Abstract
The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.
Highlights
The selective loss of dopaminergic neurons, from the SNc,3 is a hallmark of idiopathic Parkinson disease (PD)
The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death
Using both in vivo and in vitro models of PD, we found that exogenously administrated leptin can reverse dopaminergic cell loss and functional behaviors induced by the dopamine-neuron destroying toxin, 6-OHDA via the MEK/ERK signaling pathway
Summary
Cell Culture and Neurotoxin Exposure—Unless otherwise noted, chemicals were obtained from Sigma-Aldrich, including rat leptin. Gene Transfection of MN9D Cells—Transfection of shRNA or cDNA for enhanced green fluorescent protein, dominant negative, or wild-type MEK plasmids into MN9D cells was performed using the nucleofection electroporation system [27] according to the manufacturer’s instructions (Amaxa, Gaithersburg, MD). This transfection method resulted in Ͼ95% transfection efficiency in MN9D cells. Neuronal differentiated MN9D cells were transfected for 6 h with CRE decoy oligonucleotide (5Ј-TGACGTCAGAGAGCGCTCTGACGTCA-3) or control CRE mismatch sequence (5Ј-TAGCTGCAGAGAGCGCTCTCTGCAGCTA3Ј) at the concentration of 0.1 mol/liter (all linkages phosphorothioate protected, Bio-Synthesis Inc., Lewisville, TX). Statistical Analysis—All results are expressed as mean Ϯ S.E. of at least 6 –9 measurements per data point, from three independent experiments. * or # denotes p Ͻ 0.05, ** or ## p Ͻ 0.01, and *** p Ͻ 0.001 obtained from analysis of variance and protected least significant difference
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