Avi Orr-Urtreger

Mice Homozygous for the L250T Mutation in the α7 Nicotinic Acetylcholine Receptor Show Increased Neuronal Apoptosis and Die Within 1 Day of Birth

Abstract: The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in modulating neurotransmitter release and may play a role in the regulation of neuronal growth and differentiation. A threonine for leucine 247 substitution in the channel domain of the chick α7 nAChR increases agonist affinity and decreases the rate of desensitization, creating a “gain of function” model for this receptor. We have generated mice that express the analogous mutation (L250T) in the α7 nAChR using the techniques of homologous recombination and here report their characteristics. Mice heterozygous (+/T) for the L250T mutation are viable, fertile, and anatomically normal compared with wild‐type littermates. In contrast, homozygous (T/T) L250T mice die within 2‐24 h of birth. Brains of T/T mouse pups exhibit a marked reduction in α7 nAChR protein levels and show extensive apoptotic cell death throughout the somatosensory cortex. Furthermore, α7 L250T nAChRs are functionally expressed on neurons within the brains of T/T neonatal mice and have properties that are consistent with those observed for the rat α7 L250T and the chick α7 L247T mutant nAChRs expressed in oocytes. These findings indicate that neurons in the developing brain expressing only α7 L250T mutant nAChRs are susceptible to abnormal apoptosis, possibly due to increased Ca2+ influx.

Central role of fibroblast α3 nicotinic acetylcholine receptor in mediating cutaneous effects of nicotine

Smoking is associated with aberrant cutaneous tissue remodeling, such as precocious skin aging and impaired wound healing. The mechanism is not fully understood. Dermal fibroblasts (DF) are the primary cellular component of the dermis and may provide a target for pathobiologic effects of tobacco products. The purpose of this study was to characterize a mechanism of nicotine (Nic) effects on the growth and tissue remodeling function of DF. We hypothesized that the effects of Nic on DF result from its binding to specific nicotinic acetylcholine receptors (nAChRs) expressed by these cells and that downstream signaling from the receptors alters normal cell functioning, leading to changes in skin homeostasis. Using RT-PCR and Western blotting, we found that a 24-hour exposure of human DF to 10 μm Nic causes a 1.9- to 28-fold increase of the mRNA and protein levels of the cell cycle regulators p21, cyclin D1, Ki-67, and PCNA and a 1.7- to 2-fold increase of the apoptosis regulators Bcl-2 and caspase 3. Nic exposure also up-regulated expression of the dermal matrix proteins collagen type Iα1 and elastin as well as matrix metalloproteinase-1. Mecamylamine (Mec), the specific antagonist of nAChRs, abolished Nic-induced alterations, indicating that they resulted from a pharmacologic stimulation of nAChRs expressed by DF. To establish the relevance of these findings to a specific nicotinergic pathway, we studied human DF transfected with anti-α3 antisense oligonucleotides and murine DF from α3 nAChR knockout mice. In both cases, lack of α3 was associated with alterations in fibroblast growth and function that were opposite to those observed in DF treated with Nic, suggesting that the nicotinic effects on DF were mostly mediated by α3 nAChR. In addition to α3, the nAChR subunits detected in human DF were α5, α7, β2, and β4. The exposure of DF to Nic altered the relative amounts of each of these subunits, leading to reciprocal changes in [3H]epibatidine-binding kinetics. Thus, some of the pathobiologic effects of tobacco products on extracellular matrix turnover in the skin may stem from Nic-induced alterations in the physiologic control of the unfolding of the genetically determined program of growth and the tissue remodeling function of DF as well as alterations in the structure and function of fibroblast nAChRs.

Increased severity of experimental colitis in alpha5 nicotinic acetylcholine receptor subunit-deficient mice

Substantial evidence suggests a negative association between cigarette smoking and the incidence and severity of ulcerative colitis, a common human inflammatory bowel disease. Nicotine has been implicated in this association. The detection of nicotinic acetylcholine receptors in colonic epithelium, the primary tissue affected in ulcerative colitis, suggests a role for these receptors in the beneficial effect of nicotine on colonic inflammation. Using an animal model, we demonstrate for the first time that &agr;5 nicotinic acetylcholine receptor knockout mice have significantly more severe experimental colitis than wild-type controls and that nicotine significantly ameliorates its course when compared with wild-type controls. These findings suggest that &agr;5-containing nicotinic acetylcholine receptors participate in the modulation of colitis in mice, but other nicotinic acetylcholine receptor subunits also mediate the antiinflammatory effects of nicotine.

Genetic Testing in Israel: An Overview

In Israel, genetic screening and testing are widespread and are on the rise. The socialized medical system, the governmental National Program for the Detection and Prevention of Birth Defects, the central registry of genetic disorders, and the availability of medical genetic units influence the extensive utilization of genetic services. Israeli society is a complex one-ethnically, religiously, and culturally diverse, comprised of Jews of many ethnic backgrounds, as well as Christian and Moslem Arabs, Druze, and Bedouins. Multiple founder mutations have been documented in these various ethnic populations, often down to the level of specific villages or tribes. Although carrier screening and prenatal diagnostic testing are well established in the general population, the rejection of pregnancy termination by many religious communities often prevents participation in testing. Culturally appropriate genetic programs have been initiated in religious Jewish and non-Jewish communities in an attempt to overcome cultural barriers and reduce the frequency of severe genetic diseases nationwide.

Association of Sequence Alterations in the Putative Promoter of RAB7L1 With a Reduced Parkinson Disease Risk

OBJECTIVEnTo examine whether PARK16, which was recently identified as a protective locus for Parkinson disease (PD) in Asian, white, and South American populations, is also associated with PD in the genetically homogeneous Ashkenazi Jewish population.nnnDESIGNnCase-control study.nnnSETTINGnA medical center affiliated with a university. Subjectsxa0 Five single-nucleotide polymorphisms (SNPs) located between RAB7L1 and SLC41A1 were analyzed in 720 patients with PD and 642 controls, all of Ashkenazi Jewish origin.nnnMAIN OUTCOME MEASURESnHaplotypes were defined and risk estimates were determined for each SNP and haplotype. Bioinformatic analysis defined the putative promoter region of RAB7L1 and the transcription factor binding sites that are potentially affected by 2 of the tested SNPs.nnnRESULTSnAll tested SNPs were significantly associated with PD (odds ratiosxa0=xa00.64-0.76; Pxa0=xa0.0002-.014). Two of them, rs1572931 and rs823144, were localized to the putative promoter region of RAB7L1 and their sequence variations altered the predicted transcription factor binding sites of CdxA, p300, GATA-1, Sp1, and c-Ets-1. Only 0.4% of patients were homozygous for the protective rs1572931 genotype (T/T), compared with 3.0% among controls (Pxa0=xa05xa0×xa010(-5)). This SNP was included in a haplotype that reduced the risk for PD by 10- to 12-fold (Pxa0=xa0.002-.01) in all patients with PD and in a subgroup of patients who do not carry the Ashkenazi founder mutations in the GBA or LRRK2 genes.nnnCONCLUSIONSnOur data demonstrate that specific SNP variations and haplotypes in the PARK16 locus are associated with reduced risk for PD in Ashkenazim. Although it is possible that alterations in the putative promoter of RAB7L1 are associated with this effect, the role of other genes in this locus cannot be ruled out.

Screening for familial dysautonomia in Israel: evidence for higher carrier rate among Polish Ashkenazi Jews.

Familial dysautonomia (FD) is an autosomal recessive disorder characterized by hereditary sensory and autonomic neuropathies. Although extremely rare in most populations, FD is common among Ashkenazi Jews (AJ), with a calculated carrier frequency of 1 in 30, based on disease prevalence. The gene for FD was recently identified as IKBKAP. One major mutation (IVS2 + 6T --> C) is responsible in >99.5% of cases among AJ. The purpose of this study was to determine the actual frequency of FD carriers in the AJ population in Israel and to determine whether carriers are more frequent among a subpopulation of AJ from Poland. The study group included 1267 Jews of Ashkenazi origin who were referred for routine DNA screening tests. These included 1100 individuals who were full AJ and 167 who were part AJ. None had a family history of FD. Mutation analysis for (IVS2 + 6T --> C) was performed by PCR amplification followed by restriction enzyme analysis. All positive cases were confirmed by DHPLC WAVE( trade mark ). Among the 1100 full AJ tested, 34 were found to be FD carriers (1:32). The incidence of mutation carriers was significantly higher in AJ of Polish descent (1:18) compared to AJ of non-Polish descent (1:99). Among the 167 individuals who were part AJ, there were 3 carriers (1:56). The incidence of FD among AJ, particularly those of Polish background, warrants population screening. Population screening may be performed by denaturing high-performance liquid chromatography.

Array-Based Comparative Genome Hybridization in Clinical Genetics

Abnormalities in DNA copy number are frequently found in patients with multiple anomaly syndromes and mental retardation. Array-based comparative genomic hybridization (array-CGH) is a high-resolution, whole-genome technology that improves detection of submicroscopic aberrations underlying these syndromes. Eight patients with mental disability, multiple congenital anomalies, and dysmorphic features were screened for submicroscopic chromosomal imbalances using the GenoSensor Array 300 Chip. Subtelomeric aberrations previously detected by fluorescence in situ hybridization (FISH) analysis were confirmed in two patients, and accurate diagnosis was provided in two previously undiagnosed complex cases. Microdeletions at 15q11.2-q13 in a newborn with hypotonia, cryptorchidism, and hypopigmentation were detected with few discrepancies between the array results and FISH analysis. Contiguous microdeletion of GSCL, HIRA and TBX1 genes at 22q11.2 was identified in a previously undiagnosed boy with an unusual presentation of the VCF/DiGeorge spectrum. In a newborn with aniridia, a borderline false-negative WT1 deletion was observed, most probably because of differences between the size of the genomic deletion and the microarray probe. A false-positive rate of 0.2% was calculated for clone-by-clone analysis, whereas the per patient false-positive rate was 20%. Array-CGH is a powerful tool for the rapid and accurate detection of genetic disorders associated with copy number abnormalities and can significantly improve clinical genetic diagnosis and care.

Reorganization of corticostriatal circuits in healthy G2019S LRRK2 carriers

Objective: We investigated system-level corticostriatal changes in a human model of premotor Parkinson disease (PD), i.e., healthy carriers of the G2019S LRRK2 mutation that is associated with a markedly increased, age-dependent risk of developing PD. Methods: We compared 37 asymptomatic LRRK2 G2019S mutation carriers (age range 30–78 years) with 32 matched, asymptomatic nonmutation carriers (age range 30–74 years). Using fMRI, we tested the hypothesis that corticostriatal connectivity in premotor PD shifts from severely affected to less affected striatal subregions, as shown previously in symptomatic PD. Specifically, we predicted that in premotor PD, the shift in corticostriatal connectivity would follow the same gradient of striatal dopamine depletion known from overt PD, with the dorsoposterior putamen being more affected than the ventroanterior putamen. Results: The known parallel topology of corticostriatal loops was preserved in each group, but the topography of putamen connectivity shifted. In LRRK2 G2019S mutation carriers, the right inferior parietal cortex had reduced functional connectivity with the dorsoposterior putamen but increased connectivity with the ventroanterior putamen, as compared with noncarriers. This shift in functional connectivity increased with age in LRRK2 G2019S mutation carriers. Conclusions: Asymptomatic LRRK2 G2019S mutation carriers show a reorganization of corticostriatal circuits that mirrors findings in idiopathic PD. These changes may reflect premotor basal ganglia dysfunction or circuit-level compensatory changes.

Cloning and mapping of the mouse α7-neuronal nicotinic acetylcholine receptor

We report the isolation of cDNA clones for the mouse {alpha}7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind a-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse {alpha}7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The {alpha}7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 {+-}1.7 cM-Gas2-0.9 cM {+-} 0.9 cM-D7Mit70-1.8 {+-} 1.2 cM- Acra7-4.4 {+-}1.0 cM-Hras1-ps11/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14. 26 refs., 3 figs.

Increased sensitivity to nicotine-induced seizures in mice heterozygous for the L250T mutation in the α7 nicotinic acetylcholine receptor

α7 Nicotinic acetylcholine receptors (nAChRs) are sparsely distributed throughout the peripheral and central nervous systems. Several studies have suggested that central α7 nicotinic receptors may influence sensitivity to nicotine-induced seizures in mice. In order to investigate the effect of α7 nAChRs on seizure sensitivity, we tested heterozygous mice with a threonine for leucine substitution at position 250 (L250T) within the channel domain, which is known to increase current amplitude and decreases desensitization of the channel. We show that administration of low doses of nicotine to these mutant mice increased the sensitivity to nicotine-induced seizures and the mortality rate. EEG recordings showed high amplitude rhythmic activity during tonic–clonic seizures. Pretreatment with the α7 nicotinic receptor antagonist methyllycaconitine inhibited the seizures induced by nicotine. These findings further suggest an important role for α7 nAChRs in the nicotine-induced seizures model of epilepsy.