Effects of cannabidiol in post-stroke mood disorders in neonatal rats.

BACKGROUND: Neonatal rats can manifest post-stroke mood disorders (PSMD) following middle cerebral artery occlusion (MCAO). We investigated whether cannabidiol (CBD) neuroprotection, previously demonstrated in neonatal rats after MCAO, includes prevention of PSMD development. METHODS: Seven-day-old Wistar rats (P7) underwent MCAO and received either vehicle or 5 mg/kg CBD treatment. Brain damage was quantified by MRI, and neurobehavioral and histological (TUNEL) studies were performed at P14 and P37. PSMD were assessed using the tail suspension test, forced swimming test, and open field tests. The dopaminergic system was evaluated by quantifying dopaminergic neurons (TH+) in the Ventral Tegmental Area (VTA), measuring brain dopamine (DA) concentration and DA transporter expression, and assessing the expression and function D2 receptors (D2R) through [35S]GTPγS binding. Animals without MCAO served as controls. RESULTS: CBD reduced MCAO-induced brain damage and improved motor performance. At P14, MCAO induced depressive-like behavior, characterized by reduced TH+ cell population and DA levels, which CBD did not prevent. However, CBD ameliorated hyperactivity observed at P37, preventing increased DA concentration by restoring D2R function. CONCLUSIONS: These findings confirm the development of PSMD following MCAO in neonatal rats and highlight CBD as a neuroprotective agent capable of long-term functional normalization of the dopaminergic system post-MCAO. IMPACT: MCAO in neonatal rats led to post-stroke mood disorders consisting in a depression-like picture in the medium term evolving towards long-term hyperactivity, associated with an alteration of the dopaminergic system. The administration of CBD after MCAO did not prevent the development of depressive-like behavior, but reduced long-term hyperactivity, normalizing dopamine receptor function. These data point to the importance of considering the development of depression-like symptoms after neonatal stroke, a well-known complication after stroke in adults. Our work confirms the interest of CBD as a possible treatment for neonatal stroke.

Mitochondrial dysfunction in chromaffin cells from the R6/1 mouse model of Huntington's disease: Impact on exocytosis and calcium current regulation.

From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (ICa). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased ICa inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of ICa and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues.

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.

Neuroprotection with the cannabigerol quinone derivative VCE-003.2 and its analogs CBGA-Q and CBGA-Q-Salt in Parkinson's disease using 6-hydroxydopamine-lesioned mice.

The quinone derivative of the non-psychotropic cannabinoid cannabigerol (CBG), so-called VCE-003.2, has been recently investigated for its neuroprotective properties in inflammatory models of Parkinson's disease (PD) in mice. Such potential derives from its activity at the peroxisome proliferator-activated receptor-γ (PPAR-γ). In the present study, we investigated the neuroprotective properties of VCE-003.2 against the parkinsonian neurotoxin 6-hydroxydopamine (6-OHDA), in comparison with two new CBG-related derivatives, the cannabigerolic acid quinone (CBGA-Q) and its sodium salt CBGA-Q-Salt, which, similarly to VCE-003.2, were found to be active at the PPAR-γ receptor, but not at the cannabinoid CB1 and CB2 receptors. First, we investigated their cytoprotective properties in vitro by analyzing cell survival in cultured SH-SY5Y cells exposed to 6-OHDA. We found an important cytoprotective effect of VCE-003.2 at a concentration of 20 µM, which was not reversed by the blockade of PPAR-γ receptors with GW9662, supporting its activity at an alternative site (non-sensitive to classic antagonists) in this receptor. We also found CBGA-Q and CBGA-Q-Salt being cytoprotective in this cell assay, but their effects were completely eliminated by GW9662, thus indicating that they are active at the canonical site in the PPAR-γ receptor. Then, we moved to in vivo testing using mice unilaterally lesioned with 6-OHDA. Our data confirmed that VCE-003.2 administered orally (20 mg/kg) preserved tyrosine hydroxylase (TH)-positive nigral neurons against 6-OHDA-induced damage, whereas it completely attenuated the astroglial (GFAP) and microglial (CD68) reactivity found in the substantia nigra of lesioned mice. Such neuroprotective effects caused an important recovery in the motor deficiencies displayed by 6-OHDA-lesioned mice in the pole test and the cylinder rearing test. We also investigated CBGA-Q, given orally (20 mg/kg) or intraperitoneally (10 mg/kg, i.p.), having similar benefits compared to VCE-003.2 against the loss of TH-positive nigral neurons, glial reactivity and motor defects caused by 6-OHDA. Lastly, the sodium salt of CBGA-Q, given orally (40 mg/kg) to 6-OHDA-lesioned mice, also showed benefits at behavioral and histopathological levels, but to a lower extent compared to the other two compounds. In contrast, when given i.p., CBGA-Q-Salt (10 mg/kg) was poorly active. We also analyzed the concentrations of dopamine and its metabolite DOPAC in the striatum of 6-OHDA-lesioned mice after the treatment with the different compounds, but recovery in the contents of both dopamine and DOPAC was only found after the treatment with VCE-003.2. In summary, our data confirmed the neuroprotective potential of VCE-003.2 in 6-OHDA-lesioned mice, which adds to its previous activity found in an inflammatory model of PD (LPS-lesioned mice). Additional phytocannabinoid derivatives, CBGA-Q and CBGA-Q-Salt, also afforded neuroprotection in 6-OHDA-lesioned mice, but their effects were lower compared to VCE-003.2, in particular in the case of CBGA-Q-Salt. In vitro studies confirmed the relevance of PPAR-γ receptors for these effects.

ßOHB Protective Pathways in Aralar-KO Neurons and Brain: An Alternative to Ketogenic Diet.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether ß-hydroxybutyrate (ßOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that ßOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring ßOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of ßOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of ßOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by ßOHB administration, a 4-d ßOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of ßOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.SIGNIFICANCE STATEMENTAralar deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that ßOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. ßOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-knock-out (KO) mice, ßOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The ßOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.

Immunohistochemical Study of ASC Expression and Distribution in the Hippocampus of an Aged Murine Model of Alzheimer's Disease.

Neuroinflammation is involved in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), and is notably dependent on age. One important inflammatory pathway exerted by innate immune cells of the nervous system in response to danger signals is mediated by inflammasomes (IF) and leads to the generation of potent pro-inflammatory cytokines. The protein "apoptosis-associated speck-like protein containing a caspase recruitment domain" (ASC) modulates IF activation but has also other functions which are crucial in AD. We intended to characterize immunohistochemically ASC and pattern recognition receptors (PRR) of IF in the hippocampus (HP) of the transgenic mouse model Tg2576 (APP), in which amyloid-beta (Aß) pathology is directly dependent on age. We show in old-aged APP a significant amount of ASC in microglia and astrocytes associated withAß plaques, in the absence of PRR described by others in glial cells. In addition, APP developed foci with clusters of extracellular ASC granules not spatiallyrelated to Aß plaques, which density correlated with the advanced age of mice and AD development. Clusters were associated withspecific astrocytes characterized by their enlarged ring-shaped process terminals, ASC content, and frequent perivascular location. Their possible implication in ASC clearance and propagation of inflammation is discussed.

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-ß (Aß) content, phospho-Tau/Tau ratio and the number of Aß 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.

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 .

Altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington's disease is linked to over-expression of mutated huntingtin.

As the peripheral sympathoadrenal axis is tightly controlled by the cortex via hypothalamus and brain stem, the central pathological features of Hunting's disease, (HD) that is, deposition of mutated huntingtin and synaptic dysfunctions, could also be expressed in adrenal chromaffin cells. To test this hypothesis we here present a thorough investigation on the pathological and functional changes undergone by chromaffin cells (CCs) from 2-month (2 m) to 7-month (7 m) aged wild-type (WT) and R6/1 mouse model of Huntington's disease (HD), stimulated with acetylcholine (ACh) or high [K+ ] (K+ ). In order to do this, we used different techniques such as inmunohistochemistry, patch-clamp, and amperometric recording. With respect to WT cells, some of the changes next summarized were already observed in HD mice at a pre-disease stage (2 m); however, they were more pronounced at 7 m when motor deficits were clearly established, as follows: (i) huntingtin over-expression as nuclear aggregates in CCs; (ii) smaller CC size with decreased dopamine ß-hydroxylase expression, indicating lesser number of chromaffin secretory vesicles; (iii) reduced adrenal tissue catecholamine content; (iv) reduced Na+ currents with (v) membrane hyperpolarization and reduced ACh-evoked action potentials; (v) reduced [Ca2+ ]c transients with faster Ca2+ clearance; (vi) diminished quantal secretion with smaller vesicle quantal size; (vii) faster kinetics of the exocytotic fusion pore, pore expansion, and closure. On the basis of these data, the hypothesis is here raised in the sense that nuclear deposition of mutated huntingtin in adrenal CCs of R6/1 mice could be primarily responsible for poorer Na+ channel expression and function, giving rise to profound depression of cell excitability, altered Ca2+ handling and exocytosis. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14201.

Trehalose rescues glial cell dysfunction in striatal cultures from HD R6/1 mice at early postnatal development.

The pathological hallmark of Huntington disease (HD) is the intracellular aggregation of mutant huntingtin (mHTT) in striatal neurons and glia associated with the selective loss of striatal medium-sized spiny neurons. Up to the present, the role of glia in HD is poorly understood and has been classically considered secondary to neuronal disorder. Trehalose is a disaccharide known to possess many pharmacological properties, acting as an antioxidant, a chemical chaperone, and an inducer of autophagy. In this study, we analyzed at an early postnatal development stage the abnormalities observed in striatal glial cell cultures of postnatal R6/1 mice (HD glia), under baseline and stressing conditions and the protective effects of trehalose. Our data demonstrate that glial HD alterations already occur at early stages of postnatal development. After 20 postnatal days in vitro, striatal HD glia cultures showed more reactive astrocytes with increased expression of glial fibrillary acidic protein (GFAP) but with less replication capacity, less A2B5(+) glial progenitors and more microglia than wild-type (WT) cultures. HD glia had lower levels of intracellular glutathione (GSH) and was more susceptible to H2O2 and epoxomicin insults. The amount of expressed GDNF and secreted mature-BDNF by HD astrocytes were much lower than by WT astrocytes. In addition, HD glial cultures showed a deregulation of the major proteolytic systems, the ubiquitin-proteasomal system (UPS), and the autophagic pathway. This produces a defective protein quality control, indicated by the elevated levels of ubiquitination and p62 protein. Interestingly, we show that trehalose, through its capacity to induce autophagy, inhibited p62/SQSTM1 accumulation and facilitated the degradation of cytoplasmic aggregates from mHTT and α-synuclein proteins. Trehalose also reduced microglia activation and reversed the disrupted cytoskeleton of astrocytes accompanied with an increase in the replication capacity. In addition, trehalose up-regulated mature-BDNF neurotrophic factor expression and secretion, probably mediating cytoskeletal organization and helping in vesicular BDNF transport. Together, these findings indicate that glia suffers functional early changes in the disease process, changes that may contribute to HD neurodegeneration. Trehalose could be a very promising compound for treatment of HD and other diseases with abnormal protein aggregates. Furthermore our study identifies glial cells as a novel target for trehalose to induce neurotrophic and neuroprotective actions in HD.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich's ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 µg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Neuroprotective role of liver growth factor "LGF" in an experimental model of cerebellar ataxia.

Cerebellar ataxias (CA) comprise a heterogeneous group of neurodegenerative diseases characterized by a lack of motor coordination. They are caused by disturbances in the cerebellum and its associated circuitries, so the major therapeutic goal is to correct cerebellar dysfunction. Neurotrophic factors enhance the survival and differentiation of selected types of neurons. Liver growth factor (LGF) is a hepatic mitogen that shows biological activity in neuroregenerative therapies. We investigate the potential therapeutic activity of LGF in the 3-acetylpiridine (3-AP) rat model of CA. This model of CA consists in the lesion of the inferior olive-induced by 3-AP (40 mg/kg). Ataxic rats were treated with 5 µg/rat LGF or vehicle during 3 weeks, analyzing: (a) motor coordination by using the rota-rod test; and (b) the immunohistochemical and biochemical evolution of several parameters related with the olivo-cerebellar function. Motor coordination improved in 3-AP-lesioned rats that received LGF treatment. LGF up-regulated NeuN and Bcl-2 protein levels in the brainstem, and increased calbindin expression and the number of neurons receiving calbindin-positive projections in the cerebellum. LGF also reduced extracellular glutamate and GABA concentrations and microglia activation in the cerebellum. In view of these results, we propose LGF as a potential therapeutic agent in cerebellar ataxias.

Impact of Standard Versus Low Pneumoperitoneum Pressure on Peritoneal Environment in Laparoscopic Cholecystectomy. Randomized Clinical Trial.

BACKGROUND: High CO 2 pneumoperitoneum pressure during laparoscopy adversely affects the peritoneal environment. This study hypothesized that low pneumoperitoneum pressure may be linked to less peritoneal damage and possibly to better clinical outcomes. MATERIALS AND METHODS: One hundred patients undergoing scheduled laparoscopic cholecystectomy were randomized 1:1 to low or to standard pneumoperitoneum pressure. Peritoneal biopsies were performed at baseline time and 1 hour after peritoneum insufflation in all patients. The primary outcome was peritoneal remodeling biomarkers and apoptotic index. Secondary outcomes included biomarker differences at the studied times and some clinical variables such as length of hospital stay, and quality and safety issues related to the procedure. RESULTS: Peritoneal IL6 after 1 hour of surgery was significantly higher in the standard than in the low-pressure group (4.26±1.34 vs. 3.24±1.21; P =0.001). On the contrary, levels of connective tissue growth factor and plasminogen activator inhibitor-I were higher in the low-pressure group (0.89±0.61 vs. 0.61±0.84; P =0.025, and 0.74±0.89 vs. 0.24±1.15; P =0.028, respectively). Regarding apoptotic index, similar levels were found in both groups and were 44.0±10.9 and 42.5±17.8 in low and standard pressure groups, respectively. None of the secondary outcomes showed differences between the 2 groups. CONCLUSIONS: Peritoneal inflammation after laparoscopic cholecystectomy is higher when surgery is performed under standard pressure. Adhesion formation seems to be less in this group. The majority of patients undergoing surgery under low pressure were operated under optimal workspace conditions, regardless of the surgeon's expertise.

AGC1-malate aspartate shuttle activity is critical for dopamine handling in the nigrostriatal pathway.

The mitochondrial transporter of aspartate-glutamate Aralar/AGC1 is a regulatory component of the malate-aspartate shuttle. Aralar deficiency in mouse and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. A lack of neurofilament-labeled processes is detected in the cerebral cortex, but whether different types of neurons are differentially affected by Aralar deficiency is still unknown. We have now found that Aralar-knockout (Aralar-KO) post-natal mice show hyperactivity, anxiety-like behavior, and hyperreactivity with a decrease of dopamine (DA) in terminal-rich regions. The striatum is the brain region most affected in terms of size, amino acid and monoamine content. We find a decline in vesicular monoamine transporter-2 (VMAT2) levels associated with increased DA metabolism through MAO activity (DOPAC/DA ratio) in Aralar-KO striatum. However, no decrease in DA or in the number of nigral tyrosine hydroxylase-positive cells was detected in Aralar-KO brainstem. Adult Aralar-hemizygous mice presented also increased DOPAC/DA ratio in striatum and enhanced sensitivity to amphetamine. Our results suggest that Aralar deficiency causes a fall in GSH/GSSG ratio and VMAT2 in striatum that might be related to a failure to produce mitochondrial NADH and to an increase of reactive oxygen species (ROS) in the cytosol. The results indicate that the nigrostriatal dopaminergic system is a target of Aralar deficiency.

"MiR-7 controls cholesterol biosynthesis through posttranscriptional regulation of DHCR24 expression".

Dysregulation of cholesterol homeostasis is associated with several pathologies including cardiovascular diseases and neurological disorders such as Alzheimer's disease (AD). MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of cholesterol metabolism. We previously established the role of miR-7 in regulating insulin resistance and amyloidosis, which represents a common pathological feature between type 2 diabetes and AD. We show here an additional metabolic function of miR-7 in cholesterol biosynthesis. We found that miR-7 blocks the last steps of the cholesterol biosynthetic pathway in vitro by targeting relevant genes including DHCR24 and SC5D posttranscriptionally. Intracranial infusion of miR-7 on an adeno-associated viral vector reduced the expression of DHCR24 in the brain of wild-type mice, supporting in vivo miR-7 targeting. We also found that cholesterol regulates endogenous levels of miR-7 in vitro, correlating with transcriptional regulation through SREBP2 binding to its promoter region. In parallel to SREBP2 inhibition, the levels of miR-7 and hnRNPK (the host gene of miR-7) were concomitantly reduced in brain in a mouse model of Niemann Pick type C1 disease and in murine fatty liver, which are both characterized by intracellular cholesterol accumulation. Taken together, the results establish a novel regulatory feedback loop by which miR-7 modulates cholesterol homeostasis at the posttranscriptional level, an effect that could be exploited for therapeutic interventions against prevalent human diseases.

Rottlerin Stimulates Exosome/Microvesicle Release Via the Increase of Ceramide Levels Mediated by Ampk in an In Vitro Model of Intracellular Lipid Accumulation.

Exosomes/microvesicles originate from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of rottlerin, a polyphenol, on exosome/microvesicle secretion in a model of intracellular lipid trafficking impairment, and elucidated the mechanism of action. In a model of lipid trafficking impairment in C6 glia cells, rottlerin increased ceramide levels, while decreasing hexosylceramide content. This was accompanied by increased exosome/microvesicle secretion, thereby reducing the concentration of lipids in the endolysosomal compartment. The reduction of hexosylceramide levels by rottlerin was attributed to the increase of ß-glucosidase (glucosylceramidase) activity, and the effects of rottlerin were abrogated by ß-glucosidase inhibitors such as isofagomine D-tartrate and AMP-deoxynojirimycin. Moreover, treatment with ML-266, a potent activator of the ß-glucosidase enzyme, recapitulated the effects of rottlerin on the sphingolipid profile and exosome/microvesicle secretion. Finally, inhibition of AMPK (AMP-activated protein kinase) using compound C prevented both exosome/microvesicle secretion and the elimination of endolysosome lipids, which were promoted by rottlerin. The results showed that the decrease in intracellular lipid deposition induced by rottlerin was mediated by ß-glucosidase activation and exosome/microvesicle release via the AMPK pathway. Rottlerin consumption could represent an additional health benefit in lysosomal deposition diseases.

The inhibition of NCS-1 binding to Ric8a rescues fragile X syndrome mice model phenotypes.

Fragile X syndrome (FXS) is caused by the loss of function of Fragile X mental retardation protein (FMRP). FXS is one of the leading monogenic causes of intellectual disability (ID) and autism. Although it is caused by the failure of a single gene, FMRP that functions as an RNA binding protein affects a large number of genes secondarily. All these genes represent hundreds of potential targets and different mechanisms that account for multiple pathological features, thereby hampering the search for effective treatments. In this scenario, it seems desirable to reorient therapies toward more general approaches. Neuronal calcium sensor 1 (NCS-1), through its interaction with the guanine-exchange factor Ric8a, regulates the number of synapses and the probability of the release of a neurotransmitter, the two neuronal features that are altered in FXS and other neurodevelopmental disorders. Inhibitors of the NCS-1/Ric8a complex have been shown to be effective in restoring abnormally high synapse numbers as well as improving associative learning in FMRP mutant flies. Here, we demonstrate that phenothiazine FD44, an NCS-1/Ric8a inhibitor, has strong inhibition ability in situ and sufficient bioavailability in the mouse brain. More importantly, administration of FD44 to two different FXS mouse models restores well-known FXS phenotypes, such as hyperactivity, associative learning, aggressive behavior, stereotype, or impaired social approach. It has been suggested that dopamine (DA) may play a relevant role in the behavior and in neurodevelopmental disorders in general. We have measured DA and its metabolites in different brain regions, finding a higher metabolic rate in the limbic area, which is also restored with FD44 treatment. Therefore, in addition to confirming that the NCS-1/Ric8a complex is an excellent therapeutic target, we demonstrate the rescue effect of its inhibitor on the behavior of cognitive and autistic FXS mice and show DA metabolism as a FXS biochemical disease marker.

New Insights on the Regulation of the Insulin-Degrading Enzyme: Role of microRNAs and RBPs.

The evident implication of the insulin-degrading enzyme (IDE) in Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM), among its capacity to degrade insulin and amyloid-ß peptide (Aß), suggests that IDE could be an essential link in the relation between hyperinsulinemia, insulin resistance and AD. However, little is known about the cellular and molecular regulation of IDE expression, and even less has been explored regarding the post-transcriptional regulation of IDE, although it represents a great molecular target of interest for therapeutic treatments. We recently described that miR-7, a novel candidate for linking AD and T2DM at the molecular level, regulates IDE and other key genes in both pathologies, including some key genes involved in the insulin signaling pathway. Here, we explored whether other miRNAs as well as other post-transcriptional regulators, such as RNA binding proteins (RBP), could potentially participate in the regulation of IDE expression in vitro. Our data showed that in addition to miR-7, miR-125, miR-490 and miR-199 regulate IDE expression at the post-transcriptional level. Moreover, we also found that IDE contains multiple potential binding sites for several RBPs, and a narrow-down prediction analysis led us to speculate on a novel regulation of IDE by RALY and HuD. Taken together, these results demonstrate the novel players controlling IDE expression that could represent potential therapeutical targets to treat several metabolic diseases with a high impact on human health, including AD and T2DM.

Trehalose ameliorates dopaminergic and tau pathology in parkin deleted/tau overexpressing mice through autophagy activation.

Tauopathies are neurodegenerative diseases, sporadic or familial, mainly characterized by dementia and parkinsonism associated to atrophy of the frontotemporal cortex and the basal ganglia, with deposition of abnormal tau in brain. Hereditary tauopathies are related with mutations of the tau gene. Up to the present, these diseases have not been helped by any disease-modifying treatment, and patients die a few years after the onset of symptoms. We have developed and characterized a mouse model of tauopathy with parkinsonism, overexpressing human mutated tau protein with deletion of parkin (PK(-/-)/Tau(VLW)). At 3 months of age, these mice present abnormal dopamine-related behavior, severe dropout of dopamine neurons in the ventral midbrain, reduced dopamine levels in the striatum and abundant phosphorylated tau-positive neuritic plaques, neurofibrillary tangles, astrogliosis, and, at 12 months old, plaques of murine beta-amyloid in the hippocampus. Trehalose is a natural disaccharide that increases the removal of abnormal proteins through enhancement of autophagy. In this work, we tested if 1% trehalose in the drinking water reverts the PK(-/-)/Tau(VLW) phenotype. The treatment with trehalose of 3-month-old PK(-/-)/Tau(VLW) mice for 2.5 months reverted the dropout of dopamine neurons, which takes place in the ventral midbrain of vehicle treated PK(-/-)/Tau(VLW) and the reduced dopamine-related proteins levels in the midbrain and striatum. The number of phosphorylated tau-positive neuritic plaques and the levels of phosphorylated tau decreased, as well as astrogliosis in brain regions. The autophagy markers in the brain, the autophagic vacuoles isolated from the liver, and the electron microscopy data indicate that these effects of trehalose are mediated by autophagy. The treatment with trehalose for 4 months of 3-month-old PK(-/-)/Tau(VLW) mice maintained the amelioration of the tau pathology and astrogliosis but failed to revert DA-related pathology in the striatum. Furthermore, the 3-week treatment with trehalose of 14-month-old PK(-/-)/Tau(VLW) mice, at the limit of their life expectancy, improved the motor behavior and anxiety of these animals, and reduced their levels of phosphorylated tau and the number of murine beta-amyloid plaques. Trehalose is neuroprotective in this model of tauopathy. Since trehalose is free of toxic effects at high concentrations, this study opens the way for clinical studies of the effects of trehalose in human tauopathies.

Parkin deletion causes cerebral and systemic amyloidosis in human mutated tau over-expressing mice.

Deposition of proteins leading to amyloid takes place in some neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. Mutations of tau and parkin proteins produce neurofibrillary abnormalities without deposition of amyloid. Here we report that mature, parkin null, over-expressing human mutated tau (PK(-/-)/Tau(VLW)) mice have altered behaviour and dopamine neurotransmission, tau pathology in brain and amyloid deposition in brain and peripheral organs. PK(-/-)/Tau(VLW) mice have abnormal behaviour and severe drop out of dopamine neurons in the ventral midbrain, up to 70%, at 12 months and abundant phosphorylated tau positive neuritic plaques, neuro-fibrillary tangles, astrogliosis, microgliosis and plaques of murine beta-amyloid in the hippocampus. PK(-/-)/Tau(VLW) mice have organomegaly of the liver, spleen and kidneys. The electron microscopy of the liver confirmed the presence of a fibrillary protein deposits with amyloid characteristics. There is also accumulation of mouse tau in hepatocytes. These mice have lower levels of CHIP-HSP70, involved in the proteosomal degradation of tau, increased oxidative stress, measured as depletion of glutathione which, added to lack of parkin, could trigger tau accumulation and amyloidogenesis. This model is the first that demonstrates beta-amyloid deposits caused by over-expression of tau and without modification of the amyloid precursor protein, presenilins or secretases. PK(-/-)/Tau(VLW) mice provide a link between the two proteins more important for the pathogenesis of Alzheimer disease.