Morquio B disease: From pathophysiology towards diagnosis.

Morquio B disease is an attenuated phenotype within the spectrum of beta galactosidase (GLB1) deficiencies. It is characterised by dysostosis multiplex, ligament laxity, mildly coarse facies and heart valve defects due to keratan sulphate accumulation, predominantly in the cartilage. Morquio B patients have normal neurological development, setting them apart from those with the more severe GM1 gangliosidosis. Morquio B disease, with an incidence of 1:250.000 to 1:1.000.000 live births, is very rare. Here we report the clinical-biochemical data of nine patients. High amounts of keratan sulfate were detected using LC-MS/MS in the patients' urinary samples, while electrophoresis, the standard procedure of qualitative glycosaminoglycans analysis, failed to identify this metabolite in any of the patients' samples. We performed molecular analyses at gene, gene expression and protein expression levels, for both isoforms of the GLB1 gene, lysosomal GLB1, and the cell-surface expressed Elastin Binding Protein. We characterised three novel GLB1 mutations [c.75 + 2 T > G, c.575A > G (p.Tyr192Cys) and c.2030 T > G (p.Val677Gly)] identified in three heterozygous patients. We also set up a copy number variation assay by quantitative PCR to evaluate the presence of deletions/ insertions in the GLB1 gene. We propose a diagnostic plan, setting out the specific clinical- biochemical and molecular features of Morquio B, in order to avoid misdiagnoses and improve patients' management.

Type I sialidosis, a normosomatic lysosomal disease, in the differential diagnosis of late-onset ataxia and myoclonus: An overview.

Lysosomal storage diseases (LSDs) are rare to extremely rare monogenic disorders. Their incidence, however, has probably been underestimated owing to their complex clinical manifestations. Sialidosis is a prototypical LSD inherited as an autosomal recessive trait and caused by mutations in the NEU1 gene that result in a deficiency of alpha-N-acetyl neuraminidase 1 (NEU1). Two basic forms of this disease, type I and type II, are known. The dysmorphic type II form features LSD symptoms including congenital hydrops, dysmorphogenetic traits, hepato-splenomegaly and severe intellectual disability. The diagnosis is more challenging in the normosomatic type I forms, whose clinical findings at onset include ocular defects, ataxia and generalized myoclonus. Here we report the clinical, biochemical and molecular analysis of five patients with sialidosis type I. Two patients presented novel NEU1 mutations. One of these patients was compound heterozygous for two novel NEU1 missense mutations: c.530A>T (p.Asp177Val) and c.1010A>G (p.His337Arg), whereas a second patient was compound heterozygous for a known mutation and a novel c.839G>A (p.Arg280Gln) mutation. We discuss the impact of these new mutations on the structural properties of NEU1. We also review available clinical reports of patients with sialidosis type I, with the aim of identifying the most frequent initial clinical manifestations and achieving more focused diagnoses.

Exacerbation of C1q dysregulation, synaptic loss and memory deficits in tau pathology linked to neuronal adenosine A2A receptor.

Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by RNA sequencing analysis. In particular, we found that A2AR overexpression in THY-Tau22 mice led to the hippocampal upregulation of C1q complement protein-also observed in patients with frontotemporal lobar degeneration-and correlated with the loss of glutamatergic synapses, likely underlying the observed memory deficits. These data reveal a key impact of overactive neuronal A2AR in the onset of synaptic loss in tauopathies, paving the way for new therapeutic approaches.

A2A R-induced transcriptional deregulation in astrocytes: An in vitro study.

Adenosine A2A receptors (A2A R) are modulators of various physiological processes essential for brain homeostasis and fine synaptic tuning. In certain neurodegenerative conditions, notably Alzheimer's disease (AD), A2A Rs are pathologically upregulated in neurons but also in astrocytes. In that context, the use of A2A Rs inhibitors, normalizing impaired receptor function, is seen as a potential therapeutic strategy. However, the impact of A2A R alterations, particularly in astrocytes, is not fully understood. Here, we investigated the effect of A2A R overexpression on transcriptional deregulation in primary astrocytic cultures. By performing whole transcriptome analysis, we found that A2A R overexpression promotes robust transcriptional changes, mostly affecting immune response, angiogenesis, and cell activation-related genes. Importantly, we observed that treatment with SCH58261, a selective A2A R antagonist, restored the expression levels of several inflammatory and astrocytic activation-related genes, such as Interleukin-1beta and vimentin. This supports the notion that A2A R blockade could restore some astrocytic dysfunctions associated with abnormal A2A R expression, further arguing for a potential beneficial impact of receptor antagonists in A2A R-induced transcriptional deregulation, inflammation, and astrogliosis. Overall, our findings provide novel insights into the putative impact of A2A R overexpression on transcriptional deregulation in astrocytes, thereby opening novel avenues for the use of A2A R antagonists as potential therapeutic strategy in neurodegenerative diseases.

Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

Time-course of protection by the selective A2A receptor antagonist SCH58261 after transient focal cerebral ischemia.

Evidence indicates that the adenosine A2A receptor subtype is of critical importance in stroke. In previous studies, in the model of permanent middle cerebral artery occlusion (pMCAo), the adenosine A2A receptor antagonist, SCH58261, administered soon after ischemia, proved protective against excessive glutamate outflow in the first 4 h after ischemia and against neurological deficit and tissue damage evaluated 24 h after pMCAo. In the present work, we investigated if neuroprotective effect of SCH58261 was maintained 7 days after transient MCAo (tMCAo). SCH58261 (0.01 mg/kg, i.p.), administered twice/day for 7 days, protected from neurological deficit 1 day after tMCAo, but no more after 5 and 7 days. Two days after tMCAo, SCH58261 did not reduce blood cell infiltration, evaluated as HIS-48 positive cells, into ischemic striatal and cortical tissue. Moreover, 7 days after tMCAo, SCH58261 has not protected ischemic areas from damage and has not ameliorated myelin organization into the ischemic striatum. Protection by the A2A receptor antagonist 24 h after ischemia is attributable to reduced excitotoxicity. Seven days after ischemia the early protective effect of the A2A receptor antagonist likely has been overwhelmed by a secondary damage due to blood cell infiltration and neuroinflammation.

Adenosine A2A receptors modulate acute injury and neuroinflammation in brain ischemia.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A(2A) receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A(2A) receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A(2A) receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A(2A) receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A(2A) receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A(2A) receptor agonists a wide therapeutic time-window of hours and even days after stroke.

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.

Caffeine intake exerts dual genome-wide effects on hippocampal metabolism and learning-dependent transcription.

Caffeine is the most widely consumed psychoactive substance in the world. Strikingly, the molecular pathways engaged by its regular consumption remain unclear. We herein addressed the mechanisms associated with habitual (chronic) caffeine consumption in the mouse hippocampus using untargeted orthogonal omics techniques. Our results revealed that chronic caffeine exerts concerted pleiotropic effects in the hippocampus at the epigenomic, proteomic, and metabolomic levels. Caffeine lowered metabolism-related processes (e.g., at the level of metabolomics and gene expression) in bulk tissue, while it induced neuron-specific epigenetic changes at synaptic transmission/plasticity-related genes and increased experience-driven transcriptional activity. Altogether, these findings suggest that regular caffeine intake improves the signal-to-noise ratio during information encoding, in part through fine-tuning of metabolic genes, while boosting the salience of information processing during learning in neuronal circuits.

The Adenosinergic Signaling: A Complex but Promising Therapeutic Target for Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder in elderly people. AD is characterized by a progressive cognitive decline and it is neuropathologically defined by two hallmarks: extracellular deposits of aggregated ß-amyloid (Aß) peptides and intraneuronal fibrillar aggregates of hyper- and abnormally phosphorylated Tau proteins. AD results from multiple genetic and environmental risk factors. Epidemiological studies reported beneficial effects of caffeine, a non-selective adenosine receptors antagonist. In the present review, we discuss the impact of caffeine and of adenosinergic system modulation on AD, in terms of pathology and therapeutics.

Beneficial Effect of a Selective Adenosine A2A Receptor Antagonist in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease.

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) and mitigates both amyloid and Tau lesions in transgenic mouse models of the disease. While short-term treatment with A2AR antagonists have been shown to alleviate cognitive deficits in mouse models of amyloidogenesis, impact of a chronic and long-term treatment on the development of amyloid burden, associated neuroinflammation and memory deficits has never been assessed. In the present study, we have evaluated the effect of a 6-month treatment of APPsw/PS1dE9 mice with the potent and selective A2AR antagonist MSX-3 from 3 to 9-10 months of age. At completion of the treatment, we found that the MSX-3 treatment prevented the development of memory deficits in APP/PS1dE9 mice, without significantly altering hippocampal and cortical gene expressions. Interestingly, MSX-3 treatment led to a significant decrease of Aß1-42 levels in the cortex of APP/PS1dE9 animals, while Aß1-40 increased, thereby strongly affecting the Aß1-42/Aß1-40 ratio. Together, these data support the idea that A2AR blockade is of therapeutic value for AD.

Role of adenosine in oligodendrocyte precursor maturation.

Differentiation and maturation of oligodendroglial cells are postnatal processes that involve specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognized. As evaluated on OPC cultures, adenosine, by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary, by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K(+) currents (IK) that are involved in the regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma.

Low doses of the selective adenosine A2A receptor agonist CGS21680 are protective in a rat model of transient cerebral ischemia.

Evidence indicate that adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A adenosine receptors occurs at central level on neurons and microglia of ischemic striatum and cortex after focal ischemia. Adenosine A2A receptor subtype is localized not only at central level but also peripherally on blood cells, where it is known to exert antiinflammatory effect. Purpose of the present work was to investigate the putative neuroprotective effect of the adenosine A2A receptor agonist CGS21680 in a rat model of transient medial cerebral artery occlusion (MCAo). Transient cerebral ischemia was induced by 1h occlusion of MCA. CGS21680 (0.01 and 0.1mg/kg, i.p.) was administered starting 4h after ischemia according to a chronic protocol (twice/day for 7 days). CGS21680, at the dose of 0.1mg/kg transiently increased heart frequency but did not modify blood pressure. At the dose of 0.01mg/kg the drug did not modify either heart frequency or blood pressure. Following transient MCAo, CGS21680 at both doses protected from neurological deficit from the first day up to 7 days thereafter. At this time, it has reduced microgliosis, astrogliosis and improved myelin organization in the striatum and cytoarchitecture of the ischemic cortex and striatum. Two days after transient MCAo, CGS21680 has reduced the number of infiltrated granulocytes into the ischemic tissue. Data indicate that CGS21680 systemically administered is protective by immunosuppressive effects.

Adenosine is present in rat brain synaptic vesicles.

Evidences in the central nervous system are in favor that adenosine under basal conditions is released by a direct excitation-secretion modality. However, till now, there is no direct evidence that adenosine is contained in synaptic vesicles. Eight synaptic vesicle fractions were recovered on a discontinuous sucrose gradient after ultracentrifugation of the crude synaptosomal fraction (pellet P2) of rat brain. The adenosine content in each fraction was measured by high-performance liquid chromatography coupled to a fluorescence detector (minimum sensitivity 10 femtomoles). The immunoblot analysis, to detect synaptophysin, a molecular marker for the vesicle membrane, showed that fractions from 3 to 8 were rich in synaptophysin. The sum of adenosine found in fractions 3-8 was (mean ± SEM, n = 4) 3325.6 ± 94.6 pmol/mg of tissue protein. We proved that adenosine measured in synaptic vesicle fractions was not contaminated by cytosolic adenosine, as adenosine exogenously added to the P2 preferentially distributed in fractions 1 and 2 that are synaptophysin-free and did not contaminate the vesicle pellet P3. Data provide direct demonstration that adenosine is present in rat brain synaptic vesicle fractions. This information is consistent with the notion that adenosine is stored in synaptic vesicles and is released under normoxic physiological conditions by an excitation-secretion mechanism typical of neuronal cells.

Adenosine A2A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation.