A synthetic Pur-based peptide binds and alters G-quadruplex secondary structure present in the expanded RNA repeat of C9orf72 ALS/FTD.

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD.

Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity.

Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.

Nitric oxide and cyclic GMP regulate early events in agrin signaling in skeletal muscle cells.

Agrin released from motor nerve terminals directs differentiation of the vertebrate neuromuscular junction (NMJ). Activity of nitric oxide synthase (NOS), guanylate cyclase (GC), and cyclic GMP-dependent protein kinase (PKG) contributes to agrin signaling in embryonic frog and chick muscle cells. Stimulation of the NO/cyclic GMP (cGMP) pathway in embryos potentiates agrin's ability to aggregate acetylcholine receptors (AChRs) at NMJs. Here we investigated the timing and mechanism of NO and cGMP action. Agrin increased NO levels in mouse C2C12 myotubes. NO donors potentiated agrin-induced AChR aggregation during the first 20 min of agrin treatment, but overnight treatment with NO donors inhibited agrin activity. Adenoviruses encoding siRNAs against each of three NOS isoforms reduced agrin activity, indicating that these isoforms all contribute to agrin signaling. Inhibitors of NOS, GC, or PKG reduced agrin-induced AChR aggregation in mouse muscle cells by approximately 50%. However, increased activation of the GTPase Rac1, an early step in agrin signaling, was dependent on NOS activity and was mimicked by NO donors and a cGMP analog. Our results indicate that stimulation of the NO/cGMP pathway is important during the first few minutes of agrin signaling and is required for agrin-induced Rac1 activation, a key step leading to reorganization of the actin cytoskeleton and subsequent aggregation of AChRs on the surface of skeletal muscle cells.

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction.

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction.

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.

Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis).

Husbandry and health of the African clawed frog, Xenopus laevis, greatly influences the quality of oocytes produced. One factor affecting oocyte quality is the water conditions in which females are maintained. Dechlorination and adequate salt concentration are known to affect oocytes, but water hardness has not been considered an important factor in Xenopus husbandry by the research community. We found that, when females were kept in soft water or water with marine salts alone, even when it was cooled to 17 to 18 degrees C, the quality of oocytes decreased; only 20 to 25% of resulting embryos developed to tailbud stages. Survival and normal development of embryos increased significantly within one month of addition to the laboratory housing water of salts that mimic conditions in African Rift Valley lakes. These salts greatly increased water hardness; development of embryos to tailbud stages remained high (50 to 70% on average) for more than a year after their addition to the water housing females. Water from South African ponds where X. laevis are collected, and from wells used by the major suppliers of X. laevis, also was moderately to very hard. Our results suggest that X. laevis is naturally adapted to hard water, and indicate that increasing general hardness during laboratory housing is more important for oocyte quality and embryo development than is increasing carbonate hardness (alkalinity) in the water used to house females.

The role of nitric oxide signaling in the formation of the neuromuscular junction.

The formation of the vertebrate neuromuscular junction (NMJ) depends on the action of neural agrin on the muscle cell. The requirement for agrin and its receptor, muscle-specific kinase (MuSK), has been well established over the past 20 years. However, the signaling mechanisms through which agrin and MuSK cause synaptic differentiation are not well understood. New evidence from studies of muscle cells in culture and in embryos indicates that nitric oxide (NO) is an effector of agrin-induced postsynaptic differentiation at the NMJ. Cyclic GMP (cGMP) production by guanylate cyclase appears to be an important downstream step in this pathway. Nitric oxide and cGMP regulate the activity of several kinases, some of which may influence interaction of dystrophin and utrophin with the actin cytoskeleton to mediate or modulate postsynaptic differentiation in muscle cells. These signaling molecules could also play a role in retrograde signaling to influence differentiation of presynaptic nerve terminals.

Identification and characterization of novel murine cytomegalovirus M112-113 (e1) gene products.

The human cytomegalovirus (HCMV) UL112-113 gene products play important roles in viral DNA replication and transcriptional regulation. In this report, we characterize two novel transcripts originating from the homologous M112-113 (e1) region of the murine cytomegalovirus (MCMV) genome. These transcripts of 2.0 and 2.4 kb represent alternatively spliced products of the e1 gene region. Analysis of the e1 proteins demonstrates the presence of a previously unidentified 87-kDa protein that is likely encoded by the 2.4-kb transcript. All four protein products derived from the e1 gene region are expressed with early kinetics, are coordinately regulated, and localize predominantly to the nucleus of MCMV-infected cells. The expression pattern and localization of the e1 proteins show significant similarity to those of the HCMV UL112-113 proteins, signifying that MCMV e1 will serve as a useful model for assessing the role of this early gene region during viral infection.