Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Montreal Neurological Institute, McGill University

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration (Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.

Brain-derived neurotrophic factor facilitates TrkB down-regulation and neuronal injury after status epilepticus in the rat hippocampus.

There is a great deal of evidence showing the capacity of physical exercise to enhance cognitive function, reduce anxiety and depression, and protect the brain against neurodegenerative disorders. Although the effects of exercise are well documented in the mature brain, the influence of exercise in the developing brain has been poorly explored. Therefore, we investigated the morphological and functional hippocampal changes in adult rats submitted to daily treadmill exercise during the adolescent period. Male Wistar rats aged 21 postnatal days old (P21) were divided into two groups: exercise and control. Animals in the exercise group were submitted to daily exercise on the treadmill between P21 and P60. Running time and speed gradually increased over this period, reaching a maximum of 18 m/min for 60 min. After the aerobic exercise program (P60), histological and behavioral (water maze) analyses were performed. The results show that early-life exercise increased mossy fibers density and hippocampal expression of brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B, improved spatial learning and memory, and enhanced capacity to evoke spatial memories in later stages (when measured at P96). It is important to point out that while physical exercise induces hippocampal plasticity, degenerative effects could appear in undue conditions of physical or psychological stress. In this regard, we also showed that the exercise protocol used here did not induce inflammatory response and degenerating neurons in the hippocampal formation of developing rats. Our findings demonstrate that physical exercise during postnatal development results in positive changes for the hippocampal formation, both in structure and function.

Early exercise promotes positive hippocampal plasticity and improves spatial memory in the adult life of rats.

The transcription factor p53 mediates neuronal death in a variety of stress-related and neurodegenerative conditions. The proapoptotic activity of p53 is tightly regulated by the apoptosis-stimulating proteins of p53 (ASPP) family members: ASPP1 and ASPP2. However, whether ASPP1/2 play a role in the regulation of p53-dependent neuronal death in the CNS is currently unknown. To address this, we asked whether ASPP1/2 contribute to the death of retinal ganglion cells (RGCs) using in vivo models of acute optic nerve damage in mice and rats. Here, we show that p53 is activated in RGCs soon after injury and that axotomy-induced RGC death is attenuated in p53 heterozygote and null mice. We demonstrate that ASPP1/2 proteins are abundantly expressed by injured RGCs, and that short interfering (si)RNA-based ASPP1 or ASPP2 knockdown promotes robust RGC survival. Comparative gene expression analysis revealed that siASPP-mediated downregulation of p53-upregulated-modulator-of-apoptosis (PUMA), Fas/CD95, and Noxa depends on p53 transcriptional activity. Furthermore, siRNA against PUMA or Fas/CD95 confers neuroprotection, demonstrating a functional role for these p53 targets in RGC death. Our study demonstrates a novel role for ASPP1 and ASPP2 in the death of RGCs and provides evidence that blockade of the ASPP-p53 pathway is beneficial for central neuron survival after axonal injury.

ASPP1/2 regulate p53-dependent death of retinal ganglion cells through PUMA and Fas/CD95 activation in vivo.

Photoreceptor neurons (R cells) in the Drosophila eye define a map of visual space by connecting to targets in distinct layers of the optic lobe, with R1-6 cells connecting to the lamina (the first optic ganglion) and R7 and R8 cells connecting to the medulla (the second optic ganglion). Here, we show that Wengen (Wgn) directly binds Moesin (Moe) through a cytosolic membrane proximal domain and this interaction is important for mediating two distinct aspects of axonal targeting. First, we show that loss of wgn or moe function disrupts cell autonomous R8 axon targeting. Second, we report that wgn or moe mutants show defects in R2-R5 targeting that result from disruption of non-cell autonomous effects, which are secondary to the cell autonomous R8 phenotype. Thus, these studies reveal that the Wgn-Moe signaling cascade plays a key role in photoreceptor target field innervations through cell autonomous and non-cell autonomous mechanisms.

Wengen, the sole tumour necrosis factor receptor in Drosophila, collaborates with moesin to control photoreceptor axon targeting during development.

Our knowledge of the destructive events that regulate axonal degeneration is rudimentary. Here, we examine the role of caspases and their endogenous inhibitor, the X-linked inhibitor of apoptosis protein (XIAP), in axonal degeneration of dorsal root ganglion (DRG) axons. We show that caspase-3, caspase-6, and caspase-9 are present in axons and are cleaved upon nerve growth factor (NGF) withdrawal. We observed that caspase-3 activity is high in NGF-withdrawn axons and that CASP3(-/-) axons are protected from degeneration. XIAP(-/-) DRG sensory neurons degenerate more rapidly and contain more active caspase-3 than their wild-type counterparts, indicating that axonal caspases are normally regulated by XIAP. Importantly, axonal XIAP levels drop sharply after NGF withdrawal; if XIAP levels are maintained by overexpression, axonal caspase-3 activation and axonal degeneration are suppressed. Finally, we show that XIAP(-/-) embryos have stunted dermal innervation. We propose that XIAP-mediated caspase inhibition plays an important role in regulating morphogenic events that shape the nervous system during development.

XIAP regulates caspase activity in degenerating axons.

The neurotrophins play crucial roles regulating survival and apoptosis in the developing and injured nervous system. The four neurotrophins exert profound and crucial survival effects on developing peripheral neurons, and their expression and action is intimately tied to successful innervation of peripheral targets. In the central nervous system, they are dispensable for neuronal survival during development but support neuronal survival after lesion or other forms of injury. Neurotrophins also regulate apoptosis of both peripheral and central neurons, and we now recognize that there are regulatory advantages to having the same molecules regulate life and death decisions. This chapter examines the biological contexts in which these events take place and highlights the specific ligands, receptors, and signaling mechanisms that allow them to occur.

Nicolas Unsain

Department of Neurology and Neurosurgery

Montreal Neurological Institute, McGill University

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Nicolas Unsain

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Neurology and Neurosurgery

Montreal Neurological Institute, McGill University

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Department of Neurology and Neurosurgery