Effect of Ser-129 phosphorylation on interaction of α-synuclein with synaptic and cellular membranes.

In the healthy brain, less than 5% of α-synuclein (α-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of α-syn is Ser(P)-129. The effects of Ser(P)-129 modification on α-syn distribution and solubility are poorly understood. As α-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 α-syn and analyzed the effects of manipulating Ser(P)-129 levels on α-syn membrane interactions using synaptosomal membranes and neural precursor cells from α-syn-deficient mice or transgenic mice expressing human α-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either α-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT α-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A α-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 α-syn showed a preferential membrane association compared with WT Ser(P)-129 α-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of α-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129.

Expression of Semaphorin3C in axotomized rodent facial and rubrospinal neurons.

Semaphorins are a family of axonal guidance molecules that, by virtue of their chemorepulsive or chemoattractive actions, may be the important factors in determining the success or failure of axonal regeneration in the mature nervous system after injury. Here, we have used two adult mouse models of nervous system injury to evaluate the neuronal expression of Semaphorin3C (Sema3C) in regenerating (facial motoneurons) and non-regenerating (rubrospinal) neurons following axonal injury. Using in situ hybridization (ISH), we observed that uninjured facial motoneurons express Sema3C mRNA and, following axonal injury, there is a transient up-regulation in Sema3C mRNA expression in injured motoneurons. In contrast, Sema3C mRNA was not detected in uninjured rubrospinal neurons; however, following axotomy, injured rubrospinal neurons significantly up-regulate Sema3C mRNA expression. The increase in Sema3C mRNA expression in axotomized rubrospinal neurons was not limited to the mouse nervous system: serial dilution RT-PCR analysis revealed a similar increase in Sema3C mRNA expression in the axotomized rat rubrospinal nucleus, 3 days following a rubrospinal tract lesion. This demonstrates that increased Sema3C mRNA levels in axotomized rubrospinal neurons is common to both mouse and rat injury models.

Brain-derived neurotrophic factor gene transfer with adeno-associated viral and lentiviral vectors prevents rubrospinal neuronal atrophy and stimulates regeneration-associated gene expression after acute cervical spinal cord injury.

STUDY DESIGN: Experimental animal study. OBJECTIVE: To determine if viral vectors carrying the gene for brain-derived neurotrophic factor (BDNF) could be used to promote an axonal regenerative response in rubrospinal neurons after an acute cervical spinal cord injury. SUMMARY OF BACKGROUND DATA: Following axotomy in the cervical spinal cord, rubrospinal neurons undergo severe atrophy and fail to up-regulate important genes for regeneration. This can be attenuated or reversed with the infusion of BDNF to the injured cell bodies. This infusion technique, however, causes substantial parenchymal damage around the red nucleus and is limited by occlusion of the infusion pumps. This study examined whether viral vectors could be used to deliver the BDNF gene in a less damaging fashion and whether this could promote a regenerative response in injured rubrospinal neurons. METHODS: Following a cervical spinal cord injury, the viral vectors were injected into the vicinity of the injured red nucleus. The extent of parenchymal damage around the red nucleus was assessed, as was the immunoreactivity to BDNF and cellular transfection patterns. Rubrospinal neuronal cross-sectional area was measured to determine if atrophy had been reversed, and in situ hybridization for GAP-43 and Talpha1 tubulin was performed to determine if there genes, which are important for axonal regeneration, were up-regulated. RESULTS: Parenchymal damage associated with viral injection was significantly less than with previous infusion techniques. BDNF immunoreactivity around the red nucleus indicated that the BDNF transgene was expressed. Both viral vectors reversed rubrospinal neuronal atrophy and promoted the expression of GAP-43 and Talpha1 tubulin. CONCLUSIONS: Viral-mediated transfer of the BDNF gene was successful at promoting a regenerative response in rubrospinal neurons following acute cervical spinal cord injury, with significantly less parenchymal damage than previously observed when infusing the BDNF protein.

Dose-dependent beneficial and detrimental effects of ROCK inhibitor Y27632 on axonal sprouting and functional recovery after rat spinal cord injury.

Axonal regeneration within the injured central nervous system (CNS) is hampered by multiple inhibitory molecules in the glial scar and the surrounding disrupted myelin. Many of these inhibitors stimulate, either directly or indirectly, the Rho intracellular signaling pathway, providing a strong rationale to target it following spinal cord injuries. In this study, we infused either control (PBS) or a ROCK inhibitor, Y27632 (2 mM or 20 mM, 12 microl/day for 14 days) into the intrathecal space of adult rats starting immediately after a cervical 4/5 dorsal column transection. Histological analysis revealed that high dose-treated animals displayed significantly more axon sprouts in the grey matter distal to injury compared to low dose-treated rats. Only the high dose regimen stimulated sprouting of the dorsal ascending axons along the walls of the lesion cavity. Footprint analysis revealed that the increased base of support normalized significantly faster in control and high dose-treated animals compared to low dose animals. Forepaw rotation angle, and the number of footslips on a horizontal ladder improved significantly more by 6 weeks in high dose animals compared to the other two groups. In a food pellet reaching test, high dose animals performed significantly better than low dose animals, which failed to recover. There was no evidence of mechanical allodynia in any treatment group; however, the slightly shortened heat withdrawal times normalized only with the high dose treatment. Collectively, our data support beneficial effects of high dose Y27632 treatment but indicate that low doses might be detrimental.

Both positive and negative factors regulate gene expression following chronic facial nerve resection.

Previously, we reported that following a chronic nerve resection, removal of the neuroma reversed the atrophy, increased the number of countable motoneurons and resulted in the re-expression of GAP-43 and alpha tubulin mRNA. In the present study, we questioned whether this response was due to the removal of the neuroma, or a result of factors such as neurotrophins, produced at the injury site. To test this hypothesis, 10 weeks after axotomy, the axonal transport blocker colchicine or, glial derived neurotrophic factor (GDNF) was injected proximal to the neuroma. The injection of GDNF or colchicine elicited an increase in motoneuron size and in GAP-43, but not alpha tubulin, mRNA. These data suggest that in addition to factors produced at the injury site, the neuroma acts as a source of target-like repressive signals that when removed results in an increase in gene expression and motoneuron size. To analyze the regenerative potential of chronically resected motoneurons, mice without a previous nerve injury and mice with a chronic resection received a pre-degenerated segment of sciatic nerve attached to the proximal facial nerve stump. Axons from both the chronic and acute groups grew into the grafts, however, significantly more retrogradely labeled motoneurons were counted in the acute group compared to the chronic resection group. No difference in motoneuron cell size was observed between the two groups of regenerated neurons. Therefore, despite severe atrophy, many of the surviving mouse facial motoneurons retain the propensity to extend their axons when provided with the appropriate environment.