Interaction of neurotrophin signaling with Bcl‐2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth

Abstract

Enhanced spiral ganglion neuron (SGN) survival and regeneration of peripheral axons following deafness will likely enhance the efficacy of cochlear implants. Overexpression of Bcl-2 prevents SGN death, but inhibits neurite growth. Here we assessed the consequences of Bcl-2 targeted to either the mitochondria (GFP-Bcl-2-Maob) or endoplasmic reticulum (ER, GFP-Bcl-2-Cb5) on cultured SGN survival and neurite growth. Transfection of wild type GFP-Bcl-2, GFP-Bcl-2-Cb5, or GFP-Bcl-2-Maob increased SGN survival, with GFP-Bcl-2-Cb5 providing the most robust response. Paradoxically, expression of GFP-Bcl-2-Maob results in SGN death in the presence of neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF), neurotrophins that independently promote SGN survival via Trk receptors. This loss of SGNs is associated with cleavage of caspase 3 and appears specific for neurotrophin signaling, since co-expression of constitutively active mitogen activated kinase kinase (MEKΔEE) or phosphatidyl inositol-3 kinase (P110), but not other prosurvival stimuli (e.g. membrane depolarization), also results in the loss of SGNs expressing GFP-Bcl-2-Maob. MEKΔEE and P110 promote SGN survival while P110 promotes neurite growth to a greater extent than NT-3 or MEKΔEE. However wild-type GFP-Bcl-2, GFP-Bcl-2-Cb5 and GFP-Bcl-2-Maob inhibit neurite growth even in the presence of neurotrophins, MEKΔEE, or P110. Historically, Bcl-2 has been thought to act primarily at the mitochondria to prevent neuronal apoptosis. Nevertheless, our data show that Bcl-2 targeted to the ER is more effective at rescuing SGNs in the absence of trophic factors. Additionally, Bcl-2 targeted to the mitochondria results in SGN death in the presence of neurotrophins.

Influence of cAMP and protein kinase A on neurite length from spiral ganglion neurons

“…In this case the cultures were maintained in the absence of neurotrophic factors. We have previously shown that P110 and MKK-JNK1 each increase SGN neurite length (Atkinson et al, 2011; Renton et al, 2010). Expression of P110, but not MKK-JNK1 significantly increased neurite length in SGNs expressing GPKA (p<0.05) (Fig.…”

“…Activation of PI3-K or JNK are independently sufficient to increase SGN neurite length (Atkinson et al, 2011; Renton et al, 2010). We asked if activation of either of these could rescue neurite length in SGNs with activated PKA.…”

“…GPKA, GPKAnls, GPKAnes, GPKI, expression plasmids were kindly provided by Dr. Steven Green (University of Iowa, Iowa City, IA) (Bok et al, 2003). Green fluorescent protein (GFP), MKK-JNK1, and p110 expression plasmids have previously been described (Atkinson et al, 2011; Hansen et al, 2007; Renton et al, 2010). For co-transfections the ratio of plasmids was 1:1.…”

“…Images were captured of the first ~15 transfected neurons encountered as the observer scanned through each well from top to bottom. The mean neurite length for each experimental repetition and standard error of the mean (SEM) was determined using Excel software (Microsoft, Seattle, WA) (Atkinson et al, 2011; Renton et al, 2010). Significance for differences in neurite length among treatment groups was determined by a one-way ANOVA followed by a Holm-Sidak post-hoc comparison in SigmaStat (Systat Software, Chicago, IL).…”

Parecoxib Protects Mouse Cortical Neurons Against OGD/R Induced Neurotoxicity by Up-Regulating Bcl-2

“…Interestingly, accumulating evidence suggested that ER was involved in regulating apoptosis, either independent of mitochondrial, or in concert with mitochondria-initiated pathways [24]. Moreover, it is worth noting that Bcl-2 protein can localize and target to the mitochondria and ER both, and promote neuron survival [25]. It is possible that Bcl-2, as a key effector of parecoxib, inhibits OGD/R induced neuronal apoptosis via cross-talk between ER and mitochondriadependent death cascades.…”