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The antidepressant desipramine (DMI) is a selective inhibitor of norepinephrine (NE) transport that down-regulates the norepinephrine transporter (NET) protein in a concentration- and time-dependent manner in vitro. In this study, possible regulatory effects of DMI on NET mRNA and protein levels were investigated with the NET-expressing SK-N-BE(2)M17 cell line and rat brain tissue. Northern blot analysis showed that incubation of the cultured cells with DMI (5-500 nM) for 3 days reduced levels of NET mRNA in both its 5.8-kb (by up to 58%) and 3.6-kb forms (to 68%), whereas incubation for 14 days increased both levels (to 40% and 100%) in a concentration-dependent manner. In contrast, NET protein levels decreased after 3-14 days of exposure of the cells to DMI, as determined by western blotting. The in vitro findings were supported by in viva treatment of rats with DMI. Thus, in situ hybridization demonstrated initially decreased, and later increased, NET mRNA levels in locus coeruleus (LC) tissue of rats treated with DMI; whereas NET protein levels in the LC were reduced after 14 days, but unchanged after three daily DMI treatments. Thus, DMI had similar effects on NET expression in vitro and in viva, with opposite changes in NET mRNA and protein levels, suggesting that the regulatory mechanisms involved are complex and noncongruent. [ABSTRACT FROM AUTHOR]

Development of noraderenergic (NA) neurons in the vertebrate brain is dependent on the homeodomain proteins Phox2a and 2b. Here, we show that Phox2a directly controls the NA identity by activating NA-synthesizing dopamine β-hydroxylase (DBH) gene. Single point mutations in the homeodomain of Phox2a resulted in a failure to transactivate the DBH promoter in vitro and resulted in the loss of NA neurons in vivo. In addition, injection of Phox2a-specific antisense oligonucleotide induced the loss of NA neurons in developing zebrafish. Phox2a and 2b activate the DBH promoter and bind to three domains (PBD1–3). PBD1 is composed of two overlapping sites with which monomers of Phox2a can interact. In contrast, PBD2 and 3 interact with the dimeric form of Phox2a. Mutations in three or four, but not one or two, of the binding sites completely abolished activation of the DBH promoter by Phox2a or 2b, while the conversion of PBD3 to a consensus motif (ATTA) improved the DBH promoter activity by > 10-fold. Taken together, these findings establish that Phox2a and 2b control the development of NA neurons in part by directly transactivating DBH transcription through interactions with four binding sites clustered in the proximal promoter. [ABSTRACT FROM AUTHOR]

The specification of neurotransmitter identity is a critical step in neural development. Recent progresses have indicated that the closely related homeodomain factors Phox2a and 2b are essential for development of noradrenergic (NA) neuron differentiation, and may directly determine the neurotransmitter identity. With a long-term goal of understanding the regulatory cascade of NA phenotype determination, we isolated and characterized a hPhox2a genomic clone encompassing approximately 7.5 kb of the 5′ upstream promoter region, the entire exon–intron structure, and approximately 4 kb of the 3′ flanking region. Using mRNAs isolated from the Phox2a-expressing human cell line, both primer extension and 5′-rapid amplification of cDNA ends analyses identified a single transcription start site that resides 172 nucleotides upstream of the start codon. The transcription start site was preceded by a TATA-like sequence motif and transcripts from this site contained an additional G residue at the 5′ position, supporting the authenticity of this site as the transcriptional start site of hPhox2a. We assembled hPhox2a–luciferase reporter constructs containing different lengths of the 5′ upstream sequences. Transient transfection assays of these reporter constructs in both hPhox2a-positive and -negative cell lines show that 1.3-kb or longer upstream sequences of the hPhox2a gene may confer NA cell-specific reporter gene expression. Furthermore, cotransfection assays in the Phox2a-negative HeLa cell line show that forced expression of Phox2b, but not that of Phox2a or MASH1, significantly transactivates the transcriptional activity of hPhox2a. This study will provide a frame to further delineate the regulatory cascade of NA neuron differentiation. [ABSTRACT FROM AUTHOR]

Abstract Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of catecholamine synthesis and its expression is necessary for neurotransmitter specification of all catecholaminergic neurons, while dopamine β-hydroxylase (DBH) is essential for the noradrenergic phenotype. In the present study, we show that Nurr1, an orphan nuclear receptor critical for dopaminergic (DA) neuron development, directly transactivates the promoter activity of the TH gene in a cell type-dependent manner, while it does not regulate the DBH promoter. Consistent with these results, only the TH promoter contains multiple sequence motifs homologous to the known Nurr1-binding motif, NBRE. TH promoter deletional analysis indicates that < 1.0 kb upstream sequences, encompassing three NBRE-like motifs (i.e. NL1, NL2 and NL3) are mostly responsible for the effects of Nurr1. Among these potential motifs, site-directed mutational analysis showed that NL1, residing from - 35 to - 28 bp, was most critical for mediating the transactivation by Nurr1. Strikingly, however, both DNase I footprinting and electrophoretic mobility shift assays showed that NL3, but not NL1 or NL2, has high binding affinity to Nurr1. To determine whether the proximity of these motifs may be important for transactivation by Nurr1 in the transient transfection assay, we generated reporter gene constructs in which NL3 is immediately proximal to the TATA box. Indeed, NL3 was more efficient in this position than NL1 or NL2 for mediating the transactivation by Nurr1. Our results suggest that Nurr1 may play a direct role for specification of DA neurotransmitter identity by activating TH gene transcription in a cell context-dependent manner. [ABSTRACT FROM AUTHOR]

Cerebral clearance of amyloid β-protein (Aβ) is decreased in early-onset and late-onset Alzheimer's disease (AD). Aβ is cleared from the brain by enzymatic degradation and by transport out of the brain. More than 20 Aβ-degrading enzymes have been described. Increasing the degradation of Aβ offers an opportunity to decrease brain Aβ levels in AD patients. This review discusses the direct and indirect approaches which have been used in experimental systems to alter the expression and/or activity of Aβ-degrading enzymes. Also discussed are the enzymes' regulatory mechanisms, the conformations of Aβ they degrade, where in the scheme of Aβ production, extracellular release, cellular uptake, and intracellular degradation they exert their activities, and changes in their expression and/or activity in AD and its animal models. Most of the experimental approaches require further confirmation. Based upon each enzyme's effects on Aβ (some of the enzymes also possess β-secretase activity and may therefore promote Aβ production), its direction of change in AD and/or its animal models, and the Aβ conformation(s) it degrades, investigating the effects of increasing the expression of neprilysin in AD patients would be of particular interest. Increasing the expression of insulin-degrading enzyme, endothelin-converting enzyme-1, endothelin-converting enzyme-2, tissue plasminogen activator, angiotensin-converting enzyme, and presequence peptidase would also be of interest. Increasing matrix metalloproteinase-2, matrix metalloproteinase-9, cathepsin-B, and cathepsin-D expression would be problematic because of possible damage by the metalloproteinases to the blood brain barrier and the cathepsins' β-secretase activity. Many interventions which increase the enzymatic degradation of Aβ have been shown to decrease AD-type pathology in experimental models. If a safe approach can be found to increase the expression or activity of selected Aβ-degrading enzymes in human subjects, then the possibility that this approach could slow the AD progression should be examined in clinical trials., (© 2023 International Society for Neurochemistry.)

The neurovascular unit, composed of vascular endothelium, vascular smooth muscle, extracellular matrix components, pericytes, astrocytes, microglia, and neurons, allows the highly regulated exchange of molecules and the limited trafficking of cells to the brain through coordinated signaling activity. The passage of peripheral immune cells to the brain parenchyma is observed when there is clear damage to the barriers of this neurovascular unit, as occurs in traumatic brain injury. The possibility of leukocyte infiltration to the brain in neurodegenerative conditions has been proposed. In this review, we focus on describing the evidence for peripheral immune cell infiltration to the brain in the two most frequent neurodegenerative diseases: Alzheimer's and Parkinson's diseases. In particular, we address the mechanisms that promote the passage of these cells into the brain under such pathological conditions. We also discuss the relevance of the resulting cellular interactions, which provide evidence that the presence of peripheral immune cells in the brain is a key point in these neurodegenerative diseases., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

The blood-brain barrier (BBB) is a structure mainly formed by brain capillary endothelial cells (BCEC) whose role is to regulate the exchange of compounds between the blood and the brain. In this process efflux and uptake transporters play a key role. Aim of this study was to compare the two previously established cell lines hCMEC/D3 and hBMEC as BBB cell models for the application of an adenoviral system to transiently express OATP2B1 and Pgp. Comparison of hCMEC/D3 and hBMEC mRNA and protein levels of BBB markers showed a unique expression pattern for each cell line. While showing similar expression of the efflux transporter BCRP, transferrin receptor (TFRC) and of the tight junctions proteins Occludin and ZO-1, hCMEC/D3 displayed higher levels of the endothelial marker PECAM1, VE-cadherin, Von Willebrand Factor (VWF) and of the efflux transporter Pgp. Moreover, measuring integrity of the monolayer by determining the Trans-Endothelial Electrical Resistance (TEER), electrical capacitance (C Cl ), and inulin apparent permeability coefficient (P app ) revealed higher TEER and lower C Cl for hBMEC but comparable P app in the two cell lines. Following adenoviral infection, enhanced OATP2B1 and Pgp expression and functionality could be observed only in hBMEC. Importantly, the adenoviral expression system did not affect expression of BBB markers and permeability in both cell lines. Taken together, our results provide first evidence that hBMEC is an applicable human BBB cell model in which adenoviral infection can be used to transiently express and investigate transporters of interest., (© 2024 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Vitamin D has been identified as a key factor in dopaminergic neurogenesis and differentiation. Consequently, developmental vitamin D (DVD) deficiency has been linked to disorders of abnormal dopamine signalling with a neurodevelopmental basis such as schizophrenia. Here we provide further evidence of vitamin D's role as a mediator of dopaminergic development by showing that it increases neurite outgrowth, neurite branching, presynaptic protein re‐distribution, dopamine production and functional release in various in vitro models of developing dopaminergic cells including SH‐SY5Y cells, primary mesencephalic cultures and mesencephalic/striatal explant co‐cultures. This study continues to establish vitamin D as an important differentiation agent for developing dopamine neurons, and now for the first time shows chronic exposure to the active vitamin D hormone increases the capacity of developing neurons to release dopamine. This study also has implications for understanding mechanisms behind the link between DVD deficiency and schizophrenia. [ABSTRACT FROM AUTHOR]

The discovery of leptin in 1994 was an "eureka moment" in the field of neurometabolism that provided new opportunities to better understand the central control of energy balance and glucose metabolism. Rapidly, a prevalent model in the field emerged that pro‐opiomelanocortin (POMC) neurons were key in promoting leptin's anorexigenic effects and that the arcuate nucleus of the hypothalamus (ARC) was a key region for the regulation of energy homeostasis. While this model inspired many important discoveries, a growing body of literature indicates that this model is now outdated. In this review, we re‐evaluate the hypothalamic leptin–melanocortin model in light of recent advances that directly tackle previous assumptions, with a particular focus on the ARC. We discuss how segregated and heterogeneous these neurons are, and examine how the development of modern approaches allowing spatiotemporal, intersectional, and chemogenetic manipulations of melanocortin neurons has allowed a better definition of the complexity of the leptin–melanocortin system. We review the importance of leptin in regulating glucose homeostasis, but not food intake, through direct actions on ARC POMC neurons. We further highlight how non‐POMC, GABAergic neurons mediate leptin's direct effects on energy balance and influence POMC neurons. [ABSTRACT FROM AUTHOR]

Dopamine beta-hydroxylase (DβH) is an essential neurotransmitter-synthesizing enzyme that catalyzes the formation of norepinephrine (NE) from dopamine and has been extensively studied since its discovery in the 1950s. NE serves as a neurotransmitter in both the central and peripheral nervous systems and is the precursor to epinephrine synthesis in the brain and adrenal medulla. Alterations in noradrenergic signaling have been linked to both central nervous system and peripheral pathologies. DβH protein, which is found in circulation, can, therefore, be evaluated as a marker of norepinephrine function in a plethora of different disorders and diseases. In many of these diseases, DβH protein availability and activity are believed to contribute to disease presentation or select symptomology and are believed to be under strong genetic control. Alteration in the DβH protein by genetic polymorphisms may result in DβH becoming rate-limiting and directly contributing to lower NE and epinephrine levels and disease. With the completion of the human genome project and the advent of next-generation sequencing, new insights have been gained into the existence of naturally occurring DβH sequencing variants (genetic polymorphisms) in disease. Also, biophysical tools coupled with genetic sequences are illuminating structure-function relationships within the enzyme. In this review, we discuss the role of genetic variants in DβH and its role in health and disease., (© 2019 International Society for Neurochemistry.)

Parkinson's disease (PD) is a heterogeneous multi‐systemic disorder unique to humans characterized by motor and non‐motor symptoms. Preclinical experimental models of PD present limitations and inconsistent neurochemical, histological, and behavioral readouts. The 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD is the most common in vivo screening platform for novel drug therapies; nonetheless, behavioral endpoints yielded amongst laboratories are often discordant and inconclusive. In this study, we characterized neurochemically, histologically, and behaviorally three different MPTP mouse models of PD to identify translational traits reminiscent of PD symptomatology. MPTP was intraperitoneally (i.p.) administered in three different regimens: (i) acute—four injections of 20 mg/kg of MPTP every 2 h; (ii) sub‐acute—one daily injection of 30 mg/kg of MPTP for 5 consecutive days; and (iii) chronic—one daily injection of 4 mg/kg of MPTP for 28 consecutive days. A series of behavioral tests were conducted to assess motor and non‐motor behavioral changes including anxiety, endurance, gait, motor deficits, cognitive impairment, circadian rhythm and food consumption. Impairments in balance and gait were confirmed in the chronic and acute models, respectively, with the latter showing significant correlation with lesion size. The sub‐acute model, by contrast, presented with generalized hyperactivity. Both, motor and non‐motor changes were identified in the acute and sub‐acute regime where habituation to a novel environment was significantly reduced. Moreover, we report increased water and food intake across all three models. Overall, the acute model displayed the most severe lesion size, while across the three models striatal dopamine content (DA) did not correlate with the behavioral performance. The present study demonstrates that detection of behavioral changes following MPTP exposure is challenging and does not correlate with the dopaminergic lesion extent. [ABSTRACT FROM AUTHOR]

Treating central nervous system (CNS) diseases is complicated by the incapability of numerous therapeutics to cross the blood–brain barrier (BBB), mainly composed of brain endothelial cells (BECs). Genetically modifying BECs into protein factories that supply the CNS with recombinant proteins is a promising approach to overcome this hindrance, especially in genetic diseases, like Niemann Pick disease type C2 (NPC2), where both CNS and peripheral cells are affected. Here, we investigated the potential of the BEC‐specific adeno‐associated viral vector (AAV‐BR1) encoding NPC2 for expression and secretion from primary BECs cultured in an in vitro BBB model with mixed glial cells, and in healthy BALB/c mice. Transduced primary BECs had significantly increased NPC2 gene expression and secreted NPC2 after viral transduction, which significantly reversed cholesterol deposition in NPC2 deficient fibroblasts. Mice receiving an intravenous injection with AAV‐BR1‐NCP2‐eGFP were sacrificed 8 weeks later and examined for its biodistribution and transgene expression of eGFP and NPC2. AAV‐BR1‐NPC2‐eGFP was distributed mainly to the brain and lightly to the heart and lung, but did not label other organs including the liver. eGFP expression was primarily found in BECs throughout the brain but occasionally also in neurons suggesting transport of the vector across the BBB, a phenomenon also confirmed in vitro. NPC2 gene expression was up‐regulated in the brain, and recombinant NPC2 protein expression was observed in both transduced brain capillaries and neurons. Our findings show that AAV‐BR1 transduction of BECs is possible and that it may denote a promising strategy for future treatment of NPC2. [ABSTRACT FROM AUTHOR]

No robust biomarkers have yet been identified for autism spectrum disorder (ASD) or autistic traits. Familial factors likely influence biomarkers such as protein concentrations. Comparing twins with ASD or high autistic traits to the less affected co‐twin allows estimating the impact of familial confounding. We measured 203 proteins in cerebrospinal fluid (n = 86) and serum (n = 127) in twins (mean age 14.2 years, 44.9% females) enriched for ASD and other neurodevelopmental conditions. Autistic traits were assessed by using the parent‐report version of the Social Responsiveness Scale‐2. In cerebrospinal fluid, autistic traits correlated negatively with three proteins and positively with one. In serum, autistic traits correlated positively with 15 and negatively with one. Also in serum, six were positively—and one negatively—associated with ASD. A pathway analysis of these proteins revealed immune system enrichment. In within twin pair analyses, autistic traits were associated with serum B‐cell activating factor (BAFF) only, whereas Cystatin B (CSTB) remained significantly associated with ASD. These associations did not remain significant when only considering monozygotic twins. For the remainder, the within‐pair analysis indicated familial confounding, including shared environment and genes, influencing both autism and protein levels. Our findings indicate proteins involved in immunity as putative biomarkers of autistic traits and ASD with partial genetic confounding. Although some results are in line with previous studies in general, further studies are needed for replication. [ABSTRACT FROM AUTHOR]

Although cyclooxygenase (COX) role in cancer angiogenesis has been studied, little is known about its role in brain angioplasticity. In the present study, we chronically infused mice with ketorolac, a non-specific COX inhibitor that does not cross the blood-brain barrier (BBB), under normoxia or 50% isobaric hypoxia (10% O2 by volume). Ketorolac increased mortality rate under hypoxia in a dose-dependent manner. Using in vivo multiphoton microscopy, we demonstrated that chronic COX inhibition completely attenuated brain angiogenic response to hypoxia. Alterations in a number of angiogenic factors that were reported to be COX-dependent in other models were assayed at 24-hr and 10-day hypoxia. Intriguingly, hypoxia-inducible factor 1 was unaffected under COX inhibition, and vascular endothelial growth factor receptor type 2 (VEGFR2) and C-X-C chemokine receptor type 4 (CXCR4) were significantly but slightly decreased. However, a number of mitogen-activated protein kinases (MAPKs) were significantly reduced upon COX inhibition. We conclude that additional, angiogenic factor-independent mechanism might contribute to COX role in brain angioplasticity, probably including mitogenic COX effect on endothelium. Our data indicate that COX activity is critical for systemic adaptation to chronic hypoxia, and BBB COX is essential for hypoxia-induced brain angioplasticity. These data also indicate a potential risk for using COX inhibitors under hypoxia conditions in clinics. Further studies are required to elucidate a complete mechanism for brain long-term angiogenesis regulation through COX activity. [ABSTRACT FROM AUTHOR]

To evaluate the neuroprotection exerted by ketosis against acute damage of the mammalian central nervous system (CNS). Search engines were interrogated to identify experimental studies comparing the mitigating effect of ketosis (intervention) versus non-ketosis (control) on acute CNS damage. Primary endpoint was a reduction in mortality. Secondary endpoints were a reduction in neuronal damage and dysfunction, and an 'aggregated advantage' (composite of all primary and secondary endpoints). Hedges' g was the effect measure. Subgroup analyses evaluated the modulatory effect of age, insult type, and injury site. Meta-regression evaluated timing, type, and magnitude of intervention as predictors of neuroprotection. The selected publications were 49 experimental murine studies (period 1979-2020). The intervention reduced mortality (g 2.45, SE 0.48, p < .01), neuronal damage (g 1.96, SE 0.23, p < .01) and dysfunction (g 0.99, SE 0.10, p < .01). Reduction of mortality was particularly pronounced in the adult subgroup (g 2.71, SE 0.57, p < .01). The aggregated advantage of ketosis was stronger in the pediatric (g 3.98, SE 0.71, p < .01), brain (g 1.96, SE 0.18, p < .01), and ischemic insult (g 2.20, SE 0.23, p < .01) subgroups. Only the magnitude of intervention was a predictor of neuroprotection (g 0.07, SE 0.03, p 0.01 per every mmol/L increase in ketone levels). Ketosis exerts a potent neuroprotection against acute damage to the mammalian CNS in terms of reduction of mortality, of neuronal damage and dysfunction. Hematic levels of ketones are directly proportional to the effect size of neuroprotection. [ABSTRACT FROM AUTHOR]

Preclinical studies have shown that mesenchymal stem cells have a positive effect in perinatal brain injury models. The mechanisms that cause these neurotherapeutic effects are not entirely intelligible. Mitochondrial damage, inflammation, and reactive oxygen species are considered to be critically involved in the development of injury. Mesenchymal stem cells have immunomodulatory action and exert mitoprotective effects which attenuate production of reactive oxygen species and promote restoration of tissue function and metabolism after perinatal insults. This review summarizes the present state, the underlying causes, challenges and possibilities for effective clinical translation of mesenchymal stem cell therapy. [ABSTRACT FROM AUTHOR]

Several lines of evidence indicate that the propagation of misfolded α‐synuclein (α‐syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α‐syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α‐syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α‐syn degrading enzymes and has been implicated in the clearance of extracellular α‐syn. However, the physiological role of this enzyme in regulating α‐syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock‐out (Klk6−/−) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α‐syn processing. Behavioral phenotyping showed that Klk6−/− mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α‐syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α‐syn. Upon in vivo administration of α‐syn pre‐formed fibrils (PFF), α‐syn pathologic accumulations were evident both in the brains of Klk6−/−mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α‐syn levels observed in the A53T α‐syn over‐expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α‐syn fibrils in α‐syn turnover studies. [ABSTRACT FROM AUTHOR]

Glucose hypometabolism is observed in epilepsy and promotes epileptogenesis. Glucose hypometabolism in epilepsy may be attributed to decreased neuronal glucose uptake, but its molecular mechanism remains unclear. Zinc‐α2‐glycoprotein (ZAG) is related to glucose metabolism and is reported to suppress seizures. The anti‐epileptic effect of ZAG may be attributed to its regulation of neuronal glucose metabolism. This study explored the effect of ZAG on neuronal glucose uptake and its molecular mechanism via insulin‐like growth factor 1 receptor (IGF1R)‐regulated glucose transporter 3 (GLUT‐3) expression. The ZAG level was modulated by lentivirus in primary culture neurons. Neuronal seizure models were induced by Mg2+‐free artificial cerebrospinal fluid. We assessed neuronal glucose uptake by the 2‐NBDG method and Glucose Uptake Colorimetric Assay Kit. IGF1R was activated by IGF1 and blocked by AXL1717. The expression and distribution of IGF1R and GLUT‐3, together with IGF1R phosphorylation, were measured by western blot. The binding between ZAG and IGF1R was determined by coimmunoprecipitation. Neuronal glucose uptake and GLUT‐3 expression were significantly decreased by seizure or ZAG knockdown, whereas ZAG over‐expression or IGF1 treatment reversed this decrease. The effect of ZAG on neuronal glucose uptake and GLUT‐3 expression was blocked by AXL1717. ZAG increased IGF1R distribution and phosphorylation possibly by binding. Additionally, IGF1R increased GLUT‐3 activity by increasing GLUT‐3 expression. In epilepsy/seizure, neuronal glucose uptake suppression may be attributed to a decrease in ZAG, which suppresses neuronal GLUT‐3 expression by regulating the activity of IGF1R. ZAG, IGF1R, and GLUT‐3 may be novel potential therapeutic targets of glucose hypometabolism in epilepsy and seizures. [ABSTRACT FROM AUTHOR]

Glioblastomas (GBMs) are highly aggressive primary brain tumors characterized by cellular heterogeneity, insensitivity to chemotherapy and poor patient survival. Lysophosphatidic acid (LPA) is a lysophospholipid that acts as a bioactive signaling molecule and plays important roles in diverse biological events during development and disease, including several cancer types. Microglial cells, the resident macrophages of the central nervous system, express high levels of Autotaxin (ATX,Enpp2), an enzyme that synthetizes LPA. Our study aimed to investigate the role of LPA on tumor growth and invasion in the context of microglia‐GBM interaction. First, through bioinformatics studies, patient data analysis demonstrated that more aggressive GBM expressed higher levels of ENPP2, which was also associated with worse patient prognosis with proneural GBM. Using GBM‐microglia co‐culture system we then demonstrated that GBM secreted factors were able to increase LPA1 and ATX in microglia, which could be further enhanced by hypoxia. On the other hand, interaction with microglial cells also increased ATX expression in GBM. Furthermore, microglial‐induced GBM proliferation and migration could be inhibited by pharmacological inhibition of LPA1, suggesting that microglial‐derived LPA could support tumor growth and invasion. Finally, increased LPA1 expression was observed in GBM comparing with other gliomas and could be also associated with worse patient survival. These results show for the first time a microglia‐GBM interaction through the LPA pathway with relevant implications for tumor progression. A better understanding of this interaction can lead to the development of new therapeutic strategies setting LPA as a potential target for GBM treatment. [ABSTRACT FROM AUTHOR]

Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood–brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease Niemann Pick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non‐mitotic monocultures with a biological effect on NPC2‐deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa‐cells, known to persist in a mitotic state. When co‐cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non‐mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non‐viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects. Cover Image for this issue: https://doi.org/10.1111/jnc.15050. [ABSTRACT FROM AUTHOR]

Over the last two decades, a number of studies have underlined the importance of lysosomal‐based degradative pathways in maintaining the homeostasis of post‐mitotic cells, and revealed the remarkable contribution of a functional autophagic machinery in the promotion of longevity. In contrast, defects in the clearance of organelles and aberrant protein aggregates have been linked to accelerated neuronal loss and neurological dysfunction. Several neurodegenerative disorders, among which Alzheimer disease (AD), Frontotemporal dementia, and Amyotrophic Lateral Sclerosis to name a few, are associated with alterations of the autophagy and endo‐lysosomal pathways. In Parkinson disease (PD), the most prevalent genetic determinant, Leucine‐rich repeat kinase 2 (LRRK2), is believed to be involved in the regulation of intracellular vesicle traffic, autophagy and lysosomal function. Here, we review the current understanding of the mechanisms by which LRRK2 regulates lysosomal‐based degradative pathways in neuronal and non‐neuronal cells and discuss the impact of pathogenic PD mutations in contributing to lysosomal dyshomeostasis. [ABSTRACT FROM AUTHOR]

Our understanding of the biological basis of Parkinson's disease (PD) has been greatly improved in recent years by the identification of mutations that lead to inherited PD. One of the strengths of using genetics to try to understand disease biology is that it is inherently unbiased and can be applied at a genome‐wide scale. More recently, many studies have used another set of unbiased approaches, proteomics, to query the function of familial PD genes in a variety of contexts. We will discuss some specific examples, including; elucidation of protein‐protein interaction networks for two dominantly inherited genes, α‐synuclein and leucine rich‐repeat kinase 2 (LRRK2); the identification of substrates for three genes for familial PD that are also enzymes, namely LRRK2, pink1, and parkin; and changes in protein abundance that arise downstream to introduction of mutations associated with familial PD. We will also discuss those situations where we can integrate multiple proteomics approaches to nominate deeper networks of inter‐related events that outline pathways relevant to inherited PD. This article is part of the Special Issue "Proteomics". [ABSTRACT FROM AUTHOR]

The levels and conformers of alpha‐synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha‐synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha‐synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha‐synuclein species, in a manner that depends on alpha‐synuclein post‐translational modifications. Extracellular alpha‐synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha‐synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. This article is part of the Special Issue "Synuclein". [ABSTRACT FROM AUTHOR]

Abstract: Parkinson's disease can be caused by mutations in the α‐synuclein gene and is characterized by aggregates of α‐synuclein protein. We have previously shown that over‐expression of the small GTPase Rab7 can induce clearance of α‐synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson's disease, we expressed the pathogenic A53T mutant of α‐synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled‐coil domain‐containing protein 1 (FYCO1) and Rab‐interacting lysosomal protein (RILP). FYCO1‐EGFP‐decorated vesicles containing α‐synuclein. RILP‐EGFP also decorated vesicular structures, but they did not contain α‐synuclein. FYCO1 over‐expression reduced the number of cells with α‐synuclein aggregates, defined as visible particles of EGFP‐tagged α‐synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α‐synuclein protein as assayed by western blot, increased the disappearance of α‐synuclein aggregates in time‐lapse microscopy and decreased α‐synuclein‐induced toxicity assayed by the Trypan blue assay. siRNA‐mediated knockdown of FYCO1 but not RILP reduced Rab7‐induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7‐induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α‐synuclein aggregates. Extracellular α‐synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α‐synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T‐α‐synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α‐synuclein and could be beneficial in patients with Parkinson's disease. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/ [ABSTRACT FROM AUTHOR]

Abstract: Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature, post‐mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti‐depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin‐2 (NEUROG2) to generate a homogenous population of post‐mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline, the vast majority of cells are post‐mitotic, and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f, we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2‐responsive genes were determined by RNA‐Seq. FGF2‐regulated genes previously identified in non‐neuronal cell types were up‐regulated (EGR1, ETV4, SPRY4, and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron‐specific genes were also identified that may mediate FGF2‐dependent increases in synaptic efficacy including NRXN3, SYT2, and GALR1. Since several of these genes have been implicated in MDD previously, these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. [ABSTRACT FROM AUTHOR]

Abstract: Septic encephalopathy with confusion and agitation occurs early during sepsis and contributes to the severity of the disease. A decrease in the sphingosine‐1‐phosphate (S1P) blood levels has been shown in patients and in animal models of sepsis. The lipid mediator S1P is known to be involved in endothelial barrier function in a context‐dependent manner. We utilized lipopolysaccharide (LPS)‐injected mice as a model for septic encephalopathy and first performed tracer permeability assays to assess the blood–brain barrier (BBB) breakdown in vivo. At time points corresponding to the BBB breakdown post LPS injection, we aimed to characterize the regulation of the sphingolipid signaling pathway at the BBB during sepsis. We measured sphingolipid concentrations in blood, in mouse brain microvessels (MBMVs), and brain tissue. We also analyzed the expression of S1P receptors, transporters, and metabolizing enzymes in MBMVs and brain tissue. Primary mouse brain microvascular endothelial cells (MBMECs) were isolated to evaluate the effects of LPS on transendothelial electrical resistance (TEER) as a measure of permeability in vitro. We observed a relevant decrease in S1P levels after LPS injection in all three compartments (blood, MBMVs, brain tissue) that was accompanied by an increased expression of the S1P receptor type 1 and of sphingosine kinase 1 on one hand and of the S1P degrading enzymes lipid phosphate phosphatase 1 (LPP1) and S1P phosphatase 1 on the other hand, as well as a down‐regulation of sphingosine kinase 2. Application of LPS to a monolayer of primary MBMECs did not alter TEER, but serum from LPS‐treated mice lead to a breakdown of the barrier compared to serum from vehicle‐treated mice. We observed profound alterations of the sphingolipid metabolism at the BBB after LPS injection that point toward a therapeutic potential of drugs interfering with this pathway as novel approach for the detrimental overwhelming immune response in sepsis. Read the Editorial Highlight for this article on page 115. Cover Image for this Issue: doi. 10.1111/jnc.14161. [ABSTRACT FROM AUTHOR]

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine ( O-Glc NAc) regulates protein homeostasis. The O-Glc NAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-Glc NAc transferase- and O-Glc NAcase-knockout alleles have helped define the role O-Glc NAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-Glc NAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-Glc NAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-Glc NAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an ' O-Glc NAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-Glc NAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-Glc NAc in the physiology of the brain and its susceptibility to neurodegenerative injury. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus ( rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia ( DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 ( hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. . [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-β in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD. [ABSTRACT FROM AUTHOR]

Developing therapies for central nervous system ( CNS) diseases is exceedingly difficult because of the blood-brain barrier ( BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 ( AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. [ABSTRACT FROM AUTHOR]

Cocaine addiction is a chronic relapsing disorder characterized by the loss of control over drug-seeking and taking, and continued drug use regardless of adverse consequences. Despite years of research, effective treatments for psycho-stimulant addiction have not been identified. Persistent vulnerability to relapse arises from a number of long-lasting adaptations in the reward circuitry that mediate the enduring response to the drug. Recently, we reported that the activity of the canonical or Wnt/β-catenin pathway in the prefrontal cortex (PFC) is very important in the early stages of cocaine-induced neuroadaptations. In the present work, our main goal was to elucidate the relevance of this pathway in cocaine-induced long-term neuroadaptations that may underlie relapse. We found that a cocaine challenge, after a period of abstinence, induced an increase in the activity of the pathway which is revealed as an increase in the total and nuclear levels of β-catenin (final effector of the pathway) in the nucleus accumbens ( NAcc), together with a decrease in the activity of glycogen synthase kinase 3β ( GSK3β). Moreover, we found that the pharmacological modulation of the activity of the pathway has long-term effects on the cocaine-induced neuroplasticity at behavioral and molecular levels. All the results imply that changes in the Wnt/β-catenin pathway effectors are long-term neuroadaptations necessary for the behavioral response to cocaine. Even though more research is needed, the present results introduce the Wnt canonical pathway as a possible target to manage cocaine long-term neuroadaptations. [ABSTRACT FROM AUTHOR]

Glioblastomas (GBMs) are highly aggressive primary brain tumors characterized by cellular heterogeneity, insensitivity to chemotherapy and poor patient survival. Lysophosphatidic acid (LPA) is a lysophospholipid that acts as a bioactive signaling molecule and plays important roles in diverse biological events during development and disease, including several cancer types. Microglial cells, the resident macrophages of the central nervous system, express high levels of Autotaxin (ATX,Enpp2), an enzyme that synthetizes LPA. Our study aimed to investigate the role of LPA on tumor growth and invasion in the context of microglia-GBM interaction. First, through bioinformatics studies, patient data analysis demonstrated that more aggressive GBM expressed higher levels of ENPP2, which was also associated with worse patient prognosis with proneural GBM. Using GBM-microglia co-culture system we then demonstrated that GBM secreted factors were able to increase LPA 1 and ATX in microglia, which could be further enhanced by hypoxia. On the other hand, interaction with microglial cells also increased ATX expression in GBM. Furthermore, microglial-induced GBM proliferation and migration could be inhibited by pharmacological inhibition of LPA 1 , suggesting that microglial-derived LPA could support tumor growth and invasion. Finally, increased LPA 1 expression was observed in GBM comparing with other gliomas and could be also associated with worse patient survival. These results show for the first time a microglia-GBM interaction through the LPA pathway with relevant implications for tumor progression. A better understanding of this interaction can lead to the development of new therapeutic strategies setting LPA as a potential target for GBM treatment., (© 2020 International Society for Neurochemistry.)

Aside from its roles in as a classical neurotransmitter involved in regulation of behavior, noradrenaline ( NA) has other functions in the CNS. This includes restricting the development of neuroinflammatory activation, providing neurotrophic support to neurons, and providing neuroprotection against oxidative stress. In recent years, it has become evident that disruption of physiological NA levels or signaling is a contributing factor to a variety of neurological diseases and conditions including Alzheimer's disease (AD) and Multiple Sclerosis. The basis for dysregulation in these diseases is, in many cases, due to damage occurring to noradrenergic neurons present in the locus coeruleus ( LC), the major source of NA in the CNS. LC damage is present in AD, multiple sclerosis, and a large number of other diseases and conditions. Studies using animal models have shown that experimentally induced lesion of LC neurons exacerbates neuropathology while treatments to compensate for NA depletion, or to reduce LC neuronal damage, provide benefit. In this review, we will summarize the anti-inflammatory and neuroprotective actions of NA, summarize examples of how LC damage worsens disease, and discuss several approaches taken to treat or prevent reductions in NA levels and LC neuronal damage. Further understanding of these events will be of value for the development of treatments for AD, multiple sclerosis, and other diseases and conditions having a neuroinflammatory component. [ABSTRACT FROM AUTHOR]

Substantial progress has been made in the genetic basis of Parkinson's disease ( PD). In particular, by identifying genes that segregate with inherited PD or show robust association with sporadic disease, and by showing the same genes are found on both lists, we have generated an outline of the cause of this condition. Here, we will discuss what those genes tell us about the underlying biology of PD. We specifically discuss the relationships between protein products of PD genes and show that common links include regulation of the autophagy-lysosome system, an important way by which cells recycle proteins and organelles. We also discuss whether all PD genes should be considered to be in the same pathway and propose that in some cases the relationships are closer, whereas in other cases the interactions are more distant and might be considered separate. [ABSTRACT FROM AUTHOR]

Parkinson's disease is a progressive neurological disorder that is characterized by the formation of intracellular protein inclusion bodies composed primarily of a misfolded and aggregated form of the protein α-synuclein. There is growing evidence that supports the prion-like hypothesis of α-synuclein progression. This hypothesis postulates that α-synuclein is a prion-like pathological agent and is responsible for the progression of Parkinson pathology in the brain. Potential misfolding or aggregation of α-synuclein that might occur in the peripheral nervous system as a result of some insult, environmental or genetic (or more likely a combination of both) that might spread into the midbrain, eventually causing degeneration of the neurons in the substantia nigra. As the disease progresses further, it is likely that α-synuclein pathology continues to spread throughout the brain, including the cortex, leading to deterioration of cognition and higher brain functions. While it is unknown why α-synuclein initially misfolds and aggregates, a great deal has been learned about how the cell handles aberrant α-synuclein assemblies. In this review, we focus on these mechanisms and discuss them in an attempt to define the role that they might play in the propagation of misfolded α-synuclein from cell-to-cell. [ABSTRACT FROM AUTHOR]

Statins exhibit neuroprotective effects after spinal cord injury ( SCI). However, the molecular mechanism underlying these effects remains unknown. This study demonstrates that the hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin (Simv) exhibits neuroprotective effects on neuronal apoptosis and supports functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. In specific, Simv administration after SCI significantly up-regulated the expression of low density lipoprotein receptor-related protein 6 phosphorylation and β-catenin protein, increased the mRNA expression of lymphoid enhancer factor-1 and T-cell factor-1, and suppressed the expression of β-catenin phosphorylation in the spinal cord neurons. Simv enhanced motor neuronal survival in the spinal cord anterior horn and decreased the lesion of spinal cord tissues after SCI. Simv administration after SCI also evidently reduced the expression levels of Bax, active caspase-3, and active caspase-9 in the spinal cord neurons and the proportion of transferase UTP nick end labeling (TUNEL)-positive neuron cells, but increased the expression level of Bcl-2 in the spinal cord neurons. However, the anti-apoptotic effects of Simv were reduced in cultured spinal cord nerve cells when the Wnt/β-catenin signaling pathway was suppressed in the lipopolysaccharide-induced model. Furthermore, the Basso, Beattie, and Bresnahan scores indicated that Simv treatment significantly improved the locomotor functions of rats after SCI. This study is the first to report that Simv exerts neuroprotective effects by reducing neuronal apoptosis, and promoting functional and pathological recovery after SCI by activating the Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

Corticotropin releasing factor ( CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter ( NET) in vitro. SK-N- BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [3H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. [ABSTRACT FROM AUTHOR]

Cerebral infarction causes permanent neuronal loss inducing severe morbidity and mortality. Because hypertension is the main risk factor for cerebral infarction and most patients with hypertension take antihypertensive drugs daily, the neuroprotective effects and mechanisms of anti-hypertensive drugs need to be investigated. Cilnidipine, a long-acting, new generation 1,4-dihydropyridine inhibitor of both L- and N-type calcium channels, was reported to reduce oxidative stress. In this study, we investigated whether cilnidipine has therapeutic effects in an animal model of cerebral infarction. After determination of the most effective dose of cilnidipine, a total of 128 rats were subjected to middle cerebral artery occlusion. Neurobehavioral function test and brain MRI were performed, and rats with similar sized infarcts were randomized to either the cilnidipine group or the control group. Cilnidipine treatment was performed with reperfusion after 2-h occlusion. Western blots and immunohistochemistry were also performed after 24-h occlusion. Initial infarct volume on diffusion-weighted MRI was not different between the cilnidipine group and the control group; however, fluid-attenuated inversion recovery MRI at 24 h showed significantly reduced infarct volume in the cilnidipine group compared with the control group. Cilnidipine treatment significantly decreased the number of triphosphate nick end labeling-positive cells compared to the control group. Western blot and immunohistochemistry showed increased expression of phosphorylated Akt (Ser473), phosphorylated glycogen synthase kinase-3β, and Bcl-2 and decreased expression of Bax and cleaved caspase-3. These results suggest that cilnidipine, which is used for the treatment of hypertension, has neuroprotective effects in the ischemic brain through activation of the PI3K pathway. [ABSTRACT FROM AUTHOR]

Manganese (Mn) is an essential heavy metal that is naturally found in the environment. Daily intake through dietary sources provides the necessary amount required for several key physiological processes, including antioxidant defense, energy metabolism, immune function and others. However, overexposure from environmental sources can result in a condition known as manganism that features symptomatology similar to Parkinson's disease (PD). This disorder presents with debilitating motor and cognitive deficits that arise from a neurodegenerative process. In order to maintain a balance between its essentiality and neurotoxicity, several mechanisms exist to properly buffer cellular Mn levels. These include transporters involved in Mn uptake, and newly discovered Mn efflux mechanisms. This review will focus on current studies related to mechanisms underlying Mn import and export, primarily the Mn transporters, and their function and roles in Mn-induced neurotoxicity. [ABSTRACT FROM AUTHOR]

Exposure to atmospheric particulate matter PM2.5 (aerodynamic diameter < 2.5 lm) has been epidemiologically associated with respiratory illnesses. However, recent data have suggested that PM2.5 is able to infiltrate into circulation and elicit a systemic inflammatory response. Potential adverse effects of air pollutants to the central nervous system (CNS) have raised concerns, but whether PM2.5 causes neurotoxicity remains unclear. In this study, we have demonstrated that PM2.5 impairs the tight junction of endothelial cells and increases permeability and monocyte transmigration across endothelial monolayer in vitro, indicating thatPM2.5 is able to disrupt blood-brain barrier integrity and gain access to the CNS. Exposure of primary neuronal cultures to PM2.5 resulted in decrease in cell viability and loss of neuronal antigens. Furthermore, supernatants collected from PM2.5-treated macrophages and microglia were also neurotoxic. These macrophages and microglia significantly increased extracellular levels of glutamate following PM2.5 exposure, which were negatively correlated with neuronal viability. Pre-treatment with NMDA receptor antagonist MK801 alleviated neuron loss, suggesting that PM2.5 neurotoxicity is mediated by glutamate. To determine the potential source of excess glutamate production, we investigated glutaminase, the main enzyme for glutamate generation. Glutaminase was reduced in PM2.5-treated macrophages and increased in extracellular vesicles, suggesting that PM2.5 induces glutaminase release through extracellular vesicles. In conclusion, these findings indicatePM2.5 as a potential neurotoxic factor, crucial to understanding the effects of air pollution on the CNS. [ABSTRACT FROM AUTHOR]

Drug delivery to the brain for the treatment of pathologies with a CNS component is a significant clinical challenge. Pglycoprotein (PgP), a drug efflux pump in the endothelial cell membrane, is a major factor in preventing therapeutics from crossing the blood-brain barrier (BBB). Identifying PgP regulatory mechanisms is key to developing agents to modulate PgP activity. Previously, we found that PgP trafficking was altered concomitant with increased PgP activity and disassembly of high molecular weight PgP-containing complexes during acute peripheral inflammatory pain. These data suggest that PgP activity is post-translationally regulated at the BBB. The goal of the current study was to identify proteins that co-localize with PgP in rat brain microvessel endothelial cell membrane microdomains and use the data to suggest potential regulatory mechanisms. Using new density gradients of microvessel homogenates, we identified two unique pools (1,2) of PgP in membrane fractions. Caveolar constituents, caveolin1, cavin1, and cavin2, co-localized with PgP in these fractions indicating the two pools contained caveolae. A chaperone (Hsc71), protein disulfide isomerase and endosomal/lysosomal sorting proteins (Rab5, Rab11a) also co-fractionated with PgP in the gradients. These data suggest signaling pathways with a potential role in post-translational regulation of PgP activity at the BBB. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptors (PPAR)-α and -γ in astrocytes play important roles in inflammatory brain pathologies. Understanding the regulation of both activity and expression levels of PPARs is an important neuroscience issue. Toll-like receptor (TLR) agonists are inflammatory stimuli that could modulate PPAR, but the mechanisms of their control in astrocytes are poorly understood. In the present study, we report that lipopolysaccharide, peptidoglycan, and flagellin, which are agonists of TLR4, TLR1/2, and TLR5, respectively, exert time- and nuclear factor kappa-light-chain-enhancer of activated B cells-dependent suppression of mRNA, protein and activity of PPARα and PPARγ. In naïve astrocytes, PPARα and PPARγ mRNA have short turnover time (half-life about 30 min for PPARα, 75 min for PPARγ) with a nearly two-fold stabilization after TLR-activation. p38 inhibition abolished TLR-induced stabilization. The levels of PPARα and PPARγ mRNA, and protein and DNA-binding activity could be modified using c-Jun N-terminal Kinase and p38 inhibitors. In addition, the expression levels of both PPARα and PPARγ isotypes were induced after inhibition of protein synthesis. This induction signifies participation of additional regulatory proteins with short life-time. They are p38-sensitive for PPARα and c-Jun N-terminal Kinase-sensitive for PPARγ. Thus, PPARα and PPARγ are regulated in astrocytes on mRNA and protein levels, mRNA stability, and DNA-binding activity during TLR-mediated responses. [ABSTRACT FROM AUTHOR]

In a previous study, we reported that intrathecal injection of mesenchymal stem cells (MSCs) slowed disease progression in G93A mutant superoxide dismutase1 transgenic mice. In this study, we found that intrathecal MSC administration vastly increased the infiltration of peripheral immune cells into the spinal cord of Amyotrophic lateral sclerosis (ALS) mice (G93A mutant superoxide dismutase1 transgenic). Thus, we investigated the immunomodulatory effect of MSCs on peripheral blood mononuclear cells (PBMCs) in ALS patients, focusing on regulatory T lymphocytes (Treg; CD4+/CD25high/FoxP3+) and the mRNA expression of several cytokines (IFN-γ, TNF-α, IL-17, IL-4, IL-10, IL-13, and TGF-β). Peripheral blood samples were obtained from nine healthy controls (HC) and sixteen patients who were diagnosed with definite or probable ALS. Isolated PBMCs from the blood samples of all subjects were co-cultured with MSCs for 24 or 72 h. Based on a fluorescence- activated cell sorting analysis, we found that co-culture with MSCs increased the Treg/total T-lymphocyte ratio in the PBMCs from both groups according to the co-culture duration. Co-culture of PBMCs with MSCs for 24 h led to elevated mRNA levels of IFN-γ and IL-10 in the PBMCs from both groups. However, after co-culturing for 72 h, although the IFNc mRNA level had returned to the basal level in co-cultured HC PBMCs, the IFN-γ mRNA level in co-cultured ALS PBMCs remained elevated. Additionally, the levels of IL-4 and TGF-β were markedly elevated, along with Gata3 mRNA, a Th2 transcription factor mRNA, in both HC and ALS PBMCs cocultured for 72 h. The elevated expression of these cytokines in the co-culture supernatant was confirmed via ELISA. Furthermore, we found that the increased mRNA level of indoleamine 2,3-dioxygenase (IDO) in the co-cultured MSCs was correlated with the increase in Treg induction. These findings of Treg induction and increased anti-inflammatory cytokine expression in co-cultured ALS PBMCs provide indirect evidence that MSCs may play a role in the immunomodulation of inflammatory responses when MSC therapy is targeted to ALS patients. [ABSTRACT FROM AUTHOR]

Retinal ganglion cells transmit the visual signal from the retina to the brain. We have previously shown that the activator protein 2 (AP-2)δ (TFAP2D) transcription factor is expressed in one third of ganglion cells in developing retina suggesting a specialized role for these AP-2δ-expressing cells. Here, we address the role of AP-2δ in retina by in ovo electroporation of RCAS/AP-2δ retroviral constructs into the eyes of chick embryos at day 2 of gestation. Ectopic expression of AP-2δ does not affect lineage differentiation in the developing retina. However, immunostaining of retinal tissue with markers associated with axonal growth such as growth-associated protein 43 and polysialic acid-neural cell adhesion molecule (PSA-NCAM) demonstrates axonal misrouting and abnormal axonal bundling. Treatment of AP-2δ-misexpressing retinal cell cultures with endoneuraminidase, an enzyme that removes PSA from NCAM, decreases AP-2δ-induced axonal bundling. Our data suggest a role for AP-2δ in polysialylation of NCAM, with ectopic expression of AP-2δ resulting in premature bundling of emerging axons and misrouting of axons. We propose that expression of AP-2δ in a subset of ganglion cells contributes to the fine-tuning of axonal growth in the developing retina. [ABSTRACT FROM AUTHOR]

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late-onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid-β (Aβ) peptides occurs at residues 244-272 of apoE and residues 12-28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12-28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12-28P elicits a therapeutic effect on tau-related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1M146V, APPSwe and tauP30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12-28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice. [ABSTRACT FROM AUTHOR]

Genetic mutations in leucine-rich repeat kinase 2 ( LRRK2) have been linked to autosomal dominant Parkinson's disease. The most prevalent mutation, G2019S, results in enhanced LRRK2 kinase activity that potentially contributes to the etiology of Parkinson's disease. Consequently, disease progression is potentially mediated by poorly characterized phosphorylation-dependent LRRK2 substrate pathways. To address this gap in knowledge, we transduced SH- SY5Y neuroblastoma cells with LRRK2 G2019S via adenovirus, then determined quantitative changes in the phosphoproteome upon LRRK2 kinase inhibition ( LRRK2- IN-1 treatment) using stable isotope labeling of amino acids in culture combined with phosphopeptide enrichment and LC- MS/ MS analysis. We identified 776 phosphorylation sites that were increased or decreased at least 50% in response to LRRK2- IN-1 treatment, including sites on proteins previously known to associate with LRRK2. Bioinformatic analysis of those phosphoproteins suggested a potential role for LRRK2 kinase activity in regulating pro-inflammatory responses and neurite morphology, among other pathways. In follow-up experiments, LRRK2- IN-1 inhibited lipopolysaccharide-induced tumor necrosis factor alpha ( TNFα) and C-X-C motif chemokine 10 (CXCL10) levels in astrocytes and also enhanced multiple neurite characteristics in primary neuronal cultures. However, LRRK2- IN-1 had almost identical effects in primary glial and neuronal cultures from LRRK2 knockout mice. These data suggest LRRK2- IN-1 may inhibit pathways of perceived LRRK2 pathophysiological function independently of LRRK2 highlighting the need to use multiple pharmacological tools and genetic approaches in studies determining LRRK2 function. [ABSTRACT FROM AUTHOR]

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression. [ABSTRACT FROM AUTHOR]

Neuropilin tolloid-like 1 (Neto1), is a CUB domain-containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein ( APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1 HA was shown to co-immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1 HA did not co-immunoprecipitate APP695 FLAG. Co-immunoprecipitations from mammalian cells co-transfected with APP695 FLAG, Neto1 HA and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co-exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/ APP/Neto1 complex. Neto1 HA caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695 FLAG- or PSD-95αc-Myc enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1 HA but deletion of the intracellular tail resulted in a loss of Neto-1 HA co-immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/ NMDA receptor trafficking complexes. [ABSTRACT FROM AUTHOR]