Francisco Gonçalves

Depression as a Glial-Based Synaptic Dysfunction

Recent studies combining pharmacological, behavioral, electrophysiological and molecular approaches indicate that depression results from maladaptive neuroplastic processes occurring in defined frontolimbic circuits responsible for emotional processing such as the prefrontal cortex, hippocampus, amygdala and ventral striatum. However, the exact mechanisms controlling synaptic plasticity that are disrupted to trigger depressive conditions have not been elucidated. Since glial cells (astrocytes and microglia) tightly and dynamically interact with synapses, engaging a bi-directional communication critical for the processing of synaptic information, we now revisit the role of glial cells in the etiology of depression focusing on a dysfunction of the “quad-partite” synapse. This interest is supported by the observations that depressive-like conditions are associated with a decreased density and hypofunction of astrocytes and with an increased microglia “activation” in frontolimbic regions, which is expected to contribute for the synaptic dysfunction present in depression. Furthermore, the traditional culprits of depression (glucocorticoids, biogenic amines, brain-derived neurotrophic factor, BDNF) affect glia functioning, whereas antidepressant treatments (serotonin-selective reuptake inhibitors, SSRIs, electroshocks, deep brain stimulation) recover glia functioning. In this context of a quad-partite synapse, systems modulating glia-synapse bidirectional communication—such as the purinergic neuromodulation system operated by adenosine 5′-triphosphate (ATP) and adenosine—emerge as promising candidates to “re-normalize” synaptic function by combining direct synaptic effects with an ability to also control astrocyte and microglia function. This proposed triple action of purines to control aberrant synaptic function illustrates the rationale to consider the interference with glia dysfunction as a mechanism of action driving the design of future pharmacological tools to manage depression.

Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimers disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.

Behavioral Phenotyping of Parkin-Deficient Mice: Looking for Early Preclinical Features of Parkinson's Disease

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinsons disease (PD) begin many years before the appearance of the characteristic motor symptoms. Neuropsychiatric, sensorial and cognitive deficits are recognized as early non-motor manifestations of PD, and are not attenuated by the current anti-parkinsonian therapy. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient mice do not display spontaneous degeneration of the nigrostriatal pathway or enhanced vulnerability to dopaminergic neurotoxins such as 6-OHDA and MPTP. Here, we employed adult homozygous C57BL/6 mice with parkin gene deletion on exon 3 (parkin −/−) to further investigate the relevance of Parkin in the regulation of non-motor features, namely olfactory, emotional, cognitive and hippocampal synaptic plasticity. Parkin −/− mice displayed normal performance on behavioral tests evaluating olfaction (olfactory discrimination), anxiety (elevated plus-maze), depressive-like behavior (forced swimming and tail suspension) and motor function (rotarod, grasping strength and pole). However, parkin −/− mice displayed a poor performance in the open field habituation, object location and modified Y-maze tasks suggestive of procedural and short-term spatial memory deficits. These behavioral impairments were accompanied by impaired hippocampal long-term potentiation (LTP). These findings indicate that the genetic deletion of parkin causes deficiencies in hippocampal synaptic plasticity, resulting in memory deficits with no major olfactory, emotional or motor impairments. Therefore, parkin −/− mice may represent a promising animal model to study the early stages of PD and for testing new therapeutic strategies to restore learning and memory and synaptic plasticity impairments in PD.

Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure

BackgroundElevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).MethodsRetinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.ResultsWe report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.ConclusionsThis work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.

Adenosine A2A Receptors Modulate α-Synuclein Aggregation and Toxicity.

Abstract Abnormal accumulation of aggregated &agr;‐synuclein (aSyn) is a hallmark of sporadic and familial Parkinsons disease (PD) and related synucleinopathies. Recent studies suggest a neuroprotective role of adenosine A2A receptor (A2AR) antagonists in PD. Nevertheless, the precise molecular mechanisms underlying this neuroprotection remain unclear. We assessed the impact of A2AR blockade or genetic deletion (A2AR KO) on synaptic plasticity and neuronal cell death induced by aSyn oligomers. We found that impairment of LTP associated with aSyn exposure was rescued in A2AR KO mice or upon A2AR blockade, through an NMDA receptor‐dependent mechanism. The mechanisms underlying these effects were evaluated in SH‐SY5Y cells overexpressing aSyn and rat primary neuronal cultures exposed to aSyn. Cell death in both conditions was prevented by selective A2AR antagonists. Interestingly, blockade of these receptors did not interfere with aSyn oligomerization but, instead, reduced the percentage of cells displaying aSyn inclusions. Altogether, our data raise the possibility that the well‐documented effects of A2AR antagonists involve the control of the latter stages of aSyn aggregation, thereby preventing the associated neurotoxicity. These findings suggest that A2AR represent an important target for the development of effective drugs for the treatment of PD and related synucleinopathies.

Hyperglycaemia at admission in acute coronary syndrome patients: prognostic value in diabetics and non-diabetics

Objective To evaluate the impact of admission glycaemia on short-term and long-term prognosis in diabetic and non-diabetic patients admitted for acute coronary syndromes (ACS), and to identify the independent predictors of post-ACS mortality in this population. Methods This study included 1149 consecutive patients admitted to a single coronary care unit for ACS between May 2004 and December 2006. Our population was divided into four groups according to the quartiles of glycaemia at admission [Q1 > 5.77 mmol/l, Q2 (5.77–7.0) mmol/l, Q3 (7.0–9.22) mmol/l and Q4 ≥ 9.22 mmol/l]. Diabetic (n = 396) and non-diabetic (n = 753) subgroups were then separately analysed. Results Hyperglycaemia at admission was associated with worse cardiovascular risk profile, high levels of necrosis and inflammation biomarkers and low left ventricle ejection fraction. Considering overall population, in-hospital, 30-day and 3-year mortalities were higher in more elevated glycaemia quartiles. In diabetic patients, there were no significant differences in mortality among glycaemia quartiles; however, in non-diabetic group higher admission glucose levels were associated with successively higher in-hospital and 3-year mortalities. After multivariate regression analysis, glycaemia at admission ≥ 5.77 mmol/l, age ≥ 72 years, Killip class [1 and troponin I ≥ 6.0 ng/ml were independent predictors of in-hospital mortality. Conclusion This study suggests that, in a broad ACS population, hyperglycaemia at admission is a short-term and long-term bad prognosis marker, particularly in non-diabetic patients, being a strong independent predictor of in-hospital mortality.

Different danger signals differently impact on microglial proliferation through alterations of ATP release and extracellular metabolism

Microglia rely on their ability to proliferate in the brain parenchyma to sustain brain innate immunity and participate in the reaction to brain damage. We now studied the influence of different danger signals activating microglia, both internal (typified by glutamate, associated with brain damage) and external (using a bacterial lipopolysaccharide, LPS), on the proliferation of microglia cells. We found that LPS (100 ng/mL) increased, whereas glutamate (0.5 mM) decreased proliferation. Notably, LPS decreased whereas glutamate increased the extracellular levels of ATP. In contrast, LPS increased whereas glutamate decreased the extracellular catabolism of ATP into adenosine through ecto‐nucleotidases and ecto‐5’‐nucleotidase. Finally, apyrase (degrades extracellular ATP) abrogated glutamate‐induced inhibition of microglia proliferation; conversely, inhibitors of ecto‐nucleotidases (ARL67156 or α,β‐methylene ADP) and adenosine deaminase (degrades extracellular adenosine) abrogated the LPS‐induced increase of microglia proliferation, which was blocked by a selective A2A receptor antagonist, SCH58261 (50 nM). Overall, these results highlight the importance of the extracellular purinergic metabolism to format microglia proliferation and influence the spatio‐temporal profile of neuroinflammation in different conditions of brain damage. GLIA 2015;63:1636–1645

Adenosine A2b receptors control A1 receptor‐mediated inhibition of synaptic transmission in the mouse hippocampus

Adenosine is a neuromodulator mostly acting through A1 (inhibitory) and A2A (excitatory) receptors in the brain. A2B receptors (A2BR) are Gs/q‐protein‐coupled receptors with low expression in the brain. As A2BR function is largely unknown, we have now explored their role in the mouse hippocampus. We performed electrophysiological extracellular recordings in mouse hippocampal slices, and immunological analysis of nerve terminals and glutamate release in hippocampal slices and synaptosomes. Additionally, A2BR‐knockout (A2BR‐KO) and C57/BL6 mice were submitted to a behavioural test battery (open field, elevated plus‐maze, Y‐maze). The A2BR agonist BAY60‐6583 (300 nm) decreased the paired‐pulse stimulation ratio, an effect prevented by the A2BR antagonist MRS 1754 (200 nM) and abrogated in A2BR‐KO mice. Accordingly, A2BR immunoreactivity was present in 73 ± 5% of glutamatergic nerve terminals, i.e. those immunopositive for vesicular glutamate transporters. Furthermore, BAY 60‐6583 attenuated the A1R control of synaptic transmission, both the A1R inhibition caused by 2‐chloroadenosine (0.1–1 μm) and the disinhibition caused by the A1R antagonist DPCPX (100 nm), both effects prevented by MRS 1754 and abrogated in A2BR‐KO mice. BAY 60‐6583 decreased glutamate release in slices and also attenuated the A1R inhibition (CPA 100 nm). A2BR‐KO mice displayed a modified exploratory behaviour with an increased time in the central areas of the open field, elevated plus‐maze and the Y‐maze and no alteration of locomotion, anxiety or working memory. We conclude that A2BR are present in hippocampal glutamatergic terminals where they counteract the predominant A1R‐mediated inhibition of synaptic transmission, impacting on exploratory behaviour.

High sucrose consumption induces memory impairment in rats associated with electrophysiological modifications but not with metabolic changes in the hippocampus

High sugar consumption is a risk factor for metabolic disturbances leading to memory impairment. Thus, rats subject to high sucrose intake (HSu) develop a metabolic syndrome and display memory deficits. We now investigated if these HSu-induced memory deficits were associated with metabolic and electrophysiological alterations in the hippocampus. Male Wistar rats were submitted for 9 weeks to a sucrose-rich diet (35% sucrose solution) and subsequently to a battery of behavioral tests; after sacrifice, their hippocampi were collected for ex vivo high-resolution magic angle spinning (HRMAS) metabolic characterization and electrophysiological extracellular recordings in slices. HSu rats displayed a decreased memory performance (object displacement and novel object recognition tasks) and helpless behavior (forced swimming test), without altered locomotion (open field). HRMAS analysis indicated a similar hippocampal metabolic profile of HSu and control rats. HSu rats also displayed no change of synaptic transmission and plasticity (long-term potentiation) in hippocampal Schaffer fibers-CA1 pyramid synapses, but had decreased amplitude of long-term depression in the temporoammonic (TA) pathway. Furthermore, HSu rats had an increased density of inhibitory adenosine A1 receptors (A1R), that translated into a greater potency of A1R in Schaffer fiber synapses, but not in the TA pathway, whereas the endogenous activation of A1R in HSu rats was preserved in the TA pathway but abolished in Schaffer fiber synapses. These results suggest that HSu triggers a hippocampal-dependent memory impairment that is not associated with altered hippocampal metabolism but is probably related to modified synaptic plasticity in hippocampal TA synapses.

Glycemia at admission: the metabolic echocardiography in acute coronary syndrome patients

Background Ventricular dysfunction in acute coronary syndrome (ACS) patients is a recognized predictor of in-hospital and post-discharge morbidity and mortality. Recently, hyperglycemia at admission has been considered as an important marker of poor in-hospital prognosis. Aim To characterize an ACS population and to identify independent predictors of one-year mortality. Methods This study included 1179 consecutive patients admitted to a single coronary care unit for acute coronary syndrome between May 2004 and December 2006. Patients were followed up for 12 months after ACS. Results In our population, 70.9% of patients were male, with a mean age of 66.6 ± 12.5 years; 15.7% were admitted by unstable angina, 44.7% by non-ST elevation myocardial infarction and 38.5% by ST elevation myocardial infarction; 16% of patients had left ventricular systolic dysfunction during the index admission. In-hospital mortality was 3.5% and complications occurred in 5.6% of patients. Mortality rate at 1-year of follow-up was 8.8% and rehospitalization rate for heart failure was 5.5%. After multivariate regression analysis, left ventricular systolic dysfunction [odds ratio (OR): 3.58; confidence interval (CI): 1.57-8.16], glycemia at admission >137 mg/dl (OR: 3.58; CI: 1.52-8.47) and age >65 years (OR: 2.67; CI: 1.08-6.59) were independent predictors of 1-year mortality. Conclusion In this population, hyperglycemia at admission was an independent predictor of 1-year mortality, with a predictive value equivalent to that of left ventricular systolic dysfunction. This fact, never before described, emphasizes the importance of metabolic abnormalities and their control in the prognosis of ACS patients. Eur J Cardiovasc Prev Rehabil 16:164-168