Absence seizures in C3H/HeJ and knockout mice caused by mutation of the AMPA receptor subunit Gria4

Absence epilepsy, characterized by spike–wave discharges (SWD) in the electroencephalogram, arises from aberrations within the circuitry of the cerebral cortex and thalamus that regulates awareness. The inbred mouse strain C3H/HeJ is prone to absence seizures, with a major susceptibility locus, spkw1, accounting for most of the phenotype. Here we find that spkw1 is associated with a hypomorphic retroviral-like insertion mutation in the Gria4 gene, encoding one of the four amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor subunits in the brain. Consistent with this, Gria4 knockout mice also have frequent SWD and do not complement spkw1. In contrast, null mutants for the related gene Gria3 do not have SWD, and Gria3 loss actually lowers SWD of spkw1 homozygotes. Gria3 and Gria4 encode the predominant AMPA receptor subunits in the reticular thalamus, which is thought to play a central role in seizure genesis by inhibiting thalamic relay cells and promoting rebound burst firing responses. In Gria4 mutants, synaptic excitation of inhibitory reticular thalamic neurons is enhanced, with increased duration of synaptic responses—consistent with what might be expected from reduction of the kinetically faster subunit of AMPA receptors encoded by Gria4. These results demonstrate for the first time an essential role for Gria4 in the brain, and suggest that abnormal AMPA receptor-dependent synaptic activity can be involved in the network hypersynchrony that underlies absence seizures

A Missense Mutation in a Highly Conserved Alternate Exon of Dynamin-1 Causes Epilepsy in Fitful Mice

Dynamin-1 (Dnm1) encodes a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Mice heterozygous for a novel spontaneous Dnm1 mutation—fitful—experience recurrent seizures, and homozygotes have more debilitating, often lethal seizures in addition to severe ataxia and neurosensory deficits. Fitful is a missense mutation in an exon that defines the DNM1a isoform, leaving intact the alternatively spliced exon that encodes DNM1b. The expression of the corresponding alternate transcripts is developmentally regulated, with DNM1b expression highest during early neuronal development and DNM1a expression increasing postnatally with synaptic maturation. Mutant DNM1a does not efficiently self-assemble into higher order complexes known to be necessary for proper dynamin function, and it also interferes with endocytic recycling in cell culture. In mice, the mutation results in defective synaptic transmission characterized by a slower recovery from depression after trains of stimulation. The DNM1a and DNM1b isoform pair is highly conserved in vertebrate evolution, whereas invertebrates have only one isoform. We speculate that the emergence of more specialized forms of DNM1 may be important in organisms with complex neuronal function

Dynamin 1 isoform roles in a mouse model of severe childhood epileptic encephalopathy.

Dynamin 1 is a large neuron-specific GTPase involved in the endocytosis and recycling of pre-synaptic membranes and synaptic vesicles. Mutations in the gene encoding dynamin 1 (DNM1) underlie two epileptic encephalopathy syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Mice homozygous for the Dnm1 fitful mutation, a non-synonymous coding variant in an alternatively spliced exon of Dnm1 (exon 10a; isoform designation: Dnm1a(Ftfl)) have an epileptic encephalopathy-like disorder including lethal early onset seizures, locomotor and neurosensory deficits. Although fitful heterozygotes have milder recurrent seizures later in life, suggesting an additive or semi-dominant mechanism, the molecular etiology must also consider the fact that Dnm1a(Ftfl) exerts a dominant negative effect on endocytosis in vitro. Another complication is that the fitful mutation induces alterations in the relative abundance of Dnm1 splice variants; mutants have a downregulation of Dnm1a and an upregulation of Dnm1b, changes which may contribute to the epileptic pathology. To examine whether Dnm1a loss of function, Dnm1a(Ftfl) dominance or compensation by Dnm1b is the most critical for severe seizures, we studied alternate isoform-specific mutant mice. Mice lacking Dnm1 exon 10a or Dnm1 exon 10b have neither spontaneous seizures nor other overt abnormalities, suggesting that in normal conditions the major role of each isoform is redundant. However, in the presence of Dnm1a(Ftfl) only exon 10a deleted mice experience severe seizures. These results reveal functional differences between Dnm1a and Dnm1b isoforms in the presence of a challenge, i.e. toxic Dnm1(Ftfl), while reinforcing its effect explicitly in this model of severe pediatric epilepsy. Neurobiol Dis 2016; 95:1-11

Independent Neuronal Origin of Seizures and Behavioral Comorbidities in an Animal Model of a Severe Childhood Genetic Epileptic Encephalopathy.

The childhood epileptic encephalopathies (EE\u27s) are seizure disorders that broadly impact development including cognitive, sensory and motor progress with severe consequences and comorbidities. Recently, mutations in DNM1 (dynamin 1) have been implicated in two EE syndromes, Lennox-Gastaut Syndrome and Infantile Spasms. Dnm1 encodes dynamin 1, a large multimeric GTPase necessary for activity-dependent membrane recycling in neurons, including synaptic vesicle endocytosis. Dnm1Ftfl or fitful mice carry a spontaneous mutation in the mouse ortholog of DNM1 and recapitulate many of the disease features associated with human DNM1 patients, providing a relevant disease model of human EE\u27s. In order to examine the cellular etiology of seizures and behavioral and neurological comorbidities, we engineered a conditional Dnm1Ftfl mouse model of DNM1 EE. Observations of Dnm1Ftfl/flox mice in combination with various neuronal subpopulation specific cre strains demonstrate unique seizure phenotypes and clear separation of major neurobehavioral comorbidities from severe seizures associated with the germline model. This demonstration of pleiotropy suggests that treating seizures per se may not prevent severe comorbidity observed in EE associated with dynamin-1 mutations, and is likely to have implications for other genetic forms of EE. PLoS Genet 2015 Jun 30; 11(6):e100543

Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1–pHluorin imaging and membrane capacitance (C(m)) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1–pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1–pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1–pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic C(m) decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster C(m) decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic C(m) decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis

Reduced lifespan of <i>Dnm1</i>^{<i>Ftfl/flox</i>} mice crossed to different Cre driver lines.

<p>Survival curves of <i>Dnm1</i>^{<i>Ftfl/flox</i>} mice in conjunction with specific cre driver lines from postnatal day 0 until day 180. 100% of the <i>Gad2</i>-cre;<i>Dnm1</i>^{<i>Ftfl/flox</i>} mice, 100% of the <i>Dlx5/6</i>-cre;<i>Dnm1</i>^{<i>Ftfl/flox</i>} mice and 91% of the <i>Pvalb</i>-cre;<i>Dnm1</i>^{<i>Ftfl/flox</i>} mice die before weaning. The <i>Emx1</i>-cre;<i>Dnm1</i>^{<i>Ftfl/flox</i>} mice do not have a reduced lifespan.</p

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis. J Neurosci 2014 Jan 15; 34(3):705-16

Schematic of the <i>Dnm1</i> transcript and the conditional EE genotype strategy.

<p>(Top) The <i>Dnm1</i>^<i>Ftfl</i> allele is shown with the mutation in alternatively spliced exon 10a. (Middle) The <i>Dnm1</i>^<i>flox</i> allele is shown with location of the loxP sites flanking exons 2–4 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005347#pgen.1005347.ref007" target="_blank">7</a>]. (Bottom) After cre recombinase activity, exons 2–4 are deleted resulting in a frameshift and the <i>Dnm1</i>^<i>null</i> allele. Thus, the genotypes in the present studies are compound heterozygous, <i>Dnm1</i>^{<i>Ftfl/null</i>}, where the null (deleted allele) is present only in cells that express cre recombinase.</p

Summary of the effects of each cre line in combination with the <i>Dnm1</i>^{<i>Ftfl/flox</i>} genotype.

<p>Summary of the effects of each cre line in combination with the <i>Dnm1</i>^{<i>Ftfl/flox</i>} genotype.</p