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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0003
Embryonic and fetal ontogenesis of the central nervous system is realized by genetically programmed developmental processes with precise timing of onset. Both anatomical development and timing are altered in brain malformations. Keratan sulfate (KS) proteoglycan is a key axonal pathfinding guide and insulator of tracts and fascicles within the central nervous system and an essential determinant of the equilibrium of excitation and inhibition at the level of the individual neuron during development. KS is selective by repelling glutamatergic axons and facilitating GABAergic axons at sites of synapse formation. Its demonstration by immunocytochemistry provides another perspective for understanding epileptogenesis at the cellular level in brain resections for epilepsy and at autopsy, including fetal tissues. Examples of malformations in which epileptogenesis is influenced by KS are polymicrogyria, schizencephaly, focal cortical dysplasias, holoprosencephaly, and Down syndrome.
Keratan sulfate (KS) proteoglycan is a long glycosaminoglycan chain and is one of the most important of several axonal guidance molecules in the developing nervous system. Glycans are a diverse group of sugar-based polymers that coat cells and attach to proteins, forming glycoproteins. In the central nervous system (CNS), KS is secreted by astrocytes and is present mainly in the extracellular matrix (Uchimura 2015; Caterson & Melrose 2018; Pomin & Mulloy 2018; Melrose 2018). It also is expressed in the peripheral nervous system within autonomic ganglia in embryos and fetuses, as well as around differentiating ganglion cells of peripheral neuroblastic tumors (e.g., neuroblastoma, ganglioneuroblastoma, ganglioneuroma) in children, especially toddlers (Sarnat & Yu 2022 ; Sarnat et al. 2023). KS is widespread in the body with highest concentration in cornea, cartilage, and brain (Melrose 2018). It may be demonstrated by biochemical assay and immunocytochemically as granulo-filamentous deposits in the extracellular spaces but is not evident ultra-structurally by transmission electron microscopy. KS also is invisible to magnetic resonance imaging (MRI) and other present neuroimaging modalities. KS is highly conserved in evolution, expressed in all vertebrates and most invertebrates.
Nearly all malformations of the CNS, focal and generalized, can be understood best in the context of interruption of or interference with one or more developmental processes. These processes mostly occur simultaneously rather than sequentially, though a few only involve early ontogenesis, such as gastrulation and establishment of the axes of the neural tube; others mainly occur late, such as myelination. Some occur over a short period of time and others over very extended periods. In the case of myelination, for example, the corpus callosum initiates myelination at about 2–3 months postnatally by tissue stains and 3–4 months by MRI visualization; the corticospinal tract begins myelination in the corona radiata and, internal capsule and cerebral peduncles in the late third trimester of gestation and is not complete until late adolescence. The last pathway to myelinate in the human brain is the ipsilateral frontotemporal association bundle, at 32 years of age (Yakovlev & Lecours 1967).
Semantics are important for precise scientific communication and specific terms should not be degraded to general terms by applying them incorrectly (Sarnat 2007; Sarnat & Suchet 2023). Technically, the term “dysplasia” refers to abnormal growth of a tissue or cells (e.g., hemimegalencephaly; megalocytic dysplastic neurons in focal cortical dysplasia type II); “dysgenesis” refers to abnormal tissue architecture (e.g., the abnormally laminated cerebral cortex with displaced, disoriented neurons in holoprosencephaly and lissencephalies). In the literature, these two terms often are used interchangeably. “Heterotopic cells” are displaced within their organ of origin, such as subcortical white matter neurons; “ectopic cells” are displaced outside their organ of origin, such as isolated neurons within the leptomeninges.
Equally important to interference with developmental pathways is timing of events: onset of clinical expression of a genetic mutation or embryonic or fetal exposure to a teratogenic substance or congenital infection (Sarnat 2016). A distinction between etiology and pathogenesis is important to understanding both normal and abnormal neural development. A genetic mutation or a teratogenic toxin is etiological in malformations, but pathogenesis depends upon the developmental processes affected. Genetic expression and the onset and interaction of various developmental genes determine programmed progress of anatomical changes during normal ontogenesis.
The timing of onset of genetic expression in the 33 mitotic cycles of the periventricular neuroepithelium determines the difference between limited cortical lesions of focal cortical dysplasia type II and hemimegalencephaly (Sarnat 2019), further confirmed because the postzygotic somatic mutation is the same (D’Gama et al. 2015; Mühlebner et al. 2019). Another example is determining by coronal MRI whether agenesis of the corpus callosum (whose first pioneer axons traverse the midline at 10 weeks gestation) is accompanied by agenesis of the anterior commissure (whose first axons cross the midline at 7 weeks gestation). Thus callosal agenesis with a preserved anterior commissure indicates that the onset of genetic expression or an exogenous teratogenic neurotoxin occurred between 7 and 10 weeks or before 7 weeks (Sarnat 2019). Partial agenesis of the corpus callosum also is related to timing, since this commissure forms in an antero-posterior gradient.
Timing also can be problematic at times. Focal cortical dysplasia type IIIa (ILAE Neuropathological Classification) is hippocampal sclerosis associated with either focal dysplasia type I or II in the temporal neocortex (Blümcke et al. 2002, 2011; Najm et al. 2018). But the cortical lesion is congenital from fetal life, whereas hippocampal sclerosis is always a postnatally acquired lesion, often during adolescence or adult life. It poses the still debated question of whether a chronically discharging epileptic focus in the mesial temporal lobe cortex can induce hippocampal sclerosis over a long period, often years. Hippocampal sclerosis refers not to gliosis, which may also coexist to a variable degree, but rather to loss of pyramidal neurons from Ammon’s horn in particular but sometimes also from the dentate gyrus (Blümcke et al. 2002).
The principal developmental processes may be summarized as follows: gastrulation that produces cephalization and bilateral symmetry; neurulation; establishment of axes of the neural tube and genetic gradients along those axes; neural crest cell migration and differentiation; neuronogenesis and gliogenesis; neuroblast migrations (erroneously called “neuronal migration” but neurons are mature nerve cells than cannot and do not migrate); physiological apoptosis of excessive and redundant neurons; axonal pathfinding; synaptogenesis; myelination (Sarnat & Menkes 2000; Sarnat & Flores-Sarnat 2016; Sarnat et al. 2022).
Another aspect of nervous system development, in addition to tissue architecture reorganization, is maturation of individual neurons, a feature particularly relevant to epileptogenesis. Mounting evidence indicates a commitment to cellular lineage and even to the type of neuron during the “undifferentiated” neuroepithelial cell stage, though this commitment may be changeable at early stages to compensate for cellular loss as in fetal cerebral infarcts. Neuroblastic maturation involves the development of an energy supply system (adenosine triphosphatase [ATP]) to enable a polarized plasma membrane of the cell and a resting membrane potential that can become depolarized. Receptors and ion channels also form in these plasma membranes. Synaptogenesis can then occur that is selective for axons of certain types of neurotransmitters. Axonal sprouting may occur during the course of neuroblast migration, but development of dendrites usually awaits the completion of neuroblast migration and mature position of the neuron. The synthesis of neurotransmitter, axoplasmic flow to transmit the molecules to the axonal terminals, and proliferation of mitochondria to provide energy to this system must be established. Intermediate filaments and cytoplasmic proteins form; primitive early filaments of vimentin and nestin are replaced by neurofilament proteins. In neuroanatomy and neuropathology, a wide variety of proteins can be demonstrated by immunocytochemistry in tissue sections and denote neuronal maturation as well as identify specific types of neurons, such as GABAergic inhibitory interneurons using antibodies against calcium-binding proteins such as calretinin and parvalbumin (Ulfig 2002; Sarnat 2013, 2015). Such tissue markers are useful in demonstrating the ratio of selective losses of glutamatergic neurons that have arrived at the cortical plate by radial migration and GABAergic neurons that have arrived by tangential migration. Individual neurons and other neural cells may be abnormal in some malformations, especially those involving the mTOR signaling pathway and related genetic pathways, so that megalocytic, dysplastic neurons are found in focal cortical dysplasia type II, hemimegalencephaly, and tuberous sclerosis complex. Balloon cells in mTOR disorders are cells of mixed lineage, expressing both neuronal and glial proteins and other features.
An example of how neuropathological sections of developing brain can be mapped for time-linked maturation is synaptophysin, a glycoprotein that is a principal structural element of the membranes of synaptic vesicles regardless of the neurotransmitter contained within the vesicles (Sarnat 2014; Sarnat et al. 2010). In malformations of the cortex, not only is the distribution of synapses altered, but the timing of synaptogenesis also may be delayed or even too early in relation to other processes of neuronal maturation (Sarnat & Flores-Sarnat 2021b). Precocious synapse formation may lead to early formation of epileptogenic circuitry within the cortex and severe infantile epilepsies. Beneath focal cortical dysplasias types I and II are complex synaptic plexi between heterotopic neurons in the U-fiber layer that project into the cortex to join epileptic networks and thus contribute to seizure propagation, though it is uncertain whether these plexi can initiate seizures (Sarnat et al. 2018). These plexi are well demonstrated by synaptophysin immunoreactivity in surgical cortical resections for epilepsy.
Finally, some tissue markers used in neuropathology indicate an epileptic focus regardless of the presence or absence of an underlying lesion or its nature. An example is α-B-crystallin, a heat-shock protein that is normally not expressed but is activated in glial cells of both grey and white matter at an epileptic focus and often has a gradient of disappearance at 2.5 to 3.0 cm away from a focus identified by intraoperative electocorticography (Sarnat & Flores-Sarnat 2009). Potentially it may contribute to determining whether the seizure focus in the brain resection was complete, though further study and confirmation of this marker are needed.
Extracellular matrix molecules that guide embryonic and fetal axonal growth cones do so by selective attraction or repulsion of axons that will secrete specific neurotransmitters which are recognized even before transmitter synthesis is initiated by the soma of the maturing neuroblast; this process has been known for decades as chemotaxis. KS envelops both long and short axonal fascicles within the CNS to ensure that axons within tracts do not exit these tracts before their intended destination. Surrounding of fascicles also prevents random axons emanating from grey matter structures along the course of the trajectory from entering the fascicle en route (Fig. 3–1). KS thus preserves purity of tracts with axons from the same origin and similar destinations, for example, the corticospinal spinothalamic and spinocerebellar tracts.
Control 19-week gestational age (19wk GA) fetus with coronal section at level of the corpus striatum. The anterior limb of the internal capsule shows keratan sulfate enveloping axonal fascicles but ensheathing only a few individual axons. This envelopment (more...)
Keratan sulfate forms a template (but not a scaffold because is chemical, not structural) and thus forms future axonal fascicles before axons are projected into them; KS similarly is expressed in neuroblast migratory pathways at the initiation of migration, synthesized and secreted by radial glial cells (Sarnat HB, manuscript in preparation).
Axonal guidance molecules and especially KS have a property of influencing the epileptic potential of individual neurons. KS repels glutamatergic (excitatory) axons while facilitating GABAergic (inhibitory) axons at developing synaptic junctions (Blümcke et al. 2002, 2011). This property also is useful in axonal pathfinding. KS is strongly expressed in the dorsal median septum of the spinal cord, which is formed from aggregates of basal processes of roof plate ependymal cells of the central canal. It prevents rostrally growing axons of the dorsal columns from decussating improperly and ascending on the wrong side, which would confuse the brain about laterality of sensory stimuli (Snow et al. 1990). Dorsal column axons are glutamatergic, by contrast with spinothalamic tract axons which are GABAergic. After dorsal column axons synapse in the nuclei gracilis and cuneatus at the spino-medullary junction, the neurons of these nuclei project ascending secondary GABAergic axons to the thalamus. During the stage of axonal pathfinding, the neuronal soma does not yet produce or secrete neurotransmitters, but KS still is able to recognize these immature axons that are already genetically programmed to secrete specific transmitters.
Chondrocytes are intensely immunoreactive for KS both in immature cartilage (e.g., embryonic notochord) and in mature cartilage. KS is expressed in cartilaginous structures, both those derived from neural crest (e.g., cranial vault, facial bones, dorsal and lateral spines of vertebrae, ribs) and those cartilages associated with the formation of endochondral bone (e.g., cranial base, vertebral bodies, long bones of the extremities). In diagnostic pathology, KS is useful in identifying cartilage in bony dysplasias and bone tumors in which chondrocytes may be difficult to recognize histologically with certainty. The repulsion of certain axons by KS explains why peripheral nerves do not penetrate cartilage in the fetus or in the adult. Aggrecan is a small physiological modification of keratan sulfate that is essential in weight-bearing cartilages (Hayes & Melrose 2020).
Keratan should not be confused with keratin, a totally different molecule that forms fingernails and toenails, rhinoceros frontal horns and the exoskeletal shells of shrimp. Nor is keratan even similar to cytokeratin, as in cutaneous epithelium.
An important feature of KS is its ability to distinguish between glutamatergic and GABAergic immature axons before these neurotransmitter molecules are synthesized in the neuronal soma. KS surrounds neuronal membranes except for terminal dendritic ramifications and dendritic spines (Fig. 3–2) (Caterson & Melrose 2018; Pomin & Mulloy 2018; Melrose 2018). The lack of KS at dendritic spines enables axo-dendritic synapses to be glutamatergic but prevents these axons from forming axo-somatic synapses while enabling GABAergic axons for synaptogenesis at the neuronal soma. Axo-somatic synapses thus are inhibitory and axo-dendritic synapses are excitatory. KS therefore is an important determinant of the excitatory/inhibitory synaptic ratio and of epileptogenesis at the level of the individual neuron.
Normal frontal cortex of a 10-year-old girl showing by immunocytochemistry granulo-filamentous deposits of keratan sulfate in the extracellular spaces and, in particular, adherent to the somatic membranes of neurons, including proximal axons and dendritic (more...)
During normal ontogenesis, cerebral cortical KS appears first in the molecular zone during the late second trimester and later is seen in the deep cortical layers. In mature brain, the normal pattern is horizontal laminar with highest concentration in the deep cortical layers and subcortical U-fiber layer but not in the deep white matter (Sarnat 2019). The highest concentrations of KS and that appear earliest in the fetal brain are in the thalamus (Fig. 3–3) and globus pallidus, but not the corpus striatum, except for KS surrounding multiple small intrinsic bundles of striatal axons, the “pencil bundles of Wilson” (Fig. 3–1) (Sarnat 2019). KS thus envelops both large and small axonal bundles and fascicles with both long or short trajectories. In the fetal thalamus in the second trimester every astrocyte is filled with recently synthesized KS (Sarnat 2019).
Macroscopic coronal section of a 26-week gestational age (26wk GA) fetus showing intense keratan sulfate reactivity in thalamus (th), weak reactivity in the cortical plate (cp), and none in the hippocampus (hip) or germinal matrix (gm). The operculum (more...)
Polymicrogyria (PMG) is structurally more complex than simply a cluster of smaller than normal gyri that can be identified in neuroimaging, especially MRI, and neuropathologically upon macroscopic (gross) inspection of the cortex at surgery or at autopsy. PMG involves abnormal cortical lamination and fewer than normal neurons but also is characterized by discontinuities in the pial membrane between adjacent microgyria (Judkins et al. 2011; Squier & Jansen 2014; Jansen et al. 2016; Diamandis et al. 2017). Such gaps facilitate fusion of the molecular zones of microgyria and synaptic short-circuitry of axo-dendritic excitatory synapses, shifting the excitatory/inhibitory ratio in favor of excitation that results in epileptogenesis (Sarnat & Flores-Sarnat 2021a). The continuity of molecular zones of adjacent microgyria is well demonstrated by synaptophysin immunoreactivity (Fig. 3–4).
Perisylvian polymicrogyria in a 37.5-week gestational age male fetus who had spontaneous intrauterine death. The microgyria are poorly organized and gaps in the pia mater result in fusion of adjacent gyri. Synaptic short-circuitry results with continuity (more...)
PMG is a frequent cerebral malformation, and its most common location is perisylvian, involving cortex within the lateral cerebral (Sylvian) fissure of all three lips: frontal, temporal, and insular. It may be unilateral or bilateral and also within the depth of a schizencephalic cleft. PMG also occurs as a focal developmental disorder in other parts of the cerebral cortex and sometimes is generalized throughout the cortex of both cerebral hemispheres.
Hypoplastic microgyria and ulegyria also occur in the cortex surrounding porencephalic cysts and in other cortical areas that have suffered chronic fetal ischemia (Kim et al. 2006). It may be induced in experimental animals, such as with focal cortical lesions induced by freezing in neonatal mice (Dos Santis Heringer et al. 2022). Acquired microgyria should be distinguished from polymicrogyria as a primary disorder of cortical development, such as those induced by genetic mutation.
This form of focal cortical dysplasia (FCD) is less frequent than type II and, unlike type II, does not involve megalocytic dysplastic neurons and is not a defect in the mTOR signaling pathway. The typical clinical picture is intractable focal epilepsy in a young child with localization of the focus in the parieto-occipital region (Sarnat 2019).
FCD Ia is characterized histopathologically by abnormal cortical lamination with excessive micro-columns of neurons (Coras et al. 2021; Najm et al. 2018, 2022), resembling normal cortical architecture during the first half of gestation and generalized cortical neuronal micro-columns in some metabolic/genetic disease (Sarnat & Flores-Sarnat 2013). The radial micro-columns of single neurons in a row alternate with synaptic columns as shown by synaptophysin immunocytochemistry (Sarnat & Flores-Sarnat 2013). The KS expression in the cortex in FCD Ia is abnormally patchy in distribution but also contain radial columnar expression (Fig. 3–5) (Sarnat 2019).
Cerebral cortex of a 3-year-old boy with focal epilepsy since infancy, at site of focal cortical dysplasia type Ia which was highly epileptogenic. He was seizure-free after resection of this focus. The keratan sulfate distribution is abnormally patchy (more...)
This malformation of failure of embryonic prosencephalic cleavage into two distinct cerebral hemispheres is associated with severe dyslamination and disorganization of the cerebral cortex with greatest severity near to the midline and less affected cortices of more lateral regions (Sarnat 1992; Golden 1998; Sarnat & Flores-Sarnat 2001). Though one might anticipate that holoprosencephy (HPE) might be a highly epileptogenic condition from the cortical and synaptic disorganization, about 40% of infants and children with HPE do not manifest seizures, though most have abnormal electroencephalograms with multifocal spikes and slow asynchronous background rhythms.
In fetal HPE, keratan sulfate ensheaths many individual axons in addition to enveloping the fascicle itself (Fig. 3–6). This excessive insulation may protect against epilepsy by increasing the threshold for axonal membrane depolatization and propagation of action potentials (Sarnat 2019). In normal developing brain, a minority of axons may be similarly ensheathed, but in HPE they are the majority (Sarnat & Flores-Sarnat 2021b).
Fetus of 22 weeks gestational age with alobar holoprosencephaly; 46XX. This coronal section of the forebrain shows an abnormal axonal aggregate of thalamocortical projections. Individual axons are ensheathed and insulated by keratan sulfate deposits. (more...)
By contrast with a potentially protective effect of KS axonal ensheathment, synaptogenesis in the cortex of fetuses with HPE is precocious and out of synchrony with other processes of neuronal maturation (Sarnat & Flores-Sarnat 2013). This phenomenon may lead to early development of epileptic circuitry and severe infantile epilepsies in the early postnatal period (Sarnat & Flores-Sarnat 2013).
Epilepsy is infrequent in children with trisomy-21, except for a high incidence of infantile spasms, which have different and more complex epileptogenic networks than focal epilepsy (Stafstrom & Konkol 1994). One reason for the low incidence of epilepsy in Down syndrome is that postnatal infants, but not fetuses, experience a progressive atrophy of terminal dendritic arborizations and loss of dendritic spines in the molecular layer of cortex, as shown with Golgi impregnations (Takashima et al. 1981). Axo-dendritic synapses thus are diminished and axo-somatic synapses unaffected, shifting the excitatory/inhibitory ratio toward relatively greater inhibition (Sarnat & Flores-Sarnat 2021a). Adults with Down syndrome have an increasing incidence of late-onset myoclonic epilepsy in middle age and beyond, coincidental with progressive cerebral lesions of Alzheimer disease, another constant feature of Down syndrome (Altuna et al. 2021).
Keratoconus, a progressive non-inflammatory corneal dystrophy that leads to visual loss and eventual blindness, affects about 9% of children with Down syndrome (Kristianslund & Drolsum 2021), though it also occurs sporadically in patients with normal chromosomes. The molecular basis of keratoconus is a KS mutation in the stroma of the cornea (Funderburgh et al. 1989; Midura et al. 1990; Wollensak & Buddecke 1990; Sawaguchi et al. 1991). KS is important for the structure of the stromal matrix and maintenance of corneal transparency (Wentz-Hunter et al. 2001). Not only is total corneal KS usually decreased in keratoconus, though there may be a compensatory overexpression (Wentz-Hunter et al. 2001), but the filamentous KS molecule is truncated into a shorter than normal length (Midura et al. 1990). Corneal scarring may account for part of diminished corneal KS (Sawaguchi et al. 1991). At present no data are available to indicate whether cerebral KS also is diminished or altered during fetal and postnatal development in Down syndrome, but it is a topic of current investigation in our laboratory (Sarnat HB & Aronica E, unpublished).
In fetal hydrocephalus due to aqueductal stenosis, Dandy-Walker spectrum malformation, or other obstructive cause, as well as in “communicating” hydrocephalus in which no obstruction to CSF flow is evident, KS remains normally expressed in the cortex despite severe compression of the cerebral mantle by increased ventricular size and increased intraventricular pressure (Sarnat HB, unpublished data). The preserved KS in this condition may explain, at least in part, why isolated hydrocephalus without cortical malformation is not associated with epilepsy.
In cases of chronic ischaemia of either immature or mature brain and in brains of older children and adults that undergo atrophy with chronic intractable epilepsy, there is loss of KS from its normal cortical distribution and especially from the extracellular matrix. However, in these conditions KS is intensely demonstrated within astrocytes (Fig. 3–7) (Sarnat 2019). These glial cells synthesize KS and probably are trying to compensate for its loss in the neuropil by overproducing KS to re-secrete, though the effort is inadequate.
Severely atrophic lateral temporal cortex of a 16-year-old boy with poorly controlled focal epilepsy since early childhood. Keratan sulfate has disappeared from the cortical extracellular spaces, but it is strongly reactive within scattered astrocytes, (more...)
Embryonic and fetal ontogenesis of the CNS is realized by genetically programmed developmental processes with precise timing of onset. Both anatomical development and timing are altered in brain malformations. Keratan sulfate proteoglycan is an essential determinant of the equilibrium of excitation and inhibition at the level of the individual neuron during development, as well as a key axonal pathfinding guide and insulator of tracts and fascicles within the CNS. KS also is selective by repelling glutamatergic axons and facilitating GABAergic axons at sites of synapse formation. Its demonstration by immunocytochemistry provides another perspective for understanding epileptogenesis at the cellular level in brain resections for epilepsy and at autopsy including fetal tissues.
We are grateful to Dr. Weiming Yu, pediatric anatomical pathologist at Alberta Children’s Hospital, for his contributions and advice to our research program.
The authors declare no relevant conflicts.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
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