Estrogen: A multifunctional messenger to nigrostriatal dopaminergic neurons

Gonadal steroids affect a wide variety of functions in the mammalian brain ranging from the regulation of neuroendocrine systems and the modulation of behavior to the stimulation of differentiation and plasticity of distinct neuronal populations and circuits. The last decades have also demonstrated that estrogen serves as a neuroprotective factor for distinct neurodegenerative disorders. Such neuroprotective effects of estrogen are most obvious for Parkinson's and Alzheimer's disease. Despite this knowledge, little is known about the mechanisms and cellular targets by that estrogen might elicit its protective influence. In the past, we have intensively studied the effects of estrogen on midbrain dopaminergic neurons which represent the most affected cell population during Parkinson's disease. These studies were mainly performed on developing dopaminergic cells and revealed that estrogen is an important regulator of plasticity and function of this neuronal phenotype. Precisely, we found that dopaminergic neurons are direct targets for estrogen and that estrogen stimulates neurite extension/branching and the expression of tyrosine hydroxylase, the key enzyme in dopamine synthesis. Together with other in vivo studies, we might draw the conclusion that estrogen is required for the plasticity and activity of the developing and adult nigrostriatal system. The presence of the estrogen-synthesizing enzyme aromatase within the nigrostriatal system further supports this idea. Surprisingly, estrogen effects on nigrostriatal cell function are not only transmitted by classical nuclear estrogen receptors but also depend on nonclassical estrogen actions mediated through putative membrane receptors coupled to diverse intracellular signaling cascades. In the future, it has to be elucidated whether nonclassical mechanisms besides genomic actions also contribute to estrogen-mediated neuroprotection in the adult CNS.     

Ogg1 null mice exhibit age-associated loss of the nigrostriatal pathway and increased sensitivity to MPTP

Cumulative damage to cellular macromolecules via oxidative stress is a hallmark of aging and neurodegenerative disease. Whether such damage is a cause or a subsequent effect of neurodegeneration is still unknown. This paper describes the development of an age-associated mild parkinsonian model in mice that lack the DNA repair enzyme 8-oxoguanine glycosylase 1 (Ogg1). Aged OGG1 knock-out (OGG1 KO) mice show a decreased spontaneous locomotor behavior and evidence a decrease in striatal dopamine levels, a loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN), and an increase in ubiquitin-positive inclusions in their remaining SN neurons. In addition, young OGG1 KO mice are more susceptible to the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) than their wild-type (WT) counterparts. Age-associated increases in 7,8-dihydro-2'-deoxyguanine (oxo(8)dG) have been reported in brain regions and neuronal populations affected in Parkinson's disease (PD), toxin-induced parkinsonian models, and mice harboring genetic abnormalities associated with PD. Because of these increased oxo(8)dG levels, the OGG1 KO mouse strain could shed light on molecular events leading to neuronal loss as a consequence of cumulative oxidative damage to DNA during aging and after toxicological challenge.     

Disruption of LRRK2 does not cause specific loss of dopaminergic neurons in zebrafish

Mutations in LRRK2 are genetically linked to Parkinson's disease (PD) but its normal biological function is largely unknown. Sheng et al. recently reported that deletion of the WD40 domain of LRRK2 in zebrafish specifically causes PD-like loss of neurons and behavior defect. However, our similar early study and recent confirming experiments using the same reagents reported by Sheng et al. failed to reproduce the phenotype of the loss of dopaminergic neurons, although the mRNA of LRRK2 was molecularly disrupted. Our study suggests that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation.     

Attenuation of methamphetamine-induced nigrostriatal dopaminergic neurotoxicity in mice by lipopolysaccharide pretreatment

Immunological activation has been proposed to play a role in methamphetamine-induced dopaminergic terminal damage. In this study, we examined the roles of lipopolysaccharide, a pro-inflammatory and inflammatory factor, treatment in modulating the methamphetamine-induced nigrostriatal dopamine neurotoxicity. Lipopolysaccharide pretreatment did not affect the basal body temperature or methamphetamine-elicited hyperthermia three days later. Such systemic lipopolysaccharide treatment mitigated methamphetamine-induced striatal dopamine and 3,4-dihydroxyphenylacetic acid depletions in a dose-dependent manner. As the most potent dose (1 mg/kg) of lipopolysaccharide was administered two weeks, one day before or after the methamphetamine dosing regimen, methamphetamine-induced striatal dopamine and 3,4-dihydroxyphenylacetic acid depletions remained unaltered. Moreover, systemic lipopolysaccharide pretreatment (1 mg/kg) attenuated local methamphetamine infusion-produced dopamine and 3,4-dihydroxyphenylacetic acid depletions in the striatum, indicating that the protective effect of lipopolysaccharide is less likely due to interrupted peripheral distribution or metabolism of methamphetamine. We concluded a critical time window for systemic lipopolysaccharide pretreatment in exerting effective protection against methamphetamine-induced nigrostriatal dopamine neurotoxicity.     

4-Hydroxytamoxifen attenuates methamphetamine-induced nigrostriatal dopaminergic toxicity in intact and gonadetomized mice

The present study was undertaken to assess the ability of 4-hydroxytamoxifen (4-OHT) to alter methamphetamine-induced nigrostriatal dopaminergic toxicity. Three daily doses of 4-OHT (6 micro g/day) effectively attenuated methamphetamine-induced nigrostriatal dopamine depletions in both sexes of intact and gonadectomized C57BL/6 J mice. 4-OHT alone did not alter the dopamine content levels in the striatum. Both male and female mice exhibited similar Cu, Zn-superoxide dismutase protein levels in the striata whether after gonadectomy or 4-OHT treatment. Furthermore, basal body temperature and methamphetamine-induced hyperthermia were not affected by 4-OHT treatment in either sex of mice. Using a lucigenen-derived chemiluminescence assay, we found that 4-OHT by itself can serve as a potent superoxide anion radical scavenger in vitro. The protective effects of 4-OHT against methamphetamine-induced nigrostriatal dopamine depletion can be, in part, due to its antioxidative characteristics. The free radical-scavenging ability of 4-OHT calls for further investigations for its uses in clinical practice.     

Transgenic mice lacking NMDAR-dependent LTD exhibit deficits in behavioral flexibility

While most studies have focused on the role of long-term potentiation in behavior, far less is known about the role of long-term depression (LTD). To examine the potential involvement of LTD in learning and memory, we generated transgenic mice that express a fragment of the SV40 small t antigen known to inhibit protein phosphatase 2A (PP2A). Small t antigen expression blocked both stimulus-induced and chemically induced NMDAR-dependent LTD at Schaffer collateral synapses but did not affect potentiation, depotentiation, or mGluR-dependent LTD. This physiological phenotype was associated with deficits in behavioral flexibility in both the Morris water maze and a delayed nonmatch to place T-maze task, suggesting that NMDAR-dependent LTD is required for behavioral flexibility and may act by weakening previously encoded memory traces when new information is learned.     

Neuroprotective effect of the antiparkinsonian drug pramipexole against nigrostriatal dopaminergic degeneration in rotenone-treated mice

Pramipexole, an agonist for dopamine (DA) D2/D3-receptors, has been used to treat both early and advanced Parkinson's disease (PD). In this study, we examined the effect of pramipexole on DA neurons in a PD model of C57BL/6 mice, which were treated with rotenone (30 mg/kg, p.o.) daily for 28 days. Pramipexole (1 mg/kg, i.p.) was injected daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone caused a loss of DA neurons in the substantia nigra pars compacta (SNpc), motor deficits and the up-regulation of α-synuclein immunoreactivity in some surviving DA neurons. Pramipexole inhibited rotenone-induced DA neuronal death and motor deficits, and reduced immunoreactivity for α-synuclein. In addition, pramipexole inhibited the in vitro oligomerization of human wild-type α-synuclein by H₂O₂ plus cytochrome c. To examine the neuroprotective effect of pramipexole against oxidative stress, we used a DJ-1-knockdown SH-SY5Y cell line and electron spin resonance (ESR) spectrometry. Simultaneous treatment with H₂O₂ and pramipexole resulted in the significant protection of DJ-1-knockdown cells against cell death in a concentration-dependent manner. A high concentration of pramipexole directly scavenged hydroxyl radical (OH) generated from H₂O₂ and Fe²⁺. Furthermore, pramipexole increased Bcl-2 immunoreactivity in DA neurons in the SNpc. These results suggest that pramipexole may protect DA neurons against exposure to rotenone by chronic oral administration, and this effect is mediated by multiple functions including scavenging of OH and induction of Bcl-2 protein.     

Neurotoxicity and behavioral deficits associated with Septin 5 accumulation in dopaminergic neurons

Septin 5, a parkin substrate, is a vesicle- and membrane-associated protein that plays a significant role in inhibiting exocytosis. The regulatory function of Septin 5 in dopaminergic (DAergic) neurons of substantia nigra (SN), maintained at relatively low levels, has not yet been delineated. As loss of function mutations of parkin are the principal cause of a familial Parkinson's disease, a prevailing hypothesis is that the loss of parkin activity results in accumulation of Septin 5 which confers neuron-specific toxicity in SN-DAergic neurons. In vitro and in vivo models were used to support this hypothesis. In our well-characterized DAergic SN4741 cell model, acute accumulation of elevated levels of Septin 5, but not synphilin-1 (another parkin substrate), resulted in cytotoxic cell death that was markedly reduced by parkin co-transfection. A transgenic mouse model expressing a dominant negative parkin mutant accumulated moderate levels of Septin 5 in SN-DAergic neurons. These mice acquired a progressive l-DOPA responsive motor dysfunction that developed despite a 25% higher than normal level of striatal dopamine (DA) and no apparent loss of DAergic neurons. The phenotype of this animal, increased striatal dopamine and reduced motor function, was similar to that observed in parkin knockout animals, suggesting a common DAergic alteration. These data suggest that a threshold level of Septin 5 accumulation is required for DAergic cell loss and that l-DOPA-responsive motor deficits can occur even in the presence of elevated DA.     

Increased apoptosis of parasympathetic but not enteric neurons in mice lacking GFRalpha2

Enteric neurons, unlike sympathetic and sensory neurons that require target-derived neurotrophins for survival, do not undergo classical caspase-3-mediated programmed cell death (PCD) during normal development. Whether parasympathetic neurons in the pancreas, which originate from a subpopulation of enteric nervous system (ENS) precursors, or other parasympathetic neurons undergo PCD during normal mammalian development is unknown. In GFRalpha2-deficient mice, many submandibular and intrapancreatic parasympathetic neurons are missing but whether this is due to increased neuronal death is unclear. Here we show that activated caspase-3 and PGP9.5 doubly positive neurons are present in wild-type mouse pancreas between embryonic day E15 and birth. Thus, in contrast to ENS neurons, intrapancreatic neurons undergo PCD via apoptosis during normal development. We also show that, in GFRalpha2-deficient mice, most intrapancreatic neurons are lost during this late fetal period, which coincides with a period of increased apoptosis of the neurons. Since the percentage of BrdU and Phox2b doubly positive cells in the fetal pancreas and the number of intrapancreatic neurons at E15 were similar between the genotypes, impaired precursor proliferation and migration are unlikely to contribute to the loss of intrapancreatic neurons in GFRalpha2-KO mice. Caspase-3-positive neurons were also found in GFRalpha2-deficient submandibular ganglia around birth, suggesting that parasympathetic neurons depend on limited supply of (presumably target-derived neurturin) signaling via GFRalpha2 for survival.     

Cannabinoid receptor type 1 protects nigrostriatal dopaminergic neurons against MPTP neurotoxicity by inhibiting microglial activation

This study examined whether the cannabinoid receptor type 1 (CB(1)) receptor contributes to the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced significant loss of nigrostriatal DA neurons and microglial activation in the substantia nigra (SN), visualized with tyrosine hydroxylase or macrophage Ag complex-1 immunohistochemistry. Real-time PCR, ELISA, Western blotting, and immunohistochemistry disclosed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase, and subsequent reactive oxygen species production and oxidative damage of DNA and proteins in MPTP-treated SN, resulting in degeneration of DA neurons. Conversely, treatment with nonselective cannabinoid receptor agonists (WIN55,212-2 and HU210) led to increased survival of DA neurons in the SN, their fibers and dopamine levels in the striatum, and improved motor function. This neuroprotection by cannabinoids was accompanied by suppression of NADPH oxidase reactive oxygen species production and reduced expression of proinflammatory cytokines from activated microglia. Interestingly, cannabinoids protected DA neurons against 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia, but not in neuron-enriched mesencephalic cultures devoid of microglia. The observed neuroprotection and inhibition of microglial activation were reversed upon treatment with CB(1) receptor selective antagonists AM251 and/or SR14,716A, confirming the involvement of the CB(1) receptor. The present in vivo and in vitro findings clearly indicate that the CB(1) receptor possesses anti-inflammatory properties and inhibits microglia-mediated oxidative stress. Our results collectively suggest that the cannabinoid system is beneficial for the treatment of Parkinson's disease and other disorders associated with neuroinflammation and microglia-derived oxidative damage.     

Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice

Mutations in the DJ-1 gene were recently identified in an autosomal recessive form of early-onset familial Parkinson disease. Structural biology, biochemistry, and cell biology studies have suggested potential functions of DJ-1 in oxidative stress, protein folding, and degradation pathways. However, animal models are needed to determine whether and how loss of DJ-1 function leads to Parkinson disease. We have generated DJ-1 null mice with a mutation that resembles the large deletion mutation reported in patients. Our behavioral analyses indicated that DJ-1 deficiency led to age-dependent and task-dependent motoric behavioral deficits that are detectable by 5 months of age. Unbiased stereological studies did not find obvious dopamine neuron loss in 6-month- and 11-month-old mice. Neurochemical examination revealed significant changes in striatal dopaminergic function consisting of increased dopamine reuptake rates and elevated tissue dopamine content. These data represent the in vivo evidence that loss of DJ-1 function alters nigrostriatal dopaminergic function and produces motor deficits.     

Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals

Despite the identification of several mutations in familial Parkinson's disease (PD), the underlying mechanisms of dopaminergic neuronal loss in idiopathic PD are still unknown. To study whether caspase-dependent apoptosis may play a role in the pathogenesis of PD, we examined 6-hydroxydopamine (6-OHDA) toxicity in dopaminergic SH-SY5Y cells and in embryonic dopaminergic mesencephalic cultures. 6-OHDA induced activation of caspases 3, 6 and 9, chromatin condensation and cell death in SH-SY5Y cells. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethylketone (zVAD-fmk) or adenovirally mediated ectopic expression of the X-chromosomal inhibitor of apoptosis protein (XIAP) blocked caspase activation and prevented death of SH-SY5Y cells. Similarly, zVAD-fmk provided protection from 6-OHDA-induced loss of tyrosine hydroxylase-positive neurones in mesencephalic cultures. In contrast, zVAD-fmk failed to protect mesencephalic dopaminergic neurones from 6-OHDA-induced loss of neurites and reduction of [(3)H]dopamine uptake. These data suggest that, although caspase inhibition provides protection from 6-OHDA-induced death of dopaminergic neurones, the neurones may remain functionally impaired.     

Ethyl pyruvate rescues nigrostriatal dopaminergic neurons by regulating glial activation in a mouse model of Parkinson's disease

This study examined whether ethyl pyruvate (EP) promotes the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. MPTP induced degeneration of nigrostriatal DA neurons and glial activation as visualized by tyrosine hydroxylase, macrophage Ag complex-1, and/or glial fibrillary acidic protein immunoreactivity. Western blotting and immunohistochemistry showed activation of microglial NADPH oxidase and astroglial myeloperoxidase (MPO) and subsequent reactive oxygen species/reactive nitrogen species production and oxidative DNA damage in the MPTP-treated substantia nigra. Treatment with EP prevented degeneration of nigrostriatal DA neurons, increased striatal dopamine levels, and improved motor function. This neuroprotection afforded by EP was associated with the suppression of astroglial MPO expression, NADPH oxidase-, and/or inducible NO synthase-derived reactive oxygen species/reactive nitrogen species production by activated microglia. Interestingly, EP was found to protect DA neurons from 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present findings show that EP may inhibit glial-mediated oxidative stress, suggesting that EP may have therapeutic value in the treatment of aspects of Parkinson's disease related to glia-derived oxidative damage.     

PACAP27 prevents Parkinson-like neuronal loss and motor deficits but not microglia activation induced by prostaglandin J2

Neuroinflammation is a major risk factor in Parkinson's disease (PD). Alternative approaches are needed to treat inflammation, as anti-inflammatory drugs such as NSAIDs that inhibit cyclooxygenase-2 (COX-2) can produce devastating side effects, including heart attack and stroke. New therapeutic strategies that target factors downstream of COX-2, such as prostaglandin J2 (PGJ2), hold tremendous promise because they will not alter the homeostatic balance offered by COX-2 derived prostanoids. In the current studies, we report that repeated microinfusion of PGJ2 into the substantia nigra of non-transgenic mice, induces three stages of pathology that mimic the slow-onset cellular and behavioral pathology of PD: mild (one injection) when only motor deficits are detectable, intermediate (two injections) when neuronal and motor deficits as well as microglia activation are detectable, and severe (four injections) when dopaminergic neuronal loss is massive accompanied by microglia activation and motor deficits. Microglia activation was evaluated in vivo by positron emission tomography (PET) with [¹¹C](R)PK11195 to provide a regional estimation of brain inflammation. PACAP27 reduced dopaminergic neuronal loss and motor deficits induced by PGJ2, without preventing microglia activation. The latter could be problematic in that persistent microglia activation can exert long-term deleterious effects on neurons and behavior. In conclusion, this PGJ2-induced mouse model that mimics in part chronic inflammation, exhibits slow-onset PD-like pathology and is optimal for testing diagnostic tools such as PET, as well as therapies designed to target the integrated signaling across neurons and microglia, to fully benefit patients with PD.     

Leptin treatment prevents type I diabetic marrow adiposity but not bone loss in mice

Leptin is a hormone secreted by adipocytes that is implicated in the regulation of bone density. Serum leptin levels are decreased in rodent models of type 1 (T1-) diabetes and in diabetic patients. Whether leptin mediates diabetic bone changes is unclear. Therefore, we treated control and T1-diabetic mice with chronic (28 days) subcutaneous infusion of leptin or saline to elucidate the therapeutic potential of leptin for diabetic osteoporosis. Leptin prevented the increase of marrow adipocytes and the increased aP2 expression that we observed in vehicle-treated diabetic mice. However, leptin did not prevent T1-diabetic decreases in trabecular bone volume fraction or bone mineral density in tibia or vertebrae. Consistent with this finding, markers of bone formation (osteocalcin RNA and serum levels) in diabetic mice were not restored to normal levels with leptin treatment. Interestingly, markers of bone resorption (TRAP5 RNA and serum levels) were decreased in diabetic mice by leptin treatment. In summary, we have demonstrated a link between low leptin levels in T1-diabetes and marrow adiposity. However, leptin treatment alone was not successful in preventing bone loss.     

NDRG4 protein-deficient mice exhibit spatial learning deficits and vulnerabilities to cerebral ischemia

The N-myc downstream-regulated gene (NDRG) family consists of four related proteins, NDRG1-NDRG4, in mammals. We previously generated NDRG1-deficient mice that were unable to maintain myelin sheaths in peripheral nerves. This condition was consistent with human hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease type 4D, caused by a nonsense mutation of NDRG1. In contrast, the effects of genetic defects of the other NDRG members remain unknown. In this study, we focused on NDRG4, which is specifically expressed in the brain and heart. In situ mRNA hybridization on the brain revealed that NDRG4 was expressed in neurons of various areas. We generated NDRG4-deficient mice that were born normally with the expected Mendelian frequency. Immunochemical analysis demonstrated that the cortex of the NDRG4-deficient mice contained decreased levels of brain-derived neurotrophic factor (BDNF) and normal levels of glial cell line-derived neurotrophic factor, NGF, neurotrophin-3, and TGF-β1. Consistent with BDNF reduction, NDRG4-deficient mice had impaired spatial learning and memory but normal motor function in the Morris water maze test. When temporary focal ischemia of the brain was induced, the sizes of the infarct lesions were larger, and the neurological deficits were more severe in NDRG4-deficient mice compared with the control mice. These findings indicate that NDRG4 contributes to the maintenance of intracerebral BDNF levels within the normal range, which is necessary for the preservation of spatial learning and the resistance to neuronal cell death caused by ischemic stress.