The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Correlation of serum and urinary matrix metalloproteases/tissue inhibitors of metalloproteases with subclinical allograft fibrosis in renal transplantation

Progressive interstitial fibrosis and tubular atrophy (IF/TA) is a leading cause of chronic allograft dysfunction. Increased extracellular matrix remodeling regulated by matrix metalloproteases (MMPs) and their inhibitors (TIMPs) has been implicated in the development of IF/TA. The aim of this study was to investigate whether urinary/serum MMPs/TIMPs correlate with subclinical IF/TA detected in surveillance biopsies within the first 6months post-transplant. We measured eight different MMPs/TIMPs simultaneously in urine and serum samples from patients classified as normal histology (n=15), IF/TA 1 (n=15) and IF/TA 2-3 (n=10). There was no difference in urinary MMPs/TIMPs among the three groups, and only 1/8 serum MMPs/TIMPs (i.e. MMP-1) was significantly elevated in biopsies with IF/TA 2-3 (p=0.01). In addition, urinary/serum MMPs/TIMPs were not different between surveillance biopsies demonstrating an early development of IF/TA (i.e. delta IF/TA≥1 compared to a previous biopsy obtained three months before; n=11) and stable grade of IF/TA (i.e. delta IF/TA=0; n=20). Next, we investigated whether urinary/serum MMP/TIMP levels are elevated during acute subclinical tubulitis in surveillance biopsies obtained within the first 6months post-transplant (n=25). Compared to biopsies with normal histology, serum MMPs/TIMPs were not different; however, all urinary MMP/TIMP levels were numerically higher during subclinical tubulitis (MMP-1, MMP-7, TIMP-1 with p≤0.04). We conclude that urinary/serum MMPs/TIMPs do hardly correlate with existing or early developing IF/TA in surveillance biopsies obtained within the first 6months post-transplant. This could be explained by the dynamic process of extracellular matrix remodeling, which seems to be active during acute tubulo-interstitial injury/inflammation, but not in quiescent IF/TA.

Clinical relevance of brain volume measures in multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease with an inflammatory and neurodegenerative pathology. Axonal loss and neurodegeneration occurs early in the disease course and may lead to irreversible neurological impairment. Changes in brain volume, observed from the earliest stage of MS and proceeding throughout the disease course, may be an accurate measure of neurodegeneration and tissue damage. There are a number of magnetic resonance imaging-based methods for determining global or regional brain volume, including cross-sectional (e.g. brain parenchymal fraction) and longitudinal techniques (e.g. SIENA [Structural Image Evaluation using Normalization of Atrophy]). Although these methods are sensitive and reproducible, caution must be exercised when interpreting brain volume data, as numerous factors (e.g. pseudoatrophy) may have a confounding effect on measurements, especially in a disease with complex pathological substrates such as MS. Brain volume loss has been correlated with disability progression and cognitive impairment in MS, with the loss of grey matter volume more closely correlated with clinical measures than loss of white matter volume. Preventing brain volume loss may therefore have important clinical implications affecting treatment decisions, with several clinical trials now demonstrating an effect of disease-modifying treatments (DMTs) on reducing brain volume loss. In clinical practice, it may therefore be important to consider the potential impact of a therapy on reducing the rate of brain volume loss. This article reviews the measurement of brain volume in clinical trials and practice, the effect of DMTs on brain volume change across trials and the clinical relevance of brain volume loss in MS.

Functional and anatomical outcome of eyes with neovascular age-related macular degeneration treated with intravitreal ranibizumab following an exit strategy regimen.

AIMS To assess the functional and morphological outcome of eyes with neovascular AMD treated with intravitreal ranbizumab following an exit strategy treatment regime. METHODS The Bern treatment regime for neovascular AMD has a fixed injection schedule, even in the non-active stage of the disease. The regimen has been adapted from the PIER study treatment protocol. Eyes with non-active AMD will receive 4 injections in the first year, and 2 injections in the second year of follow-up before treatment stops. Patients that received ranibizumab for treatment and reached the exit criteria were identified, and charts were reviewed to assess functional and morphological outcome. RESULTS Only 2.6% of all patients (15 out of 575 patients) reached the exit criteria. Mean change in best corrected ETDRS visual acuity (VA) was 4.5±16.9 letters when comparing baseline VA to 4 weeks after the last injection (p=0.32). OCT mean foveal thickness was significantly thinner after last treatment (247.9±43.0 µm) compared to baseline (332.5±83.1 µm, p=0.002). The mean total number of ranibizumab injections was 15.6±8.0, and the mean total treatment period was 40.9±18.3 months. Twenty percent of eyes had geographic atrophy present at baseline versus 46.6% at the end of treatment. CONCLUSIONS Even with a fixed treatment regime and a defined treatment exit strategy, only a small percentage of patients reach exit criteria. Retinal thickness has been significantly reduced by repeated intravitreal ranibizumab injections, and geographic atrophy became more frequent.

Vaginal cytokines do not differ between postmenopausal women with and without symptoms of vulvovaginal irritation.

OBJECTIVE The aim of this exploratory pilot study was to determine if there are differences in vaginal cytokine levels between postmenopausal women with and without vulvovaginal irritative symptoms (itching, burning, or pain). METHODS Postmenopausal women (n = 34) not using hormone therapy and presenting with or without symptoms of vulvovaginal irritation were asked to volunteer for this study. Each participant underwent a vaginal examination and screening for vaginitis using Amsel criteria, pH, and light microscopy. A vaginal lavage with 5.0 mL of sterile saline was carried out, and a peripheral blood sample was obtained. The vaginal lavage and serum samples were assayed for interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α by specific enzyme-linked immunosorbent assays. Results were adjusted for total protein concentration and presented as the amount of cytokines per protein (pg/μg protein). Statistical analysis was performed using SAS version 9.3 (SAS Institute, Cary, NC). The means and SDs of all variables among women with and without vulvovaginal irritation were compared using independent-samples Student's t test. RESULTS A total of 26 postmenopausal women were enrolled into the study (symptomatic, n = 15; asymptomatic, n = 11). The mean (SD) vaginal pH for all participants was 5.9 (1.2). There were no significant differences (P > 0.05) in age, age at menopause, vaginal pH, and vaginal and serum cytokines and chemokines (IL-1β, IL-6, IL-8, and tumor necrosis factor-α) among symptomatic versus asymptomatic women. IL-8 was the most abundant vaginal cytokine, with mean (SD) vaginal IL-8 levels being 4.1 (3.4) and 3.1 (3.9) pg/μg protein in the symptomatic versus asymptomatic groups, respectively (P = 0.55). There were no significant linear correlations (P > 0.05) between serum and vaginal cytokine levels for all endpoints. CONCLUSIONS The presence or absence of postmenopausal vulvovaginal symptoms does not significantly differentiate vaginal inflammatory markers. Serum and vaginal cytokines are not significantly linearly correlated among postmenopausal women with and without symptoms commonly associated with vaginal atrophy, implying that this is a local reaction.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Combined systemic and local morpholino treatment rescues the phenotype of the SMA Delta 7 mouse model

Spinal muscular atrophy (SMA) is a childhood fatal motor neuron disease caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, currently without effective treatment. One possible therapeutic approach is the use of antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to increase the production of functional SMN protein. A range of ASOs with different chemical properties is suitable for these applications, including a morpholino (MO) variant, which has a particularly excellent safety, and efficacy profile. We used a 25- nt MO oligomer sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D(-10-34)) with superior efficacy to previously described sequences also in transgenic SMA Δ7 mice. The combined local and systemic administration of MO (bare or conjugated to octa-guanidine) is necessary to increase full-length SMN expression, leading to robust neuropathological features improvement and survival rescue. Additionally, several snRNA levels that are dysregulated in SMA mice could be restored by MO treatment. These results demonstrate that MO therapy is efficacious and can result in phenotypic rescue. These data provide important insights for the development of therapeutic strategies in SMA patients.

Congenital cervical kyphosis in two young sighthounds

Introduction: Cervical vertebral (C) malformation is rarely reported in large breed dogs. Congenital cervical kyphosis (CCK) may result from defects of vertebral segmentation, failure of formation or both. This report describes two cases of C3-C4 CCK in young sighthounds, treated surgically. Case description: An 18-month-old female Deerhound and a six-week-old female Borzoi dog were presented because of the complaints of reluctance to exercise and signs of of neck pain. Both dogs were neurologically normal. Diagnostic imaging revealed C3-C4 deformity, moderate kyphosis, and spinal canal stenosis associated with chronic spinal cord pressure atrophy. Both dogs underwent surgical treatment. Results: A staged two-step surgery starting with dorsal decompression was elected in the Deerhound. After the first surgical procedure, the dog developed focal myelomalacia and phrenic nerve paralysis and was euthanatized. A ventral distraction-fusion technique with two locking plates was performed in the Borzoi. This patient recovered uneventfully and long-term follow-up computed tomography revealed complete spondylodesis. Clinical significance: Until now, CCK has only been described in sighthounds. Congenital cervical kyphosis might be considered a differential diagnosis in these breeds that are presented with signs of cervical pain. Ventral realignment-fusion and bone grafting may be considered for surgical treatment, although the earliest age at which this procedure can and should be performed remains unclear.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.

An ARHGEF10 deletion is highly associated with a juvenile-onset inherited polyneuropathy in Leonberger and Saint Bernard dogs

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10-10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3'-end of exon 17 and six nucleotides from the 5'-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3'-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [3]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma (FUS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [4]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation, RNA splicing, mRNA transport in neurons and microRNA processing [5] Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [6]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [7]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [8,9] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently established protocol [10] and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy. [1] Cleveland, D.W. et al. (2001) Nat Rev Neurosci 2(11): 806-819 [2] Sathasivam, S. (2010) Singapore Med J 51(5): 367-372 [3] Schymick, J.C. et al. (2007) Hum Mol Genet Vol 16: 233-242 [4] Pratt, A.J. et al. (2012). Degener Neurol Neuromuscul Dis 2012(2): 1-14 [5] Lagier-Tourenne, C. Hum Mol Genet, 2010. 19(R1): p. R46-64 [6] Mochizuki, Y. et al. (2012) J Neurol Sci 323(1-2): 85-92 [7] Dormann, D. et al. (2010) EMBO J 29(16): 2841-2857 [8] Hockemeyer, D. et al. (2011) Nat Biotech 29(8): 731-734 [9] Joung, J.K. and J.D. Sander (2013) Nat Rev Mol Cell Biol 14(1): 49-55 [10]Amoroso, M.W. et al. (2013) J Neurosci 33(2): 574-586.

The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations

Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n = 1267) and number of breeds studied (n = 22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p < 0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p < 0.0001) and pancreatic acinar atrophy (PAA, p = 0.04) in German shepherds.

HMG-CoA Reductase Inhibition Promotes Neurological Recovery, Peri-Lesional Tissue Remodeling, and Contralesional Pyramidal Tract Plasticity after Focal Cerebral Ischemia

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors are widely used for secondary stroke prevention. Besides their lipid-lowering activity, pleiotropic effects on neuronal survival, angiogenesis, and neurogenesis have been described. In view of these observations, we were interested whether HMG-CoA reductase inhibition in the post-acute stroke phase promotes neurological recovery, peri-lesional, and contralesional neuronal plasticity. We examined effects of the HMG-CoA reductase inhibitor rosuvastatin (0.2 or 2.0 mg/kg/day i.c.v.), administered starting 3 days after 30 min of middle cerebral artery occlusion for 30 days. Here, we show that rosuvastatin treatment significantly increased the grip strength and motor coordination of animals, promoted exploration behavior, and reduced anxiety. It was associated with structural remodeling of peri-lesional brain tissue, reflected by increased neuronal survival, enhanced capillary density, and reduced striatal and corpus callosum atrophy. Increased sprouting of contralesional pyramidal tract fibers crossing the midline in order to innervate the ipsilesional red nucleus was noticed in rosuvastatin compared with vehicle-treated mice, as shown by anterograde tract tracing experiments. Western blot analysis revealed that the abundance of HMG-CoA reductase was increased in the contralesional hemisphere at 14 and 28 days post-ischemia. Our data support the idea that HMG-CoA reductase inhibition promotes brain remodeling and plasticity far beyond the acute stroke phase, resulting in neurological recovery.

Quality control for retinal OCT in multiple sclerosis: validation of the OSCAR-IB criteria.

BACKGROUND Retinal optical coherence tomography (OCT) permits quantification of retinal layer atrophy relevant to assessment of neurodegeneration in multiple sclerosis (MS). Measurement artefacts may limit the use of OCT to MS research. OBJECTIVE An expert task force convened with the aim to provide guidance on the use of validated quality control (QC) criteria for the use of OCT in MS research and clinical trials. METHODS A prospective multi-centre (n = 13) study. Peripapillary ring scan QC rating of an OCT training set (n = 50) was followed by a test set (n = 50). Inter-rater agreement was calculated using kappa statistics. Results were discussed at a round table after the assessment had taken place. RESULTS The inter-rater QC agreement was substantial (kappa = 0.7). Disagreement was found highest for judging signal strength (kappa = 0.40). Future steps to resolve these issues were discussed. CONCLUSION Substantial agreement for QC assessment was achieved with aid of the OSCAR-IB criteria. The task force has developed a website for free online training and QC certification. The criteria may prove useful for future research and trials in MS using OCT as a secondary outcome measure in a multi-centre setting.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.