Peter B. Crino

The clinicopathologic spectrum of focal cortical dysplasias: A consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission†

Purpose:  Focal cortical dysplasias (FCD) are localized regions of malformed cerebral cortex and are very frequently associated with epilepsy in both children and adults. A broad spectrum of histopathology has been included in the diagnosis of FCD. An ILAE task force proposes an international consensus classification system to better characterize specific clinicopathological FCD entities.

Molecular Characterization of the Dendritic Growth Cone: Regulated mRNA Transport and Local Protein Synthesis

The molecular mechanisms that regulate growth cone guidance of dendrite outgrowth remain to be elucidated. We hypothesized that mRNA localization in dendritic growth cones and their local protein synthesis may be important for growth cone functioning. The appearance of 23 of 31 growth cone mRNAs was developmentally regulated. Also, alteration of growth cone morphology affected the relative levels of three mRNAs. Finally, using single dendrite transfection, it was shown that local protein synthesis occurs in dendrites and growth cones. A heterogeneous population of mRNAs exists in dendritic growth cones of cultured hippocampal neurons whose relative abundances are developmentally regulated and can vary with changes in growth cone physiology. The demonstration of protein synthesis in growth cones suggests that translation of the localized mRNAs may contribute to regulation of growth cone motility and dendrite outgrowth.

The Tuberous Sclerosis Complex

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that results from mutations in the TSC1 or TSC2 genes and is associated with hamartoma formation in multiple organ systems. The neurological manifestations of TSC are particularly challenging and include infantile spasms, intractable epilepsy, cognitive disabilities, and autism. Progress over the past 15 years has demonstrated that the TSC1 or TSC2 encoded proteins modulate cell function via the mTOR signaling cascade and serve as keystones in regulating cell growth and proliferation. The mTOR pathway provides an intersection for an intricate network of protein cascades that respond to cellular nutrition, energy levels, and growth‐factor stimulation. In the brain, TSC1 and TSC2 have been implicated in cell body size, dendritic arborization, axonal outgrowth and targeting, neuronal migration, cortical lamination, and spine formation. Antagonism of the mTOR pathway with rapamycin and related compounds may provide new therapeutic options for TSC patients.

mTOR cascade activation distinguishes tubers from focal cortical dysplasia.

Balloon cells (BCs) in focal cortical dysplasia (FCD) and giant cells (GCs) in tubers of the tuberous sclerosis complex (TSC) share phenotypic similarities. TSC1 or TSC2 gene mutations in TSC lead to mTOR pathway activation and p70S6kinase (phospho‐S6K) and ribosomal S6 (phospho‐S6) protein phosphorylation. Phospho‐S6K, phospho‐S6, and phospho‐S6K–activated proteins phospho‐STAT3 and phospho‐4EBP1 were detected immunohistochemically in GCs, whereas only phospho‐S6 was observed in BCs. Expression of four candidate gene families (cell signaling, cell adhesion, growth factor/receptor, and transcription factor mRNAs) was assayed in single, microdissected phospho‐S6–immunolabeled BCs and GCs as a strategy to define whether BCs and GCs exhibit differential transcriptional profiles. Among 60 genes, differential expression of 24 mRNAs distinguished BCs from GCs and only 4 genes showed similar expression profiles between BCs and GCs. Tuberin mRNA levels were reduced in GCs from TSC patients with TSC2 gene mutations but were unchanged in BCs. Phospho‐S6K, ‐S6, ‐STAT3, and ‐4EBP1 expression in GCs reflects loss of hamartin‐tuberin–mediated mTOR pathway inhibition. Phospho‐S6 expression alone in BCs does not support mTOR cascade activation in FCD. Differential gene expression profiles in BCs and GCs supports the hypothesis that these cell types derive by distinct pathogenic mechanisms. Ann Neurol 2004

PREPARATION OF CDNA FROM SINGLE CELLS AND SUBCELLULAR REGIONS

Phenotypic characterization of cells in conjunction with single-cell mRNA analysis, which yields information regarding expression of multiple genes in individual neurons, facilitates a detailed and comprehensive view of neuronal cell biology. More specifically, the aRNA amplification method has provided an approach to analyze mRNA levels in single cells that have been phenotypically characterized on the basis of electrophysiology, morphology, and/or protein expression. In this way, relative mRNA abundances can be directly assayed from a well-defined population of neurons. The concept of expression profiling led to the development of robotics methods for arraying thousands of cDNAs on microarrays. These cDNA arrays can be screened with labeled aRNA or cDNA to generate a molecular fingerprint of a specific cell type, disease state, or therapeutic efficacy. A broad view of how gene expression is altered in single neurons affected by a particular disease process may provide clues to pathogenetic disease mechanisms or avenues for therapeutic interventions. The use of mRNA profiles to produce diagnostics and therapeutics is called transcript-aided drug design (TADD). When coupled with single-cell resolution, TADD promises to be an important tool in diagnosis of disease states, as well as provide a blueprint on which to develop therapeutic strategies. For example, mRNA abundances in an individual diseased cell may increase, decrease, or remain constant, and thus it is possible that a pharmaceutical alone or in combination with other drugs may be specifically designed to restore mRNA abundances to a normal state. Alternatively, if functional protein levels parallel the mRNA level changes, then drugs targeting the function of the proteins translated from these altered mRNAs may prove to be therapeutic. One promise of such an approach is that information about mRNA abundances that are altered in a diseased cell may provide new therapeutic indications for existing drugs. For example, if the abundance of mRNA for the beta-adrenergic receptor is altered as shown by the microarrays for a particular disease, already available adrenergic receptor agonists or antagonists that had not previously been used in this particular disease paradigm may prove to be therapeutically efficacious. The expression profile of a given cell is a measure of the potential for protein expression. Proteins are generally the functional entities within cells and differences in protein function often result in disease. The ability to monitor the coordinate changes in gene expression, in single phenotypically identified cells, that correlate with disease will provide unique insight into the expressed genetic variability of cells and will likely furnish unforeseen insight into the underlying cellular mechanisms that produce disease etiology.

Inflammation in epilepsy: Clinical observations

Over the past decade, an increasing number of observations indicate that activation of inflammatory processes occurs in variety of focal epilepsies. Understanding the feature and consequences of neuroinflammation, including the contribution to development and perpetuation of seizures, as well as to mood or cognitive dysfunction, is a major requisite for delineating its role in epilepsy. The present article discusses the most recent observations supporting the involvement of the inflammatory response in human focal epilepsy. It also evaluates emerging evidence concerning the possibility to identify epilepsy‐associated inflammatory biomarkers in cerebrospinal fluid and serum, as well as the potential application of neuroimaging approaches to study the inflammatory reactions in chronic epilepsy patients in vivo, aiming to improve the recognition of appropriate patient populations who might benefit from antiinflammatory or immunomodulatory therapies.

Impaired glial glutamate transport in a mouse tuberous sclerosis epilepsy model

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte‐specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT‐1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC. Ann Neurol 2003

mTOR: A pathogenic signaling pathway in developmental brain malformations

The mTOR signaling network functions as a pivotal regulatory cascade during the development of the cerebral cortex. Aberrant hyperactivation of mTOR as a consequence of loss-of-function gene mutations encoding mTOR inhibitor proteins such as TSC1, TSC2, PTEN and STRADα has been recently linked to developmental cortical malformations associated with epilepsy and neurobehavioral disabilities. Investigation of mTOR signaling in these disorders provides for the first time exciting future avenues for assessment of biomarkers, patient stratification and prognostic measures as well as the opportunity for targeted therapy to regulate mTOR activity across all age groups. As we learn more about mTOR and its activity in the developing brain, many challenges will arise that must be overcome before widespread clinical therapeutics can be implemented.

Selective alterations in glutamate and GABA receptor subunit mRNA expression in dysplastic neurons and giant cells of cortical tubers

The molecular pharmacologic basis of epileptogenesis in cortical tubers in the tuberous sclerosis complex is unknown. Altered transcription of genes encoding glutamatergic and γ‐aminobutyric acid (GABA)‐ergic receptors and uptake sites may contribute to seizure initiation and may occur selectively in dysplastic neurons and giant cells. Arrays containing GABA A (GABAAR), GluR, NMDA receptor (NR) subunits, GAD65, the vesicular GABA transporter (VGAT), and the neuronal glutamate transporter (EAAC1) cDNAs were probed with amplified poly (A) mRNA from tubers or normal neocortex to identify changes in gene expression. Increased levels of EAAC1, and NR2B and 2D subunit mRNAs and diminished levels of GAD65, VGAT, GluR1, and GABAAR α1 and α2 were observed in tubers. Ligand‐binding experiments in frozen tuber homogenates demonstrated an increase in functional NR2B‐containing receptors. Arrays were then probed with poly (A) mRNA from single, microdissected dysplastic neurons, giant cells, or normal neurons (n = 30 each). Enhanced expression of GluR 3, 4, and 6 and NR2B and 2C subunit mRNAs was noted in the dysplastic neurons, whereas only the NR2D mRNA was upregulated in giant cells. GABAAR α1 and α2 mRNA levels were reduced in both dysplastic neurons and giant cells compared to control neurons. Differential expression of GluR, NR, and GABAAR mRNAs in tubers reflects cell‐specific changes in gene transcription that argue for a distinct molecular phenotype of dysplastic neurons and giant cells and suggests that dysplastic neurons and giant cells make differential contributions to epileptogenesis in the tuberous sclerosis complex. Ann Neurol 2001;49:67–78

Differential expression of glutamate and GABA-A receptor subunit mRNA in cortical dysplasia

Objective: Focal cortical dysplasia is characterized by disorganized cortical lamination, dysplastic and heterotopic neurons, and an association with epilepsy. The contribution that dysplastic and heterotopic neurons make to epileptogenesis in focal cortical dysplasia is unknown and the phenotype of these cells may be distinct. The authors hypothesized that the expression of genes encoding glutamatergic (glutamate [GluR] and N -methyl-d-aspartate NMDA receptors [NR]) and γ-aminobutyric acid A receptor (GABA A R) subunits is distinct in dysplastic and heterotopic neurons and that changes in receptor gene expression could be defined in a cell-specific pattern. Methods: Single immunohistochemically labeled dysplastic and heterotopic neurons were microdissected from human focal cortical dysplasia specimens obtained during epilepsy surgery. Pyramidal neurons were microdissected from postmortem control cortex and from temporal cortex without dysplasia resected during temporal lobectomy. Poly (A) messenger RNA (mRNA) from single neurons was amplified, radiolabeled, and used to probe complementary DNA (cDNA) arrays containing GluR 1–6 , NR 1A,1B , NR 2A–D , and GABA A Rα 1–6 , and -Rβ 1–3 subunit cDNAs. The relative hybridization intensities of each mRNA-cDNA hybrid were quantified by phosphorimaging. Results: GluR, NR, and GABA A R subunit mRNA expression did not differ between control neurons and nondysplastic epilepsy specimens. Expression of GluR 4 , NR 2B , and NR 2C subunit mRNA was increased, and NR 2A and GABA A Rβ 1 subunit mRNA was decreased in dysplastic compared with pyramidal and heterotopic neurons. In contrast, GABA A Rα 1 , -Rα 2 , and -Rβ 2 as well as GluR 1 mRNA levels were reduced in both dysplastic and heterotopic neurons. Conclusions: Differential expression of GluR, NR, and GABA A R mRNA in dysplastic and heterotopic neurons demonstrates cell specific gene transcription changes in focal cortical dysplasia. These results suggest that dysplastic and heterotopic neurons may be pharmacologically distinct and make differential contributions epileptogenesis in focal cortical dysplasia.