Nigrostriatal degeneration determines dynamics of glial inflammatory and phagocytic activity.

Glial cells are key players in the initiation of innate immunity in neurodegeneration. Upon damage, they switch their basal activation state and acquire new functions in a context and time-dependent manner. Since modulation of neuroinflammation is becoming an interesting approach for the treatment of neurodegenerative diseases, it is crucial to understand the specific contribution of these cells to the inflammatory reaction and to select experimental models that recapitulate what occurs in the human disease. Previously, we have characterized a region-specific activation pattern of CD11b+ cells and astrocytes in the α-synuclein overexpression mouse model of Parkinson´s disease (PD). In this study we hypothesized that the time and the intensity of dopaminergic neuronal death would promote different glial activation states. Dopaminergic degeneration was induced with two administration regimens of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), subacute (sMPTP) and chronic (cMPTP). Our results show that in the sMPTP mouse model, the pro-inflammatory phenotype of striatal CD11b+ cells was counteracted by an anti-inflammatory astrocytic profile. In the midbrain the roles were inverted, CD11b+ cells exhibited an anti-inflammatory profile and astrocytes were pro-inflammatory. The overall response generated resulted in decreased CD4 T cell infiltration in both regions. Chronic MPTP exposure resulted in a mild and prolonged neuronal degeneration that generated a pro-inflammatory response and increased CD4 T cell infiltration in both regions. At the onset of the neurodegenerative process, microglia and astrocytes cooperated in the removal of dopaminergic terminals. With time, only microglia maintained the phagocytic activity. In the ventral midbrain, astrocytes were the main phagocytic mediators at early stages of degeneration while microglia were the major phagocytic cells in the chronic state. In this scenario, we questioned which activation pattern recapitulates better the features of glial activation in PD. Glial activation in the cMPTP mouse model reflects many pathways of their corresponding counterparts in the human brain with advanced PD. Altogether, our results point toward a context-dependent cooperativity of microglia/myeloid cells and astrocytes in response to neuronal damage and the relevance of selecting the right experimental models for the study of neuroinflammation.

An MRI assessment of prostate cancer local recurrence using the PI-RR system: diagnostic accuracy, inter-observer reliability among readers with variable experience, and correlation with PSA values.

OBJECTIVES: The Prostate Imaging for Recurrence Reporting (PI-RR) system has been recently proposed to promote standardisation in the MR assessment of prostate cancer (PCa) local recurrence after radical prostatectomy (RP) and radiation therapy (RT). This study aims to evaluate PI-RR's diagnostic accuracy, assess the inter-observer reliability among readers with variable experience, and correlate imaging results with anatomopathological and laboratory parameters. METHODS: Patients who underwent a pelvic MRI for suspicion of PCa local recurrence after RP or RT were retrospectively enrolled (October 2017-February 2020). PI-RR scores were independently assessed for each patient by five readers with variable experience in prostate MRI (two senior and three junior radiologists). Biochemical data and histopathological features were collected. The reference standard was determined through biochemical, imaging, or histopathological follow-up data. Reader's diagnostic performance was assessed using contingency tables. Cohen's kappa coefficient (κ) and intraclass correlation coefficient (ICC) were calculated to measure inter-observer reliability. RESULTS: The final cohort included 120 patients (median age, 72 years [IQR, 62-82]). Recurrence was confirmed in 106 (88.3%) patients. Considering a PI-RR score ≥ 3 as positive for recurrence, minimum and maximum diagnostic values among the readers were as follows: sensitivity 79-86%; specificity 64-86%; positive predictive value 95-98%; negative predictive value 33-46%; accuracy 79-87%. Regardless of reader's level of experience, the inter-observer reliability resulted good or excellent (κ ranges across all readers: 0.52-0.77), and ICC was 0.8. Prostate-specific antigen (PSA) velocity, baseline-PSA, and trigger-PSA resulted predictive of local recurrence at imaging. CONCLUSIONS: The PI-RR system is an effective tool for MRI evaluation of PCa local recurrence and facilitates uniformity among radiologists. CLINICAL RELEVANCE STATEMENT: This study confirmed the PI-RR system's good diagnostic accuracy for the MRI evaluation of PCa local recurrences. It showed high reproducibility among readers with variable experience levels, validating it as a promising standardisation tool for assessing patients with biochemical recurrence. KEY POINTS: • In this retrospective study, the PI-RR system revealed promising diagnostic performances among five readers with different experience (sensitivity 79-86%; specificity 64-86%; accuracy 79-87%). • The inter-observer reliability among the five readers resulted good or excellent (κ ranges: 0.52-0.77) with an intraclass correlation coefficient of 0.8. • The PI-RR assessment score may facilitate standardisation and generalizability in the evaluation of prostate cancer local recurrence among radiologists.

Exploratory Analysis of the Role of Radiomic Features in the Differentiation of Oncocytoma and Chromophobe RCC in the Nephrographic CT Phase.

In diagnostic imaging, distinguishing chromophobe renal cell carcinomas (chRCCs) from renal oncocytomas (ROs) is challenging, since they both present similar radiological characteristics. Radiomics has the potential to help in the differentiation between chRCCs and ROs by extracting quantitative imaging. This is a preliminary study of the role of radiomic features in the differentiation of chRCCs and ROs using machine learning models. In this retrospective work, 38 subjects were involved: 19 diagnosed with chRCCs and 19 with ROs. The CT nephrographic contrast phase was selected in each case. Three-dimensional segmentations of the lesions were performed and the radiomic features were extracted. To assess the reliability of the features, the intraclass correlation coefficient was calculated from the segmentations performed by three radiologists with different degrees of expertise. The selection of features was based on the criteria of excellent intraclass correlation coefficient (ICC), high correlation, and statistical significance. Three machine learning models were elaborated: support vector machine (SVM), random forest (RF), and logistic regression (LR). From 105 extracted features, 41 presented an excellent ICC and 6 were not highly correlated with each other. Only two features showed significant differences according to histological type and machine learning models were developed with them. LR was the better model, in particular, with an 83% precision.

Gynaecological Causes of Acute Pelvic Pain: Common and Not-So-Common Imaging Findings.

In female patients, acute pelvic pain can be caused by gynaecological, gastrointestinal, and urinary tract pathologies. Due to the variety of diagnostic possibilities, the correct assessment of these patients may be challenging. The most frequent gynaecological causes of acute pelvic pain in non-pregnant women are pelvic inflammatory disease, ruptured ovarian cysts, ovarian torsion, and degeneration or torsion of uterine leiomyomas. On the other hand, spontaneous abortion, ectopic pregnancy, and placental disorders are the most frequent gynaecological entities to cause acute pelvic pain in pregnant patients. Ultrasound (US) is usually the first-line diagnostic technique because of its sensitivity across most common aetiologies and its lack of radiation exposure. Computed tomography (CT) may be performed if ultrasound findings are equivocal or if a gynaecologic disease is not initially suspected. Magnetic resonance imaging (MRI) is an extremely useful second-line technique for further characterisation after US or CT. This pictorial review aims to review the spectrum of gynaecological entities that may manifest as acute pelvic pain in the emergency department and to describe the imaging findings of these gynaecological conditions obtained with different imaging techniques.

Pilot Study for the Assessment of the Best Radiomic Features for Bosniak Cyst Classification Using Phantom and Radiologist Inter-Observer Selection.

Since the Bosniak cysts classification is highly reader-dependent, automated tools based on radiomics could help in the diagnosis of the lesion. This study is an initial step in the search for radiomic features that may be good classifiers of benign-malignant Bosniak cysts in machine learning models. A CCR phantom was used through five CT scanners. Registration was performed with ARIA software, while Quibim Precision was used for feature extraction. R software was used for the statistical analysis. Robust radiomic features based on repeatability and reproducibility criteria were chosen. Excellent correlation criteria between different radiologists during lesion segmentation were imposed. With the selected features, their classification ability in benignity-malignity terms was assessed. From the phantom study, 25.3% of the features were robust. For the study of inter-observer correlation (ICC) in the segmentation of cystic masses, 82 subjects were prospectively selected, finding 48.4% of the features as excellent regarding concordance. Comparing both datasets, 12 features were established as repeatable, reproducible, and useful for the classification of Bosniak cysts and could serve as initial candidates for the elaboration of a classification model. With those features, the Linear Discriminant Analysis model classified the Bosniak cysts in terms of benignity or malignancy with 88.2% accuracy.

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models.

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

Microglia and astrocyte activation is region-dependent in the α-synuclein mouse model of Parkinson's disease.

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.

Efficacy of a Contextualized Measurement of Life Satisfaction: A Pilot Study on the Assessment of Progress in Eating Disorder Therapy.

Eating disorders strongly affect psychological distress and its perception. However, most of the existing instruments for assessing life satisfaction rely on a point-estimation method that is biased due to the circumstantial conditions around the time of assessment. The main goal of this study was to apply a different kind of instrument-the Life Satisfaction Chart-that situates the current state of life satisfaction in the context of personal history and describes the life stages through a graph. The assessment was applied to a sample of 29 adolescent women (average age of 17.88) who were enrolled in a clinical program to treat their eating disorders. The results showed that their estimation of their current life satisfaction was almost identical to the estimation provided by a therapist for those who were in therapy phases 1, 2, and 3 (of four), while patients' point-estimation satisfaction showed statistically significant differences when compared with the situated estimations. In therapy phase 4, significant discrepancies were observed between the therapist's perception and the patients' perception, because the therapist focused only on eating disorder recovery, whilst the patients evaluated their lives under almost-normal conditions, taking into account further dimensions. The Life Satisfaction Chart is a new approach to life-satisfaction measurement that showed promising measurement and therapeutical properties.

Haemodynamic-dependent arrest of circulating tumour cells at large blood vessel bifurcations as new model for metastasis.

Homing of circulating tumour cells (CTC) at distant sites represents a critical event in metastasis dissemination. In addition to physical entrapment, probably responsible of the majority of the homing events, the vascular system provides with geometrical factors that govern the flow biomechanics and impact on the fate of the CTC. Here we mathematically explored the distribution of velocities and the corresponding streamlines at the bifurcations of large blood vessel and characterized an area of low-velocity at the carina of bifurcation that favours the residence of CTC. In addition to this fluid physics effect, the adhesive capabilities of the CTC provide with a biological competitive advantage resulting in a marginal but systematic arrest as evidenced by dynamic in vitro recirculation in Y-microchannels and by perfusion in in vivo mice models. Our results also demonstrate that viscosity, as a main determinant of the Reynolds number that define flow biomechanics, may be modulated to limit or impair CTC accumulation at the bifurcation of blood vessels, in agreement with the apparent positive effect observed in the clinical setting by anticoagulants in advanced oncology disease.

Mental Health Screening of Healthcare Professionals Who Are Candidates for Psychological Assistance during the COVID-19 Pandemic.

Healthcare professionals (HCPs) are among those most affected by the COVID-19 health emergency, with many presenting symptoms of anxiety and depression. Research shows that one of the factors involved in mitigating the impact of stressful situations is the use of cognitive emotional regulation mechanisms. The aims of this study were (a) to describe the functional and dysfunctional cognitive emotional regulation mechanisms (FRMs and DRMs) by gender, (b) to screen the main group of healthcare professionals who are candidates to receive psychological assistance based on FRMs and DRMs, and (c) to determine the HCP profile of candidates for psychological assistance. A cross-sectional descriptive study was conducted. Data were obtained from an adhoc questionnaire-the Cognitive Emotional Regulation Questionnaire (CERQ-18), the Generalized Anxiety Disorder scale (GAD-7), and the nine-item Patient Health Questionnaire (PHQ-9). The representative sample comprised 1452 HCPs. The results revealed significant differences between men and women in the use of DRMs. Women showed a higher use of catastrophizing (≤0.001) and rumination (0.008). The screening procedure detected that 7.5% (109 cases) of the HCPs were candidates to receive psychological support. According to the results of this study, age group (30-39 years old), professional activity (being a nurse or nursing assistant), and having psychological symptoms of anxiety and depression are variables that independently increase the probability of requiring psychological assistance. The gender variable was not found to be an independent factor when it comes to receiving psychological support. In conclusion, it is necessary to consider the influence of cognitive emotional regulation strategies employed by HCPs in the screening of candidates for psychological assistance and design effective interventions to reverse the emotional distress caused by COVID-19.

Expression of Endothelial NOX5 Alters the Integrity of the Blood-Brain Barrier and Causes Loss of Memory in Aging Mice.

Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown.

Understanding the Potential Role of Sirtuin 2 on Aging: Consequences of SIRT2.3 Overexpression in Senescence.

Sirtuin 2 (SIRT2) has been associated to aging and age-related pathologies. Specifically, an age-dependent accumulation of isoform 3 of SIRT2 in the CNS has been demonstrated; however, no study has addressed the behavioral or molecular consequences that this could have on aging. In the present study, we have designed an adeno-associated virus vector (AAV-CAG-Sirt2.3-eGFP) for the overexpression of SIRT2.3 in the hippocampus of 2 month-old SAMR1 and SAMP8 mice. Our results show that the specific overexpression of this isoform does not induce significant behavioral or molecular effects at short or long term in the control strain. Only a tendency towards a worsening in the performance in acquisition phase of the Morris Water Maze was found in SAMP8 mice, together with a significant increase in the pro-inflammatory cytokine Il-1ß. These results suggest that the age-related increase of SIRT2.3 found in the brain is not responsible for induction or prevention of senescence. Nevertheless, in combination with other risk factors, it could contribute to the progression of age-related processes. Understanding the specific role of SIRT2 on aging and the underlying molecular mechanisms is essential to design new and more successful therapies for the treatment of age-related diseases.

Gut microbial dysbiosis after traumatic brain injury modulates the immune response and impairs neurogenesis.

The influence of the gut microbiota on traumatic brain injury (TBI) is presently unknown. This knowledge gap is of paramount clinical significance as TBI patients are highly susceptible to alterations in the gut microbiota by antibiotic exposure. Antibiotic-induced gut microbial dysbiosis established prior to TBI significantly worsened neuronal loss and reduced microglia activation in the injured hippocampus with concomitant changes in fear memory response. Importantly, antibiotic exposure for 1 week after TBI reduced cortical infiltration of Ly6Chigh monocytes, increased microglial pro-inflammatory markers, and decreased T lymphocyte infiltration, which persisted through 1 month post-injury. Moreover, microbial dysbiosis was associated with reduced neurogenesis in the dentate gyrus 1 week after TBI. By 3 months after injury (11 weeks after discontinuation of the antibiotics), we observed increased microglial proliferation, increased hippocampal neuronal loss, and modulation of fear memory response. These data demonstrate that antibiotic-induced gut microbial dysbiosis after TBI impacts neuroinflammation, neurogenesis, and fear memory and implicate gut microbial modulation as a potential therapeutic intervention for TBI.

CB2 Receptors and Neuron-Glia Interactions Modulate Neurotoxicity Generated by MAGL Inhibition.

Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron-glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures; mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron-glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions.

Midbrain microglia mediate a specific immunosuppressive response under inflammatory conditions.

BACKGROUND: Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. METHODS: To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4+ T cells purified from OT-II transgenic mice. RESULTS: Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4+ T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in co-inhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFß, and the increase in infiltrating regulatory T cells. CONCLUSIONS: These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability.

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.

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). In vitro 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 in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo 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 ex vivo 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.

Cannabinoid pharmacology/therapeutics in chronic degenerative disorders affecting the central nervous system.

The endocannabinoid system (ECS) exerts a modulatory effect of important functions such as neurotransmission, glial activation, oxidative stress, or protein homeostasis. Dysregulation of these cellular processes is a common neuropathological hallmark in aging and in neurodegenerative diseases of the central nervous system (CNS). The broad spectrum of actions of cannabinoids allows targeting different aspects of these multifactorial diseases. In this review, we examine the therapeutic potential of the ECS for the treatment of chronic neurodegenerative diseases of the CNS focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. First, we describe the localization of the molecular components of the ECS and how they are altered under neurodegenerative conditions, either contributing to or protecting cells from degeneration. Second, we address recent advances in the modulation of the ECS using experimental models through different strategies including the direct targeting of cannabinoid receptors with agonists or antagonists, increasing the endocannabinoid tone by the inhibition of endocannabinoid hydrolysis, and activation of cannabinoid receptor-independent effects. Preclinical evidence indicates that cannabinoid pharmacology is complex but supports the therapeutic potential of targeting the ECS. Third, we review the clinical evidence and discuss the future perspectives on how to bridge human and animal studies to develop cannabinoid-based therapies for each neurodegenerative disorder. Finally, we summarize the most relevant opportunities of cannabinoid pharmacology related to each disease and the multiple unexplored pathways in cannabinoid pharmacology that could be useful for the treatment of neurodegenerative diseases.

Stereological Estimates of Glutamatergic, GABAergic, and Cholinergic Neurons in the Pedunculopontine and Laterodorsal Tegmental Nuclei in the Rat.

The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.

The expression of cannabinoid type 1 receptor and 2-arachidonoyl glycerol synthesizing/degrading enzymes is altered in basal ganglia during the active phase of levodopa-induced dyskinesia.

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB1 receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB1 receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.