Impairment of mitochondria dynamics by human A53T α-synuclein and rescue by NAP (davunetide) in a cell model for Parkinson's disease.

The formation of oligomers and aggregates of overexpressed or mutant α-synuclein play a role in the degeneration of dopaminergic neurons in Parkinson's disease by causing dysfunction of mitochondria, reflected in their disturbed mobility and production of ROS. The mode of action and mechanisms underlying this mitochondrial impairment is still unclear. We have induced stable expression of wild-type, A30P or A53T α-synuclein in neuronally differentiated SH-SY5Y neuroblastoma cells and studied anterograde and retrograde mitochondrial trafficking in this cell model for Parkinson's disease. In contrast to wild-type and A30P, A53T α-synuclein significantly inhibited mitochondrial trafficking, at first retrogradely and in a later stage anterogradely. Accordingly, A53T α-synuclein also caused the highest increase in ROS production in the dysmobilized mitochondria in comparison to wild-type or A30P α-synuclein. Treatment with NAP, the eight amino acid peptide identified as the active component of activity-dependent neuroprotective protein (ADNP), completely annihilated the adverse effects of A53T on mitochondrial dynamics. Our results reveal that A53T α-synuclein (oligomers or aggregates) leads to the inhibition of mitochondrial trafficking, which can be rescued by NAP, suggesting the involvement of microtubule disruption in the pathophysiology of Parkinson's disease.

Early transient presence of implanted bone marrow stem cells reduces lesion size after cerebral ischaemia in adult rats.

AIMS: Previous studies on the therapeutic time window for intravascular administration of bone marrow stem cells (BMSCs) after stroke have shown that early intervention (from 3 h after onset) in the middle cerebral artery occlusion (MCAO) rat model is the most effective approach to reduce ischaemic lesion size. We have confirmed these observations but noticed that 2 weeks after transplantation, almost none of the grafted BMSCs could be detected in or around the lesion. The present experiments aimed to assess the fate and kinetics of intravascularly injected BMSCs shortly after administration in correlation to the development of the ischaemic lesion after MCAO. METHODS: We administered a syngeneic suspension of complete (haematopoietic and mesenchymal) BMSCs via the carotid artery to rats at 2 h after MCAO onset. We examined the distribution and tissue location of BMSCs within the first 24 h after arterial administration by perfusion-fixating rats and performing immunohistochemical analysis at different time points. RESULTS: The vast majority (>95%) of BMSCs appeared to become trapped in the spleen shortly after injection. Six hours after implantation, together with the appearance of activated microglia, the first BMSCs could be detected in and around the lesion; their number gradually increased during the first 12 h after implantation but started to decrease at 24 h. The implanted BMSCs were surrounded by activated and phagocytotic microglia. CONCLUSION: Our results show that ischaemic lesion size reduction can already be achieved by the early transient presence at the lesion site of intravascularly implanted BMSCs, possibly mediated via activated microglia.

p75NTR independent oligodendrocyte death in cuprizone-induced demyelination in C57BL/6 mice.

Feeding C57Bl/6 J mice the copper chelator cuprizone leads to selective apoptosis of mature oligodendrocytes and concomitant demyelination predominantly in the corpus callosum. The process of oligodendrocyte apoptosis in this animal model for multiple sclerosis (MS) involves early microglial activation, but no infiltration of T-lymphocytes. Therefore, this model could mimic early stages of oligodendrocyte degeneration Affected oligodendrocytes express the common neurotrophin receptor, p75(NTR), a 'stress-receptor' which under certain circumstances can induce apoptosis. Only affected oligodendrocytes in MS lesions and MS animal models express this receptor. In order to study the significance of p75(NTR) in the fate of oligodendrocytes, we have exposed wild-type as well as p75(NTR)-knockout mice to a 0.2% (w/w) cuprizone diet and performed a comparative immunohistochemical analysis of the corpus callosum at various time points. Surprisingly, our results show that the absence of p75(NTR) did not alter cuprizone-induced oligodendrocyte death (and subsequent de- or remyelination). Apparently, intracellular apoptosis pathways in adult oligodendrocytes do not require p75(NTR) activated signal transduction in the absence of T-lymphocytes and T-lymphocyte derived cytokines.

Functionally deficient neuronal differentiation of mouse embryonic neural stem cells in vitro.

Embryonic mouse neural stem cells (NSCs) were isolated from E14 mice, multiplied in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and plated in laminin-coated wells in basic serum-free neurobasal medium. After 7 days in vitro, approximately 20% of the embryonic mouse NSCs developed into morphologically and biochemically fully maturated neurons, with extensive dendrites and multiple synaptic contacts. However, even after 22 days of culture, none of these neurons developed voltage-dependent sodium-channels characteristic for a functional neuron. Apparently, the morphological differentiation and the electrophysiological maturation of an embryonic mouse NSC into a neuron are independently regulated.

Reduced p75NTR expression delays disease onset only in female mice of a transgenic model of familial amyotrophic lateral sclerosis.

hSOD1 (G93A) transgenic mice develop pathological changes similar to those in patients with familial amyotrophic lateral sclerosis (FALS). In particular, the progressive degeneration of motoneurons is charactered in this mouse model. One feature of stressed motoneurons in ALS and the hSOD1 mice is the induction of the p75 neurotrophin receptor, which is thought, under certain circumstances, to be a death-signaling molecule. We have studied disease progression of hSOD1 (G93A) mice in the absence of the p75NTR receptor and we monitored histological changes in the ventral spinal cord. Whereas female double transgenics showed prolonged survival, this effect was not observed in males. Improved survival in female mice was not correlated with increased motoneuronal survival, but with less astrocytic activation in lumbar ventral spinal cord, as shown by glial fibrillary acidic protein immunohistochemistry. These data suggest that p75NTR is not directly involved in the mechanism leading to motoneuron degeneration. More likely, an indirect process, presumably via regulation of astrocytes, might be responsible for the increased survival responses of female double transgenic mice.

Expression of the low affinity neurotrophin receptor p75 in spinal motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a lethal neurodegenerative disorder involving motoneuron loss in the cortex, brainstem and spinal cord, resulting in progressive paralysis. Aberrant neurotrophin signalling via the low affinity neurotrophin receptor p75 has been suggested to be involved in the motoneuron death by the activation of apoptotic pathways. In order to investigate the involvement of neurotrophin receptor p75 in the amyotrophic lateral sclerosis related motoneuron degeneration process, we have studied the expression of this receptor in the spinal cord of transgenic mice carrying a mutated human Cu, Zn superoxide dismutase gene. Mutations in the superoxide dismutase gene are one of the genetic causes for familiar amyotrophic lateras sclerosis and human superoxide dismutase-1 transgenic mice develop symptoms and pathology similar to those in human amyotrophic lateras sclerosis. Our study shows that in these mice, spinal motoneurons, which normally do not contain the neurotrophin receptor p75 receptor, express this receptor during the progress of the disease. Expression of the neurotrophin receptor p75 receptor coincides with the expression of activating transcription factor 3, a member of the activating transcription factor/cyclic AMP family of stress transcription factors. Only a minority of these spinal motoneurons actually showed co-expression of neurotrophin receptor p75 with caspase-3 activity, suggesting that expression of the neurotrophin receptor p75 receptor is not directly related to the execution phase of the apoptosis process.

Elevated levels of neurotrophins in human biceps brachii tissue of amyotrophic lateral sclerosis.

Previous studies suggest that neurotrophins support regeneration and survival of injured motoneurons. Based on these findings, brain-derived neurotrophic factor (BDNF) has been clinically investigated for its therapeutic potential in amyotrophic lateral sclerosis (ALS), a rapidly progressing and fatal motoneuronal disease. We questioned whether imbalances of neurotrophic levels are indeed involved in the pathology of ALS. Therefore the expression of nerve growth factor (NGF), BDNF, neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) was investigated in postmortem muscle tissue of the biceps from 15 patients with neuropathologically confirmed sporadic ALS and 15 age-matched controls. Using mRNA analysis techniques and quantitative protein measurements, we have demonstrated that both mRNA and protein levels of all four neurotrophins are increased in muscle tissue of ALS patients. The production levels displayed a disease duration dependency and different expression patterns emerged for the four neurotrophins. Whereas the early phase of the disease was characterized by a strong upregulation of BDNF, levels of NGF, NT-3, and NT-4/5 gradually increased in the course of the disorder, peaking at later stages. We conclude that decreased neurotrophic support from muscle tissue is most likely not the cause of motoneuron degeneration in ALS. On the contrary, our results suggest that degenerating motoneurons in ALS are exposed to elevated levels of muscle-derived neurotrophins.