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.
Zhang
Dan
D
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
Hu
Xiaoming
X
Wei
Sung-Jen
SJ
Liu
Jie
J
Gao
Huiming
H
Qian
Li
L
Wilson
Belinda
B
Liu
Gengtao
G
Hong
Jau-Shyong
JS
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