TNFR-1 and GDF-15 Are Associated With Plasma Neurofilament Light Chain and Progranulin Among Community-Dwelling Older Adults: A Secondary Analysis of the MAPT Study.

There is growing evidence that cognitive decline can be affected by both nutritional aspects and inflammation. Plasma neurodegenerative biomarkers stand out as minimally invasive useful measures to monitor the potential risk of cognitive decline. This study aimed to investigate the associations between biomarkers of neurodegeneration, nutrition, and inflammation among community-dwelling older adults, and to verify if associations differed according to apolipoprotein E (APOE) ε4 status. This cross-sectional analysis included 475 participants ≥70 years old from the Multidomain Alzheimer Preventive Trial (MAPT), mean age 76.8 years (SD = 4.5), 59.4% women. Biomarkers of neurodegeneration (plasma amyloid-ß 42/40-Aß 42/40, neurofilament light chain-NfL, progranulin), nutrition (erythrocyte docosahexaenoic acid, eicosapentaenoic acid, omega-3 index; plasma homocysteine-Hcy, 25 hydroxyvitamin D), inflammation (plasma tumor necrosis factor receptor 1-TNFR-1, monocyte chemoattractant protein 1-MCP-1, interleukin 6-IL-6), and cellular stress (plasma growth differentiation factor 15-GDF-15) were assessed. Linear regression analyses were performed to investigate the associations between nutritional and inflammatory biomarkers (independent variables) and neurodegenerative biomarkers (dependent variables), with adjustments for age, sex, education, body mass index, physical activity, allocation to MAPT groups, and APOE ε4 status. After adjusting for confounders, Aß 42/40 was not associated with nutritional or inflammatory markers. NfL was positively associated with GDF-15, TNFR-1, IL-6, and Hcy. Progranulin was positively associated with GDF-15, TNFR-1, and MCP-1. Analyses restricted to APOE ε4 carriers (n = 116; 26.9%) or noncarriers were mostly similar. Our cross-sectional study with community-dwelling older adults corroborates previous evidence that inflammatory pathways are associated to plasma markers of neurodegeneration. Clinical Trials Registration Number: NCT00672685.

Characterization of Biological Material Adsorption to the Surface of Nanoparticles without a Prior Separation Step: a Case Study of Glioblastoma-Targeting Peptide and Lipid Nanocapsules.

PURPOSE: Current preclinical therapeutic strategies involving nanomedicine require increasingly sophisticated nanosystems and the characterization of the complexity of such nanoassemblies is becoming a major issue. Accurate characterization is often the factor that can accelerate the translational approaches of nanomedicines and their pharmaceutical development to reach the clinic faster. We conducted a case study involving the adsorption of the NFL-TBS.40-63 (NFL) peptide (derived from neurofilaments) to the surface of lipid nanocapsules (LNCs) (a combined nanosystem used to target glioblastoma cells) to develop an analytical approach combining the separation and the quantification in a single step, leading to the characterization of the proportion of free peptide and thus the proportion of peptide adsorbed to the lipid nanocapsule surface. METHODS: LNC suspensions, NFL peptide solution and LNC/NFL peptide mixtures were characterized using a Size-Exclusion Chromatography method (with a chromatographic apparatus). In addition, this method was compared to centrifugal-filtration devices, currently used in literature for this case study. RESULTS: Combining the steps for separation and characterization in one single sequence improved the accuracy and robustness of the data and led to reproducible results. Moreover the data deviation observed for the centrifugal-filtration devices demonstrated the limits for this increasingly used characterization approach, explained by the poor separation quality and highlighting the importance for the method optimization. The high potential of the technique was shown, proving that H-bond and/or electrostatic interactions mediate adsorption of the NFL peptide to the surface of LNCs. CONCLUSIONS: Used only as a characterization tool, the process using chromatographic apparatus is less time and solvent consuming than classical Size-Exclusion Chromatography columns only used for separation. It could be a promising tool for the scientific community for characterizing the interactions of other combinations of nanosystems and active biological agents.

Glassy Dynamics and Memory Effects in an Intrinsically Disordered Protein Construct.

Glassy, nonexponential relaxations in globular proteins are typically attributed to conformational behaviors that are missing from intrinsically disordered proteins. Yet, we show that single molecules of a disordered-protein construct display two signatures of glassy dynamics, logarithmic relaxations and a Kovacs memory effect, in response to changes in applied tension. We attribute this to the presence of multiple independent local structures in the chain, which we corroborate with a model that correctly predicts the force dependence of the relaxation. The mechanism established here likely applies to other disordered proteins.

Theoretical characterization of Al(III) binding to KSPVPKSPVEEKG: Insights into the propensity of aluminum to interact with key sequences for neurofilament formation.

Classical molecular dynamic simulations and density functional theory are used to unveil the interaction of aluminum with various phosphorylated derivatives of the fragment KSPVPKSPVEEKG (NF13), a major multiphosphorylation domain of human neurofilament medium (NFM). Our calculations reveal the rich coordination chemistry of the resultant structures with a clear tendency of aluminum to form multidentate structures, acting as a bridging agent between different sidechains and altering the local secondary structure around the binding site. Our evaluation of binding energies allows us to determine that phosphorylation has an increase in the affinity of these peptides towards aluminum, although the interaction is not as strong as well-known chelators of aluminum in biological systems. Finally, the presence of hydroxides in the first solvation layer has a clear damping effect on the binding affinities. Our results help in elucidating the potential structures than can be formed between this exogenous neurotoxic metal and key sequences for the formation of neurofilament tangles, which are behind of some of the most important degenerative diseases.

Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in ß-Tubulin Isotypes.

Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/ßI- and αI/ßIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two ß-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the ßI isotype, but not the ßIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the ßI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress ßIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association.

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation.

Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method.

Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy.

Early expression of the high molecular weight neurofilament subunit attenuates axonal neurite outgrowth.

Phospho-dependent interactions of the C-terminal region of the high molecular weight NF subunit (NF-H) with each other and with other cytoskeletal elements stabilize the axonal cytoskeleton and contribute to an increase in axonal caliber. The same kinase cascades that mediate axonal pathfinding via growth cone dynamics are those that foster NF-mediated axonal stabilization, yet there is a developmental delay in the accumulation of NF C-terminal phosphorylation. Moreover, the phospho-mediated C-terminal NF-H interactions that stabilize the axonal cytoskeleton also inhibit axonal elongation. We hypothesized that a delay in expression and/or accumulation of NF-H within developing axons is essential to allow axonal elongation and pathfinding. We tested this hypothesis in differentiating NB2a/d1 cells. The first 3 days of differentiation of NB2a/d1 cells is normally accompanied by rapid elongation of axonal neurites. This period is followed by the accumulation of C-terminally phosphorylated NF-H, cessation of axonal elongation and an increase in axonal caliber. Herein, overexpression of GFP-tagged NF-H simultaneously with induction of differentiation fostered accumulation of C-terminally phosphorylated NF-H within developing axonal neurites within 48hr, which was accompanied by retardation of axonal elongation and a hastened increase in caliber. These effects were prevented by treatment with inhibitors of kinases that mediate the association of NFs with other cytoskeletal elements. Overexpression of GFP-NF-H lacking the C-terminal 187 amino acids (which mediate NF-NF interactions) did not retard elongation nor increase caliber. These findings support the hypothesis that a developmental delay in NF-H C-terminal phosphorylation is essential to allow appropriate axonal elongation prior to stabilization.

Investigating the Structural Variability and Binding Modes of the Glioma Targeting NFL-TBS.40-63 Peptide on Tubulin.

NFL-TBS.40-63 is a 24 amino acid peptide corresponding to the tubulin-binding site located on the light neurofilament subunit, which selectively enters glioblastoma cells, where it disrupts their microtubule network and inhibits their proliferation. We investigated its structural variability and binding modes on a tubulin heterodimer using a combination of NMR experiments, docking, and molecular dynamics (MD) simulations. Our results show that, while lacking a stable structure, the peptide preferentially binds on a specific single site located near the ß-tubulin C-terminal end, thus giving us precious hints regarding the mechanism of action of the NFL-TBS.40-63 peptide's antimitotic activity at the molecular level.

Comparative study of protein unfolding in aqueous urea and dimethyl sulfoxide solutions: surface polarity, solvent specificity, and sequence of secondary structure melting.

Elucidation of possible pathways between folded (native) and unfolded states of a protein is a challenging task, as the intermediates are often hard to detect. Here, we alter the solvent environment in a controlled manner by choosing two different cosolvents of water, urea, and dimethyl sulfoxide (DMSO) and study unfolding of four different proteins to understand the respective sequence of melting by computer simulation methods. We indeed find interesting differences in the sequence of melting of α helices and ß sheets in these two solvents. For example, in 8 M urea solution, ß-sheet parts of a protein are found to unfold preferentially, followed by the unfolding of α helices. In contrast, 8 M DMSO solution unfolds α helices first, followed by the separation of ß sheets for the majority of proteins. Sequence of unfolding events in four different α/ß proteins and also in chicken villin head piece (HP-36) both in urea and DMSO solutions demonstrate that the unfolding pathways are determined jointly by relative exposure of polar and nonpolar residues of a protein and the mode of molecular action of a solvent on that protein.

Heterogeneity in the properties of NEFL mutants causing Charcot-Marie-Tooth disease results in differential effects on neurofilament assembly and susceptibility to intervention by the chaperone-inducer, celastrol.

Aberrant aggregation of neurofilament proteins is a common feature of neurodegenerative diseases. For example, neurofilament light protein (NEFL) mutants causing Charcot-Marie-Tooth disease induce misassembly of neurofilaments. This study demonstrated that mutations in different functional domains of NEFL have different effects on filament assembly and susceptibility to interventions to restore function. The mouse NEFL mutants, NEFL(Q333P) and NEFL(P8R), exhibited different assembly properties in SW13-cells, cells lacking endogenous intermediate filaments, indicating different consequences of these mutations on the biochemical properties of NEFL. The p.Q333P mutation caused reversible misfolding of the protein. NEFL(Q333P) could be refolded and form coil-coiled dimers, in vitro using chaotropic agent, and in cultured cells by induction of HSPA1 and HSPB1. Celastrol, an inducer of chaperone proteins, induced HSPA1 expression in motor neurons and prevented the formation of neurofilament inclusions and mitochondrial shortening induced by expression of NEFL(Q333P), but not in sensory neurons. Conversely, celastrol had a protective effect against the toxicity of NEFL(P8R), a mutant which is sensitive to HSBP1 but not HSPA1 chaperoning, only in large-sized sensory neurons, not in motor neurons. Importantly, sensory and motor neurons do not respond identically to celastrol and different chaperones are upregulated by the same treatment. Thus, effective therapy of CMT not only depends on the identity of the mutated gene, but the consequences of the specific mutation on the properties of the protein and the neuronal population targeted.

Structure-function analysis of the glioma targeting NFL-TBS.40-63 peptide corresponding to the tubulin-binding site on the light neurofilament subunit.

We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either ß-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation.

Brain tumour targeting strategies via coated ferrociphenol lipid nanocapsules.

In this study, a new active targeting strategy to favour ferrociphenol (FcdiOH) internalisation into brain tumour cells was developed by the use of lipid nanocapsules (LNCs) coated with a cell-internalising peptide (NFL-TBS.40-63 peptide) that interacts with tubulin-binding sites. In comparison, OX26 murine monoclonal antibodies (OX26-MAb) targeting transferrin receptors were also inserted onto the LNC surface. The incorporation of OX26 or peptide did not influence the in vitro antiproliferative effect of FcdiOH-LNCs on the 9L cells since their IC50 values were found in the same range. In vivo, intracerebral administration of OX26-FcdiOH-LNCs or peptide-FcdiOH-LNCs by convection enhanced delivery did not enhance the animal median survival time in comparison with untreated rats (25 days). Interestingly, intra-carotid treatment with peptide-FcdiOH-LNCs led to an ameliorated survival time of treated rats with the presence of animals surviving until days 35, 40 and 44. Such results were not obtained with OX26-MAbs, demonstrating the benefit of NFL-TBS.40-63 peptide as an active ligand for peripheral drug delivery to the brain tumours.

The vimentin-tubulin binding site peptide (Vim-TBS.58-81) crosses the plasma membrane and enters the nuclei of human glioma cells.

Cell-penetrating peptides (CPPs) can translocate through the plasma membrane and localize in different cell compartments providing a promising delivery system for peptides, proteins, nucleic acids, and other products. Here we describe features of a novel cell-penetrating peptide derived from the intermediate filament protein vimentin, called Vim-TBS.58-81. We show that it enters cells from a glioblastoma line via endocytosis where it distributes throughout the cytoplasm and nucleus. Moreover, when coupled to the pro-apoptogenic peptide P10, it localizes to the nucleus inhibiting cell proliferation. Thus, the Vim-TBS.58-81 peptide represents an effective vector for delivery of peptides and potentially a broad range of cargos to the nucleus.

[Neurofilament immune fluorescence staining: a new method for observing the morphology of the motor endplate].

OBJECTIVE: To investigate the effect of neurofilament (NF) immune fluorescence staining on observing morphology of the motor endplate. METHODS: Six SPF rats were used and their bilateral soleus muscles harvested under anesthesia. Then the samples were fixed with polyphosphate formaldehyde, and made frozen sections. Finally the neurofilament immunofluorescence staining was performed. The morphology of the motor endplate was observed under fluorescence microscope and laser scanning confocal fluorescence microscope. RESULTS: The claw-shape motor endplate was seen clearly under the fluorescent microscope and laser scanning confocal fluorescence microscope, and the images observed used to reconstruct the three-dimensional structure of motor endplate. CONCLUSION: Neurofilament immune fluorescence staining is a useful method for the morphology study of the motor endplate.

Mutation of charged residues to neutral ones accelerates urea denaturation of HP-35.

Following the studies of urea denaturation of ß-hairpins using molecular dynamics, in this paper, molecular dynamics simulations of two peptides, a 35 residue three helix bundle villin headpiece protein HP-35 and its doubly norleucine-substituent mutant (Lys24Nle/Lys29Nle) HP-35 NleNle, were undertaken in urea solutions to understand the molecular mechanism of urea denaturation of α-helices. The mutant HP-35 NleNle was found to denature more easily than the wild type. During the expansion of the small hydrophobic core, water penetration occurs first, followed by that of urea molecules. It was also found that the initial hydration of the peptide backbone is achieved through water hydrogen bonding with the backbone CO groups during the denaturation of both polypeptides. The mutation of the two charged lysine residues to apolar norleucine enhances the accumulation of urea near the hydrophobic core and facilitates the denaturation process. Urea also interacts directly with the peptide backbone as well as side chains, thereby stabilizing nonnative conformations. The mechanism revealed here is consistent with the previous study on secondary structure of ß-hairpin polypeptide, GB1, PEPTIDE 1, and TRPZIP4, suggesting that there is a general mechanism in the denaturation of protein backbone hydrogen bonds by urea.

Aluminum induces neurofilament aggregation by stabilizing cross-bridging of phosphorylated c-terminal sidearms.

Exposure to neurotoxin aluminum neurotoxicity is accompanied by the perikaryal accumulation of tangles of phosphorylated neurofilaments (NFs). We examined their formation and reversibility under cell-free conditions. AlCl3 induced dose-dependent formation of NF aggregates, ultimately incorporating 100% of detectable NFs. The same concentration of CaCl2 induced approximately 25% of NFs to form longitudinal dimers and did not induce aggregation. AlCl3 induced similar percentages of aggregates in the presence or absence of CaCl2, and CaCl2 could not reduce pre-formed aggregates. CaCl(2)-induced dimers and AlCl(3)-induced aggregates were prevented by prior NF dephosphorylation. While CaCl(2)-induced dimers were dissociated by phosphatase treatment, AlCl(3)-induced aggregates were only reduced by approximately 50%, suggesting that aggregates may sequester phosphorylation sites. Since phosphatases regulate NF phosphorylation within perikarya, inhibition of NF dephosphorylation by aluminum would promote perikaryal NF phosphorylation and foster precocious phospho-dependent NF-NF associations. These findings are consistent with the notion that prolonged interactions induced among phospho-NFs by the trivalent aluminum impairs axonal transport and promotes perikaryal aggregation.

MS characterization of qualitative protein polymorphisms in the spinal cords of inbred mouse strains.

The spinal cord proteomes of two inbred mouse strains with different susceptibility to experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, were investigated by 2-DE and MALDI-MS. A proteome map comprising 304 different protein species was established. Using 2-D fluorescence difference gel electrophoresis, a comparison of the mouse strains revealed 26 qualitatively polymorphic proteins with altered electrophoretic mobility. MS analyses and DNA sequencing were applied to characterize their structural differences and 14 single amino acid substitutions were identified. Moreover, analysis of selectively enriched phosphopeptides from the neurofilament heavy polypeptide of both mouse strains revealed a high degree of diversity in the phosphorylated C-terminal domains of this protein. The described approach is capable to structurally characterize qualitative protein polymorphisms, whereas their functional significance remains to be elucidated. For some proteins formerly associated with experimental autoimmune encephalomyelitis and/or multiple sclerosis structural polymorphisms are described here, which may be subjected to further investigations. In addition, this work should be of general interest for proteomic analysis of inbred strains, because it shows potentials and constraints in the use of 2-DE analysis and MALDI-MS to detect and characterize structural protein polymorphisms.

Comparative study of CSF neurofilaments in HTLV-1-associated myelopathy/tropical spastic paraparesis and other neurological disorders.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive CNS disease leading to corticospinal tract degeneration. Various degenerative diseases have increased neurofilament subunit concentration in cerebrospinal fluid (CSF), frequently showing hyperphosphorylation in neurofilaments. The aim of this study was to determine if there were elevated concentrations of neurofilament light subunit (NFL) and phosphorylated forms of neurofilament heavy subunit (PNFH) in HAM/TSP CSF. NF concentrations were compared with those of controls and patients with neurodegenerative diseases associated with other retroviruses (HIV-associated dementia, HAD) and a form of prion disease (familiar Creutzfeldt-Jakob, FCJD). Western blotting of CSF with antibodies against NFL showed two immunoreactive bands of 66 and 59 kDa, the latter probably corresponding to a partially degraded NFL form. The concentration of the 59-kDa form was not different in HAM/TSP compared with controls, but it was significantly increased in HAD and FCJD groups. ELISA assay for PNFH did not show differences among HAM/TSP, HAD, and control groups, while PNFH concentration was significantly elevated in FCJD. Our results show that CSF NFL and PNFH are not molecular markers of axonal damage for HAM/TSP probably due to the slow progression of this disease. NFL phosphorylation studies required previous immunoprecipitation from CSF for mass spectrometric analysis. This preliminary analysis indicated phosphorylation at S472 and at some other residues.

Rapamycin rescues TDP-43 mislocalization and the associated low molecular mass neurofilament instability.

TDP-43 is a nuclear protein involved in exon skipping and alternative splicing. Recently, TDP-43 has been identified as the pathological signature protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis. In addition, TDP-43-positive inclusions are present in Parkinson disease, dementia with Lewy bodies, and 30% of Alzheimer disease cases. Pathological TDP-43 is redistributed from the nucleus to the cytoplasm, where it accumulates. An approximately 25-kDa C-terminal fragment of TDP-43 accumulates in affected brain regions, suggesting that it may be involved in the disease pathogenesis. Here, we show that overexpression of the 25-kDa C-terminal fragment is sufficient to cause the mislocalization and cytoplasmic accumulation of endogenous full-length TDP-43 in two different cell lines, thus recapitulating a key biochemical characteristic of TDP-43 proteinopathies. We also found that TDP-43 mislocalization is associated with a reduction in the low molecular mass neurofilament mRNA levels. Notably, we show that the autophagic system plays a role in TDP-43 metabolism. Specifically, we found that autophagy inhibition increases the accumulation of the C-terminal fragments of TDP-43, whereas inhibition of mTOR, a key protein kinase involved in autophagy regulation, reduces the 25-kDa C-terminal fragment accumulation and restores TDP-43 localization. Our results suggest that autophagy induction may be a valid therapeutic target for TDP-43 proteinopathies.