Virginia Barone

Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning

Deletion of the ryanodine receptor type 3 (RyR3) results in specific changes in hippocampal synaptic plasticity, without affecting hippocampal morphology, basal synaptic transmission or presynaptic function. Robust long‐term potentiation (LTP) induced by repeated, strong tetanization in the CA1 region and in the dentate gyrus was unaltered in hippocampal slices in vitro, whereas weak forms of plasticity generated by either a single weak tetanization or depotentiation of a robust LTP were impaired. These distinct physiological deficits were paralleled by a reduced flexibility in re‐learning a new target in the water‐maze. In contrast, learning performance in the acquisition phase and during probe trial did not differ between the mutants and their wild‐type littermates. In the open‐field, RyR3−/− mice displayed a normal exploration and habituation, but had an increased speed of locomotion and a mild tendency to circular running. The observed physiological and behavioral effects implicate RyR3‐mediated Ca2+ release in the intracellular processes underlying spatial learning and hippocampal synaptic plasticity.

Requirement for the ryanodine receptor type 3 for efficient contraction in neonatal skeletal muscles

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post‐natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3−/− mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation–contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).

Frequency of Ret mutations in long- and short-segment Hirschsprung disease

Hirschsprung disease, or congenital aganglionic megacolon, is a genetic disorder of neural crest development affecting 1:5,000 newborns. Mutations in the RET proto‐oncogene, repeatedly identified in the heterozygous state in both long‐ and short‐segment Hirschsprung patients, lead to loss of both transforming and differentiating capacities of the activated RET through a dominant negative effect when expressed in appropriate cellular systems. The approach of single‐strand conformational polymorphism analysis established for all the 20 exons of the RET proto‐oncogene, and previously used to screen for point mutations in Hirschsprung patients allowed us to identify seven additional mutations among 39 sporadic and familial cases of Hirschsprung disease (detection rate 18%). This relatively low efficiency in detecting mutations of RET in Hirschsprung patients cannot be accounted by the hypothesis of genetic heterogeneity, which is not supported by the results of linkage analysis in the pedigrees analyzed so far. Almost 74% of the point mutations in our series, as well as in other patient series, were identified among long segment patients, who represented only 25% of our patient population. The finding of a C620R substitution in a patient affected with total colonic aganglionosis confirms the involvement of this mutation in the pathogenesis of different phenotypes (i.e., medullary thyroid carcinoma and Hirschsprung). Finally the R313Q mutation identified for the first time in homozygosity in a child born of consanguineous parents is associated with the most severe Hirschsprung phenotype (total colonic aganglionosis with small bowel involvement). Hum Mutat 9:243–249, 1997.

Increased Susceptibility to Cortical Spreading Depression in the Mouse Model of Familial Hemiplegic Migraine Type 2

Familial hemiplegic migraine type 2 (FHM2) is an autosomal dominant form of migraine with aura that is caused by mutations of the α2-subunit of the Na,K-ATPase, an isoform almost exclusively expressed in astrocytes in the adult brain. We generated the first FHM2 knock-in mouse model carrying the human W887R mutation in the Atp1a2 orthologous gene. Homozygous Atp1a2R887/R887 mutants died just after birth, while heterozygous Atp1a2+/R887 mice showed no apparent clinical phenotype. The mutant α2 Na,K-ATPase protein was barely detectable in the brain of homozygous mutants and strongly reduced in the brain of heterozygous mutants, likely as a consequence of endoplasmic reticulum retention and subsequent proteasomal degradation, as we demonstrate in transfected cells. In vivo analysis of cortical spreading depression (CSD), the phenomenon underlying migraine aura, revealed a decreased induction threshold and an increased velocity of propagation in the heterozygous FHM2 mouse. Since several lines of evidence involve a specific role of the glial α2 Na,K pump in active reuptake of glutamate from the synaptic cleft, we hypothesize that CSD facilitation in the FHM2 mouse model is sustained by inefficient glutamate clearance by astrocytes and consequent increased cortical excitatory neurotransmission. The demonstration that FHM2 and FHM1 mutations share the ability to facilitate induction and propagation of CSD in mouse models further support the role of CSD as a key migraine trigger.

A novel mutation in the ATP1A2 gene causes alternating hemiplegia of childhood

Alternating hemiplegia of childhood (AHC, MIM 104290) is a rare syndrome, characterised by early onset of episodic hemi- or quadriplegia lasting minutes to days. This disorder, first reported by Verret and Steel in 1971,1 has historically been thought to represent a migraine equivalent1 or an unusual form of epilepsy or a movement disorder,2 as it typically presents with complex and variable clinical features. In most patients, the earliest manifestations clearly related to AHC are tonic–dystonic attacks and paroxysmal nystagmus associated with autonomic changes and paroxysmal dyspnoea and usually appear between 3 and 6 months of age. The hemiplegic episodes develop before 18 months of age lasting anywhere from minutes to days at a time and involving either side of the body or shifting from one side to the other during the same episode with a period of bilateral weakness when the second side becomes involved.3 Some attacks are characterised by a bilateral involvement, which is apparent from the beginning, and do not follow an hemiplegic episode,3 which determines extreme hypotonia of the whole body with inability to move and a level of consciousness markedly depressed. A characteristic feature of AHC is the disappearance of all abnormalities when the child falls asleep.3 With increasing age, hemiplegic episodes follow a general pattern of initially increasing frequency and duration, followed by a plateau, and finally by a decrease in the number and duration of attacks. Analogously, the paroxysmal manifestations associated with hemiplegia tend to decrease in frequency and intensity with time and usually disappear after 5–7 years. Development before the onset of hemiplegic episodes may be normal or delayed. Epileptic seizures are reported in a variable percentage of sporadic patients.3–5 An association with migraine has also been noticed since the earliest description.1 Indeed, for …

Contribution of Ryanodine Receptor Type 3 to Ca 2 Sparks in Embryonic Mouse Skeletal Muscle

The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.

Molecular cloning and functional characterization of a GABA/betaine transporter from human kidney.

The human homologue of the canine GABA/betaine transporter (BGT‐1) was isolated from a kidney inner medulla cDNA library. The coding sequence predicts a 614 amino acids protein with the typical features of neurotransmitter transporter family. The gene maps to chromosome 12p13 and, in addition to kidney, is also expressed in brain, liver, heart, skeletal muscle, and placenta. Functional studies reveal a K m = 20 μM for GABA transport and a coupling to Na+ and Cl− with a stoichiometry 3 Na+:2 Cl−:1 GABA. At 500 μM the GABA transport was inhibited by various compounds with the following potency order: quinidine > verapamil > phloretin > betaine.

Intracellular Ca2+ release channels in evolution

Intracellular Ca(2+)-release channels (ICRCs) form a superfamily of genes that encompasses two distinct subfamilies: the inositol trisphosphate receptor and the ryanodine receptor genes, which encode the largest ion channels known today. During evolution from nematodes to man, mechanisms of gene duplication and divergence have increased the number of known ICRC genes, which have been gradually co-opted to contribute to the increasing complexity of intracellular Ca(2+) signalling required for regulation of specialised eukaryotic cell activities.

Two de novo mutations in the Na,K-ATPase gene ATP1A2 associated with pure familial hemiplegic migraine

Familial hemiplegic migraine (FHM) is a rare autosomal dominantly inherited subtype of migraine, in which hemiparesis occurs during the aura. The majority of the families carry mutations in the CACNA1A gene on chromosome 19p13 (FHM1). About 20% of FHM families is linked to chromosome 1q23 (FHM2), and has mutations in the ATP1A2 gene, encoding the α2-subunit of the Na,K-ATPase. Mutation analysis in a Dutch and a Turkish family with pure FHM revealed two novel de novo missense mutations, R593W and V628M, respectively. Cellular survival assays support the hypothesis that both mutations are disease-causative. The identification of the first de novo mutations underscores beyond any doubt the involvement of the ATP1A2 gene in FHM2.

Homer proteins and InsP 3 receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.