A human mitofusin 2 mutation can cause mitophagic cardiomyopathy.

Cardiac muscle has the highest mitochondrial density of any human tissue, but mitochondrial dysfunction is not a recognized cause of isolated cardiomyopathy. Here, we determined that the rare mitofusin (MFN) 2 R400Q mutation is 15-20× over-represented in clinical cardiomyopathy, whereas this specific mutation is not reported as a cause of MFN2 mutant-induced peripheral neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). Accordingly, we interrogated the enzymatic, biophysical, and functional characteristics of MFN2 Q400 versus wild-type and CMT2A-causing MFN2 mutants. All MFN2 mutants had impaired mitochondrial fusion, the canonical MFN2 function. Compared to MFN2 T105M that lacked catalytic GTPase activity and exhibited normal activation-induced changes in conformation, MFN2 R400Q and M376A had normal GTPase activity with impaired conformational shifting. MFN2 R400Q did not suppress mitochondrial motility, provoke mitochondrial depolarization, or dominantly suppress mitochondrial respiration like MFN2 T105M. By contrast to MFN2 T105M and M376A, MFN2 R400Q was uniquely defective in recruiting Parkin to mitochondria. CRISPR editing of the R400Q mutation into the mouse Mfn2 gene induced perinatal cardiomyopathy with no other organ involvement; knock-in of Mfn2 T105M or M376V did not affect the heart. RNA sequencing and metabolomics of cardiomyopathic Mfn2 Q/Q400 hearts revealed signature abnormalities recapitulating experimental mitophagic cardiomyopathy. Indeed, cultured cardiomyoblasts and in vivo cardiomyocytes expressing MFN2 Q400 had mitophagy defects with increased sensitivity to doxorubicin. MFN2 R400Q is the first known natural mitophagy-defective MFN2 mutant. Its unique profile of dysfunction evokes mitophagic cardiomyopathy, suggesting a mechanism for enrichment in clinical cardiomyopathy.

Mitochondria are organelles with an essential role in providing energy to the cells of the body. If damaged, they are repaired by fusing and exchanging contents with sister mitochondria in a process that requires mitofusin proteins. While mutations in the gene for mitofusin 2 have been linked to nerve damage, they do not appear to affect the heart ­ despite high concentrations of mitochondria in heart muscle cells. However, previous research showed that experimentally disrupting the programmed removal of mitochondria, a process also regulated by mitofusin 2, can cause heart muscle disease known as cardiomyopathy. This suggests that mutations affecting different mitofusin 2 roles might harm individual cell types in different ways. To investigate, Franco et al. carried out a genetic screen of people with cardiomyopathy, identifying a rare mitofusin 2 mutation, called R400Q, that was more common in this group. Experiments showed that R400Q caused cardiomyopathy in mice and affected mitochondrial repair and replacement, but not movement. By contrast, a mutation linked to Charcot-Marie-Tooth disease type 2A ­ which causes nerve damage ­ affected mitochondrial movement but not clearance, leading to nerve cell damage but not cardiomyopathy. This led Franco et al. to suggest that mitochondrial movement is central to nerve cell health, whereas mitochondrial repair and replacement plays an important role in cardiac development. Genetic cardiomyopathies affect around 1 in 500 people, but only half of the gene mutations responsible are known. These results suggest that mutations affecting mitochondrial quality control factors could be involved, highlighting a direction for future studies into modifiers of cardiomyopathy.

Generation and validation of novel conditional flox and inducible Cre alleles targeting fibroblast growth factor 18 (Fgf18).

BACKGROUND: Fibroblast growth factor 18 (FGF18) functions in the development of several tissues, including the lung, limb bud, palate, skeleton, central nervous system, and hair follicle. Mice containing a germline knockout of Fgf18 (Fgf18 -/- ) die shortly after birth. Postnatally, FGF18 is being evaluated for pathogenic roles in fibrosis and several types of cancer. The specific cell types that express FGF18 have been difficult to identify, and the function of FGF18 in postnatal development and tissue homeostasis has been hampered by the perinatal lethality of Fgf18 null mice. RESULTS: We engineered a floxed allele of Fgf18 (Fgf18 flox ) that allows conditional gene inactivation and a CreERT2 knockin allele (Fgf18 CreERT2 ) that allows the precise identification of cells that express Fgf18 and their lineage. We validated the Fgf18 flox allele by targeting it in mesenchymal tissue and primary mesoderm during embryonic development, resulting in similar phenotypes to those observed in Fgf18 null mice. We also use the Fgf18 CreERT2 allele, in combination with a conditional fluorescent reporter to confirm known and identify new sites of Fgf18 expression. CONCLUSION: These alleles will be useful to investigate FGF18 function during organogenesis and tissue homeostasis, and to target specific cell lineages at embryonic and postnatal time points.

Neurotransmission selectively regulates synapse formation in parallel circuits in vivo.

Activity is thought to guide the patterning of synaptic connections in the developing nervous system. Specifically, differences in the activity of converging inputs are thought to cause the elimination of synapses from less active inputs and increase connectivity with more active inputs. Here we present findings that challenge the generality of this notion and offer a new view of the role of activity in synapse development. To imbalance neurotransmission from different sets of inputs in vivo, we generated transgenic mice in which ON but not OFF types of bipolar cells in the retina express tetanus toxin (TeNT). During development, retinal ganglion cells (RGCs) select between ON and OFF bipolar cell inputs (ON or OFF RGCs) or establish a similar number of synapses with both on separate dendritic arborizations (ON-OFF RGCs). In TeNT retinas, ON RGCs correctly selected the silenced ON bipolar cell inputs over the transmitting OFF bipolar cells, but were connected with them through fewer synapses at maturity. Time-lapse imaging revealed that this was caused by a reduced rate of synapse formation rather than an increase in synapse elimination. Similarly, TeNT-expressing ON bipolar cell axons generated fewer presynaptic active zones. The remaining active zones often recruited multiple, instead of single, synaptic ribbons. ON-OFF RGCs in TeNT mice maintained convergence of ON and OFF bipolar cells inputs and had fewer synapses on their ON arbor without changes to OFF arbor synapses. Our results reveal an unexpected and remarkably selective role for activity in circuit development in vivo, regulating synapse formation but not elimination, affecting synapse number but not dendritic or axonal patterning, and mediating independently the refinement of connections from parallel (ON and OFF) processing streams even where they converge onto the same postsynaptic cell.

A proteomic approach for the discovery of protease substrates.

Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. "Protease proteomics" may therefore represent an important tool for the discovery of the native substrates of a variety of proteases.

ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain.

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.

A putative ariadne-like E3 ubiquitin ligase (PAUL) that interacts with the muscle-specific kinase (MuSK).

Formation of the postsynaptic membrane at the skeletal neuromuscular junction (NMJ) requires activation of the muscle-specific receptor tyrosine kinase (MuSK). Few intracellular mediators or modulators of MuSK actions are known. E3 ubiquitin ligases may serve this role, because activities of several receptor tyrosine kinases, G-protein-coupled receptors and channels are modulated by ubiquitination. Here, we report identification of a putative Ariadne-like ubiquitin ligase (PAUL) that binds to the cytoplasmic domain of MuSK. PAUL is expressed in numerous tissues of developing and adult mice, and is present at NMJs in muscle fibers but is not confined to them.

Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase.

Activity-dependent and -independent signals collaborate to regulate synaptogenesis, but their relative contributions are unclear. Here, we describe the formation of neuromuscular synapses at which neurotransmission is completely and specifically blocked by mutation of the neurotransmitter-synthesizing enzyme choline acetyltransferase. Nerve terminals differentiate extensively in the absence of neurotransmitter, but neurotransmission plays multiple roles in synaptic differentiation. These include influences on the numbers of pre- and postsynaptic partners, the distribution of synapses in the target field, the number of synaptic sites per target cell, and the number of axons per synaptic site. Neurotransmission also regulates the formation or stability of transient acetylcholine receptor-rich processes (myopodia) that may initiate nerve-muscle contact. At subsequent stages, neurotransmission delays some steps in synaptic maturation but accelerates others. Thus, neurotransmission affects synaptogenesis from early stages and coordinates rather than drives synaptic maturation.