Neuroglobin involvement in respiratory chain function and retinal ganglion cell integrity

Abstract

Neuroglobin is a member of the globin superfamily expressed in vertebrate brain and retina. The protein is thought to be involved in neuronal protection from hypoxia or oxidative stress and could represent a key element of Alzheimer disease pathogenesis. Our aim was to determine whether neuroglobin could be directly associated with mitochondrial metabolism and integrity. We identified three different forms of neuroglobin in the retina, varying in their apparent molecular masses; all forms are abundant in mitochondrial fractions. This indicates that a significant fraction of the protein localizes within the organelle either in the matrix or in the matrix side of the inner membrane. Since neuroglobin was especially abundant in the ganglion cell layer, we transduced retinal ganglion cells with an anti-neuroglobin short hairpin RNA using in vivo electroporation. Neuroglobin knockdown leads to reduced activities of respiratory chain complexes I and III, degeneration of retinal ganglion cells, and impairment of visual function. The deleterious effect on cell survival was confirmed in primary retinal ganglion cells subjected to inhibition of neuroglobin expression. Hence, neuroglobin should be considered as a novel mitochondrial protein involved in respiratory chain function which is essential for retinal ganglion cell integrity.

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…This phenomenon of dimerization, resistant to heat denaturation and reduction, has been observed previously for Ngb in whole protein extracts from mouse brain and retinas [45][46][47][48]. Furthermore, it has been shown that three forms of apparent molecular masses (∼17, ∼19 and ∼21 kDa), specifically recognized by anti-Ngb antibodies, were identified in mitochondrial enriched fractions of mouse retina [49]. This pattern of three different molecular mass forms is similar to the one that is seen after expression of NGB in Sf9 insect cells (Fig.…”

“…3A). These forms might represent atypical migrations of stable protein conformations and it has been hypothesized that rupture/ formation of the intra molecular (CD7-B5) disulfide bond of NGB [50] can give rise to different apparent molecular mass forms [49]. Posttranslational modifications could also occur, such as covalent links of cofactors or phosphorylation.…”

Effect of light on global gene expression in the neuroglobin-deficient mouse retina

“…A number of studies have reported high levels of Ngb in the retina and adverse effects of Ngb knockdown on neuronal survival, leading the hypothesis that Ngb is important for retinal oxygen homeostasis and neuronal survival (7)(8)(9)(10)(11)(12)(13)(14)(30)(31)(32). Recently, Chan et al (13) found that transgenic overexpression of Ngb conferred protection against retinal ischemia via a decrease in oxidative stress levels.…”

“…Recently, Chan et al (13) found that transgenic overexpression of Ngb conferred protection against retinal ischemia via a decrease in oxidative stress levels. Similarly, Lechauve et al (14) showed that siRNA knockdown of Ngb reduced mitochondrial activity, induced cell death and had an adverse effect on visual performance. In the present study, genome-wide transcriptional Expression was normalized to hypoxanthine phosphoribosyltransferase 1 (Htpr1) as the internal reference (n=9-10 in each group).…”

“…Previously, several studies have implicated a neuroprotective function of Ngb in retinal diseases (9)(10)(11)(12). Furthermore, transgenic mice overexpressing Ngb were found to be resistant to retinal ischemia (13) and in vivo knockdown of Ngb using small interfering RNA (siRNA) induced the degradation of retina ganglion cells and behavioral effects associated with compromised visual performance (14). In contrast to these studies, we recently showed limited Ngb immunoreactivity in the retina and no effect of Ngb deficiency on neuronal survival using genetically Ngb-deficient (Ngb-null) mice (15).…”