Arnulfo Quesada

Estrogen interacts with the IGF-1 system to protect nigrostriatal dopamine and maintain motoric behavior after 6-hydroxdopamine lesions

The most prominent neurochemical hallmark of Parkinsons disease (PD) is the loss of nigrostriatal dopamine (DA). Animal models of PD have concentrated on depleting DA and therapies have focused on maintaining or restoring DA. Within this context estrogen protects against 6‐hydroxdopamine (6‐OHDA) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) lesions of the nigrostriatal DA pathway. Present studies tested the hypothesis that neuroprotective estrogen actions involve activation of the insulin‐like growth factor‐1 (IGF‐1) system. Ovariectomized rats were treated with either a single subcutaneous injection of 17β‐estradiol benzoate or centrally or peripherally IGF‐1. All rats were infused unilaterally with 6‐OHDA into the medial forebrain bundle (MFB) to lesion the nigrostriatal DA pathway. Tyrosine hydroxylase (TH) immunocytochemistry confirmed that rats injected with 6‐OHDA had a massive loss of TH immunoreactivity in both the ipsilateral substantia nigra compacta (60% loss) and the striatum (>95% loss) compared to the contralateral side. Loss of TH immunoreactivity was correlated with loss of asymmetric forelimb movements, a behavioral assay for motor deficits. Pretreatment with estrogen or IGF‐1 significantly prevented 6‐OHDA‐induced loss of substantia nigra compacta neurons (20% loss) and TH immunoreactivity in DA fibers in the striatum (<20% loss) and prevented the loss of asymmetric forelimb use. Blockage of IGF‐1 receptors by intracerebroventricular JB‐1, an IGF‐1 receptor antagonist, attenuated both estrogen and IGF‐1 neuroprotection of nigrostriatal DA neurons and motor behavior. These findings suggest that IGF‐1 and estrogen acting through the IGF‐1 system may be critical for neuroprotective effects of estrogen on nigrostriatal DA neurons in this model of PD.

PI3 Kinase/Akt Activation Mediates Estrogen and IGF-1 Nigral DA Neuronal Neuroprotection Against a Unilateral Rat Model of Parkinson's Disease

Recently, using the medial forebrain bundle (MFB) 6‐hydroxydopmaine (6‐OHDA) lesion rat model of Parkinsons disease (PD), we have demonstrated that blockade of central IGF‐1 receptors (IGF‐1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6‐OHDA lesions in IGF‐1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107–116). This suggested that the IGF‐1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF‐1R‐induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF‐1, the mitogen‐activated protein kinase (MAPK/ERK), and phosphatidyl‐inositol‐3‐kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF‐1 neuroprotection of SNpc DA neurons after a unilateral administration of 6‐OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF‐1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF‐1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF‐1‐mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.

Distribution and localization Patterns of Estrogen Receptor-β and Insulin-Like Growth Factor-1 Receptors in Neurons and Glial Cells of the Female Rat Substantia Nigra

Although several studies have focused on the neuroprotective effects of estrogen (E2) on stroke, there have been tantalizing reports on the potential neuroprotective role of E2 in degenerative neuronal diseases such as Alzheimers and Parkinsons (PD). In animal models of PD, E2 protects the nigrostriatal dopaminergic (DA) system against neurotoxins. However, little is known about the cellular and molecular mechanism(s) involved by which E2 elicits its neuroprotective effects on the nigrostriatal DA system. A preferred mechanism for neuroprotection is the interaction of E2 with specific neuroprotective growth factors and receptors. One such neuroprotective factor/receptor system is insulin‐like growth factor‐1 (IGF‐1). E2 neuroprotective effects in the substantia nigra (SN) DA system have been shown to be dependent on IGF‐1. To determine whether E2 also interacts with the IGF‐1 receptor (IGF‐1R) and to determine the cellular localization of estrogen receptor (ER) and IGF‐1R, we compared the distribution of ER and IGF‐1R in the SN. Stereological measurements revealed that 40% of the subpopulation of tyrosine hydroxylase‐immunoreactive (TH‐ir) SN pars compacta (SNpc) DA neurons are immunoreactive for estrogen receptor‐β (ERβ). No immunolabeling for ERα was observed. In situ hybridization and immunocytochemistry studies confirmed the expression of IGF‐1R mRNA and revealed that almost all TH‐ir SNpc DA neurons were immunoreactive for IGF‐1R, respectively. Moreover, one‐third of glial fibrillary acidic protein (GFAP‐ir) cells in the SN were ERβ‐ir, and 67% of GFAP‐ir cells expressed IGF‐1R‐ir. Therefore, the localization of ERβ and IGF‐1R on SNpc DA neurons and astrocytes suggests a modulatory role of E2 on IGF‐1R, and this modulation may affect neuroprotection. J. Comp. Neurol. 503:198–208, 2007.

T-type calcium channel antagonists suppress tremor in two mouse models of essential tremor.

Essential tremor is a common disorder that lacks molecular targets for therapeutic development. T-type calcium channel activation has been postulated to underlie rhythmicity in the olivo-cerebellar system that is implicated in essential tremor. We therefore tested whether compounds that antagonize T-type calcium channel currents suppress tremor in two mouse models that possess an essential tremor-like pharmacological response profile. Tremor was measured using digitized spectral motion power analysis with harmaline-induced tremor and in the GABA(A) receptor α1 subunit-null model. Mice were given ethosuximide, zonisamide, the neuroactive steroid (3β,5α,17β)-17-hydroxyestrane-3-carbonitrile (ECN), the 3,4-dihydroquinazoline derivative KYS05064, the mibefradil derivative NNC 55-0396, or vehicle. In non-sedating doses, each compound reduced harmaline-induced tremor by at least 50% (range of maximal suppression: 53-81%), and in the GABA(A) α1-null model by at least 70% (range 70-93%). Because the T-type calcium channel Cav3.1 is the dominant subtype expressed in the inferior olive, we assessed the tremor response of Cav3.1-deficient mice to harmaline, and found that null and heterozygote mice exhibit as much tremor as wild-type mice. In addition, ECN and NNC 55-0396 suppressed harmaline tremor as well in Cav3.1-null mice as in wild-type mice. The finding that five T-type calcium antagonists suppress tremor in two animal tremor models suggests that T-type calcium channels may be an appropriate target for essential tremor therapy development. It is uncertain whether medications developed to block only the Cav3.1 subtype would exhibit efficacy.

C‐terminal mechano‐growth factor induces heme oxygenase‐1‐mediated neuroprotection of SH‐SY5Y cells via the protein kinase Cϵ/Nrf2 pathway

Recently, a variant of insulin‐like growth factor‐1, mechano‐growth factor (MGF), has been discovered whose 24‐amino‐acid carboxy end is protective in models of stroke, nerve injury, and amyotrophic lateral sclerosis, suggesting broad‐spectrum neuroprotective properties. Moreover, we recently demonstrated in vitro and in vivo that a modified protease‐resistant 24‐amino‐acid MGF derivative (MGF24) protects dopaminergic neurons from oxidative stress‐induced apoptosis via induction of the stress response protein heme oxygenase‐1. However, the underlying mechanism by which MGF24 up‐regulates heme oxygenase‐1 expression is unknown. In this study, we demonstrate that MGF24‐induced heme oxygenase‐1 up‐regulation is dependent on activation of protein kinase Cϵ and NF‐E2‐related factor‐2 (Nrf2). MGF24 induces nuclear translocation of Nrf2, and siRNA knockdown of Nrf2 or of heme oxygenase‐1 prevents MGF24‐induced heme oxygenase‐1 up‐regulation and neuroprotection of SH‐SY5Y cells against 6‐hydroxydopamine‐induced cell death. Pharmacological inhibition of ERK, p38 MAPK, PI3K/Akt, or PKC signaling revealed that only PKC inhibition by GF109203X prevents MGF24s ability to protect against 6‐hydroxydopamine‐induced cell death. GF109203X also prevented MGF24‐induced Nrf2 nuclear translocation and heme oxygenase‐1 up‐regulation. siRNA knockdown of protein kinase Cϵ blocks MGF24‐induced Nfr2 nuclear translocation, heme oxygenase‐1 expression, and neuroprotection. Taken together, these results demonstrate that PKC activity is needed for MGF24s activation of Nrf2, which in turn increases heme oxygenase‐1 expression, a critical event in mediating MGF24s neuroprotection against 6‐hydroxydopamine‐induced apoptosis. Published 2011 Wiley‐Liss, Inc.

C-terminal mechano growth factor protects dopamine neurons: a novel peptide that induces heme oxygenase-1.

To assess potential efficacy of mechano growth factor (MGF) for chronic neurodegenerative disorders, we studied whether MGF protects dopamine (DA) neurons subjected to neurotoxic stress. We show that a short 24-amino acid C-terminal peptide of MGF (MGF24) upregulates heme oxygenase-1 (HO-1) expression and protects SH-SY5Y cells against apoptosis and cell loss induced by three DA cell-specific neurotoxins: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP(+)), and rotenone. MGF24 maintains the mitochondrial membrane potential and blocks the release of mitochondrial apoptotic-inducing factor into the cytoplasm induced by 6-OHDA, MPP(+), and rotenone. Chemical inhibition of HO-1 with zinc protoporphyrin-IX prevents neuroprotection by MGF24 against the three neurotoxins. MGF24 does not activate Akt signaling nor does Akt inhibition block MGF24 protection of SH-SY5Y cells. In 6-OHDA-lesioned rats, central or peripheral MGF24 administration protects against the development of contralateral forelimb under-utilization, reduces ipsilateral nigral DA cell body loss, and attenuates tyrosine hydroxylase fiber loss in the ipsilateral striatum, independent of IGF-1 receptor activation. Peripheral MGF24 administration upregulates HO-1 expression in striatal and midbrain tissue. This report is the first to demonstrate that a small peptide, MGF24, upregulates HO-1, an important cell defense mediator, and protects DA cells, suggesting new strategies for neuroprotection in Parkinsons disease.

The Mibefradil Derivative NNC55-0396, a Specific T-Type Calcium Channel Antagonist, Exhibits Less CYP3A4 Inhibition than Mibefradil

A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca2+ channel inhibitor without L-type Ca2+ channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s—CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1—and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC50 = 33 ± 3 nM, Ki = 23 ± 0.5 nM) and Ro40-5966 (IC50 = 30 ± 7.8 nM, Ki = 21 ± 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC50 = 300 ± 30 nM, Ki = 210 ± 6 nM). More dramatically, mibefradil (IC50 = 566 ± 71 nM, Ki = 202 ± 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6β-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC50 = 11 ± 1.1 μM, Ki = 3.9 ± 0.4 μM). Loss of testosterone 6β-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (KI = 3.87 μM, Kinact = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (KI = 83 nM, Kinact = 0.048/min). In contrast, NNC55-0396 (IC50 = 29 ± 1.2 nM, Ki = 2.8 ± 0.3 nM) and Ro40-5966 (IC50 = 46 ± 11 nM, Ki = 4.5 ± 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC50 = 129 ± 21 nM, Ki = 12.7 ± 0.9 nM). Our results suggest that NNC55–0396 could be a more favorable T-type Ca2+ antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.

Estrogen and CCK1 receptor modification of μ-opioid receptor binding in the cortex of female rats

Cholecystokinin (CCK) in the nervous system has effects opposite to those of opioids. However, the mechanism by which CCK opposes the effect of opioids at the receptor or cellular level is still unknown. In the brain, distributions of CCK receptors and opioid receptors have been demonstrated to overlap. The present study was undertaken to determine the mechanism of CCK-opioid interactions in the cortex of ovariectomized rats. Furthermore, because estrogen is a powerful regulator of CCK and opioid activity, we examined whether estrogen state also modulates the interactions of these neuropeptides. mu-Opioid (MOP) receptor binding was examined in cortical membranes that were preincubated with CCK-8S and CCK receptor agonist and antagonist followed with 3H-DAMGO. Pharmacological results revealed that CCK-8S suppressed 3H-DAMGO binding in cortical membranes of ovariectomized rats. The same result was obtained using a CCK1 receptor agonist (JMV-180), whereas a CCK2 receptor agonist (CCK-4) failed to suppress 3H-DAMGO binding. Antagonism of the CCK1 receptor by JMV-179 blocked both CCK-8S and JMV-180 suppression of 3H-DAMGO binding. Furthermore, estrogen treatment to female rats resulted in a suppression of 3H-DAMGO binding in cortical membranes. These results demonstrate an estrogen regulation of the MOP receptor and a protein-protein interaction between CCK1 receptor and MOP receptor.

Octanoic Acid Suppresses Harmaline-Induced Tremor in Mouse Model of Essential Tremor

Recent work exploring the use of high-molecular weight alcohols to treat essential tremor (ET) has identified octanoic acid as a potential novel tremor-suppressing agent. We used an established harmaline-based mouse model of ET to compare tremor suppression by 1-octanol and octanoic acid. The dose-related effect on digitized motion power within the tremor bandwidth as a fraction of overall motion power was analyzed. Both 1-octanol and octanoic acid provided significant reductions in harmaline tremor. An 8-carbon alkyl alcohol and carboxylic acid each suppress tremor in a pre-clinical mouse model of ET. Further studies are warranted to determine the safety and efficacy of such agents in humans with ET.

Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor.

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29μM respectively), and also less potent at inhibiting testosterone 6-β-hydroxylation (Ki: 0.71 and 0.12μM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20mg/kg), suppressed tremor while NNC 55-0396 at 12.5mg/kg suppressed harmaline-induced tremor by half by 20-100min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.