Effects Of DSP-4 Research Articles

Opposing effects of β-2 and β-1 adrenergic receptor signaling on neuroinflammation and dopaminergic neuron survival in α-synuclein-mediated neurotoxicity

BackgroundNoradrenergic neurons in the locus coeruleus (LC) are the primary source of norepinephrine (NE) in the brain and degeneration of these neurons is reported in the early stages of Parkinson’s disease (PD), even prior to dopaminergic neuron degeneration in the substantia nigra (SN), which is a hallmark of PD pathology. NE depletion is generally associated with increased PD pathology in neurotoxin-based PD models. The effect of NE depletion in other models of PD-like α-synuclein-based models is largely unexplored. In PD models and in human patients, β-adrenergic receptors’ (AR) signaling is associated with a reduction of neuroinflammation and PD pathology. However, the effect of NE depletion in the brain and the extent of NE and β-ARs signaling involvement in neuroinflammation, and dopaminergic neuron survival is poorly understood.MethodsTwo mouse models of PD, a 6OHDA neurotoxin-based model and a human α-synuclein (hα-SYN) virus-based model of PD, were used. DSP-4 was used to deplete NE levels in the brain and its effect was confirmed by HPLC with electrochemical detection. A pharmacological approach was used to mechanistically understand the impact of DSP-4 in the hα-SYN model of PD using a norepinephrine transporter (NET) and a β-AR blocker. Epifluorescence and confocal imaging were used to study changes in microglia activation and T-cell infiltration after β1-AR and β2-AR agonist treatment in the hα-SYN virus-based model of PD.ResultsConsistent with previous studies, we found that DSP-4 pretreatment increased dopaminergic neuron loss after 6OHDA injection. In contrast, DSP-4 pretreatment protected dopaminergic neurons after hα-SYN overexpression. DSP-4-mediated protection of dopaminergic neurons after hα-SYN overexpression was dependent on β-AR signaling since using a β-AR blocker prevented DSP-4-mediated dopaminergic neuron protection in this model of PD. Finally, we found that the β-2AR agonist, clenbuterol, reduced microglia activation, T-cell infiltration, and dopaminergic neuron degeneration, whereas xamoterol a β-1AR agonist showed increased neuroinflammation, blood brain barrier permeability (BBB), and dopaminergic neuron degeneration in the context of hα-SYN-mediated neurotoxicity.ConclusionsOur data demonstrate that the effects of DSP-4 on dopaminergic neuron degeneration are model specific, and suggest that in the context of α-SYN-driven neuropathology, β2-AR specific agonists may have therapeutic benefit in PD.

Effects of DSP4 on the Noradrenergic Phenotypes and Its Potential Molecular Mechanisms in SH-SY5Y Cells

Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.

Effects of DSP4 and methylphenidate on spatial memory performance in rats

In this experiment, we have investigated the spatial memory performance of rats following a central noradrenaline depletion induced by three different doses of the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) and following administration of three different doses of methylphenidate (MPH). The rats were required to find food pellets hidden on a holeboard. The sole administration of DSP4 induced only minor cognitive deficits. However, the treatment with MPH increased the reference memory error, the impulsivity and the motor activity of the DSP4-treated rats. Since the noradrenergic terminals in a DSP4-treated rat are significantly reduced, the administration of MPH has little effect on the noradrenergic system and increases dopaminergic rather than noradrenergic activity, resulting in an imbalance with relatively high dopaminergic and low noradrenergic activities. It is suggested that a reduction of noradrenaline and an increase of dopamine induce ADHD-related deficits and that the depletion of noradrenaline is not sufficient for an appropriate rat model of ADHD.

Effects of DSP4 and dizolcipine on connectivity of solid E19 cortical grafts to ablated SmI region of adult rats; an in vivo electrophysiological study

The functional connectivity of an embryonic graft implanted into the lesioned somatosensory cortex and the effect of DSP4 (a selective noradrenergic neurotoxin to noradrenergic terminals) and dizolcipine (a non-competitive NMDA receptor antagonist), was studied electrophysiologically. The forepaw representational area of the rat primary somatosensory cortex was lesioned unilaterally and, 3–4 weeks later, tissue from the same region of E19 rat embryos was implanted into the cavity. At 7–9 months later, the rats were anaesthetized and single unit activity was recorded from the grafts in response to contralateral forepaw, ipsilateral hindpaw and contralateral hindpaw stimulation and compared with that obtained in control rats, in rats pretreated with dizolcipine immediately after lesioning and in rats given DSP4 24 h before transplantation. Neurones within the graft were integrated into the host brain and developed a pattern of representation similar to that of intact rats, but with a reduced proportion of neurones exhibiting short-latency response to contralateral forepaw stimulation and an increased proportion responding to stimulation of more than one paw. Pretreatment with dizolcipine did not increase short-latency responses to stimulation of contralateral forepaw stimulation however pretreatment with DSP4 reduced such responses and increased proportion of inhibitory responses. It was concluded that the noardrenergic system plays an important role in establishing host–graft connectivity. The importance of further pharmacological studies on host–graft connectivity and the relation of such connections to neural plasticity were discussed.

Effects of DSP-4 on monoamine and monoamine metabolite levels and on beta adrenoceptor binding kinetics in rat brain at different times after administration.

Effects of DSP-4 on noradrenaline (NA), 3-methoxy-4-hydroxyphenyl glycol (MHPG), serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) levels and on beta adrenoceptor binding kinetics (Bmax and KD) in rat hippocampus, cortex and hypothalamus were studied between 24 hours and 14 days after systemic administration. Beta adrenoceptor numbers in hippocampus and cortex, but not in hypothalamus, were significantly increased after DSP-4. No significant changes in KD values were observed in hypothalamus, but significant increases in this parameter were measured in hippocampus and cortex. NA and MHPG levels were significantly decreased in all three brain regions, but MHPG/NA ratios were increased in hippocampus, decreased in cortex and unchanged in hypothalamus. Very prominent increases in 5-HIAA levels were observed in all three brain regions, but only at one day after DSP-4. The greatest increases in 5-HIAA levels occurred in the hippocampus, but this effect of DSP-4 appeared to be slightly diminished by pre-treatment with fluoxetine. In cortex and hippocampus 5-HT levels were slightly, but significantly decreased after DSP-4.

Conflict behavior in maudsley reactive and nonreactive rats: Effects of noradrenergic neuronal destruction

The present studies were designed to examine the effects of treatment with the noradrenergic neurotoxin N-(2-chloroethyl)- n-ethyl-2-bromobenzylamine HCl (DSP4; 65 mg/kg, IP) on conflict behavior in the Maudsley reactive (MR) and nonreactive (MNRA) rat strains. In daily 10-min sessions, water-restricted rats were trained to drink water from a tube that was ocassionally electrified; electrification was signaled by the presence of a tone (7-s duration; ISI = 30 s). Consistent with previous reports, the number of shocks accepted by rats of the MR and MNRA strains did not differ initially, but MNRA rats exhibited a dramatic increase in punished responding relative to their MR counterparts over the course of several weeks of conflict testing. This MR vs. MNRA strain difference in punished responding did not exhibit extinction following discontinuation of CSD conflict behavior testing for a period of 6 weeks. Whether it was administered after conflict training or before, DSP4 treatment did not reduce the MR vs. MNRA strain difference in conflict behavior; rather, DSP4 treatment tended to increase the magnitude of the MR vs. MNRA difference in conflict behavior. The effects of DSP4 on norepinephrine (NE) and 5-hydroxytrypamine (5-HT) concentrations in the pons medulla region were determined in one group of conflict-experienced MR and MNRA rats (35 weeks after administration) and in a second group of naive MR and MNRA rats (3 weeks after administration). There were no MR vs. MNRA strain differences in NE or 5-HT concentrations in vehicle-treated rats. DSP4 treatment significantly reduced NE, but not 5-HT, concentrations when compared to control values; rats that were sacrificed 3 weeks following DSP4 administration exhibited a greater NE depletion than did rats sacrificed 35 weeks after DSP4 administration. Finally, there were no significant correlations between pons medulla region NE concentrations and conflict behavior in either strain alone or when the data from the two strains were combined. The present results are not consistent with the hypothesis that the MR vs. MNRA strain difference in conflict behavior is the result of strain differences in brain NE function.

The effects of noradrenergic neurotoxin, DSP-4, on the performance of young and aged rats in spatial navigation task

The present study investigated whether an overactive noradrenergic system is related to the impairment in learning/memory in aged subjects. The effects of partial noradrenaline depletion (using the noradrenergic neurotoxin DSP-4) on the acquisition of a water maze task was investigated in young and aged rats, and hippocampal noradrenaline content was correlated with spatial learning performance in similar rats. DSP-4 treatment impaired markedly the acquisition of the water maze task in aged rats, but improved it slightly in young rats. DSP-4 treatment decreased swimming speed, and this effect tended to be more marked in young rats. In the group of control rats, hippocampal noradrenaline tended to correlate positively with spatial bias in aged rats (the rats with the highest noradrenaline content in the hippocampus tended to have the best spatial learning/memory), but negatively in young rats. These results do not support the hypothesis that spatial learning/memory impairment is due to an overactive noradrenergic system in aged rats. Further studies are needed to clarify the reasons of the marked age-related difference in the effects of DSP-4 on the performance of water maze task in rats.

DSP-4-induced depletion of brain norepinephrine produces opposite effects on exploratory behavior 3 and 14 days after treatment

Exploratory behavior of a complex novel environment was examined 3 and 14 days following treatment with the noradrenergic-selective neurotoxin, DSP-4. This toxin significantly decreased norepinephrine concentrations in neocortex and hippocampus but not hypothalamus. DSP-4 significantly increased exploratory behavior in animals tested 3 days after treatment. In contrast, exploratory behavior was decreased in animals tested 14 days after treatment. The effect of DSP-4 at 3 days is similar to treatments that act to inhibit noradrenergic function such as administration of the alpha 1-antagonist, prazosin, or the alpha 2-agonist, clonidine. The effect of DSP-4 at 14 days resembles that observed following treatment with the alpha 1-agonist, phenylephrine, or the alpha 2-antagonist, idazoxan. These data provide additional support for a role of noradrenergic systems in exploratory behavior. The simplest explanation for the time dependent effects of DSP-4 on exploratory behavior is the occurrence of the slow development of a supersensitivity of cerebral systems affected by norepinephrine.

Direct effects of the adrenergic neurotoxin DSP4 on central opiate receptors: implications for neuroendocrine studies.

We have shown that the noradrenergic neurotoxin DSP4 can acutely abolish naloxone-induced secretion of LH in the rat. A part of this influence is clearly related to an unexpected interaction with hypothalamic opiate ( [3H]-naloxone) binding sites. Injection of DSP4 (50 mg/kg) can severely reduce opiate binding assayed in vitro. In addition, the drug is very potent in blocking opiate receptors in vitro, as determined in slice and homogenate assays. Consideration must now be given to the possibility that many previous studies on acute effects of DSP4 undoubtedly involved the opiatergic system in addition to noradrenergic terminals. Thus, DSP4 is not the drug of choice for experiments designed to probe catecholamine-opiate interactions in the control of LH release.

Effects of the noradrenaline neurotoxin DSP4 on the postnatal development of central noradrenaline neurons in the rat

The effect of the noradrenaline neurotoxin DSP4 on the postnatal development of central noradrenergic neurons in the rat has been investigated using neurochemical techniques. The results demonstrated a preferential effect of DSP4 on the locus coeruleus noradrenergic neuron system without any notable effects on the dopamine and adrenaline neurons and only a minor neurotoxic effect on the serotonin neurons. The effect of DSP4 on the serotonin neurons could be completely prevented by pretreatment with the uptake blocker zimelidine, without affecting the action of DSP4 on noradrenergic neurons. Neonatal DSP4 treatment systemically led to permanent depletions of noradrenaline in the cerebral cortex and spinal cord and marked increases of noradrenaline in the cerebellum and ponsmedulla. These effects of DSP4 were dose-dependent and could be blocked by pretreatment with the noradrenaline uptake blocker desipramine. The alterations in endogenous noradrenaline levels were quantitatively similar to changes observed in [ 3H]noradrenaline uptake in slices in vitro. There were no significant changes of these noradrenergic parameters when analysing the whole CNS after neonatal DSP4 treatment, in spite of marked regional changes in both directions. Administration of DSP4 to rats of different ages produced acutely marked depletions of noradrenaline in all regions including the pons-medulla and the cerebellum at all developmental stages. Marked and permanent depletions of noradrenaline were found in the distant noradrenergic nerve terminal projections after treatment at all ages, whereas increases in noradrenaline levels in the pons-medulla and cerebellum were only observed in rats treated with DSP4 up to the age of 3–5 days, whereas a DSP4 administration in older rats led to substantial and permanent depletions of noradrenaline in both these regions. The results indicate that the alteration of the postnatal development of noradrenergic neurons after treatment of rats up to the age of 3–5 days is mainly related to a ‘pruning effect’ of DSP4, in which prevention of the development of distant nerve terminal projections causes an increased outgrowth of nerves in collateral systems spared by the neurotoxin. The results indicate that DSP4 may be a useful denervation tool for studying various aspects of noradrenergic neurotransmission of developing locus coeruleus neurons.