Cullin 5 regulates Dab1 protein levels and neuron positioning during cortical development

Many laminated regions of the mammalian brain develop by the migration of neuronal precursor cells, whose final positions are coordinated by signals from the secreted molecule Reelin. Early events in Reelin signaling have been identified, but the mechanism of signal down-regulation has been unclear. A possible source of negative feedback is the Reelin-induced degradation of the critical intracellular signaling component, Disabled-1 (Dab1). Here we show that degradation of Dab1 depends on Dab1 phosphorylation at specific tyrosine residues and on the E3 ubiquitin ligase component Cullin 5 (Cul5). Cul5 forms complexes with SOCS (suppressors of cytokine signaling) proteins, which bind to phosphorylated Dab1 and target it for degradation in tissue culture cells. Ablation of Cul5 in migrating neurons causes an accumulation of active Dab1 protein and a unique cortical layering defect, characterized by excess migration and buildup of neurons at the top of the cortical plate. The results implicate Cul5 and SOCS proteins in down-regulation of Dab1 in vivo and show that Cul5 plays an essential role in regulating neuron migrations during cortical development, possibly by opposing a promigratory effect of Dab1.     

Development of cortical GABAergic circuits and its implications for neurodevelopmental disorders

GABAergic interneurons powerfully control the function of cortical networks. In addition, they strongly regulate cortical development by modulating several cellular processes such as neuronal proliferation, migration, differentiation and connectivity. Not surprisingly, aberrant development of GABAergic circuits has been implicated in many neurodevelopmental disorders including schizophrenia, autism and Tourette's syndrome. Unfortunately, efforts directed towards the comprehension of the mechanisms regulating GABAergic circuits formation and function have been impaired by the strikingly heterogeneity, both at the morphological and functional level, of GABAergic interneurons. Recent technical advances, including the improvement of interneurons-specific labelling techniques, have started to reveal the basic principles underlying this process. This review summarizes recent findings on the mechanisms underlying the construction of GABAergic circuits in the cortex, with a particular focus on potential implications for brain diseases with neurodevelopmental origin.     

Abnormal development of the human cerebral cortex

This review presents an overview of human cortical malformation based on the insights gained from examination of human fetal brains. Examination at early stages of fetal brain development allows the identification of the specific pathways which are disrupted in human cortical malformation. Detailed examination of human fetal brains in parallel with studies of genetics and animal models is leading to new concepts of cortical malformations. Here we review a range of human cortical malformations based on a simple classification according to the developmental process thought to be disrupted: neuroblast proliferation, undermigration, overmigration, cortical maturation and destructive lesions. A single case example of a dated intrauterine injury illustrates the spectrum of malformations which may result at a single period in development. The recommended methods of examination of human fetal brain are described together with some of their pitfalls. Detailed neuropathological observations indicate the need for caution in the classification of malformations; radiological findings and pathology of the mature brain do not reflect the specific disruptive pathways of cortical malformations. While many insults may lead to the same pattern of malformation, a single insult can lead to multiple patterns of malformation. Our detailed studies of the human fetal brain suggest that the interface between the meninges and the radial glial end feet may be an intriguing new focus of interest in understanding cortical development.     

Expanding the spectrum of TUBA1A-related cortical dysgenesis to Polymicrogyria

De novo mutations in the TUBA1A gene are responsible for a wide spectrum of neuronal migration disorders, ranging from lissencephaly to perisylvian pachygyria. Recently, one family with polymicrogyria (PMG) and mutation in TUBA1A was reported. Hence, the purpose of our study was to determine the frequency of TUBA1A mutations in patients with PMG and better define clinical and imaging characteristics for TUBA1A-related PMG. We collected 95 sporadic patients with non-syndromic bilateral PMG, including 54 with perisylvian PMG and 30 PMG with additional brain abnormalities. Mutation analysis of the TUBA1A gene was performed by sequencing of PCR fragments corresponding to TUBA1A-coding sequences. Three de novo missense TUBA1A mutations were identified in three unrelated patients with PMG representing 3.1% of PMG and 10% of PMGs with complex cerebral malformations. These patients had bilateral perisylvian asymmetrical PMG with dysmorphic basal ganglia cerebellar vermian dysplasia and pontine hypoplasia. These mutations (p.Tyr161His; p.Val235Leu; p.Arg390Cys) appear distributed throughout the primary structure of the alpha-tubulin polypeptide, but their localization within the tertiary structure suggests that PMG-related mutations are likely to impact microtubule dynamics, stability and/or local interactions with partner proteins. These findings broaden the phenotypic spectrum associated with TUBA1A mutations to PMG and further emphasize that additional brain abnormalities, that is, dysmorphic basal ganglia, hypoplastic pons and cerebellar dysplasia are key features for the diagnosis of TUBA1A-related PMG.     

Acetylcholine release from rat cortical slices during postnatal development and aging

Acetylcholine release from cortical slices superfused with choline-enriched Krebs solution containing physostigmine was investigated at birth, at 7, 20 and 30 days, and at 3 and 24 months of age, in order to assess age influence on the functional efficiency of the cortical cholinergic network. The slices were electrically stimulated at frequencies from 1 to 10 Hz for 5 min periods, preceded and followed by rest periods. The superfusate was collected every 5 min and acetylcholine content quantified by bioassay. In the newborn and 7 day-old pups acetylcholine release was approximately 50% lower than that of the 3 month-old rats at all frequencies tested. The highest release was elicited in the 30 day-old rats. Beginning with this age the evoked ACh release underwent a decline which in the 24 month-old rats brought it back to the same level as in the newborn ones. The blockade of the muscarinic autoreceptors by atropine 1.5 × 10^?8 M caused an increase in acetylcholine release at 20 day, 3 and 24 months of age but not in the newborn and 7 day-old pups. Adenosine 3 × 10^?5 M decreased acetylcholine output in newborn and adult but had no effect in the senescent rats.     

Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.     

Slit2/Robo1信号对鸡胚早期神经管及体节发育的影响

目的: 探讨Slit2/Robo1对鸡胚早期神经管和体节发育的影响。 方法: 显微注射法将质粒注射入HH10期胚胎神经管内,活体胚胎细胞电穿孔方法转染胚胎半侧神经管,以另一侧神经管为对照侧,原位杂交及免疫荧光方法观察转染10 h后神经管的发育和神经嵴细胞迁移至体节的情况。结果: 下调Robo1侧神经管发育较正常对照侧异常,同时发现Slug表达和神经嵴细胞迁移至体节路线发生改变。结论: Slit2/Robo1信号可能通过影响Slug基因表达,对胚胎早期神经管闭合、神经嵴细胞正常产生及迁移方向以及体节分化有重要作用。     

Neuropeptide Y Y5 receptor protein in the cortical/limbic system and brainstem of the rat: expression on gamma-aminobutyric acid and corticotropin-releasing hormone neurons

Neuropeptide Y displays diverse modes of action in the CNS including the modulation of cortical/limbic function. Some of these physiological actions have been at least partially attributed to actions of neuropeptide Y on the Y5 receptor subtype. We utilized an antibody raised against the Y5 receptor to characterize the distribution of this receptor subtype in the rat cortical/limbic system and brainstem. Y5-like immunoreactivity was located primarily in neuronal cell bodies and proximal dendritic processes throughout the brain. In the cortex, Y5 immunoreactivity was limited to a subpopulation of small γ-aminobutyric-acid interneurons (approximately 15 μm diameter) scattered throughout all cortical levels. Double label immunofluorescence was also used to demonstrate that all of the Y5 immunoreactive neurons in the cortex displayed intense corticotropin releasing hormone immunoreactivity. The most intense Y5 immunoreactive staining in the hippocampus was located in the pyramidal cell layer of the small CA2 subregion and the fasciola cinerea, with lower levels of staining in the hilar region of the dentate gyrus and CA3 subregion of the pyramidal cell layer. Nearly all of the Y5 immunoreactive neurons in the hilar region of the hippocampus displayed γ-aminobutyric-acid immunoreactivity. In the brainstem, Y5 immunoreactivity was most intense in the Edinger–Westphal nucleus, locus coeruleus and the mesencephalic trigeminal nucleus.

The present study provides neuroanatomical evidence for the possible sites of action of the neuropeptide Y/Y5 receptor system in the control of cortical/limbic function. The presence of Y5 immunoreactivity on cell bodies and proximal dendritic processes in specific regions of the hippocampus suggests that this receptor functions to modulate postsynaptic activity. These data also suggest that the neuropeptide Y/Y5 system may play a role in the modulation of a specific population of GABAergic neurons in the cortex, namely those that contain corticotropin-releasing hormone. The location of the Y5 receptor immunoreactivity fits with the known physiological actions of neuropeptide Y and this receptor.     

Comparative aspects of cortical neurogenesis in vertebrates

The mammalian neocortex consists of six layers. By contrast, the reptilian and avian cortices have only three, which are believed to be equivalent to layers I, V and VI of mammals. In mammals, the majority of cortical cell proliferation occurs in the ventricular and subventricular zones, but there are a small number of scattered individual divisions throughout the cortex. Neurogenesis in the cortical subventricular zone is believed to contribute to the supragranular layers. To estimate the proportions of different forms of divisions in reptiles and birds, we examined the site of proliferation in embryonic turtle (stages 18-25) and chick (embryonic days 8-15) brains using phospho-histone H3 (a G2 and M phase marker) immunohistochemistry. In turtle, only few scattered abventricular H3-immunoreactive cells were found outside the ventricular zone; the majority of the H3-immunoreactive cells were located in the ventricular zone throughout the entire turtle brain. Ventricular zone cell proliferation peaks at stages 18 and 20, before an increase of abventricular proliferation at stages 23 and 25. In turtle cortex, however, abventricular proliferation at any given stage never exceeded 17.5+/-2.47% of the total division and the mitotic profiles did not align parallel to the ventricular zone. Phospho-histone H3 immunoreactivity in embryonic chick brains suggests the lack of subventricular zone in the dorsal cortex, but the presence of subventricular zone in the ventral telencephalon. We were able to demonstrate that the avian subventricular zone is present in both pallial and subpallial regions of the ventral telencephalon during embryonic development, and we characterize the spatial and temporal organization of the subventricular zone. Comparative studies suggest that the subventricular zone was involved in the laminar expansion of the cortex to six layers in mammals from the three-layered cortex found in reptiles and birds. Within mammals, the number of neurons in a cortical column appears to be largely constant; nevertheless, there are considerable differences between the germinal zones in mammalian species. It is yet to be determined whether these elaborations of the subventricular zone may have contributed to cell diversity, tangential expansion or gyrus formation of the neocortex and whether it might have been the major driving force behind the evolution of the six-layered neocortex in mammals.     

Postnatal analysis of the effect of embryonic knockdown and overexpression of candidate dyslexia susceptibility gene homolog Dcdc2 in the rat

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.     

New insights into the development of the human cerebral cortex

The cerebral cortex constitutes more than half the volume of the human brain and is presumed to be responsible for the neuronal computations underlying complex phenomena, such as perception, thought, language, attention, episodic memory and voluntary movement. Rodent models are extremely valuable for the investigation of brain development, but cannot provide insight into aspects that are unique or highly derived in humans. Many human psychiatric and neurological conditions have developmental origins but cannot be studied adequately in animal models. The human cerebral cortex has some unique genetic, molecular, cellular and anatomical features, which need to be further explored. The Anatomical Society devoted its summer meeting to the topic of Human Brain Development in June 2018 to tackle these important issues. The meeting was organized by Gavin Clowry (Newcastle University) and Zoltán Molnár (University of Oxford), and held at St John's College, Oxford. The participants provided a broad overview of the structure of the human brain in the context of scaling relationships across the brains of mammals, conserved principles and recent changes in the human lineage. Speakers considered how neuronal progenitors diversified in human to generate an increasing variety of cortical neurons. The formation of the earliest cortical circuits of the earliest generated neurons in the subplate was discussed together with their involvement in neurodevelopmental pathologies. Gene expression networks and susceptibility genes associated to neurodevelopmental diseases were discussed and compared with the networks that can be identified in organoids developed from induced pluripotent stem cells that recapitulate some aspects of in vivo development. New views were discussed on the specification of glutamatergic pyramidal and γ‐aminobutyric acid (GABA)ergic interneurons. With the advancement of various in vivo imaging methods, the histopathological observations can be now linked to in vivo normal conditions and to various diseases. Our review gives a general evaluation of the exciting new developments in these areas. The human cortex has a much enlarged association cortex with greater interconnectivity of cortical areas with each other and with an expanded thalamus. The human cortex has relative enlargement of the upper layers, enhanced diversity and function of inhibitory interneurons and a highly expanded transient subplate layer during development. Here we highlight recent studies that address how these differences emerge during development focusing on diverse facets of our evolution.     

Proteolytic activity during cortical development is distinct from that involved in hypoxic ischemic injury

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in brain development and the etiology of adult cerebral injuries. In this study, we determined the MMP-2 and 9 responses following hypoxic ischemia (HI) injury in the developing brain. First, we characterized the developmental changes of MMP activity in the rat brain from embryonic day 18 (E18) to postnatal day 120 (P120). MMP-2 activity was high from E18 to P3 and decreased with age (P< or =0.001), while MMP-9 activity was not detectable. MMP-2 immunoreactivity was closely associated with differentiating cortical plate and subplate neurons. Next, we characterized the proteolytic changes after unilateral HI brain injury in 3- (P3) and 21- (P21) day-old rats. Zymography revealed that in the P21 rat brain, MMP-9 activity (150 and 92 kDa forms) was increased at 6 h and remained elevated 24 h post-injury in the ipsilateral injured hemisphere (P< or =0.001), whereas there was a gradual increase in MMP-2 (65 kDa) activity, reaching a peak at 5 days (P< or =0.001). Similarly, quantitative real time polymerase chain reaction (qRT-PCR) indicated significant elevations in MMP-9 and MMP-2 mRNA expression in the injured cortex (P< or =0.05) and hippocampus (P< or =0.05) at 1 and 5 days post-injury, respectively in the P21 rat brain. In the P3 rat brain, zymography results revealed that both pro (92 kDa) and cleaved (87 kDa) MMP-9 activities were upregulated in the ipsilateral injured hemisphere from 6 h to 1 day after injury (P< or =0.001). In contrast, cleaved MMP-2 (60 kDa) was only moderately upregulated at 6 h (P< or =0.01), while pro MMP-2 (65 kDa) levels were unaffected. MMP-9 mRNA expression was also increased at 6 h (P< or =0.05) following injury at P3, whereas MMP-2 expression remained unchanged compared with the uninjured contralateral hemisphere. Immunohistochemistry indicated that MMP-9 protein expression was localized predominantly to neurons and peri-vascular astrocytes in the affected regions at early time points, whereas MMP-2 was present on reactive astrocytes surrounding the infarct at later time points. Together, these results indicate that MMP-2 may be primarily associated with the development and differentiation of cortical plate neurons and wound recovery processes. Conversely, MMP-9 appeared to be associated with more acute processes during the period of lesion development.     

Localization of CRMP5 mRNA by in situ hybridisation during development of the mouse forebrain

The expression of the collapse response mediator protein CRMP5 in the prenatal mouse is largely unknown. Evidence suggests that CRMP family members play important roles in neurite outgrowth, and CRMP5 is known to modulate outgrowth of processes in oligodendrocytes through signalling via neuropilin-1 and SemaA. Furthermore, CRMP family members function in axon regeneration after injury and are implicated in the early stages of Alzheimer's disease. Despite these findings relatively little is known about the specific roles these proteins play. The aim of the present study was to evaluate CRMP5 expression in the developing mouse forebrain using in situ hybridisation. Serial coronal sections of brain from E12.5 to E18.5 were analysed. We found highly specific patterns of expression which were restricted to the post-mitotic layers of both the ganglionic eminence and neocortex, and an additional domain of strong expression in the pyramidal layers of the hippocampus in all prenatal ages. Our results are therefore consistent with a role for CRMP5 in process extension. Interestingly, our results also revealed a temporal switch in high-expression levels from the ganglionic eminence to the cortex at a critical time during tangential cell migration. However, the pattern of expression appeared more representative of a general permissiveness for neurite outgrowth rather than one which is restricted to a particular cell subset or cell class. Additionally, expression was also found during periods predominated by neurogenesis and not neurite extension. We conclude that expression of CRMP5 is consistent with a dynamic implicit role in forebrain development.     

Slow-oscillatory transcranial direct current stimulation can induce bidirectional shifts in motor cortical excitability in awake humans

Constant transcranial direct stimulation (c-tDCS) of the primary motor hand area (M1_HAND) can induce bidirectional shifts in motor cortical excitability depending on the polarity of tDCS. Recently, anodal slow oscillation stimulation at a frequency of 0.75 Hz has been shown to augment intrinsic slow oscillations during sleep and theta oscillations during wakefulness. To embed this new type of stimulation into the existing tDCS literature, we aimed to characterize the after effects of slowly oscillating stimulation (so-tDCS) on M1_HAND excitability and to compare them to those of c-tDCS. Here we show that so-tDCS at 0.8 Hz can also induce lasting changes in corticospinal excitability during wakefulness. Experiment 1. In 10 healthy awake individuals, we applied c-tDCS or so-tDCS to left M1_HAND on separate days. Each tDCS protocol lasted for 10 min. Measurements of motor evoked potentials (MEPs) confirmed previous work showing that anodal c-tDCS at an intensity of 0.75 mA (maximal current density 0.0625 mA/cm2) enhanced corticospinal excitability, while cathodal c-tDCS at 0.75 mA reduced it. The polarity-specific shifts in excitability persisted for at least 20 min after c-tDCS. Using a peak current intensity of 0.75 mA, neither anodal nor cathodal so-tDCS had consistent effects on corticospinal excitability. Experiment 2. In a separate group of ten individuals, peak current intensity of so-tDCS was raised to 1.5 mA (maximal current density 0.125 mA/cm2) to match the total amount of current applied with so-tDCS to the amount of current that had been applied with c-tDCS at 0.75 mA in Experiment 1. At peak intensity of 1.5 mA, anodal and cathodal so-tDCS produced bidirectional changes in corticospinal excitability comparable to the after effects that had been observed after c-tDCS at 0.75 mA in Experiment 1. The results show that so-tDCS can induce bidirectional shifts in corticospinal excitability in a similar fashion as c-tDCS if the total amount of applied current during the tDCS session is matched.     

Spatial extent of coherent sensory-evoked cortical activity

Summary Analysis of published values for the depth profile of evoked potentials in primary sensory cortex of cat and monkey provide a consistent estimate for the net current dipole moment per unit area of cortical surface. Comparison with values of the total current dipole moment obtained from neuromagnetic studies on human subjects indicates that coherent neuronal activity giving rise to long-latency sensory evoked components recorded in the human electroencephalogram or magnetoencephalogram extends over a cortical area that is typically 40–400 mm².     

微管相关蛋白Doublecortin在成年大鼠神经元前体细胞发生中的表达

目的:探索神经元前体细胞的微管相关蛋白Doublecortin(DCX)在成年大鼠神经元前体细胞发生中的表达,为神经元前体细胞的增殖和迁移研究提供理论及实验基础。方法:将大鼠脑组织行冰冻切片,采用免疫组化在光镜下观察DCX免疫阳性细胞的表达部位和形态特点。结果:神经元前体细胞的标志物DCX主要分布在4个神经发生相关区域:室下区、齿状回的粒下层、吻侧迁移流和嗅球。然而在皮质等无神经发生的区域只有极少量的DCX免疫阳性细胞。DCX免疫阳性细胞在形态上均呈梭形的胞体和单个的前导突起。结论:DCX免疫阳性细胞的形态符合神经元前体细胞的形态特征。DCX作为神经元前体细胞的标志物可以用来研究神经元前体细胞的增殖和迁移。     

Regulation of early spontaneous network activity and GABAergic neurons development by thyroid hormone

Early in development spontaneous activity modulates survival and connectivity of neurons and thus plays a crucial role in the formation of neural networks. The emergence of synchronous activity in cultured neocortical networks initially is driven by large GABAergic interneurons. Here we studied the impact of thyroid hormone on early network development and especially on the development of large GABAergic neurons. Triiodothyronine enhances the frequency of early spontaneous synchronous network activity and an overall increase in network connectivity is indicated by the increased density of glutamatergic and GABAergic synapses. The hormone-induced increase of activity parallels cell type–specific changes in neuronal soma size and cell density, with strong effects on somatic and axonal growth of large GABAergic interneurons. Interestingly, large GABAergic neuron growth is both activity- and hormone-regulated. Blocking neuronal activity by tetrodotoxin or the glutamate receptor blockers D-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione disodium reveals a direct contribution of triiodothyronine to somatic growth, which also precedes the formation of synchronous network activity. The hormone-mediated effects on spontaneous activity and on large GABAergic neurons growth can be blocked by the nuclear thyroid hormone receptors antagonist 1-850. Thus, our data suggest that triiodothyronine actions result in functional maturation of early cortical networks and cell type–specific structural alterations. The increase in spontaneous activity might initially follow the growth of the large GABAergic neurons, which show an exquisite sensitivity to the presence of thyroid hormones. For the most part, however, the hormone-mediated growth of the GABAergic neurons relies strongly on the maturation of glutamatergic synaptic activity.     

Impacts of methotrexate on survival,dendrite development,and synapse formation of cortical neurons

Methotrexate (MTX) is an anti-metabolite that has been used for the treatment of patients of acute lymphocytic leukemia or non-Hodgikin lymphoma for decades. In some cases, MTX-treated patients suffer from neurological side effects, including seizures and cognitive dysfunctions. While most patients are at developmental stages, information of the mechanisms of the side effects of MTX treatment on the developing neurons has been limited. Neurons develop in five steps in the human brain: neurogenesis, polarity formation, dendrite and axon development, synapse formation, and neuronal death. Except for neurogenesis, these processes can be recapitulated in the primary culture system of cortical neurons. Using primary cultured cortical neurons, we studied the impact of MTX treatment on dendrite development, synapse formation, and neuronal death in the present report. MTX treatment impaired neuronal survival, dendrite development, and synapse formation. Interestingly, half maximal effective concentrations (EC₅₀s) of MTX for all three processes are at the similar range and lower than the MTX concentration in the cerebrospinal fluid in treated patients. Our results provide possible mechanisms of neurological side effects in treated patients.