Striatal expression of GDNF and differential vulnerability of midbrain dopaminergic cells

Glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-beta superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRalpha1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro-gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro-gold, and after colchicine injection in the lateral ventricle, they become GDNF-immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6-OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons

Cloning and functional expression of a new epithelial sodium channel delta subunit isoform differentially expressed in neurons of the human and monkey telencephalon.

Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them

The neuronal-specific SGK1.1 kinase regulates {delta}-epithelial Na+ channel independently of PY motifs and couples it to phospholipase C signaling

The delta-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that delta-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of delta-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that delta-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of deltabetagamma-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when delta-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of delta-ENaC. Our data support a physiological role for SGK1.1 in the regulation of delta-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel

Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD

Dopamine transporter glycosylation correlates with the vulnerability of midbrain dopaminergic cells in Parkinson's disease

The dopamine transporter (DAT) is a membrane glycoprotein responsible for dopamine (DA) uptake, which has been involved in the degeneration of DA cells in Parkinson's disease (PD). Given that DAT activity depends on its glycosylation status and membrane expression, and that not all midbrain DA cells show the same susceptibility to degeneration in PD, we have investigated a possible relationship between DAT glycosylation and function and the differential vulnerability of DA cells. Glycosylated DAT expression, DA uptake, and DAT V(max) were significantly higher in terminals of nigrostriatal neurons than in those of mesolimbic neurons. No differences were found in non-glycosylated DAT expression and DAT K(m), and DA uptake differences disappeared after deglycosylation of nigrostriatal synaptosomes. The expression pattern of glycosylated DAT in the human midbrain and striatum showed a close anatomical relationship with DA degeneration in parkinsonian patients. This relationship was confirmed in rodent and monkey models of PD, and in HEK cells expressing the wild-type and a partially deglycosylated DAT form. These results strongly suggest that DAT glycosylation is involved in the differential vulnerability of midbrain DA cells in PD

DRD3 (dopamine receptor D3) but not DRD2 activates autophagy through MTORC1 inhibition preserving protein synthesis

Growing evidence shows that autophagy is deficient in neurodegenerative and psychiatric diseases, and that its induction may have beneficial effects in these conditions. However, as autophagy shares signaling pathways with cell death and interferes with protein synthesis, prolonged use of autophagy inducers available nowadays is considered unwise. The search for novel autophagy inducers indicates that DRD2 (dopamine receptor 2)-DRD3 ligands may also activate autophagy, though critical aspects of the action mechanisms and effects of dopamine ligands on autophagy are still unknown. In order to shed light on this issue, DRD2- and DRD3-overexpressing cells and drd2 KO, drd3 KO and wild-type mice were treated with the DRD2-DRD3 agonist pramipexole. The results revealed that pramipexole induces autophagy through MTOR inhibition and a DRD3-dependent but DRD2-independent mechanism. DRD3 activated AMPK followed by inhibitory phosphorylation of RPTOR, MTORC1 and RPS6KB1 inhibition and ULK1 activation. Interestingly, despite RPS6KB1 inhibition, the activity of RPS6 was maintained through activation of the MAPK1/3-RPS6KA pathway, and the activity of MTORC1 kinase target EIF4EBP1 along with protein synthesis and cell viability, were also preserved. This pattern of autophagy through MTORC1 inhibition without suppression of protein synthesis, contrasts with that of direct allosteric and catalytic MTOR inhibitors and opens up new opportunities for G protein-coupled receptor ligands as autophagy inducers in the treatment of neurodegenerative and psychiatric diseases. Abbreviations: AKT/Protein kinase B: thymoma viral proto-oncogene 1; AMPK: AMP-activated protein kinase; BECN1: beclin 1; EGFP: enhanced green fluorescent protein; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GPCR; G protein-coupled receptor; GFP: green fluorescent protein; HEK: human embryonic kidney; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP2K/MEK: mitogen-activated protein kinase kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MDA: malonildialdehyde; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PPX: pramipexole; RPTOR/raptor: regulatory associated protein of MTOR, complex 1; RPS6: ribosomal protein S6; RPS6KA/p90S6K: ribosomal protein S6 kinase A; RPS6KB1/p70S6K: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.This work was supported by the Spanish Ministerio de Economía y Competitividad [BFU2016-77363-R] and Gobierno Autónomo de Canarias [2018-00000034] to T.G.H. D.L.R. was supported by the “Programa Agustín de Betancourt” (Cabildo Insular de Tenerife). F.F.R. and A.F.C. were supported by the Programme Ayudas para contratos predoctorales para la formación de doctores”, Spanish Ministerio de Economía y Competitividad [BES-2014-067781 and BES-2017‐079923, respectively]; Consejería de Educación, Universidades y Sostenibilidad, Gobierno de Canarias [2018-00000034]; Ministerio de Economía, Industria y Competitividad, Gobierno de España [BFU2016-77363-R]; Ministerio de Economía, Industria y Competitividad, Gobierno de España [BES-2014-067781]; Ministerio de Economía, Industria y Competitividad, Gobierno de España [BES-2017‐079923]; Cabildo de Tenerife (ES) [Programa Agustín de Betancourt]

MAGIC upper limits on the GRB 090102 afterglow

Indications of a GeV component in the emission from gamma-ray bursts (GRBs) are known since the Energetic Gamma-Ray Experiment Telescope observations during the 1990s and they have been confirmed by the data of the Fermi satellite. These results have, however, shown that our understanding of GRB physics is still unsatisfactory. The new generation of Cherenkov observatories and in particular the MAGIC telescope, allow for the first time the possibility to extend the measurement of GRBs from several tens up to hundreds of GeV energy range. Both leptonic and hadronic processes have been suggested to explain the possible GeV/TeV counterpart of GRBs. Observations with ground-based telescopes of very high energy (VHE) photons (E > 30 GeV) from these sources are going to play a key role in discriminating among the different proposed emission mechanisms, which are barely distinguishable at lower energies. MAGIC telescope observations of the GRB 090102 (z = 1.547) field and Fermi Large Area Telescope data in the same time interval are analysed to derive upper limits of the GeV/TeV emission. We compare these results to the expected emissions evaluated for different processes in the framework of a relativistic blastwave model for the afterglow. Simultaneous upper limits with Fermi and a Cherenkov telescope have been derived for this GRB observation. The results we obtained are compatible with the expected emission although the difficulties in predicting the HE and VHE emission for the afterglow of this event makes it difficult to draw firmer conclusions. Nonetheless, MAGIC sensitivity in the energy range of overlap with space-based instruments (above about 40 GeV) is about one order of magnitude better with respect to Fermi. This makes evident the constraining power of ground-based observations and shows that the MAGIC telescope has reached the required performance to make possible GRB multiwavelength studies in the VHE range