Myelin and oligodendrocyte lineage cell dysfunctions: New players in the etiology and treatment of depression and stress-related disorders.

Depressive disorders are complex, multifactorial disorders that have been traditionally attributed exclusively to neuronal abnormalities. However, recent studies have increased our understanding of the contribution of glial cells-and particularly of oligodendroglia-to the pathogenesis and treatment outcome of depression and stress-related disorders. This review scrutinizes recent studies focusing on the neurosupportive functions exerted by myelin and oligodendrocyte lineage cells and their disruption in depression and stress-related disorders. It also illustrates how myelin and oligodendroglia respond to antidepressants and non-pharmacological treatment alternatives and proposes oligodendroglia-directed approaches as novel therapeutic options for depressive disorders.

In vivo silencing of miR-125a-3p promotes myelin repair in models of white matter demyelination.

In the last decade, microRNAs have been increasingly recognized as key modulators of glial development. Recently, we identified miR-125a-3p as a new player in oligodendrocyte physiology, regulating in vitro differentiation of oligodendrocyte precursor cells (OPCs). Here, we show that miR-125a-3p is upregulated in active lesions of multiple sclerosis (MS) patients and in OPCs isolated from the spinal cord of chronic experimental autoimmune encephalomyelitis (EAE) mice, but not in those isolated from the spontaneously remyelinating corpus callosum of lysolecithin-treated mice. To test whether a sustained expression of miR-125a-3p in OPCs contribute to defective remyelination, we modulated miR-125a-3p expression in vivo and ex vivo after lysolecithin-induced demyelination. We found that lentiviral over-expression of miR-125a-3p impaired OPC maturation, whereas its downregulation accelerated remyelination. Transcriptome analysis and luciferase reporter assay revealed that these effects are partly mediated by the direct interaction of miR-125a-3p with Slc8a3, a sodium-calcium membrane transporter, and identified novel candidate targets, such as Gas7, that we demonstrated necessary to correctly address oligodendrocytes to terminal maturation. These findings show that miR-125a-3p upregulation negatively affects OPC maturation in vivo, suggest its role in the pathogenesis of demyelinating diseases and unveil new targets for future promyelinating protective interventions.

KRIT1 Deficiency Promotes Aortic Endothelial Dysfunction.

Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/- mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.

Preventive motor training but not progenitor grafting ameliorates cerebellar ataxia and deregulated autophagy in tambaleante mice.

Treatment options for degenerative cerebellar ataxias are currently very limited. A large fraction of such disorders is represented by hereditary cerebellar ataxias, whose familiar transmission facilitates an early diagnosis and may possibly allow to start preventive treatments before the onset of the neurodegeneration and appearance of first symptoms. In spite of the heterogeneous aetiology, histological alterations of ataxias often include the primary degeneration of the cerebellar cortex caused by Purkinje cells (PCs) loss. Thus, approaches aimed at replacing or preserving PCs could represent promising ways of disease management. In the present study, we compared the efficacy of two different preventive strategies, namely cell replacement and motor training. We used tambaleante (tbl) mice as a model for progressive ataxia caused by selective loss of PCs and evaluated the effectiveness of the preventive transplantation of healthy PCs into early postnatal tbl cerebella, in terms of PC replacement and functional preservation. On the other hand, we investigated the effects of motor training on PC survival, cerebellar circuitry and their behavioral correlates. Our results demonstrate that, despite a good survival rate and integration of grafted PCs, the adopted grafting protocol could not alleviate the ataxic symptoms in tbl mice. Conversely, preventive motor training increases PCs survival with a moderate positive impact on the motor phenotype.

Early phenotypic asymmetry of sister oligodendrocyte progenitor cells after mitosis and its modulation by aging and extrinsic factors.

Oligodendrocyte progenitor cells (OPCs) persist in the adult central nervous system and guarantee oligodendrocyte turnover throughout life. It remains obscure how OPCs avoid exhaustion during adulthood. Similar to stem cells, OPCs could self-maintain by undergoing asymmetric divisions generating a mixed progeny either keeping a progenitor phenotype or proceeding to differentiation. To address this issue, we examined the distribution of stage-specific markers in sister OPCs during mitosis and later after cell birth, and assessed its correlation with distinct short-term fates. In both the adult and juvenile cerebral cortex a fraction of dividing OPCs gives rise to sister cells with diverse immunophenotypic profiles and short-term behaviors. Such heterogeneity appears as cells exit cytokinesis, but does not derive from the asymmetric segregation of molecules such as NG2 or PDGFRa expressed in the mother cell. Rather, rapid downregulation of OPC markers and upregulation of molecules associated with lineage progression contributes to generate early sister OPC asymmetry. Analyses during aging and upon exposure to physiological (i.e., increased motor activity) and pathological (i.e., trauma or demyelination) stimuli showed that both intrinsic and environmental factors contribute to determine the fraction of symmetric and asymmetric OPC pairs and the phenotype of the OPC progeny as soon as cells exit mitosis.

Oligodendrocyte Progenitors in Glial Scar: A Bet on Remyelination.

Oligodendrocyte progenitor cells (OPCs) represent a subtype of glia, giving rise to oligodendrocytes, the myelin-forming cells in the central nervous system (CNS). While OPCs are highly proliferative during development, they become relatively quiescent during adulthood, when their fate is strictly influenced by the extracellular context. In traumatic injuries and chronic neurodegenerative conditions, including those of autoimmune origin, oligodendrocytes undergo apoptosis, and demyelination starts. Adult OPCs become immediately activated; they migrate at the lesion site and proliferate to replenish the damaged area, but their efficiency is hampered by the presence of a glial scar-a barrier mainly formed by reactive astrocytes, microglia and the deposition of inhibitory extracellular matrix components. If, on the one hand, a glial scar limits the lesion spreading, it also blocks tissue regeneration. Therapeutic strategies aimed at reducing astrocyte or microglia activation and shifting them toward a neuroprotective phenotype have been proposed, whereas the role of OPCs has been largely overlooked. In this review, we have considered the glial scar from the perspective of OPCs, analysing their behaviour when lesions originate and exploring the potential therapies aimed at sustaining OPCs to efficiently differentiate and promote remyelination.

Mecp2 knock-out astrocytes affect synaptogenesis by interleukin 6 dependent mechanisms.

Synaptic abnormalities are a hallmark of several neurological diseases, and clarification of the underlying mechanisms represents a crucial step toward the development of therapeutic strategies. Rett syndrome (RTT) is a rare neurodevelopmental disorder, mainly affecting females, caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene, leading to a deep derangement of synaptic connectivity. Although initial studies supported the exclusive involvement of neurons, recent data have highlighted the pivotal contribution of astrocytes in RTT pathogenesis through non-cell autonomous mechanisms. Since astrocytes regulate synapse formation and functionality by releasing multiple molecules, we investigated the influence of soluble factors secreted by Mecp2 knock-out (KO) astrocytes on synapses. We found that Mecp2 deficiency in astrocytes negatively affects their ability to support synaptogenesis by releasing synaptotoxic molecules. Notably, neuronal inputs from a dysfunctional astrocyte-neuron crosstalk lead KO astrocytes to aberrantly express IL-6, and blocking IL-6 activity prevents synaptic alterations.

Distinct roles of Nogo-a and Nogo receptor 1 in the homeostatic regulation of adult neural stem cell function and neuroblast migration.

In the adult mammalian subventricular zone (SVZ), GFAP-positive neural stem cells (NSCs) generate neuroblasts that migrate tangentially along the rostral migratory stream (RMS) toward the olfactory bulb (OB). In the mouse brain, we found that the plasticity inhibitors Nogo-A and Nogo receptor 1 (NgR1) are differentially expressed in the SVZ-OB system, in which Nogo-A identifies immature neuroblasts and NgR1 germinal astrocytes. We therefore examined the role of Nogo-A and NgR1 in the regulation of neurogenesis. Pharmacological experiments show that Nogo-66/NgR1 interaction reduces the proliferation of NSCs. This is consistent with a negative-feedback loop, in which newly generated neurons modulate cell division of SVZ stem cells. Moreover, the Nogo-A-Δ20 domain promotes neuroblast migration toward the OB through activation of the Rho/ROCK (Rho-associated, coiled-coil containing protein kinase) pathway, without the participation of NgR1. Our findings reveal a new unprecedented function for Nogo-A and NgR1 in the homeostatic regulation of the pace of neurogenesis in the adult mouse SVZ and in the migration of neuroblasts along the RMS.

Molecular and functional heterogeneity in dorsal and ventral oligodendrocyte progenitor cells of the mouse forebrain in response to DNA damage.

In the developing mouse forebrain, temporally distinct waves of oligodendrocyte progenitor cells (OPCs) arise from different germinal zones and eventually populate either dorsal or ventral regions, where they present as transcriptionally and functionally equivalent cells. Despite that, developmental heterogeneity influences adult OPC responses upon demyelination. Here we show that accumulation of DNA damage due to ablation of citron-kinase or cisplatin treatment cell-autonomously disrupts OPC fate, resulting in cell death and senescence in the dorsal and ventral subsets, respectively. Such alternative fates are associated with distinct developmental origins of OPCs, and with a different activation of NRF2-mediated anti-oxidant responses. These data indicate that, upon injury, dorsal and ventral OPC subsets show functional and molecular diversity that can make them differentially vulnerable to pathological conditions associated with DNA damage.

Expression of the new P2Y-like receptor GPR17 during oligodendrocyte precursor cell maturation regulates sensitivity to ATP-induced death.

The P2Y-like receptor GPR17 is expressed by adult neural progenitor cells, suggesting a role in lineage determination. Here, we characterized GPR17 expression and function in mouse cortical primary astrocytes/precursor cell cultures. GPR17 is expressed by a subpopulation of oligodendrocyte precursor cells (OPCs), but not by astrocytes. This expression pattern was also confirmed in vivo. In vitro, GPR17 expression was markedly influenced by culturing conditions. In the presence of growth factors (GFs), no significant GPR17 expression was found. When cultures were shifted to a differentiating medium, a dramatic, time-dependent increase in the number of highly branched GPR17-positive cells was observed. Under these conditions, GPR17 was induced in the totality of O4-positive immature oligodendrocytes. Instead, in cultures originally grown in the absence of GFs, GPR17 was already expressed in morphologically more mature OPCs. Shifting of these cultures to differentiating conditions induced GPR17 only in a subpopulation of O4-positive cells. Under both culture protocols, appearance of more mature CNPase- and MBP-positive cells was associated to a progressive loss of GPR17. GPR17 expression also sensitized cells to adenine nucleotide-induced cytotoxicity, whereas activation with uracil nucleotides promoted differentiation towards a more mature phenotype. We suggest that GFs may keep OPCs in a less differentiated stage by restraining GPR17 expression, and that, under permissive conditions, GPR17 contributes to OPCs differentiation. However, upon high extracellular adenine nucleotide concentrations, as during trauma and ischemia, GPR17 sensitizes cells to cytotoxicity. This double-edged sword role may be exploited to unveil new therapeutic approaches to acute and chronic brain disorders.

The GPR17 receptor in NG2 expressing cells: focus on in vivo cell maturation and participation in acute trauma and chronic damage.

NG2-expressing cells comprise a population of cycling precursors that can exit the cell cycle and differentiate into mature oligodendrocytes. As a whole, they display heterogeneous properties and behaviors that remain unresolved at the molecular level, although partly interpretable as distinct maturation stages. To address this issue, we analyzed the expression of the GPR17 receptor, recently shown to decorate NG2-expressing cells and to operate as an early sensor of brain damage, in immature and adult oligodendrocyte progenitors in the intact brain and after injury. In both the early postnatal and adult cerebral cortex, distinct GPR17 protein localizations and expression levels define different stages of oligodendroglial maturation, ranging from the precursor phase to the premyelinating phenotype. As soon as cells exit mitosis, a fraction of NG2-expressing cells displays accumulation of GPR17 protein in the Golgi apparatus. GPR17 expression is subsequently upregulated and distributed to processes of cells that stop dividing, progressively lose NG2 positivity and assume premyelinating features. Absence of colabeling with mature markers or myelin proteins indicates that GPR17 is downregulated when cells complete their final maturation. BrdU-based fate-mapping demonstrated that a significant fraction of newly generated oligodendrocyte progenitors transiently upregulates GPR17 during maturation. Importantly, we also found that GPR17 does not participate to the early reaction of NG2-expressing cells to damage, while it is induced at postacute stages after injury. These findings identify GPR17 as a marker for progenitor progression within the oligodendroglial lineage and highlight its participation to postacute reactivity of NG2 cells in different injury paradigms.

Elovl5 Expression in the Central Nervous System of the Adult Mouse.

ELOVL5 (Elongase of Very-Long Fatty Acid 5) gene encodes for an enzyme that elongates long chain fatty acids, with a marked preference for polyunsaturated molecules. In particular, it plays an essential role in the elongation of omega-3 and omega-6 fatty acids, precursors for long-chain polyunsaturated fatty acids (PUFAs). Mutations of ELOVL5 cause the spino-cerebellar ataxia type 38 (SCA38), a rare autosomal neurological disease characterized by gait abnormality, dysarthria, dysphagia, hyposmia and peripheral neuropathy, conditions well represented by a mouse model with a targeted deletion of this gene (Elovl5-/- mice). However, the expression pattern of this enzyme in neuronal and glial cells of the central nervous system (CNS) is still uninvestigated. This work is aimed at filling this gap of knowledge by taking advantage of an Elovl5-reporter mouse line and immunofluorescence analyses on adult mouse CNS sections and glial cell primary cultures. Notably, Elovl5 appears expressed in a region- and cell type-specific manner. Abundant Elovl5-positive cells were found in the cerebellum, brainstem, and primary and accessory olfactory regions, where mitral cells show the most prominent expression. Hippocampal pyramidal cells of CA2/CA3 where also moderately labeled, while in the rest of the telencephalon Elovl5 expression was high in regions related to motor control. Analysis of primary glial cell cultures revealed Elovl5 expression in oligodendroglial cells at various maturation steps and in microglia, while astrocytes showed a heterogeneous in vivo expression of Elovl5. The elucidation of Elovl5 CNS distribution provides relevant information to understand the physiological functions of this enzyme and its PUFA products, whose unbalance is known to be involved in many pathological conditions.

c-Jun N-terminal kinase 1 (JNK1) modulates oligodendrocyte progenitor cell architecture, proliferation and myelination.

During Central Nervous System ontogenesis, myelinating oligodendrocytes (OLs) arise from highly ramified and proliferative precursors called oligodendrocyte progenitor cells (OPCs). OPC architecture, proliferation and oligodendro-/myelino-genesis are finely regulated by the interplay of cell-intrinsic and extrinsic factors. A variety of extrinsic cues converge on the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/MAPK) pathway. Here we found that the germinal ablation of the MAPK c-Jun N-Terminal Kinase isoform 1 (JNK1) results in a significant reduction of myelin in the cerebral cortex and corpus callosum at both postnatal and adult stages. Myelin alterations are accompanied by higher OPC density and proliferation during the first weeks of life, consistent with a transient alteration of mechanisms regulating OPC self-renewal and differentiation. JNK1 KO OPCs also show smaller occupancy territories and a less complex branching architecture in vivo. Notably, these latter phenotypes are recapitulated in pure cultures of JNK1 KO OPCs and of WT OPCs treated with the JNK inhibitor D-JNKI-1. Moreover, JNK1 KO and WT D-JNKI-1 treated OLs, while not showing overt alterations of differentiation in vitro, display a reduced surface compared to controls. Our results unveil a novel player in the complex regulation of OPC biology, on the one hand showing that JNK1 ablation cell-autonomously determines alterations of OPC proliferation and branching architecture and, on the other hand, suggesting that JNK1 signaling in OLs participates in myelination in vivo.

Exposure to fine particulate matter (PM2.5) hampers myelin repair in a mouse model of white matter demyelination.

Epidemiological studies show a strong association between exposure to air pollution - and particularly to particulate matter (PM) -, increased prevalence of Multiple Sclerosis (MS) and higher rates of hospital admissions for MS and MS relapses. Besides having immunomodulatory effects and sustaining a systemic oxidative-inflammatory response, PM may participate in MS pathogenesis by targeting also Central Nervous System (CNS)-specific processes, such as myelin repair. Here we show that, in a mouse model of lysolecithin-induced demyelination of the subcortical white matter, post-injury exposure to fine PM hampers remyelination, disturbs oligodendroglia differentiation dynamics and promotes astroglia and microglia reactivity. These findings support the view that exposure to fine PM can contribute to demyelinating pathologies by targeting the endogenous regenerative capability of the CNS tissue.

Mouse brain expression patterns of Spg7, Afg3l1, and Afg3l2 transcripts, encoding for the mitochondrial m-AAA protease.

BACKGROUND: The m-AAA (ATPases Associated with a variety of cellular Activities) is an evolutionary conserved metalloprotease complex located in the internal mitochondrial membrane. In the mouse, it is a hetero-oligomer variably formed by the Spg7, Afg3l1, and Afg3l2 encoded proteins, or a homo-oligomer formed by either Afg3l1 or Afg3l2. In humans, AFG3L2 and SPG7 genes are conserved, whereas AFG3L1 became a pseudogene. Both AFG3L2 and SPG7 are involved in a neurodegenerative disease, namely the autosomal dominant spinocerebellar ataxia SCA28 and a recessive form of spastic paraplegia, respectively. RESULTS: Using quantitative RT-PCR, we measured the expression levels of Spg7, Afg3l1, and Afg3l2 in the mouse brain. In all regions Afg3l2 is the most abundant transcript, followed by Spg7, and Afg3l1, with a ratio of approximately 5:3:1 in whole-brain mRNA. Using in-situ hybridization, we showed that Spg7, Afg3l1 and Afg3l2 have a similar cellular pattern of expression, with high levels in mitral cells, Purkinje cells, deep cerebellar nuclei cells, neocortical and hippocampal pyramidal neurons, and brainstem motor neurons. However, in some neuronal types, differences in the level of expression of these genes were present, suggesting distinct degrees of contribution of their proteins. CONCLUSIONS: Neurons involved in SCA28 and hereditary spastic paraplegia display high levels of expression, but similar or even higher expression is also present in other types of neurons, not involved in these diseases, suggesting that the selective cell sensitivity should be attributed to other, still unknown, mechanisms.

Understanding the effects of air pollution on neurogenesis and gliogenesis in the growing and adult brain.

Exposure to air pollution - and particularly to particulate matter (PM) - is strongly associated with higher risk of neurodevelopmental disorders, poor mental health and cognitive defects. In animal models, disruption of CNS development and disturbances of adult neurogenesis contribute to PM neurotoxicity. Recent studies show that gestational PM exposure not only affects embryonic neurodevelopment, but also disturbs postnatal brain growth and maturation, by interfering with neurogenic/gliogenic events, myelination and synaptogenesis. Similarly, adult neurogenesis is affected at many levels, from neural stem cell amplification up to the maturation and integration of novel neurons in the adult brain parenchyma. The underlying mechanisms are still by and large unknown. Beyond microglia activation and neuroinflammation, recent studies propose a role for novel epigenetic mechanisms, including DNA methylation and extracellular vesicles-associated microRNAs.

KRIT1 loss-mediated upregulation of NOX1 in stromal cells promotes paracrine pro-angiogenic responses.

Cerebral cavernous malformation (CCM) is a cerebrovascular disorder of proven genetic origin characterized by abnormally dilated and leaky capillaries occurring mainly in the central nervous system, with a prevalence of 0.3-0.5% in the general population. Genetic studies have identified causative mutations in three genes, CCM1/KRIT1, CCM2 and CCM3, which are involved in the maintenance of vascular homeostasis. However, distinct studies in animal models have clearly shown that CCM gene mutations alone are not sufficient to cause CCM disease, but require additional contributing factors, including stochastic events of increased oxidative stress and inflammation. Consistently, previous studies have shown that up-regulation of NADPH oxidase-mediated production of reactive oxygen species (ROS) in KRIT1 deficient endothelium contributes to the loss of microvessel barrier function. In this study, we demonstrate that KRIT1 loss-of-function in stromal cells, such as fibroblasts, causes the up-regulation of NADPH oxidase isoform 1 (NOX1) and the activation of inflammatory pathways, which in turn promote an enhanced production of proangiogenic factors, including vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2). Furthermore and importantly, we show that conditioned media from KRIT1 null fibroblasts induce proliferation, migration, matrix metalloproteinase 2 (MMP2) activation and VE-cadherin redistribution in wild type human endothelial cells. Taken together, our results demonstrate that KRIT1 loss-of-function in stromal cells affects the surrounding microenvironment through a NOX1-mediated induction and release of angiogenic factors that are able to promote paracrine proangiogenic responses in human endothelial cells, thus pointing to a novel role for endothelial cell-nonautonomous effects of KRIT1 mutations in CCM pathogenesis, and opening new perspectives for disease prevention and treatment.

Selection of reference genes for quantitative real-time RT-PCR studies in mouse brain.

Since a growing number of studies based on the real-time reverse transcriptase polymerase chain reaction (RT-PCR) continue to be published in order to highlight genes specifically involved in brain development, maturation, and function, the identification of reference genes suitable for this kind of experiments is now an urgent need in the neuroscience field. The aim of this work was to verify the suitability of some very common housekeeping genes (such as Gapdh, 18s, and B2m) and of some relatively new control genes (such as Pgk1, Tfrc, and Gusb) during mouse brain maturation. We tested the candidate reference genes in mouse whole brain, cerebellum, brain stem, hippocampus, medial septum, frontal neocortex, and olfactory bulb. Moreover, we reported the first complete study of Pgk1 expression throughout the development and the aging of mouse brain. Although no tested gene showed to be the optimal reference for all mouse brain regions, in general, the new housekeeping genes were highly stable in most of the analyzed regions. Above all, with few exceptions, Pgk1 showed to be a reliable control for the analyzed mouse brain regions during development, maturation, and aging.

Emerging pharmacological approaches to promote neurogenesis from endogenous glial cells.

Neurodegenerative disorders are emerging as leading contributors to the global disease burden. While some drug-based approaches have been designed to limit or prevent neuronal loss following acute damage or chronic neurodegeneration, regeneration of functional neurons in the adult Central Nervous System (CNS) still remains an unmet need. In this context, the exploitation of endogenous cell sources has recently gained an unprecedented attention, thanks to the demonstration that, in some CNS regions or under specific circumstances, glial cells can activate spontaneous neurogenesis or can be instructed to produce neurons in the adult mammalian CNS parenchyma. This field of research has greatly advanced in the last years and identified interesting molecular and cellular mechanisms guiding the neurogenic activation/conversion of glia. In this review, we summarize the evolution of the research devoted to understand how resident glia can be directed to produce neurons. We paid particular attention to pharmacologically-relevant approaches exploiting the modulation of niche-associated factors and the application of selected small molecules.