Identification of a Novel 1,4,8-Triazaphenanthrene Derivative as a Neuroprotectant for Dopamine Neurons Vulnerable in Parkinson's Disease.

Parkinson's disease (PD) is a chronic degenerative disorder characterized by typical motor symptoms caused by the death of dopamine (DA) neurons in the midbrain and ensuing shortage of DA in the striatum, at the level of nerve terminals. No curative treatment is presently available for PD in clinical practice. In our search for neuroprotectants in PD, we generated new 1,4,8-triazaphenanthrenes by combining 6-endo-dig-cycloisomerization of propargylquinoxalines and Suzuki or Sonogashira cross-coupling reactions. Neuroprotection assessment of newly synthesized 1,4,8-triazaphenanthrenes in a PD cellular model resulted in the discovery of a new hit compound PPQ (5m). Neuroprotection by 5m was concentration-dependent and the result of a combined effect on intracellular calcium release channels and astroglial cells. Of interest, 5m also counteracted DA cell loss in a mouse model of PD, making this molecule a promising candidate for PD treatment.

A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro.

Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1ß and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.

Signaling Mechanisms in the Nitric Oxide Donor- and Amphetamine-Induced Dopamine Release in Mesencephalic Primary Cultured Neurons.

Previous research has shown that nitric oxide (NO) synthase inhibitors prevent rodents' sensorimotor gating impairments induced by dopamine releasing drugs, such as amphetamine (Amph) and methylphenidate. The mechanisms of this effect have not been entirely understood. In the present work, we investigated some possible mechanisms by which the NO donor, NOC-12 (3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene), influence spontaneous and Amph-induced dopamine release, using rat mesencephalic primary cultured neurons preparations. Our results showed that NOC-12 increased dopamine release in a concentration-dependent manner and potentiated the Amph-induced one. Dopamine release induced by NOC-12 was disrupted by N-acetyl-L-cystein (NAC-a free radical scavenger) and MK-801, a NMDA (N-methyl-D-aspartate) non-competitive antagonist, and was concentration dependently affected by oxadiazolo[4,3]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC). In contrast, dopamine released by Amph was facilitated by NAC and by MK-801 and not affected by nifedipine (a L-type-Ca(+2) channel blocker), which enhanced NOC-12-induced dopamine release. The present work demonstrates that DA release induced by NOC-12 is partially dependent on sGC and on NMDA activation, and is modulated by L-type Ca(+2) channel and the antioxidant NAC. This mechanism differs from the Amph-induced one, which appears not to depend on L-type Ca(+2) channel and seems to be facilitated by NMDA channel blocking and by NAC. These results suggest that Amph and NOC-12 induce dopamine release through complementary pathways, which may explain the potentiation of Amph-induced dopamine release by NOC-12. These findings contribute to understand the involvement of NO in dopamine-related neuropsychiatric and neurodegenerative diseases.

Sparing of orexin-A and orexin-B neurons in the hypothalamus and of orexin fibers in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated macaques.

Several studies conducted in patients with Parkinson's disease have reported that the degeneration of substantia nigra dopaminergic neurons, which are essential for motor control, is associated with the loss of hypothalamic orexin neurons, which are involved in sleep regulation. In order to better explore the mutual interactions between these two systems, we wished to determine in macaques: (i) if the two orexin peptides, orexin-A and orexin-B, are distributed in the same hypothalamic cells and if they are localized in nerve terminals that project onto nigral dopaminergic neurons, and (ii) if there is a loss of orexin neurons in the hypothalamus and of orexin fibers innervating nigral dopaminergic neurons in macaques rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. We showed that virtually all cells stained for orexin-A in the hypothalamus co-expressed orexin-B. Numerous terminals stained for both orexin-A and orexin-B immunoreactivity that innervated the whole extent of the ventral tegmental area and substantia nigra pars compacta were found in close proximity to tyrosine hydroxylase-immunoreactive dendrites. These data indicate that orexin-A and orexin-B peptides are in a position to play a role in controlling the activity of nigral dopaminergic neurons. However, no loss of orexin-A or orexin-B neurons in the hypothalamus and no loss of orexin fibers in the substantia nigra pars compacta was found in MPTP-treated macaques when compared with control macaques. We conclude that a relatively selective dopaminergic lesion, such as that performed in MPTP-treated macaques, is not sufficient to induce a loss of hypothalamic orexin neurons.

Bee venom and its component apamin as neuroprotective agents in a Parkinson disease mouse model.

Bee venom has recently been suggested to possess beneficial effects in the treatment of Parkinson disease (PD). For instance, it has been observed that bilateral acupoint stimulation of lower hind limbs with bee venom was protective in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. In particular, a specific component of bee venom, apamin, has previously been shown to have protective effects on dopaminergic neurons in vitro. However, no information regarding a potential protective action of apamin in animal models of PD is available to date. The specific goals of the present study were to (i) establish that the protective effect of bee venom for dopaminergic neurons is not restricted to acupoint stimulation, but can also be observed using a more conventional mode of administration and to (ii) demonstrate that apamin can mimic the protective effects of a bee venom treatment on dopaminergic neurons. Using the chronic mouse model of MPTP/probenecid, we show that bee venom provides sustained protection in an animal model that mimics the chronic degenerative process of PD. Apamin, however, reproduced these protective effects only partially, suggesting that other components of bee venom enhance the protective action of the peptide.

Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical parkinsonism in Guadeloupe.

In Guadeloupe, epidemiological data have linked atypical parkinsonism with fruit and herbal teas from plants of the Annonaceae family, particularly Annona muricata. These plants contain a class of powerful, lipophilic complex I inhibitors, the annonaceous acetogenins. To determine the neurotoxic potential of these substances, we administered annonacin, the major acetogenin of A. muricata, to rats intravenously with Azlet osmotic minipumps (3.8 and 7.6 mg per kg per day for 28 days). Annonacin inhibited complex I in brain homogenates in a concentration-dependent manner, and, when administered systemically, entered the brain parenchyma, where it was detected by matrix-associated laser desorption ionization-time of flight mass spectrometry, and decreased brain ATP levels by 44%. In the absence of evident systemic toxicity, we observed neuropathological abnormalities in the basal ganglia and brainstem nuclei. Stereological cell counts showed significant loss of dopaminergic neurones in the substantia nigra (-31.7%), and cholinergic (-37.9%) and dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32)-immunoreactive GABAergic neurones (-39.3%) in the striatum, accompanied by a significant increase in the number of astrocytes (35.4%) and microglial cells (73.4%). The distribution of the lesions was similar to that in patients with atypical parkinsonism. These data are compatible with the theory that annonaceous acetogenins, such as annonacin, might be implicated in the aetiology of Guadeloupean parkinsonism and support the hypothesis that some forms of parkinsonism might be induced by environmental toxins.

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease.

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.