken together, these results suggest that Ca²⁺ clearance deficits are a feature of glaucoma-induced RGC dysfunction that precede neuronal death.

Video S1. Light-evoked Ca²⁺ responses of healthy RGC in living mice, related to Figure 1

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from sham control retina. The green horizontal bar indicates the light stimulus.

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Video S2. Early glaucomatous damage disrupts light-evoked RGC Ca²⁺ dynamics, related to Figure 1

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC at 2 weeks following glaucoma induction. The green horizontal bar indicates the light stimulus.

Download video file^{(6MB, mp4)}

Impaired Ca²⁺ clearance is conserved across RGC subtypes

To confirm our in vivo findings and to investigate whether OHT affected Ca²⁺ dynamics across RGC classes, we conducted a large-scale analysis of Ca²⁺ recordings in Thy1-GCaMP6 retinal explants to sample a wide number of neurons in all retinal locations.³² RGC Ca²⁺ signals were imaged at two weeks of glaucoma induction in response to full-field flashes, color stimuli, and bars moving in eight different directions using TPLSM (Figure 2A). Individual RGC responses corresponding to stimulus synchronization pulses were extracted with suite2P, aligned, and analyzed using a customized MATLAB script (Figure 2B). We performed principal component analysis and clustering of Ca²⁺ responses, which resulted in the identification of seven functionally distinct groups (Figure 2C). Among these, three groups were ON-RGCs characterized by increased Ca²⁺ signals at the onset of a moving bar stimulus, and four groups were ON-OFF RGCs displaying increased Ca²⁺ signal at both the onset and offset of moving bar stimuli (Figures 2D and S2A–S2G). Similar clusters have been identified in previous Ca²⁺ imaging studies⁹^,³³ except for OFF-RGCs, which were not detected in Thy1-GCaMP6 retinas.

Next, we examined whether RGC Ca²⁺ responses were altered at two weeks after OHT induction. ON-RGCs (clusters 1–3) exhibited a marked delay in Ca²⁺ signal decay relative to sham uninjured controls (Figures 2E and 2F), consistent with our in vivo imaging studies. Quantitative analysis confirmed a significant increase in the decay τ for responses in ON-RGCs in glaucomatous retinas as compared to their sham counterparts (Figure 2G). We saw small, but significant differences in the rise rate and peak amplitude of ON-RGC responses in glaucomatous retinas (Figures 2H and 2I). We next sought to characterize ON-OFF RGCs; however, we were unable to accurately measure the ON-decay and OFF-rise components of their light responses due to the speed at which the moving bar entered and exited the receptive field. Instead, we used their ON-rise and OFF-decay components to characterize their Ca²⁺ dynamics. Similar to ON-RGCs, ON-OFF RGCs (clusters 4–7) displayed a substantial delay in Ca²⁺ signal decay during OHT stress (Figures 2J–2L), while the Ca²⁺ rise rate remained unchanged (Figure 2M) and the amplitude decreased slightly (Figure 2N). Importantly, analysis of Ca²⁺ transient parameters in the entire RGC population sampled, which included all clusters (n = 1,119 RGCs), demonstrated a considerable increase in Ca²⁺ decay at two weeks of OHT (Figure 2O). Globally, there was no significant change in the Ca²⁺ rise rate (Figure 2P), and only a modest reduction in the peak amplitude response was detected (Figure 2Q). Lastly, we asked whether the overall preference for ON or OFF contrast changes was altered by high intraocular pressure. To do this, we computed the ON-OFF index³² for each RGC and compared histograms of these indices between OHT and sham conditions. These distributions were qualitatively similar yet showed a statistically significant reduction in OFF-dominated RGC responses (Figure 2R), consistent with previous findings.⁹ Collectively, our data indicate that impaired Ca²⁺ clearance across many RGC subtypes is altered during OHT stress.

OHT leads to ER stress and SERCA2 downregulation

Ca²⁺ homeostasis relies on the rapid redistribution of Ca²⁺ ions into subcellular organelles including the ER, which serves as the most important Ca²⁺ store in neurons.³⁴ ER stress is characterized by the activation of the unfolded protein response (UPR), which involves signaling by protein kinase RNA-like ER kinase (PERK), eukaryotic initiation factor 2α-subunit (eIF2α), and activating transcription factor 4 (ATF4).³⁵ Under persistent stress, ATF4 promotes the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which can mediate cell death.³⁶^,³⁷^,³⁸^,³⁹ To investigate whether RGC Ca²⁺ clearance deficits were associated with OHT-induced ER stress, we examined protein expression levels of phosphorylated (active) PERK and eIF2α (pPERK and peIF2α, respectively) as well as ATF4, and CHOP selectively in RGCs. Glaucomatous and sham retinas were collected, dissociated, fixed, permeabilized, and labeled with antibodies against UPR proteins and the RGC-specific marker RBPMS, and protein expression was quantified by flow cytometry (Figures S3A–S3E). Our data show that, after two weeks of OHT, RGCs displayed a marked increase in pPERK, peIF2α, ATF4, and CHOP relative to sham-operated controls (Figures 3A–3D).

The UPR pathway attempts to restore ER homeostasis by reducing protein translation and increasing the degradation of proteins at the ER membrane.⁴⁰ We reasoned that proteins involved in Ca²⁺ regulation by the ER, notably the SERCA2 Ca²⁺ pump, are likely to be affected by OHT-induced stress. First, we confirmed by immunohistochemistry that both SERCA2a and SERCA2b isoforms are abundantly expressed in naive mouse RGCs, as previously reported¹⁹^,²⁰ (Figures S3F and S3G). Quantitative reverse-transcription PCR (RT-PCR) analysis of mRNA extracted from whole retinas or fluorescence-activated cell (FAC)-sorted RGCs demonstrated that SERCA2 (ATP2A2) gene expression was markedly reduced during OHT (Figures 3E and 3F, and S3H–S3N). Flow cytometry protein expression profiling using an antibody that recognizes both SERCA2a and SERCA2b confirmed that SERCA2 protein substantially decreased in RGCs subjected to glaucomatous damage (Figures 3G and 3H). To validate these findings, we performed immunohistochemistry of mouse retinas using SERCA2a/b-specific antibodies and found significant SERCA2 downregulation in RGCs during OHT (Figures 3I–3K). Furthermore, we analyzed retinal sections from postmortem eye specimens from donor patients diagnosed with primary open-angle glaucoma (Table S1). We observed a substantial decrease of SERCA2 labeling in RGCs from individuals with glaucoma relative to age-matched controls (Figures 3L and 3M). Quantification of SERCA2-positive epifluorescence in individual RGCs confirmed decreased SERCA2 activity in patients with glaucoma (Figure 3N). Taken together, these data demonstrate signs of ER stress at the early stages of OHT-induced damage accompanied by SERCA2 downregulation in both glaucomatous mice and humans, suggesting that these responses are conserved across species.

SERCA2 downregulation is consistent with Ca²⁺ clearance deficits, and pharmacological activation of SERCA2 restores Ca²⁺ dynamics and promotes neuronal survival

To explore whether SERCA2 deficiency played a role in altered Ca²⁺ dynamics in glaucoma, we recorded light-evoked Ca²⁺ transients during pharmacological modulation of SERCA2 function in vivo. First, we performed loss-of-function experiments using cyclopiazonic acid (CPA), a specific inhibitor of SERCA.⁴¹ Single-RGC Ca²⁺ responses were longitudinally recorded before and after intravitreal injection of CPA or vehicle (PBS) in naive Thy1-GCaMP6 mice. Eyes that received CPA exhibited long Ca²⁺ decay dynamics (Figure 4A) that resembled responses observed in OHT (Figures 1 and 2). Quantification of Ca²⁺ dynamics confirmed that SERCA2 inhibition dramatically hindered Ca²⁺ clearance, whereas the rise rate and peak amplitude response did not change (Figures 4B–4D). Intravitreal administration of PBS did not alter any of the Ca²⁺ transient parameters measured (Figures 4E–4H) ruling out confounding effects stemming from the intraocular injection.

In complementary experiments, we investigated the effect of CDN1163, a small-molecule activator of SERCA.⁴² CDN1163 binds to the SERCA enzyme to activate Ca²⁺-ATPase activity via an allosteric mechanism.⁴²^,⁴³ We longitudinally recorded Ca²⁺ dynamics before and after CDN1163 injection at two weeks after OHT, when there are visible alterations in Ca²⁺ homeostasis. CDN1163 treatment effectively reduced Ca²⁺ decay time, consistent with faster Ca²⁺ clearance, in RGCs subjected to glaucoma (Figure 4I). Quantitative analysis confirmed that Ca²⁺ clearance was significantly improved by CDN1163, while there was no change in the Ca²⁺ rise rate and amplitude of the responses (Figures 4J–4L). Vehicle-treated control eyes showed no change in Ca²⁺ dynamics (Figures 4M–4P). Next, we asked whether improved Ca²⁺ dynamics with CDN1163 impacted RGC survival during OHT. For this purpose, a single intravitreal injection of CDN1163 was performed at two weeks after OHT induction and RGC density was quantified a week later (3 weeks OHT), a time when there is significant RGC loss in our glaucoma model to allow the assessment of neuroprotection.²⁴ CDN1163 promoted RGC survival and, strikingly, supported neuronal density to levels found in uninjured sham-operated control, whereas pronounced neuronal death was observed in vehicle-treated retinas (Figures 4Q–4T).

To determine whether the role of SERCA2 activation with CDN1163 was specific to OHT damage, we also examined its effect after ONC, a model of traumatic optic nerve injury that triggers selective and rapid RGC loss in an intraocular pressure-independent manner⁴⁴^,⁴⁵^,⁴⁶^,⁴⁷^,⁴⁸^,⁴⁹ (Figures 5A–5C). In this model, we observed a reduction of SERCA2 protein expression in RGCs (Figures 5D–5F) accompanied by reduced Ca²⁺ clearance at 4 days post lesion, prior to RGC death (Figures 5G–5K). Ca²⁺ dynamics were longitudinally recorded before and after intravitreal injection of CDN1163, which was administered 4 days after ONC. Similar to the glaucoma model, our data show that CDN1163 improved Ca²⁺ clearance (Figure 5L) whereas the rates of Ca²⁺ rise and amplitude were not statistically different among groups (Figures 5M–5O). Control eyes treated with vehicle did not display changes in Ca²⁺ dynamics (Figures 5P–5S). Importantly, a single injection of CDN1163 at 4 days post lesion promoted significant RGC survival at one week after ONC relative to vehicle-treated controls (Figures 5T–5W). Collectively, these data indicate that SERCA2 loss of function contributes to Ca²⁺ clearance deficits and that pharmacological SERCA activation restores Ca²⁺ homeostasis and promotes RGC survival in glaucoma and traumatic optic nerve injury.

SERCA2 activation restores Ca²⁺ dynamics and promotes RGC survival after traumatic optic nerve injury

(A and B) Schematic of the mouse optic nerve crush (ONC) model, which does not affect intraocular pressure (IOP) (N = 12 mice/group, two-way ANOVA with Sidak’s multiple comparison post hoc test, n.s.: not significant).

(C) Axonal injury leads to significant RGC loss starting at 1 week after ONC (sham: N = 5 mice/group; one-way ANOVA with Dunnett’s post hoc test, ∗∗∗p < 0.001, n.s.: not significant).

(D and E) Immunohistochemical analysis of retinal cross-sections with antibodies against SERCA2, RBPMS, and calreticulin confirms a significant reduction in SERCA2 expression in the ER of injured RGCs.

(F) Quantification of epifluorescence intensity in SERCA2-positive RGCs demonstrates a decrease in SERCA2 activity at 4 days post ONC relative to sham controls (sham: N = 3 mice, n = 51 RGCs; ONC: N = 4 mice, n = 66 RGCs; two-tailed Student’s t test, ∗∗∗p < 0.001).

(G) In naive (uninjured) RGC soma, light stimulation elicits a brief Ca²⁺ transient with fast signal decay. Exponential fits show the decay time constant (τ).

(H) Injured ON-RGCs from ONC eyes display slower Ca²⁺ responses characterized by increased signal decay time.

(I–K) Quantitative analysis of Ca²⁺ signal decay (I), rise rate (J), and amplitude (K) from sham and ONC retinas (sham: N = 6 mice, n = 55 RGCs; ONC: N = 6 mice, n = 38 RGCs; two-tailed Welch’s t test, ∗p < 0.05, n.s.: not significant).

(L) Representative traces of light-evoked RGC Ca²⁺ responses from ONC Thy1-GCaMP6f retinas before and after treatment with the SERCA activator CDN1163.

(M–O) Responses from the same neuron before and after treatment with CDN1163 show that SERCA activation rescues Ca²⁺ dynamics after ONC (N = 5 mice, n = 10 RGCs; Wilcoxon matched-pairs signed rank test, ∗p < 0.01, n.s.: not significant).

(P) Representative traces of light-evoked Ca²⁺ responses of single RGCs in Thy1-GCaMP6f retinas with ONC before and after PBS control treatment.

(Q–S) Longitudinal analysis of responses from the same injured RGC before and PBS administration shows no change in light-evoked Ca²⁺ dynamics (N = 5 mice, n = 11 RGCs; Wilcoxon matched-pairs signed rank test, n.s.: not significant).

(T–W) Representative images of RBPMS-labeled flat-mounted retinas from sham (T), and ONC eyes treated with PBS (U) or CDN1163 (V). (T) Quantification of RBPMS-positive RGCs shows that CDN1163 promotes RGC neuroprotection at 1 week of ONC (N = 5 mice/group, one-way ANOVA with Tukey’s multiple comparisons post hoc test, ∗∗∗p < 0.001). Data are presented as mean values ± SEM. The cartoon in this figure was generated with BioRender (https://biorender.com).

SERCA2 gene supplementation rescues Ca²⁺ dynamics during OHT- and ONC-induced stress

To rule out possible pan-retinal or off-target effects of pharmacological SERCA modulation, we sought to increase SERCA2 function selectively in RGCs using a serotype 2 AAV (AAV2) encoding mCherry-tagged murine SERCA2 under the control of the CAMKII promoter (AAV.SERCA2). Thy1-GCaMP6 mice received a single intravitreal injection of AAV.SERCA2, and RGC-specific SERCA2 expression was confirmed by co-localization of mCherry and RBPMS in retinal flat mounts and cross-sections (Figures 6A and 6B). Quantification of the number of RGCs transfected by AAV.SERCA2 showed that ∼50% of all RBPMS-positive RGCs co-expressed AAV-mediated SERCA2 (Figure 6C), and this level of expression was sustained for over 1 year after AAV.SERCA2 delivery (Figure S4A). To further confirm that AAV increased SERCA2 expression above intrinsic levels in RGCs, we carried out flow cytometry to quantify SERCA2 protein in RGCs co-labeled with mCherry and RBPMS (Figures S4B and S4C). We found a significant increase in AAV-mediated SERCA2 relative to a control virus carrying only mCherry (AAV.Ctl) (Figure 6D).

Next, we investigated whether RGCs transfected by AAV.SERCA2 showed an improvement in light-evoked Ca²⁺ dynamics using TPLSM live imaging. AAV.SERCA2 or AAV.Ctl was injected into Thy1-GCaMP6 mice one week prior to OHT induction, and TPLSM-based Ca²⁺ imaging was performed two weeks after OHT induction (total OHT exposure: 2 weeks). Our analysis focused on RGCs transfected with AAV.SERCA2 that also expressed GCaMP6f, which were readily identified by TPLSM (Figure S4D). Glaucomatous eyes that received control AAV.Ctl showed a substantial delay in Ca²⁺ clearance compared to the rapid signal decay observed in sham-operated eyes (Figures 6E and 6F, 6H–6I and Video S3). In contrast, AAV.SERCA2 markedly reduced Ca²⁺ decay time in RGCs from glaucomatous eyes, indicative of faster responses and increased Ca²⁺ clearance capacity (Figures 6G–6J and Video S4). Quantification of Ca²⁺ transient parameters confirmed that AAV.SERCA2 reduced Ca²⁺ decay time to levels like those found in sham-operated mice, while control AAV.Ctl had no effect (Figure 6K). The rise rate and peak amplitude of the responses did not change across groups (Figures 6L and 6M).

Video S3. Light-evoked RGC Ca²⁺ responses in glaucomatous retina treated with control AAV.Ctl

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from glaucomatous retinas treated with AAV.Ctl. The green horizontal bar indicates the light stimulus.

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Video S4. AAV.SERCA2 treatment enhances light-evoked Ca²⁺ dynamics in injured RGCs, related to Figure 5

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from glaucomatous retinas treated with AAV.SERCA2. The green horizontal bar indicates the light stimulus.

Download video file^{(1.6MB, mp4)}

To validate these findings, we examined the global effect of AAV.SERCA2 on light-evoked Ca²⁺ changes in all RGC clusters identified in retinal explants. Consistent with our in vivo observations, SERCA2 gene augmentation increased the Ca²⁺ clearance rate in RGCs from glaucomatous eyes, while there was no effect in neurons treated with control AAV.Ctl (Figures 6N–6P). Notably, quantification of Ca²⁺ transient parameters confirmed that AAV.SERCA2 restored Ca²⁺ clearance to values like those found in uninjured sham eyes, while AAV.Ctl had no effect (Figure 6Q). The rise rate and the peak amplitude of the responses did not change between glaucomatous eyes treated with AAV.SERCA2 or AAV.Ctl (Figures 6R and 6S). In addition, we examined whether AAV-mediated SERCA2 supplementation impacted ON-OFF responses in OHT. AAV.SERCA2 restored the balance of ON-OFF predominance to levels found in non-injured, sham-operated control eyes, while AAV.Ctl had no effect (Figures 6T and 6U). Lastly, we investigated the effect of AAV.SERCA2 administration in the ONC model. AAV.SERCA2 or AAV.Ctl was injected into Thy1-GCaMP6 mice one week prior to optic nerve injury, and TPLSM-based Ca²⁺ imaging was performed one week post lesion. Our data show that, similar to OHT, AAV.SERCA2 shortened Ca²⁺ decay time in RGCs from ONC-injured eyes relative to AAV.Ctl (Figures 6V–6Y). There were no statistically significant differences between the Ca²⁺ rise rate and signal amplitude across groups (Figures 6Z and 6AA). We conclude that SERCA2 supplementation is sufficient to improve Ca²⁺ decay rates by enhancing Ca²⁺ clearance, which positively impacts RGC function during glaucomatous or traumatic stress.

SERCA2 gene therapy attenuates ER stress, promotes neuronal survival, and prevents loss of visual function

To investigate whether SERCA2 gene transfer influenced ER stress, we examined pPERK, peIF2α, ATF4, and CHOP protein levels in RGCs from eyes injected with AAV.SERCA2 or AAV.Ctl during OHT damage. Analysis of flow cytometry data revealed that SERCA2 supplementation substantially reduced pPERK, peIF2α, ATF4, and CHOP protein expression in RGCs relative to control eyes (Figures 7A–7D), suggesting that enhancing SERCA2 activity reduced ER stress in these neurons. Next, we evaluated the effect of SERCA2 gene therapy on neuronal survival by quantifying RGC density after intravitreal injection of AAV.SERCA2 and AAV.Ctl at three and four weeks of OHT to assess neuroprotection. AAV.SERCA2 promoted RGC survival and, strikingly, preserved neuronal density to levels found in non-injured, sham-operated control eyes (100% survival), while substantial RGC death was observed in control retinas treated with AAV.Ctl (Figures 7E–7H). In the ONC model, we evaluated the effect of SERCA2 gene therapy at one and two weeks after the lesion (Figures 7I–7L). We found that AAV.SERCA2 conferred robust neuroprotection promoting the survival of 60% of all RGCs at two weeks post injury relative to only 18% of neurons that remained in AAV.Ctl-treated eyes (Figures 7I–7L).

To assess retina-brain connectivity, we measured the optomotor reflex response, a visual behavior characterized by direction-selective head movements used for image stabilization triggered by RGC inputs.⁵⁰ Mice from all groups were subjected to longitudinal evaluation of the stereotyped behavior evoked by grating light stimuli (Figure 7M), and visual acuity was calculated. Visual acuity decreased in eyes with high intraocular pressure in a time-dependent manner, becoming significantly lower than sham controls starting at three weeks after OHT induction (Figure 7N); therefore, we evaluated the effect of SERCA2 gene transfer at three and four weeks of glaucomatous damage. Our data show that AAV.SERCA2 treatment markedly improved visual acuity in mice with glaucoma relative to AAV.Ctl-treated eyes at three and four weeks of glaucoma induction (Figure 7O). Together, these results reveal significant beneficial effects of SERCA2 gene augmentation that attenuate ER stress, promote neuronal survival, re-establish circuit connectivity, and prevent loss of visual function.

Discussion

The precise balance of Ca²⁺ influx and efflux plays a vital role in regulating RGC excitation and inhibition to accurately convey visual information from the retina to the brain. Maintaining cytosolic Ca²⁺ within neurons at extremely low levels is required for dynamic signaling and the proper function of neuronal circuits. Indeed, Ca²⁺ signaling is fast and efficient due to the steep Ca²⁺ concentration gradient, which can be as large as 10⁵-fold, between the extracellular and intracellular spaces.⁵¹ This gradient is also conserved between different organelles and the cytosol, which facilitates a wide range of Ca²⁺-specific signaling events.⁵¹ An abnormal rise in intracellular Ca²⁺ can alter synaptic function, increase reactive oxygen species, disrupt energy metabolism, and activate apoptotic pathways.⁵²^,⁵³^,⁵⁴ How is this finely tuned intracellular Ca²⁺ homeostasis affected during glaucomatous RGC neurodegeneration? Our study reveals that OHT leads to ER stress and downregulation of SERCA2, which disrupts cytosolic Ca²⁺ clearance leading to aberrant neuronal function and RGC death. Remarkably, SERCA2 gene augmentation was sufficient to enhance Ca²⁺ clearance capacity, attenuate ER stress, rescue light-evoked Ca²⁺ signal responses, promote RGC survival, and prevent loss of visual function.

Ca²⁺ clearance is compromised in injured RGCs

A key finding stemming from our two in vivo models of RGC damage, OHT and ONC, as well as ex vivo experiments is that the capacity of RGCs to clear cytosolic Ca²⁺ is compromised at the early stages of neurodegeneration. In our study, deficits in Ca²⁺ clearance were observed across all RGC subgroups identified in Thy1-GCaMP6f mice including ON and ON-OFF RGCs. An elegant study by Li and colleagues, conducting non-invasive Ca²⁺ retinal imaging in a silicon oil-based model of experimental glaucoma, reported the presence of nine clusters including four OFF RGC subgroups.⁹ There are differences between this study and ours that could account for these findings. For example, they used an AAV encoding the Ca²⁺ sensor jGCaMP7s rather than Thy1-GCaMP6f mice used here, which may lead to differences in the pattern of GCaMP expression. We could not assess OFF RGC responses in the Thy1-GCaMP6f mice because this neuronal subtype is not well labeled in this mouse strain. In addition, Li et al. noted differences in the responses of the same RGC subtype in different retinal regions.⁹ We routinely imaged from the dorsotemporal quadrant of the retina because the GCaMP6f expression pattern in RGCs in the Thy1-GCaMP6f mouse line is most abundant in this region. Both studies report changes in RGC Ca²⁺ responses preceding cell death and found that some RGC subtypes shift their Ca²⁺ response to light after injury. Overall, the reduced capacity of RGCs to maintain low concentrations of cytosolic Ca²⁺ is predicted to activate stress and apoptotic pathways increasing the vulnerability of damaged neurons. Taken together, these findings strongly suggest that compromised Ca²⁺ clearance is a conserved feature of RGC dysfunction that may prime these neurons for neurodegeneration.

ER stress and loss of SERCA2 function are key features of glaucomatous neurodegeneration

Recent studies suggest a close association between ER stress and neuronal pathology in cellular and animal models as well as in patients affected by neurodegenerative conditions.¹⁷ The unique cellular structure of RGCs coupled with their high energy demands makes these neurons susceptible to ER stress.⁵⁵^,⁵⁶ We demonstrate that, early after OHT induction and prior to overt neuronal loss, there is activation of the UPR response featuring increased levels of pPERK, peIF2α, ATF4, and CHOP selectively in RGCs (Figures 3A–3D). This is consistent with previous studies showing ER stress in RGCs after traumatic optic nerve injury, high intraocular pressure, and optic neuritis.⁵⁷^,⁵⁸^,⁵⁹ Moreover, CHOP gene deletion or small-molecule CHOP inhibitors protected RGCs in models of optic neuropathies,⁵⁷^,⁵⁸^,⁵⁹^,⁶⁰^,⁶¹ supporting the role of ER stress in RGC death.

The relationship between ER stress and Ca²⁺ handling in injured RGCs, however, has not been explored. Based on the critical role of the ER as a major Ca²⁺ storage in neurons¹⁶ and our finding that Ca²⁺ clearance is defective in glaucoma, we put forward the hypothesis that the mechanisms responsible for Ca²⁺ uptake by the ER were compromised early in the pathology. We focused on SERCA2 because of its high affinity for Ca²⁺ and enhanced capacity to restore cytosolic Ca²⁺ concentrations to resting levels in neurons.⁶² In addition, SERCA2 is highly conserved and abundantly expressed by RGCs across species.¹⁹^,²⁰ We show that SERCA2 protein is downregulated in RGCs from mice subjected to OHT and ONC as well as in patients with glaucoma (Figures 3I–3N, 5D–5F), suggesting that loss of SERCA2 function disrupts Ca²⁺ clearance by the ER in this disease. SERCA2 dysregulation has been implicated in several disorders including schizophrenia, Alzheimer’s disease, and cerebral ischemia.⁶² For example, studies in mammalian cell lines and X. laevis oocytes showed that mutations in the presenilin (PS) gene decreased SERCA2 activity and that modulating SERCA function altered amyloid β production.⁶³ Additional in vitro studies showed that familial PS mutations resulted in reduced SERCA2 activity leading to intracellular Ca²⁺ dysregulation.⁶⁴ SERCAs are ATPases that transport two Ca²⁺ ions from the cytoplasm to the ER lumen per ATP molecule hydrolyzed.⁶⁵ RGCs undergo metabolic stress in glaucoma characterized by low levels of ATP and nicotinamide adenine dinucleotide as well as adenosine monophosphate kinase hyperactivity.²⁵^,²⁸^,⁶⁶^,⁶⁷ Thus, in addition to SERCA2 downregulation in glaucomatous RGCs, it is possible that energy deficits contribute to the loss of SERCA2 function in these neurons.

Selective SERCA2 activation promotes RGC survival and restores visual function

The ultimate goal of therapies for neurodegenerative conditions is to enhance neuronal viability and restore circuit function.⁶⁸^,⁶⁹ Our data demonstrate that strategies that enhance SERCA2 activity during pressure-related stress or traumatic optic nerve injury have a major beneficial effect on RGC survival and function. Both pharmacological SERCA2 activation and SERCA2 (ATP2A2) gene augmentation fully recovered light-evoked single-RGC Ca²⁺ dynamics, notably Ca²⁺ clearance capacity, while exerting a potent and lasting neuroprotective effect. Although the transfection rate with AAV.SERCA2 was 50% (Figure 5C), RGC survival during OHT was similar to that in sham uninjured retinas (100% survival). This finding suggests that improving Ca²⁺ homeostasis in targeted RGCs also exerts a beneficial effect on neighboring, non-targeted neurons. Of interest, a positive neuroprotective bystander effect was observed in zebrafish motoneurons after spinal cord injury, a response that was attributed to enhanced gap junction coupling between neurons that increased Ca²⁺ homeostasis.⁷⁰

We show that AAV-mediated SERCA2 gene transfer returned ER stress markers to pre-injury levels. The maintenance of functional Ca²⁺ stores in the ER is essential for this organelle to fulfill its functions, and depletion of Ca²⁺ from the ER has been associated with neuronal pathology.⁷¹^,⁷² Therefore, our data suggest that, in addition to reducing neuronal cytosolic Ca²⁺ levels, AAV.SERCA2 enhances Ca²⁺ uptake into the ER hence alleviating organelle stress. In addition, we found a modest shift in RGC contrast preferences toward ON responses during glaucomatous damage. These changes suggest increased vulnerability of RGCs with dendritic stratification in the OFF sublayer, which may be caused by heightened organelle stress in this neuronal subtype. This is consistent with previous studies showing more pronounced histological and functional defects in OFF and ON-OFF RGCs, characterized by a reduction in RGC dendritic area or length in the OFF sublamina in mouse glaucoma models.⁹^,⁷³^,⁷⁴^,⁷⁵^,⁷⁶^,⁷⁷ In our study, AAV.SERCA2 shifted ON-OFF preference to normal values, suggesting that Ca²⁺ homeostasis plays a critical role in maintaining RGC synaptic connectivity and circuit balance. Remarkably, AAV.SERCA2-treated eyes also displayed a significant recovery in retinal function and visual acuity, evidenced by improved optomotor responses. Taken together, our findings underscore the importance of SERCA2 dysfunction during RGC neurodegeneration and provide clear evidence that restoring Ca²⁺ balance in these neurons is a valuable strategy to enhance visual function in glaucoma and traumatic optic neuropathies.

Limitations of the study

Despite robust RGC neuroprotection with AAV.SERCA2, we observed partial recovery of visual function. The reason behind this finding is not clear, but may reflect the intrinsic nature of the optomotor response test used here. The optokinetic reflex occurs when ON direction-selective RGCs (oDSGCs) detect slow, global image motions on the retina.⁷⁸ Hence, this assay captures only the response of oDSGCs and, as such, it is possible that these neurons had lower AAV.SERCA2 transfection rates or responded less efficiently to SERCA2 activation resulting in partial functional recovery warranting additional investigation. In addition, we cannot exclude that other Ca²⁺ efflux pathways, from sources in mitochondria and cell membranes,⁶ might also play a role in Ca²⁺ clearance defects in the context of glaucoma and traumatic optic neuropathy. Thus, exploring the contribution and therapeutic potential of alternative Ca²⁺ efflux pathways in future studies would be useful. From a translational perspective, developing strategies that selectively target neurons and restore Ca²⁺ homeostasis is likely to be widely applied to boost neuronal health in neurodegenerative diseases with relatively few harmful side effects.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Adriana Di Polo (adriana.di.polo@umontreal.ca).

Materials availability

All non-commercial reagents or mouse lines used in this paper are available from the lead contact upon request.

Data and code availability

•
All data reported in this paper will be shared by the lead contact upon request.
•
All original code used in this paper is available from the lead contact upon request.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

We thank Dominique Gauchat and Philippe St-Onge (Cytometry platform) and Aurélie Cleret-Buhot (Imaging platform) at the University of Montreal Hospital Research Center for technical support. This work was funded by grants from the Alcon Research Institute (A.D.P.), Canadian Institutes of Health Research (CIHR) (A.D.P. and A.K.), and the Natural Sciences and Engineering Council of Canada (A.K.). A.D.P. holds a Canada Research Chair (Tier 1), and A.K. holds a Canada Research Chair (Tier 2). Y.S. was supported by postdoctoral fellowships from the Fonds de Recherche du Québec - Santé (FRQS), CIHR, and the Uehara Memorial Foundation. A.G.R.O. was supported by a graduate scholarship from FRQS. H.Q. was supported by a postdoctoral fellowship from the Mexican National Council of Science and Technology (CONACYT).

Author contributions

Conceptualization, Y.S., A.K., and A.D.P.; methodology, Y.S., A.G.R.O., S.E.H., N.B., and L.A.-M.; investigation/experimentation, Y.S., A.G.R.O., S.E.H., N.B., H.Q., and F.D.; data analyses, Y.S., A.G.R.O., S.E.H., A.K., and A.D.P.; writing – original draft, Y.S., A.K., and A.D.P.; writing – review and editing, Y.S., A.G.R.O., S.E.H., N.B., H.Q., F.D., L.A.-M., A.K., and A.D.P.; visualization, Y.S., A.G.R.O., and S.E.H.; supervision, A.D.P.; funding acquisition, A.D.P.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Guinea Pig polyclonal anti-RBPMS	PhosphoSolutions	Cat# 1832-RBPMS; RRID: AB_2492226
Rabbit polyclonal anti-GFP	Invitrogen	Cat# A11122; RRID: AB_221569
Mouse monoclonal anti-AP2α	Developmental Studies Hybridoma Bank	Cat# 3b5; RRID: AB_2313947
Mouse monoclonal non-phosphorylated anti-Neurofilament H	BioLegend	Cat# 801701; RRID: AB_2564642
Goat polyclonal anti-Choline Acetyltransferase	Millipore	Cat# AB144P; RRID: AB_2079751
Mouse monoclonal anti-SERCA2 ATPase (IID8)	Invitrogen	Cat# MA3-910; RRID: AB_2227681
Mouse monoclonal anti-SERCA2 ATPase (2A7-A1)	Invitrogen	Cat# MA3-919; RRID: AB_325502
Rabbit polyclonal anti-Calreticulin	Invitrogen	Cat# PA3-900; RRID: AB_325990
Mouse monoclonal anti-mCherry (1C51)	Abcam	Cat# ab125096; RRID: AB_11133266
Rabbit polyclonal anti- Phospho-PERK (Thr982)	Invitrogen	Cat# PA5-40294; RRID: AB_2576881
Rabbit monoclonal anti- Phospho-eIF2α (Ser51)	Cell Signaling Technology	Cat# 3398; RRID: AB_2096481
Rabbit monoclonal anti-ATF4 (D4B8)	Cell Signaling Technology	Cat# 11815; RRID: AB_2616025
Mouse monoclonal anti-CHOP (L63F7)	Cell Signaling Technology	Cat# 2895; RRID: AB_2089254
Rat monoclonal anti-mCherry (16D7)	Invitrogen	Cat# M11240; RRID: AB_2536614
Rat monoclonal anti-Thy1.2	BD Biosciences	Cat# 740005; RRID: RRID: AB_2739777
Mouse monoclonal anti-GFAP	Invitrogen	Cat# A21295; RRID: AB_2535832
Mouse monoclonal anti-CD45.2	BD Biosciences	Cat# 560693; RRID: AB_1727491
Rat monoclonal anti-CD34	Biolegend	Cat# 119308; RRID: AB_493400
Rat monoclonal anti-CD11b	BioLegend	Cat# 101216; RRID: AB_312799
Hamster monoclonal anti-CD11c	BD Biosciences	Cat # 550261; RRID: AB_398460

Bacterial and virus strains

AAV2-CaMKII-mATP2A2/mCherry	Vector Biolabs	N/A Lot# 211227
AAV2-CaMKII-mCherry	Vector Biolabs	N/A Lot# 210315

Chemicals, peptides, and recombinant proteins

Cyclopiazonic acid	Sigma-Aldrich	Cat# C175
CDN1163	Sigma-Aldrich	Cat# SML1682
Propidium iodide	Invitrogen	Cat# R37108

Experimental models: Organisms/strains

Mouse: Thy1-GCaMP6f (GP5.17)	The Jackson Laboratory	JAX: 025393; RRID: IMSR_JAX:025393
Mouse: CD1	The Charles River Laboratory	CRL:022; RRID: IMSR_CRL:022
Mouse: Thy1-GCaMP6f x CD1	Di Polo Laboratory	N/A
Mouse: C57BL/6	The Charles River Laboratory	CRL:027; RRID: IMSR_CRL:027

Oligonucleotides

ATP2A2 (Mm01201431_m1)	Life Technologies	Cat# 4331182
Actb (Mm02619580_g1)	Life Technologies	Cat# 4331182

Software and algorithms

Fiji/ImageJ	Schneider et al., 2012⁷⁹	https://imagej.nih.gov/ij/
Imaris 8.1	Oxford Instruments	https://imaris.oxinst.com/
R 4.2.0	The R Foundation	https://www.r-project.org/
MATLAB 2020b	MathWorks	https://www.mathworks.com/
Suite2P	Carsen Stringer et al., 2020	https://www.suite2p.org/
FlowJo	BD Biosciences	https://www.flowjo.com/
GraphPad 6.0	Dotmatics	https://www.graphpad.com/scientific-software/prism/

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Experimental models and subject details

Experimental animals

All procedures were approved by the animal protection committee of the University of Montreal Hospital Research Center and followed the Canadian Council on Animal Care guidelines. Experiments included female and male adult mice (2–5 months) expressing the Ca²⁺ indicator GCaMP6f (fast kinetics) downstream of the Thy1 promoter to visualize light-evoked Ca²⁺ dynamics selectively in RGCs (Thy1-GCaMP6f, Jackson Laboratory, 025393). For live retinal imaging, this line was backcrossed to an albino background (CD1, Charles River, Saint-Constant, Canada, 022) for at least six generations. For optomotor response assay, C57BL/6 mice (Charles River, 027) were used. Animals were housed in 12h light/12h dark cyclic light conditions, with an average in-cage illumination level of 10 lux, and fed ad libitum. Ambient temperature and humidity were maintained at 21°C–22°C and 45–55%, respectively. All procedures were performed under general anesthesia with ketamine (200 mg/kg), xylazine (2 mg/kg), and acepromazine (0.4 mg/kg), except for the experiments involving longitudinal two-photon microscope imaging in live animals, which required ketamine (80 mg/kg) and xylazine (10 mg/kg).

Mouse glaucoma model

Unilateral elevation of intraocular pressure was performed by a single injection of magnetic microbeads into the mouse anterior chamber.²⁴ Animals were anesthetized and a drop of tropicamide was applied on the cornea to induce pupil dilation (Mydriacyl, Alcon, Mississauga, ON, Canada). A custom-made sharpened microneedle attached to a microsyringe pump (World Precision Instruments, Sarasota, FL) was loaded with a magnetic microbead solution (1.5 μL: 2.4 x 10⁶ beads, diameter: 4.5 μm, Dynabeads M-450 Epoxy, Thermo Fisher Scientific, Waltham, MA). Using a micromanipulator, the tip of the microneedle was gently pushed through the cornea to inject the microbeads into the anterior chamber. The microbeads were immediately attracted to the iridocorneal angle using a hand-held magnet. Sham controls received an injection of PBS. Only one eye was operated on and an antibiotic drop was applied immediately after the surgery (Tobrex, Tobramycin 0.3%, Alcon, Geneva, Switzerland). Intraocular pressure was measured in awake animals before and after the procedure, and biweekly thereafter always at the same time (10 a.m.- 12 p.m.), using a calibrated TonoLab rebound tonometer (Icare, Vantaa, Finland). For this purpose, a drop of proparacaine hydrochloride (0.5%, Alcon) was applied to the cornea and a minimum of 10 consecutive readings were taken per eye and averaged.

Optic nerve crush model

Optic nerve crush was performed to induce selective axonal injury in RGCs as previously described.⁴⁸^,⁴⁹ The optic nerve was exposed intraorbitally and crushed with a microforceps at ∼2 mm posterior to the optic disc for a duration of 3 s. During the procedure, care was taken to avoid injury to the ophthalmic artery and the vasculature of the retina was routinely monitored post-operatively by fundus examination to confirm normal blood circulation. Antibiotic drops were applied on the cornea of the treated eye. All surgeries were conducted by an experienced experimenter who visually verified optic nerve crush throughout the procedure, and only one eye was operated.

Human glaucoma specimens and processing

Following institutional research board approval, glaucoma human retina specimens and controls were obtained from the Human Eye Biobank for Research (HEBR, St. Michael’s Hospital, Toronto, ON). Inclusion criteria for surgical glaucoma specimens were a history of primary open-angle glaucoma and histopathological demonstration of optic nerve head excavation. Age-matched controls were surgical specimens from individuals without any eye pathology. A total of 21 donor retinas were examined (13 glaucoma specimens and 8 age-matched controls, Table S1). Paraffin retinal sections were heated for 20 min in citrate buffer (80°C–90°C) followed by incubation in blocking solution (10% normal donkey serum, 1% bovine serum albumin, 0.5% Triton X-100) for 1 h. Retinal sections were incubated with primary antibodies: SERCA2 (4 μg/mL, Invitrogen), Calreticulin (1:250, Invitrogen), and RBPMS (0.25 μg/mL, PhosphoSolutions), followed by anti-mouse Alexa 647, anti-rabbit Alexa Fluor 594, or anti-guinea pig Alexa Fluor 488 (2–4 μg/mL, Molecular Probes). Glaucoma and age-matched control retinas were processed simultaneously under identical conditions. All RGCs in each retinal section were analyzed and three retinal sections per sample case were examined. Quantification of fluorescence was carried out using ImageJ from single in-focus plane images. The contour of individual RGC was outlined, and circularity, area, and mean fluorescence were measured along with adjacent background readings. The total corrected cellular fluorescence (TCCF) was calculated using the formula TCCF = integrated density – (area of selected cell × mean fluorescence of background readings).²⁵

Method details

Retinal immunohistochemistry

Animals were anesthetized and transcardially perfused with ice-cold 4% paraformaldehyde (PFA) in phosphate saline buffer (PBS). Eyes were immediately collected, post-fixed in PFA, and processed to generate cryosections or retinal flat mounts as described.⁸⁰^,⁸¹ Retinas were incubated in the following primary antibodies overnight at 4°C overnight (cross-sections) or 3 days (whole-mount retinas): RBPMS (0.25 μg/mL, PhosphoSolutions, Aurora, CO), GFP (2 μg/mL, Invitrogen), Ap2α (0.77 μg/mL, Developmental Studies Hybridoma Bank, Iowa City, IA), non-phosphorylated NF-H (1 μg/mL, BioLegend, San Diego, CA), Choline Acetyltransferase (1:200, Millipore, Burlington, MA), SERCA2 (4 μg/mL, Invitrogen, Waltham, MA), Calreticulin (1:250, Invitrogen) or mCherry (2 μg/mL, Abcam, Cambridge, FL) followed by fluorophore-conjugated secondary antibodies (2–4 μg/mL, Invitrogen). Sections were rinsed and mounted in antifade solution (SlowFade, Molecular Probes, Eugene, OR) with DAPI. Images were acquired with an Axio Imager M2 optical sectioning microscope (Zeiss, Oberkochen, Germany) or a confocal microscope (Zeiss LSM 900 Airyscan 2, Zeiss, Oberkochen, Germany). Fluorescence intensity was measured in regions of interest (ROIs) with ImageJ (National institute of Health: NIH, Bethesda, MD). A minimum of six stereological sections per eye were analyzed as described.⁸⁰^,⁸¹^,⁸²

Two-photon microscopy live Ca²⁺ recordings

TPLSM live retinal imaging was performed as previously described.²⁶^,²⁷^,²⁸ Mice were anesthetized and placed on a custom-made setup designed to accommodate light stimulation during imaging. The sclera was exposed and the conjunctiva gently teased to place a 5-mm diameter coverslip (Harvard apparatus, Holliston, MA) to generate a flat plane for imaging (field of view: 400 × 400 μm) with a multiphoton microscope controlled by Zen software (LSM780, Zeiss). For excitation, we used a mode-locked Ti:sapphire laser (Chameleon Ultra, Coherent, Santa Clara, CA) through a water-immersion objective (20x, NA = 1.0, Zeiss). For light-triggered visual stimulation, we generated a flash stimulus (10² cd/m², 6 msec) with a Powerlab unit (ADInstruments, Colorado Springs, CO) presented using a white light-emitting diode centered relative to the pupil and located 5 mm away from the corneal apex. RGCs expressing GCaMP6f were scanned at 12 Hz and Ca²⁺ signals were analyzed in circular regions of interest (ROIs) encompassing the entire soma. For longitudinal imaging, mice were randomly assigned to experimental or control groups and Ca²⁺ dynamics were recorded to obtain baseline responses. Two weeks after glaucoma induction or 15 min of intravitreal injection of pharmacological reagents, a second recording was captured. To identify the same RGCs, landmark blood vessels were labeled by tail vein injection of Texas Red coupled dextran (70 kDa, 1 mg/mL in 100 μL, Sigma). Stimulus onset and TPLSM imaging recordings were synchronized offline by identifying the frame at which the light stimulus was registered. Light-evoked Ca²⁺ responses were analyzed by averaging the fluorescence intensity of all pixels within each ROI using ImageJ after background subtraction. The following Ca²⁺ transient parameters were calculated: i) rise rate: defined as the slope of the rising phase (ΔF/F₀/Δtime) determined by best fit linear regression model due to fast Ca²⁺ kinetics during this phase, ii) decay time: defined as the exponential decay time constant (Tau) of Ca²⁺ signals obtained from single exponential curve fitting, and iii) peak amplitude: defined as ΔF/F₀ peak, where F₀ is the baseline fluorescence signal averaged over a 2 s period before the beginning of the light stimulus. The rising slope and amplitude are attributed to Ca²⁺ influx from extracellular sources and internal stores, while the signal decay is mediated by Ca²⁺ clearance mechanisms.⁷^,⁸³ A freely available custom R routine (version 4.2.0) was used for data analyses.

Ca²⁺ recordings in retinal explants

Ca²⁺ imaging was performed as previously described.⁸⁴ Briefly, mice were dark adapted for at least 2 h, euthanized, and then retinas rapidly dissected under infrared illumination into oxygenated (95% O₂; 5% CO₂) Ames solution (MilliporeSigma, Oakville, ON). Next, retinas were mounted onto a filter paper (MilliporeSigma) with the RGC layer facing up, placed in a recording chamber, mounted on the stage of a custom-built two-photon microscope, and perfused with oxygenated Ames solution warmed to 32°C–34°C. Responses of GCaMP6f-positive RGCs to visual stimuli delivered through the objective were imaged at 920 nm and collected at 45 Hz. Each image plane (323.74 μm × 323.74 μm) of the movie contained GCaMP fluorescence, sulphorhodamine 101 (SR101) fluorescence, stage coordinates, and visual stimulus synchronization pulses to permit offline analysis. SR101 (2 mg/mL, MilliporeSigma) was added to the recording chamber to label blood vessels and a map of the main blood vessels emanating from the optic disk. Data acquisition was performed with ScanImage synced to the visual stim using a photodiode sync pulse and a random pulse using custom made code in MATLAB 2020b.

Visual stimuli

A DLP light crafter (Texas Instruments, Dallas, TX) was used to project dichromatic (405 nm, 520 nm) visual stimuli through a custom lens assembly that steered stimulus patterns into the back of a 16× objective.⁸⁵ All visual stimuli were written in MATLAB using the psychophysics toolbox and displayed with a background intensity set to 1 × 10⁴ R∗/rod/s. Custom electronics were made to synchronize the projector LED to the scan retrace of the two-photon microscope. Moving bar stimulus consisted of a bright bar moving along its long axis in one of eight directions. The bar was 300 μm wide, 1500 μm long moving at 1000 μm/s. Full-field stimulus was presented with increment and decrement of light that lasted 2s. Full-field chirp consisted of a full contrast sinusoidal intensity modulation from 1 to 16Hz. Full-field contrast consisted of a 4Hz synodal with 8-bit contrast increase per cycle. Full-field color stimuli were delivered using full flashes with only UV or green LEDs that lasted 3s. For all full-field stimuli a 1200μm circle in a gray background was used. Each stimulus was repeated three times for each recorded field.

Data pre-processing

Individual RGC Ca²⁺ responses corresponding to stimulus were extracted with suite2P⁸⁶ and subsequently aligned with stimuli using custom made MATLAB code. Signals were smoothed to remove noise with moving mean of 10 and expressed as ΔF/F, where the median of the peristimulus baseline was used to calculate the difference. For further analysis, Ca²⁺ signals were filtered by quality to discard unresponsive cells.

Principal component analysis (PCA) and clustering

PCA analysis was implemented in R to extract features from our 5 different stimuli. A total of 7 features were extracted from the stimuli, 2 features from the mean response to the full-field (averaging across trials), 1 from the mean response to the color stimulus and 4 features for the moving bar stimulus. These features were further used for functional clustering using a diagonal, varying volume, and shape Gaussian mixture model (GMM). This model was evaluated using the Bayesian information criterion to select the optimal number of clusters resulting in 7 groups. PCA loadings and GMM from sham controls were implemented for dimensional reduction and cluster assignment of other experimental conditions. Manifold Approximation and Projection (UMAP) was applied for cluster visualization.

Like in vivo Ca²⁺ imaging, mice were randomly assigned into experimental or control groups and rise rate, decay time, and peak amplitude of average RGC responses analyzed in MATLAB using the curve fitting toolbox. The ON-OFF index was calculated using the following equation³².

On−Off Index = ONresp−Offresp/ONresp+Offresp

where ONresp and OFFresp are the mean cell response over a bright and dark full-field stimulus, respectively.

Intravitreal injections

The following reagents were administered intraocularly: i) cyclopiazonic acid (CPA, 10 μM, Sigma), ii) CDN1163 (10 μM, Sigma), iii) serotype 2 AAV (AAV2) encoding mCherry-labeled murine SERCA2 under control of the CaMKII promoter (AAV.SERCA2, 2.4 x 10¹² genome copies (GC)/mL, Vector BioLabs, Malvern, PA), or iv) control AAV2 carrying only mCherry (AAV.Ctl, 7.0 x 10¹² GC/mL, Vector BioLabs). A sharpened custom-made glass micropipette was inserted through the sclera into the posterior chamber to reach the vitreous cavity and deliver a total volume of 2 μL of each reagent. AAVs were injected one week prior to magnetic microbead-induced glaucoma or optic nerve crush lesion. All injections were done into the superior-temporal quadrant of the eye and were carefully conducted to avoid injury to ocular structures or retinal detachment as described by us.⁸⁷^,⁸⁸

FAC-sorting and gene expression analysis

Fresh retinas were cut into small pieces using scissors and incubated in a dispase solution (5 U/mL) (Stemcell Technologies, Vancouver, Canada) containing DNase I (2000 U/ml) (Worthington BioChemical, Lakewood, NJ) for 20–25 min at 37°C in an Eppendorf thermomixer (Eppendorf, Mississauga, ON) with shaking (350 rpm). Enzymatic digestion was stopped using a blocking solution (HBSS, 2% BSA) and the dissociated cells were then incubated with the following primary antibodies: Thy1.2 (0.25 μg/μL, BD Biosciences, Franklin Lakes, NJ), glial fibrillary acidic protein (GFAP, 1 μg/μL, Invitrogen), CD45.2 (0.25 μg/μL, 103126, Biolegend, San Diego, CA), CD34 (0.2 μg/μL, Biolegend), CD11b (0.2 μg/μL, Biolegend), and CD11c (0.2 μg/μL, BD Biosciences) as previously described.²⁸ Propidium iodide (PI, Invitrogen) was used to assess cell viability and was added to the cells just before sorting. Dead cells and doublets were discarded, and only Thy1.2⁺ GFAP⁻ CD45.2^- CD34⁻ CD11b⁻ CD11c⁻ were selected for further processing. Sorted RGC were collected directly in lysis buffer and RNA was extracted using the RNAeasy micro plus kit (Qiagen, Hilden, Germany). Nucleotide concentration was measured using a bioanalyzer and only samples with high RNA integrity were included. Total RNA (100 pg) was transcribed to cDNA using the RevertAid kit (Invitrogen) and quantitative PCR was performed using TaqMan primers against following transcripts: SERCA2 (Mm01201431_m1) and β-actin (Mm02619580_g1) (Thermo Fisher Scientific). Amplification was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, Waltham, MA) with the following cycle conditions: 95°C 15 s, 60°C 1min, 72°C 1min. Reactions were run in triplicates for each sample, and the 2^−ΔΔCt method was used to calculate relative gene expression.

Flow cytometry for protein quantification in RGCs

Total protein in RGCs

Fresh retinas were cut into small pieces using scissors and incubated in a dispase (5 U/ml, Stemcell Technologies, Vancouver, BC) and DNase I (2000 U/ml, Worthington Biochemical, Lakewood, NJ) solution for 20–25 min at 37°C in an Eppendorf thermomixer (Eppendorf, Mississauga, ON) with shaking (350 rpm). Enzymatic digestion was stopped using a blocking solution (HBSS, 2% BSA) and dissociated cells were washed twice with the same solution. The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, MA) was used to determine the viability of cells prior to the fixation and permeabilization required for intracellular antibody staining. Cells were washed in fluorescence-activated cell sorting (FACS) buffer (PBS with 1% fetal bovine serum and 0.1% sodium azide), fixed and permeabilized in a paraformaldehyde (PFA, 1.5%)/saponin buffer prewarmed at 37^o C. Cells were incubated in primary antibodies against RBPMS (0.4 μg/μL, PhosphoSolutions, Aurora, CO) and SERCA2 (1 μg/μL, Invitrogen) as well as validated antibodies against ATF4 (0.068 μg/μL, Cell Signaling Technology) and CHOP (0.575 μg/μL, Cell Signaling Technology).⁸⁹^,⁹⁰^,⁹¹^,⁹² Cells were then incubated in secondary antibodies: Alexa Fluor 647 anti-guinea pig for RBPMS, Alexa Fluor 488 anti-mouse for CHOP, and Alexa Fluor 750 anti-rabbit for ATF4 (0.4 μg/μL, Invitrogen) and washed twice prior to flow cytometry acquisitions. AAV.SERCA2 treated retinas were incubated with an antibody against mCherry to enhance the signal (1 μg/μL, Invitrogen).

Phosphorylated protein expression in RGCs

Dissociated retinal cells were fixed in PFA (1.5%) for 10 min at 37°C, permeabilized in Phosflow Perm Buffer III (BD Biosciences), and incubated with validated antibodies against anti-phospho-PERK (pPERK, 1 μg/μL, Invitrogen), phosho-eIF2α (peIF2α, 0.037 μg/μL, Cell Signaling Technology),⁸⁹^,⁹⁰^,⁹²^,⁹³^,⁹⁴ and RBPMS (0.4 μg/μL, PhosphoSolutions). Cells were washed twice in FACS buffer and incubated with the following secondary antibodies for 30 min: Alexa Fluor 647 anti-guinea pig for RBPMS, Alexa Fluor 488 anti-rabbit for pPERK or peIF2α (0.4 μg/μL, Invitrogen), followed by two additional washes. Control cells were incubated with only secondary antibodies or with primary antibodies using a secondary antibody from different species to rule out non-specific binding. Data were acquired using the BD LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo).

Quantification of RGC survival and AAV transfection rate

Mice were transcardially perfused with 4% PFA, retinas were dissected out and incubated for three days with an antibody against RBPMS (0.25 μg/mL, PhosphoSolutions) or mCherry (2 μg/mL, Abcam, Cambridge, FL) followed by an Alexa 488 or Alexa 647 conjugated secondary antibody (2–4 μg/mL, Invitrogen). Retinas were washed and mounted using an antifade reagent (SlowFade, Molecular Probes). Images were obtained using an Axio Imager M2 optical sectioning microscope (20× objective, Zeiss) equipped with an automated stage for X-, Y-, and z axis movement, a color camera (Axiocam 509 mono, Zeiss), and image analysis software (Zen, Zeiss). We used an unbiased stereological approach based on systematic uniform random sampling from 3D-dissectors (stacks) across the entire retina, and images were acquired using identical exposure time and gain settings for all experimental and control groups as described.²⁶^,²⁸ RGC somas were quantified using a custom-made quadrant dissector in Fiji/ImageJ. For assessment of AAV.SERCA2 transfection rate only RGC co-labeled with RBPMS and mCherry-labeled SERCA2 were quantified.

Optomotor response assay

Visual acuity was evaluated by measuring the optomotor reflex using the OptoMotry virtual reality system (Cerebral Mechanics Inc., Medicine Hat, AB, Canada), which allows the quantification of visuomotor behaviors in response to visual stimuli.²⁸ The animals were placed on an elevated platform in the center of a testing arena with walls composed of computer monitors displaying a rotating vertical black and white sinusoidal grating pattern. The staircase method was used to determine the spatial frequency of applying sinusoidal steps. Both contrast (100%) and rotation speed (12°/s) were kept constant. Cameras were used to record the animals, and an observer blinded to treatment monitored their tracking behavior. A behavioral response was considered positive when the motor response (head movement) was concordant with the direction of the visual stimulus (moving bars). Individual scores from each mouse were collected before and after each treatment.

Statistical analysis

Detailed information regarding the number of animals or cells analyzed in each experiment is indicated in the figure legends. In each graph, values are provided as the mean ± standard error of the mean (S.E.M), and individual values are presented in each bar. All cohorts were evaluated with normality (Shapiro-Wilk test) and variance (F-test) tests. Statistical analyses were performed using GraphPad Instat software (GraphPad Software Inc., San Diego, CA) by a Student’s t-test or Welch’s t-test or by a one-way analysis of variance (ANOVA) followed by a Dunnett’s or Tukey’s post hoc tests, or two-way ANOVA followed by a Sidak’s post hoc test, or Wilcoxon matched-pairs signed rank test. A p-value of ≤0.05 was considered significant.

Published: November 29, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2024.101839.

Supplemental Information

Document S1. Figures S1–S4 and Table S1

mmc1.pdf^{(1.2MB, pdf)}

Document S2. Article plus supplemental information

mmc6.pdf^{(10.2MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Light-evoked Ca²⁺ responses of healthy RGC in living mice, related to Figure 1

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from sham control retina. The green horizontal bar indicates the light stimulus.

Download video file^{(1.6MB, mp4)}

Video S2. Early glaucomatous damage disrupts light-evoked RGC Ca²⁺ dynamics, related to Figure 1

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC at 2 weeks following glaucoma induction. The green horizontal bar indicates the light stimulus.

Download video file^{(6MB, mp4)}

Video S3. Light-evoked RGC Ca²⁺ responses in glaucomatous retina treated with control AAV.Ctl

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from glaucomatous retinas treated with AAV.Ctl. The green horizontal bar indicates the light stimulus.

Download video file^{(6MB, mp4)}

Video S4. AAV.SERCA2 treatment enhances light-evoked Ca²⁺ dynamics in injured RGCs, related to Figure 5

TPLSM Ca²⁺ imaging of a representative GCaMP6f-positive RGC from glaucomatous retinas treated with AAV.SERCA2. The green horizontal bar indicates the light stimulus.

Download video file^{(1.6MB, mp4)}

Document S1. Figures S1–S4 and Table S1

mmc1.pdf^{(1.2MB, pdf)}

Document S2. Article plus supplemental information

mmc6.pdf^{(10.2MB, pdf)}

Data Availability Statement

•
All data reported in this paper will be shared by the lead contact upon request.
•
All original code used in this paper is available from the lead contact upon request.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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PERMALINK

Endoplasmic reticulum stress-related deficits in calcium clearance promote neuronal dysfunction that is prevented by SERCA2 gene augmentation

Yukihiro Shiga

Aline Giselle Rangel Olguin

Sana El Hajji

Nicolas Belforte

Heberto Quintero

Florence Dotigny

Luis Alarcon-Martinez

Arjun Krishnaswamy

Adriana Di Polo

Summary

Graphical abstract

Highlights

Introduction

Results

Ca2+ clearance deficits precede overt neuronal death during glaucomatous stress

Figure 1.

Impaired Ca2+ clearance is conserved across RGC subtypes

Figure 2.

OHT leads to ER stress and SERCA2 downregulation

Figure 3.

SERCA2 downregulation is consistent with Ca2+ clearance deficits, and pharmacological activation of SERCA2 restores Ca2+ dynamics and promotes neuronal survival

Figure 4.

Figure 5.

SERCA2 gene supplementation rescues Ca2+ dynamics during OHT- and ONC-induced stress

Figure 6.

SERCA2 gene therapy attenuates ER stress, promotes neuronal survival, and prevents loss of visual function

Figure 7.

Discussion

Ca2+ clearance is compromised in injured RGCs

ER stress and loss of SERCA2 function are key features of glaucomatous neurodegeneration

Selective SERCA2 activation promotes RGC survival and restores visual function

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

STAR★Methods

Key resources table

Experimental models and subject details

Experimental animals

Mouse glaucoma model

Optic nerve crush model

Human glaucoma specimens and processing

Method details

Retinal immunohistochemistry

Two-photon microscopy live Ca2+ recordings

Ca2+ recordings in retinal explants

Visual stimuli

Data pre-processing

Principal component analysis (PCA) and clustering

Intravitreal injections

FAC-sorting and gene expression analysis

Flow cytometry for protein quantification in RGCs

Total protein in RGCs

Phosphorylated protein expression in RGCs

Quantification of RGC survival and AAV transfection rate

Optomotor response assay

Statistical analysis

Footnotes

Supplemental Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Ca²⁺ clearance deficits precede overt neuronal death during glaucomatous stress

Impaired Ca²⁺ clearance is conserved across RGC subtypes

SERCA2 downregulation is consistent with Ca²⁺ clearance deficits, and pharmacological activation of SERCA2 restores Ca²⁺ dynamics and promotes neuronal survival

SERCA2 gene supplementation rescues Ca²⁺ dynamics during OHT- and ONC-induced stress

Ca²⁺ clearance is compromised in injured RGCs

Two-photon microscopy live Ca²⁺ recordings

Ca²⁺ recordings in retinal explants