Gamma-secretase-regulated proteolysis of the Notch receptor by mitochondrial intermediate peptidase.

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.

RPE-derived factors modulate photoreceptor differentiation: a possible role in the retinal stem cell niche.

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, approximately 63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.

Glutamate stimulates neurotrophin expression in cultured Müller cells.

The uptake of excess extracellular glutamate and the secretion of neurotrophins by glial cells have been suggested to protect CNS neurons from glutamate-induced toxicity. In the retina, perturbation of glutamate transport and decreased retrograde transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may contribute to ganglion cell death in experimental glaucoma. Although many studies show a clear relationship between glutamate and neurotrophic factors, such relationship has not been thoroughly investigated in the retinal environment. In the following study, we determined the effects of glutamate on early passaged rat Müller cells, specifically their expression of neurotrophic factors including BDNF, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial-cell line derived neurotrophic factor (GDNF); and of glutamate receptors and transporters using immunoblots or enzyme-linked immunosorbent assays. Binding of BDNF to its cognate receptor TrkB was also determined using co-immunoprecipitation studies. Cultured Müller cells grown in the presence of glutamate were also assayed for survival using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Our study showed that while glutamate treatment did not promote cell death, it upregulated secretion of BDNF, NGF, NT-3, NT-4, and GDNF by Müller cells. While solitary bands at approximately 13-14 kDa were observed for NGF, NT-3, and NT-4; two BDNF-reactive bands were observed in immunoblots: a faster migrating band at the reported size of the BDNF monomer (approximately 13 kDa); and a more intense band at approximately 36 kDa. GDNF-reactive bands were observed at approximately 22, approximately 28, and approximately 55 kDa. Glutamate also induced significant changes in glutamate receptor and transporter proteins, as well maintained the association of BDNF to TrkB in Müller cells. The decreased N-methyl-D-aspartate receptor (NMDAR) levels and sustained activation of TrkB by BDNF could serve as protective mechanisms for Müller cell survival. Moreover, the increased secretion of neurotrophic factors and upregulation of L-glutamate/L-aspartate transporter (GLAST) expression in Müller cells may protect retinal neurons from glutamate toxicity.

Expression of p75(NTR) in photoreceptor cells of dystrophic rat retinas.

Although a gene mutation in the Royal College of Surgeons (RCS) dystrophic rat results in defective phagocytosis and in accumulation of debris in the subretinal space, the molecular mechanisms leading to photoreceptor cell death remain unclear. In this study, the expression of p75(NTR), the low-affinity neurotrophin receptor incriminated in the apoptosis of developing neurons, was investigated at various stages of retinal degeneration in dystrophic rats using immunohistochemistry, in situ reverse transcription polymerase chain reaction (RT-PCR), Western blot, and relative RT-PCR. In normal adult retinas, p75(NTR) immunolabeling was observed mainly in the outer limiting membrane, with punctate labeling in the inner nuclear and ganglion cell layers. In 18- to 30-day-old dystrophic retinas, the immunostaining appeared to increase especially in the photoreceptor outer and inner segments. Dense staining was also observed in the retinal pigment epithelium (RPE) and choroid. In 60-day-old dystrophic rat retinas, the density of immunolabeling for p75(NTR) increased dramatically in the remaining inner retina, especially in the inner nuclear, inner plexiform, and ganglion cell layers. Post-embedding immunogold labeling of normal retinas verified the distribution of p75(NTR) in photoreceptor cells within the inner segments, cell bodies, and outer segments. The apparent increased intensity in p75(NTR) immunostaining in dystrophic retinas was verified by Western blots and densitometric analyses. In situ RT-PCR and relative RT-PCR further established increased synthesis of p75(NTR) in dystrophic retinas. The increased levels of p75(NTR) in the RPE and photoreceptor cells, the initial sites of injury, during retinal degeneration in dystrophic rats strongly suggest that altered expression of p75(NTR) may be directly involved in photoreceptor death.

Photic injury promotes cleavage of p75NTR by TACE and nuclear trafficking of the p75 intracellular domain.

The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor superfamily that paradoxically mediates neuronal survival and differentiation or apoptotic cell death. Cleavage of p75NTR by a constitutively active metalloprotease could result in shedding of its extracellular domain (p75ECD) and generation of a pro-apoptotic intracellular domain (p75ICD). In this study, we established that exposure of a transgenic mouse photoreceptor cell line to intense light upregulated the expression of p75NTR and of the disintegrin metalloprotease tumor necrosis factor-converting enzyme (TACE) and resulted in apoptotic cell death. Light damage promoted TACE cleavage of p75NTR resulting in shedding of the soluble p75ECD and nuclear translocation of the p75ICD. Overexpression of TACE and p75NTR-induced p75NTR cleavage and secretion of p75ECD, but not nuclear transport of p75ICD. Light-induced cleavage of p75NTR, nuclear localization of p75ICD, and apoptosis were inhibited by IC-3, a metalloprotease inhibitor. Increased levels of p75NTR and TACE were observed in photoreceptor cells of animals with photic injury. Our findings support a role for TACE in the proteolytic cleavage of p75NTR and light-induced apoptosis.

Microglia-derived pronerve growth factor promotes photoreceptor cell death via p75 neurotrophin receptor.

Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a approximately 32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The approximately 32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR(+)/trkA(-) 661w cells. Our study shows that a approximately 32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.