Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.     

2-(Cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one, a novel compound with neuroprotective and neurotrophic effects in vitro

Focusing on development of novel drug candidates for the treatment of neurodegenerative diseases, we developed and synthesized a new compound, 2-(cyclohexylamino)-1-(4-cyclopentylpiperazin-1-yl)-2-methylpropan-1-one (amido-piperizine 1). The compound demonstrated robust neuroprotective properties after both glutamate excitotoxicity and peroxide induced oxidative stress in primary cortical cultures. Furthermore, amido-piperizine 1 was found to significantly induce neurite outgrowth in vitro which could suggest central reparative and regenerative potential of the compound.With these potential beneficial effects in CNS, the ability of the amido-piperizine 1 to penetrate the blood-brain barrier was tested using MDR1-MDCK cells. Amido-piperizine 1 was found not to be a P-gp substrate and to have a high blood-brain barrier penetration potential, indicating excellent availability to the CNS. Moreover, amido-piperizine 1 had a fast metabolic clearance rate in vitro, suggesting that parenteral in vivo administration seems preferable.As an attempt to elucidate a possible mechanism of action, we found that amido-piperizine 1 bound in nano-molar range to the sigma-1 receptor, which could explain the observed neuroprotective and neurotrophic properties, and with a 100-fold lower affinity to the sigma-2 receptor. These results propose that amido-piperizine 1 may hold promise as a drug candidate for the treatment of stroke/traumatic brain injury or other neurodegenerative diseases.     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

A novel PIP2 binding of epsilonPKC and its contribution to the neurite induction ability

Protein kinase C-epsilon (epsilonPKC) induces neurite outgrowth in neuroblastoma cells but molecular mechanism of the epsilonPKC-induced neurite outgrowth is not fully understood. Therefore, we investigated the ability of phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding of epsilonPKC and its correlation with the neurite extension. We found that full length epsilonPKC bound to PIP(2) in a 12-omicron-tetradecanoylphorbol-13-acetate dependent manner, while the regulatory domain of epsilonPKC (epsilonRD) bound to PIP(2) without any stimulation. To identify the PIP(2) binding region, we made mutants lacking several regions from epsilonRD, and examined their PIP(2) binding activity. The mutants lacking variable region 1 (V1) bound to PIP(2) stronger than intact epsilonRD, while the mutants lacking pseudo-substrate or common region 1 (C1) lost the binding. The PIP(2) binding ability of the V3-deleted mutant was weakened. Those PIP(2) bindings of epsilonPKC, epsilonRD and the mutants well correlated to their neurite induction ability. In addition, a chimera of pleckstrin homology domain of phospholipase Cdelta and the V3 region of epsilonPKC revealed that PIP(2) binding domain and the V3 region are sufficient for the neurite induction, and a first 16 amino acids in the V3 region was important for neurite extension. In conclusion, epsilonPKC directly binds to PIP(2) mainly through pseudo-substrate and common region 1, contributing to the neurite induction activity.     

Acetylcholinesterase modulates neurite outgrowth on fibronectin

Acetylcholinesterase (AChE) has been reported to be involved in the modulation of neurite outgrowth. To understand the role played by different domains, we transfected neuroblastoma cells with three constructs containing the invariant region of AChE, differing in the exon encoding the C-terminus and therefore in AChE cellular fate and localization. All isoforms increased neurite extension, suggesting the involvement of the invariant domain [A. De Jaco, G. Augusti-Tocco, S. Biagioni, Alternative AChE molecular forms exhibit similar ability to induce neurite outgrowth, J. Neurosci. Res. 70 (2002) 756-765]. The peripheral anionic site (PAS) is encoded by invariant exons and represents the domain involved in non-cholinergic functions of AChE. Masking of PAS with fasciculin results in a significant decrease of neurite outgrowth in all clones overexpressing AChE. A strong reduction was also observed when clones were cultured on fibronectin. Treatment of clones with fasciculin, therefore masking PAS, abolished the fibronectin-induced reduction. The inhibition of the catalytic site cannot revert the fibronectin effect. Finally, when clones were cultured on fibronectin in the presence of heparin, a ligand of fibronectin, the inhibitory effect was completely reversed. Our results indicate that PAS could directly or indirectly mediate AChE/fibronectin interactions.     

ALK activation induces Shc and FRS2 recruitment: Signaling and phenotypic outcomes in PC12 cells differentiation

Activation of the neuronal receptor tyrosine kinase ALK (anaplastic lymphoma kinase) promoted the neuron-like differentiation of PC12 cells through specific activation of the ERK MAP-kinase pathway. However, the nature of primary signaling events initiated is still poorly documented. Here, we established that Shc and FRS2 adaptors were recruited and phosphorylated following antibody-based ALK activation. We further demonstrated that Shc was recruited to the consensus phosphotyrosine site NPTpY(1507) and FRS2 was likely recruited to a novel non-orthodox phosphotyrosine site within ALK. Finally, we characterized a functional role for Shc and likely FRS2 in ALK-dependant MAP-kinase activation and neuronal differentiation of PC12 cells. These findings hence open attractive perspectives concerning specific characteristics of ALK in the control of the mechanisms driving neuronal differentiation.     

Phospholipase D1 activation through Src and Ras is involved in basic fibroblast growth factor-induced neurite outgrowth of H19-7 cells

Phospholipase D (PLD) is implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. We found that PLD1 is activated when basic fibroblast growth factor (bFGF) stimulates neurite outgrowth of an immortalized hippocampal cell line (H19-7). Overexpression of PLD1 in H19-7 cells dramatically elongated bFGF-induced neurite outgrowth and increased PLD activity. Transfection of DN-rPLD1 blocked bFGF-induced PLD activation and completely inhibited neurite outgrowth induced by bFGF, suggesting that PLD1 activation is important in bFGF-induced neurite outgrowth of H19-7 cells. PLD activation and neurite outgrowth induced by bFGF was dependent on phospholipase C gamma (PLC-gamma) and Ca2+, but not protein kinase C (PKC). Furthermore, inhibition of Src and Ras partially blocked bFGF-induced PLD activation and neurite outgrowth, respectively. Coinhibition of Src and Ras completely blocked bFGF-induced PLD activation, suggesting that Src and Ras independently regulate PLD1 activation. Interestingly, bFGF-induced PLD activation and neurite outgrowth did not require ERK1/2 activated by Ras. Taken together, this study demonstrates that bFGF activates PLD1 through PLC-gamma activation, which leads to neurite outgrowth in H19-7 cells. Furthermore, our results show that PLD1 activation by bFGF is regulated by Src and Ras independently.