Expression of LIM domain-binding 3 (LDB3), a striated muscle Z-band alternatively spliced PDZ-motif protein in the nervous system.

LIM domain-binding 3 (LDB3) is a member of the Enigma family of PDZ-LIM proteins. LDB3 has been reported as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mechanosensory actin cytoskeleton remodeling. This study shows that LDB3 is broadly expressed in the central and peripheral nervous system of human and mouse. LDB3 is predominantly expressed in the adult stages compared to early development and at a significantly higher level in the spinal cord than in the brain. As in skeletal muscle and heart, LDB3 is extensively alternatively spliced in the neurons. Three novel splice isoforms were identified suggesting splicing-dependent regulation of LDB3 expression in the nervous system. Expression of LDB3 in the motor cortex, cerebellum, spinal motor neuron, peripheral nerve, and neuromuscular junction in addition to skeletal muscle indicates important roles for this PDZ-LIM family protein in motor planning and execution. Moreover, expression in the hippocampal neurons suggests roles for LDB3 in learning and memory. LDB3 interactors filamin C and myotilin are also expressed in the spinal motor neuron, nerve, and neuromuscular junction, thereby providing the basis for neurogenic manifestations in myopathies associated with mutations in these so-called muscle proteins.

Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation.

Mechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle.

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course.

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.

CUG-BP, Elav-like family member 1 (CELF1) is required for normal myofibrillogenesis, morphogenesis, and contractile function in the embryonic heart.

BACKGROUND: CUG-BP, Elav-like family member 1 (CELF1) is a multifunctional RNA binding protein found in a variety of adult and embryonic tissues. In the heart, CELF1 is found exclusively in the myocardium. However, the roles of CELF1 during cardiac development have not been completely elucidated. RESULTS: Myofibrillar organization is disrupted and proliferation is reduced following knockdown of CELF1 in cultured chicken primary embryonic cardiomyocytes. In vivo knockdown of Celf1 in developing Xenopus laevis embryos resulted in myofibrillar disorganization and a trend toward reduced proliferation in heart muscle, indicating conserved roles for CELF1 orthologs in embryonic cardiomyocytes. Loss of Celf1 also resulted in morphogenetic abnormalities in the developing heart and gut. Using optical coherence tomography, we showed that cardiac contraction was impaired following depletion of Celf1, while heart rhythm remained unperturbed. In contrast to cardiac muscle, loss of Celf1 did not disrupt myofibril organization in skeletal muscle cells, although it did lead to fragmentation of skeletal muscle bundles. CONCLUSIONS: CELF1 is required for normal myofibril organization, proliferation, morphogenesis, and contractile performance in the developing myocardium. Developmental Dynamics 245:854-873, 2016. © 2016 Wiley Periodicals, Inc.

Identification of Targets of CUG-BP, Elav-Like Family Member 1 (CELF1) Regulation in Embryonic Heart Muscle.

CUG-BP, Elav-like family member 1 (CELF1) is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP) for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in MYH7B as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased MYH7B levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.

Identification of transcripts regulated by CUG-BP, Elav-like family member 1 (CELF1) in primary embryonic cardiomyocytes by RNA-seq.

CUG-BP, Elav-like family member 1 (CELF1) is a multi-functional RNA binding protein that regulates pre-mRNA alternative splicing in the nucleus, as well as polyadenylation status, mRNA stability, and translation in the cytoplasm [1]. Dysregulation of CELF1 has been implicated in cardiomyopathies in myotonic dystrophy type 1 and diabetes [2-5], but the targets of CELF1 regulation in the heart have not been systematically investigated. We previously demonstrated that in the developing heart CELF1 expression is restricted to the myocardium and peaks during embryogenesis [6-8]. To identify transcripts regulated by CELF1 in the embryonic myocardium, RNA-seq was used to compare the transcriptome of primary embryonic cardiomyocytes following siRNA-mediated knockdown of CELF1 to that of controls. Raw data files of the RNA-seq reads have been deposited in NCBI's Gene Expression Omnibus [9] under the GEO Series accession number GSE67360. These data can be used to identify transcripts whose levels or alternative processing (i.e., alternative splicing or polyadenylation site usage) are regulated by CELF1, and should provide insight into the pathways and processes modulated by this important RNA binding protein during normal heart development and during cardiac pathogenesis.

RNA binding proteins in the regulation of heart development.

In vivo, RNA molecules are constantly accompanied by RNA binding proteins (RBPs), which are intimately involved in every step of RNA biology, including transcription, editing, splicing, transport and localization, stability, and translation. RBPs therefore have opportunities to shape gene expression at multiple levels. This capacity is particularly important during development, when dynamic chemical and physical changes give rise to complex organs and tissues. This review discusses RBPs in the context of heart development. Since the targets and functions of most RBPs--in the heart and at large--are not fully understood, this review focuses on the expression and roles of RBPs that have been implicated in specific stages of heart development or developmental pathology. RBPs are involved in nearly every stage of cardiogenesis, including the formation, morphogenesis, and maturation of the heart. A fuller understanding of the roles and substrates of these proteins could ultimately provide attractive targets for the design of therapies for congenital heart defects, cardiovascular disease, or cardiac tissue repair.

Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFß3.

BACKGROUND: Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor ß (TGFß) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFß-dependent endocardial cell invasion. RESULTS: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFß3 after it is no longer required to induce basal levels of invasion. TGFß3, but not TGFß2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFß3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFß3 production in the endocardium. More TGFß3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFß3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFß3 secretion, and early exposure to excess TGFß3 induces precocious invasion. MBNL1 expression precedes TGFß3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFß3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFß is necessary and sufficient to mediate this effect. CONCLUSIONS: Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFß3 production.

Beta-glucosidase 1 (GBA1) is a second bile acid ß-glucosidase in addition to ß-glucosidase 2 (GBA2). Study in ß-glucosidase deficient mice and humans.

Beta-glucosidase 1 (GBA1; lysosomal glucocerebrosidase) and ß-glucosidase 2 (GBA2, non-lysosomal glucocerebrosidase) both have glucosylceramide as a main natural substrate. The enzyme-deficient conditions with glucosylceramide accumulation are Gaucher disease (GBA-/- in humans), modelled by the Gba-/- mouse, and the syndrome with male infertility in the Gba2-/- mouse, respectively. Before the leading role of glucosylceramide was recognised for both deficient conditions, bile acid-3-O-ß-glucoside (BG), another natural substrate, was viewed as the main substrate of GBA2. Given that GBA2 hydrolyses both BG and glucosylceramide, it was asked whether vice versa GBA1 hydrolyses both glucosylceramide and BG. Here we show that GBA1 also hydrolyses BG. We compared the residual BG hydrolysing activities in the GBA1-/-, Gba1-/- conditions (where GBA2 is the almost only active ß-glucosidase) and those in the Gba2-/- condition (GBA1 active), with wild-type activities, but we used also the GBA1 inhibitor isofagomine. GBA1 and GBA2 activities had characteristic differences between the studied fibroblast, liver and brain samples. Independently, the hydrolysis of BG by pure recombinant GBA1 was shown. The fact that both GBA1 and GBA2 are glucocerebrosidases as well as bile acid ß-glucosidases raises the question, why lysosomal accumulation of glucosylceramide in GBA1 deficiency, and extra-lysosomal accumulation in GBA2 deficiency, are not associated with an accumulation of BG in either condition.

In silico and functional studies of the regulation of the glucocerebrosidase gene.

In Gaucher disease (GD), the inherited deficiency of glucocerebrosidase results in the accumulation of glucocerebroside within lysosomes. Although almost 300 mutations in the glucocerebrosidase gene (GBA) have been identified, the ability to predict phenotype from genotype is quite limited. In this study, we sought to examine potential GBA transcriptional regulatory elements for variants that contribute to phenotypic diversity. Specifically, we generated the genomic sequence for the orthologous genomic region ( approximately 39.4kb) encompassing GBA in eight non-human mammals. Computational comparisons of the resulting sequences, using human sequence as the reference, allowed the identification of multi-species conserved sequences (MCSs). Further analyses predicted the presence of two putative clusters of transcriptional regulatory elements upstream and downstream of GBA, containing five and three transcription factor-binding sites (TFBSs), respectively. A firefly luciferase (Fluc) reporter construct containing sequence flanking the GBA gene was used to test the functional consequences of altering these conserved sequences. The predicted TFBSs were individually altered by targeted mutagenesis, resulting in enhanced Fluc expression for one site and decreased expression for seven others sites. Gel-shift assays confirmed the loss of nuclear-protein binding for several of the mutated constructs. These identified conserved non-coding sequences flanking GBA could play a role in the transcriptional regulation of the gene contributing to the complexity underlying the phenotypic diversity seen in GD.

Ralfinamide administered orally before hindpaw neurectomy or postoperatively provided long-lasting suppression of spontaneous neuropathic pain-related behavior in the rat.

Ralfinamide is analgesic when applied as a single dose in rodent models of stimulus-evoked chronic pain. However, it is unknown whether its chronic application after nerve injury can suppress spontaneous chronic pain, the main symptom driving patients to seek treatment. In this study ralfinamide was administered to rats at doses producing plasma levels similar to those causing analgesia in pain patients. The analgesic effect was tested on autotomy, a behavior of self-mutilation of a denervated paw that models spontaneous neuropathic pain. Sprague-Dawley male rats (N=10-20/group) underwent transection of the sciatic and saphenous nerves unilaterally. Ralfinamide or its vehicle were administered per os for 7 days preoperatively (80 mg/kg; bid), followed by the vehicle or Ralfinamide, until postoperative d42. Autotomy was scored daily until d63. Lasting 'preemptive analgesia' was found in rats treated with ralfinamide preoperatively, expressed by delayed autotomy onset (P=0.009) and reduced scores on d63 (P=0.01). Rats treated with ralfinamide (30 or 60 mg/kg; bid) from the operation till d42, but not preoperatively, also showed delayed autotomy (P=0.05, P=0.006), and reduced autotomy scores lasting till d63 (P=0.02, P=0.01), for the two doses, respectively. Combining ralfinamide treatments for 7 days preoperatively and 42 days postoperatively also resulted in significantly suppressed scores on d42 and d63 (P=0.005, P=0.001, respectively). Suppression of neuropathic pain-related behavior was likely caused by a combination of mechanisms reported for ralfinamide, including inhibition of Na+ and Ca++ currents in Nav1.3, Nav1.7, Nav1.8, and Cav2.2 channels in rat DRG neurons, inhibition of substance P release from spinal cord synaptosomes, NMDA receptor antagonism and neuroprotection.

State-dependent action of cocaine on brain temperature and movement activity: implications for movement sensitization.

Because neural activity is highly energy consuming and heat producing, brain temperature offers a reliable, real-time measure of an animal's activity state and its changes induced by environmental and drug challenges. Therefore, it allows evaluation of the activity state of an animal preceding drug administration and its relation to subsequent drug-induced neural effects. This approach was used to explore the state dependency of cocaine's effects. Brain and body temperatures, as well as locomotion were measured simultaneously in rats during repeated, daily administration of cocaine (15 mg/kg i.p., daily for 5 days) under different experimental conditions. The drug was administered via (a). a chronically implanted catheter in quiet resting conditions, (b). an injection made under quiet rest or (c). an injection under activated conditions associated with placement in the cage. Although brain temperature and movement increased after cocaine administration in each condition, cocaine's action (evaluated as cocaine-saline difference for both parameters) was situational. Catheter-administered cocaine induced the strongest movement activation and robust, monophasic temperature increase, which remained relatively stable following each subsequent drug infusion. Cocaine injected during quiet and, especially, activated conditions, induced a weaker locomotor activation, while the temperature response (evaluated as drug-saline difference) had a biphasic pattern. Cocaine initially inhibited the temperature increases seen in saline-treated animals (0-20 min) and then induced a more prolonged hyperthermia, which was about twofold weaker than that seen after catheter-administered drug. Although movement activation gradually increased following repeated treatment in activated conditions, the magnitude of this sensitized motor response barely reached the levels induced by the initial cocaine administration via catheter. These data suggest that both the acute effects of cocaine in the brain and their change following repeated drug administration are dependent upon the ongoing neural activity state of the animal. Cocaine's interaction with this activity state is a crucial factor determining the behavioral effects of this drug, including state-dependent motor sensitization.