Daniel L. Burgess

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13–qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

Mutation of the Ca2+ Channel β Subunit Gene Cchb4 Is Associated with Ataxia and Seizures in the Lethargic (lh) Mouse

Ca2+ channel beta subunits regulate voltage-dependent calcium currents through direct interaction with alpha 1 subunits. The beta- and alpha 1-binding motifs are conserved, and all beta subunits can stimulate current amplitude, voltage dependence, and kinetics when coexpressed with various alpha 1 subunits. We used a positional candidate approach to determine that the ataxia and seizures in the lethargic (lh) mouse arise from mutation of the beta-subunit gene Cchb4 on mouse chromosome 2. A four-nucleotide insertion into a splice donor site results in exon skipping, translational frameshift, and protein truncation with loss of the alpha 1-binding site. The lethargic phenotype is the first example of a mammalian neurological disease caused by an inherited defect in a non-pore-forming subunit of a voltage-gated ion channel.

A Triplet Repeat Expansion Genetic Mouse Model of Infantile Spasms Syndrome, Arx(GCG)10+7, with Interneuronopathy, Spasms in Infancy, Persistent Seizures, and Adult Cognitive and Behavioral Impairment

Infantile spasms syndrome (ISS) is a catastrophic pediatric epilepsy with motor spasms, persistent seizures, mental retardation, and in some cases, autism. One of its monogenic causes is an insertion mutation [c.304ins (GCG)7] on the X chromosome, expanding the first polyalanine tract of the interneuron-specific transcription factor Aristaless-related homeobox (ARX) from 16 to 23 alanine codons. Null mutation of the Arx gene impairs GABA and cholinergic interneuronal migration but results in a neonatal lethal phenotype. We developed the first viable genetic mouse model of ISS that spontaneously recapitulates salient phenotypic features of the human triplet repeat expansion mutation. Arx(GCG)10+7 (“Arx plus 7”) pups display abnormal spasm-like myoclonus and other key EEG features, including multifocal spikes, electrodecremental episodes, and spontaneous seizures persisting into maturity. The neurobehavioral profile of Arx mutants was remarkable for lowered anxiety, impaired associative learning, and abnormal social interaction. Laminar decreases of Arx+ cortical interneurons and a selective reduction of calbindin-, but not parvalbumin- or calretinin-expressing interneurons in neocortical layers and hippocampus indicate that specific classes of synaptic inhibition are missing from the adult forebrain, providing a basis for the seizures and cognitive disorder. A significant reduction of calbindin-, NPY (neuropeptide Y)-expressing, and cholinergic interneurons in the mutant striatum suggest that dysinhibition within this network may contribute to the dyskinetic motor spasms. This mouse model narrows the range of critical pathogenic elements within brain inhibitory networks essential to recreate this complex neurodevelopmental syndrome.

β Subunit Reshuffling Modifies N- and P/Q-Type Ca2+Channel Subunit Compositions in Lethargic Mouse Brain

Neuronal voltage-dependent Ca2+ channels are heteromultimers of alpha1, beta, and alpha2delta subunits, and any one of five alpha1 subunits (alpha1A-E) may associate with one of four beta subunits (beta1-4). The specific alpha1-beta combination assembled determines single-channel properties, while variation in the proportion of each combination contributes to the functional diversity of neurons. The mouse mutant lethargic (lh) exhibits severe neurological defects due to a mutation that deletes the alpha1 subunit interaction domain of the beta4 subunit. Since beta subunits regulate critical alpha1 subunit properties in heterologous expression systems, loss of beta4 in lethargic could dramatically alter channel localization and behavior unless beta1-3 subunits can be used as substitutes in vivo. Here we demonstrate increased steady-state associations of alpha1A and alpha1B with the remaining beta1-3 subunits, without significant changes in beta1-3 mRNA abundance. The immunolocalization of alpha1A and alpha1B protein in lethargic brain is indistinguishable from wild-type by light microscopy. Furthermore, the measurement of large-amplitude P-type currents in dissociated lethargic Purkinje neurons indicates that these alpha1A-containing channels retain regulation by beta subunits. We conclude that several properties of alpha1A and alpha1B proteins are not uniquely regulated by beta4 in vivo and may be rescued by beta1-3 subunit reshuffling. The complex neurological manifestation of the lethargic mutation therefore emerges from loss of beta4 coupled with the widespread pairing of surrogate beta subunits with multiple Ca2+ channel subtypes. The existence of beta subunit reshuffling demonstrates that molecular plasticity of Ca2+ channel assembly, a normal feature of early brain development, is retained in the mature brain.

Single gene defects in mice: the role of voltage-dependent calcium channels in absence models.

Nineteen genes encoding alpha1, beta, gamma, or alpha2delta voltage-dependent calcium channel subunits have been identified to date. Recent studies have found that three of these genes are mutated in mice with generalised cortical spike-wave discharges (models of human absence epilepsy), emphasising the importance of calcium channels in regulating the expression of this inherited seizure phenotype. The tottering (tg) locus encodes the calcium channel alpha1 subunit gene Cacna1a, lethargic (lh) encodes the beta subunit gene Cacnb4, and stargazer (stg) encodes the gamma subunit gene Cacng2. These calcium channel mutants should provide important insights into the basic mechanisms of neuronal synchronisation, and the genes may be considered candidates for involvement in similar human disorders. The mutant models offer an important opportunity to elucidate the molecular, developmental, and physiological mechanisms underlying one subtype of absence epilepsy. Since calcium channels are involved in numerous cellular functions, including proliferation and differentiation, membrane excitability, neurite outgrowth and synaptogenesis, signal transduction, and gene expression, their role in generating the absence epilepsy phenotype may be complex. A comparative analysis of channel function and neural excitability patterns in tottering, lethargic, and stargazer brain should be useful in identifying the common elements of calcium channel involvement in these absence models.

Loss of the Potassium Channel β‐Subunit Gene, KCNAB2, Is Associated with Epilepsy in Patients with 1p36 Deletion Syndrome

Summary:  Purpose: Clinical features associated with chromosome 1p36 deletion include characteristic craniofacial abnormalities, mental retardation, and epilepsy. The presence and severity of specific phenotypic features are likely to be correlated with loss of a distinct complement of genes in each patient. We hypothesize that hemizygous deletion of one, or a few, critical gene(s) controlling neuronal excitability is associated with the epilepsy phenotype. Because ion channels are important determinants of seizure susceptibility and the voltage‐gated K+ channel β‐subunit gene, KCNAB2, has been localized to 1p36, we propose that deletion of this gene may be associated with the epilepsy phenotype.

Stargazin and Other Transmembrane AMPA Receptor Regulating Proteins Interact with Synaptic Scaffolding Protein MAGI-2 in Brain

The spatial coordination of neurotransmitter receptors with other postsynaptic signaling and structural molecules is regulated by a diverse array of cell-specific scaffolding proteins. The synaptic trafficking of AMPA receptors by the stargazin protein in some neurons, for example, depends on specific interactions between the C terminus of stargazin and the PDZ [postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1] domains of membrane-associated guanylate kinase scaffolding proteins PSD-93 or PSD-95. Stargazin [Cacng2 (Ca2+ channel γ2 subunit)] is one of four closely related proteins recently categorized as transmembrane AMPA receptor regulating proteins (TARPs) that appear to share similar functions but exhibit distinct expression patterns in the CNS. We used yeast two-hybrid screening to identify MAGI-2 (membrane associated guanylate kinase, WW and PDZ domain containing 2) as a novel candidate interactor with the cytoplasmic C termini of the TARPs. MAGI-2 [also known as S-SCAM (synaptic scaffolding molecule)] is a multi-PDZ domain scaffolding protein that interacts with several different ligands in brain, including PTEN (phosphatase and tensin homolog), dasm1 (dendrite arborization and synapse maturation 1), dendrin, axin, β- and δ-catenin, neuroligin, hyperpolarization-activated cation channels, β1-adrenergic receptors, and NMDA receptors. We confirmed that MAGI-2 coimmunoprecipitated with stargazin in vivo from mouse cerebral cortex and used in vitro assays to localize the interaction to the C-terminal -TTPV amino acid motif of stargazin and the PDZ1, PDZ3, and PDZ5 domains of MAGI-2. Expression of stargazin recruited MAGI-2 to cell membranes and cell–cell contact sites in transfected HEK-293T cells dependent on the presence of the stargazin -TTPV motif. These experiments identify MAGI-2 as a strong candidate for linking TARP/AMPA receptor complexes to a wide range of other postsynaptic molecules and pathways and advance our knowledge of protein interactions at mammalian CNS synapses.

Allelic mutations of the sodium channel SCN8A reveal multiple cellular and physiological functions

Allelic mutations of Scn8a in the mouse have revealed the range of neurological disorders that can result from alternations of one neuronal sodium channel. Null mutations produce the most severe phenotype, with motor neuron failure leading to paralysis and juvenile lethality. Two less severe mutations cause ataxia, tremor, muscle weakness, and dystonia. The electrophysiological effects have been studied at the cellular level by recording from neurons from the mutant mice. The data demonstrate that Scn8a is required for the complex spiking of cerebellar Purkinje cells and for persistent sodium current in several classes of neurons, including some with pacemaker roles. The mouse mutations of Scn8a have also provided insight into the mode of inheritance of channelopathies, and led to the identification of a modifier gene that affects transcript splicing. These mutations demonstrate the value of mouse models to elucidate the pathophysiology of human disease.

Voltage‐Dependent Calcium Channel Mutations in Neurological Disease

ABSTRACT: Calcium ion channel mutations disrupt channel function and create recognizable disease phenotypes in the nervous system. The broad array of underlying cellular alterations is commensurate with the expanding genetic diversity of the voltage‐gated calcium ion channel complex and its critical role in regulating cell function. Currently, 16 calcium channel genes are known, and mutations in 7 of these are associated with distinct inherited neurological disorders. These mutations provide new insight into the structure and function of the channels, and link specific subunits to cellular disease processes, including altered excitability, synaptic signaling, and cell death. Studies of mutant channel behavior, subunit interactions, and the differentiation of neural networks demonstrate unique patterns of downstream rearrangement. Developmental analysis of molecular plasticity in these mutants is a critical step to define the intervening mechanisms that translate aberrant ion channel behavior into the diverse clinical phenotypes observed.

Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15

Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (< 20 kb) deletion was detected at the insertion site, with no evidence of additional rearrangement of the chromosomal DNA. Noncomplementation of the transgene-induced mutation and med was demonstrated in a cross with medJ/+mice. The new allele is designated medTgNA4Bs (medtg). The homologous human locus MED was assigned to chromosome 12. Synaptotagmin 1 and contactin 1 were eliminated as candidate genes for the med mutation. The transgene-induced allele provides molecular access to the med gene, whose function is required for synaptic transmission at the neuromuscular junction and long-term survival of cerebellar Purkinje cells.