Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum.

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1-5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.

Protection of dopamine neurons by CDNF and neurturin variant N4 against MPP+ in dissociated cultures from rat mesencephalon.

Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 µM) decreased tyrosine hydroxylase-positive cells to 41.7 ± 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 ± 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 ± 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.

The Molecular Chaperone Hsc70 Interacts with Tyrosine Hydroxylase to Regulate Enzyme Activity and Synaptic Vesicle Localization.

We previously reported that the vesicular monoamine transporter 2 (VMAT2) is physically and functionally coupled with Hsc70 as well as with the dopamine synthesis enzymes tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase, providing a novel mechanism for dopamine homeostasis regulation. Here we expand those findings to demonstrate that Hsc70 physically and functionally interacts with TH to regulate the enzyme activity and synaptic vesicle targeting. Co-immunoprecipitation assays performed in brain tissue and heterologous cells demonstrated that Hsc70 interacts with TH and aromatic amino acid decarboxylase. Furthermore, in vitro binding assays showed that TH directly binds the substrate binding and carboxyl-terminal domains of Hsc70. Immunocytochemical studies indicated that Hsc70 and TH co-localize in midbrain dopaminergic neurons. The functional significance of the Hsc70-TH interaction was then investigated using TH activity assays. In both dopaminergic MN9D cells and mouse brain synaptic vesicles, purified Hsc70 facilitated an increase in TH activity. Neither the closely related protein Hsp70 nor the unrelated Hsp60 altered TH activity, confirming the specificity of the Hsc70 effect. Overexpression of Hsc70 in dopaminergic MN9D cells consistently resulted in increased TH activity whereas knockdown of Hsc70 by short hairpin RNA resulted in decreased TH activity and dopamine levels. Finally, in cells with reduced levels of Hsc70, the amount of TH associated with synaptic vesicles was decreased. This effect was rescued by addition of purified Hsc70. Together, these data demonstrate a novel interaction between Hsc70 and TH that regulates the activity and localization of the enzyme to synaptic vesicles, suggesting an important role for Hsc70 in dopamine homeostasis.

Protection of cultured dopamine neurons from MPP(+) requires a combination of neurotrophic factors.

Parkinson's disease is a progressive neurodegenerative disorder, caused in part by the loss of dopamine (DA) neurons in the substantia nigra (SN). Neurotrophic factors have been shown to increase the basal survival of DA neurons in vitro, as well as to protect the neurons from some toxins under certain in vitro conditions and in animal models. Although these factors have often been tested individually, they have rarely been studied in combinations. We therefore examined the effect of such combinations after acute exposure to the toxin 1-methyl-4-phenylpyridinium (MPP(+) ) using dissociated postnatal rat midbrain cultures isolated from SN and ventral tegmental area (VTA). We found that significant loss of DA neurons in the SN occurred with an LC50 of between 1 and 10 µm, whereas the LC50 of DA neurons from the VTA was approximately 1000-fold higher. We did not observe neuroprotection against MPP(+) by individual exposure to glial cell-line derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), transforming growth factor beta (TGFß), basic fibroblast growth factor (FGF-2) or growth/differentiation factor 5 (GDF5) at concentrations of 100 or 500 ng/mL. Combinations of two, three or four neurotrophic factors were also ineffective. However, when the SN cultures were exposed to a combination of all five neurotrophic factors, each at a concentration of 100 ng/mL, we observed a 30% increase in DA neuron survival in the presence of 10 and 500 µm MPP(+) . These results may be relevant to the use of neurotrophic factors as therapeutic treatments for Parkinson's disease.

ERK1, 2, and 5 expression and activation in dopaminergic brain regions during postnatal development.

Degeneration and dysfunctioning of dopaminergic neurons in the midbrain have been associated with serious neurodegenerative and neuropsychiatric disorders. Elucidating the underlying neurobiology of these neurons during early postnatal development may provide important information regarding the etiology of these disorders. Cellular signaling pathways have been shown to regulate postnatal neuronal development. Among several signaling pathways, extracellular-regulated mitogen kinases (ERK) 1, 2, and 5 have been shown to be crucial for the survival and function of dopaminergic neurons. In this study, the basal expression and activation of ERK1, 2, and 5 were studied during postnatal development in regions rich in DA cells and terminals. In the striatum (STR) and ventral mesencephalon regions of the substantia nigra (SN) and ventral tegmental area (VTA), ERK5 expression and activation were high during early postnatal days and declined with aging. Interestingly, sharp increases in phosphorylated or activated ERK1 and ERK2 were observed at postnatal day (PND) 7 in the SN and VTA. In contrast, in the STR, the levels of phosphorylated ERK1 and 2 were significantly higher at PND0 than at any other PND examined. Overall, the understanding of alterations in ERK signaling in regions rich in DA cells and DA terminals during postnatal neuronal development may provide information about their role in regulation of dopamine neuronal development which may ultimately provide insight into the underlying mechanisms of dopamine neurodegeneration.

Comparison of GDF5 and GDNF as neuroprotective factors for postnatal dopamine neurons in ventral mesencephalic cultures.

Loss of dopamine neurons is associated with the motor deficits that occur in Parkinson's disease. Although many drugs have proven to be useful in the treatment of the symptoms of this disease, none has been shown to have a significant impact on the development of the disease. However, we believe that several neurotrophic factors have the potential to reduce its progression. Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-ß superfamily of neurotrophic factors, has been extensively studied in this regard. Less attention has been paid to growth/differentiation factor 5 (GDF5), another member of the same superfamily. This study compares GDNF and GDF5 in dissociated cultures prepared from ventral mesencephalon and in organotypic co-cultures containing substantia nigra, striatum, and neocortex. We report that both GDNF (10-500 ng/ml) and GDF5 (100-500 ng/ml) promoted the survival of dopamine neurons from the substantia nigra of postnatal rats, although GDNF was considerably more potent than GDF5. In contrast, neither factor had any significant effect on the survival of dopamine neurons from the rat ventral tegmental area. Using organotypic co-cultures, we also compared GDF5 with GDNF as chemoattractants for the innervation of the striatum and the neocortex by dopamine neurons from the substantia nigra. The addition of either GDF5 or GDNF (100-500 ng/ml) caused innervation by dopamine neurons into the cortex as well as the striatum, which did not occur in untreated cultures. Our results are consistent with similar findings suggesting that GDF5, like GDNF, deserves attention as a possible therapeutic intervention for Parkinson's disease.

Role of ERK1, 2, and 5 in dopamine neuron survival during aging.

Extracellular signal-regulated kinases (ERKs) 1, 2, and 5 have been shown to play distinct roles in proliferation, differentiation, and neuronal viability. In this study, we examined ERK1, 2, and 5 expression and activation in the substantia nigra (SN), striatum (STR), and ventral tegmental area (VTA) during aging. An age-related decrease in phosphorylated ERK5 was observed in the SN and STR, whereas an increase in total ERK1 was observed in all 3 regions. In primary cultures of the SN and VTA, inhibition of ERK5 but not ERK1 and 2 decreased dopamine neuronal viability significantly. These data suggest that ERK5 is essential for the basal survival of SN and VTA dopaminergic neurons. This is the first study to examine ERK1, 2, and 5 expression and activation in the SN, STR, and VTA during aging, and the relative roles of ERK1, 2, and 5 in basal survival of SN and VTA dopaminergic neurons. These data raise the possibility that a decline in ERK5 signaling may play a role in age-related impairments in dopaminergic function.

Assaying multiple biochemical variables from the same tissue sample.

Experiments often involve multiple analyses, such as assays of neurotransmitters and proteins, and this can require different initial sample preparations. Typically, this is accomplished by using different animals or different tissue samples from the same animal. Either approach renders comparisons between assays more variable and greatly increases the effort and/or cost. Using tissue collected from rat striatum and molecules of special relevance to studies of Parkinson's disease, we show that tissue sonication in water prior to aliquoting into the appropriate concentrated solutions (e.g. HClO(4) and lysis buffers) permits several types of measurements to be made from the same initial samples. Dopamine and its metabolite homovanillic acid, serotonin and its metabolite 5-hydroxyindoleacetic acid, tyrosine hydroxylase and its phosphorylation at Ser19 and Ser31, and the dopamine transporter were unaffected. However, phospho-Akt levels fell slightly and phospho-ERK1/2 tended to drop. We also present a simple technique to preserve phosphorylation state of proteins such as ERK1/2 by perfusing animals through the heart with a phosphatase inhibitor, NaF. Dopamine metabolite dihydroxyphenyl acetic acid (DOPAC) levels were raised with both techniques, however. The general principles reported here are likely to apply to other brain regions, facilitate multiple comparisons of variables, increase efficiency, and decrease costs.

Neuroprotective role of ERK1/2 and ERK5 in a dopaminergic cell line under basal conditions and in response to oxidative stress.

Loss of motor function in Parkinson's disease is due in part to degeneration of dopamine (DA) neurons. Pharmacological evidence suggests that the mitogen-activated protein kinase signaling pathways involving extracellular signal-regulated kinases (ERKs) play important roles in neuroprotection of DA neurons. However, the relative roles of the several ERK isoforms in the viability of DA neurons have not yet been determined. In the present study, we investigated the contributions of ERK5, as well as ERK1/2, to MN9D cell survival under basal conditions and in response to 6-hydroxydopamine (6-OHDA). We observed that U0126, an inhibitor of ERK activation, decreased basal survival of these cells. To differentiate between ERK1/2 and ERK5, cells were transfected with a dominant negative form of either ERK5 or MEK1, the upstream activator of ERK1/2. Transfection of MN9D cells with either dominant negative construct mimicked U0126, reducing cell survival. Moreover, transfection of the cells in such a way as to increase ERK5 or ERK1/2 activity inhibited 6-OHDA-induced cell death, although this effect was significant only in the case of ERK1/2 activation. These studies suggest that activations of ERK5 and ERK1/2 both promote basal DA cell survival and that ERK1/2 also protects DA cells from oxidative stress. These are the first studies to demonstrate a role for ERK5 in DA neuronal survival and to investigate the relative roles of ERK1/2 and ERK5 in basal DA survival and neuroprotection from oxidative stress.

Dopaminergic innervation of forebrain by ventral mesencephalon in organotypic slice co-cultures: effects of GDNF.

Numerous studies have verified the ability of glial cell line-derived neurotrophic factor (GDNF) to protect or rescue neurons in models of Parkinson's disease. However, the role of GDNF in the development of dopaminergic (DA) neurons remains unclear. We investigated the hypothesis that GDNF is a target protein for the DA neurons of the mesencephalon forming the nigrostriatal pathway in an in vitro rat model. Organotypic slice cultures were prepared from tissue isolated from postnatal rat pups including but not limited to the substantia nigra (SN), striatum, and cerebral cortex. These cultures were maintained for up to 100 days in vitro. In the absence of exogenous GDNF, DA neurons from the SN grew into the striatum but not the cerebral cortex or hippocampus as determined by immunostaining for tyrosine hydroxylase. The addition of exogenous GDNF increased the survival of DA neurons and also enhanced the number of dopaminergic processes innervating the striatum. GDNF also induced DA innervation of the cerebral cortex but not hippocampus. In conclusion, our studies indicate that the normal pattern of innervation by DA neurons of the mesencephalon can be recapitulated with organotypic co-cultures and that this pattern can be altered by GDNF.

Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line-derived neurotrophic factor.

6-Hydroxydopamine (6-OHDA)-induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6-OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6-OHDA (40-100 microm, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6-OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6-OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non-specific concentrations of 6-OHDA. The effects of 6-OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z-DEVD-fmk (150 microm), a caspase inhibitor. Glial cell line-derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6-OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can selectively destroy DA neurons in post-natal cultures of SN, acting at least in part by initiating caspase-dependent apoptosis, and this effect can be attenuated early but not late by GDNF.