analyzing-parkinson-s-disease-mouse-model-induced-adeno-associated

Analyzing the Parkinson's Disease Mouse Model Induced by Adeno-associated Viral Vectors Encoding Human &#945;-Synuclein

Parkinson&#39;s disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, ...

Fundación Ciencia & Vida

Evidence from inflammatory bowel diseases (IBD) patients and animal models has indicated that gut inflammation is driven by effector CD4 T-cell, including Th1 and Th17. Conversely, Treg seem to be dysfunctional in IBD. Importantly, dopamine, which is abundant in the gut mucosa under homoeostasis, undergoes a sharp reduction upon intestinal inflammation. Here we analysed the role of the high-affinity dopamine receptor D3 (DRD3) in gut inflammation. Our results show that Drd3 deficiency confers a stronger immunosuppressive potency to Treg, attenuating inflammatory colitis manifestation in mice. Mechanistic analyses indicated that DRD3-signalling attenuates IL-10 production and limits the acquisition of gut-tropism. Accordingly, the ex vivo transduction of wild-type Treg with a siRNA for Drd3 induced a potent therapeutic effect abolishing gut inflammation. Thus, our findings show DRD3-signalling as a major regulator of Treg upon gut inflammation.

Dopaminergic signalling limits suppressive activity and gut homing of regulatory T cells upon intestinal inflammation.

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9), a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed. 

Methylation of histone H3 lysine 9 occurs during translation.

Pharmacological evidence associates type I dopamine receptors, including subtypes D1 and D5, with learning and memory. Analyses using genetic approaches have determined the relative contribution of dopamine receptor D1 (D1R) in cognitive tasks. However, the lack of drugs that can discriminate between D1R and D5R has made the pharmacological distinction between the two receptors difficult. Here, we aimed to determine the role of D5R in learning and memory. In this study we tested D5R knockout mice and wild-type littermates in a battery of behavioral tests, including memory, attention, locomotion, anxiety and motivational evaluations. Our results show that genetic deficiency of D5R significantly impairs performance in the Morris water maze paradigm, object location and object recognition memory, indicating a relevant role for D5R in spatial memory and recognition memory. Moreover, the lack of D5R resulted in decreased exploration and locomotion. In contrast, D5R deficiency had no impact on working memory, anxiety and depressive-like behavior, measured using the spontaneous alternation, open-field, tail suspension test, and forced swimming test. Electrophysiological analyses performed on hippocampal slices showed impairment in long-term-potentiation in mice lacking D5R. Further analyses at the molecular level showed that genetic deficiency of D5R results in a strong and selective reduction in the expression of the NMDA receptor subunit NR2B in the hippocampus. These findings demonstrate the relevant contribution of D5R in memory and suggest a functional interaction of D5R with hippocampal glutamatergic pathways. 

Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory.

Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells.

Dopamine Receptor D3 Signaling on CD4+ T Cells Favors Th1- and Th17-Mediated Immunity.

The dual potential to promote tolerance or inflammation to self-antigens makes dendritic cells (DCs) fundamental players in autoimmunity. Previous results have shown that stimulation of dopamine receptor D5 (DRD5) in DCs potentiates their inflammatory behaviour, favouring the development of experimental autoimmune encephalomyelitis (EAE). Here, we aimed to decipher the underlying mechanism and to test its relevance in multiple sclerosis (MS) patients. Our data shows that DRD5-deficiency confined to DCs in EAE mice resulted in reduced frequencies of CD4 T-cell subsets with inflammatory potential in the central nervous system, including not only Th1 and Th17 cells but also granulocyte-macrophage colony-stimulating factor producers. Importantly,  depletion of dopamine from DCs resulted in a dramatic reduction of EAE severity, highlighting the relevance of an autocrine loop promoting inflammation . Mechanistic analyses indicated that DRD5-signalling in both mouse DCs and human monocytes involves the attenuation of signal transducer and activator of transcription 3-activation, a transcription factor that limits the production of the inflammatory cytokines interleukin (IL)-12 and IL-23. Furthermore, we found an exacerbated expression of all dopamine receptors in peripheral blood pro-inflammatory monocytes obtained from MS patients. These findings illustrate a novel mechanism by which myeloid antigen-presenting cells may trigger the onset of their inflammatory behaviour promoting the development of autoimmunity.

Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Encephalomyelitis.

Neuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4 T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4 T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4 T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4 T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp).  analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4 T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4 T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4 T-cells obtained from PD patients. The results obtained from  experiments performed in mice show that the transference of CD4 T-cells treated  with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4 T-cells transduced  with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4 T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment.

Dopamine Receptor D3 Expression Is Altered in CD4 T-Cells From Parkinson's Disease Patients and Its Pharmacologic Inhibition Attenuates the Motor Impairment in a Mouse Model.

Dendritic cells (DCs) have the ability to induce tolerance or inflammation in response to self-antigens, which makes them fundamental players in autoimmunity. In this regard, immunogenic DCs produce IL-12 and IL-23 favouring the acquisition of Th1 and Th17 inflammatory phenotypes, respectively, by autoreactive CD4 T-cells, thus promoting autoimmunity. Conversely, tolerogenic DCs produce IL-10 and TGF-, inducing the generation of CD4 T-cells with suppressive activity (Treg), which promote tolerance to self-constituents. Previous studies have shown that STAT3 signalling in DCs attenuates the production of proinflammatory cytokines, whilst NF-B activation promotes it. In this study, we aimed to generate DCs displaying strong and constitutive tolerogenic profile to be used as immunotherapy in autoimmunity. To this end, we transduced bone marrow-derived DCs with lentiviral particles codifying for a constitutively active version of STAT3 (constitutively active STAT3 (STAT3ca)) or with a constitutive repressor of NF-B (IB superrepressor (IBSR)), and their therapeutic potential was evaluated in a mouse model of arthritis induced by collagen (CIA). Our results show that STAT3ca transduction favoured the production of the anti-inflammatory mediator IL-10, whereas IBSR transduction attenuated the expression of the proinflammatory cytokine IL-23 in DCs. Moreover, both STAT3ca-transduced and IBSR-transduced DCs separately exerted a mild but significant therapeutic effect reducing the severity of CIA development. Furthermore, when DCs were transduced with both STAT3ca and IBSR together, they reduced CIA manifestation significantly stronger than when transduced with only STAT3ca or IBSR separately. These results show STAT3 and NF-B as two important and complementary regulators of the tolerogenic behaviour of DCs, which should be considered as molecular targets in the design of DC-based suppressive immunotherapies for the treatment of autoimmune disorders.

STAT3 Activation in Combination with NF-KappaB Inhibition Induces Tolerogenic Dendritic Cells with High Therapeutic Potential to Attenuate Collagen-Induced Arthritis.

CD4 T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4β7 are necessary and sufficient to imprint colonic homing on CD4 T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation.

The Heteromeric Complex Formed by Dopamine Receptor D and CCR9 Leads the Gut Homing of CD4 T Cells Upon Inflammation.

Fluoxetine is often prescribed to treat depression during pregnancy. Rodent studies have shown that fluoxetine exposure during early development can induce persistent changes in the emotional behavior of the offspring. However, the effects of prenatal fluoxetine on memory have not been elucidated. This study evaluates the memory of adult male offspring from rat dams orally administered with a clinically relevant dose of 0.7 mg/kg fluoxetine from 9 weeks before pregnancy to 1 week before delivery. Hippocampal-dependent (Morris Water Maze, MWM) and non-hippocampal-dependent (Novel Object Recognition, NOR) memory paradigms were assessed. Anxiety- and depressive-like symptoms were also evaluated using the Open Field Test, Tail Suspension Test and Sucrose Preference Test. Male rats exposed to fluoxetine during gestation displayed NOR memory impairments during adulthood, as well as increased anxiety- and depressive-like symptoms. In the MWM, the offspring of fluoxetine-treated dams did not show learning deficits. However, a retention impairment was found on remote memory, 15 days after the end of training. Molecular analyses showed increased expression of NMDA subunit NR, and a decrease in NR-to- NR ratio in the temporal cortex, but not in the hippocampus, suggesting changes in NMDA receptor composition. These results suggest that in utero exposure to fluoxetine induces detrimental effects on non-hippocampal memory and in remote retention of hippocampal-dependent memory, which is believed to be stored in the temporal cortex, possibly due to changes in cortical NMDA receptor subunit stoichiometry. The present results warrant the need for studies on potential remote memory deficits in human offspring exposed to fluoxetine in utero.

Article	Year
Dopaminergic signalling limits suppressive activity and gut homing of regulatory T cells upon intestinal inflammation. Mucosal immunology\| PubMed ID: 33184477	2021
Methylation of histone H3 lysine 9 occurs during translation. Nucleic acids research\| PubMed ID: 26405197	2015
Dopamine receptor D5 deficiency results in a selective reduction of hippocampal NMDA receptor subunit NR2B expression and impaired memory. Neuropharmacology\| PubMed ID: 26714288	2016
Dopamine Receptor D3 Signaling on CD4+ T Cells Favors Th1- and Th17-Mediated Immunity. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 27183640	2016
Dopaminergic Stimulation of Myeloid Antigen-Presenting Cells Attenuates Signal Transducer and Activator of Transcription 3-Activation Favouring the Development of Experimental Autoimmune Enc... Frontiers in immunology\| PubMed ID: 29619030	2018
Dopamine Receptor D3 Expression Is Altered in CD4 T-Cells From Parkinson's Disease Patients and Its Pharmacologic Inhibition Attenuates the Motor Impairment in a Mouse Model. Frontiers in immunology\| PubMed ID: 31118938	2019
STAT3 Activation in Combination with NF-KappaB Inhibition Induces Tolerogenic Dendritic Cells with High Therapeutic Potential to Attenuate Collagen-Induced Arthritis. Journal of immunology research\| PubMed ID: 31355296	2019
The Heteromeric Complex Formed by Dopamine Receptor D and CCR9 Leads the Gut Homing of CD4 T Cells Upon Inflammation. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 33864900	2021
Prenatal fluoxetine impairs non-hippocampal but not hippocampal memory in adult male rat offspring. Neuropharmacology\| PubMed ID: 34375626	2021

Valentina Ugalde

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