To understand how mutations in Matrin 3 (MATR3) cause amyotrophic lateral sclerosis (ALS) and distal myopathy, we used transcriptome and interactome analysis, coupled with microscopy. Over-expression of wild-type (WT) or F115C mutant MATR3 had little impact on gene expression in neuroglia cells. Only 23 genes, expressed at levels of >100 transcripts showed ≥1.6-fold changes in expression by transfection with WT or mutant MATR3:YFP vectors. We identified ~123 proteins that bound MATR3, with proteins associated with stress granules and RNA processing/splicing being prominent. The interactome of myopathic S85C and ALS-variant F115C MATR3 were virtually identical to WT protein. Deletion of RNA recognition motif (RRM1) or Zn finger motifs (ZnF1 or ZnF2) diminished the binding of a subset of MATR3 interacting proteins. Remarkably, deletion of the RRM2 motif caused enhanced binding of >100 hundred proteins. In live cells, MATR3 lacking RRM2 (ΔRRM2) formed intranuclear spherical structures that fused over time into large structures. Our findings in the cell models used here suggest that MATR3 with disease-causing mutations is not dramatically different from WT protein in modulating gene regulation or in binding to normal interacting partners. The intra-nuclear localization and interaction network of MATR3 is strongly modulated by its RRM2 domain.

Mutations in more than 80 different positions in superoxide dismutase 1 (SOD1) have been associated with amyotrophic lateral sclerosis (fALS). There is substantial evidence that a common consequence of these mutations is to induce the protein to misfold and aggregate. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. In the present study, we have mutagenized Glu residues at positions 40 and 133 that are involved in stabilizing the β-barrel structure of the native protein and a critical Zn binding domain, respectively, to examine how specific mutations may cause SOD1 misfolding and aggregation. Mutations associated with ALS as well as experimental mutations were introduced into these positions. We used an assay in which mutant SOD1 was fused to yellow fluorescent protein (SOD1:YFP) to visualize the formation of cytosolic inclusions by mutant SOD1. We then used existing structural data on SOD1, to predict how different mutations might alter local 3D conformation. Our findings reveal an association between mutant SOD1 aggregation and amino acid substitutions that are predicted to introduce steric strain, sometimes subtly, in the 3D conformation of the peptide backbone.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the "strain" of the conformer that is propagating.

RNA binding proteins associated with amyotrophic lateral sclerosis (ALS) and muscle myopathy possess sequence elements that are low in complexity, or bear resemblance to yeast prion domains. These sequence elements appear to mediate phase separation into liquid-like membraneless organelles. Using fusion proteins of matrin 3 (MATR3) to yellow fluorescent protein (YFP), we recently observed that deletion of the second RNA recognition motif (RRM2) caused the protein to phase separate and form intranuclear liquid-like droplets. Here, we use fusion constructs of MATR3, TARDBP43 (TDP43) and FUS with YFP or mCherry to examine phase separation and protein colocalization in mouse C2C12 myoblast cells. We observed that the N-terminal 397 amino acids of MATR3 (tagged with a nuclear localization signal and expressed as a fusion protein with YFP) formed droplet-like structures within nuclei. Introduction of the myopathic S85C mutation into NLS-N397 MATR3:YFP, but not ALS mutations F115C or P154S, inhibited droplet formation. Further, we analyzed interactions between variants of MATR3 lacking RRM2 (ΔRRM2) and variants of TDP43 with disabling mutations in its RRM1 domain (deletion or mutation). We observed that MATR3:YFP ΔRRM2 formed droplets that appeared to recruit the TDP43 RRM1 mutants. Further, coexpression of the NLS-397 MATR3:YFP construct with a construct that encodes the prion-like domain of TDBP43 produced intranuclear droplet-like structures containing both proteins. Collectively, our studies show that N-terminal sequences in MATR3 can mediate phase separation into intranuclear droplet-like structures that can recruit TDP43 under conditions of low RNA binding.

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis (fALS) induce the protein to misfold and aggregate. Missense mutations at more than 80 different amino acid positions have been associated with disease. How these mutations heighten the propensity of SOD1 to misfold and aggregate is unclear. With so many mutations, it is possible that more than one mechanism of aggregation may be involved. Of many possible mechanisms to explain heightened aggregation, one that has been suggested is that mutations that eliminate charged amino acids could diminish repulsive forces that would inhibit aberrant protein:protein interactions. Mutations at twenty-one charged residues in SOD1 have been associated with fALS, but of the 11 Lys residues in the protein, only 1 has been identified as mutated in ALS patients. Here, we examined whether loss of positively charged surface Lys residues in SOD1 would induce misfolding and formation of intracellular inclusions. We mutated four different Lys residues (K30, K36, K75, K91) in SOD1 that are not particularly well conserved, and expressed these variants as fusion proteins with yellow fluorescent protein (YFP) to assess inclusion formation. We also assessed whether these mutations induced binding to a conformation-restricted SOD1 antibody, designated C4F6, which recognizes non-natively folded protein. Although we observed some mutations to cause enhanced C4F6 binding, we did not observe that mutations that reduce charge at these positions caused the protein to form intracellular inclusions. Our findings may have implications for the low frequency of mutations at Lys residues SOD1 in ALS patients.

Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed inDrosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here we describeretro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation inDrosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validateretro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of theDrosophilahemibrain. Our experiments establishretro-Tango as a key method for circuit tracing in neuroscience research.