The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here, we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates, and found them enriched for components of the nuclear pore complex (NPC) and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins (Nups) and transport factors (TFs), and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts, and iPSC-derived neurons. Nuclear pore pathology is present in brain tissue from sporadic ALS cases (sALS) and those with genetic mutations in TARDBP (TDP-ALS) and C9orf72 (C9-ALS). Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.

There is ample evidence for localization of messenger RNAs (mRNAs) and protein synthesis in neuronal dendrites; however, demonstrations of these processes in presynaptic terminals are limited. We used expansion microscopy to resolve pre- and postsynaptic compartments in rodent neurons. Most presynaptic terminals in the hippocampus and forebrain contained mRNA and ribosomes. We sorted fluorescently labeled mouse brain synaptosomes and then sequenced hundreds of mRNA species present within excitatory boutons. After brief metabolic labeling, >30% of all presynaptic terminals exhibited a signal, providing evidence for ongoing protein synthesis. We tested different classic plasticity paradigms and observed distinct patterns of rapid pre- and/or postsynaptic translation. Thus, presynaptic terminals are translationally competent, and local protein synthesis is differentially recruited to drive compartment-specific phenotypes that underlie different forms of plasticity.

Spinal muscular atrophy (SMA) is a neurodegenerative disease primarily affecting spinal motor neurons. It is caused by reduced levels of the survival of motor neuron (SMN) protein, which plays an essential role in the biogenesis of spliceosomal small nuclear ribonucleoproteins in all tissues. The etiology of the specific defects in the motor circuitry in SMA is still unclear, but SMN has also been implicated in mediating the axonal localization of mRNA-protein complexes, which may contribute to the axonal degeneration observed in SMA. Here, we report that SMN deficiency severely disrupts local protein synthesis within neuronal growth cones. We also identify the cytoskeleton-associated growth-associated protein 43 (GAP43) mRNA as a new target of SMN and show that motor neurons from SMA mouse models have reduced levels of GAP43 mRNA and protein in axons and growth cones. Importantly, overexpression of two mRNAbinding proteins, HuD and IMP1, restores GAP43 mRNA and protein levels in growth cones and rescues axon outgrowth defects in SMA neurons. These findings demonstrate that SMN plays an important role in the localization and local translation of mRNAs with important axonal functions and suggest that disruption of this function may contribute to the axonal defects observed in SMA.

Local protein synthesis is a ubiquitous feature of neuronal pre-and postsynaptic compartments.One sentence summary: Protein synthesis occurs in all synaptic compartments, including excitatory and inhibitory axon terminals. Presynaptic terminals from mouse cortex and hippocampus contain translation machineryEfforts to localize molecules or cell biological events to neuronal pre-or postsynaptic compartments using fluorescence microscopy are limited by the tight association of the axonal bouton and the dendritic spine or synapse; the synaptic cleft, the only clear region of separation, is only ~ 20 nm wide. Here, in order to increase the resolving power to visualize mRNA molecules in pre-and postsynaptic compartments, we optimized fluorescence in situ hybridization (FISH) and nascent protein detection methods for use with expansion microscopy (22) ( Fig. 1A; see Methods). We used adult mouse brain slices or rat cultured hippocampal neurons and found that expansion resulted in an enlargement of both pre-and postsynaptic compartments, with an average expansion of ~3.4 fold. This yielded a clear separation between the pre-and postsynaptic compartments. To evaluate whether ribosomes and mRNA species are present in defined presynaptic compartments, we used immunolabelling for either excitatory (vGLUT1; (23, 24) or inhibitory (vGAT; (25, 26)) nerve terminals in expanded mouse brain sections (both cortex and hippocampus) ( Fig. 1B-E) or rat cultured hippocampal neurons. We took care to identify the molecules-of-interest within individual z-sections positively labeled for excitatory or inhibitory terminals. We noted that signal detected outside of immunolabeled compartments corresponded to signal arising from nearby unlabeled cells. We detected ribosomes in a large majority (>75%) of both excitatory and inhibitory presynaptic nerve terminals, using antibodies directed against either a small (RPS11) or a large (RPL26) ribosomal protein ( Fig. 1 B-E). Next, we used FISH probes to detect 18s and 28s rRNA as well as polyadenylated mRNA (detected with a poly d(T) probe) in expanded samples ( Fig. 1B-E). Consistent with the abundance of ribosomal proteins, we detected rRNA in over 80% of both excitatory and inhibitory nerve terminals ( Fig. 1B-E). RNase treatment effectively reduced all rRNA signal. In cultured neurons, we also noted that poly(A) mRNA was abundant, as expected, in dendritic spines. In addition, we used an anti-tau antibody to label axons and detected both 18s and 28s rRNA in axonal segments. Thus, mRNAs and ribosomes were abundant in excitatory and inhibitory presynaptic nerve terminals from both mouse brain slices and rat hippocampal cultured neurons.

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Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.

No abstract

Myc has been characterized as a transcription factor that activates expression of genes involved in pluripotency and cancer, and as a component of the replication complex. Here we find that Myc is present at promoters and enhancers of D. melanogaster genes during interphase. Myc co-localizes with Orc2, which is part of the pre-replication complex, during G1. As is the case in mammals, Myc associates preferentially with paused genes, suggesting that it may also be involved in the release of RNAPII from promoter proximal pausing in Drosophila. Interestingly, about 40% of Myc sites present in interphase persists during mitosis. None of the Myc mitotic sites correspond to enhancers and only some correspond to promoters. The rest of mitotic Myc sites overlap with binding sites for multiple insulator proteins that are also maintained in mitosis. These results suggest alternative mechanisms to explain the role of Myc in pluripotency and cancer.