New insights into a spectrum of developmental malformations related to mTOR dysregulations: challenges and perspectives.

In recent years the role of the mammalian target of rapamycin (mTOR) pathway has emerged as crucial for normal cortical development. Therefore, it is not surprising that aberrant activation of mTOR is associated with developmental malformations and epileptogenesis. A broad spectrum of malformations of cortical development, such as focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC), have been linked to either germline or somatic mutations in mTOR pathway-related genes, commonly summarised under the umbrella term 'mTORopathies'. However, there are still a number of unanswered questions regarding the involvement of mTOR in the pathophysiology of these abnormalities. Therefore, a monogenetic disease, such as TSC, can be more easily applied as a model to study the mechanisms of epileptogenesis and identify potential new targets of therapy. Developmental neuropathology and genetics demonstrate that FCD IIb and hemimegalencephaly are the same diseases. Constitutive activation of mTOR signalling represents a shared pathogenic mechanism in a group of developmental malformations that have histopathological and clinical features in common, such as epilepsy, autism and other comorbidities. We seek to understand the effect of mTOR dysregulation in a developing cortex with the propensity to generate seizures as well as the aftermath of the surrounding environment, including the white matter.

Review: The international consensus classification of Focal Cortical Dysplasia - a critical update 2018.

The Diagnostic Methods commission of the International League against Epilepsy (ILAE) released a first international consensus classification of Focal Cortical Dysplasia (FCD) in 2011. Since that time, this FCD classification has been widely used in clinical diagnosis and research (more than 740 papers cited in Pubmed between 1/1/2012 and 7/1/2017). Herein, we review the new data that will inform and revise the FCD classification. Many recent papers described molecular-genetic characteristics in FCD type II including multiple mutations in the mTOR pathway. In addition, the electro-clinico-imaging phenotype and surgical outcomes of FCD type II (in particular type IIb) were further defined and validated. These results pave the way for the design of an integrated clinico-pathological and genetic classification system, as recently recommended by the WHO for the classification of malignant brain tumours. On the other hand, little new information was acquired on FCD types I and III. Focal cortical dysplasia type I subtypes are still lacking a comprehensive description of clinical phenotypes, reproducible imaging characteristics, and specific molecular/genetic biomarkers. Associated FCD III subtypes also became rare in published literature. Despite temporal lobe epilepsy being the most common focal epilepsy in adults, we have not identified neurophysiological, imaging, histopathological and/or genetic biomarkers to reliably classify FCD III with or without hippocampal sclerosis. In respect of pathogenesis, FCD adjacent to a non-developmental, postnatally acquired lesion is difficult to explain and perhaps does not exist. This update may help foster shared efforts towards a better understanding of FCD, potential future updates of classification and novel targeted treatments.

[A proposal for a molecular genetic classification of the malformations of the nervous system]. / Propuesta para una clasificación genética molecular de las malformaciones del sistema nervioso.

OBJECTIVE: This proposal is a first attempt to incorporate the recent molecular genetic data that explains programming of development etiologically. DEVELOPMENT: Traditional schemes of classifying nervous system malformations are based upon descriptive morphogenesis of anatomical processes of ontogeny, such as neurulation, neuroblast migration and axonal pathfinding; such anatomical schemes do not allow for the incorporation of multiple genetic etiologies that lead to the same anatomical result, such as holoprosencephaly or lissencephaly. A scheme based purely on genetic mutations also is impractical because several genes might be involved sequentially in a cascade, the same genes serve different functions at different stages and are involved in multiple organ systems. Some complex malformations result from several unrelated defective genes, again citing the example of holoprosencephaly. Finally, a pure genetic classification would be too inflexible to incorporate anatomical criteria and also acquired lesions of the fetal brain that lead to secondary focal dysgeneses. The basis for the proposed scheme is, therefore, disturbances in patterns of genetic expression: polarity gradients of the axes of the neural tube (e.g. upregulation or downregulation of genetic influences); segmentation (e.g. deletions of specific neuromeres; ectopic expression); mutations that cause change in cell lineage (e.g. dysplastic gangliocytoma of cerebellum; myofiber differentiation within brain); and specific genes or molecules that mediate neuroblast migration in its early (e.g. filamin 1), middle (e.g. LIS1; doublecortin) or late course (e.g. reelin; L1 CAM). CONCLUSIONS: The classification schemes that served so well throughout the 20th century no longer are adequate for the 21st century. The proposed scheme undoubtedly will undergo many future revisions, but it provides a starting point using currently available data.

Propuesta para una clasificación genética molecular de las malformaciones del sistema nervioso / A proposal for a molecular genetic classification of the malformations of the nervous system

Objetivos. Esta propuesta es un primer intento para incorporar los datos recientes de genética molecular que explican la programación del desarrollo en un contexto etiológico. Desarrollo. Los esquemas tradicionales para clasificar las malformaciones del sistema nervioso se basan en la morfogénesis descriptiva como si fueran procesos aislados de la ontogenia, como la neurulación, la migración celular, el crecimiento axonal, etc. Estos esquemas anatómicos son inadecuados para incluir las disgenesias de etiologías múltiples con resultados anatómicos similares, por ejemplo la holoprosencefalia y la lisencefalia. Un esquema basado solamente en las mutaciones genéticas no es práctico porque pueden estar involucrados varios genes en forma secuencial o secundaria. Los mismos genes sirven para diferentes funciones en diferentes etapas y están relacionados con múltiples órganos y sistemas. Algunas malformaciones complejas, otra vez, como ejemplo, la holoprosencefalia, son causadas por varios genes defectuosos no relacionados. Además, una clasificación puramente genética sería muy inflexible para incorporar algunos criterios anatómicos o para incluir malformaciones focales secundarias a lesiones adquiridas en la vida fetal. Por lo tanto, la base para el esquema propuesto son los trastornos en los modelos de expresión genética: los gradientes de polaridad de los ejes del tubo neural (sobre regulación y baja regulación de influencias genéticas), la segmentación (deleciones de neurómeros específicos; expresión ectópica), mutaciones que causan cambios en la estirpe celular (gangliocitoma displásico del cerebelo; diferenciación de fibras musculares estriados en el cerebro), y genes o moléculas específicos que regulan la migración de los neuroblastos en su curso inicial (filamin-1), medio (LIS1; DCX) o tardío (reelin; L1-CAM). Conclusiones. Las clasificaciones de las disgenesias cerebrales que sirvieron bien para todo el siglo XX, ya no serán adecuadas para el siglo XXI. Sin duda este esquema va a tener muchas revisiones en el futuro, pero por lo menos es un punto de partida al utilizar los datos disponibles en la actualidad (AU)

Congenital malformations of the nervous system: a neuropathologic perspective.

Neurologists and neuropathologists face new diagnostic challenges in the era of molecular genetics and should strive to integrate descriptive morphologic data with the emerging understanding of genetic programming of ontogenesis. This integration is of particular importance in defining and understanding malformations of the developing brain and spinal cord. Both medical specialists have much to contribute to the clinical neurobiology of the twenty-first century, but new tools and perspectives are required.

Neuropathologic research strategies in holoprosencephaly.

Hypotheses are presented to explain the pathogenesis of several clinical features of holoprosencephaly, and neuropathologic approaches to testing these hypotheses are suggested. The traditional morphologic classification of holoprosencephaly into alobar, semilobar, and lobar forms is grades of severity, and each occurs in all of the genetic mutations known. Of the four defective genes identified as primary in human holoprosencephaly, three exhibit a ventrodorsal gradient of expression (SHH, SIX3, and TGIF) and one a dorsoventral gradient (ZIC2). But, in addition to the vertical axis, genes expressed in the neural tube also may have rostrocaudal and mediolateral gradients in the other axes. These other gradients may be equally as important as the vertical. If the rostrocaudal gradient extends as far as the mesencephalic neuromere, it may interfere with the formation, migration, or apoptosis of the mesencephalic neural crest, which forms membranous bones of the face, orbits, nose, and parts of the eyes, and may explain the midfacial hypoplasia seen in many, but not all, children with holoprosencephaly. This rostrocaudal gradient also causes noncleavage of the caudate nucleus, thalamus, and hypothalamus and contributes to the formation of the dorsal cyst of holoprosencephaly, which is probably derived from an expanded suprapineal recess of the 3rd ventricle with secondary dilation of the telencephalic monoventricle and at times may produce a unique transfontanellar encephalocele. The extent of the mediolateral gradient may explain the severe disorganization of cerebral cortical architecture in medial parts of the forebrain and normal cortex in lateral parts, including the radial glial fibers. This preserved lateral cortex may explain why some children with holoprosencephaly have better intellectual function than expected and may also be important in the pathogenesis of epilepsy, by contrast with malformations such as lissencephaly, in which the entire cerebral cortex is involved. Epilepsy in some, but not all, cases also may be related to the sequential maturation of axonal terminals in relation to the neurons they innervate. Diabetes insipidus is a complication in a majority of patients; other neuroendocrinopathies occur less frequently. Secondary down-regulation of the OTP gene or of downstream genes such as BRN2 or SIM1 may result in failure of terminal differentiation of magnocellular neurons of the supraoptic and paraventricular hypothalamic nuclei. Disoriented radial glial fibers or abnormal ependyma may allow aberrant migration of neuroepithelial cells into the ventricle. A new classification of holoprosencephaly is needed to integrate morphologic and genetic criteria.

Molecular genetic classification of central nervous system malformations.

Traditional schemes of classifying nervous system malformations are based on descriptive morphogenesis of anatomic processes of ontogenesis, such as neurulation, neuroblast migration, and axonal pathfinding. This proposal is a first attempt to incorporate the recent molecular genetic data that explain programming of development etiologically. A scheme based purely on genetic mutations would not be practical, in part because only in a few dysgeneses are the specific defects known, but also because several genes might be involved sequentially and many genes inhibit or augment the expression of others. The same genes serve different functions at different stages and are involved in multiple organ systems. Some complex malformations, such as holoprosencephaly, result from several unrelated defective genes. Finally, a pure genetic classification would be too inflexible to incorporate some anatomic criteria. The basis for the proposed scheme is, therefore, disturbances in patterns of genetic expression; polarity gradients of the axes of the neural tube (eg, upregulation or downregulation of genetic influences); segmentation (eg, deletions of specific neuromeres, ectopic expression); mutations that cause change in cell lineage (eg, dysplastic gangliocytoma of cerebellum, myofiber differentiation within brain); and specific genes or molecules that mediate neuroblast migration in its early (eg, filamin-1), middle (eg, LIS1, double-cortin), or late course (eg, reelin, L1-CAM). The proposed scheme undoubtedly will undergo many future revisions, but it provides a starting point using currently available data.

Kearns-Sayre syndrome with a novel mitochondrial DNA deletion.

We describe a 17-year-old boy with a clinical neurologic picture consistent with Kearns-Sayre syndrome. His manifestations included progressive external ophthalmoplegia, bilateral ptosis, retinitis pigmentosa, and muscle weakness. He was found to harbor an abundant novel deletion in skeletal muscle mitochondrial DNA. Biochemical analysis of the patient's biopsied skeletal muscle showed that the specific activities of all four respiratory complexes with mitochondrial DNA-encoded subunits were markedly reduced in contrast to normal activity levels of entirely nuclear DNA-encoded enzyme activities (eg, complex II and citrate synthase). Ultrastructural analysis also indicated the presence of strikingly abnormal mitochondria with both unusual cristae and frequent paracrystalline inclusions. The great amount of the deleted mitochondrial DNA in this patient's muscle, as well as the concomitant reduction in specific respiratory complex activity, suggests that the mitochondrial DNA deletion plays a role in the pathogenesis of this neurologic disease.

Skeletal muscle mitochondrial defects in nonspecific neurologic disorders.

A group of 25 children (5 months to 20 years of age) presenting with intractable seizures, developmental delay, and severe hypotonia, who did not fall into the known categories of mitochondrial encephalomyopathies, underwent muscle biopsy for evaluation of mitochondrial function and were compared with age-matched control subjects. Biopsied skeletal muscle was analyzed for six mitochondrial enzyme-specific activities, mitochondrial DNA point mutations and deletions, and mitochondrial DNA levels. The data reveal a high incidence of specific mitochondrial enzyme activity defects. Reduced activity levels were evident in complex I (11 patients), III (24 patients), IV (nine patients), and V (10 patients). Two patients also exhibited pronounced reduction in mitochondrial DNA levels (80% reduction compared with control subjects). Two patients manifested increased levels of 5-kb and 7.4-kb mitochondrial DNA deletions. Pathogenic mutations previously described in association with mitochondrial encephalomyopathies were not evident. The data suggest that mitochondrial dysfunction, including extensive defects in specific enzyme activities, may be frequently present in children with seizures, developmental delay, and hypotonia that do not fall within the known mitochondrial encephalomyopathies. These mitochondrial deficiencies can be primarily ascertained by biochemical analysis and are rarely accompanied by mitochondrial ultrastructural changes. The molecular basis of these defects, their role in these disorders, and potential treatment warrant further study.

[How to construct a neural tube: molecular genetics of neuro-embryological development]. / Cómo construir un tubo neural: la genética molecular del desarrollo neuroembriológico.

INTRODUCTION: The 'new neuroembryology' combines classical morphogenesis with new molecular genetic data on the programming of neural differentiation and the interactions of transcription products of various developmental genes. DEVELOPMENT: The neuroepithelium is generated from the primitive (Hensen's) node in birds and mammals, homologous with the dorsal lip of the amphibian gastula. The developing neural placode is 'induced' by the notochord, which initiates the differentiation of the floor plate, a ventral midline ependyma. This induction is effected by a gene called Sonic hedgehog, which also is a strong ventralizing influence and induces motor neuron differentiation. Various families of genes program neural tube differentiation with dorsoventral or ventrodorsal gradients, rostrocaudal gradients and mediolateral gradients. Genes that establish the primordial axes of differentiation are 'organizer genes' and those involved with the identity of specific structures are 'regulatory genes'. Some developmental genes continue to be expressed in adult life and preserve the unique identity of specific cellular types. The neural tube is divided into compartments or segments known as neuromeres (or rhombomeres in the hindbrain) with physical and chemical barriers that limit cell migration between segments; the entire spinal cord is formed from rhombomere 8. Both extrinsic and intrinsic mechanical factors contribute to bend the neural placode to form the neural tube [REV NEUROL 1999; 28: 110-6].

Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system.

Synaptophysin is a protein of synaptic vesicles and may be demonstrated in tissue sections of human brain and spinal cord by immunocytochemistry using a monoclonal antibody. Synaptophysin immunoreactivity was studied in paraffin-embedded sections of the central nervous system (CNS) in 14 normal human fetuses and neonates ranging in age from 8 to 41 weeks gestation, and in three brains with heterotopic neurons or malformations. A progressive expression of synaptophysin is seen in axonal terminals within grey matter in various parts of the CNS, beginning in the ventral horns of the spinal cord and brainstem tegmentum at 12-14 weeks. In the cerebellum, the molecular layer shows a band of reactivity from 18 weeks; by term two parallel bands of synaptophysin are seen in the molecular layer and reactivity also is demonstrated in the Purkinje and internal granular layers. In the cerebral neocortex, the molecular zone has weak synaptophysin reactivity as early as 10 weeks, though reactivity is not detected in the deep layers of the cortical plate until 19 weeks and in layers 2-4 until 25 weeks gestation. Synaptophysin reactivity is strong at the surface of neurons but not detected in their somatic cytoplasm; coarsely beaded reactivity within the neuropil probably corresponds to synaptic vesicles in terminal axons. Similar granular synaptophysin reactivity is seen around heterotopic neurons in the subcortical white matter, in dysgenesis of the cerebellar cortex and in the residual anencephalic forebrain. Thermal intensification by heating the incubating solution in a microwave oven often enhances immunoreactivity because of more complete antigen retrieval and is recommended for tissue stored in formalin or in paraffin for long periods. Synaptophysin provides a useful tissue marker of synaptogenesis during normal development and in cerebral dysgeneses, and may provide useful correlations with functional imaging of the brain in living patients. Used in conjunction with other neuronal markers, the expression of synaptophysin in terminal axons of distant neurons, in temporal relation to the maturation of the neurons they innervate, may provide clues to the pathogenesis of epilepsy in early infancy.

[Neuroblastic migration: embryological aspects and mechanisms]. / La migración neuroblástica: aspectos embriológicos y mecanismos.

INTRODUCTION: The migration of immature neurons of the cerebrum is genetically programmed from the primitive neuroepithelium before the end of the final mitotic cycle. The orientation of the mitotic spindle determines when a neuroepithelial cell is ready to start migration and the proportion of major genetic material it is destined to receive. DEVELOPMENT: The gene LIS1, defective in lissencephaly type 1 of Miller and Dieker, is expressed in the neuroepithelial cells, in the ependyma and the Cajal-Retzius neurons. These transitory fetal cells are the first neurons of the cerebral cortex. Most of the neurons of the cortical plate arrive by means of glial radial cells which guide them towards their destination. Cell adhesion molecules from the neuroblasts themselves, the glial radial cell, the extracellular matrix and perhaps the ependymal cells are important in adhering the neuroblasts to the glial radial cells. Genetic deficiency of these molecules results in defective migration. The mechanism of cellular movement is still not fully understood. Disorders of migration may also be induced by non-genetic factors, such as infarcts or other lesions which damage or destroy the glial radial fibres during the fetal period.

Histochemistry and immunocytochemistry of the developing ependyma and choroid plexus.

The adult human ependyma expresses no intermediate filament proteins or secretory proteins; the fetal ependyma shows strong immunocytochemical (ICC) expression of vimentin, glial fibrillary acidic protein (GFAP), cytokeratins (CKs) of high molecular weight, glycoproteins, and S-100beta protein. Each has a precise and specific spatial distribution within the developing ependyma and a predictable time of appearance and regression in each region of the ventricular system. Several are coexpressed, but some appear earlier or persist longer than others. Secretory proteins of ependymal cells are important in several developmental processes such as the guidance of axonal growth cones. GFAP is not expressed in the floor plate ependyma at any stage of development, unlike vimentin and CK. The choroid plexus epithelium is a specialized ependyma, with an ICC profile that differs from the surface ependyma: vimentin, CK, and S-100beta protein continue to be expressed throughout fetal and adult life, but GFAP is not expressed. Certain cerebral malformations are associated with specific ICC abnormalities: ependymal S-100beta protein continues to be immunoreactive in disorders of neuroblast migration; ependymal vimentin is focally upregulated in Chiari malformations and congenital aqueductal stenosis. Other mammalian and nonmammalian species have characteristic profiles of ependymal immunoreactivity to the same proteins expressed in humans but exhibit interspecific differences.

Neuronal nuclear antigen (NeuN): a marker of neuronal maturation in early human fetal nervous system.

Neuronal nuclear antigen (NeuN) immunocytochemistry was studied in 15 normal human fetal nervous systems of 8-24 weeks gestation and in four term neonates. Material was derived from products of conception or from autopsy. Antigen retrieval was enhanced for immunocytochemistry by microwave heating of formalin-fixed paraffin sections. NeuN appears highly specific as a marker of neuronal nuclei in human fetal brain. Only rare nuclei are recognized in the germinal matrix. Cerebellar external granule cells are more strongly immunoreactive than postmigratory internal granule cells until 24 weeks gestation; by term most internal and only a few external granule cells are recognized by NeuN antibody. In the cerebrum, some reactive nuclei are demonstrated along radial glial fibers, particularly near the cortical plate. Within the cortical plate, only deep neurons (future layers 4-6) are marked at 19-22 weeks, but by 24 weeks most neurons in the cortical plate exhibit immunoreactivity, though at term some in layer 2 are still non-reactive. Some neurons fail to be recognized by NeuN at all ages: Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells are examples. Despite their common origin in the cerebellar tubercle, basal pontine neurons are strongly reactive even before midgestation, hence NeuN does not predict embryonic origin. Neurons of dorsal root and cranial nerve ganglia are reactive even at 8 weeks. This study of normal fetal central nervous system provides a basis for neuropathological evaluation and as a prelude to applications in cerebral dysgeneses.

The pachygyria-polymicrogyria spectrum of cortical dysplasia in X-linked hydrocephalus.

Neural cell adhesion molecules (CAM) play important roles in neural development, neurite outgrowth, axonal guidance, fasciculation and synapse formation. Neuropathological studies of X-linked hydrocephalus (XLH) associated with L1 CAM mutations emphasize marked hypoplasia of the pyramidal tract, agenesis of the corpus callosum and septum pellucidum, and a thin cerebral mantle with hypoplastic white matter, but there are no detailed studies of the cerebral cortex in the literature. We report clinical, neuroimaging, and neuropathological findings in three boys with XLH. All had severe congenital hydrocephalus with marked thinning of the cerebral mantle and severe development disabilities. The brain specimens from the three boys showed both pachygyria and polymicrogyria, hypoplasia of the medullary pyramids, hypoplasia of the corpus callosum, small anterior commissure, hypoplasia and poorly differentiated hippocampi. A small but patent aqueduct was present in all three brains. Despite the extensive cerebral malformations, the cortex in all three brains showed normal-appearing laminar cortical neuronal architecture and absence of gliosis. In XLH, it is likely that the poor developmental outcome of spasticity, contractures and severe mental retardation results from a disturbance of neuronal connectivity, fasciculation, and synapse formation rather than aqueductal stenosis, increased intracranial pressure, or abnormal neuroblast migration.