Support of Retinal Ganglion Cell Survival and Axon Regeneration by Lithium through a Bcl-2-Dependent Mechanism

Abstract

purpose. To explore whether lithium, a long-standing mood-stabilizing drug, can be used to induce expression of Bcl-2 and support the survival and regeneration of axons of retinal ganglion cells (RGCs).

methods. Levels of expression of Bcl-2 in the retina were assessed with quantitative reverse transcription-polymerase chain reaction. To determine whether lithium directly supports the survival of and axon-regenerative functions of RGCs, various amounts of lithium were added to cultures of isolated RGCs. Anti-Thy1.2 antibodies-conjugated to magnetic beads were used to isolate the RGCs. In addition, retina-brain slice cocultures were prepared from tissues of Bcl-2-deficient or Bcl-2-transgenic mice and treated with various amounts of lithium. The effects of the expression of Bcl-2 on lithium-mediated functions were then analyzed.

results. Normal mouse retina expressed very low levels of Bcl-2 after birth. Addition of lithium in the culture increased mRNA levels of Bcl-2 in retinas of postnatal mice in a dose-dependent manner. Moreover, lithium promoted not only the survival of RGCs but also the regeneration of their axons. Depleting or forcing the expression of Bcl-2 in RGCs eliminated the effects of lithium.

conclusions. Lithium supports both the survival and regeneration of RGC axons through a Bcl-2-dependent mechanism. This suggests that lithium may be used to treat glaucoma, optic nerve neuritis, the degeneration of RGCs and their nerve fibers, and other brain and spinal cord disorders involving nerve damage and neuronal cell loss. To achieve full regeneration of the severed optic nerve, it may be essential to combine lithium therapy with other drugs that mediate induction of a permissive environment in the mature central nervous system.

After injury, the postnatal mammalian optic nerve, similar to many other axonal pathways in the central nervous system (CNS), regenerates poorly. Most often, injured retinal ganglion cells (RGCs) undergo apoptotic cell death. ^{1 2} Although this regenerative failure has long been attributed to extrinsic inhibitors in the environment of the mature brain, ^{3 4} our research has demonstrated that an intrinsic component that initiates axonal growth after injury is absent in mature RGCs. ⁵ We have identified this intrinsic component as the antiapoptotic protein Bcl-2. ⁶ The expression of Bcl-2 in RGCs coincides with the regenerative capacity of their axons after injury. RGCs of wild-type mice lose the ability to regenerate axons at embryonic day 18, when expression of Bcl-2 declines to a very low level. Furthermore, in the absence of Bcl-2 knockout mice, RGC axons fail to regenerate, even in the early embryonic stages. In contrast, overexpression of Bcl-2 in postnatal mice not only supports the survival of RGCs, but also facilitates a robust regeneration of severed axons, when the CNS environment is maintained in a condition permissive of the extension of axons (e.g., in the neonatal stage). ^{5 6 7}  

Taken together, the findings in these studies suggest that a prerequisite for successful regeneration of severed optic nerves in adult mammals is the activation of an intrinsic mechanism for regeneration of RGC axons, such as the induction of the expression of Bcl-2 in neurons. Moreover, our work also suggests that the CNS environment has strong inhibitory mechanisms that block regeneration of axons in adults. Therefore, for RGC axons to regenerate in adults, an induction of Bcl-2 with a simultaneous manipulation of the mature CNS environment is necessary. Because little is known about the molecular mechanisms and signals that regulate expression of Bcl-2, inducing expression in postnatal RGCs remains a challenge. 

Recent studies have shown that lithium (Li⁺), a simple monovalent cation that has been used safely in the treatment of bipolar disorder in humans for more than 30 years, robustly increases the levels of Bcl-2 protein. ^{8 9 10} Although Li⁺ exerts effects on a number of intracellular signaling cascades, including regulating turnover of phosphoinositide, protein kinase C, and glycogen synthase kinase-3β (GSK-3β), ^{10 11} its therapeutic effects are observed only after long-term administration. The lag period between the onset of treatment and the appearance of therapeutic effects has fueled research to identify genes with expression that is regulated by prolonged administration of Li⁺. These studies have led to the unexpected finding that Li⁺ robustly increases expression of Bcl-2 in various areas of the rodent brain and in cells of human neuronal origin. ^{8 9 10} Consistent with its induction of Bcl-2, Li⁺, at therapeutically relevant concentrations, exerts cytoprotection against the deleterious effects of a variety of insults, including glutamate, activation of the N-methyl-d-aspartate receptor, deprivation of serum and nerve growth factors, radiation, infusion of striatal quinolinic acid, or middle cerebral artery occlusion. ^{10 12 13} Subsequent studies, using magnetic resonance spectroscopy, have also shown that prolonged treatment with Li⁺ increases the levels of N-acetyl aspartate (NAA, a putative marker of neuronal viability and function) ¹⁰ and significantly increases total gray matter content in the human brain. ¹⁴ Taken together, the preclinical and clinical data suggest that the therapeutic effects of Li⁺ are promoted by the upregulation of Bcl-2. 

RGCs have long been used as a model for the study of CNS neurons, but the effect of Li⁺ on the retina has never been studied. We hypothesized that, if Li⁺ induces expression of Bcl-2 in neurons, it may not only prevent injury-induced degeneration of RGCs and other neurons in the CNS, but may also promote the regeneration of the axons in these cells. Therefore, in this study, we used RGCs as a model to examine whether Li⁺ affects both neuronal survival and regeneration of axons in the CNS and how these effects are related to its role in inducing the expression of Bcl-2. 

Methods

Animals

Adult C57BL/6J mice deficient in Bcl-2 ¹⁵ and mice that overexpress the Bcl-2 transgene driven by a neuron-specific enolase promoter ¹⁶ were maintained in the mouse facility of the Schepens Eye Research Institute (SERI). Mouse genotypes were determined after death, by use of standard polymerase chain reaction (PCR) methodology on tail DNA. All experimental procedures and use of animals were approved by SERI’s Animal Care and Use Committee and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Reverse Transcription-Polymerase Chain Reaction

For RT-PCR, total retinal RNA was isolated with extraction reagent (TRIzol; Gibco; Rockville, MD) according to the manufacturer’s instructions. One microgram total RNA was subjected to reverse transcription in a total volume of 20 μL of reaction mixture containing 4 μL RT buffer, 1 μg oligo-dT primer, 0.5 mM of each of the dNTPs, 10 mM dithiothreitol, and 5 U RNase inhibitor (all from Gibco). The reaction was performed at 42°C for 45 minutes with 1 U of reverse transcriptase (Superscript II; Gibco) and terminated by incubating at 75°C for 15 minutes. 

Each PCR reaction contained equivalent amounts of cDNA. For relative quantification, as used in this study, the relative amount of target gene Bcl-2 in differing samples was determined and compared with the amount of the internal standard control gene, glyceraldehyde-3-phosphate dehydrogenase (G3PDH). PCR primers for detection of mouse Bcl-2 were designed to span the second intron, according to Bcl-2 gene sequence (GenBank accession number: NM009741; http://www.ncbi.nlm.nih.gov/Genbank; provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD), so that the amplification of potentially contaminating genomic DNA would produce PCR fragments that were substantially larger than the cDNA PCR products. The DNA sequences of forward and reverse primers were as follows: Bcl-2 sense 5′-ATGTGTGTGGAGAGCGTCAAC-3′, antisense 5′-AGACAGCCAGGAGAAATCAAAC, with a resultant 148-bp product; G3PDH sense 5′-AGAACATCATCCCTGCATCC-3′, and antisense 5′-AGCCGTATTCATTGTCATACC-3′, with a resultant 317-bp product. 

The PCR mixture consisted of 2.5 μL PCR buffer (Applied Biosystems, Foster City, CA), 1.5 mM Mg²⁺, 0.2 mM dNTP, 25 nM primers, and 1 U Taq DNA polymerase (Applied Biosystems). PCR reactions were performed with the following program: 1 cycle of incubation at 94°C for 4 minutes followed by 32 cycles of denaturing at 94°C for 1 minute; annealing at 55°C for 30 seconds; and extension at 72°C for 45 seconds. PCR products were resolved in 2% agarose gel by electrophoresis and stained with ethidium bromide. The resolved PCR products were imaged by UV illuminator and digitally photographed (DC120 digital camera; Eastman Kodak; Rochester, NY). The intensities of the DNA bands were quantified by computerized image analysis and NIH Image software, with the gel-analyzer function of Image J ver. 1.17 (http://www.nih.gov/; National Institutes of Health, Bethesda, MD). 

Isolation of RGCs

We adopted a magnetic-bead separation method ¹⁷ to isolate RGCs from mice, using an antibody against a RGC-specific marker, Thy1.2, as the primary antibody. Rabbit anti-Thy1.2 conjugated with micrometal beads (CD90) was purchased from Multinyi Biotech (Auburn, CA). In brief, mouse pups were anesthetized by hypothermia and killed. The retinas were dissected in Mg²⁺/Ca²⁺-free Hanks’ balanced salt solution (HBSS) and dissociated by incubating for 10 minutes at 37°C in HBSS containing 1% papain (Worthington Biochemicals, Lakewood, NJ) and 5 U/mL DNase (Gibco). Retinal cells were then transferred to a solution with the papain inhibitor 1% ovomucoid (Worthington Biochemicals) and triturated. Dissociated cells were treated for 15 minutes at 4°C with rabbit anti-Thy1.2 antibody conjugated to the micrometal beads in elution buffer (phosphate-buffered saline with 0.5% bovine serum albumin and 2 mM EDTA; Sigma, St. Louis, MO). The cell suspensions were loaded onto a metal column and separated with the elution buffer in the presence and absence of a magnetic field. 

Characterization of the Isolated RGCs

Mouse pups were anesthetized by hypothermia. A retrograde fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, 6% dissolved in dimethylformamide; Molecular Probes, Eugene, OR) was injected into the superior colliculus (SC) to cover all the target areas of the optic nerve. After 24 to 48 hours, mouse pups were reanesthetized and killed and retinas were dissected and examined under a fluorescence microscope to ensure proper retrograde transport of DiI. RGCs were then isolated with magnetic bead separation and seeded in culture. The percentage of cells with DiI labeling was recorded. 

In addition, RGCs were fixed with 4% paraformaldehyde and blocked for 15 minutes at room temperature in PBS containing 2.5% fetal bovine serum, 2.5% goat serum, and 0.2% Triton X-100. They were then incubated at 4°C overnight with rabbit anti-Thy1.2 (PharMingen, San Diego, CA) followed by incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibody IgG (1:200; Chemicon, Temecula, CA) for 2 hours at room temperature. Cultures were observed under a microscope equipped with fluorescence illumination, and the number of FITC-positive cells was counted. 

Culture Conditions for Isolated RGCs and Cell Viability Assay

Twenty-four-well plates were precoated with 100 μg/mL poly-d-lysine (Sigma) for 2 hours followed by a 2-hour coating with 2 μg/mL human merosin (Gibco). Approximately 1 × 10⁵ purified RGCs were seeded onto each well in culture medium (NeuroBasal Medium; Gibco), supplemented with B27 (Gibco), 100 U/mL penicillin-streptomycin, 0.5 mM glutamine, and 12.5 μM glutamate. The cultures were treated with 1 mM LiCl and incubated at 37°C in humidified 5% CO₂ and 95% air. 

Cell viability was determined with a cytotoxicity staining kit (Live/Dead; Molecular Probes). RGC cultures were incubated at room temperature for 45 minutes in PBS containing calcein (10 μg/mL) and ethidium D (5 μg/mL; Molecular Probes). Live cells cleave calcein, which yields cytoplasmic green fluorescence, and ethidium D labels nucleic acids of dead cells with red fluorescence. The cultures were visualized with an inverted microscope equipped with fluorescence illumination (TE300; Nikon) and phase contrast, and the numbers of green (live) and red (dead) cells were counted. 

Retina-Brain Slice Cocultures

Retina-brain slice cocultures were prepared as described previously. ^{5 6} Briefly, 2-day-old mouse pups were anesthetized by hypothermia and killed, and their tails were collected for genotyping. The retinas and brains were dissected in HBSS. Coronal brain slices (300 μm) were prepared, and those containing the SC were selected and placed to abut retinal explants in six-well culture inserts (BD Biosciences, Franklin Lakes, NJ). The cocultures were maintained in medium (NeuroBasal Medium; Gibco) supplemented with B27 (Gibco), 0.5 mM glutamine, and 12.5 μM glutamate. LiCl was chosen as the lithium salt (0.2–5.0 mM) and was added to the culture on the day of plating. After 5 days of incubation, cultures were fixed with 4% paraformaldehyde. Retinal axons were labeled by placing four crystals of the fluorescent tracer DiI into each retinal explant. After 2 weeks, allowing time for the dye to diffuse, the cultures were visualized with the microscope. Using phase contrast, the retinal explant and SC and their boundaries were clearly identified by the differing intensities of light transmission through the tissues. The number of labeled axons that regenerated into the brain slices was then quantified with the aid of a fluorescence microscope. 

Statistical Analysis

All data were expressed as the mean ± SD, and statistical significance, defined by P < 0.05, was determined by Student’s t-test. 

Results

Induction of Bcl-2 in the Mouse Retina by Lithium

It has been reported that addition of Li⁺ to cultured CNS neurons induces the expression of Bcl-2, a central regulator for neuronal survival and regeneration of RGC axons. ^{18 19} These data prompted us to determine whether Li⁺ can also induce expression of Bcl-2 in the mouse retina. Retina-brain slice cocultures were prepared and incubated in the absence and presence of LiCl (0.2–5.0 mM). After 5 days of coculturing, retinal RNA was collected, and the levels of expression of Bcl-2 were analyzed by quantitative RT-PCR. Because the clinical therapeutic effect of Li⁺ is normally obtained at 0.5 to 1.2 mM, we examined six different concentrations of LiCl in culture, from 0.2 to 5.0 mM. We found that all the cultures exposed to LiCl showed dose-dependent increases in the level of Bcl-2 mRNA. The upregulation of Bcl-2 in the retina was observed when the concentration of LiCl was as low as 0.2 mM, and it was three times higher than that in a nontreated culture when 1.0 mM LiCl was applied (Fig. 1) . These results indicate that Li⁺ also acts on the retina to induce expression of Bcl-2 mRNA. 

Evaluation of Purity and Yield of Isolated RGCs

The antiapoptotic functions of Bcl-2 are well documented. ^{20 21} We expected, therefore, that if Li⁺ induced expression of Bcl-2 in the retina, it would promote retinal neuron survival in culture. To determine whether Li⁺ directly promotes RGC survival, we first adopted a method to purify RGCs from the postnatal day (P)2 mouse retina, using antibody against the RGC-specific marker Thy1.2 conjugated to magnetic beads. Both Thy1.2⁺ and Thy1.2⁻ cell populations were collected and seeded in culture. This procedure yielded approximately 90,000 to 120,000 isolated Thy1.2⁺ cells, which accounted for almost all the RGCs in a P2 retina. ^{22 23 24}  

To confirm that isolated cells were RGCs, we prelabeled the RGCs by injecting the retrograde tracer DiI into the SC. After 1 to 3 days, allowing time for dye transportation, mice were anesthetized again and killed, and Thy1.2⁺ cells were isolated. The number of fluorescent Thy1.2⁺ cells was counted by epifluorescence microscopy. The isolated Thy1.2⁺ cells consisted of 92.5% ± 20.8% (or 111,000 ± 25,000 cells) of the cells that were determined to be RGCs, as evidenced by DiI fluorescence (Figs. 2A 2B) . In contrast, the Thy1.2 cell population had very few DiI-labeled cells. This confirms that most RGCs were in the isolated population of Thy1.2⁺ cells. The purity of the RGCs was further verified by immunofluorescent staining for expression of the RGC-specific marker Thy1.2 antigen. Consistent with the DiI-labeling results, 93.7% ± 4.7% of the isolated cells were Thy1.2⁺ (Fig. 2C) . Furthermore, the isolated cells revealed a morphology similar to that of the RGCs described by Barres et al., ²² which were purified by antibody panning. These results show that the magnetic-bead separation method yields an accurate and efficient purification of most RGCs in the mouse retina. 

RGC Survival in Culture

In isolated RGC cultures, we investigated whether Li⁺ directly promotes the survival of RGCs. RGCs were purified from P2 pups and cultured in the absence and presence of LiCl (1.0 mM). After 5 days of incubation, survival of RGCs was assessed with a cytotoxicity staining kit (Live/Dead; Molecular Probes). In the absence of LiCl, the majority of RGCs died within 5 days; only 11.2% ± 2.8% survived (Figs. 3A 3C) . Addition of LiCl increased the survival of RGCs twofold, a significant effect (P < 0.05) and led to a 22.3% ± 5.8% survival rate of RGCs after 5 days of incubation (Figs. 3B 3C) . These results suggest that Li⁺ acts directly on RGCs to exert a neuroprotective effect. 

Axon Regeneration in the Retina-Brain Slice Coculture System

We have reported that Bcl-2 protects RGCs from apoptotic cell death and promotes regeneration of RGC axons. ⁶ In this study, we sought to determine whether the addition of Li⁺ would have an effect similar to that of Bcl-2 on regeneration of RGC axons. To study regeneration, we used a previously established model of retina-brain slice cocultures, in which a retinal explant was placed directly against a brain slice containing the target area of RGC axons, the SC. All RGC axons connecting to the brain were axotomized before culturing. Therefore, any fibers growing from retinal explants into brain slices after culturing would represent an active regeneration of RGC axons (the only type of retinal fibers that would innervate the brain). The coculture model offers many advantages over the in vivo models and the cell culture system for studying drug activities in regulating regeneration in the CNS. 

Taking advantages of this model, we used tissues derived from P2 wild-type (WT) mice to investigate whether Li⁺ would support the regeneration of RGC axons (Fig. 4A) . The number and lengths of neurites extending from the retina into the brain slices were quantified in the absence and presence of different concentrations of LiCl (Fig. 4B) . We found that, in the absence of LiCl, cultured retinal explants sent 18 ± 7.4 neurites into brain slices, with an average length of 220 ± 43 μm (n = 8). With increasing amounts of LiCl (0.2–5.0 mM), the number and length of neurites that extended into brain slices increased in a dose-dependent manner. The effect of LiCl peaked at 1.0 mM, with an average of 40 ± 9.8 neurites, with average axonal length of 780 ± 319 μm (n = 8), yielding an approximate threefold increase in both the number and length of regenerating axons over those in the untreated control. In previous clinical studies, ²⁵ Li⁺ has been shown to exert a toxic effect at concentrations higher than 3.5 mM. This was similar to the significantly reduced neurite outgrowth we observed in coculture preparations with concentration of LiCl greater than 5.0 mM. Thus, we conclude that Li⁺ is able to support the regeneration of retinal axons at its established therapeutic concentrations (0.5–1.2 mM). 

Bcl-2-Dependent Promotion of Regeneration

In this study, we noted a close correlation between the Li⁺-induced increase in expression of Bcl-2 and the increase in the number of surviving RGCs and the regeneration of axons, suggesting that regulation of expression of Bcl-2 is at least one of the mechanisms through which Li⁺ mediates RGC functions. To determine whether Bcl-2 is an essential component of Li⁺-mediated survival of RGCs and axonal regeneration, we studied genetically engineered mice that were either deficient in function of Bcl-2 (knockout, KO) or that had overexpression of the Bcl-2 transgene (TG). 

We first examined retina-brain slice cocultures prepared from Bcl-2 KO mice to determine whether Bcl-2 is necessary for Li⁺-induced function of RGCs. Bcl-2 KO mice were obtained from heterozygous breeding. Within each litter, only 25% of the mice were Bcl-2 homozygous KO, 50% were heterozygous for Bcl-2, and the other 25% were WT. Cocultures were prepared and scored blindly before mouse genotypes were determined by standard PCR methodology on mouse-tail DNAs. In agreement with our previous report, ⁶ in the absence of LiCl, cultures derived from Bcl-2 KO mice displayed much less vigorous axonal growth from retina into the brain slices than did those derived from WT and/or heterozygous littermates (Fig. 5) . There was more than a threefold reduction in the number of regenerating axons in cultures prepared from Bcl-2 homozygous KO mice compared with those from WT and heterozygous littermate control animals. Treatment with 1.0 mM LiCl failed to promote regeneration of retinal axons in cultures prepared from Bcl-2 KO mice, whereas it induced an approximate twofold increase in axon regeneration in cultures prepared from both WT and heterozygous mice (Fig. 5) . These results indicate that Bcl-2 is an essential contributor to Li⁺-induced regeneration of retinal axons. 

To determine whether induction of expression of Bcl-2 was the only factor contributing to the Li⁺-induced regeneration of RGC axons, we next studied mice that overexpress Bcl-2. Bcl-2 TG mice were obtained from matings of WT C57BL/6J females with males carrying a Bcl-2 transgene under the control of a neuron-specific enolase promoter (line 73a). ¹⁶ Thus, within each litter, half of the offspring were Bcl-2 TG, whereas the other half were WT mice to serve as littermate control animals. Retina-brain slice cocultures were prepared before mouse genotypes were decoded. Consistent with our previous findings, cocultures prepared from Bcl-2 TG mice without any treatment exhibited robust retinal axon regeneration in comparison with the cultures prepared from the WT littermates (Fig. 6) . Overexpression of Bcl-2 stimulated more than a fourfold increase in regeneration of axons from the retina into the brain slices over those of the WT control. The addition of LiCl (1.0 mM) to the cultures promoted extension of RGC axons into the brain sections in cultures prepared from WT mice, but had no effect in cultures prepared from Bcl-2 TG mice. Taken together, the findings led us to conclude that Li⁺ mediates the survival of RGCs and axonal regeneration through induction of expression of Bcl-2. 

Discussion

Our study showed that Li⁺, acting directly on RGCs, supported both neuronal survival and axonal regeneration at established therapeutic concentrations (0.5–1.2 mM). We further demonstrated that addition of LiCl induced expression of Bcl-2 mRNA in mouse retinas. Depletion (Bcl-2 KO) or overexpression (Bcl-2 TG) of Bcl-2 both eliminated the regenerative-promotional effect of LiCl in culture, indicating an essential role for Bcl-2 in this process. These results suggest, for the first time, that Li⁺ may be used as a therapeutic drug for treating retinal and optic nerve neurodegeneration (e.g., glaucoma and optic nerve neuritis) and conditions involving optic nerve damage and/or RGC loss. They also offer new clues toward a better understanding of the regulation of regeneration in the retina and CNS. 

Investigation of a drug’s effect on optic nerve regeneration in vivo is often impeded by the low efficiency of drug delivery, inconsistent surgical conditions, and other complex problems. In contrast, primary cell and tissue culture systems, although they do not present the same problems as does an in vivo model, present largely discrepant results compared with in vivo observations when applied to the study of regeneration of the optic nerve and CNS. These discrepancies are normally caused by disruption of cell-cell interactions, and the supplementation of artificial substrate in culture dishes. Our retina-brain slice coculture system is designed to circumvent these problems by use of retinal explants that maintain intercellular interactions and provide a natural environment (brain slice) for regenerating axons to navigate. Thus, retina-brain slice cocultures offer advantages over primary culture systems, in that they resemble the in vivo environment of severed axons, facilitating the drug screening process. 

In the present study, we demonstrate that Li⁺ supports both RGC survival and axonal regeneration, although less effectively (∼50%) than overexpression of Bcl-2 in the transgenic mouse model, which carries 10 to 20 copies of the transgene. We have demonstrated in coculture experiments that the level of expression of Bcl-2 in the retinal slices (not in the brain slices) influences the number of RGC axons regenerated. ⁶ This, taken together with results of purified RGC cultures, shows that Li⁺ acts directly on RGCs to promote their growth and survival. 

The similarity in the actions of Li⁺ and Bcl-2 on both survival of RGCs and axonal regeneration suggests a parallel mechanism at work. Consistent with this observation and with the reports by Manji et al. ¹⁰ and Chen and Chuang, ⁹ in our study Li⁺ induced Bcl-2 transcription in retinas. Most important, Li⁺ lost its regenerative effect on RGCs in the absence of Bcl-2, indicating an essential role for Bcl-2 in this process. Furthermore, we hypothesized that, if Li⁺ exerts its regenerative effect through induction of expression of Bcl-2, then overexpression of Bcl-2 in these neurons would also attenuate the effect of Li⁺. Using mice that overexpress Bcl-2 in neurons, we confirmed this hypothesis. Although the possibility of “ceiling effects” (the inability of neurons to enhance regeneration beyond a certain maximal level) cannot be entirely excluded, the data strongly suggest that induction of expression of Bcl-2 is a fundamental element in Li⁺-mediated regenerative function. 

Evidence is emerging that Bcl-2 is a key player in regulating both neural survival and axonal regeneration. ^{26 27 28 29} Moreover, the mechanism of Bcl-2 supporting the intrinsic growth potential of CNS axons is distinct from Bcl-2’s control of apoptosis. ^{6 30 31 32 33} We suggest two parallel mechanisms at work that influence regeneration in the CNS of adult mammals: loss of intrinsic, Bcl-2 supported growth potential by CNS axons and appearance of inhibitory molecules in the mature brain environment. We have shown in retina-brain slice cocultures that mature RGCs taken from Bcl-2-overexpressing mice show robust regeneration of axons in mice of all ages when cocultured with an embryonic (permissive) brain environment; however, they fail to regenerate axons if cocultured with adult brain slices (a nonpermissive environment). ⁶ Our recent studies further indicate that this inhibitory nature of the brain environment matures at approximately P5. Overexpression of Bcl-2 is sufficient to promote optic nerve regeneration if the injury is incurred at P3 in mice, but it fails at P5 or after (Ma HF, Chen DF, unpublished results, 2002). These data are supported by the reports of Chierzi et al. ³⁴ and Lodovichi et al. ⁷ showing no regeneration of the optic nerve in P5 and adult Bcl-2-overexpressing mice in vivo. Therefore, we suggest that for optic nerve regeneration to occur in the adult, it is essential to induce expression of Bcl-2 (e.g., by LiCl) in RGCs and to manipulate the CNS environment to make it permissive. 

Li⁺ continues to be one of the primary treatments for bipolar disorder, but the precise mechanisms by which this cation exerts its therapeutic effects have remained unclear. Although it is known that Li⁺ directly inhibits inositol monophosphatases and GSK-3β, ^{11 12 13} it is likely that the long-term changes in gene expression mediated by Li⁺ are responsible for its therapeutic efficacy. ^{35 36} In this context, recent evidence demonstrating that Li⁺ robustly upregulates Bcl-2, promotes cell survival, and enhances hippocampal neurogenesis has generated considerable excitement within the clinical neuroscience community. ^{37 38}  

Although it is still unclear how Li⁺ induces the expression of Bcl-2 in neurons, emerging evidence suggests three possibilities. First, Li⁺ has been shown to regulate directly the DNA binding activity of certain transcription factors, such as cAMP response element-binding protein, which binds the cAMP response element in the Bcl-2 gene promoter. ^{39 40} Second, Li⁺ may stimulate a phosphatidylinositol-3-kinase/Akt signaling cascade and, consequently, upregulate expression of Bcl-2 through activation of the cAMP response element-binding protein. ^{41 42} Third, Li⁺ may also elicit changes in expression of Bcl-2 by inhibiting the expression of p53, an established negative regulator of expression of Bcl-2. ^{9 43}  

Regardless of the mechanisms by which Li⁺ induces expression of Bcl-2 in RGCs, our results suggest that Li⁺ is a potential treatment for optic nerve injury, glaucoma, or other CNS degenerative processes involving neuronal cell loss and nerve damage. In view of Li’s well-established safety profile in humans and the fact that robust effects are observed at well-tolerated levels, clinical trails should be undertaken to investigate novel treatments for these devastating illnesses. 

 Supported by Grant R01 EY012983 from the National Eye Institute and grants from the Department of the Army, Lilly’s Foundation for Aging Research, the 50th Anniversary Program for Scholars in Medicine of Harvard Medical School, the Juvenile Diabetes Research Foundation, the Minda de Gunzburg Research Center for Retinal Transplantation, and the Ocular Gene Therapy Program, at the Schepens Eye Research Institute (DFC).

 Submitted for publication February 26, 2002; revised July 25, 2002; accepted August 9, 2002.

 Commercial relationships policy: P (HM, DFC); N (all others).

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Li⁺ induced endogenous expression of Bcl-2 in the mouse retina in a dose-dependent manner. (A) Photomicrograph shows the representative results of two replicate experiments of quantitative RT-PCR assessing the mRNA levels of Bcl-2 and G3PDH, a house-keeping gene, from cultured mouse retinas that were treated with differing concentrations of lithium. Levels of Bcl-2 mRNA increased markedly, in contrast to the consistent G3PDH levels. (B) Quantitative analysis of Bcl-2 mRNA levels in relation to the levels of G3PDH, by quantitative RT-PCR.

Figure 2.

 

Isolation of RGCs with Thy1.2 antibody conjugated with magnetic beads. Photomicrographs show isolated RGCs prelabeled with DiI and plated in culture for 1 hour. (A) Phase-contrast photomicrograph shows the morphology of isolated RGCs (arrowheads). (B, C) Epifluorescence photomicrographs reveal positive DiI labeling (B, arrowheads) and anti-Thy1.2 immunofluorescence staining (C, arrowheads) of isolated RGCs. Scale bar, 50 μm.

Figure 3.

 

Li⁺ supported survival of RGCs in culture. (A, B) Phase-contrast photomicrographs show isolated RGCs in culture in the absence (A) and presence (B) of LiCl (1 mM). Arrowheads: surviving RGCs. An increased number of surviving RGCs was noted in the Li⁺-treated culture in comparison with that of untreated cells. (C) Quantification of surviving RGCs in the absence and presence of LiCl after 5 days of incubation. Percentage of surviving RGCs was determined by dividing the number of surviving RGCs, counted at day 5 of culturing, by the number of RGCs originally plated. Data are presented as the mean ± SD (*P < 0.05). Scale bar, 50 μm.

Figure 4.

 

Li⁺ promoted RGC axon regeneration in a dose-dependent manner in culture. (A) Epifluorescence photomicrographs of representative retina-brain slice cocultures in the absence and presence of LiCl. Regenerating axons were labeled by placing DiI into retinal explants and were visualized by fluorescence microscope. Arrowheads: labeled axons growing into the brain slices. (B, C) Dose-response curve of Li⁺ on axon regeneration. (B) The number of labeled axons extending into the brain slices. (C) Quantification of the longest distances of axon regeneration into the brain slices, measured from the interfaces of the retinal explants and brain slices. (*P < 0.05 compared with control).

Figure 5.

 

The regeneration-promoting effect of Li⁺ was dependent on the expression of Bcl-2. The number (A) and length (B) of regenerating axons is shown in retina-brain slice cocultures that were prepared from postnatal mice: wild-type (+/+), Bcl-2 heterozygous (+/−), or Bcl-2 homozygous knockout (−/−). The cultures were maintained in the absence or presence of 1 mM LiCl. Error bars, SD.

Figure 6.

 

The regeneration-promoting effect of Li⁺ was attenuated by overexpression of Bcl-2. The number (A) and length (B) of regenerating axons were recorded from retina-brain slice cocultures prepared from tissues of WT mice or mice that overexpress the Bcl-2 transgene (Bcl-2 tg). Error bars, SD.