Julia W. Chang

Dendritic Translocation of RC3/Neurogranin mRNA in Normal Aging, Alzheimer Disease and Fronto-Temporal Dementia

RC3/neurogranin is a postsynaptic protein kinase C (PKC)-/calmodulin-binding substrate implicated in long-term potentiation (LTP) forms of synaptic plasticity. Our previous digoxigenin in situ hybridization (DIG-ISH) studies detected RC3 mRNA in apical dendrites and cell bodies of neurons in the rat cerebral cortex and hippocampus. This observation suggested that RC3 mRNA is selectively translocated to dendrites, where it may be translated locally in response to synaptic activity. To test this hypothesis further, we isolated a full-length cDNA clone of the homologous human RC3 mRNA from a human cortex γGT11 library, determined its nucleotide and predicted amino acid sequences, and performed mRNA expression studies in cerebral cortex from normal human patients and from patients with Alzheimer disease (AD) and fronto-temporal dementia (FTD). The human cDNA clone detects a single ≈1.3 kb mRNA whose nucleotide sequence is 73% similar to the rat nucleotide sequence and 96% similar to its amino acid sequence. DIG-ISH studies detect robust staining of RC3 mRNA in cell bodies of numerous neurons throughout Layers II-VI and in both apical and basal dendrites of pyramidal neurons in human neocortex (temporal/frontal). We conclude that dendritic targeting of RC3 mRNA is conserved in human brain. In AD neocortex tissue, there is little or no evidence for RC3 mRNA translocation to dendrites, while in FTD neocortex, targeting of RC3 mRNA to apical dendrites is preserved. Comparative studies in AD and FTD point to the potential importance of synapse integrity and the dendritic cytoskeleton in RC3 mRNA targeting in the human neocortex.

Enhanced GABAergic Network and Receptor Function in Pediatric Cortical Dysplasia Type IIB Compared with Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) and cortical dysplasia Type IIB (CDIIB) share histopathologic features that suggest similar epileptogenic mechanisms. This study compared the morphological and electrophysiological properties of cortical cells in tissue from pediatric TSC (n=20) and CDIIB (n=20) patients using whole-cell patch clamp recordings and biocytin staining. Cell types were normal-appearing and dysmorphic-cytomegalic pyramidal neurons, interneurons, and giant/balloon cells, including intermediate neuronal-glial cells. In the cortical mantle, giant/balloon cells occurred more frequently in TSC than in CDIIB cases, whereas cytomegalic pyramidal neurons were found more frequently in CDIIB. Cell morphology and membrane properties were similar in TSC and CDIIB cases. Except for giant/balloon and intermediate cells, all neuronal cell types fired action potentials and displayed spontaneous postsynaptic currents. However, the frequency of spontaneous glutamatergic postsynaptic currents in normal pyramidal neurons and interneurons was significantly lower in CDIIB compared with TSC cases and the GABAergic activity was higher in all neuronal cell types in CDIIB. Further, acutely dissociated pyramidal neurons displayed higher sensitivity to exogenous application of GABA in CDIIB compared with TSC cases. These results indicate that, in spite of similar histopathologic features and basic cell membrane properties, TSC and CDIIB display differences in the topography of abnormal cells, excitatory and inhibitory synaptic network properties, and GABA(A) receptor sensitivity. These differences support the notion that the mechanisms of epileptogenesis could differ in patients with TSC and CDIIB. Consequently, pharmacologic therapies should take these findings into consideration.

Differential expression of interferon-γ and chemokine genes distinguishes Rasmussen encephalitis from cortical dysplasia and provides evidence for an early Th1 immune response

BackgroundRasmussen encephalitis (RE) is a rare complex inflammatory disease, primarily seen in young children, that is characterized by severe partial seizures and brain atrophy. Surgery is currently the only effective treatment option. To identify genes specifically associated with the immunopathology in RE, RNA transcripts of genes involved in inflammation and autoimmunity were measured in brain tissue from RE surgeries and compared with those in surgical specimens of cortical dysplasia (CD), a major cause of intractable pediatric epilepsy.MethodsQuantitative polymerase chain reactions measured the relative expression of 84 genes related to inflammation and autoimmunity in 12 RE specimens and in the reference group of 12 CD surgical specimens. Data were analyzed by consensus clustering using the entire dataset, and by pairwise comparison of gene expression levels between the RE and CD cohorts using the Harrell-Davis distribution-free quantile estimator method.ResultsConsensus clustering identified six RE cases that were clearly distinguished from the CD cases and from other RE cases. Pairwise comparison showed that seven mRNAs encoding interferon-γ, CCL5, CCL22, CCL23, CXCL9, CXCL10, and Fas ligand were higher in the RE specimens compared with the CD specimens, whereas the mRNA encoding hypoxanthine-guanine phosphoribosyltransferase was reduced. Interferon-γ, CXCL5, CXCL9 and CXCL10 mRNA levels negatively correlated with time from seizure onset to surgery (P <0.05), whereas CCL23 and Fas ligand transcript levels positively correlated with the degree of tissue destruction and inflammation, respectively (P <0.05), as determined from magnetic resonance imaging (MRI) T2 and FLAIR images. Accumulation of CD4+ lymphocytes in leptomeninges and perivascular spaces was a prominent feature in RE specimens resected within a year of seizure onset.ConclusionsActive disease is characterized by a Th1 immune response that appears to involve both CD8+ and CD4+ T cells. Our findings suggest therapeutic intervention targeting specific chemokine/chemokine receptors may be useful in early stage RE.

In Rasmussen Encephalitis, Hemichannels Associated with Microglial Activation are linked to Cortical Pyramidal Neuron Coupling: A Possible Mechanism for Cellular Hyperexcitability

Rasmussen encephalitis (RE) is a rare but devastating condition, mainly in children, characterized by sustained brain inflammation, atrophy of one cerebral hemisphere, epilepsy, and progressive cognitive deterioration. The etiology of RE‐induced seizures associated with the inflammatory process remains unknown.

Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures

Brain and primary neuron fractions enriched in synaptic terminals are important tools for neuroscientists in biochemical, neuroanatomical and physiological studies. We describe an annotated updated micro-method for preparing synaptoneurosomes (SNs) enriched in presynaptic and postsynaptic elements. An easy to follow, step-by-step, protocol is provided for making SNs from small amounts of mammalian brain tissue. This includes novel applications for material obtained from human neurosurgical procedures and primary rat neuronal cultures. Our updated method for preparing SNs using smaller amounts of tissue provides a valuable new tool and expands the capabilities of neuroscientists.

Evidence for Resident Memory T Cells in Rasmussen Encephalitis

Rasmussen encephalitis (RE) is a rare pediatric neuroinflammatory disease of unknown etiology characterized by intractable seizures, and progressive atrophy usually confined to one cerebral hemisphere. Surgical removal or disconnection of the affected cerebral hemisphere is currently the only intervention that effectively stops the seizures. Histopathological evaluation of resected brain tissue has shown that activated brain resident macrophages (microglia) and infiltrating T cells are involved in the inflammatory reaction. Here, we report that T cells isolated from seven RE brain surgery specimens express the resident memory T cell (TRM) marker CD103. CD103 was expressed by >50% of CD8+ αβ T cells and γδ T cells irrespective of the length of time from seizure onset to surgery, which ranged from 0.3 to 8.4 years. Only ~10% of CD4+ αβ were CD103+, which was consistent with the observation that few CD4+ T cells are found in RE brain parenchyma. Clusters of T cells in brain parenchyma, which are a characteristic of RE histopathology, stained for CD103. Less than 10% of T cells isolated from brain specimens from eight surgical cases of focal cortical dysplasia (FCD), a condition that is also characterized by intractable seizures, were CD103+. In contrast to the RE cases, the percent of CD103+ T cells increased with the length of time from seizure onset to surgery. In sections of brain tissue from the FCD cases, T cells were predominantly found around blood vessels, and did not stain for CD103. The presence of significant numbers of TRM cells in RE brain irrespective of the length of time between clinical presentation and surgical intervention supports the conclusion that a cellular immune response to an as yet unidentified antigen(s) occurs at an early stage of the disease. Reactivated TRM cells may contribute to disease progression.