Frings Mouse Research Articles

Anticonvulsive properties of soticlestat, a novel cholesterol 24-hydroxylase inhibitor.

ObjectiveThe formation of 24S‐hydroxycholesterol is a brain‐specific mechanism of cholesterol catabolism catalyzed by cholesterol 24‐hydroxylase (CYP46A1, also known as CH24H). CH24H has been implicated in various biological mechanisms, whereas pharmacological lowering of 24S‐hydroxycholesterol has not been fully studied. Soticlestat is a novel small‐molecule inhibitor of CH24H. Its therapeutic potential was previously identified in a mouse model with an epileptic phenotype. In the present study, the anticonvulsive property of soticlestat was characterized in rodent models of epilepsy that have long been used to identify antiseizure medications.MethodsThe anticonvulsive property of soticlestat was investigated in maximal electroshock seizures (MES), pentylenetetrazol (PTZ) acute seizures, 6‐Hz psychomotor seizures, audiogenic seizures, amygdala kindling, PTZ kindling, and corneal kindling models. Soticlestat was characterized in a PTZ kindling model under steady‐state pharmacokinetics to relate its anticonvulsive effects to pharmacodynamics.ResultsAmong models of acutely evoked seizures, whereas anticonvulsive effects of soticlestat were identified in Frings mice, a genetic model of audiogenic seizures, it was found ineffective in MES, acute PTZ seizures, and 6‐Hz seizures. The protective effects of soticlestat against audiogenic seizures increased with repetitive dosing. Soticlestat was also tested in models of progressive seizure severity. Soticlestat treatment delayed kindling acquisition, whereas fully kindled animals were not protected. Importantly, soticlestat suppressed the progression of seizure severity in correlation with 24S‐hydroxycholesterol lowering in the brain, suggesting that 24S‐hydroxycholesterol can be aggressively reduced to produce more potent effects on seizure development in kindling acquisition.SignificanceThe data collectively suggest that soticlestat can ameliorate seizure symptoms through a mechanism distinct from conventional antiseizure medications. With its novel mechanism of action, soticlestat could constitute a novel class of antiseizure medications for treatment of intractable epilepsy disorders such as developmental and epileptic encephalopathy.

Targeted Blockade of TARP-γ8-Associated AMPA Receptors: Anticonvulsant Activity with the Selective Antagonist LY3130481 (CERC-611).

6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]- 3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise, LY3130481 attenuated convulsions in mice and rats with long-term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP γ-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient. Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP γ-8 is sufficient for these protective effects.

ADGRV1 is implicated in myoclonic epilepsy.

To investigate the significance of variation in ADGRV1 (also known as GPR98, MASS1, and VLGR1), MEF2C, and other genes at the 5q14.3 chromosomal locus in myoclonic epilepsy. We studied the epilepsy phenotypes of 4 individuals with 5q14.3 deletion and found that all had myoclonic seizures. We then screened 6 contiguous genes at 5q14.3, MEF2C, CETN3, MBLAC2, POLR3G, LYSMD3, and ADGRV1, in a 95-patient cohort with epilepsy and myoclonic seizures. Of these genes, point mutations in MEF2C cause a phenotype involving seizures and intellectual disability. A role for ADGRV1 in epilepsy has been proposed previously, based on a recessive mutation in the Frings mouse model of audiogenic seizures, as well as a shared homologous region with another epilepsy gene, LGI1. Six patients from the myoclonic epilepsy cohort had likely pathogenic ultra-rare ADGRV1 variants, and statistical analysis showed that ultra-rare variants were significantly overrepresented when compared to healthy population data from the Genome Aggregation Database. Of the remaining genes, no definite pathogenic variants were identified. Our data suggest that the ADGRV1 variation contributes to epilepsy with myoclonic seizures, although the inheritance pattern may be complex in many cases. In patients with 5q14.3 deletion and epilepsy, ADGRV1 haploinsufficiency likely contributes to seizure development. The latter is a shift from current thinking, as MEF2C haploinsufficiency has been considered the main cause of epilepsy in 5q14.3 deletion syndrome. In cases of 5q14.3 deletion and epilepsy, seizures likely occur due to haploinsufficiency of one or both of ADGRV1 and MEF2C.

FV-082: A safer orally active broad-spectrum antiepileptic drug candidate

Broad spectrum Anti-Epileptic Drugs (AED) are valued for their potential to treat refractory epilepsy and as general ‘neurostabilizers’ have been employed for a variety of neurological diseases including migrane, biopolar disorder and neuropathic pain. However, the benefits of historical AEDs have been constrained by unintended CNS side effects. Combining proprietary breakthrough chemistry with Fluorinov Pharma and NIH’s in vivo phenotypic screening strategy identified three completely novel candidate drugs, including FV-082 with superior safety and efficacy profiles. FV-082 was screened across available in vitro and rodent seizure models at ASP, and was found to display an excellent spectrum of anti-seizure activity and safety profiles superior to that produced by Depakote, Neurontin and Keppra. FV-082 has an oral ED50 of 20.1 ± 0.7 and 17.4 ± 0.6 mg/kg in the rat MES and mouse 6Hz (ip) assays, respectively. FV-082 prevented seizures induced by sound in the Audiogenic Seizure (AGS)-susceptible Frings mouse model (i.p) with an ED50 value of 13.0 ± 2.0 mg/kg. In addition, FV-082 displayed significant efficacy in the electrically induced corneally kindled mouse, kindled rat amygdala and hippocampal seizures models. Importantly, FV-082 was also found to limit seizure spread and elevate seizure threshold in preclinical animal models, therefore, it has the potential to be effective in refractory, or pharmacoresistant, epilepsy. Also, the anticonvulsant pharmacology suggests applicability for treating multiple forms of epilepsy, such as generalized tonic−clonic, complex partial, and myoclonic seizures, at dose levels that confer a favorable safety margin (therapeutic index ≥ 34). Safety assessment of FV-082 suggests no interaction with the major CYP isoenzymes when tested up to 500 μM and no functional hERG activity when tested up to 20 μM. Further, acute toxicity studies revealed that FV082 exhibited no adverse effects at oral and intravenous doses in rats and mice with a maximally tolerated dose greater than 600 mg/kg in the mice (i.p) and rats (p.o). Also, there was a pronounced safety margin between behavioral efficacy (rat oral MES neurotoxicity and (rat TD50 > 600 mg/kg) for a protective index (rat oral TD50/MES ED50) of >34-fold. Hence, FV-082 displays a considerably broader CNS therapeutic index superior to historical AEDs. Additionally, a convenient and cost effective manufacturing process for FV-082 has been developed allowing for access to multi-kilogram scale synthesis. In-vivo pharmacokinetic studies in rat and dog revealed that FV-082 had excellent oral bioavailability and good oral half-life, and excellent plasma drug levels across multiple models of epilepsy and pain. In addition, drug exposure as measured by Cmax and AUC was shown to be linear with dose. Mechanistically, broad ion channel profiling in combination with a CEREP broad profiling screen (10 μM) across 90 known GPCR, ion channel, transporter and enzyme targets studies suggest that several mechanisms contribute to the observed pharmacological profile of FV-082 but that no single mechanism is likely to be a major contributor. In fact, in whole-cell patch-clamp recordings FV-082 inhibited voltage-gated Na + channel Nav1.7, and the CEREP panel indicated that FV-082 interacted with androgen receptor (AR) and (h) recombinant enzyme monoamine-oxidase-B (MAO-B). The anticonvulsant profile of FV-082 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. Furthermore, in models of neuropathic pain (spinal nerve injury [SNI] and formalin inflammatory pain, administration of a single dose showed robust efficacy (SNI: 50 mg/kg ip; and formalin: 71 mg/kg ip). Based on these promising preclinical data, FV-082 warrants clinical investigation.

FV-137 candidate drug: Novel and selective ion channel modulation for epilepsy and pain

Broad spectrum Anti-Epileptic Drugs (AED) are valued for their potential to treat refractory epilepsy and as general ‘neurostabilizers’ have been employed for a variety of neurological diseases including migrane, biopolar disorder and neuropathic pain. However, the benefits of historical AEDs have been constrained by unintended CNS side effects of these non-selective medicines. Combining proprietary breakthrough chemistry with Fluorinov Pharma and NIH’s in vivo phenotypic screening strategy identified three completely novel candidate drugs, including FV-137 with superior safety and efficacy profiles targeting selective ion channels. FV-137 is a novel, orally administered AED, structurally distinct than any known AED, displaying promising preclinical efficacy and safety. FV-137 emerged from a program whose goal was to identify broad-spectrum anti-epileptic drugs (AED) using gold standard benchmarks such as Depakote ® , Neurontin ® and Keppra ® as primary comparators. FV-137 was extensively screened across all available in vitro and rodent seizure models at ASP, and was found to display an excellent spectrum of anti-seizure activity and safety profiles superior to that produced by Depakote, Neurontin and Keppra. FV-137 has an oral ED50 of 22.7 ± 0.6 and 104.9±4.3 mg/kg in the rat MES (po), mouse PTZ (ip) assays, respectively. FV-137 also produced a dose related protection of a psychomotor seizure syndrome induced by a 6-Hz electrical stimulus with an oral ED50 of 48.5 ± 1mg/kg, and prevented seizures induced by sound in the Audiogenic Seizure (AGS)-susceptible Frings mouse model (i.p) with an ED50 value of 28.9 ± 3.0 mg/kg. In addition, FV-137 displayed significant efficacy in the electrically induced corneally kindled mouse, kindled rat amygdala, kindled rat hippocampal and lamotrigineresistant kindled rat seizures models. Additionally, FV-137 also increased seizure threshold in a dose– dependent manner, therefore, it has the potential to be effective in refractory, or pharmacoresistant, epilepsy. In-vivo pharmacokinetic studies in rat revealed that FV-137 had excellent oral bioavailability (F% = 85%), moderate oral half-life (~3h), and predicted plasma drug levels in the range 5-50μM across multiple models of epilepsy. In addition, drug exposure as measured by Cmax and AUC was shown to be linear with dose. Safety assessment of FV-137 suggests no interaction with the major CYP isoenzymes when tested up to 500 μM, except for 1A2 and 2D6 with IC50 values of 19 uM and 156 uM respectively and no functional hERG activity when tested up to 50 μM. Irwin and acute toxicity studies suggest a maximally tolerated oral dose of greater than 500 mg/kg in the mice and rats. Mechanistically, in whole-cell patch-clamp recordings FV-137 inhibited P/Q-type Ca 2+ Channels, Cav2.1/β4/α2δ1 and Cav2.2/β3/α2δ1, and voltage-gated Na + channels, Nav1.6 and Nav1.7 and no inhibition up to 100 μM in other related ion channels. Importantly, no significant affinities were observed in a CEREP broad profiling screen (10 μM) across 90 known GPCR, ion channel, transporter and enzyme targets. The anticonvulsant profile of FV-137 suggests that it may be useful for treating multiple forms of epilepsy (generalized tonic-clonic, complex partial, absence seizures), including refractory (or pharmacoresistant) epilepsy, at dose levels that confer a good safety margin. Furthermore, in models of neuropathic pain (spinal nerve injury [SNI] and spinal nerve ligation models [SNL]) and formalin inflammatory pain, administration of a single dose showed robust efficacy (SNI: 23 mg/kg ip; SNL: 60mg/kg and formalin: 41 mg/kg ip). On the basis of its pharmacology and superior drug-like properties, FV-137 was identified as a promising AED candidate.

Very large G protein-coupled receptor 1 regulates myelin-associated glycoprotein via Gαs/Gαq-mediated protein kinases A/C

VLGR1 (very large G protein-coupled receptor 1), also known as MASS1 (monogenic audiogenic seizure susceptible 1), is an orphan G protein-coupled receptor that contains a large extracellular N terminus with 35 calcium-binding domains. A truncating mutation in the Mass1 gene causes autosomal recessive, sound-induced seizures in the Frings mouse. However, the function of MASS1 and the mechanism underlying Frings mouse epilepsy are not known. Here, we found that MASS1 protein is enriched in the myelinated regions of the superior and inferior colliculi, critical areas for the initiation and propagation of audiogenic seizures. Using a panel of myelin antibodies, we discovered that myelin-associated glycoprotein (MAG) expression is dramatically decreased in Frings mice. MASS1 inhibits the ubiquitylation of MAG, thus enhancing the stability of this protein, and the calcium-binding domains of MASS1 are essential for this regulation. Furthermore, MASS1 interacts with Gαs/Gαq and activates PKA and PKC in response to extracellular calcium. Suppression of signaling by MASS1 RNAi or a specific inhibitor abrogates MAG up-regulation. We postulate that MASS1 senses extracellular calcium and activates cytosolic PKA/PKC pathways to regulate myelination by means of MAG protein stability in myelin-forming cells of the auditory pathway. Further work is required to determine whether MAG dysregulation is a cause or consequence of audiogenic epilepsy and whether there are other pathways regulated by MASS1.

Lacosamide, a novel anti-convulsant drug, shows efficacy with a wide safety margin in rodent models for epilepsy

This paper comprises a series of experiments in rodent models of partial and generalized epilepsy which were designed to describe the anti-convulsant profile of the functionalized amino acid lacosamide. Lacosamide was effective against sound-induced seizures in the genetically susceptible Frings mouse, against maximal electroshock test (MES)-induced seizures in rats and mice, in the rat hippocampal kindling model of partial seizures, and in the 6Hz model of psychomotor seizures in mice. The activity in the MES test in both mice (4.5mg/kg i.p.) and rats (3.9 mg/kg p.o.) fell within the ranges previously reported for most clinically available anti-epileptic drugs. At both the median effective dose for MES protection, as well as the median toxic dose for rotorod impairment, lacosamide elevated the seizure threshold in the i.v. pentylenetetrazol seizure test, suggesting that it is unlikely to be pro-convulsant at high doses. Lacosamide was inactive against clonic seizures induced by subcutaneous administration of the chemoconvulsants pentylenetetrazol, bicuculline, and picrotoxin, but it did inhibit NMDA-induced seizures in mice and showed full efficacy in the homocysteine model of epilepsy. In summary, the overall anti-convulsant profile of lacosamide appeared to be unique, and the drug displayed a good margin of safety in those tests in which it was effective. These results suggest that lacosamide may have the potential to be clinically useful for at least the treatment of generalized tonic-clonic and partial-onset epilepsies, and support ongoing clinical trials in these indications.

Auditory Deficits Associated with the Frings Mgr1 (Mass1) Mutation in Mice

The gene responsible for the audiogenic seizure (AGS) phenotype in Frings mice, which was identified and originally designated Mass1, is now referred to as Mgr1. Although the function of the gene product is not known, the expression pattern suggests a role in the developing CNS. Hearing impairment is often observed in AGS-susceptible rodents and is thought to contribute to the pathology of AGS. We therefore hypothesized that the Frings mouse exhibits early-onset hearing impairment and that the Frings Mgr1 mutation is responsible for the hearing impairment phenotype that leads to the development of AGS susceptibility. Auditory brainstem response (ABR) thresholds were used to evaluate auditory function in mice carrying the Frings Mgr1 allele and were compared with other AGS-susceptible and -resistant mice. ABR testing demonstrated that mice possessing the Frings Mgr1 allele exhibit a mild to moderate level of hearing impairment that is present during the days following hearing onset. Furthermore, the hearing impairment resulting from the Frings Mgr1 allele is relatively stable, which explains the long duration of AGS susceptibility exhibited by Frings mice compared with other AGS-susceptible mice.

The Mass1frings mutation underlies early onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher syndrome IIC

The human ortholog of the gene responsible for audiogenic seizure susceptibility in Frings and BUB/BnJ mice (mouse gene symbol Mass1) recently was shown to underlie Usher syndrome type IIC (USH2C). Here we report that the Mass1 frings mutation is responsible for the early onset hearing impairment of BUB/BnJ mice. We found highly significant linkage of Mass1 with ABR threshold variation among mice from two backcrosses involving BUB/BnJ mice with mice of strains CAST/EiJ and MOLD/RkJ. We also show an additive effect of the Cdh23 locus in modulating the progression of hearing loss in backcross mice. Together, these two loci account for more than 70% of the total ABR threshold variation among the backcross mice at all ages. The modifying effect of the strain-specific Cdh23 ahl variant may account for the hearing and audiogenic seizure differences observed between Frings and BUB/BnJ mice, which share the Mass1 frings mutation. During postnatal cochlear development in BUB/BnJ mice, stereocilia bundles develop abnormally and remain immature and splayed into adulthood, corresponding with the early onset hearing impairment associated with Mass1 frings . Progressive base–apex hair cell degeneration occurs at older ages, corresponding with the age-related hearing loss associated with Cdh23 ahl . The molecular basis and pathophysiology of hearing loss suggest BUB/BnJ and Frings mice as models to study cellular and molecular mechanisms underlying USH2C auditory pathology.

C-Fos immunohistochemical mapping of the audiogenic seizure network and tonotopic neuronal hyperexcitability in the inferior colliculus of the Frings mouse

The Frings mouse is a model of audiogenic seizure (AGS) susceptibility. The genetic locus responsible for the AGS phenotype in the Frings mouse has been named monogenic audiogenic seizure-susceptible ( MASS1). MASS1 is unique in that it is one of only two identified seizure loci that are not associated with an ion channel mutation. Furthermore, Frings mice display a robust AGS phenotype demonstrating very high and prolonged susceptibility to sound-induced tonic extension seizures. The purpose of this investigation was to use c-Fos immunohistochemistry to map the brain structures involved in the Frings AGS and to examine neuronal hyperexcitability in the inferior colliculus, the brain structure that is recognized as the site of AGS initiation. AGS mapping revealed that intense seizure-induced neuronal activation was mostly limited to structures involved in a brainstem seizure network, including the external and dorsal nuclei of the inferior colliculus, as observed in other AGS rodents. Acoustically induced c-Fos expression in the central nucleus of the inferior colliculus to sub-AGS threshold tone stimulations displayed a greater level of neuronal activation in AGS-susceptible Frings, DBA/2J and noise-primed C57BL/6J mice compared to AGS-resistant C57BL/6J and CF1 mice. The AGS-susceptible mice also displayed c-Fos immunoreactivity that was more focused within the tonotopic response domain of the inferior colliculus compared to AGS-resistant mice. Furthermore, Frings mice displayed significantly greater tonotopic hyper-responsiveness compared to other AGS-susceptible mice.

Characterization of the anticonvulsant profile and enantioselective pharmacokinetics of the chiral valproylamide propylisopropyl acetamide in rodents

1. Propylisopropyl acetamide (PID) is a new chiral amide derivative of valproic acid. The purpose of this study was to evaluate the anticonvulsant activity of PID in rodent models of partial, secondarily generalized and sound-induced generalized seizures which focus on different methods of seizure induction, both acute stimuli, and following short-term plastic changes as a result of kindling, and to assess enantioselectivity and enantiomer-enantiomer interactions in the pharmacokinetics and pharmacodynamics of racemic PID and its pure enantiomers in rodents. 2. Anticonvulsant activity of (S)-PID, (R)-PID and racemic PID was evaluated in the 6 Hz psychomotor seizure model in mice, in the hippocampal kindled rat, and in the Frings audiogenic seizure susceptible mouse. The pharmacokinetics of (S)-PID and (R)-PID was studied in mice and rats. 3. In mice (S)-PID, (R)-PID and racemic PID were effective in preventing the 6 Hz seizures with (R)-PID being significantly (P < 0.05) more potent (ED(50) values 11 mg kg(-1), 46 mg kg(-1) and 57 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively) than (S)-PID (ED(50) values 20 mg kg(-1), 73 mg kg(-1) and 81 mg kg(-1) at stimulation intensities of 22, 32 and 44 mA, respectively). (S)-PID, (R)-PID and racemic PID also blocked generalized seizures in the Frings mice (ED(50) values 16 mg kg(-1), 20 mg kg(-1) and 19 mg kg(-1) respectively). 4. In the hippocampal kindled rat a dose of 40 mg kg(-1) of (R)- and (S)-PID prevented the secondarily generalized seizure, whereas racemic PID also blocked the expression of partial seizures following an i.p. dose of 40 mg kg(-1). Racemic PID also significantly increased the seizure threshold in this model. 5. Mechanistic studies showed that PID did not affect voltage-sensitive sodium channels or kainate-, GABA- or NMDA- evoked currents. 6. The pharmacokinetics of PID was enantioselective following i.p. administration of individual enantiomers to mice, with (R)-PID having lower clearance and longer half-life than (S)-PID. In rats and mice, no enantioselectivity in the pharmacokinetics of PID was observed following administration of the racemate, which may be due to enantiomer-enantiomer interaction. 7. This study demonstrated that PID has both enantioselective pharmacokinetics and pharmacodynamics. The better anticonvulsant potency of (R)-PID in comparison to (S)-PID may be due to its more favorable pharmacokinetic profile. The enhanced efficacy of the racemate over the individual enantiomers in the kindled rat may be explained by a pharmacokinetic enantiomer-enantiomer interaction in rats. This study also showed the importance of studying the pharmacokinetics and pharmacodynamics of chiral drugs following administration of the individual enantiomers as well as the racemic mixture.

A nonsense mutation of the MASS1 gene in a family with febrile and afebrile seizures.

A naturally occurring mutation of the mass1 (monogenic audiogenic seizure-susceptible) gene recently has been reported in the Frings mouse strain, which is prone to audiogenic seizures. The human orthologous gene, MASS1, was mapped to chromosome 5q14, for which we previously have reported significant evidence of linkage to febrile seizures (FEB4). We screened for MASS1 mutations in individuals from 48 families with familial febrile seizures and found 25 DNA alterations. None of nine missense polymorphic alleles was significantly associated with febrile seizures; however, a nonsense mutation (S2652X) causing a deletion of the C-terminal 126 amino acid residues was identified in one family with febrile and afebrile seizures. Our results suggest that a loss-of-function mutation in MASS1 might be responsible for the seizure phenotypes, though it is not likely that MASS1 contributed to the cause of febrile seizures in most of our families.

A common protein interaction domain links two recently identified epilepsy genes.

Until recently, all genes found to be mutated in hereditary idiopathic epilepsies encoded subunits of ion channels, leading to the view of this class of diseases as channelopathies. Two apparent exceptions to this rule are the MASS1 gene, which is mutated in the Frings mouse model of audiogenic epilepsy, and the LGI1 gene, which is mutated in autosomal dominant partial epilepsy with auditory features (ADPEAF). Careful sequence analysis of the two protein products encoded by those genes shows a common feature: both sequences harbour a novel homology domain consisting of a 7-fold repeated 44-residue motif. The architecture and structural features of this new domain make it a likely member of the growing class of protein interaction domains with a seven-bladed beta-propeller fold. In the MASS1 gene product, which has recently been shown to be a fragment of the very large G-protein-coupled receptor VLGR1, this EAR domain (for epilepsy-associated repeat) is part of the ligand-binding ectodomain. LGI1, as well as a number of newly identified LGI1 relatives, is predicted to be a secreted protein, and consists of an N-terminal leucine-rich repeat region and a C-terminal EAR region. The known portion of the human genome encodes six EAR proteins, some of which map to chromosome regions associated with seizure disorders. The EAR domain is likely to play an important role in the pathogenesis of epilepsy, either by binding to an unknown anti-epileptic ligand, or more likely by interfering with axon guidance or synaptogenesis.

A novel epilepsy gene

Clues to the causes of epilepsy have come from work on Frings mice – spontaneously occurring mutants that are susceptible to seizures triggered by loud noises. Unlike many rodent models of audiogenic-seizure susceptibility (AGS) which are polygenic, recent work by Skradski and colleagues has revealed that the Frings phenotype is due to the autosomal recessive inheritance of a single gene, Mass1. Mutant mice are homozygous for a single basepair deletion in the Mass1 gene, resulting in premature termination of the encoded protein product. This novel protein product provides a new mechanism for the cause of epilepsy. The MASS1 protein is likely to have a role in the metabolism of iron or other metals, oxidative stress and/or neurotransmitter processing, whereas to date most AGS genes have encoded ion channels. (Skradski, S.L. et al. [2001] Neuron 31, 537–544) AI

A Novel Gene Causing a Mendelian Audiogenic Mouse Epilepsy

Frings mice are a model of generalized epilepsy and have seizures in response to loud noises. This phenotype is due to the autosomal recessive inheritance of a single gene on mouse chromosome 13. Here we report the fine genetic and physical mapping of the locus. Sequencing of the region led to identification of a novel gene; mutant mice are homozygous for a single base pair deletion that leads to premature termination of the encoded protein. Interestingly, the mRNA levels of this gene in various tissues are so low that the cDNA has eluded detection by standard library screening approaches. Study of the MASS1 protein will lead to new insights into regulation of neuronal excitability and a new pathway through which dysfunction can lead to epilepsy.

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D-aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure.

Glutamate receptor GluR1 expression is altered selectively by chronic audiogenic seizures in the Frings mouse brain

The audiogenic seizure-susceptible mouse, Frings, is genetically susceptible to sound-induced seizures and provides a reliable model of reflex epilepsy that lasts throughout the life span of the animal. We used immunohistochemistry to examine if the expression of the non-N-methyl-D-aspartate glutamate receptor (GluR) subunits GluR1, GluR2, or GluR3 were altered subsequent to multiple seizures. Following a regimen of one seizure per day for 3 weeks, GluR1 immunoreactivity, but not GluR2 or GluR3, was substantially elevated in the outer shell of the nucleus accumbens in 21 of 31 chronically seized Frings mice. No other brain regions such as the hippocampus exhibited any qualitative changes in expression of these subunits. In 9 of the 21 Frings mice exhibiting increased GluR1, but in none of the controls, bilateral structural lesions were observed in the lateral hypothalamus. These results support a model where highly localized changes in the expression of GluR1 occur in response to repeated audiogenic seizure. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 209–216, 1998

Genetic Mapping of a Locus (mass1) Causing Audiogenic Seizures in Mice

Frings audiogenic seizure-susceptible mice are a model for sensory-evoked reflex seizures. Their seizure phenotype is characterized by wild running, loss of righting reflex, tonic flexion, and tonic extension in response to high-intensity sound stimulation. The Frings mice represent an inbred colony that has not been genetically characterized. This investigation studied the mode of inheritance for audiogenic seizures by crossing the Frings mouse with the seizure-resistant C57BL/6J mouse. Among the backcross progeny generated by crossing (Frings × C57BL/6J)F1 mice with the Frings strain, 391 of the 836 N2 progeny were audiogenic seizure susceptible, a finding consistent with monogenic inheritance. Genetic mapping and linkage analysis of hybrid mice using MIT microsatellite marker sequences localized the seizure gene, namedmass1for monogenic audiogenic seizure susceptible, to an approximately 3.6 cM interval in the middle of mouse chromosome 13. Linkage ofmass1to chromosome 13 is an important step in identifying the gene associated with a monogenic seizure disorder in mice, which may ultimately lead to a better understanding of the pathophysiology of human seizure disorders.

Preclinical characterization of MDL 27,192 as a potential broad spectrum anticonvulsant agent with neuroprotective properties.

The compound 5-(4-chlorophenyl)-2,4-dihydro-4-ethyl-3H-1,2,4-triazol-3-one (MDL 27,192) was evaluated in a variety of rodent models to assess its anticonvulsant profile and its potential neuroprotective activity. MDL 27,192 demonstrated anticonvulsant activity in a wide range of epilepsy models that are genetically-based (audiogenic seizures in the seizure susceptible DBA/2J or Frings mouse; spike wave seizures in genetic absence epilepsy rats of Strasbourg (GAERS), electrically-based (MES seizures in mice and rats, corneally-kindled seizures in rats) and chemically-based (bicuculline, PTZ, picrotoxin, 3-mercaptopropionic acid, quinolinic acid and strychnine). When compared to valproate, orally administered MDL 27,192 was 17-48-fold more potent as an anticonvulsant and showed a safety index one to three-fold greater. Following a timed intravenous administration of PTZ to mice, MDL 27,192, but not phenytoin or carbamazepine, consistently increased the latencies to first twitch and clonus. MDL 27,192 was active in a genetic model of absence epilepsy, the GAERS rat model. These data indicate that MDL 27,192 likely exerts its anticonvulsant action by affecting seizure spread and by raising seizure threshold. MDL 27,192 did not display any signs of tolerance following subchronic (15 day) administration. In tests of neuroprotective potential, MDL 27,192 reduced infarct volume in a permanent middle cerebral artery occlusion model of focal cerebral ischemia in rats and reduced the loss of hippocampal dentate hilar neurons in an animal model of unilateral head injury. In summary, MDL 27,192 possesses a broad-spectrum anticonvulsant profile. The potential for reduced tolerance and neuroprotective activity are additional positive features of MDL 27,192's preclinical profile.

Interleukin-1α in the Brain Is Induced by Audiogenic Seizure

We examined the expression of the sleep-inducing cytokine interleukin-1α (IL-1α) in the brains of audiogenic seizure-susceptible mice subsequent to the induction of sound-induced seizure. Animal models of epilepsy often require lesioning or trauma that may nonspecifically alter IL-1α expression. To avoid this, we employed the Frings mouse strain; a model of auditory-evoked reflex epilepsy. Frings mice were exposed to a high-intensity sound stimulus to induce a tonic extension seizure, and the expression of IL-1α transcripts in different brain regions was measured thereafter. Compared to control animals, IL-1α transcripts were elevated 6 to 8 h postseizure in the hypothalamus, but not hippocampus, by a dexamethasone-sensitive pathway. Similar results were obtained from the genetically distinct DBA/2J audiogenic seizure-susceptible mouse strain. These findings demonstrate that the expression of IL-1α is altered following generalized seizure activity, induced by noninvasive sensory stimulation, in a brain-region-specific manner.