Seizure-related Gene Research Articles

Integrative analysis of epilepsy-associated genes reveals expression-phenotype correlations

Epilepsy is a highly prevalent neurological disorder characterized by recurrent seizures. Patients exhibit broad genetic, molecular, and clinical diversity involving mild to severe comorbidities. The factors that contribute to this phenotypic diversity remain unclear. Here we used publicly available datasets to systematically interrogate the expression pattern of 230 epilepsy-associated genes across human tissues, developmental stages, and central nervous system (CNS) cellular subtypes. We grouped genes based on their curated phenotypes into 3 broad classes: core epilepsy genes (CEG), where seizures are the dominant phenotype, developmental and epileptic encephalopathy genes (DEEG) that are associated with developmental and epileptic encephalopathy, and seizure-related genes (SRG), which are characterized by the presence of seizures and gross brain malformations. We find that compared to the other two groups of genes, DEEGs are highly expressed within the adult CNS, exhibit the highest and most dynamic expression in various brain regions across development, and are significantly enriched in GABAergic neurons. Our analysis provides an overview of the expression pattern of epilepsy-associated genes with spatiotemporal resolution and establishes a broad expression-phenotype correlation in epilepsy.

Differential brain expression pattern of Sez6 alternative splicing isoform with deleted transmembrane domain

Seizure-related gene 6 (Sez6) is a transmembrane protein specifically localized on neuronal dendrites and responsible for dendritic branching and synapse formation. Alternative splicing produces three isoforms of Sez6 mRNAs: the dominant isoform encodes a transmembrane-type protein, whereas the two recessive isoforms encode transmembrane and secretory proteins. In the present study, to clarify the differential functions of these isoforms, the expression patterns resulting from Sez6 splicing isoforms were investigated in the mouse brain as well as in cultured neurons. The whole brains were sliced into coronal sections of 1-mm thickness, and brain areas were punched out from these coronal sections. The mRNA levels of each Sez6 isoform in the prefrontal cortex, cingulate cortex, striatum, hippocampus, and amygdala, where Sez6 expression has been reported previously, were analyzed using a qPCR technique, and primary neurons cultured under different treatment conditions were assessed in terms of increased Sez6 gene expression.Our results show that the splicing patterns of Sez6 were modulated in a brain area-specific manner. In particular, the striatum showed a characteristic splicing pattern of recessive isoforms. Moreover, neuronal activation by convulsant drug stimulation increased recessive isoforms like the dominant isoform in cultured cortical neurons at 5 or 10 days in vitro.In conclusion, alternative splicing of Sez6, as well as of other proteins expressed specifically in the brain, results in brain area-specific expression patterns. Furthermore, the alternative splicing of Sez6 may be modulated by drugs that elevate Sez6 gene expression.

Seizurogenic effect of perfluorooctane sulfonate in zebrafish larvae

While the developmental neurotoxicity of perfluorooctane sulfonate (PFOS) has been reported, its seizurogenic potential has not been investigated. Behavior assessment was conducted in zebrafish larvae exposed to PFOS at concentrations of 0, 0.1, 1, 5, 10, and 20 μM. Changes in electrophysiological signals and in the concentration of 20 neurochemicals were measured. Behavior assessment revealed that PFOS altered larval behaviors and significantly increased the counts and duration of bursting (an irregular high-speed movement). Electrophysiological analysis showed that the number of seizure-like events and duration of seizure-like signals were significantly increased, corresponding to results observed using pentylenetetrazol as a positive seizurogenic agent. The outbreak of seizures detected via abnormal electrophysiological signals was confirmed by the increased expression of c-fos and bdnf, which are typical seizure-related genes. Analysis of neurochemicals indicated that PFOS dysregulated overall neurotransmission systems, and aberrant endogenous concentrations of various neurochemicals in the amino acid, cholinergic, dopaminergic, serotonergic and kynurenergic, and GABAergic systems were associated with seizure-like behavior and signals. This study, the first to demonstrate that exposure to PFOS provokes a seizurogenic effect in developing zebrafish larvae, should stimulate further research on the association between PFOS exposure and neurodevelopmental toxicity or neurological disorders.

Size-dependent seizurogenic effect of polystyrene microplastics in zebrafish embryos

The effects of polystyrene microplastic (PS-MP) size on neurotoxicity remain to be evaluated at various microsizes, and the seizurogenic effects of PS-MPs are unknown. This study aimed to evaluate the swimming behavior of zebrafish larvae under light–dark transitions after exposure to four PS-MP sizes (i.e., 1, 6, 10, and 25 μm) at concentrations of 500, 5,000, and 50,000 particles/mL. Changes in electroencephalographic signals, seizure-related gene expression, and neurochemical concentrations were measured. Locomotor activity was inhibited only by 10-μm PS-MPs. According to electroencephalographic signals, the number and total duration of seizure-like events significantly increased by 10-μm PS-MPs, which was confirmed by the altered expression of seizure-related genes c-fos and pvalb5. Additionally, an increase in the levels of neurochemicals choline, betaine, dopamine, 3-methoxytyramine, and gamma-aminobutyric acid indicated that the observed hypoactivity and seizure-like behavior were associated with the dysregulation of the cholinergic, dopaminergic, and GABAergic systems. Overall, these findings demonstrate that exposure to PS-MPs can potentially cause seizurogenic effects in developing zebrafish embryos, and we highlight that PS-MPs 10 µm in size dominantly affect neurotoxicity.

BACE inhibitor treatment of mice induces hyperactivity in a Seizure-related gene 6 family dependent manner without altering learning and memory

BACE inhibitors, which decrease BACE1 (β-secretase 1) cleavage of the amyloid precursor protein, are a potential treatment for Alzheimer’s disease. Clinical trials using BACE inhibitors have reported a lack of positive effect on patient symptoms and, in some cases, have led to increased adverse events, cognitive worsening and hippocampal atrophy. A potential drawback of this strategy is the effect of BACE inhibition on other BACE1 substrates such as Seizure-related gene 6 (Sez6) family proteins which are known to have a role in neuronal function. Mice were treated with an in-diet BACE inhibitor for 4–8 weeks to achieve a clinically-relevant level of amyloid-β40 reduction in the brain. Mice underwent behavioural testing and postmortem analysis of dendritic spine number and morphology with Golgi-Cox staining. Sez6 family triple knockout mice were tested alongside wild-type mice to identify whether any effects of the treatment were due to altered cleavage of Sez6 family proteins. Wild-type mice treated with BACE inhibitor displayed hyperactivity on the elevated open field, as indicated by greater distance travelled, but this effect was not observed in treated Sez6 triple knockout mice. BACE inhibitor treatment did not lead to significant changes in spatial or fear learning, reference memory, cognitive flexibility or anxiety in mice as assessed by the Morris water maze, context fear conditioning, or light–dark box tests. Chronic BACE inhibitor treatment reduced the density of mushroom-type spines in the somatosensory cortex, regardless of genotype, but did not affect steady-state dendritic spine density or morphology in the CA1 region of the hippocampus. Chronic BACE inhibition for 1–2 months in mice led to increased locomotor output but did not alter memory or cognitive flexibility. While the mechanism underlying the treatment-induced hyperactivity is unknown, the absence of this response in Sez6 triple knockout mice indicates that blocking ectodomain shedding of Sez6 family proteins is a contributing factor. In contrast, the decrease in mature spine density in cortical neurons was not attributable to lack of shed Sez6 family protein ectodomains. Therefore, other BACE1 substrates are implicated in this effect and, potentially, in the cognitive decline in longer-term chronically treated patients.

Genetic inactivation of the sigma-1 chaperone protein results in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures

There is a growing body of evidence demonstrating the significant involvement of the sigma-1 chaperone protein in the modulation of seizures. Several sigma-1 receptor (Sig1R) ligands have been demonstrated to regulate the seizure threshold in acute and chronic seizure models. However, the mechanism by which Sig1R modulates the excitatory and inhibitory pathways in the brain has not been elucidated. The aim of this study was to compare the susceptibility to seizures of wild type (WT) and Sig1R knockout (Sig1R−/−) mice in intravenous pentylenetetrazol (PTZ) and (+)-bicuculline (BIC) infusion-induced acute seizure and Sig1R antagonist NE-100-induced seizure models. To determine possible molecular mechanisms, we used quantitative PCR, Western blotting and immunohistochemistry to assess the possible involvement of several seizure-related genes and proteins. Peripheral tissue contractile response of WT and Sig1R−/− mice was studied in an isolated vasa deferentia model. The most important finding was the significantly decreased expression of the R2 subunit of the GABA-B receptor in the hippocampus and habenula of Sig1R−/− mice. Our results demonstrated that Sig1R−/− mice have decreased thresholds for PTZ- and BIC-induced tonic seizures. In the NE-100-induced seizure model, Sig1R−/− animals demonstrated lower seizure scores, shorter durations and increased latency times of seizures compared to WT mice. Sig1R-independent activities of NE-100 included downregulation of the gene expression of iNOS and GABA-A γ2 and inhibition of KCl-induced depolarization in both WT and Sig1R−/− animals. In conclusion, the results of this study indicate that the lack of Sig1R resulted in decreased expression of the R2 subunit of the GABA-B receptor and increased susceptibility to seizures. Our results confirm that Sig1R is a significant molecular target for seizure modulation and warrants further investigation for the development of novel anti-seizure drugs.

The Role of EZH2 Inhibitor, GSK-126, in Seizure Susceptibility.

GSK-126 is recognized as an inhibitor of enhancer of zeste homolog-2 (EZH2) activity. Because of its inhibition of EZH2 activation, GSK-126 is considered a potential anti-tumor drug. EZH2 is a histone methyltransferase that catalyzes histone 3 tri-methylation at lysine 27 (H3K27me3), resulting in gene silencing. A previous report showed that decreased H3K27me3 levels in the hippocampus may promote seizure susceptibility, possibly restricting the clinical application of GSK-126. The role of GSK-126 in seizure susceptibility was investigated in this study. We first determined a critical concentration of pentamethazol (PTZ) under which mice exhibit no seizures. We then found that mice pretreated with GSK-126 and injected with the same concentration of PTZ experienced marked convulsions. Peripheral injections of GSK-126 decreased H3K27me3 levels in the hippocampus of mice, while some seizure-related genes (Oasl1, Sox7, armcx5, Ncx3, etc.) were found to be differentially expressed in the hippocampus of those mice . These differences in the expression levels might reflect the crucial role of these genes and related pathways in the promotion of seizure susceptibility. Our results suggest that GSK-126 promotes seizure susceptibility due to its role as an EZH2 inhibitor. These findings may provide evidence to support the development of GSK-126 as a clinical drug.

New BACE1 Chimeric Peptide InhibitorsSelectively Prevent AβPP-β Cleavage Decreasing Amyloid-β Production and Accumulation in Alzheimer's Disease Models.

A disease-modifying therapy for Alzheimer's disease (AD) is still an unmet clinical need. The formation of amyloid-β (Aβ) requires the initial cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (beta-site AβPP cleaving enzyme 1), which is a prime therapeutic target for AD. We aimed to design and develop a selective BACE1 inhibitor suitable to AD treatment. The new BACE1 inhibitors consist on a chimeric peptide including a sequence related to the human Swedish mutant form of AβPP (AβPPswe) conjugated with the TAT carrier that facilitates cell membrane permeation and the crossing of the blood-brain barrier. Additionally to the chimeric peptide in the L-form, we developed a D-retroinverso chimeric peptide. The latter strategy, never used with BACE1 inhibitors, is considered to favor a significantly higher half-life and lower immunogenicity. We found that both chimeric peptides inhibit recombinant BACE1 activity and decrease Aβ40/42 production in Neuro-2a (N2A) cells expressing AβPPswe without inducing cytotoxicity. The intraperitoneal administration of these peptides to 3xTg-AD mice decreased plasma and brain Aβ40/42 levels, as well as brain soluble AβPPβ production. Also, a reduction of insoluble Aβ was observed in the brain after chronic treatment. Noteworthy, the chimeric peptides selectively inhibited the AβPP-β cleavage relatively to the proteolysis of other BACE1 substrates such as close homologue of L1 (CHL1) and seizure-related gene 6 (SEZ6). Overall these new BACE1 chimeric peptideshold promising potential as a selective disease-modifying therapy for AD.

Lack of Sez6 Family Proteins Impairs Motor Functions, Short-Term Memory, and Cognitive Flexibility and Alters Dendritic Spine Properties.

Seizure-related gene 6 (Sez6), Sez6-Like (Sez6L), and Sez6-Like 2 (Sez6L2) comprise a family of homologous proteins widely expressed throughout the brain that have been linked to neurodevelopmental and psychiatric disorders. Here, we use Sez6 triple knockout (TKO) mice, which lack all three Sez6 family proteins, to demonstrate that Sez6 family proteins regulate dendritic spine structure and cognitive functions, motor learning, and maintenance of motor functions across the lifespan. Compared to WT controls, we found that Sez6 TKO mice had impaired motor learning and their motor coordination was negatively affected from 6weeks old and declined more rapidly as they aged. Sez6 TKO mice had reduced spine density in the hippocampus and dendritic spines were shifted to more immature morphologies in the somatosensory cortex. Cognitive testing revealed that they had enhanced stress responsiveness, impaired working, and spatial short-term memory but intact spatial long-term memory in the Morris water maze albeit accompanied by a reversal deficit. Our study demonstrates that the lack of Sez6 family proteins results in phenotypes commonly associated with neuropsychiatric disorders making it likely that Sez6 family proteins contribute to the complex etiologies of these disorders.

Gene expression profiling in a mouse model of Dravet syndrome

Dravet syndrome is a severe, early-onset epileptic encephalopathy frequently resulting from de novo mutations of SCN1A. Mice with heterozygous deletion of Scn1a (Scn1a+/−) model many features of Dravet syndrome, including spontaneous seizures and premature lethality. Scn1a+/− mice exhibit variable phenotype penetrance and expressivity dependent upon the strain background. On the 129S6/SvEvTac (129) strain, Scn1a+/− mice do not display an overt phenotype. However Scn1a+/− mice on the [129S6xB6]F1 strain (F1.Scn1a+/−) exhibit juvenile-onset spontaneous seizures and premature lethality. QTL mapping identified several modifier loci responsible for strain-dependent differences in survival of Scn1a+/− mice, but these loci do not account for all the observed phenotypic variance. Global RNA-seq analysis was performed to identify additional genes and pathways that may contribute to variable phenotypes. Hippocampal gene expression was analyzed in wild-type (WT) and Scn1a+/− mice on both F1 and 129 strains, at two time points during disease development. There were few gene expression differences between 129.WT and 129.Scn1a+/− mice and approximately 100 genes with small expression differences (6–36%) between F1.WT and F1.Scn1a+/− mice. Strain-specific gene expression differences were more pronounced, with dozens of genes with >1.5-fold expression differences between 129 and F1 strains. Age-specific and seizure-related gene expression differences were most prominent, with hundreds of genes with >2-fold differences in expression identified between groups with and without seizures, suggesting potential differences in developmental trajectory and/or homeostatic plasticity during disease onset. Global expression differences in the context of Scn1a deletion may account for strain-dependent variation in seizure susceptibility and survival observed in Scn1a+/− mice.

Exome sequencing in an Italian family with Alzheimer\u2019s disease points to a role for seizure-related gene 6 (SEZ6) rare variant R615H

BackgroundThe typical familial form of Alzheimer’s disease (FAD) accounts for about 5% of total Alzheimer’s disease (AD) cases. Presenilins (PSEN1 and PSEN2) and amyloid-β (A4) precursor protein (APP) genes carry all reported FAD-linked mutations. However, other genetic loci may be involved in AD. For instance, seizure-related gene 6 (SEZ6) has been reported in brain development and psychiatric disorders and is differentially expressed in the cerebrospinal fluid of AD cases.MethodsWe describe a targeted exome sequencing analysis of a large Italian kindred with AD, negative for PSEN and APP variants, that indicated the SEZ6 heterozygous mutation R615H is associated with the pathology.ResultsWe overexpressed R615H mutation in H4-SW cells, finding a reduction of amyloid peptide Aβ(1–42). Sez6 expression decreased with age in a mouse model of AD (3xTG-AD), but independently from transgene expression.ConclusionsThese results support a role of exome sequencing for disease-associated variant discovery and reinforce available data on SEZ6 in AD models.

Functions of the Alzheimer's Disease Protease BACE1 at the Synapse in the Central Nervous System.

Inhibition of the protease β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a promising treatment strategy for Alzheimer’s disease, and a number of BACE inhibitors are currently progressing through clinical trials. The strategy aims to decrease production of amyloid-β (Aβ) peptide from the amyloid precursor protein (APP), thus reducing or preventing Aβ toxicity. Over the last decade, it has become clear that BACE1 proteolytically cleaves a number of substrates in addition to APP. These substrates are not known to be involved in the pathogenesis of Alzheimer’s disease but have other roles in the developing and/or mature central nervous system. Consequently, BACE inhibition and knockout in mice results in synaptic and other neuronal dysfunctions and the key substrates responsible for these deficits are still being elucidated. Of the BACE1 substrates that have been validated to date, a number may contribute to the synaptic deficits seen with BACE blockade, including neuregulin 1, close homologue of L1 and seizure-related gene 6. It is important to understand the impact that BACE blockade may have on these substrates and other proteins detected in substrate screens and, if necessary, develop substrate-selective BACE inhibitors.

N-glycosylation modulates filopodia-like protrusions induced by sez-6 through regulating the distribution of this protein on the cell surface

Seizure-related gene 6 (sez-6) is a trans-membrane protein expressed by neuronal cells that modulates dendritic branching. It has three clusters of eleven possible N-glycosylation sites in the extracellular domain region: sugar chain (SC)1-3, SC4-7, and SC8-11. Recent reports suggest that N-glycosylation modulates the membrane trafficking and function of trans-membrane proteins. Here, we studied the role of N-glycosylation in sez-6 function. We transfected mutants lacking one, two, or all N-glycosylation clusters into neuro2a cells. A mutant lacking all N-glycosylation was transported to the cell membrane. Mutants lacking one cluster (sez-6 ΔSC1-3, ΔSC4-7, ΔSC8-11) were evenly distributed on the cell membrane and secreted into the conditioned medium, as in wild-type sez-6; in contrast, the unglycosylated mutant, sez-6 ΔSC1-11, and mutants having only one cluster (sez-6 SC1-3, SC8-11) were localized in some portions on the cell membrane. Despite sez-6 SC4-7 having only one cluster, it was transported like the wild type. Among mutants behaving like the wild type, sez-6 ΔSC1-3 and ΔSC4-7 reduced neurite formation. Interestingly, mutants lacking SC4-7 (sez-6 ΔSC4-7) did not affect the formation of filopodia-like protrusions. In contrast, other mutants as well as the wild type induced it, suggesting that SC4-7 is crucial for filopodia-like protrusions. Our results indicate that N-glycosylation regulates cell morphology through modulating the cell surface distribution of sez-6 protein.

Insights from zebrafish and mouse models on the activity and safety of ar-turmerone as a potential drug candidate for the treatment of epilepsy.

In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

Correlation between human seizure-related gene 6 variants and idiopathic generalized epilepsy in a Southern Chinese Han population.

This study sought to analyze the genotype and gene mutations of human seizure-related gene 6 in 98 patients with idiopathic generalized epilepsy (non-febrile seizures), who were selected from three generations of the Chinese Han population living in Shanghai, Zhejiang Province, Wuxi of Jiangsu Province, and Jiangxi Province of Southern China. Twenty-six patients’ parents were available as a first-degree relatives group and 100 biologically unrelated healthy controls were collected as the control group. Based on the age of onset and seizure type, the patients were divided into six subgroups. Polymerase chain reaction and DNA direct sequencing analysis showed that the most frequent mutations c.1249dupC (p.Gly418Argfx31) and c.1636A > G (p.Thr546Ala) were detected in some idiopathic generalized epilepsy patients and their asymptomatic first-degree relatives (30.6% vs. 19.2% and 11.2% vs. 26.9%). A novel mutation c.1807G > A (p.Val603Met) was found in a patient with late-onset idiopathic generalized epilepsy. There was no significant difference in the incidence of these three mutations among the different subgroups of idiopathic generalized epilepsy and controls. Thus, further analysis of a larger population is needed to confirm the assumption that human seizure-related gene 6 is a susceptibility gene for idiopathic generalized epilepsy with various sub-syndromes.

Sez-6 May Play an Important Role in Neurite Outgrowth through the PKCγ Signaling Pathways

Seizure-related gene 6 (sez-6) was originally identified in a study of pentylenetetrazole-treated cortical neurons. Further studies on the structure and expression pattern suggested that Sez-6 may play an important role in neuronal development and function. In the present study, PC12 cells were used as a model to investigate the role of Sez-6 in neurite outgrowth. After a period of NGF treatment, the expression of Sez-6 in PC12 cells was increased. When Sez-6 expression was suppressed by the addition of an effective short hairpin RNA (shRNA) plasmid, the neurite outgrowth was significantly inhibited. In addition, we detected the expression level of protein kinase Cgamma (PKCgamma), and found that the PKCgamma protein level was reduced in the differentiated PC12 cells but increased in PC12 cells lacking Sez-6. Taken together, our results indicate that Sez-6 acts on the neurite outgrowth of PC12 cells likely through the PKCgamma signaling pathways.

The modulation of neuronal morphology by the interaction between a mental retardation gene, motopsin/neurotrypsin, and seizure related gene (Sez)-6

The modulation of neuronal morphology by the interaction between a mental retardation gene, motopsin/neurotrypsin, and seizure related gene (Sez)-6

The distribution of the seizure-related gene 6 (Sez-6) protein during postnatal development of the mouse forebrain suggests multiple functions for this protein: An analysis using a new antibody

The seizure-related gene 6 ( Sez-6) encodes a transmembrane protein that is expressed in neuronal cells. A Sez-6-deficient mouse exhibits impaired spatial memory, motor deficits, and decreased anxiety levels. To understand the function of Sez-6 during the postnatal development of the forebrain, the spatiotemporal pattern of distribution of the Sez-6 protein was immunohistochemically analyzed using a new anti-Sez-6 antibody. Western blot analysis confirmed the specificity of this new antibody, and showed that the content of the Sez-6 protein in the cerebral cortex was highest during the neonatal period and decreased gradually thereafter. Immunohistochemical analysis revealed that Sez-6 immunoreactivity (IR) was detected in various brain regions, such as the hippocampus, cerebral cortex, piriform cortex, striatum, lateral amygdala, and olfactory tubercle. The expression patterns of Sez-6 in these brain regions was divided into three groups: i) in the cerebral cortex, hippocampus, and lateral amygdala, moderate-to-strong Sez-6 IR was detected in the first postnatal week and decreased gradually thereafter; ii) Sez-6 IR was not observed during the neonatal period in the striatum and the intensity of the signal increased gradually toward adulthood; and iii) strong Sez-6 IR was observed in the olfactory tubercle, regardless of the developmental stage. Furthermore, Sez-6 IR was detected in dendrites of hippocampal and cortical pyramidal neurons neonatally, whereas it localized around the soma after postnatal day 10. These spatiotemporal alterations of the regional and intracellular distribution of the Sez-6 protein suggest multiple functions for this protein during the postnatal development of the forebrain.

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

BackgroundSeizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development.Methodology/Principal FindingsThe distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in AII glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear “bright spot” similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG).ConclusionsIn summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected.