Squamosamide derivative FLZ protects dopaminergic neurons against inflammation-mediated neurodegeneration through the inhibition of NADPH oxidase activity.
BACKGROUND
Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD) through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases.


METHODS
For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS)- and 1-methyl-4-phenylpyridinium-(MPP+)-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP-) induced PD mouse model was used.


RESULTS
FLZ showed potent efficacy in protecting dopaminergic (DA) neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH) immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and prostaglandin E2 (PGE2). Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX), the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1) FLZ's protective effect was reduced in cultures from PHOX-/- mice, and 2) FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal-glial cultures was further substantiated by an in vivo study, which showed that FLZ significantly protected against MPTP-induced DA neuronal loss, microglial activation and behavioral changes.


CONCLUSIONS
Taken together, our results clearly demonstrate that FLZ is effective in protecting against LPS- and MPTP-induced neurotoxicity, and the mechanism of this protection appears to be due, at least in part, to inhibition of PHOX activity and to prevention of microglial activation.
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. zhangd2@niehs.nih.gov
Dan
D
Zhang
Xiaoming
X
Hu
Sung-Jen
SJ
Wei
Jie
J
Liu
Huiming
H
Gao
Li
L
Qian
Belinda
B
Wilson
Gengtao
G
Liu
Jau-Shyong
JS
Hong
D000818:Animals; D000978:Antiparkinson Agents / Q000494:pharmacology / Q000627:therapeutic use*; D044482:Benzeneacetamides / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D002478:Cells, Cultured / Q000187:drug effects; D015232:Dinoprostone / Q000096:biosynthesis / Q000235:genetics; D004298:Dopamine / Q000378:metabolism*; D005260:Female; D008070:Lipopolysaccharides / Q000633:toxicity; D008297:Male; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000187:drug effects / Q000502:physiology*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics; D009410:Nerve Degeneration / Q000517:prevention & control*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use*; D020734:Parkinsonian Disorders / Q000188:drug therapy*; D010636:Phenols / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D011247:Pregnancy; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011916:Rats, Inbred F344; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D000978:Antiparkinson Agents; D044482:Benzeneacetamides; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D018696:Neuroprotective Agents; D010636:Phenols; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C084439:squamosamide; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D015232:Dinoprostone; D004298:Dopamine