4-Hydroxy-2-Nonenal Induces Mitochondrial Dysfunction and Aberrant Axonal Outgrowth in Adult Sensory Neurons that Mimics Features of Diabetic Neuropathy

Modification of proteins by 4-hydroxy-2-nonenal (4-HNE) has been proposed to cause neurotoxicity in a number of neurodegenerative diseases, including distal axonopathy in diabetic sensory neuropathy. We tested the hypothesis that exposure of cultured adult rat sensory neurons to 4-HNE would result in the formation of amino acid adducts on mitochondrial proteins and that this process would be associated with impaired mitochondrial function and axonal regeneration. In addition, we compared 4-HNE-induced axon pathology with that exhibited by neurons isolated from diabetic rats. Cultured adult rat dorsal root ganglion (DRG) sensory neurons were incubated with varying concentrations of 4-HNE. Cell survival, axonal morphology, and level of axon outgrowth were assessed. In addition, video microscopy of live cells, western blot, and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE and to localize actively respiring mitochondria. 4-HNE induced formation of protein adducts on cytoskeletal and mitochondrial proteins, and impaired axon regeneration by approximately 50% at 3 μM while having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures and caused the accumulation of mitochondria in these dystrophic structures. Neurons treated with 4-HNE exhibited a distal loss of active mitochondria. Finally, the distal axonopathy and the associated aberrant axonal structures generated by 4-HNE treatment mimicked axon pathology observed in DRG sensory neurons isolated from diabetic rats and replicated aspects of neurodegeneration observed in human diabetic sensory neuropathy.     

Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk-1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk-1 receptor and showed an increased mRNA expression and immunoreactivity for flk-1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk-1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk-1-positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene-related peptide (CGRP). Small IB4-positive neurons were devoid of flk-1 immunoreactivity. Most flk-1-positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin-1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF-induced axonal outgrowth was blocked by the flk-1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.     

Role of phosphatidylinositol 3-kinase in neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia explants

Adult ganglionic peripheral neurons have lost dependence on target-derived neurotrophin signaling for survival and regeneration after injury. To understand the mechanisms required to sustain such processes at maturity, we are studying neuronal survival and axonal outgrowth of adult mouse dorsal root ganglia (DRG) explants. We have here examined the role of phosphatidylinositol 3-kinase (PI3-K) activity. Both neuronal survival and axonal outgrowth of spontaneously growing preparations were decreased significantly by the PI3-K inhibitor LY294002 as was the increased outgrowth caused by nerve growth factor or glial cell line-derived factor. Inhibition of PI3-K activity promoted neuronal cell death to the same extent in the presence as in the absence of a growth factor, whereas inhibition of mitogen-activated protein kinase, MAPK, lacked effect. Using a compartmentalized system, it could be shown that only axonal outgrowth was decreased when the outgrowth region only was exposed to LY294002. Already-formed growth cones showed morphological changes within 5-10 min after exposure to LY294002. Akt (PKB) is one downstream effector of PI3-K. Immunofluorescence revealed the presence of activated Akt in DRG cell bodies and in axonal growth cones. Immunoreactivity was decreased by PI3-K inhibition. The results suggest that Akt is constitutively active in adult DRG neurons, and that PI3-K mediated processes are involved in neuronal survival of one or more DRG neuronal subpopulations and also in axonal elongation. The possible significance of Akt signaling for these effects is discussed.     

体外低频电刺激促进背根神经元突起的生长：上调钙离子介导的脑源性神经营养因子表达可为其途径

摘要 背景：短时低频电刺激已被证明可显著促进周围神经系统损伤后轴突的再生,目前对电刺激是如何促进其突起生长还有待证实。 目的：体外培养背根神经元,观察短时低频电刺激对神经元突起生长的影响,探讨电刺激发挥作用可能的细胞信号分子。 设计、时间及地点：体外培养背根神经元及离体电刺激处理,于2007-05/2008-10在上海交通大学医学院完成。 材料：新生48h Sprague-Dawley大鼠20只(中科院上海生命科学研究所动科所)。 方法：体外培养背根神经元,随机分为两组,正常对照组(n = 6)及电刺激组(n = 8)。电刺激组施予离体电刺激(20Hz, 100μs, 3V),持续作用1h。为探讨电刺激发挥作用经由的细胞信号分子,在施予电刺激前预先加入钙离子通道阻滞剂Nifedipine孵育4小时,再给予电刺激,再次检测各组神经元突起的生长情况。 主要观察指标：β-tubulin染神经元,测量各组神经元突起的长度。RT-PCR、 western blot和ELISA分别检测神经元BDNF的表达和分泌。 结果：短时低频电刺激促进神经元突起的生长,增强其表达和分泌BDNF (P < 0.05)。Nifedipine的使用削弱了电刺激对神经元突起生长及BDNF合成的促进作用 (P < 0.05)。 结论：短时低频电刺激促进体外培养的背根神经元突起的生长及BDNF的合成,初步认为电刺激对神经元突起生长的促进作用,至少通过促发钙内流所致BDNF表达和分泌增多所致。 关键词：电刺激;背根神经元;突起生长;BDNF;Ca2+     

Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.     

Regulation of mitochondrial dynamics and distribution by synapse position and neuronal activity in the axon

Proper distribution of axonal mitochondria is critical for multiple neuronal functions. To understand the underlying mechanisms for population behavior, quantitative characterisation of elemental dynamics on multiple time scales is required. Here we investigated the stability and transport of axonal mitochondria using live‐cell imaging of cultured mouse hippocampal neurons. We first characterised the long‐term stability of stationary mitochondria. At a given moment, about 10% of the mitochondria were in a state of transport and the remaining 90% were stationary. Among these stationary mitochondria, 40% of them remained in the same position over several days. The rest of the mitochondria transited to mobile state stochastically and this process could be detected and quantitatively analysed by time‐lapse imaging with intervals of 30 min. The stability of axonal mitochondria increased from 2 to 3 weeks in culture, was decreased by tetrodotoxin treatment, and was higher near synapses. Stationary mitochondria should be generated by pause of moving mitochondria and subsequent stabilisation. Therefore, we next analysed pause events of moving mitochondria by repetitive imaging at 0.3 Hz. We found that the probability of transient pause increased with field stimulation, decreased with tetrodotoxin treatment, and was higher near synapses. Finally, by combining parameters obtained from time‐lapse imaging with different time scales, we could estimate transition rates between different mitochondrial states. The analyses suggested specific developmental regulation in the probability of paused mitochondria to transit into stationary state. These findings indicate that multiple mitochondrial behaviors, especially those regulated by neuronal activity and synapse location, determine their distribution in the axon.     

The Janus role of c-Jun: cell death versus survival and regeneration of neonatal sympathetic and sensory neurons

We investigated the functional outcome of c-Jun activation in sympathetic and sensory neurons of neonatal rat superior cervical ganglion (SCG) and dorsal root ganglion (DRG), respectively. Distinctly different roles of c-Jun activation have been suggested for these two types of neurons. In dissociated sympathetic neurons, c-Jun has been demonstrated to promote apoptosis, whereas in sensory neurons it stimulates axonal outgrowth. In organ-cultured ganglia, we found that c-Jun was activated within 24 h of explantation in both types of neurons, and that the JNK inhibitor SP600125 could mitigate this response. In both types of neurons, c-Jun activation was also reduced by NGF treatment. Inhibition of c-Jun activation did not affect the viability of sympathetic neurons, whereas the number of apoptotic sensory neurons increased. Furthermore, inhibition of c-Jun reduced axonal outgrowth from both SCG and DRG. Thus, in organ culture, c-Jun activation may be required for axonal outgrowth and, at least in sensory neurons, it promotes survival. The role of ATF3, a neuronal marker of injury and a c-Jun dimerization partner, was also examined. We found an ATF3 induction in both SCG and DRG neurons, a response, which was reduced by JNK inhibition. The reduction of ATF3 upon JNK inhibition was much larger in DRG than in SCG, a result which might account for the higher number of apoptotic neurons in JNK inhibitor exposed DRG. Taken together, and contrary to our expectations, neonatal sympathetic and sensory neurons seem to respond to axonal injury similarly with respect to c-Jun activation, and in no case was this activation pro-apoptotic.     

Mouse olfactory ensheathing glia enhance axon outgrowth on a myelin substrate in vitro

Olfactory ensheathing glia (OEG) express cell adhesion molecules and secrete growth factors that support newly generated olfactory axons and are a promising therapeutic treatment to facilitate axonal regeneration after spinal cord injury (SCI). To study the molecular mechanisms underlying the ability of OEG to enhance axonal outgrowth, we designed an outgrowth assay using spinal cord myelin as a substrate to mimic an injury environment. We asked if olfactory bulb-derived OEG could enhance outgrowth of dorsal root ganglion (DRG) axons on myelin. When grown on myelin alone DRG axons have limited outgrowth potential. However, when OEG are co-cultured with DRG on myelin, twice as many neurons generate axons and their average length is almost twice that grown on myelin alone. We used this OEG/DRG co-culture to determine if a cell adhesion molecule expressed by OEG, L1, and a factor secreted by OEG, brain-derived neurotrophic factor (BDNF), contribute to the ability of OEG to enhance axonal outgrowth on myelin. Using OEG and DRG from L1 mutant mice we found that L1 expression does not contribute to OEG growth promotion. However, both BDNF and its receptor, TrkB, contribute to OEG-enhanced axon regeneration as function-blocking antisera against either component significantly decreased outgrowth of DRG axons. Additional BDNF further enhanced DRG axon growth on myelin alone and on myelin co-cultured with OEG. This simple mouse outgrowth model can be used to determine the molecules that contribute to OEG-enhancement of axonal outgrowth, test therapeutic compounds, and compare the outgrowth potential of other treatments for SCI.     

Developmental changes in trak-mediated mitochondrial transport in neurons

Previous studies established that the kinesin adaptor proteins, TRAK1 and TRAK2, play an important role in mitochondrial transport in neurons. They link mitochondria to kinesin motor proteins via a TRAK acceptor protein in the mitochondrial outer membrane, the Rho GTPase, Miro. TRAKs also associate with enzyme, O-linked N-acetylglucosamine transferase (OGT), to form a quaternary, mitochondrial trafficking complex. A recent report suggested that TRAK1 preferentially controls mitochondrial transport in axons of hippocampal neurons whereas TRAK2 controls mitochondrial transport in dendrites. However, it is not clear whether the function of any of these proteins is exclusive to axons or dendrites and if their mechanisms of action are conserved between different neuronal populations and also, during maturation. Here, a comparative study was carried out into TRAK-mediated mitochondrial mobility in axons and dendrites of hippocampal and cortical neurons during maturation in vitro using a shRNA gene knockdown approach. It was found that in mature hippocampal and cortical neurons, TRAK1 predominantly mediates axonal mitochondrial transport whereas dendritic transport is mediated via TRAK2. In young, maturing neurons, TRAK1 and TRAK2 contribute similarly in mitochondrial transport in both axons and dendrites in both neuronal types. These findings demonstrate maturation regulation of mitochondrial transport which is conserved between at least two distinct neuronal subtypes.     

NP1 regulates neuronal activity-dependent accumulation of BAX in mitochondria and mitochondrial dynamics

In cultured cerebellar granule neurons, low neuronal activity triggers the intrinsic program of apoptosis, which requires protein synthesis-dependent BAX translocation to mitochondria, a process that may underlie neuronal damage in neurodegeneration. However, the mechanisms that link neuronal activity with the induction of the mitochondrial program of apoptosis remain unclear. Neuronal pentraxin 1 (NP1) is a pro-apoptotic protein induced by low neuronal activity that is increased in damaged neurites in Alzheimer's disease-affected brains. Here we report that NP1 facilitates the accumulation of BAX in mitochondria and regulates mitochondrial dynamics during apoptosis in rat and mouse cerebellar granule neurons in culture. Reduction of neuronal activity increases NP1 protein levels in mitochondria and contributes to mitochondrial fragmentation in a Bax-dependent manner. In addition, NP1 is involved in mitochondrial transport in healthy neurons. These results show that NP1 is targeted to mitochondria acting upstream of BAX and uncover a novel function for NP1 in the regulation of mitochondrial dynamics and trafficking during apoptotic neurodegeneration.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Amphiregulin acts as an autocrine survival factor for adult sensory neurons

We studied the effect of amphiregulin on axonal outgrowth and survival in sensory neurons in organ cultured and dissociated mouse dorsal root ganglia (DRG). Amphiregulin at 20 ng/ml stimulated axonal outgrowth in both preparations. The EGF receptor inhibitor AG1478 inhibited outgrowth at 10 microM but not at 1 microM, where it abolished the stimulatory effects of amphiregulin. Fluoro-Jade staining and neuronal counting showed that more neurons survived in culture in the presence of amphiregulin while AG1478 at 10 microM but not 1 microM increased cell death. Small and medium sized neurons were immunopositive for both amphiregulin and the EGF receptor. Taken together these results suggest that amphiregulin can act as an autocrine survival factor for sensory neurons and stimulate axonal outgrowth through the EGF receptor.     

Utilization of an HSV-based amplicon vector encoding the axonal marker hPLAP to follow neurite outgrowth in cultured DRG neurons

Delivery of genes into DRG neurons by viral vectors is a powerful tool for the study of axonal outgrowth. In order to achieve efficient transfer of growth-related genes and simultaneously label neuronal processes, we have utilized the HSV-based amplicon vector system. A bicistronic expression cassette encoding the growth associated protein-43 (GAP-43) and the axonal marker human placental alkaline phosphatase (hPLAP) reporter gene under translation control of an internal ribosomal entry site was cloned into the HGCX amplicon vector. This hPLAP reporter enabled efficient labeling of neurites in both dissociated adult DRG neurons and embryonic DRG explants. Using this reporter, the effect of GAP-43 on neurite outgrowth in transduced DRG neurons could be demonstrated. HSV-based amplicon vectors can contribute to the study of axonal growth and guidance in cultured neurons.     

Alpha-lipoic acid prevents mitochondrial damage and neurotoxicity in experimental chemotherapy neuropathy

The study investigates if alpha-lipoic acid is neuroprotective against chemotherapy induced neurotoxicity, if mitochondrial damage plays a critical role in toxic neurodegenerative cascade, and if neuroprotective effects of alpha-lipoic acid depend on mitochondria protection.We used an in vitro model of chemotherapy induced peripheral neuropathy that closely mimic the in vivo condition by exposing primary cultures of dorsal root ganglion (DRG) sensory neurons to paclitaxel and cisplatin, two widely used and highly effective chemotherapeutic drugs. This approach allowed investigating the efficacy of alpha-lipoic acid in preventing axonal damage and apoptosis and the function and ultrastructural morphology of mitochondria after exposure to toxic agents and alpha-lipoic acid. Our results demonstrate that both cisplatin and paclitaxel cause early mitochondrial impairment with loss of membrane potential and induction of autophagic vacuoles in neurons. Alpha-lipoic acid exerts neuroprotective effects against chemotherapy induced neurotoxicity in sensory neurons: it rescues the mitochondrial toxicity and induces the expression of frataxin, an essential mitochondrial protein with anti-oxidant and chaperone properties. In conclusion mitochondrial toxicity is an early common event both in paclitaxel and cisplatin induced neurotoxicity. Alpha-lipoic acid protects sensory neurons through its anti-oxidant and mitochondrial regulatory functions, possibly inducing the expression of frataxin. These findings suggest that alpha-lipoic acid might reduce the risk of developing peripheral nerve toxicity in patients undergoing chemotherapy and encourage further confirmatory clinical trials.     

Evidence for the direct role of acetylcholinesterase in neurite outgrowth in primary dorsal root ganglion neurons

Dorsal root ganglion (DRG) neurons show a transient peak expression of acetylcholinesterase (AChE) during periods of axonal outgrowth prior to synaptogenesis, suggesting that AChE has a non-enzymatic role during development. We have previously shown that perturbation of cell surface AChE in cultured embryonic rat DRG neurons results in decreased neurite outgrowth and neurite detachment. In this report, we demonstrate a direct correlation between endogenous AChE content and neurite outgrowth in primary DRG neurons. Adenoviral vectors were constructed using full-length rat AChE(T) cDNA in either the sense or antisense orientations to overexpress or knock down AChE expression, respectively. Treatment with the sense-expressing vector produced a 2.5-fold increase in AChE expression and a 2-fold increase in neurite length compared with either untreated or null virus-treated control cells. Conversely, treatment with the antisense-expressing vector reduced AChE expression by 40% and resulted in a reduction in neurite length of similar magnitude. We also observed that overexpression of AChE resulted in greater branching at the distal tips of each primary neurite as well as an increase in cell body size. These findings further indicate that AChE expressed on the axonal surface of developing DRG neurons may modulate their adhesive properties and thereby support axonal development.