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Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice.

Mitton KP, Guzman AE, Deshpande M, Byrd D, DeLooff C, Mkoyan K, Zlojutro P, Wallace A, Metcalf B, Laux K, Sotzen J, Tran T - Mol. Vis. (2014)

Bottom Line: Nrl gene expression was decreased by 50%, while Crx gene expression was not affected.Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28.While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model.

View Article: PubMed Central - PubMed

Affiliation: Control of Gene Expression Laboratory and Pediatric Retinal Research Laboratory, Eye Research Institute, Oakland University, Rochester, MI.

ABSTRACT

Purpose: The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA's adoption as an anticonvulsant. This generated speculation for VPA's potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA's potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA's effects on the rate of photoreceptor loss in two strains of mice, Pde6b(rd1/rd1) and Pde6b(rd10/rd10) .

Methods: The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6b(rd1/rd1) mice. Pde6b(rd1/rd1) mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6b(rd10/rd10) mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6b(rd10/rd10) mice to measure retinal function.

Results: In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9-P21) dramatically slowed the loss of rod photoreceptors in Pde6b(rd1/rd1) mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6b(rd10/rd10) mice (P17-P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6b(rd10/rd10) mice earlier (P9-P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9-P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals.

Conclusions: A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6b(rd1/rd1) mice (P9-P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA's efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17-P28) started when the central retina was mature.

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Effect of a single systemic dose of valproic acid (VPA) on neurotrophic factor gene expression and rod-specific gene expression in the immature retina of wild-type mice. A: Expression of the Gdnf, Bdnf, and Cntf genes in the neural retinas of wild-type mice (C57BL/6) was examined at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after one systemic VPA dose. Littermates at each age received a high dose (415 mg/kg), low dose (250 mg/kg), or PBS. The graph shows the average of these experiments (mean±standard deviation [SD]). Gene expression was normalized to endogenous Actb expression and is plotted relative to control PBS-injected littermates (t test, * p<0.01, ** p<0.001, relative to PBS). B: Retinal Nrl gene expression at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after a single systemic injection (IP) of VPA. Littermates within each age received a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or PBS only (control). The graph shows the average of the two experiments (mean±SD; t test, *p<0.05 relative to PBS). C: Rod-specific Mef2c gene expression at age P15, 18 h after treatment with a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or control (PBS) (t test, *p<0.05 relative to PBS, n = 3/group). D: Rhodopsin gene (Rho) expression in the P12 neural retina, 18 h after injection (IP) with a high or low dose of VPA compared to PBS only (mean±SD; n = 2/group).
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f1: Effect of a single systemic dose of valproic acid (VPA) on neurotrophic factor gene expression and rod-specific gene expression in the immature retina of wild-type mice. A: Expression of the Gdnf, Bdnf, and Cntf genes in the neural retinas of wild-type mice (C57BL/6) was examined at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after one systemic VPA dose. Littermates at each age received a high dose (415 mg/kg), low dose (250 mg/kg), or PBS. The graph shows the average of these experiments (mean±standard deviation [SD]). Gene expression was normalized to endogenous Actb expression and is plotted relative to control PBS-injected littermates (t test, * p<0.01, ** p<0.001, relative to PBS). B: Retinal Nrl gene expression at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after a single systemic injection (IP) of VPA. Littermates within each age received a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or PBS only (control). The graph shows the average of the two experiments (mean±SD; t test, *p<0.05 relative to PBS). C: Rod-specific Mef2c gene expression at age P15, 18 h after treatment with a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or control (PBS) (t test, *p<0.05 relative to PBS, n = 3/group). D: Rhodopsin gene (Rho) expression in the P12 neural retina, 18 h after injection (IP) with a high or low dose of VPA compared to PBS only (mean±SD; n = 2/group).

Mentions: The expression of neurotrophic factor genes in the neural retinas of wild-type mice (C57BL/6) were examined at two postnatal ages, P12 and P15, just 18 h after a single systemic injection of VPA. Littermates at each age were injected (IP) with either a high dose of VPA (415 mg/kg), low dose of VPA (250 mg/kg), or vehicle (PBS) as controls. Results were the same at both ages, and a graph showing the average of these experiments is presented in Figure 1A. There was a dose response to VPA, with statistically significant increases in the relative expression of Gdnf and Bdnf as measured by real-time PCR. At the higher VPA dose, Gdnf (OMIM 600837) expression increased 12-fold (p<0.001, t test) and Bdnf expression increased twofold (p<0.01), compared to PBS-injected littermates. Cntf (OMIM 118945) gene expression decreased 80% (p<0.001) compared to PBS-injected littermates. In contrast to younger neural retinas, the retinal expression of these genes in adult retinas was not perturbed substantially by a single dose. While there was a trend to shift the expression of neurotrophic factors, the changes were not judged to be statistically significant compared to PBS-injected animals (data not shown).


Different effects of valproic acid on photoreceptor loss in Rd1 and Rd10 retinal degeneration mice.

Mitton KP, Guzman AE, Deshpande M, Byrd D, DeLooff C, Mkoyan K, Zlojutro P, Wallace A, Metcalf B, Laux K, Sotzen J, Tran T - Mol. Vis. (2014)

Effect of a single systemic dose of valproic acid (VPA) on neurotrophic factor gene expression and rod-specific gene expression in the immature retina of wild-type mice. A: Expression of the Gdnf, Bdnf, and Cntf genes in the neural retinas of wild-type mice (C57BL/6) was examined at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after one systemic VPA dose. Littermates at each age received a high dose (415 mg/kg), low dose (250 mg/kg), or PBS. The graph shows the average of these experiments (mean±standard deviation [SD]). Gene expression was normalized to endogenous Actb expression and is plotted relative to control PBS-injected littermates (t test, * p<0.01, ** p<0.001, relative to PBS). B: Retinal Nrl gene expression at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after a single systemic injection (IP) of VPA. Littermates within each age received a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or PBS only (control). The graph shows the average of the two experiments (mean±SD; t test, *p<0.05 relative to PBS). C: Rod-specific Mef2c gene expression at age P15, 18 h after treatment with a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or control (PBS) (t test, *p<0.05 relative to PBS, n = 3/group). D: Rhodopsin gene (Rho) expression in the P12 neural retina, 18 h after injection (IP) with a high or low dose of VPA compared to PBS only (mean±SD; n = 2/group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4225157&req=5

f1: Effect of a single systemic dose of valproic acid (VPA) on neurotrophic factor gene expression and rod-specific gene expression in the immature retina of wild-type mice. A: Expression of the Gdnf, Bdnf, and Cntf genes in the neural retinas of wild-type mice (C57BL/6) was examined at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after one systemic VPA dose. Littermates at each age received a high dose (415 mg/kg), low dose (250 mg/kg), or PBS. The graph shows the average of these experiments (mean±standard deviation [SD]). Gene expression was normalized to endogenous Actb expression and is plotted relative to control PBS-injected littermates (t test, * p<0.01, ** p<0.001, relative to PBS). B: Retinal Nrl gene expression at two postnatal ages, P12 (n = 2/group) and P15 (n = 3/group), 18 h after a single systemic injection (IP) of VPA. Littermates within each age received a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or PBS only (control). The graph shows the average of the two experiments (mean±SD; t test, *p<0.05 relative to PBS). C: Rod-specific Mef2c gene expression at age P15, 18 h after treatment with a high dose (VPA 415 mg/kg), low dose (VPA 250 mg/kg), or control (PBS) (t test, *p<0.05 relative to PBS, n = 3/group). D: Rhodopsin gene (Rho) expression in the P12 neural retina, 18 h after injection (IP) with a high or low dose of VPA compared to PBS only (mean±SD; n = 2/group).
Mentions: The expression of neurotrophic factor genes in the neural retinas of wild-type mice (C57BL/6) were examined at two postnatal ages, P12 and P15, just 18 h after a single systemic injection of VPA. Littermates at each age were injected (IP) with either a high dose of VPA (415 mg/kg), low dose of VPA (250 mg/kg), or vehicle (PBS) as controls. Results were the same at both ages, and a graph showing the average of these experiments is presented in Figure 1A. There was a dose response to VPA, with statistically significant increases in the relative expression of Gdnf and Bdnf as measured by real-time PCR. At the higher VPA dose, Gdnf (OMIM 600837) expression increased 12-fold (p<0.001, t test) and Bdnf expression increased twofold (p<0.01), compared to PBS-injected littermates. Cntf (OMIM 118945) gene expression decreased 80% (p<0.001) compared to PBS-injected littermates. In contrast to younger neural retinas, the retinal expression of these genes in adult retinas was not perturbed substantially by a single dose. While there was a trend to shift the expression of neurotrophic factors, the changes were not judged to be statistically significant compared to PBS-injected animals (data not shown).

Bottom Line: Nrl gene expression was decreased by 50%, while Crx gene expression was not affected.Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28.While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model.

View Article: PubMed Central - PubMed

Affiliation: Control of Gene Expression Laboratory and Pediatric Retinal Research Laboratory, Eye Research Institute, Oakland University, Rochester, MI.

ABSTRACT

Purpose: The histone-deacetylase inhibitor activity of valproic acid (VPA) was discovered after VPA's adoption as an anticonvulsant. This generated speculation for VPA's potential to increase the expression of neuroprotective genes. Clinical trials for retinitis pigmentosa (RP) are currently active, testing VPA's potential to reduce photoreceptor loss; however, we lack information regarding the effects of VPA on available mammalian models of retinal degeneration, nor do we know if retinal gene expression is perturbed by VPA in a predictable way. Thus, we examined the effects of systemic VPA on neurotrophic factor and Nrl-related gene expression in the mouse retina and compared VPA's effects on the rate of photoreceptor loss in two strains of mice, Pde6b(rd1/rd1) and Pde6b(rd10/rd10) .

Methods: The expression of Bdnf, Gdnf, Cntf, and Fgf2 was measured by quantitative PCR after single and multiple doses of VPA (intraperitoneal) in wild-type and Pde6b(rd1/rd1) mice. Pde6b(rd1/rd1) mice were treated with daily doses of VPA during the period of rapid photoreceptor loss. Pde6b(rd10/rd10) mice were also treated with systemic VPA to compare in a partial loss-of-function model. Retinal morphology was assessed by virtual microscopy or spectral-domain optical coherence tomography (SD-OCT). Full-field and focal electroretinography (ERG) analysis were employed with Pde6b(rd10/rd10) mice to measure retinal function.

Results: In wild-type postnatal mice, a single VPA dose increased the expression of Bdnf and Gdnf in the neural retina after 18 h, while the expression of Cntf was reduced by 70%. Daily dosing of wild-type mice from postnatal day P17 to P28 resulted in smaller increases in Bdnf and Gdnf expression, normal Cntf expression, and reduced Fgf2 expression (25%). Nrl gene expression was decreased by 50%, while Crx gene expression was not affected. Rod-specific expression of Mef2c and Nr2e3 was decreased substantially by VPA treatment, while Rhodopsin and Pde6b gene expression was normal at P28. Daily injections with VPA (P9-P21) dramatically slowed the loss of rod photoreceptors in Pde6b(rd1/rd1) mice. At age P21, VPA-treated mice had several extra rows of rod photoreceptor nuclei compared to PBS-injected littermates. Dosing started later (P14) or dosing every second day also rescued photoreceptors. In contrast, systemic VPA treatment of Pde6b(rd10/rd10) mice (P17-P28) reduced visual function that correlated with a slight increase in photoreceptor loss. Treating Pde6b(rd10/rd10) mice earlier (P9-P21) also failed to rescue photoreceptors. Treating wild-type mice earlier (P9-P21) reduced the number of photoreceptors in VPA-treated mice by 20% compared to PBS-treated animals.

Conclusions: A single systemic dose of VPA can change retinal neurotrophic factor and rod-specific gene expression in the immature retina. Daily VPA treatment from P17 to P28 can also alter gene expression in the mature neural retina. While daily treatment with VPA could significantly reduce photoreceptor loss in the rd1 model, VPA treatment slightly accelerated photoreceptor loss in the rd10 model. The apparent rescue of photoreceptors in the rd1 model was not the result of producing more photoreceptors before degeneration. In fact, daily systemic VPA was toxic to wild-type photoreceptors when started at P9. However, the effective treatment period for Pde6b(rd1/rd1) mice (P9-P21) has significant overlap with the photoreceptor maturation period, which complicates the use of the rd1 model for testing of VPA's efficacy. In contrast, VPA treatment started after P17 did not cause photoreceptor loss in wild-type mice. Thus, the acceleration of photoreceptor loss in the rd10 model may be more relevant where both photoreceptor loss and VPA treatment (P17-P28) started when the central retina was mature.

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Related in: MedlinePlus