Background Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract

The product of an integrated transgene provides a convenient and cell-specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in-frame fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy-chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin-mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer.

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Scant data exist on intracellular events during aminoglycoside-induced postantibiotic effect (PAE). We examined DNA, RNA, and protein syntheses after tobramycin exposure using [3H1thymidine, [14C]uracil, and [14CJalanine incorporation in a clinical Escherichia coli strain. Late-log-phase bacteria in oxygenated minimal salts medium at 37°C were exposed to tobramycin (7.5 ,ug/ml) The persistent suppression of bacterial growth observed after brief exposure to an antimicrobial agent has been termed the postantibiotic effect (PAE) (9, 24). Aminoglycosides, quinolones, tetracyclines, and rifampin induce a PAE against gram-negative bacilli (2,8,9,18,25,30). The mechanisms by which antibiotics induce a PAE have not been defined. Inhibition of DNA synthesis has been shown to correlate with the time required for quinolone-treated Escherichia coli to resume growth (14). ,B-Lactam exposure suppresses thymidine uptake with decreased DNA synthesis in gram-positive cocci during PAE (13, 26). The saturable linear dose-response curve of the PAE for macrolides, tetracyclines, and rifampin suggests a drug-receptor interaction, such as with subunits of susceptible bacterial ribosomes (9).Aminoglycoside antibiotics exhibit pleiotropic effects on bacteria, including inhibition of ribosomal and plasma membrane functions (12, 15). The precise mechanism of aminoglycoside action has not been elucidated for either bactericidal effects (11,16,22,31) or PAEs (13). Moreover, to our knowledge, no studies comparing DNA, RNA, and protein syntheses before, during, and after aminoglycoside-induced PAE have been reported. The aim of this study was to correlate these synthetic functions with PAE in E. coli exposed to tobramycin in vitro. MATERIALS AND METHODSBacterial strain, media, and MIC determination. A clinical isolate of E. coli capable of growth in a minimal salts medium was stored in 15% glycerol at -70°C. Bacteria were grown and all studies were performed with bubbling in a carbon * Corresponding author. minimal medium consisting of the following (per liter): NH4Cl, 2 g; Na2HPO4, 6 g; KH2PO4, 3 g; NaCl, 3 g; MgCl2-6H20, 0.062 g; Na2SO4, 0.08 g; and glucose, 6 g (31). A few preliminary experiments were also performed in Mueller-Hinton broth (Difco, Madison, Wis.).PAE determination. The PAE was measured in the standard fashion (9) by comparing regrowth curves determined by serial viable counts of exposed and unexposed control organisms. Bacteria from an overnight culture were inoculated into fresh minimal medium and allowed to grow to log phase and an optical density at 540 nm of 0.2. An aliquot was diluted 100-fold (to approximately 106 CFU/ml) and designated the control. Tobramycin (7.5 ,1g/ml) was added to the remaining cells (twice the MIC determined for this E. coli strain in this medium). After a 30-min exposure to tobramycin, bacteria were filtered with a 0.45-,um-pore-size filter (Gelman Sciences, Ann Arbor, Mich.) and resuspended in fresh minimal medium for further study. In all PAE experiments, zero hour designated the start of...

CGG repeat expansions in the FMR1 5’UTR cause the neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). These repeats form stable RNA secondary structures that support aberrant translation in the absence of an AUG start codon (RAN translation), producing aggregate-prone peptides that accumulate within intranuclear neuronal inclusions and contribute to neurotoxicity. Here, we show that the most abundant RAN translation product, FMRpolyG, is markedly less toxic when generated from a construct with a non-repetitive alternating codon sequence in place of the CGG repeat. While exploring the mechanism of this differential toxicity, we observed a +1 translational frameshift within the CGG repeat from the arginine to glycine reading frame. Frameshifts occurred within the first few translated repeats and were triggered predominantly by RNA sequence and structural features. Short chimeric R/G peptides form aggregates distinct from those formed by either pure arginine or glycine, and these chimeras induce toxicity in cultured rodent neurons. Together, this work suggests that CGG repeats support translational frameshifting and that chimeric RAN translated peptides may contribute to CGG repeat-associated toxicity in FXTAS and related disorders.