A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.

BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.

The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK.

The intrinsic apoptosis pathway, regulated by the BCL-2 protein family, is essential for embryonic development. Using mice lacking all known apoptosis effectors, BAX, BAK and BOK, we have previously defined the processes during development that require apoptosis. Rare Bok-/- Bax-/- Bak-/- triple knockout (TKO) mice developed to adulthood and several tissues that were thought to require apoptosis during development appeared normal. This raises the question if all apoptosis had been abolished in the TKO mice or if other BCL-2 family members could act as effectors of apoptosis. Here, we investigated the role of BID, generally considered to link the extrinsic and intrinsic apoptosis pathways, acting as a BH3-only protein initiating apoptosis upstream of BAX and BAK. We found that Bok-/- Bax-/- Bak-/- Bid-/- quadruple knockout (QKO) mice have additional developmental anomalies compared to TKO mice, consistent with a role of BID, not only upstream but also in parallel to BAX, BAK and BOK. Mitochondrial experiments identified a small cytochrome c-releasing activity of full-length BID. Collectively, these findings suggest a new effector role for BID in the intrinsic apoptosis pathway.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Recording of Electrically Evoked Neural Activity and Bladder Pressure Responses in Awake Rats Chronically Implanted With a Pelvic Nerve Array.

Bioelectronic medical devices are well established and widely used in the treatment of urological dysfunction. Approved targets include the sacral S3 spinal root and posterior tibial nerve, but an alternate target is the group of pelvic splanchnic nerves, as these contain sacral visceral sensory and autonomic motor pathways that coordinate storage and voiding functions of the bladder. Here, we developed a device suitable for long-term use in an awake rat model to study electrical neuromodulation of the pelvic nerve (homolog of the human pelvic splanchnic nerves). In male Sprague-Dawley rats, custom planar four-electrode arrays were implanted over the distal end of the pelvic nerve, close to the major pelvic ganglion. Electrically evoked compound action potentials (ECAPs) were reliably detected under anesthesia and in chronically implanted, awake rats up to 8 weeks post-surgery. ECAP waveforms showed three peaks, with latencies that suggested electrical stimulation activated several subpopulations of myelinated A-fiber and unmyelinated C-fiber axons. Chronic implantation of the array did not impact on voiding evoked in awake rats by continuous cystometry, where void parameters were comparable to those published in naïve rats. Electrical stimulation with chronically implanted arrays also induced two classes of bladder pressure responses detected by continuous flow cystometry in awake rats: voiding contractions and non-voiding contractions. No evidence of tissue pathology produced by chronically implanted arrays was detected by immunohistochemical visualization of markers for neuronal injury or noxious spinal cord activation. These results demonstrate a rat pelvic nerve electrode array that can be used for preclinical development of closed loop neuromodulation devices targeting the pelvic nerve as a therapy for neuro-urological dysfunction.

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin.

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology.

Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet-On cells. Over-expression of SOCS2 in PC12 Tet-On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over-expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2-induced neurite outgrowth in PC12 Tet-On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.

A small peptide mimetic of brain-derived neurotrophic factor promotes peripheral myelination.

The expression of the neurotrophins and their receptors is essential for peripheral nervous system development and myelination. We have previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts contrasting influences upon Schwann cell myelination in vitro - promoting myelination via neuronally expressed p75NTR, but inhibiting myelination via neuronally expressed TrkB. We have generated a small peptide called cyclo-dPAKKR that structurally mimics the region of BDNF that binds p75NTR. Here, we have investigated whether utilizing cyclo-dPAKKR to selectively target p75NTR is an approach that could exert a unified promyelinating response. Like BDNF, cyclo-dPAKKR promoted myelination of nerve growth factor-dependent neurons in vitro, an effect dependent on the neuronal expression of p75NTR. Importantly, cyclo-dPAKKR also significantly promoted the myelination of tropomyosin-related kinase receptor B-expressing neurons in vitro, whereas BDNF exerted a significant inhibitory effect. This indicated that while BDNF exerted a contrasting influence upon the myelination of distinct subsets of dorsal root ganglion (DRG) neurons in vitro, cyclo-dPAKKR uniformly promoted their myelination. Local injection of cyclo-dPAKKR adjacent to the developing sciatic nerve in vivo significantly enhanced myelin protein expression and significantly increased the number of myelinated axons. These results demonstrate that cyclo-dPAKKR promotes peripheral myelination in vitro and in vivo, suggesting it is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases.

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling.

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.