Matthew P. Anderson

Nucleoside triphosphates are required to open the CFTR chloride channel.

The CFTR Cl- channel contains two predicted nucleotide-binding domains (NBD1 and NBD2); therefore, we examined the effect of ATP on channel activity. Once phosphorylated by cAMP-dependent protein kinase (PKA), channels required cytosolic ATP to open. Activation occurred by a PKA-independent mechanism. ATP gamma S substituted for ATP in PKA phosphorylation, but it did not open the channel. Several hydrolyzable nucleotides (ATP greater than GTP greater than ITP approximately UTP greater than CTP) reversibly activated phosphorylated channels, but nonhydrolyzable analogs and Mg(2+)-free ATP did not. Studies of CFTR mutants indicated that ATP controls channel activity independent of the R domain and suggested that hydrolysis of ATP by NBD1 may be sufficient for channel opening. The finding that nucleoside triphosphates regulate CFTR begins to explain why CF-associated mutations in the NBDs block Cl- channel function.

Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2

One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel.

Cystic fibrosis transmembrane conductance regulator (CFTR) generates cAMP-regulated Cl- channels; mutations in CFTR cause defective Cl- channel function in cystic fibrosis epithelia. We used the patch-clamp technique to determine the single channel properties of Cl- channels in cell expressing recombinant CFTR. In cell-attached patches, an increase in cellular cAMP reversibly activated low conductance Cl- channels. cAMP-dependent regulation is due to phosphorylation, because the catalytic subunit of cAMP-dependent protein kinase plus ATP reversibly activated the channel in excised, cell-free patches of membrane. In symmetrical Cl- solutions, the channel had a channel conductance of 10.4 +/- 0.2 (n = 7) pS and a linear current-voltage relation. The channel was more permeable to Cl- than to I- and showed no appreciable time-dependent voltage effects. These biophysical properties are consistent with macroscopic studies of Cl- channels in single cells expressing CFTR and in the apical membrane of secretory epithelia. Identification of the single channel characteristics of CFTR-generated channels allows further studies of their regulation and the mechanism of ion permeation.

Cystic fibrosis transmembrane conductance regulator : a chloride channel with novel regulation

Michael J. Welsh,* Matthew P. Anderson,* Devra P. Rich,* Herbert A. Berger,* Gerene M. Denning,* Lynda S. Ostedgaard,* David N. Sheppard,* Seng H. Cheng,+ Richard J. Gregory,+ and Alan E. Smith+ *Howard Hughes Medical Institute Department of internal Medicine Department of Physiology and Biophysics University of Iowa College of Medicine Iowa City, Iowa 52242 +Genzyme Corporation Framingham, Massachusetts 01701

Increased Gene Dosage of Ube3a Results in Autism Traits and Decreased Glutamate Synaptic Transmission in Mice

Tripling the dosage of an autism- and Angelman syndrome–related gene in mice results in reduced glutamatergic synaptic transmission and autism behavioral traits. Linking a Ligase to Autism Autism spectrum disorder is highly heritable, but the genetic contribution is complex and heterogeneous. One chromosomal region, 15q11-13, when duplicated or tripled, often causes autism, accounting for up to 3% of autism risk. But we still cannot explain how even this relatively powerful autism predictor leads to the behavioral changes seen in the disease. To begin to understand these gene-to-behavior links, the authors created mice that carry, like the 15q11-13 autistic patients, double and triple doses of an E3 ubiquitin protein ligase called Ube3a. This gene is not the only one in this region but it is of particular interest because it is expressed solely from the maternal allele in neurons and is deficient in Angelman syndrome. Mice carrying a triple dose of Ube3a showed abnormalities in behavior that are the mouse equivalent of the three cardinal features of autism: Like patients, the mice preferred less social interaction with their peers than controls. They also communicated with others infrequently, which in mice takes the form of fewer vocalizations upon encountering a new mouse of the same sex. And the stereotyped, repetitive behaviors of autism spectrum disorder were mimicked in the mice by increased self-grooming. The authors also showed that synaptic transmission in the brain cortex was abnormal, a clue as to how a ubiquitin ligase might alter behaviors, although we will certainly need to know which proteins are serviced by Ube3a to begin to trace the pathway from gene to behavior. There are scores of identified and yet-to-be identified autism-associated genetic variations that individually or together may result in the behavioral and physical symptoms that make up autism spectrum disorder. Re-creation of some of these symptoms in mice by tripling the gene dosage of Ube3a reveals a key piece of the puzzle, but there are many yet to find and fit together. People with autism spectrum disorder are characterized by impaired social interaction, reduced communication, and increased repetitive behaviors. The disorder has a substantial genetic component, and recent studies have revealed frequent genome copy number variations (CNVs) in some individuals. A common CNV that occurs in 1 to 3% of those with autism—maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15)—affects several genes that have been suggested to underlie autism behavioral traits. To test this, we tripled the dosage of one of these genes, the ubiquitin protein ligase Ube3a, which is expressed solely from the maternal allele in mature neurons, and reconstituted the three core autism traits in mice: defective social interaction, impaired communication, and increased repetitive stereotypic behavior. The penetrance of these autism traits depended on Ube3a gene copy number. In animals with increased Ube3a gene dosage, glutamatergic, but not GABAergic, synaptic transmission was suppressed as a result of reduced presynaptic release probability, synaptic glutamate concentration, and postsynaptic action potential coupling. These results suggest that Ube3a gene dosage may contribute to the autism traits of individuals with maternal 15q11-13 duplication and support the idea that increased E3A ubiquitin ligase gene dosage results in reduced excitatory synaptic transmission.

Arrested maturation of excitatory synapses in autosomal dominant lateral temporal lobe epilepsy

A subset of central glutamatergic synapses are coordinately pruned and matured by unresolved mechanisms during postnatal development. We report that the human epilepsy gene LGI1, encoding leucine-rich, glioma-inactivated protein-1 and mutated in autosomal dominant lateral temporal lobe epilepsy (ADLTE), mediates this process in hippocampus. We created transgenic mice either expressing a truncated mutant LGI1 (835delC) found in ADLTE or overexpressing a wild-type LGI1. We discovered that the normal postnatal maturation of presynaptic and postsynaptic functions was arrested by the 835delC mutant LGI1, and contrastingly, was magnified by excess wild-type LGI1. Concurrently, mutant LGI1 inhibited dendritic pruning and increased the spine density to markedly increase excitatory synaptic transmission. Inhibitory transmission, by contrast, was unaffected. Furthermore, mutant LGI1 promoted epileptiform discharge in vitro and kindling epileptogenesis in vivo with partial γ-aminobutyric acidA (GABAA) receptor blockade. Thus, LGI1 represents a human gene mutated to promote epilepsy through impaired postnatal development of glutamatergic circuits (pages 1126–1127).

Fulminant JC virus encephalopathy with productive infection of cortical pyramidal neurons

The polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy and of JCV granule cell neuronopathy. We present a human immunodeficiency virus–negative patient who experienced development of multiple cortical lesions, aphasia, and progressive cognitive decline after chemotherapy for non–small‐cell lung cancer. Brain biopsy and cerebrospinal fluid polymerase chain reaction demonstrated JCV, and she had a rapidly fatal outcome. Postmortem analysis showed diffuse cortical lesions and areas of necrosis at the gray–white junction. Immunostaining showed a productive JCV infection of cortical pyramidal neurons, confirmed by electron microscopy, with limited demyelination. This novel gray matter syndrome expands the scope of JCV clinical presentation and pathogenesis. Ann Neurol 2009;65:742–748

Transplanted Hypothalamic Neurons Restore Leptin Signaling and Ameliorate Obesity in db/db Mice

Neurons transplanted from healthy donor mice can repair brain circuitry and partially normalize metabolism in obese mice. Evolutionarily old and conserved homeostatic systems in the brain, including the hypothalamus, are organized into nuclear structures of heterogeneous and diverse neuron populations. To investigate whether such circuits can be functionally reconstituted by synaptic integration of similarly diverse populations of neurons, we generated physically chimeric hypothalami by microtransplanting small numbers of embryonic enhanced green fluorescent protein–expressing, leptin-responsive hypothalamic cells into hypothalami of postnatal leptin receptor–deficient (db/db) mice that develop morbid obesity. Donor neurons differentiated and integrated as four distinct hypothalamic neuron subtypes, formed functional excitatory and inhibitory synapses, partially restored leptin responsiveness, and ameliorated hyperglycemia and obesity in db/db mice. These experiments serve as a proof of concept that transplanted neurons can functionally reconstitute complex neuronal circuitry in the mammalian brain.

Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N‐type calcium channels

Sensory signals of widely differing dynamic range and intensity are transformed into a common firing rate code by thalamocortical neurons. While a great deal is known about the ionic currents, far less is known about the specific channel subtypes regulating thalamic firing rates. We hypothesized that different K+ and Ca2+ channel subtypes control different stimulus–response curve properties. To define the channels, we measured firing rate while pharmacologically or genetically modulating specific channel subtypes. Inhibiting Kv3.2 K+ channels strongly suppressed maximum firing rate by impairing membrane potential repolarization, while playing no role in the firing response to threshold stimuli. By contrast, inhibiting Kv1 channels with α‐dendrotoxin or maurotoxin strongly increased firing rates to threshold stimuli by reducing the membrane potential where action potentials fire (Vth). Inhibiting SK Ca2+‐activated K+ channels with apamin robustly increased gain (slope of the stimulus–response curve) and maximum firing rate, with minimum effects on threshold responses. Inhibiting N‐type Ca2+ channels with ω‐conotoxin GVIA or ω‐conotoxin MVIIC partially mimicked apamin, while inhibiting L‐type and P/Q‐type Ca2+ channels had small or no effects. EPSC‐like current injections closely mimicked the results from tonic currents. Our results show that Kv3.2, Kv1, SK potassium and N‐type calcium channels strongly regulate thalamic relay neuron sensory transmission and that each channel subtype controls a different stimulus–response curve property. Differential regulation of threshold, gain and maximum firing rate may help vary the stimulus–response properties across and within thalamic nuclei, normalize responses to diverse sensory inputs, and underlie sensory perception disorders.

Autism gene Ube3a and seizures impair sociability by repressing VTA Cbln1

Maternally inherited 15q11-13 chromosomal triplications cause a frequent and highly penetrant type of autism linked to increased gene dosages of UBE3A, which encodes a ubiquitin ligase with transcriptional co-regulatory functions. Here, using in vivo mouse genetics, we show that increasing UBE3A in the nucleus downregulates the glutamatergic synapse organizer Cbln1, which is needed for sociability in mice. Epileptic seizures also repress Cbln1 and are found to expose sociability impairments in mice with asymptomatic increases in UBE3A. This Ube3a–seizure synergy maps to glutamate neurons of the midbrain ventral tegmental area (VTA), where Cbln1 deletions impair sociability and weaken glutamatergic transmission. We provide preclinical evidence that viral-vector-based chemogenetic activation of, or restoration of Cbln1 in, VTA glutamatergic neurons reverses the sociability deficits induced by Ube3a and/or seizures. Our results suggest that gene and seizure interactions in VTA glutamatergic neurons impair sociability by downregulating Cbln1, a key node in the expanding protein interaction network of autism genes.