A paradoxical relationship between mitochondrial calcium regulation and retinal ganglion cell degeneration after axon damage

Reviewer #1 (Public review):

Summary:

This study investigates how Ca2+ levels inside the RGCs' mitochondria relate to whether these cells survive or die after injury to the optic nerve. The authors used advanced in vivo fundus live imaging techniques in mice to watch these changes unfold in real time, combined with genetic and drug-based tools to alter calcium flow into these compartments. Their central finding is a striking paradox: cells that naturally survive injury tend to have higher baseline calcium levels in these compartments, yet experimentally reducing calcium entry protects the broader population of cells from death.

Strengths:

The authors are applying sophisticated biosensors to track cellular chemistry in living animals over days and weeks. The tools and methods are creative and direct to detect the longitudinal RGC degeneration with mito-Ca2+ imaging. The topic and research aspect are novel and attractive. The results are significant, showing a clear relationship between the mito-Ca2+ regulatory machinery and cell survival.

Weaknesses:

The details of the mitochondrial-located signal of the Ca2+ sensor need to be further proved in the mito-matrix or between the mito-membranes. The study primarily describes a correlation and a surprising experimental outcome without fully explaining the underlying biological reasons for the paradox. While the evidence supporting the phenomenon is good, the mechanistic insight into why high calcium is linked to survival, or why lowering it helps after injury, remains limited.

Reviewer #2 (Public review):

Summary:

The manuscript by McCraken and colleagues provides a continuation of their 2023 study (Cell Reports 42:113165) characterizing calcium regulation in retinal ganglion cells (RGCs) after acute optic nerve damage (a 10s crush using an intraorbital approach). This work is principally focused on how mitochondrial calcium stores change in both RGCs that are resilient and susceptible to injury. They report that resilient RGCs typically exhibited high calcium levels, but paradoxically, manipulating mitoCa2+ levels was more protective when the stores were reduced. Overall, regardless of susceptibility, mitoCa2+ levels decreased after injury, which is opposite to other reports that mitoCa2+ increases in degenerating neurons. The manipulation of mitoCa2+ was conducted both pharmacologically (Ru265) and by overexpression or knockdown of a primary calcium uniporter MCU. The evaluation of mitoCa2+ was conducted by using a reporter (Twitch2b) that was targeted to the mitochondria.

Strengths:

Many of the experiments are elegant and well-performed.

Weaknesses:

(1) Some experiments require further controls to validate that reagents are doing what they are intended to do.

(2) Some findings can have alternate interpretations that are not considered.

(3) There is a broad generalization to the biology of all RGCs that may not be biologically relevant to different RGC subtypes.

Reviewer #3 (Public review):

Summary:

Following previous work that demonstrated a relationship between higher homeostatic cytosolic calcium and lower retinal ganglion cell (RGC) apoptosis following injury to their axons, McCracken et al. investigated whether homeostatic calcium levels of the endoplasmic reticulum (ER) or mitochondria provide additional insights into the mechanisms by which calcium influences RGC survival. Their study reveals that homeostatic mitochondrial calcium shows a similar positive correlation with RGC survival. Despite that correlation, pharmacologic or genetic methods to lower mitochondrial calcium improved, rather than reduced, the survival of injured RGCs, while a genetic approach intended to increase mitochondrial calcium resulted in more RGC loss. These findings highlight the complexities of calcium regulation in modulating neuronal survival and raise important questions of how homeostatic levels of mitochondrial calcium affect stress responses that themselves can be either neuroprotective or neurodegenerative.

Strengths:

This study tackles an intriguing hypothesis that differences in calcium ion homeostasis in specific organelles may contribute to differences in survival of various RGC subtypes after optic nerve injury. This is a technically demanding question, and a primary strength of this work is its attention to, and meticulous reporting of, appropriate controls and, where applicable, seemingly contradictory results. Among these are careful evaluation of the effects of drug (or vehicle) delivery and genetic manipulations with and without injury and over extended time courses. The combination of thoughtful pharmacologic and genetic approaches makes for a thorough analysis of a challenging set of questions. The result is a study that provides a helpful perspective on the complicated roles that calcium, and especially mitochondrial calcium, can play across neuronal insults, neuronal types, and neuronal subtypes.

Weaknesses:

Given the paradoxical results, it would be helpful to have a clearer picture of how strongly the overexpression and knockdown of MCU altered the mitochondrial calcium levels. There may be potential for extraordinarily strong effects that would need to be tuned by using different shRNAs or promoters to more closely align with the observed differences between surviving RGCs and those that die. The investigation includes a relatively small number of resilient RGC subtypes, using the markers SPP1 and TBR2, raising questions of how generalizable the trend is between mitochondrial calcium levels and RGC resilience. The analysis and implications of Figure 3D might benefit from including not only the provided 50:50 split between "high" and "low" but also views of the data after splitting into thirds, fourths, and perhaps even fifths. The authors' inference that higher homeostatic calcium in more resilient RGCs may result in chronic mitochondrial stress is intriguing and worthy of more experimental investigation than is currently provided.