Kiely Grundahl

Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin

Synaptotagmin is an abundant synaptic vesicle-associated protein proposed to be involved in calcium-mediated neurotransmitter release. Our molecular and genetic results demonstrate that, although synaptotagmin is required for the proper function of the presynaptic nerve terminal in C. elegans, some neurotransmitter release persists in synaptogamin mutants. In C. elegans neurons, synaptotagmin is localized to regions known to be rich in synapses and appears to be associated with synaptic vesicles. Mutants defective in the synaptotagmin gene, called snt-1, exhibit severe behavioral abnormalities that are characteristic of deficiencies in synaptic function, including severe locomotion, feeding, and defecation defects. The mutants are defective in exocytosis, since they accumulate acetylcholine, and are resistant to cholinesterase inhibitors, but they nevertheless remain sensitive to cholinergic receptor agonists. In spite of these exocytic defects, snt-1 mutants are capable of coordinated motor movements, indicating that the mutants do not have a complete block of neurotransmitter release.

Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjögren’s syndrome

Sjögrens syndrome is a common autoimmune disease (affecting ∼0.7% of European Americans) that typically presents as keratoconjunctivitis sicca and xerostomia. Here we report results of a large-scale association study of Sjögrens syndrome. In addition to strong association within the human leukocyte antigen (HLA) region at 6p21 (Pmeta = 7.65 × 10−114), we establish associations with IRF5-TNPO3 (Pmeta = 2.73 × 10−19), STAT4 (Pmeta = 6.80 × 10−15), IL12A (Pmeta = 1.17 × 10−10), FAM167A-BLK (Pmeta = 4.97 × 10−10), DDX6-CXCR5 (Pmeta = 1.10 × 10−8) and TNIP1 (Pmeta = 3.30 × 10−8). We also observed suggestive associations (Pmeta < 5 × 10−5) with variants in 29 other regions, including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2 and PHIP, among others. These results highlight the importance of genes that are involved in both innate and adaptive immunity in Sjögrens syndrome.

Comparison of the American-European Consensus Group Sjögren's syndrome classification criteria to newly proposed American College of Rheumatology criteria in a large, carefully characterised sicca cohort

Abstract Objective To compare the performance of the American–European Consensus Group (AECG) and the newly proposed American College of Rheumatology (ACR) classification criteria for Sjögrens Syndrome (SS) in a well-characterised sicca cohort, given ongoing efforts to resolve discrepancies and weaknesses in the systems. Methods In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria in 646 participants. Global gene expression profiles were compared in a subset of 180 participants. Results Application of the AECG and ACR criteria resulted in classification of 279 and 268 participants with SS, respectively. Both criteria were met by 244 participants (81%). In 26 of the 35 AECG+/ACR participants, the minor salivary gland biopsy focal score was ≥1 (74%), while nine had positive anti-Ro/La (26%). There were 24 AECG−/ACR+ who met ACR criteria mainly due to differences in the scoring of corneal staining. All patients with SS, regardless of classification, had similar gene expression profiles, which were distinct from the healthy controls. Conclusions The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting either set of criteria are more similar to other SS participants than to healthy controls. Thus, there is no clear evidence for increased value of the new ACR criteria over the old AECG criteria from the clinical or biological perspective. It is our contention, supported by this report, that improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available.

Identification of a Systemic Lupus Erythematosus Susceptibility Locus at 11p13 between PDHX and CD44 in a Multiethnic Study

Systemic lupus erythematosus (SLE) is considered to be the prototypic autoimmune disease, with a complex genetic architecture influenced by environmental factors. We sought to replicate a putative association at 11p13 not yet exceeding genome-wide significance (p < 5 × 10(-8)) identified in a genome-wide association study (GWAS). Our GWA scan identified two intergenic SNPs located between PDHX and CD44 showing suggestive evidence of association with SLE in cases of European descent (rs2732552, p = 0.004, odds ratio [OR] = 0.78; rs387619, p = 0.003, OR = 0.78). The replication cohort consisted of >15,000 subjects, including 3562 SLE cases and 3491 controls of European ancestry, 1527 cases and 1811 controls of African American (AA) descent, and 1265 cases and 1260 controls of Asian origin. We observed robust association at both rs2732552 (p = 9.03 × 10(-8), OR = 0.83) and rs387619 (p = 7.7 × 10(-7), OR = 0.83) in the European samples with p(meta) = 1.82 × 10(-9) for rs2732552. The AA and Asian SLE cases also demonstrated association at rs2732552 (p = 5 × 10(-3), OR = 0.81 and p = 4.3 × 10(-4), OR = 0.80, respectively). A meta-analysis of rs2732552 for all racial and ethnic groups studied produced p(meta) = 2.36 × 10(-13). This locus contains multiple regulatory sites that could potentially affect expression and functions of CD44, a cell-surface glycoprotein influencing immunologic, inflammatory, and oncologic phenotypes, or PDHX, a subunit of the pyruvate dehydrogenase complex.

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans.

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ∼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

The cho-1 gene in Caenorhabditis elegans encodes a high-affinity plasma-membrane choline transporter believed to be rate limiting for acetylcholine (ACh) synthesis in cholinergic nerve terminals. We found that CHO-1 is expressed in most, but not all cholinergic neurons in C. elegans. cho-1 null mutants are viable and exhibit mild deficits in cholinergic behavior; they are slightly resistant to the acetylcholinesterase inhibitor aldicarb, and they exhibit reduced swimming rates in liquid. cho-1 mutants also fail to sustain swimming behavior; over a 33-min time course, cho-1 mutants slow down or stop swimming, whereas wild-type animals sustain the initial rate of swimming over the duration of the experiment. A functional CHO-1∷GFP fusion protein rescues these cho-1 mutant phenotypes and is enriched at cholinergic synapses. Although cho-1 mutants clearly exhibit defects in cholinergic behaviors, the loss of cho-1 function has surprisingly mild effects on cholinergic neurotransmission. However, reducing endogenous choline synthesis strongly enhances the phenotype of cho-1 mutants, giving rise to a synthetic uncoordinated phenotype. Our results indicate that both choline transport and de novo synthesis provide choline for ACh synthesis in C. elegans cholinergic neurons.

Interaction between innate immunity and Ro52-induced antibody causes Sjögren's syndrome-like disorder in mice

Objectives Autoantibodies reactive with Ro52 are often found in sera of patients with Sjögrens syndrome (SS). This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS. Methods New Zealand Mixed (NZM) 2758 mice were immunised with Ro52 in alum adjuvant. Control mice were immunised either with maltose-binding protein or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine-induced salivation. Antibody binding to salivary gland (SG) cells was analysed in vivo and in vitro by immunofluorescence. Sera from immunised mice were passively transferred into untreated or alum injected NZM2758 mice. Results By day 30 post-immunisation, Ro52 immunised mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunised and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunised mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalised by a SG cell line and this uptake was inhibited by cytochalasin D treatment. Conclusions Our data show for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

Previous diagnosis of Sjögren's Syndrome as rheumatoid arthritis or systemic lupus erythematosus

OBJECTIVE The diagnosis of SS is often difficult and many patients are symptomatic for years with other diagnoses before confirmation of SS. Our aim was to determine whether overlapping clinical and serologic features with RA and SLE may in part drive the misdiagnoses. METHODS A total of 1175 sicca patients were evaluated in a multidisciplinary clinic and classified as having SS based on the American-European Consensus Group Criteria. They were interrogated for a past history of suspicion or diagnosis of RA, SLE or SSc. These diseases were confirmed or ruled out by applying the corresponding classification criteria if the patients responded affirmatively. RESULTS Of these, 524 (44.6%) subjects reported previous diagnosis or suspicion of RA, SLE or SSc, which was confirmed in 130 (24.8%) but excluded in 394 (75.2%) subjects. Of those previously diagnosed with another illness, 183 (34.9%) met the criteria for primary SS. RF was present in 70/191 patients with previous diagnosis of RA compared with 445/845 without a prior RA diagnosis (P = 3.38E-05), while 128/146 with a diagnosis of SLE had positive ANA compared with 622/881 without the diagnosis (P = 8.77E-06). Age also influenced former diagnoses: people with suspected RA were older than those without the diagnosis (P = 5.89E-06), while patients with SLE suspicion were younger (P = 0.0003). Interestingly, the previous diagnoses did not significantly delay a final classification of SS. CONCLUSION Among subjects classified as SS, the presence of a positive ANA or RF was associated with a previous, apparently erroneous diagnosis of SLE or RA, respectively.

Brief Report: Patients With Primary Sjögren's Syndrome Who Are Positive for Autoantibodies to Tripartite Motif-Containing Protein 38 Show Greater Disease Severity.

Autoantibodies reactive with Ro52 (tripartite motif−containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjögrens syndrome (SS). TRIM21 belongs to a 34‐member C‐IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C‐IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti‐TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease.

Primary Sjögren's syndrome patients with autoantibodies against TRIM38 show greater severity of disease

Autoantibodies reactive with Ro52 (tripartite motif−containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjögrens syndrome (SS). TRIM21 belongs to a 34‐member C‐IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C‐IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti‐TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease.