Neuronal Cell Lineages Research Articles

NRSF silencing induces neuronal differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells that have the potential to differentiate into the neuronal cell lineage. Here, we describe the highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from human bone marrow-derived mesenchymal stem cells by NRSF silencing. Cells that have the characteristics of MSCs were obtained from human bone marrow. Lentiviral vectors were used to deliver small interference NRSF RNA (siNRSF) into MSCs. After being infected with lentivirus containing siNRSF, MSCs were successfully induced to differentiate into neuronal cells, which exhibited neuron-like morphology and formed nissl bodies. These differentiated cells expressed multiple neuron-specific genes including brain-derived neurotrophic factor (BDNF), neurogenin 1 (NGN1), neuron-specific enolase (NSE), synaptophysin (SYP), and neuron-specific growth-associated protein (SCG10), as well as expressing mature neuronal marker proteins, such as β-tubulin III, NSE, microtubule-associated protein type 2 (MAP-2), and neurofilament-200 (NF-200), yet did not express the glial markers glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (Olig2), as verified by immunofluorescence staining. The whole cell patch-clamp technique recorded TTX-sensitive Na + currents and action potential from these differentiated cells. Thus, our results demonstrate that NRSF silencing can activate some neuronal genes and induce neuronal differentiation of mesenchymal stem cells.

First Signature of Islet β-Cell-Derived Naturally Processed Peptides Selected by Diabetogenic Class II MHC Molecules

The diversity of Ags targeted by T cells in autoimmune diabetes is unknown. In this study, we identify and characterize a limited number of naturally processed peptides from pancreatic islet beta-cells selected by diabetogenic I-A(g7) molecules of NOD mice. We used insulinomas transfected with the CIITA transactivator, which resulted in their expression of class II histocompatibility molecules and activation of diabetogenic CD4 T cells. Peptides bound to I-A(g7) were isolated and examined by mass spectrometry: some peptides derived from proteins present in secretory granules of endocrine cells, and a number were shared with cells of neuronal lineage. All proteins to which peptides were identified were expressed in beta cells from normal islets. Peptides bound to I-A(g7) molecules contained the favorable binding motif characterized by acidic amino acids at the P9 position. The draining pancreatic lymph nodes of prediabetic NOD mice contained CD4 T cells that recognized three different natural peptides. Furthermore, four different peptides elicited CD4 T cells, substantiating the presence of such self-reactive T cells. The overall strategy of identifying natural peptides from islet beta-cells opens up new avenues to evaluate the repertoire of self-reactive T cells and its role in onset of diabetes.

Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal β III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

Novel neural stem cell systems

Neural stem cells are cells that can self-renew ad infinitum and have the potential to generate immature precursors and mature cells of both neuronal and glial lineages. During recent years, neural stem cells gained attention as major candidates for regenerative and cell replacement therapies in various pathological brain conditions; however, they have recently revealed unforeseen valuable characters. Here the authors review on the state of the field, with a particular focus on the most recent results on the optimisation of neural stem cells isolation/derivation and homogeneous long-term expansion, highlighting advantages of this resource and outlining their potential applications. Bearing in mind that stem-cell based therapies for the diseased and injured brain are still far from being a reality, these cells have been recently opened up to valuable exploitation for disease modelling, drug discovery and toxicology tests as short-term applications.

Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants

The transcription factor Sox2 is active in neural stem cells, and Sox2 'knockdown' mice show defects in neural stem/progenitor cells in the hippocampus and eye, and possibly some neurons. In humans, heterozygous Sox2 deficiency is associated with eye abnormalities, hippocampal malformation and epilepsy. To better understand the role of Sox2, we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of 'knockdown' mutants. Sox2 expression is high in undifferentiated cells, and declines with differentiation, but remains visible in at least some of the mature neurons. In mutant cells, neuronal, but not astroglial, differentiation was profoundly affected. beta-Tubulin-positive cells were abundant, but most failed to progress to more mature neurons, and showed morphological abnormalities. Overexpression of Sox2 in neural cells at early, but not late, stages of differentiation, rescued the neuronal maturation defect. In addition, it suppressed GFAP expression in glial cells. Our results show an in vitro requirement for Sox2 in early differentiating neuronal lineage cells, for maturation and for suppression of alternative lineage markers. Finally, we examined newly generated neurons from Sox2 ;knockdown' newborn and adult mice. GABAergic neurons were greatly diminished in number in newborn mouse cortex and in the adult olfactory bulb, and some showed abnormal morphology and migration properties. GABA deficiency represents a plausible explanation for the epilepsy observed in some of the knockdown mice, as well as in SOX2-deficient individuals.

The Expression of CD56 Is Frequently Accompanied with the Expression of Neuronal Cell Markers and Its Down-Regulation Is Induced by IL-6 in Human Myeloma Cells.

Human myeloma cells have the marked phenotypic heterogeneity of surface marker expressions, possibly because of loss of PAX-5 expression. Especially, ectopic expression of CD56, one of non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. However, only 2 (NOP2 and AMO1) out of 10 myeloma cell lines were CD56(+). In primary myeloma cells as well as CD56(−) myeloma cell lines, the treatment with forskolin could induce the expression of CD56 in the in vitro culture. In most CD56(+) primary myeloma cells as well as myeloma cell lines, the expressions of neuronal cell markers such as neuron specific enolase (NSE), nestin, β-tubulin III or chromogranin A were found coincidentally. By gene expression profiling, CD56(+) myeloma cell lines showed the marked expressions of transcription factors involved in neuronal cell lineage. On the other hand, addition of IL-6 down-regulated the expression of CD56 in CD56(+) myeloma cell lines in the in vitro culture. In 13 out of 60 patients with overt myeloma, these myeloma cells showed CD56(−) and their values of plasma CRP were significantly increased and MPC-1(−)CD45(+) immature myeloma cells were also increased compared to those in CD56(+) myeloma cases. Therefore, these results indicate that the expression of CD56 is possibly due to phenotypic changes into neuronal cell lineage, and IL-6 can block these phenotypic changes, keeping PAX-5(−) myeloma cells being uncommitted cells to any lineage.

Neurofibromatosis-1 Regulates Neuronal and Glial Cell Differentiation from Neuroglial Progenitors In Vivo by Both cAMP- and Ras-Dependent Mechanisms

Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

Bmi1 and Cell of Origin Determinants of Brain Tumor Phenotype

Glioblastomas frequently express oncogenic EGFR and loss of the Ink4a/Arf locus. Bmi1, a positive regulator of stem cell self renewal, may be critical to drive brain tumor growth. In this issue of Cancer Cell, Bruggeman and colleagues suggest that brain tumors with these molecular alterations can be initiated in both neural precursor and differentiated cell compartments in the absence of Bmi1; however, tumorigenicity is reduced, and tumors contain fewer precursor cells. Surprisingly, tumors appear less malignant when initiated in precursor cells. Bmi1-deficient tumors also had fewer neuronal lineage cells, suggesting a role for Bmi1 in determination of cell lineage and tumor phenotype.

The inhibitor of DNA binding 2 is mainly expressed in oligodendrocyte lineage cells in adult rat brain

The inhibitor of DNA binding 2 (Id2) plays an important role in the brain both during embryogenesis and adulthood. But in adult rat brain, it is still unknown whether Id2 immunoreactivity mainly exhibits in neuronal, astrocytic and/or oligodendrocyte lineage cells. It is also unclear where and when Id2 immunoreactivity mainly exhibits in oligodendrocyte lineage cells. The present study showed 90% of Id2-immunoreactivity in oligodendrocyte lineage cells in such brain regions as the corpus callosum, optic chiasm, the longitudinal fasciculus of pons, the medial septal nucleus, the fimbria of hippocampus, the anterior commissure, and the pyramidal tract. Five percent of Id2-immunoreactivity was found in astrocytes. Id2 immunoreactivity was localized in neurons of only a few brain regions. Seventy percent of Id2 immunoreactivity was found in CC-1-positive mature oligodendrocytes. These observations suggest that Id2 may be mainly involved in terminal maturation of oligodendrocytes and myelination.

Reversible effect of X‐irradiation on proliferation, neurogenesis, and cell death in the dentate gyrus of adult mice

Therapeutic cranial X-irradiation causes cognitive deficits in adult and pediatric patients, in particular, when the exposed area includes the medial temporal lobes. Effects on adult neurogenesis within the hippocampus may be related to such deficits. To investigate this relation, we irradiated the brain of young adult C57Bl/6j mice with a single dose of 4 Gy at a dose-rate of 27.5 cGy/min. We observed an approximately 80% decrease in the number of cells immunoreactive for the proliferation marker Ki67, 16 and 48 h after exposure, which was restored to control values after 1 week. The number of doublecortin- and NeuroD-immunoreactive cells of neuronal lineage was reduced by 60-70% up to 1 week after irradiation, but not after 1 month. The number of pyknotic cells increased approximately 2.5 fold after 16 h, decreased to approximately 50% of control numbers after 48 h and 1 week, and was again at normal levels after 1 month. Granule cell number did not differ between different groups and time points. There was no apparent activation of microglia or astrocytes. Our findings consist of an acute and reversible effect of X-irradiation on proliferation, neurogenesis, and cell death. Transient changes of neurogenesis may play a role in transient impairments of cognitive performance of patients exposed to X-irradiation. We present an experimental approach to temporarily alter adult hippocampal neurogenesis (AhN), allowing mechanistic investigations of AhN and its relevance to cognitive performances. The work also represents a step toward optimized radiotherapy schedules.

Characterization and differentiation of equine umbilical cord-derived matrix cells

Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton’s jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses.

Roles of leptin in prenatal and perinatal brain development

Leptin is a hormone that reduces food intake and increases energy expenditure by acting on the arcuate nucleus in the hypothalamus. Recent studies indicated that the neuronal circuit related to food intake in the hypothalamus is formed in the neonatal period and that leptin is necessary for the formation of this circuit. Our studies have further suggested that leptin may act on the fetal cerebral cortex, including the cingulate cortex, which is involved in motor and cognitive processes, and that leptin may affect maintenance and differentiation of neural stem cells, glial-restricted progenitor cells and/or neuronal lineage cells. These recent studies showed that leptin not only has homeostatic functions in adults, but also regulates brain development in the prenatal and neonatal periods. These findings suggest that leptin is related to formation of the normal brain structure and regenerative potency of neural cells as well as the predisposition to homeostatic dysfunction, low locomotor activity or impairment of cognitive function.

Isolation of Amniotic Stem Cell Lines With Potential for Therapy

Amniotic fluid contains multiple cell types derived from the developing fetus, including some that can give rise to differentiated adipose, muscle, bone, and neuronal cell lines. The present investigators have identified lines of broadly multipotent amniotic fluid-derived stem (AFS) cells that can give rise to a wide range of lineages including those in all embryonic germ layers, thereby meeting the criterion for pluripotent stem cells. Immunoselection with magnetic microspheres was used to isolate, from cultures of human amniocentesis specimens taken for prenatal genetic diagnosis, cells bearing the surface antigen c-Kit, the receptor for stem cell factor. Flow cytometry served to assess markers expressed by human AFS cells. AFS cells from 19 amniocentesis donors were able to differentiate along adipogenic, osteogenic, myogenic, endothelial, neurogenic, and hepatic pathways. Induced differentiation along multiple pathways was documented by the expression of mRNAs for lineage-specific genes. Multilineage differentiation was characteristic of AFS cells that were cloned by limiting dilution. Studies based on marking with a retroviral vector confirmed that cloned AFS cells and their differentiated derivatives did in fact descend from a single cell, and that the AFS cells are pluripotent stem cells. Feeders were not necessary for the undifferentiated AFS cells to expand extensively. The cells doubled in 36 hours and were not tumorigenic. Lines maintained for more than 250 population doublings continued to have long telomeres and normal karyotypes. The differentiated cells derived from AFS cells included neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells that formed tissue-engineered bone. These studies affirm that stem cells capable of extensive self-renewal can routinely be obtained from human amniotic fluid. AFS cells can serve as precursors to a broad range of differentiated cell types that potentially have therapeutic applications. Banking of cells that would otherwise be discarded could provide a convenient source not only for autologous treatment later in life, but for matching of histocompatible donor cells with prospective recipients.

CREB activity modulates neural cell proliferation, midbrain–hindbrain organization and patterning in zebrafish

Neural stem/progenitor cells (NPCs) self-renew and differentiate, generating neuronal and non-neuronal (glial) cell lineages. Although a number of factors, including transcription factors, have been shown to be important in the regulation of NPC proliferation and differentiation, the precise molecular networks remain to be identified. The cAMP Response Element-Binding protein (CREB) is a transcription factor important for neuronal survival, differentiation and plasticity. Recent work suggests that CREB activation, via serine phosphorylation in the kinase inducible domain, is important for neurogenesis in the adult rodent brain. We sought to further investigate CREB function in neurogenesis, using the zebrafish ( Danio rerio). Structural and functional analysis of the zebrafish CREB orthologue showed high conservation with mammalian CREB. Activated (phosphorylated) CREB (pCREB) was localised to all known proliferation zones in the adult zebrafish brain, including actively cycling cells. Furthermore, we found that modulating CREB activity during early zebrafish development caused significant defects in neural proliferation, midbrain–hindbrain organization and body patterning. These findings reveal broader and stage-specific physiological roles of CREB function during vertebrate neural development and proliferation.

Establishment of autologous embryonic stem cells derived from preantral follicle culture and oocyte parthenogenesis

To evaluate whether autologous embryonic stem cells can be established without generating clone embryos. Prospective model study. Gamete and stem cell biotechnology laboratory in Seoul National University, Seoul, Korea. F1 hybrid B6D2F1 mice. Preantral follicles were cultured, and oocytes matured in the follicles were parthenogenetically activated. Preimplantation development and stem cell characterization. More intrafollicular oocytes that were retrieved from secondary follicles matured and developed into blastocysts after parthenogenesis than those that were retrieved from primary follicles. Of those 35 blastocysts derived from 193 parthenotes, one line of colony-forming cells was established from the culturing of early secondary follicles. The established cells were positive for embryonic stem cell-specific markers and had normal diploid karyotype and telomerase activity. They differentiated into embryoid bodies in vitro and teratomas in vivo. Inducible differentiation of the established cells into neuronal lineage cells also was possible. Autologous embryonic stem cells can be established by preantral follicle culture and oocyte parthenogenesis. A combined technique of follicle culture and oocyte parthenogenesis that does not use developmentally competent oocytes has the potential to replace somatic cell nuclear transfer for autologous cell therapy.

The Doublecortin-Expressing Population in the Developing and Adult Brain Contains Multipotential Precursors in Addition to Neuronal-Lineage Cells

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However, the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus, which expressed low levels of DCX (DCX(low)), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly, the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells, which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX(low) cells retain their multipotentiality.

Le tissu adipeux : un donneur de cellules souches ?

Several laboratories have shown that the cells from the stroma-vascular fraction of the human adipose tissue express, depending on cell culture conditions, biochemical markers of multiple cell lineages including adipogenic, osteogenic, chondrogenic, myogenic, endothelial, neuronal and epithelial cell lineage. Furthermore, various in vivo approaches revealed the ability of the cells derived from the stroma-vacsular fraction of adipose tissue to repair ischemic or damaged tissues. Altogether these data strongly suggest that the stroma-vascular fraction of the human adipose tissue contains cells that exhibit properties like stem/progenitor cells. However, the exact nature of the cells, their potentiality to lead to differentiated cells in vivo as well as the mechanisms involved in their repair capability remain to be characterized to consider their use in regenerative and reparative medicine.

The proneural determinant MASH1 regulates forebrainDlx1/2expression through the I12b intergenic enhancer

Establishment of neuronal networks is an extremely complex process involving the interaction of a diversity of neuronal cells. During mammalian development, these highly organized networks are formed through the differentiation of multipotent neuronal progenitors into multiple neuronal cell lineages. In the developing forebrain of mammals, the combined function of the Dlx1, Dlx2, Dlx5 and Dlx6 homeobox genes is necessary for the differentiation of the GABAergic interneurons born in the ventricular and subventricular zones of the ventral telencephalon, as well as for the migration of these neurons to the hippocampus, cerebral cortex and olfactory bulbs. The 437 bp I12b enhancer sequence in the intergenic region of the Dlx1/2 bigene cluster is involved in the forebrain regulation of Dlx1/2. Using DNase I footprinting, we identified six regions of I12b potentially bound by transcription factors. Mutagenesis of each binding site affected the expression of reporter constructs in transgenic mice. However, the effects of impairing protein-DNA interactions were not uniform across the forebrain Dlx1/2 expression domains, suggesting that distinct regulatory interactions are taking place in the different populations of neuronal precursors. Analyses of protein-DNA interactions provide evidence of a direct role for MASH1 in Dlx1/2 regulation in the forebrain. DLX proteins play a crucial role in the maintenance of their own expression, as shown by transgenic and co-transfection experiments. These studies suggest that the seemingly continuous domains of Dlx gene expression in the telencephalon and diencephalon are in fact the combination of distinct cell populations within which different genetic regulatory interactions take place.

Segregation of postembryonic neuronal and glial lineages inferred from a mosaic analysis of the Drosophila larval brain

Due to its intermediate complexity and its sophisticated genetic tools, the larval brain of Drosophila is a useful experimental system to study the mechanisms that control the generation of cell diversity in the CNS. In order to gain insight into the neuronal and glial lineage specificity of neural progenitor cells during postembryonic brain development, we have carried an extensive mosaic analysis throughout larval brain development. In contrast to embryonic CNS development, we have found that most postembryonic neurons and glial cells of the optic lobe and central brain originate from segregated progenitors. Our analysis also provides relevant information about the origin and proliferation patterns of several postembryonic lineages such as the superficial glia and the medial-anterior Medulla neuropile glia. Additionally, we have studied the spatio-temporal relationship between gcm expression and gliogenesis. We found that gcm expression is restricted to the post-mitotic cells of a few neuronal and glial lineages and it is mostly absent from postembryonic progenitors. Thus, in contrast to its major gliogenic role in the embryo, the function of gcm during postembryonic brain development seems to have evolved to the specification and differentiation of certain neuronal and glial lineages.

Brain Transplantation of Immortalized Human Neural Stem Cells Promotes Functional Recovery in Mouse Intracerebral Hemorrhage Stroke Model

We have generated stable, immortalized cell lines of human NSCs from primary human fetal telencephalon cultures via a retroviral vector encoding v-myc. HB1.F3, one of the human NSC lines, expresses a normal human karyotype of 46, XX, and nestin, a cell type-specific marker for NSCs. F3 has the ability to proliferate continuously and differentiate into cells of neuronal and glial lineage. The HB1.F3 human NSC line was used for cell therapy in a mouse model of intracerebral hemorrhage (ICH) stroke. Experimental ICH was induced in adult mice by intrastriatal administration of bacterial collagenase; 1 week after surgery, the rats were randomly divided into two groups so as to receive intracerebrally either human NSCs labeled with beta-galactosidase (n = 31) or phosphate-buffered saline (PBS) (n = 30). Transplanted NSCs were detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemistry or double labeling with beta-galactosidase (beta-gal) and mitogen-activated protein (MAP)2, neurofilaments (both for neurons), or glial fibrillary acidic protein (GFAP) (for astrocytes). Behavior of the animals was evaluated for period up to 8 weeks using modified Rotarod tests and a limb placing test. Transplanted human NSCs were identified in the perihematomal areas and differentiated into neurons (beta-gal/MAP2(+) and beta-gal/NF(+)) or astrocytes (beta-gal/GFAP(+)). The NSC-transplanted group showed markedly improved functional performance on the Rotarod test and limb placing after 2-8 weeks compared with the control PBS group (p < .001). These results indicate that the stable immortalized human NSCs are a valuable source of cells for cell replacement and gene transfer for the treatment of ICH and other human neurological disorders. Disclosure of potential conflicts of interest is found at the end of this article.