Growth arrest and DNA damage protein 45b (Gadd45b) protects retinal ganglion cells from injuries.

We hypothesize that neurons have protective mechanisms against adverse local conditions that improve the chances of cell survival. In the present study, we find that growth arrest and DNA damage protein 45b (Gadd45b), a previously unknown molecule in neurons of any type, is neuroprotective in retinal ganglion cells (RGCs) in the retina. Gadd45b is upregulated in RGCs in response to oxidative stress, aging and elevated intraocular pressure. Using Gadd45b siRNA, we show that Gadd45b protects RGCs from dying against different neuronal injuries including oxidative stress, TNFalpha cytotoxicity, and glutamate excitotoxicity in vitro. Using Gadd45b knockout mice, we find that Gadd45b protects RGCs from dying against oxidative stress in vivo. Our data suggest that Gadd45b is an important component of the intrinsic neuroprotective mechanisms of RGC neurons in the retina and, perhaps in the CNS as well.

Evaluation of HCN2 abnormalities as a cause of juvenile audiogenic seizures in Black Swiss mice.

Epilepsy is an often-debilitating disease with many etiologies. Genetic predisposition is common for many of the generalized epilepsy syndromes, and mutations in genes encoding neuronal ion channels are causative in many cases. We previously identified a locus for juvenile audiogenic monogenic seizures (jams1) in the Black Swiss mouse strain, delimited by the gene basigin (Bsg) and the marker D10Mit140. This region includes Hcn2, the gene encoding the hyperpolarization-activated cyclic nucleotide-gated channel subunit 2 (HCN2), an ion channel implicated in epilepsy. By sequencing genomic DNA, we found that Black Swiss mice have a single polymorphism in exon 2 within the Hcn2 gene. This single G/C to A/T base change alters the third position of a codon specifying alanine residue 293, without changing the predicted amino acid sequence. Furthermore, we found no detectable differences in HCN2 protein expression in the brains of Black Swiss mice, compared to control mice. We therefore reason that juvenile audiogenic seizures in Black Swiss mice are unlikely to be due to abnormalities of HCN2 channel function.

A laser-induced mouse model of chronic ocular hypertension to characterize visual defects.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

TGFß signaling induces expression of Gadd45b in retinal ganglion cells.

PURPOSE: Growth arrest and DNA damage protein 45b (Gadd45b) functions as an intrinsic neuroprotective molecule protecting retinal ganglion cells (RGCs) from injury. This study was performed to elucidate further the induction pathway of Gadd45b expression in RGCs. METHODS: The induction of Gadd45b expression in response to TGFßNFκB signaling was investigated in RGC5 cultures in vitro and murine retina in vivo. Gadd45b mRNA and protein expression were detected by quantitative real-time RT-PCR, immunoblot assay, immunohistochemistry, and immunocytochemistry. Activation of NFκB and TGFß/Gadd45b signaling were assessed by measuring phosphorylation of NFκB and using specific inhibitors. Gadd45b siRNA was transfected into RGC5 to silence Gadd45b mRNA expression. RESULTS: Expression of TGFß receptors I and II was detected in RGC5 in vitro and RGCs in vivo. TGFß induced abundant Gadd45b mRNA and protein expression, exhibiting a dose-dependent response in vitro. Exogenous TGFß1 induced upregulation of Gadd45b expression in RGCs in murine retina in vivo. TGFß stimulated phosphorylation of NFκB, and inhibition of NFκB phosphorylation blocked induction of Gadd45b by TGFß in RGC5 cells. Induction of Gadd45b by TGFß increased the resistance of RGC5 cells against TNFα cytotoxicity and paraquat oxidative stress. CONCLUSIONS: TGFß signaling induced Gadd45b expression in RGCs. Modulation of the TGFß/NFκB/Gadd45b signaling pathway may provide a means to enhance the neuroprotective effect of Gadd45b in RGCs.

Dysfunction of the retinal pigment epithelium with age: increased iron decreases phagocytosis and lysosomal activity.

PURPOSE: Iron accumulation with age in the retinal pigment epithelium (RPE) may be one important source of oxidative stress that contributes to age-related macular degeneration (AMD). Young and old rodent RPE/choroid were compared to assess iron homeostasis during normal aging and the effects of increased iron on the functions of retinal pigment epithelial cells. METHODS: The iron level, mRNA expression, and protein level of iron-regulatory molecules in RPE/choroid were quantitatively compared between young and old animals. To test the effects of increased intracellular iron on the functions of retinal pigment epithelial cells, in vitro ARPE-19 cells were treated with high levels of iron and assessed for phagocytosis activity and lysosomal activity. RESULTS: Iron level was significantly increased in the aged RPE/choroid. Ferritin and ceruloplasmin mRNAs were significantly increased in the aged RPE/choroid, whereas transferrin, transferrin receptor, and ferroportin mRNAs did not change with age. At the protein level, decreased transferrin and transferrin receptor, increased ferritin and ceruloplasmin, and unchanged ferroportin were observed in the aged RPE/choroid. Exposure of ARPE-19 cells to increased iron markedly decreased phagocytosis activity, interrupted cathepsin D processing, and reduced cathepsin D activity in retinal pigment epithelial cells. CONCLUSIONS: The RPE/choroid of aged animals demonstrates iron accumulation and associated alterations in iron homeostasis. Iron accumulation with age may impair the phagocytosis and lysosomal functions of retinal pigment epithelial cells in the aged RPE/choroid. Therefore, age-related changes of iron homeostasis in the RPE could increase the susceptibility of the tissue to genetic mutations associated with AMD.

Changes in iron-regulatory proteins in the aged rodent neural retina.

Iron accumulation is associated with age-related neurodegenerations and may contribute to age-related increased susceptibility of neurons to damage. We compared young and old rodent retinas to assess iron homeostasis during normal aging and the effects of increased iron on the susceptibility of retinal neurons to degeneration. Retinal iron was significantly increased with age. Quantitative RT-PCR showed that transferrin and ferritin genes were upregulated in the aged retina. At the protein level, we found decreased transferrin, and increased transferrin receptor, ferritin, ferroportin, and ceruloplasmin in the aged retina. These results support an increased steady state of iron with age in the retina. We tested susceptibility of retinal neurons with increased intracellular iron to damage in vitro. Exposure of RGC-5 cells to increased iron potentiated the neurotoxicity induced by paraquat, glutamate, and TNFalpha. Our results demonstrate that iron homeostasis in the retina is altered with age and suggest that iron accumulation, due to altered levels of iron-regulatory proteins in the aged retina, could be a susceptibility factor in age-related retinal diseases.