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31 p.-12 fig.-1 tab. We dedicate this paper to Takashi Umekawa, who generated the HI model at the Karolinska Institute, and unfortunately passed away in 2018., Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin. Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4’,6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks’ balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1. © 2023 The Author(s)., This work was supported by grants from the Spanish Ministry of Science and Innovation Competitiveness MCIN/AEI/10.13039/501100011033 (https://www.ciencia.gob.es/) and ERDF “A way to make Europe” (RTI2018-099267-B-I00 and RYC-2013-12817 to AS; RTI2018–097948-A-100 and RYC-2016–20480 to OP), a Tatiana Foundation Award (P-048-FTPGB 2018) to AS a Basque Government Department of Education project (PIBA 2020_1_0030; http://www.euskadi.eus/basque-government/department-education/) to AS, a Basque Government Department of Economic development, Sustainability and environment (ELKARTEK KK-2020/00034; https://www.spri.eus/en/) to ECZ, and . SB is recipient of predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness, and VST is recipient of predoctoral fellowship from the Basque Government.

Objective: The aim is to assess the effectiveness of a biodegradable antireflux ureteral stent with heparin coating in a comparative study (BraidStent®-H) in an animal model for the treatment of ia...

The brain extracellular space (ECS) is a vast interstitial reticulum of extreme morphological complexity, composed of narrow gaps separated by local expansions, enabling interconnected highways between neural cells. Constituting on average 20% of brain volume, the ECS is key for intercellular communication, and understanding its diffusional properties is of paramount importance for understanding the brain. Within the ECS, neuroactive substances travel predominantly by diffusion, spreading through the interstitial fluid and the extracellular matrix scaffold after being focally released. The nanoscale dimensions of the ECS render it unresolvable by conventional live tissue compatible imaging methods, and historically diffusion of tracers has been used to indirectly infer its structure. Novel nanoscopic imaging techniques now show that the ECS is a highly dynamic compartment, and that diffusivity in the ECS is more heterogeneous than anticipated, with great variability across brain regions and physiological states. Diffusion is defined primarily by the local ECS geometry, and secondarily by the viscosity of the interstitial fluid, including the obstructive and binding properties of the extracellular matrix. ECS volume fraction and tortuosity both strongly determine diffusivity, and each can be independently regulated e.g. through alterations in glial morphology and the extracellular matrix composition. Here we aim to provide an overview of our current understanding of the ECS and its diffusional properties. We highlight emerging technological advances to respectively interrogate and model diffusion through the ECS, and point out how these may contribute in resolving the remaining enigmas of the ECS. The authors acknowledge funding from the Spanish Ministry of Science and Innovation (PID2020-115896RJ-I00, PID2020-113894RB-I00, PCI2022-135040-2), the Basque Government (GIC21/76, GIU21/048), CIBERNED, Human Frontier Science Program (RGP0036/2020) and Aligning Science Across Parkinson's (ASAP-020505) through the Michael J. Fox Foundation for Parkinson's Research (MJFF).

Objective: The aim was to assess a new biodegradable and antireflux intraureteral stent (BraidStent®) design in a swine model after ureteral laparoscopic operation. Materials and Methods: A total of 24 female pigs underwent initial endoscopic, nephrosonographic, and contrast fluoroscopy assessment of the urinary tract. Afterward, unilateral ureteropelvic junction obstruction was performed by laparoscopic approach. Six weeks later, the animals underwent laparoscopic Anderson-Hynes pyeloplasty, and were randomly assigned to Group-I, in which a double-pigtail ureteral stent was inserted for 6 weeks, or Group-II, in which a BraidStent®, a biodegradable intraureteral stent design, was placed. Follow-up assessments were performed at 3 and 6 weeks and 5 months. Results: In terms of therapeutic success, complete resolution was observed in 91.6% of Group-I animals and 88.8% in Group-II. No evidence of vesicoureteral reflux (VUR) was observed in Group-II animals and statistical significance in VUR and ureteral orifice damage were observed between groups. BraidStent® degradation occurred in a controlled manner between 3 and 6 weeks, without obstructive fragments. Distal ureteral peristalsis was maintained in 66.6% and 83.3% in Group-II at 3 and 6 weeks of follow-up, respectively. In Group-II, the positive bacteriuria rate was 41.6% and the migration rate 25%. Pathological assessment showed a significant improvement in ureteral healing in Group-II vs Group-I. Conclusions: The results of this comparative study in a porcine model indicate that the intraureteral BraidStent performed similarly to conventional ureteral stents. It avoids complete ureteral length intubation, the adverse effects associated with conventional ureteral stents, and maintains a high level of distal ureteral peristalsis. Moreover, the BraidStent® exhibited a predictable and controlled degradation rate and did not cause any obstructive fragments. However, further studies are needed to improve the anchoring system and reduce the risk of bacterial colonization.

International audience; Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, associated with the accumulation of misfolded α-synuclein and lysosomal impairment, two events deemed interconnected. Protein aggregation is linked to defects in degradation systems such as the autophagy-lysosomal pathway, while lysosomal dysfunction is partly related to compromised acidification. We have recently proven that acidic nanoparticles (aNPs) can re-acidify lysosomes and ameliorate neurotoxin-mediated dopaminergic neurodegeneration in mice. However, no lysosome-targeted approach has yet been tested in synucleinopathy models in vivo. Here, we show that aNPs increase α-synuclein degradation through enhancing lysosomal activity in vitro. We further demonstrate in vivo that aNPs protect nigral dopaminergic neurons from cell death, ameliorate α-synuclein pathology, and restore lysosomal function in mice injected with PD patient-derived Lewy body extracts carrying toxic α-synuclein aggregates. Our results support lysosomal re-acidification as a disease-modifying strategy for the treatment of PD and other age-related proteinopathies.

STED microscopy is one of several fluorescence microscopy techniques that permit imaging at higher spatial resolution than what the diffraction-limit of light dictates. STED imaging is unique among these super-resolution modalities in being a beam-scanning microscopy technique based on confocal or 2-photon imaging, which provides the advantage of superior optical sectioning in thick samples. Compared to the other super-resolution techniques that are based on widefield microscopy, this makes STED particularly suited for imaging inside live brain tissue, such as in slices or in vivo. Notably, the 50nm resolution provided by STED microscopy enables analysis of neural morphologies that conventional confocal and 2-photon microscopy approaches cannot resolve, including all-important synaptic structures. Over the course of the last 20years, STED microscopy has undergone extensive developments towards ever more versatile use, and has facilitated remarkable neurophysiological discoveries. The technique is still not widely adopted for live tissue imaging, even though one of its particular strengths is exactly in resolving the nanoscale dynamics of synaptic structures in brain tissue, as well as in addressing the complex morphologies of glial cells, and revealing the intricate structure of the brain extracellular space. Not least, live tissue STED microscopy has so far hardly been applied in settings of pathophysiology, though also here it shows great promise for providing new insights. This review outlines the technical advantages of STED microscopy for imaging in live brain tissue, and highlights key neurobiological findings brought about by the technique. The authors acknowledge funding for their general work from the Spanish Ministry of Science and Innovation (SAF-2017-83776-R, RYC-2014-15994 and IJCI-2017-32114), the Basque Government (PIBA19-0065 and PIBA-2020-1-0061), and the University of the Basque Country (GIU18/094 and INF19-29)

The major neurodegenerative diseases, like Alzheimer’s disease (AD), accumulate neuropathogenic proteins that compromise autophagic function. In AD, autophagy contributes to intracellular APP processing and amyloid beta (Aβ) generation by mutant presenilin-1 (PS1). However, how extracellular soluble Aβ oligomers (Aβo) impact intracellular autophagy is not well understood. The primary cilium (PC), a signaling organelle on the surface of mature neurons and glia, is able to bind Aβ. Since PC signaling pathways knowingly modify autophagy in non-brain cells, we here investigated the role of neuronal PC in the modulation of autophagy during acute extracellular Aβo overload. Our results show that, in vivo, recombinant Aβo require the presence of neuronal PC to modulate early autophagy and to induce the accumulation of autophagic vacuoles in an age-dependent manner. We show that activated Akt mediates these effects in an age-dependent manner, and that ciliary p75NTR receptor is required to block autophagy by Aβo. These findings demonstrate that neuronal PC in the adult brain participates in the deleterious effects mediated by soluble Aβo. The PC should therefore be considered as a target organelle to modulate autophagy for the treatment of neurodegenerative diseases.HighlightsAβo requires the neuronal PC to impair learning in young and old mice.Autophagy in whole hippocampus differs from autophagy response in hippocampal neurons.Aβo induce autophagolysosome accumulation through primary cilia- and age-dependent Akt phosphorylation.

Background Double J ureteral stents are widely used on urological patients to provide drainage of the upper urinary tract. Unfourtunately, ureteral stents are not free from complications, as bacterial colonization and require a second procedure for removal. The purpose of the current comparative experimental study is to evaluate a new heparin-coated biodegradable antireflux ureteral stent (BraidStent®-H) to prevent urinary bacterial colonization. Methods A total of 24 female pigs were underwent determination of bacteriuria and nephrosonographic, endoscopic and contrast fluoroscopy assessment of the urinary tract. Afterward, were randomly assigned animals to Group-I, in which a 5Fr double-pigtail ureteral stent was placed for 6 weeks, or Group-II, in which a BraidStent®-H was placed. Follow-up assessments were performed at 1, 3, 6, 8, 12 weeks. The final follow-up includes the above methods and an exhaustive pathological study of the urinary tract was accomplished after 20 weeks. Results Bacteriuria findings in the first 48 h were significant between groups at 6 h and 12 h. Asymptomatic bacteriuria does not reach 100% of the animals in Group-II until 48 h versus Group-I where it appears at 6 h. The weekly bacteriuria mean rate was 27.7% and 44.4% in Group I and II respectively, without statistical significance. In Group II there were no animals with vesicoureteral reflux, with statistical significance at 3 and 6 weeks with Group-I. The 91.2% of stents in Group-II were degraded between 3 and 6 weeks, without obstructive fragments. Distal ureteral peristalsis was maintained in 66.6–75% in Group-II at 1–6 weeks. Conclusions The heparin coating of BraidStent® allows an early decrease of bacterial colonization, but its effectiveness is low at the long term. Heparin coating did not affect scheduled degradation rate or size of stents fragments. BraidStent®-H avoids the side effects associated with current ureteral stents, thus should cause less discomfort to patients.

Background: We assessed an antireflux biodegradable heparin-coated ureteral stent (BraidStent (R)-H) in an animal model comparative study after endoscopic treatment of ureteral strictures. Methods: A total of 24 female pigs underwent initial endoscopic, nephrosonographic, and contrast fluoroscopy assessment of the urinary tract. Afterward, unilateral laparoscopic ureteral stricture model was performed. Three weeks later, the animals underwent laser endoureterotomy and were randomly assigned to Group-I, in which a double-pigtail stent was placed for 6 weeks, or Group-II, in which a BraidStent (R)-H was placed. Follow-up was carried out by ultrasonography, contrast fluoroscopy, ureteroscopy, urinalysis and bacteriuria assessment at 3, 6, 12 and 5 months. Finally, a pathological study of the urinary system was performed. Results: There were no animals in Group-II with vesicoureteral reflux, with significance at 6 weeks with Group-I. Distal ureteral peristalsis was maintained in 50-75% in Group-II at 1-6 weeks. The 91.7% of stents in Group-II were degraded between 3-6 weeks, without obstructive fragments. Bacteriuria in Group II was 33.3-50% at 3 and 6weeks. The global success rate by groups was 91.6% and 87.5% in groups I and II, respectively, with no statistical significance. Conclusions: BraidStent (R)-H has been shown to be as efficacious as current ureteral stents in the treatment of benign ureteral strictures following laser endoureterotomy. In addition, it reduces the morbidity associated with current stents and has a homogeneous and predictable degradation rate of about 6 weeks, with no obstructive fragments. Future studies are required to improve the antibacterial coating to reduce BraidStent (R)-H contamination in view of the results obtained with the heparin coating.

International audience; Most neurodegenerative disorders are characterized by deposits of misfolded proteins and neuronal degeneration in specific brain regions. Growing evidence indicates that lysosomal impairment plays a primary pathogenic role in these diseases, in particular, the occurrence of increased lysosomal pH. Thus, therapeutic development aiming at restoring lysosomal function represents a novel, precise, and promising strategy for the treatment of these pathologies. Herein we demonstrate that acidic oil-in-water nanoemulsions loaded with poly(dl-lactide- co-glycolide) (PLGA) are able to rescue impaired lysosomal pH in genetic cellular models of Parkinson's disease. For in vivo assays, nanoemulsions were labeled with an original synthetic hydrophobic far red-emitting dye to allow fluorescence monitoring. Following stereotaxic injection in the mouse brain, widespread diffusion of the nanocarrier was observed, up to 500 μm from the injection site, as well as internalization into the lysosomal compartment in brain cells. Finally, promising preliminary assays of systemic administration demonstrate that a fraction of the formulation crosses the blood brain barrier, penetrates the brain parenchyma, is internalized by cells, and colocalizes with lysosomal markers. Overall, these results suggest the feasibility and the therapeutic potential of this new nanoformulation as an effective drug delivery tool to the brain, with the potential to rescue pathological lysosomal deficits.

Luminescent single wall carbon nanotubes are now well established, as unique nanoreporters to probe the brain extracellular space[1]. On the imaging side, this comes from their rich near-infrared optical properties, which also eventually be improved by sp3-chemical functionalization[2]. In addition, their uncommon 1D morphology is an important asset for tissue penetration, yet understanding their diffusion behavior in the complex brain extracellular network is challenging and necessitates dedicated analysis tools. To this aim, we have developed two novel approaches (i) based on the local analysis of trajectory contours to locally measure the nanoscale dimensions of the brain network [3] and (ii) based on the transient evaluation of anomalous carbon nanotube diffusion to delineate the ECS molecular diffusion landscape [4]. The application of these analytical tools to extract relevant biological parameters in physiological and pathological brain models will be presented [5]. References [1] Godin et al Nat. Nanotechnol. 12 (2017) 238-243 ; Gao, et al, Nanomaterials 7, 11, (2017) 393 ; Danné et al ACS Photonics, 5, 2, (2018) 359-364 [2] Mandal et al. Scientific Reports, 10 (2020) 5286 [3] Paviolo et al Methods 174 (2020) 91-99 [4] Lee et al, in preparation [5] Soria et al Nat. Commun., 11 (2020) 3440

The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP(+) astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g-ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381-1395.

Glutamate excitotoxicity contributes to oligodendroglial and axonal damage in multiple sclerosis pathology. Extracellular glutamate concentration in the brain is controlled by cystine/glutamate antiporter (system xc-), a membrane antiporter that imports cystine and releases glutamate. Despite this, the system xc(-) activity and its connection to the inflammatory reaction in multiple sclerosis (MS) is largely unknown.Longitudinal in vivo magnetic resonance (MRI) and positron emission tomography (PET) imaging studies with 2-[(18)F]Fluoro-2-deoxy-D-glucose ([(18)F]FDG), [(11)C]-(R)-(1-(2-chlorophenyl)-N-methyl-N-1(1-methylpropyl)-3-isoquinolinecarboxamide ([(11)C]PK11195) and (4S)-4-(3-(18)F-fluoropropyl)-L-glutamate ([(18)F]FSPG) were carried out during the course of experimental autoimmune encephalomyelitis (EAE) induction in rats.[(18)F]FSPG showed a significant increase of system xc(-) function in the lumbar section of the spinal cord at 14 days post immunization (dpi) that stands in agreement with the neurological symptoms and ventricle edema formation at this time point. Likewise, [(18)F]FDG did not show significant changes in glucose metabolism throughout central nervous system and [(11)C]PK11195 evidenced a significant increase of microglial/macrophage activation in spinal cord and cerebellum 2 weeks after EAE induction. Therefore, [(18)F]FSPG showed a major capacity to discriminate regions of the central nervous system affected by the MS in comparison to [(18)F]FDG and [(11)C]PK11195. Additionally, clodronate-treated rats showed a depletion in microglial population and [(18)F]FSPG PET signal in spinal cord confirming a link between neuroinflammatory reaction and cystine/glutamate antiporter activity in EAE rats.Altogether, these results suggest that in vivo PET imaging of system xc(-) could become a valuable tool for the diagnosis and treatment evaluation of MS.

Miniaturization of ureteroscopy materials is intended to decrease tissue damage. However, tissue hypoxia and the gross and microscopic effects on tissue have not been adequately assessed. We compared the gross and microscopic effects of micro-ureteroscopy (m-URS) and conventional ureteroscopy (URS) on the urinary tract. We employed 14 pigs of the Large White race. URS was performed in one of the ureters with an 8/9.8 F ureteroscope, while a 4.85 F m-URS sheath was used in the contralateral ureter. Gross assessment of ureteral wall damage and ureteral orifice damage was performed. For microscopic assessment hematoxylin-eosin staining and immunohistochemistry for detection of tissue hypoxia were conducted. Regarding the macroscopic assessment of ureteral damage, substantial and significant differences were recorded using URS (C = 0.8), but not with m-URS. Microscopic assessment after staining with hematoxylin-eosin revealed greater epithelial desquamation in the URS group (p

Synaptic protein -synuclein (-SYN) modulates neurotransmission in a complex and poorly understood manner and aggregates in the cytoplasm of degenerating neurons in Parkinsons disease. Here, we report that -SYN present in dopaminergic nigral afferents is essential for the normal cycling and maintenance of neural stem cells (NSCs) in the brain subependymal zone of adult male and female mice. We also showthat premature senescence of adult NSCs into non-neurogenic astrocytes in mice lacking-SYN resemblesthe effects of dopaminergic fiber degeneration resulting from chronic exposure to 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine or intranigral inoculation of aggregated toxic -SYN. Interestingly, NSC loss in -SYN-deficient mice can be prevented by viral delivery of human -SYN into their sustantia nigra or by treatment with L-DOPA, suggesting that -SYN regulates dopamine availability to NSCs. Our data indicatethat-SYN, presentin dopaminergic nerveterminals supplyingthe subependymal zone, acts as a niche componentto sustainthe neurogenic potential of adult NSCs and identify -SYN and DA as potential targets to ameliorate neurogenic defects in the aging and diseased brain., This work was supported by Ministerio de Economía y Competitividad Spanish Grants SAF2014-54581 and SAF2016-77541 and Instituto de Salud Carlos III (PI12/02574, PI13/01390, Centro de Investigacio´n Biome´dica en Red de Enfermedades Neurodegenerativas, Centro de Investigacio´n Biome´dica en Red de Salud Mental and RETIC Tercel)toM.V.,J.J.T-A., A.B., and I.F;Junta de Andalucía(Proyectos de Excelencia)toJ.J.T-A.; Generalitat Valenciana (Programa Prometeo) to I.F.; European Regional Development Fund; and European Union (E.U.) to A.B. R.S.-C. and G.B. received Spanish Ministerio de Educación FPU predoctoral fellowships. Work in the I.F. laboratory is supported by Fundacio´n Botín-Banco Santander.

International audience; Mutations in the GBA1 gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) are important risk factors for Parkinson's disease (PD). In vitro, altered GBA1 activity promotes alpha-synuclein accumulation whereas elevated levels of alpha-synuclein compromise GBA1 function, thus supporting a pathogenic mechanism in PD. However, the mechanisms by which GBA1 deficiency is linked to increased risk of PD remain elusive, partially because of lack of aged models of GBA1 deficiency. As knocking-out GBA1 in the entire brain induces massive neurodegeneration and early death, we generated a mouse model of GBA1 deficiency amenable to investigate the long-term consequences of compromised GBA1 function in dopaminergic neurons. DAT-Cre and GBA1-floxed mice were bred to obtain selective homozygous disruption of GBA1 in midbrain dopamine neurons (DAT-GBA1-KO). Mice were followed for motor function, neuronal survival, alpha-synuclein phosphorylation and glial activation. Susceptibility to nigral viral vector-mediated overexpression of mutated (A53T) alpha-synuclein was assessed. Despite loss of GBA1 and substrate accumulation, DAT-GBA1-KO mice displayed normal motor performances and preserved dopaminergic neurons despite robust microglial activation in the substantia nigra, without accumulation of endogenous alpha-synuclein with respect to wild-type mice. Lysosomal function was only marginally affected. Screening of micro-RNAs linked to the regulation of GBA1, alpha-synuclein or neuroinflammation did not reveal significant alterations. Viral-mediated overexpression of A53T-alpha-synuclein yielded similar neurodegeneration in DAT-GBA1-KO mice and wild-type mice. These results indicate that loss of GBA1 function in mouse dopaminergic neurons is not critical for alpha-synuclein accumulation or neurodegeneration and suggest the involvement of GBA1 deficiency in other cell types as a potential mechanism.

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.

Much of the cell death following episodes of anoxia and ischemia in the mammalian central nervous system has been attributed to extracellular accumulation of glutamate and ATP, which causes a rise in [Ca2+]i, loss of mitochondrial potential, and cell death. However, restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury (the oxygen paradox). Herein we describe a novel signaling pathway that is activated during ischemia-like conditions (oxygen and glucose deprivation; OGD) and contributes to ischemia-induced oligodendroglial cell death. OGD induced a retarded and sustained increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation after restoring glucose and O2 (reperfusion-like conditions). Blocking the ERK1/2 pathway with the MEK inhibitor UO126 largely protected oligodendrocytes against ischemic insults. ERK1/2 activation was blocked by the high-affinity Zn2+ chelator TPEN, but not by antagonists of AMPA/kainate or P2X7 receptors that were previously shown to be involved in ischemic oligodendroglial cell death. Using a high-affinity Zn2+ probe, we showed that ischemia induced an intracellular Zn2+ rise in oligodendrocytes, and that incubation with TPEN prevented mitochondrial depolarization and ROS generation after ischemia. Accordingly, exposure to TPEN and the antioxidant Trolox reduced ischemia-induced oligodendrocyte death. Moreover, UO126 blocked the ischemia-induced increase in poly-[ADP]-ribosylation of proteins, and the poly[ADP]-ribose polymerase 1 (PARP-1) inhibitor DPQ significantly inhibited ischemia-induced oligodendroglial cell death—demonstrating that PARP-1 was required downstream in the Zn2+-ERK oligodendrocyte cell death pathway. Chelation of cytosolic Zn2+, blocking ERK signaling, and antioxidants may be beneficial for treating CNS white matter ischemia-reperfusion injury. Importantly, all the inhibitors of this pathway protected oligodendrocytes when applied after the ischemic insult. © 2012 Wiley Periodicals, Inc.

International audience; Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes.

The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP(+) astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g-ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381-1395.

It has been traditionally accepted that the gonadotropins (GtHs), follicle stimulating hormone (FSH) and luteinizing hormone (LH), are synthesized and secreted only by the pituitary. However, the presence of theses hormones in extrapituitary tissues has been demonstrated in mammals, and more recently also in fish. In this study, we cloned the cDNAs and characterized the expression of FSH-β, LH-β, and glycoprotein hormone α (GPH-α) subunits from brain and gonads of male and female pejerrey Odontesthes bonariensis at different stages of gonadal maturation. In situ hybridization revealed that, in addition to their classical location in pituitary cells, the three GtH transcripts were also located in the gonads. FSH-β and GPH-α subunits were found in the cytoplasm of oogonia, previtellogenic and vitellogenic oocytes in ovaries. LH-β expression was detected in previtellogenic and vitellogenic oocytes but not in oogonia. In males, the three subunits were expressed in spermatogonia and to a lesser extent in spermatocytes. Exposure of fish to high water temperatures that impair pejerrey reproduction also induced a decrease of extrahypophyseal expression of GtH subunits. Fil: Elisio, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Soria, Federico Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Fernandino, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Strüssmann, Carlos Augusto. Tokyo University; Japón Fil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Miranda, Leandro Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Background Glutamate excitotoxicity contributes to oligodendrocyte and tissue damage in multiple sclerosis (MS). Intriguingly, glutamate level in plasma and cerebrospinal fluid of MS patients is elevated, a feature which may be related to the pathophysiology of this disease. In addition to glutamate transporters, levels of extracellular glutamate are controlled by cystine/glutamate antiporter xc -, an exchanger that provides intracellular cystine for production of glutathione, the major cellular antioxidant. The objective of this study was to analyze the role of the system xc - in glutamate homeostasis alterations in MS pathology. Methods Primary cultures of human monocytes and the cell line U-937 were used to investigate the mechanism of glutamate release. Expression of cystine glutamate exchanger (xCT) was quantified by quantitative PCR, Western blot, flow cytometry and immunohistochemistry in monocytes in vitro, in animals with experimental autoimmune encephalomyelitis (EAE), the animal model of MS, and in samples of MS patients. Results and discussion We show here that human activated monocytes release glutamate through cystine/glutamate antiporter xc - and that the expression of the catalytic subunit xCT is upregulated as a consequence of monocyte activation. In addition, xCT expression is also increased in EAE and in the disease proper. In the later, high expression of xCT occurs both in the central nervous system (CNS) and in peripheral blood cells. In particular, cells from monocyte-macrophage-microglia lineage have higher xCT expression in MS and in EAE, indicating that immune activation upregulates xCT levels, which may result in higher glutamate release and contribution to excitotoxic damage to oligodendrocytes. Conclusions Together, these results reveal that increased expression of the cystine/glutamate antiporter system xc - in MS provides a link between inflammation and excitotoxicity in demyelinating diseases.

The aim of this research was to study how high water temperatures impair the reproductive activity of pejerrey Odontesthes bonariensis, an inland-water atherinopsid fish species from the pampasic region of Argentina. Adult pejerrey of both sexes were kept under a control-temperature regime (19 degrees C) and two experimental temperatures (23 degrees and 27 degrees C) for 8 d. The effect of elevated temperature on the pituitary-gonad axis was analyzed in terms of the expression of gonadotropin (GtH) subunits: follicle-stimulating hormone beta, luteinizing hormone beta (LH-beta), glycoprotein hormone alpha, and GtH receptors FSH-R and LH-R by semiquantitative reverse transcriptase polymerase chain reaction, plasma levels of sexual steroids by radioimmunoassay, and reproductive status by gonadal histology. The results of this work clearly indicate that short periods of exposure to high water temperatures disrupt pejerrey reproduction. This effect was observed in spawning activity, at the histological level, and in the reduction of plasma estradiol in females and testosterone in males. The mRNA levels of GtH subunits and GtH receptors generally decreased in proportion to the increase in temperature for both sexes. However, the differences between groups were statistically significant only for LH-beta and for FSH-R expression in pejerrey females. Thus, the gonads of pejerrey appear to be the primary target of high water temperature. Analysis of the air temperature in this region over the past 40 yr indicated an increase of 1.74 degrees C in mean annual temperature. This increase, coupled with the mechanism of high-temperature sensitivity shown in this study, may be one of the reasons for the decline in pejerrey populations observed in this region over recent decades.

International audience; The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organisation represents a technological challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheological properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent probes to map with nanoscale precision the local organization and rheology of the ECS. Here we expand our method by tracking single nanotubes through super-resolution imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheological properties can change drastically within few nanometres. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.