The Role of the Dopamine System in Post-Stroke Mood Disorders in Newborn Rats

Post-stroke mood disorders (PSMD) affect disease prognosis in adults. Adult rodent models underlie the importance of the dopamine (DA) system in PSMD pathophysiology. There are no studies on PSMD after neonatal stroke. We induced neonatal stroke in 7-day-old (P7) rats by temporal left middle cerebral artery occlusion (MCAO). Performance in the tail suspension test (TST) at P14 and the forced swimming test (FST) and open field test (OFT) at P37 were studied to assess PSMD. DA neuron density in the ventral tegmental area, brain DA concentration and DA transporter (DAT) expression as well as D2 receptor (D2R) expression and G-protein functional coupling were also studied. MCAO animals revealed depressive-like symptoms at P14 associated with decreased DA concentration and reduced DA neuron population and DAT expression. At P37, MCAO rats showed hyperactive behavior associated with increased DA concentration, normalization of DA neuron density and decreased DAT expression. MCAO did not modify D2R expression but reduced D2R functionality at P37. MCAO-induced depressive-like symptoms were reversed by the DA reuptake inhibitor GBR-12909. In conclusion, MCAO in newborn rats induced depressive-like symptoms and hyperactive behavior in the medium and long term, respectively, that were associated with alterations in the DA system.This research was funded by the PI19/00927, PID2019-106404RB-I00 and RD21/0012/0023 projects, integrated in the Spanish Plan for R + D + I, AES 2017–2020; funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) “A way to make Europe” (J.M.-O.) and the Irycis Chromatographic Services and Nervous System Markers Unit, UCS (2018/0135) (M.J.C.)

Coulomb dissociation of 16O into 4He and 12C

We measured the Coulomb dissociation of 16O into 4He and 12C at the R3B setup in a first campaign within FAIR Phase 0 at GSI Helmholtzzentrum für Schwerionenforschung, Darmstadt. The goal was to improve the accuracy of the experimental data for the 12C(a,?)16O fusion reaction and to reach lower center-ofmass energies than measured so far. The experiment required beam intensities of 109 16O ions per second at an energy of 500 MeV/nucleon. The rare case of Coulomb breakup into 12C and 4He posed another challenge: The magnetic rigidities of the particles are so close because of the same mass-To-charge-number ratio A/Z = 2 for 16O, 12C and 4He. Hence, radical changes of the R3B setup were necessary. All detectors had slits to allow the passage of the unreacted 16O ions, while 4He and 12C would hit the detectors' active areas depending on the scattering angle and their relative energies. We developed and built detectors based on organic scintillators to track and identify the reaction products with sufficient precision

Liver Growth Factor “LGF” as a Therapeutic Agent for Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive degenerative disorder and the most common cause of dementia in aging populations. Although the pathological hallmarks of AD are well defined, currently no effective therapy exists. Liver growth factor (LGF) is a hepatic albumin–bilirubin complex with activity as a tissue regenerating factor in several neurodegenerative disorders such as Parkinson’s disease and Friedreich’s ataxia. Our aim here was to analyze the potential therapeutic effect of LGF on the APPswe mouse model of AD. Twenty-month-old mice received intraperitoneal (i.p.) injections of 1.6 µg LGF or saline, twice a week during three weeks. Mice were sacrificed one week later, and the hippocampus and dorsal cortex were prepared for immunohistochemical and biochemical studies. LGF treatment reduced amyloid-β (Aβ) content, phospho-Tau/Tau ratio and the number of Aβ plaques with diameter larger than 25 µm. LGF administration also modulated protein ubiquitination and HSP70 protein levels, reduced glial reactivity and inflammation, and the expression of the pro-apoptotic protein Bax. Because the administration of this factor also restored cognitive damage in APPswe mice, we propose LGF as a novel therapeutic tool that may be useful for the treatment of AD.R.G.-G. was the recipient of a Research Supporting Staff Grant Contract (Spanish Health Research Fund (FIS)).Peer reviewe

Cx3cr1‐deficiency exacerbates alpha‐synuclein‐A53T induced neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by the degeneration of dopaminergic neurons of the substantia nigra and the accumulation of protein aggregates, called Lewy bodies, where the most abundant is alpha-synuclein (α-SYN). Mutations of the gene that codes for α-SYN (SNCA), such as the A53T mutation, and duplications of the gene generate cases of PD with autosomal dominant inheritance. As a result of the association of inflammation with the neurodegeneration of PD, we analyzed whether overexpression of wild-type α-SYN (α-SYNWT ) or mutated α-SYN (α-SYNA53T ) are involved in the neuronal dopaminergic loss and inflammation process, along with the role of the chemokine fractalkine (CX3CL1) and its receptor (CX3CR1). We generated in vivo murine models overexpressing human α-SYNWT or α-SYNA53T in wild type (Cx3cr1+/+ ) or deficient (Cx3cr1-/- ) mice for CX3CR1 using unilateral intracerebral injection of adeno-associated viral vectors. No changes in CX3CL1 levels were observed by immunofluorescence or analysis by qRT-PCR in this model. Interestingly, the expression α-SYNWT induced dopaminergic neuronal death to a similar degree in both genotypes. However, the expression of α-SYNA53T produced an exacerbated neurodegeneration, enhanced in the Cx3cr1-/- mice. This neurodegeneration was accompanied by an increase in neuroinflammation and microgliosis as well as the production of pro-inflammatory markers, which were exacerbated in Cx3cr1-/- mice overexpressing α-SYNA53T. Furthermore, we observed that in primary microglia CX3CR1 was a critical factor in the modulation of microglial dynamics in response to α-SYNWT or α-SYNA53T. Altogether, our study reveals that CX3CR1 plays an essential role in neuroinflammation induced by α-SYNA53T.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (Grants refs. SAF2016–76520-R and BFU2017–82407-R). TLR was supported by a Tatiana Perez de Guzmán el Bueno Foundation Fellowship.Peer reviewe

Striatal Infusion of Glial Conditioned Medium Diminishes Huntingtin Pathology in R6/1 Mice

<div><p>Huntington's disease is a neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin gene which produces widespread neuronal and glial pathology. We here investigated the possible therapeutic role of glia or glial products in Huntington's disease using striatal glial conditioned medium (GCM) from fetus mice (E16) continuously infused for 15 and 30 days with osmotic minipumps into the left striatum of R6/1 mice. Animals infused with GCM had significantly less huntingtin inclusions in the ipsilateral cerebral cortex and in the ipsilateral and contralateral striata than mice infused with cerebrospinal fluid. The numbers of DARPP-32 and TH positive neurons were also greater in the ipsilateral but not contralateral striata and substantia nigra, respectively, suggesting a neuroprotective effect of GCM on efferent striatal and nigro-striatal dopamine neurons. GCM increases activity of the autophagic pathway, as shown by the reduction of autophagic substrate, p-62, and the augmentation of LC3 II, Beclin-1 and LAMP-2 protein levels, direct markers of autophagy, in GCM infused mice. GCM also increases BDNF levels. These results suggest that CGM should be further explored as a putative neuroprotective agent in Huntington's disease.</p></div

GCM treatment during 15 days activates autophagy pathways.

<p>(<b>A</b>) Representative bands and western blot quantification of LC3 II/LC3 I ratio. The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3-II are used as an indicator of autophagy. (<b>B</b>) Representative bands and quantification of Beclin-1 antibody, direct marker of the autophagosome formation. (<b>C</b>) Western blots representative bands and quantification of p-62 specific antibody, as a substrate of autophagy and (<b>D</b>) LAMP-2 quantification, corrected by β-actin. Values are from the hemibrains, expressed as mean ± SEM (n = 6 in each group). Statistical analysis performed by t student. *p<0.05 GCM <i>vs</i> control CSF group and +p<0.05 right <i>vs</i> left hemispheres. (<b>E</b>) Representative microphotographs of Lysosomal-associated membrane protein, LAMP-2A antibody, present in lysosomes and endosomes, which implies autophagy activation. LAMP-2A staining (green) and nuclei (Hoescht, blue) immunofluorescence in striatum of R6/1 mice, with CSF and GCM infusion. (40× magnification, scale bar  = 10 µm). (<b>F</b>) Quantification of number of cells with LAMP-2A positive vesicle distribution in the perinuclear region, around the nucleus cell. Numbers of cells counted in 6 random fields of the striatum at 20× magnification. Mean ± SEM (n = 6 in each group). Statistical analysis performed by student's t test. *p<0.05, **p<0.01 GCM <i>vs</i> control CSF group; +p<0.05, +++p<0.001 right <i>vs</i> left hemispheres.</p

GCM treatment (30 days) diminishes the number of htt inclusions in cortex and striatum.

<p>(<b>A</b>) Microphotographs of the right and left cortex with the specific antibody anti-HTT inclusions and (<b>B</b>) quantification of both cortex, left and right. (<b>C</b>) Microphotographs of the right and left striatum with the specific antibody anti-HTT inclusions and (<b>D</b>) quantification of both striatum, left and right. The values are expressed as the mean ± SEM (n = 6 mice in each experimental group). The statistical analysis was performed by student's t test. *p<0.05, **p<0.01 GCM <i>vs</i> control CSF groups.</p

30⁺ cells in SNpc of the striatum <i>versus</i> CSF controls.

<p>(<b>A</b>) Stitched 10x images in order to encompass the whole Substantia Nigra Pars Compact (SNpc) in the striatum with TH positive cells (scale bar  = 100 µm). (<b>B</b>) Quantification of the number of TH cells positive cells/SNpc area. The values are expressed as the mean ± SEM (n = 3–6 mice in each experimental group). The statistical analysis was performed by student's t test. *p<0.05 GCM <i>vs</i> control CSF groups.</p

Method and implantation coordinates into striatum.

<p>(<b>A</b>) Paxinos coordinates chosen for striatal infusion in mice aiming the centre of the left striatum at AP: +0.62 mm; ML: −1.75 mm to bregma a v d DV: −3.5 mm to the skull. The injection rate was 0.25 μl/hour for both experimental times. (<b>B</b>) Image of the implantation point in the mouse brain. (<b>C, D</b>) Methylene blue staining in the striatum, after 15 and 30 days of pump implantation. (<b>E</b>) HD mouse with pump installed subQ on the back and the brain infusion kit fixed with methacrylate to the skull. (<b>F</b>) High-affinity [³H]DA uptake in GCM “fresh” (immediately thawed), GCM incubated at 37° C, for 15 days compared with Defined medium (DM). The values are expressed as the mean ± SEM (n = 5). Statistical analysis was performed by one-way ANOVA, followed by Newman–Keuls multiple comparison test. ***p<0.001 GCM <i>vs</i> DM.</p

GCM treatment (30 days) increases the number of DARPP-32 cells.

<p>(<b>A</b>) Microphotographs of the left and right striatum with DARPP-32 positive cells and (<b>B</b>) quantification of the DARPP-32⁺ cells. A portion of the micrographs has been magnified to show cell DARPP-32 positive staining. The values are expressed as the mean ± SEM (n = 6 mice in each experimental group). The statistical analysis was performed by student's t test. *p<0.05 GCM <i>vs</i> control CSF groups.</p