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.

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.

"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.

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.

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.

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.

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.

Liver Growth Factor Induces Glia-Associated Neuroprotection in an In Vitro Model of Parkinson´s Disease.

Parkinson's disease is a neurodegenerative disorder characterized by the progressive death of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a loss of the neurotransmitter dopamine in the basal ganglia. Current treatments relieve the symptoms of the disease, but none stop or delay neuronal degeneration. Liver growth factor (LGF) is an albumin-bilirubin complex that stimulates axonal growth in the striatum and protects DA neurons in the SN of 6-hydroxydopamine-lesioned rats. Our previous results suggested that these effects observed in vivo are mediated by microglia and/or astrocytes. To determine if these cells are LGF targets, E14 (embryos from Sprague Dawley rats of 14 days) rat mesencephalic glial cultures were used. Treatment with 100 pg/mL of LGF up-regulated the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response element binding protein (CREB) phosphorylation in glial cultures, and it increased the microglia marker Iba1 and tumor necrosis factor alpha (TNF-alpha) protein levels. The treatment of E14 midbrain neurons with a glial-conditioned medium from LGF-treated glial cultures (GCM-LGF) prevented the loss of DA neurons caused by 6-hydroxy-dopamine. This neuroprotective effect was not observed when GCM-LGF was applied in the presence of a blocking antibody of TNF-alpha activity. Altogether, our findings strongly suggest the involvement of microglia and TNF-alpha in the neuroprotective action of LGF on DA neurons observed in vitro.

Lipidomic Alterations in the Mitochondria of Aged Parkin Null Mice Relevant to Autophagy.

Mitochondrial quality control is important in neurological diseases, but in genetic Parkinson's disease caused by mutations in PINK and parkin mitochondrial degradation through autophagy is crucial. Reductions in autophagy and mitophagy are implicated in aging, age related diseases and Parkinson. The parkin null mice (PK-KO) show only a subtle phenotype, apparent with age or with stressors. We have studied the changes in the lipidomic composition of the mitochondrial membranes isolated from the brains of young and old PK-KO mice and compared them to wild type in order to determine possible implications for Parkinson's disease pathology. We observed an increase in the levels of phosphatidylethanolamine in the young PK-KO mice that is lost in the old and correlate to changes in the phosphatidylserine decarboxylase. PK-KO old mice mitochondria showed lower phosphatidylglicerol and phosphatidylinositol levels and higher levels of some forms of hydroxylated ceramides. Regarding cardiolipins there were changes in the degree of saturation mainly with age. The lipidomic composition discriminates between the study groups using partial least square discriminant analysis. We discuss the relevance of the lipid changes for the autophagic activity, the mitophagy, the mitochondrial activity and the Parkinson's disease pathology in absence of parkin.

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.

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.

Trehalose improves human fibroblast deficits in a new CHIP-mutation related ataxia.

In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient's fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is an individual case based on only one patient and the statistical comparisons are not valid between controls and patient, the low variability among controls and the obvious differences with this patient allow us to conclude that trehalose, through its autophagy activation capacity, anti-aggregation properties, anti-oxidative effects and lack of toxicity, could be very promising for the treatment of CHIP-mutation related ataxia, and possibly a wide spectrum of neurodegenerative disorders related to protein disconformation.

Trehalose reverses cell malfunction in fibroblasts from normal and Huntington's disease patients caused by proteosome inhibition.

Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor, cognitive and psychiatric deficits, associated with predominant loss of striatal neurons and is caused by polyglutamine expansion in the huntingtin protein. Mutant huntingtin protein and its fragments are resistant to protein degradation and produce a blockade of the ubiquitin proteasome system (UPS). In HD models, the proteasome inhibitor epoxomicin aggravates protein accumulation and the inductor of autophagy, trehalose, diminishes it. We have investigated the effects of epoxomicin and trehalose in skin fibroblasts of control and HD patients. Untreated HD fibroblasts have increased the levels of ubiquitinized proteins and higher levels of reactive oxygen species (ROS), huntingtin and the autophagy marker LAMP2A. Baseline replication rates were higher in HD than in controls fibroblasts but that was reverted after 12 passages. Epoxomicin increases the activated caspase-3, HSP70, huntingtin, ubiquitinated proteins and ROS levels in both HD and controls. Treatment with trehalose counteracts the increase in ROS, ubiquitinated proteins, huntingtin and activated caspase-3 levels induced by epoxomicin, and also increases the LC3 levels more in HD fibroblast than controls. These results suggest that trehalose could revert protein processing abnormalities in patients with Huntington's Disease.

Striatal infusion of glial conditioned medium diminishes huntingtin pathology in r6/1 mice.

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.

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.

Protection by glia-conditioned medium in a cell model of Huntington disease.

The physiological role of huntingtin and the pathogenic mechanisms that produce the disease are unknown. Mutant huntingtin changes its normal localization and produces cytoplasmic and intranuclear inclusions, changes gene transcription, alters synaptic transmission, impairs mitochondrial activity and activates caspases and other pro-apoptotic molecules, promotes excitotoxicity, energy deficits, synthesis and release reduction of neurotrophic factors and oxidative stress. Previous studies confirm that the mutant huntingtin difficult neurotrophic function of astrocytes leading to neuronal dysfunction in Huntington's disease. Our objective was to study the neuroprotective potential role of glia-conditioned medium (GCM) in an in vitro model of Huntington's disease. We used conditionally-immortalized striatal neuronal progenitor cell lines (STHdhQ7/Q7 and STHdhQ111/Q111) expressing endogenous levels of normal and mutant huntingtin with 7 and 111 glutamines, respectively. We studied the protection of fetal and postnatal glia conditioned medium (GCM) on H2O2 (2 µM), glutamate (5 mM) and 3-nitropropionic acid (2.5 mM) related toxicity. We also compared the neuroprotective effects of GCM versus that of the growth factors bFGF, BDNF and GDNF. Fetal GCM protects from every toxin, reducing the cell death and increasing the cell survival. Fetal GCM reduces the caspases fragmentation of the protein PARP, the expression of chaperone Hsp70 and the accumulation of ROS and polyubiquitinated proteins. In addition, in Q111 striatal cells treated with H2O2 (2 µM) for 24 hours, the intracellular GSH levels are higher in the presence of GCM. Notably, the 13-day and 2-month postnatal GCM, totally protects from H2O2 induced cell death in mutant striatal cells. GCM neuroprotective effects are more potent than those of the already identified neurotrophic factors. We conclude that GCM protects Q111 cells from neuronal neurotoxins and the effects of GCM are more potent than those of any known neurotrophic factor. GCM may contain new and more potent, as yet unidentified, neurotrophic molecules, potentially useful in patients with Huntington's disease.