Expression of RAD9B in the mesostriatal system of rats and humans: Overexpression in a 6-OHDA rat model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that affects primarily the dopaminergic (DAergic) neurons of the mesostriatal system, among other nuclei of the brain. Although it is considered an idiopathic disease, oxidative stress is believed to be involved in DAergic neuron death and therefore plays an important role in the onset and development of the disease. RAD9B is a paralog of the RAD9 checkpoint, sharing some similar functions related to DNA damage resistance and apoptosis, as well as the ability to form 9-1-1 heterotrimers with RAD1 and HUS1. METHODS: In addition to immunohistochemistry, immunofluorescence and Western-blot analysis, we implemented Quantitative RT-PCR and in situ hybridization techniques. RESULTS: We demonstrated RAD9B expression in rat and human mesencephalic DAergic cells using specific markers. Additionally, we observed significant overexpression of RAD9B mRNA (p<0.01) and protein (p<0.01) in the midbrain 48 h after inducing damage with 150 µg of 6-hydroxydopamine (6-OHDA) injected in a rat model of PD. Regarding protein expression, the increased levels were observed in neurons of the mesostriatal system and returned to normal 5 days post-injury. CONCLUSIONS: This response to a neurotoxin, known to produce oxidative stress specifically on DAergic neurons indicates the potential importance of RAD9B in this highly vulnerable population to cell death. In this model, RAD9B function appears to provide neuroprotection, as the induced lesion resulted in only mild degeneration. This observation highlights the potential of RAD9B checkpoint protein as a valuable target for future therapeutic interventions aimed at promoting neuroprotection.

Reinfection rate and disease severity of the BA.5 Omicron SARS-CoV-2 lineage compared to previously circulating variants of concern in the Canary Islands (Spain).

ABSTRACTThe emergence of the Omicron SARS-CoV-2 variant of concern has changed the COVID-19 scenario as this variant is characterized by high transmissibility and immune evasion ability. To evaluate the impact of this variant on the Canary Islands (Spain) population, we determined the reinfection rates and disease severity associated with the Omicron sublineages and the previously circulating variants of concern. We performed a retrospective observational study on 21,745 SARS-CoV-2 viral genomes collected from December 2020 to July 2022 in the Canary Islands (Spain). We compared the reinfection rates between lineages using pairwise proportion and Fisher's exact tests. To assess disease severity, we studied the association of Alpha, Delta, BA.1, BA.2, BA.5, and other risk factors on 28-day hospital mortality using logistic regression and Cox proportional hazard models. We observed 127 bona fide reinfection cases throughout the study period. We found that BA.5 had the highest reinfection rate compared to other lineages (vs. Delta p = 2.89 × 10-25; vs. BA.1 p = 5.17 × 10-11; vs. BA.2 p = 0.002). Among the 1,094 hospitalized patients, multivariate logistic regression showed that Alpha (Odds Ratio [OR] = 0.45, 95% Confidence Interval [CI] = 0.23-0.87, p = 0.02), BA.2 (OR = 0.38, 95% CI = 0.22-0.63, p = 1.91 × 10-4), and BA.5 (OR = 0.30, 95% CI = 0.16-0.55, p = 1.05 × 10-4) had lower 28-day hospital mortality compared to Delta. These results were confirmed by using Cox proportional hazard models. Omicron lineages, and in particular BA.5, were associated with higher reinfection rates and lower disease severity (28-day hospital mortality) than previously circulating variants of concern.

Bioinformatic approaches to draft the viral genome sequence of Canary Islands cases related to the multicountry mpox virus 2022-outbreak.

On July 23, 2022, monkeypox disease (mpox) was declared a Public Emergency of International Concern (PHEIC) by the World Health Organization (WHO) due to a multicountry outbreak. In Europe, several cases of mpox virus (MPXV) infection related to this outbreak were detected in the Canary Islands (Spain). Here we describe the combination of viral DNA sequencing and bioinformatic approaches, including methods for de novo genome assembly and short- and long-read technologies, used to reconstruct the first MPXV genome isolated in the Canary Islands on the 31st of May 2022 from a male adult patient with mild symptoms. The same sequencing and bioinformatic approaches were then validated with three other positive cases of MPXV infection from the same mpox outbreak. We obtained the best results using a reference-based approach with short reads, evidencing 46-79 nucleotide variants against viral sequences from the 2018-2019 mpox outbreak and placing the viral sequences in the new B.1 sublineage of clade IIb of the MPXV classification. This study of MPXV demonstrates the potential of metagenomics sequencing for rapid and precise pathogen identification.

Prolonged dopamine D3 receptor stimulation promotes dopamine transporter ubiquitination and degradation through a PKC-dependent mechanism.

The dopamine transporter (DAT) is a membrane glycoprotein in dopaminergic neurons, which modulates extracellular and intracellular dopamine levels. DAT is regulated by different presynaptic proteins, including dopamine D2 (D2R) and D3 (D3R) receptors. While D2R signalling enhances DAT activity, some data suggest that D3R has a biphasic effect. However, despite the extensive therapeutic use of D2R/D3R agonists in neuropsychiatric disorders, this phenomenon has been little studied. In order to shed light on this issue, DAT activity, expression and posttranslational modifications were studied in mice and DAT-D3R-transfected HEK cells. Consistent with previous reports, acute treatment with D2R/D3R agonists promoted DAT recruitment to the plasma membrane and an increase in DA uptake. However, when the treatment was prolonged, DA uptake and total striatal DAT protein declined below basal levels. These effects were inhibited in mice by genetic and pharmacological inactivation of D3R, but not D2R, indicating that they are D3R-dependent. No changes were detected in mesostriatal tyrosine hydroxylase (TH) protein expression and midbrain TH and DAT mRNAs, suggesting that the dopaminergic system is intact and DAT is posttranslationally regulated. The use of immunoprecipitation and cell surface biotinylation revealed that DAT is phosphorylated at serine residues, ubiquitinated and released into late endosomes through a PKCß-dependent mechanism. In sum, the results indicate that long-term D3R activation promotes DAT down-regulation, an effect that may underlie neuroprotective and antidepressant actions described for some D2R/D3R agonists.

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.

Pramipexole reduces soluble mutant huntingtin and protects striatal neurons through dopamine D3 receptors in a genetic model of Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansion of the polyglutamine tract in the huntingtin protein (HTT). The toxicity of mutant HTT (mHTT) is associated with intermediate mHTT soluble oligomers that subsequently form intranuclear inclusions. Thus, interventions promoting the clearance of soluble mHTT are regarded as neuroprotective. Striatal neurons are particularly vulnerable in HD. Their degeneration underlies motor symptoms and striatal atrophy, the anatomical hallmark of HD. Recent studies indicate that autophagy may be activated by dopamine D2 and D3 receptor (D2R/D3R) agonists. Since autophagy plays a central role in the degradation of misfolded proteins, and striatal neurons express D2R and D3R, D2R/D3R agonists may promote the clearance of mHTT in striatal neurons. Here, this hypothesis was tested by treating 8-week old R6/1 mice with the D2R/D3R agonist pramipexole for 4weeks. Pramipexole reduced striatal levels of soluble mHTT and increased the size of intranuclear inclusions in R6/1 mice. Furthermore, striatal DARPP-32 levels and motor functions were recovered. These effects were accompanied by an increase in LC3-II and a decrease in p62 in the striatum. Tollip, a selective adaptor of ubiquitinated polyQ proteins to LC3, was also reduced in the striata of R6/1mice but not in their wild-type littermates. No changes were detected in the cerebral cortex where D3R expression is very low, and behavioral and biochemical effects in the striatum were prevented by a D3R antagonist. The findings indicate that PPX protects striatal neurons by promoting the clearance of soluble mHTT through a D3R-mediated mechanism. The evidence of autophagy markers suggests that autophagy is activated, although it is not efficient at removing all mHTT recruited by the autophagic machinery as indicated by the increase in the size of intranuclear inclusions.

Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis.

Prolonged treatment with pramipexole promotes physical interaction of striatal dopamine D3 autoreceptors with dopamine transporters to reduce dopamine uptake.

The dopamine (DA) transporter (DAT), a membrane glycoprotein expressed in dopaminergic neurons, clears DA from extracellular space and is regulated by diverse presynaptic proteins like protein kinases, α-synuclein, D2 and D3 autoreceptors. DAT dysfunction is implicated in Parkinson's disease and depression, which are therapeutically treated by dopaminergic D2/D3 receptor (D2/D3R) agonists. It is, then, important to improve our understanding of interactions between D3R and DAT. We show that prolonged administration of pramipexole (0.1mg/kg/day, 6 to 21 days), a preferential D3R agonist, leads to a decrease in DA uptake in mouse striatum that reflects a reduction in DAT affinity for DA in the absence of any change in DAT density or subcellular distribution. The effect of pramipexole was absent in mice with genetically-deleted D3R (D3R(-/-)), yet unaffected in mice genetically deprived of D2R (D2R(-/-)). Pramipexole treatment induced a physical interaction between D3R and DAT, as assessed by co-immunoprecipitation and in situ proximity ligation assay. Furthermore, it promoted the formation of DAT dimers and DAT association with both D2R and α-synuclein, effects that were abolished in D3R(-/-) mice, yet unaffected in D2R(-/-) mice, indicating dependence upon D3R. Collectively, these data suggest that prolonged treatment with dopaminergic D3 agonists provokes a reduction in DA reuptake by dopaminergic neurons related to a hitherto-unsuspected modification of the DAT interactome. These observations provide novel insights into the long-term antiparkinson, antidepressant and additional clinical actions of pramipexole and other D3R agonists.

Striatal vessels receive phosphorylated tyrosine hydroxylase-rich innervation from midbrain dopaminergic neurons.

Nowadays it is assumed that besides its roles in neuronal processing, dopamine (DA) is also involved in the regulation of cerebral blood flow. However, studies on the hemodynamic actions of DA have been mainly focused on the cerebral cortex, but the possibility that vessels in deeper brain structures receive dopaminergic axons and the origin of these axons have not been investigated. Bearing in mind the evidence of changes in the blood flow of basal ganglia in Parkinson's disease (PD), and the pivotal role of the dopaminergic mesostriatal pathway in the pathophysiology of this disease, here we studied whether striatal vessels receive inputs from midbrain dopaminergic neurons. The injection of an anterograde neuronal tracer in combination with immunohistochemistry for dopaminergic, vascular and astroglial markers, and dopaminergic lesions, revealed that midbrain dopaminergic axons are in close apposition to striatal vessels and perivascular astrocytes. These axons form dense perivascular plexuses restricted to striatal regions in rats and monkeys. Interestingly, they are intensely immunoreactive for tyrosine hydroxylase (TH) phosphorylated at Ser19 and Ser40 residues. The presence of phosphorylated TH in vessel terminals indicates they are probably the main source of basal TH activity in the striatum, and that after activation of midbrain dopaminergic neurons, DA release onto vessels precedes that onto neurons. Furthermore, the relative weight of this "vascular component" within the mesostriatal pathway suggests that it plays a relevant role in the pathophysiology of PD.

Vulnerability of mesostriatal dopaminergic neurons in Parkinson's disease.

The term vulnerability was first associated with the midbrain dopaminergic neurons 85 years ago, before they were identified as monoaminergic neurons, when Foix and Nicolesco (1925) reported the loss of neuromelanin containing neurons in the midbrain of patients with post-encephalitic Parkinson's disease (PD). A few years later, Hassler (1938) showed that degeneration is more intense in the ventral tier of the substantia nigra compacta than in its dorsal tier and the ventral tegmental area (VTA), outlining the concept of differential vulnerability of midbrain dopaminergic (DA-) neurons. Nowadays, we know that other neuronal groups degenerate in PD, but the massive loss of nigral DA-cells is its pathological hallmark, having a pivotal position in the pathophysiology of the disease as it is responsible for the motor symptoms. Data from humans as well as cellular and animal models indicate that DA-cell degeneration is a complex process, probably precipitated by the convergence of different risk factors, mediated by oxidative stress, and involving pathogenic factors arising within the DA-neuron (intrinsic factors), and from its environment and distant interconnected brain regions (extrinsic factors). In light of current data, intrinsic factors seem to be preferentially involved in the first steps of the degenerative process, and extrinsic factors in its progression. A controversial issue is the relative weight of the impairment of common cell functions, such as energy metabolism and proteostasis, and specific dopaminergic functions, such as pacemaking activity and DA handling, in the pathogenesis of DA-cell degeneration. Here we will review the current knowledge about the relevance of these factors at the beginning and during the progression of PD, and in the differential vulnerability of midbrain DA-cells.

The dopamine transporter is differentially regulated after dopaminergic lesion.

The dopamine transporter (DAT) is a transmembrane glycoprotein responsible for dopamine (DA) uptake, which has been shown to be involved in DA-cell degeneration in Parkinson's disease (PD). At the same time, some studies suggest that DAT may be regulated in response to dopaminergic injury. We have investigated the mechanisms underlying DAT regulation after different degrees of dopaminergic lesion. DAT is persistently down-regulated in surviving midbrain DA-neurons after substantial (62%) loss of striatal DA-terminals, and transiently after slight (11%) loss of DA-terminals in rats. Transient DAT down-regulation consisted of a decrease of glycosylated (mature) DAT in the plasma membrane with accumulation of non-glycosylated (immature) DAT in the endoplasmic reticulum-Golgi (ERG) compartment, and recovery of the normal expression pattern 5 days after lesion. DAT redistribution to the ERG was also observed in HEK cells expressing rat DAT exposed to MPP(+), but not after exposure to DAT-unrelated neurotoxins. In contrast to other midbrain DA-cells, those in the ventrolateral region of the substantia nigra do not regulate DAT and degenerate shortly after slight DA-lesion. These data suggest that DAT down-regulation is a post-translational event induced by DA-analogue toxins, consisting of a stop in its glycosylation and trafficking to the plasma membrane. Its persistence after substantial DA-lesion may act as a compensatory mechanism helping maintain striatal DA levels. The fact that neurons which do not regulate DAT die shortly after lesion suggests a relationship between DAT down-regulation and neuroprotection.

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.