The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1’s functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70’s functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70’s interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions. The cell adhesion molecule L1 regulates important functions in the developing and adult nervous system under physiological and pathological conditions ,3. In huStudies in mice have shown that L1 acts through homophilic and heterophilic interactions and that the proteolytic cleavage of L1 is crucial for its neural functions ,29,30,31Stimulation of L1 signal transduction by the function-triggering L1 antibody 557 generates a transmembrane C-terminal fragment of 70 kDa (L1-70)  via a prAnother transmembrane C-terminal L1 fragment of 55 kDa (L1-55) is generated by metalloproteases, the β-site of amyloid precursor protein cleaving enzyme and γ-secretase ,38. InteBy mass spectrometric analysis upon affinity chromatography using the intracellular domain of L1 (L1-ICD) and a nuclear brain extract we have identified TOP1 as a nuclear molecule that could be associated with L1-ICD . To follWe also utilized affinity chromatography to find other nuclear and cytoplasmic molecules that could interact directly or indirectly with L1-ICD, including splicing factor proline/glutamine-rich (SFPQ), non-POU domain-containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), histone H1.4 (HistH1e), nucleoporin 93 kDa (Nup93), heat shock cognate protein 71 kDa (Hsc70), synaptotagmin 1 (SYT1), and importin β1 (impβ1) . ImmunopFor the present study, we investigated whether the aforementioned molecules interact with L1-70. Using immunoprecipitation and proximity ligation assay, we found that TOP1, PPARγ and NDUFV2, but not the other tested molecules, interact with L1-70. Thus, these interactions were chosen for further investigation of their functional consequences.ELISA showed that the interactions of TOP1 and PPARγ with L1-ICD are mediated via distinct L1-ICD sequences. Reduction of TOP1, PPARγ and NDUFV2 expression by siRNAs or inhibition of topoisomerase activity with topotecan and irinotecan reduced the L1-dependent neurite outgrowth as well as neuronal survival and migration. Reduction of L1 levels by siRNA downregulated the expression levels of the long autism genes Nrxn1 and Nlgn1 and of the mitochondrial gene ND2. Nrxn1 and Nlgn1 mRNA levels were also reduced in mutant mice deficient in L1-70. Our results indicate that the interactions of L1-70 with TOP1, PPARγ and NDUFV2 regulate L1-dependent neuronal functions.Because of the functional importance of the L1-70 fragment ,34,35,36Because we were able to show, through ELISA, that NDUFV2 directly binds to L1-ICD , we thenWe were able to show that L1-70 was not generated in gene-edited mice expressing either L1 with a mutated dibasic sequence in the third FNIII domain at position 858–863  or L1 with a mutation of arginine 687  . Thus, wWhen performing proximity ligation with L1 antibody and antibodies against SFPQ, ERα and RXR, which forms heterodimers with PPARγ , we obseTo gain more evidence for L1-70’s association with TOP1, PPARγ and NDUFV2, immunoprecipitation was performed with immobilized antibodies and a non-nuclear brain fraction of wild-type and L1-70-lacking mutant mice. Western blot analysis indicates one L1-immunopositive band of 70 kDa in the TOP1, PPARγ and NDUFV2 immunoprecipitates of the non-nuclear wild-type brain fraction, whereas no 70 kDa band was seen in the immunoprecipitates of the non-nuclear brain fractions from the L1 mutants c. No banTo investigate whether the interaction of L1 with TOP1, PPARγ and NDUFV2 plays a role in L1-mediated neurite outgrowth, cortical neurons were mock-transfected or transfected with TOP1, PPARγ and NDUFV2 siRNAs or control siRNA and then treated without and with L1 antibody 557 which induces L1-dependent neurite outgrowth ,28,29. Tn = 3) in neurons after transfection with the corresponding siRNA relative to the levels in neurons transfected with control siRNA (n = 3), respectively  the interactions of TOP1, PPARγ and NDUFV2 are reduced when the MBP-mediated generation of L1-70 is inhibited by the serine protease inhibitor aprotinin; (2) TOP1, PPARγ and NDUFV2 co-immunoprecipitate L1-70 from brain fractions of wild-type, but not L1-70-lacking mutant mice; and (3) L1 antibody 557-induced generation of L1-70 ,28 leadsWe have previously reported that NDUFV2 binds to the KDET motif in L1-ICD . We showSince TOP1 inhibitors and knockdown of TOP1 have been shown to reduce the expression levels of the Nrxn1 and Nlgn1 genes ,43,44, aTOP1 is a key transcriptional regulator in postmitotic neurons ,44 and dTOP1 has also been found in mitochondria, and TOP1-deficient murine embryonic fibroblasts showed a higher mitochondrial membrane potential, increased expression of mitochondrial genes, including ND2, decreased ATP levels and enhanced levels of reactive oxygen species (ROS) . BecauseIn utero electroporation of NDUFV2 shRNA in the cerebral cortex of mouse embryos resulted in an impaired neuronal migration and accumulation of ectopic neurons in lower cortical layers: most NDUFV2 shRNA-expressing neurons were found in the intermediate and ventricular zones, whereas control cortical neurons migrated into the upper cortical plate . MoreoveIn mice lacking the cytoplasmic polyadenylation element binding protein 1, the expression of NDUFV2 in total brain extracts and the amount of NDUFV2 in mitochondrial complex 1 was reduced . This rePPARγ agonists have been considered for the treatment of Alzheimer’s and Parkinson’s disease ,79,80,81There is increasing evidence that mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of many neurodegenerative diseases and neurodevelopmental disorders, such as autism, attention deficit hyperactivity disorder, schizophrenia, Down’s syndrome and Fragile X syndrome. In particular, abnormalities in the mitochondrial respiratory electron-transport chain during development in utero, infancy, childhood and adulthood may be involved in the etiology of neurodevelopmental disorders and neurodegenerative diseases ,109. TheL1-deficient mice  and geneThe following antibodies were from Santa Cruz Biotechnology : mouse L1 antibody C-2  against the intracellular L1 domain, mouse PPARγ antibody E-8 , mouse Topo I antibody C-21 , mouse NDUFV2 antibody F-5 , mouse importin β1 (karyopherin β1) antibody H-7 , goat TOP1 (Topo I) antibody C-15 , goat SYT (synaptotagmin 1) antibody N-19 , rabbit SFPQ (PSF) antibody H80 , rabbit WDR5 antibody H-36 , rabbit Nup93 antibody H-300 , rabbit AR (androgen receptor) antibody N-20 , rabbit VDR (vitamin D receptor) antibody C-20 , rabbit RXR (retinoid X receptors) antibody C-20 , and rabbit ER (estrogen receptor) α antibody HC-20 . Rabbit antibodies against PSPC1 , NonO , NDUFV2 , Hsc70 , and Histone H1  were from ChromoTek and Proteintech Germany . Rabbit antibodies against PPARγ , MeCP2 , HP1ɣ , and GAPDH  were acquired from Cell Signaling Technology Europe . Anti-L1CAM antibody ab12399 against the intracellular L1 domain was acquired from Abcam . Rat monoclonal function-triggering L1 antibody 557 has been described . SecondaProduction and purification of recombinant His-tagged L1-ICD  has been described .Recombinant full-length human NDUFV2  was from OriGene Technologies . Recombinant human DNA Topoisomerase-I  and recombinant full-length human PPARγ  were from Biozol .® HD Transfection Reagent (Cat #E2311) was from Promega .Synthetic L1 peptides P1 (CFIKRSKGGKYSVKDKEDTQVDSEARPMKDETFGE), P2 (RPMKDETFGEYRSLESDNEEKAFGSSQPSLNGDIK), P3 (GDIKPLGSDDSLADYGGSVD), P4 (CSVDVQFNEDGSFIGQYSGK), P5 (CSGKKEKEAAGGNDSSGATSPINPAVALE) and tat-P4 (YGRKKRRQRRRSVDVQFNEDGSFIGQYSGK) were from Schafer-N . Mouse L1 siRNA , mouse TOP1 siRNA , mouse PPARγ siRNA , mouse NDUFV2 siRNA (sc-149892), and control siRNA  were from Santa Cruz Biotechnology. FuGENEAprotinin (Cay14716-10), topotecan hydrochloride (Cay14129-10), and irinotecan hydrochloride (Cay14180-25) were from Biomol  and 4′,6-diamidino-2-phenylindole (DAPI) was from Thermo Fisher Scientific .2 and CaCl2  (PBS), treated with blocking solution  for 2  h, washed again with PBS containing 0.005% Tween 20 (PBST), and incubated with increasing concentrations of recombinant His-tagged L1-ICD as ligand for 1 h under gentle agitation. For competition ELISA, 2.5 µM L1-ICD were preincubated for 1 h without or with a five-fold molar excess of L1 peptides P1, P2, P3, P4 or P5. The mixtures were then incubated with substrate-coated recombinant proteins. After washing two times with PBS and three times with PBST, L1 antibody C-2 (1:500) in blocking solution was applied for 1 h followed by two washes with PBS and three washes with PBST and incubation with horseradish peroxidase-coupled anti-mouse antibody (diluted 1:2000 in blocking solution) for 1 h. Wells were washed again with PBST and 1 mg/mL ortho-phenylenediamine dihydrochloride (Thermo Fisher Scientific) was used for detection of bound L1-ICD. The reaction was terminated by addition of 25 µL 2.5 M sulphuric acid. Absorbance was measured at 492  nm with an ELISA reader .For ELISA, 25 µL of 10 or 20 µg/mL recombinant proteins were incubated overnight at 4 °C in 384-well microtiter plates with high binding surface . The following steps were performed at room temperature. Wells were washed with Dulbecco’s phosphate-buffered saline with MgClg for 15 min. Cells were then diluted in Neurobasal A medium  supplemented with 2 mM L-glutamine (Invitrogen), 4 nM L-thyroxine (Sigma-Aldrich), 0.1 mg/mL BSA (Sigma-Aldrich), 12.5 μg/mL insulin (Sigma-Aldrich), 30 nM sodium selenite (Sigma-Aldrich), 100 μg/mL transferrin, 0.1 mg/mL streptomycin and 100 U/mL penicillin (Invitrogen). For the proximity ligation assay, cells were seeded onto poly-L-lysine-coated 12 mm glass coverslips in a 24-well plate at a density of 2.5 × 105 cells per well. For determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich), and for neuronal survival analysis cells were seeded at a density 1.25 × 105 cells per well of a 48-well plate coated with poly-L-lysine. For analysis of RNA levels cells were seeded at a density of 3 × 106 cells per well of a 6-well plate coated with poly-L-lysine.Cerebellar neurons were prepared from cerebella of six- to eight-day-old mice. Cerebella were incubated with 10 mg/mL trypsin and 0.5 mg/mL DNase I (Sigma-Aldrich) in Hanks’ balanced salt solution (HBSS) for 15 min at 37 °C, washed with HBSS, mechanically dissociated and centrifuged at 100× Cerebellar explants were prepared from six- to seven-day-old mice. Cerebella were forced through Nitrex nets with pore sizes of 300, 200 and 100 μm and tissue pieces were plated on plastic coverslips coated with poly-L-lysine and maintained for 16 h in culture medium containing 20% horse serum. The culture medium was then replaced by serum-free medium.5 cells per well. For determination of neurite outgrowth, cells were seeded at a density of 5 × 104 cells per well of a 48-well plate coated with poly-L-lysine (Sigma-Aldrich). For Western blot analysis, cells were seeded onto poly-L-lysine-coated 12-well plates at a density of 1.5 × 106 cells per well and for analysis of RNA levels cells were seeded at a density of 3 × 106 cells per well of a 6-well plate coated with poly-L-lysine.For culturing of cortical neurons, cerebral cortices were dissected from 15.5- to 16.5-day-old embryos and incubated in 0.025% trypsin (Sigma-Aldrich) in HBSS at 37 °C for 30 min. The cortices were then incubated in HBSS containing 1% BSA (Sigma-Aldrich) and 1% trypsin inhibitor  at 37 °C for 5 min. After washing in HBSS, the tissue was mechanically dissociated and the dissociated cells were cultured in Neurobasal medium (Invitrogen) supplemented with 1% B-27 (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 U/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). For the proximity ligation assay and immunostaining, cells were seeded onto poly-L-lysine-coated 12 mm glass coverslips in a 24-well plate at a density of 1.25 × 10For stimulating L1 functions, neurons were treated with 50 µg/mL L1 antibody 557. For aprotinin treatment, neurons were treated 2 h after seeding with 1 µM aprotinin. Treatment of neurons with 50 µg/mL tat-P4 peptide was performed 30 min after seeding. Treatment with 300 nM topotecan or irinotecan was performed 30 min after seeding (neurite outgrowth) or 30 min before addition of hydrogen peroxide . Explants were treated with 300 nM topotecan and irinotecan directly after medium change and then 30 min later with 50 µg/mL L1 antibody 557. Following this, explants were maintained for a further 32 h.For transfection of cortical neurons, cortical neurons were seeded onto poly-L-lysine-coated coverslips in 24-well plates (proximity ligation and immunostaining), or onto 6-well pates , 12-well plates  and 48-well plates (neurite outgrowth) and maintained for 2 h before transfection with 1 µL (48-well), 2 µL (24-well), 4 µL (12-well) or 8 µL (6-well) 10 µM siRNA and 1–4 µL FuGENE transfection reagent per well. L1 antibody 557 (50 µg/mL) was added to the cultures 24 h after transfection  or 2 h after transfection (neurite outgrowth), and 48 h after transfection the cells were analyzed for neurite outgrowth and cell survival, or used for immunostaining, Western blot analysis, RNA isolation or proximity ligation assay.https://imagej.nih.gov/ij/index.html; RRID:SCR_003070, accessed on 8 March 2022). Numbers of red dots and numbers of DAPI-stained nuclei were determined using ImageJ and the number of red dots per image was divided by the number of nuclei per image. The average values of red dots per cell (=nucleus) were determined in ten images per condition.Cultures were fixed for 15 min at room temperature in 4% formaldehyde, washed with PBS and used for immunostaining or subjected to proximity ligation assay using Duolink PLA products according to the manufacturer’s protocol  with minor modifications. For the proximity ligation assay, cells were incubated with 1% Triton X-100 in PBS for 30 min, washed once with PBS and blocked with Duolink Blocking solution for 30 min before being incubated for 24 h at 4 °C with mouse L1 antibody C-2 and goat or rabbit antibodies against TOP1, SYT, SFPQ, WDR5, Nup93, PSPC1, NonO, NDUFV2, HistH1e, ERα, RXR, PPARγ, AR or VDR or with rabbit L1 antibody ab12399 and mouse antibodies against impβ1, TOP1, PPARγ and NDUFV2, all diluted 1:10 in Duolink Antibody Diluent. Cells were washed twice using Duolink Wash Buffer A and incubated with a mixture of secondary antibodies conjugated with oligonucleotides (Duolink PLA Anti-Rabbit or Anti-Goat Probe MINUS and Duolink Anti-Mouse PLA Probe PLUS). The proximity ligation reaction was then performed according to the manufacturer’s protocol using the Duolink In Situ Detection Reagent RED. Thereafter, the coverslips were incubated in 5 µg DAPI/mL PBS for 15 min, washed twice with PBS and mounted in Immuno-Mount (Thermo Fisher Scientific). Ten images per condition were taken using an Olympus F1000 confocal microscope  and analyzed using ImageJ software .Cells and explants were washed gently with pre-warmed culture medium, fixed in 2.5% glutaraldehyde for 30 min at room temperature, and stained with 1% toluidine blue and 1% methylene blue in 1% sodium tetraborate for 30 min at room temperature. Neurite outgrowth and neuronal migration were analyzed by measuring the total length of neurites in an Axiovert microscope with the AxioVision 4.6 imaging system  or with Image J counting the number of cells that had migrated out of the explant core. For neurite outgrowth analysis for each condition at least 100 neurons were counted and migrating cells were counted from 12–15 explants per condition.2O2 for 24 h. Live and dead cells were then stained with calcein-AM (Thermo Fisher Scientific) and propidium iodide (Sigma-Aldrich) and imaged with a Zeiss AxioObserver.A1 microscope (Carl Zeiss) with a 20× objective (aperture 0.4) and the AxioVision 4.6 software (Carl Zeiss). Live and dead cells were counted in five images (containing 350–400 cells each) from each of three wells per condition and experiment using ImageJ.To determine cell death, neurons were maintained overnight in serum-free medium and then treated with 50 µg/mL of L1 antibody 557 and exposed to oxidative stress by the addition of 10 µM HFor the preparation of nuclear and non-nuclear fractions, the Subcellular Protein Fractionation Kit for Tissue (Thermo Fisher Scientific) was used. Fractions in cytoplasmic extraction buffer (CEB) and membrane extraction buffer (MEB) were pooled and taken as non-nuclear fraction. Fractions in nuclear extraction buffer (NEB) were taken as nuclear fraction. Nuclear and non-nuclear fractions were used for immunoprecipitation.For immunoprecipitation, Protein G magnetic beads (25 μL per sample) were washed twice in phosphate-buffered saline (PBS), pH 7.4, and incubated in dilution buffer  for 10 min at 4 °C under rotation. The mouse NDUFV2, PPARγ and TOP1 antibodies (10 μg per sample) were diluted in dilution buffer. The diluted antibody solutions were incubated with the beads for 1 h at 4 °C under rotation. The beads were washed in dilution buffer for 5 min at 4 °C under rotation. A freshly prepared 13 mg/mL stock solution of dimethyl pimelimidate (DMP) was 1:1 diluted with wash buffer (0.2 M triethanolamine in PBS). After washing the beads in PBS, the diluted DMP solution (pH 8–9) was added to the beads which were then incubated for 30 min at room temperature (20–24 °C) under rotation. The beads were then washed in wash buffer for 5 min at room temperature under rotation and incubated in DMP solution and wash buffer twice more. Finally, the beads were incubated twice in quenching buffer (50 mM ethanolamine in PBS) for 5 min at room temperature under rotation. After washing, the beads were incubated with brain fractions overnight at 4 °C under rotation. The beads were then washed twice with lysis buffer and once with PBS before being boiled for 5 min in sample buffer .® TGX™ Precast Protein Gels . PageRuler™ Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as molecular weight marker. Proteins were then transferred to 0.45 μm Protran™ nitrocellulose membranes  and stained with Ponceau S to control protein loading. The membranes were then incubated for 1 h in blocking solution   with 0.05% Tween 20 (TBST) and then incubated overnight with primary antibodies (1:1000) in blocking solution at 4 °C with shaking. After washing five times for 5 min in TBST, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody . Bands were detected using enhanced chemiluminescent solution  and a CCD camera .For Western blot analysis, samples were run on 4%–20% Mini-PROTEAN® II reverse transcriptase (Thermo Fisher Scientific) was used. qPCR was performed in triplicate using reverse transcribed mRNA, the 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific), the qPCR kit QuantiNova SYBR Green , primers for determination of the mRNA levels of Nrxn1 , Nlgn1 , Cntnap2 , Dlg2 , COX1 , COX2 , COX3 , CytB , ATP6 , ND1 , ND2 , ND3 , ND4 , ND4L , ND5 , ND6 , and the reference gene actin . All primers were from Metabion . The SDS 2.4 software  was used for analysis of the qPCR data. The mRNA levels of Nrxn1, Nlgn1, Cntnap2, Dlg2, COX1, COX2, COX3, CytB, ATP6, ND1, ND2, ND3, ND4, ND4L, ND5, and ND6 relative to the mRNA levels of the reference gene actin were calculated. Data of relative gene expression  were used for statistical analysis.For reverse transcription, oligoT18 primer and SuperScript6) were scraped off in 300 µL buffer A  and incubated for 15 min on ice. After centrifugation for 8 min at 1000× g and 4 °C, the pellet was resuspended in 300 µL buffer B , and 100 µL 4 M NaCl was added. An aliquot was saved for determination of the protein concentrations. After incubation for 30 min on ice, the samples were centrifuged for 10 min at 9000× g and 4 °C. The supernatant was collected and 400 µL glycerol was added, and the samples were thoroughly mixed and stored as nuclear extracts at −80 °C until use.Cultured neurons  for 30 min at 37 °C. Stopping buffer was added to a final concentration of 1% sodium dodecyl sulfate, 15% glycerol, 50 mM EDTA, pH 8, and 0.5% bromophenol blue, and the samples were subjected to electrophoresis on a 1% agarose gel  and to ethidium bromide staining.In a total volume of 25 µL, nuclear extracts (50–100 ng protein) were incubated with 100 ng supercoiled pUC19 plasmid DNA  in reaction buffer . The types of tests are indicated in the legends.


The analysis of potassium PMFs in terms of forces revealed a conflicting influence on permeation of van der Waals and electrostatic forces that both undergo an inversion of their direction as pore charge is increased. Even if the most important transition involves the interplay between the electrostatic forces exerted by graphene and water, the simulations also revealed an important role of the changing distribution of potassium and chloride ions. The influence of pore charge on the orientation of water molecules was also found to affect the van der Waals forces they exert on potassium.Selective permeation through graphene nanopores is attracting increasing interest as an efficient and cost-effective technique for water desalination and purification. In this work, using umbrella sampling and molecular dynamics simulations with constant electric field, we analyze the influence of pore charge on potassium and chloride ion permeation. As pore charge is increased, the barrier of the potential of mean force (PMF) gradually decreases until it turns into a well split in two subminima. While in the case of K Increase of graphene pore charge determines decrease of PMF barrier that turns into well: current increases, reaches plateau and declines. This technology paves the way to a wide range of potential applications in the fields of desalination of seawater,8 wastewater purification9 and DNA sequencing.10 These applications take advantage of the ultra-thinness of graphene which allows fast water transport while excluding ions or selecting specific ion types. The performance of these technologies critically relies on precise control of pore size. In fact, atomically precise pore sizes would allow the development of molecular sieves capable of separating compounds with atomic scale differences in size and shape. Significant advances in this field have been achieved by Gilbert et al.11 who demonstrated the fabrication of individual nanopores in hexagonal boron nitride with atomically precise control of pore shape and size, and by Thirumaran et al.12 who, using controlled Ga+ ion irradiation, introduced a population of sub-nanometer pores in a MoS2 membrane, realizing atomic transport measurements. Despite these recent achievements, experiments on transport across nanopores in 2D materials such as graphene, boron nitride, molybdenum disulfide and tungsten disulfide, are still lacking and most studies infer the conductance and sub-nanometer pore diameters indirectly from computational modelling.Graphene is a thin membrane consisting of spet al.13 showed that for small nanopores the sieving effect can be ascribed to steric hindrance of the pore edge. More specifically, it was shown that in order to effectively exclude salt ions, the diameter of a graphene nanopore cannot exceed 5.5 Å. Since the creation of small nanopores is technologically more challenging than drilling larger ones,14,15 alternative selectivity strategies suitable for larger pores have been actively sought. In particular, it was shown that placement of charges on the pore edge by chemical functionalization makes the pore selective to counter-ions. Using this strategy Sint et al.8 showed that replacement of carbon atoms of the graphene pore with nitrogen and fluorine makes the pore cation selective while addition of hydrogen make it anion selective. Moreover, Zhao et al.16 showed computationally that even when the pore radius is much larger than the hydrated radius of the ion, negatively charged nanopores still exhibit remarkable selectivity. This finding was confirmed by Konatham et al.17 that however observed that the selectivity induced by charged groups becomes less effective as the pore diameter is increased.In a pioneering Molecular Dynamics (MD) study Cohen-Tanugi 18 For instance, K+ channels like KcsA pass potassium and sodium with a ratio 1000 : 1  of KcsA. A similar approach was employed by He et al.22 who designed bio-inspired graphene nanopores containing four carbonyl groups (4CO) mimicking KcsA SF, or four carboxylate groups (4COO) arranged as in the SF of the NavAb Na+ channel.The main research question addressed by the many research groups working on graphene nanopores is the following: what is the ideal functionalization providing high water flows while discriminating against specific solutes? Biological ion channels, shaped through millions of years of evolution, are characterized by flow rates comparable to the free diffusion limit while retaining high selectivity.1000 : 1  while Na22 that the 4CO construct by He et al., as expected, was potassium selective. Surprisingly however, the 4COO construct that was expected to mimick the properties of NavAb, was not sodium but potassium selective and the selectivity of another construct with three carboxylate groups turned out to be voltage-tunable. Moreover, when the pore diameter is less than 5 nm, the confined liquid structure in the nanopore affects various transport properties of the ions.23 Since such properties may deviate from the bulk medium behaviour, ion dynamics in a nanopore confined region or an atomically thin membrane is still not well understood. Another open problem is related to the fact that functionalization can lead to a high density of charge along the pore rim. This in turn, leads to the accumulation of a concentration polarization layer in the neighborhood of the graphene membrane.24 The influence of this Debye layer on ion current is still not clear.The design principles of biological ion channels, however, are far from being completely understood. For instance, MD simulations showed+ and Cl− ion permeation through a pore drilled in a graphene monolayer. This enabled us to identify general trends useful for the design of functionalized nanopores. The current–voltage curves derived from MD simulations in the presence of a constant electric field were analyzed in terms of the potential of mean force (PMF) computed through umbrella sampling (US) simulations.25 We discovered that, as pore charge is increased, the barrier of K+ PMF gradually decreases until it turns into a well split in two sub-minima. An analysis of electrostatic energy and desolvation of the ion biased in US simulations, showed that the approximately constant desolvation cost is better and better compensated by the energy of electrostatic interaction with the charged pore. Thus this scenario, appears to be consistent with theoretical models by Eisenman26,27 and Zwolak.28,29 However, the analysis of the forces acting on the biased ion revealed a richer picture where a relevant role is also played by the potassium and chloride ions whose tendency to accumulate in concentration polarization layers significantly affects the electrostatic forces. Also, we observed that pore charge influences the orientation of water molecules affecting the van der Waals force they exert on the solvated K+ ion. Finally, the study of Cl− permeation revealed that, even if the PMF profiles are extremely powerful tools for the analysis of ion permeation, they must be used with extreme care. In fact, asymmetric ion distributions arising from the application of high voltages, cannot be accounted for by a PMF computed in the V = 0 regime, but they have a profound influence on ionic currents.In this paper we study the dependence on pore charge of KI–V curves and PMF profiles is reconciled in terms of the effect of voltage-induced asymmetric ion distributions. Finally, we draw conclusions.The paper is organized as follows. After the description of our computational methodology, we study potassium permeation devoting particular attention to the analysis of the forces and orientational effects that are discussed in specific subsections. The Results section concludes with the analysis of chloride permeation where a seeming discrepancy between rp 4.5 Å. The pore is thus larger than those of typical biological ion channels. However, the radius is below the 5.5 Å cutoff for unselective ion transport16 so that permeation and selectivity are mainly determined by the rim charge. The pore was generated choosing a reference carbon atom with coordinates  and removing all the graphene atoms whose distance from the reference atom  was below the chosen pore radius. The pore was either left uncharged or assigned an overall charge −1e, −2e, −3e, −4e, −5e, −6e or −7e. The pore charge was evenly spread over all the atoms lining the pore rim  run for 0.5, 1.0 and 2.0 ns respectively. Each equilibration stage was preceded by 1000 steps of conjugate gradient minimization. The short-range van der Waals and electrostatic interactions were cut off at 12.0 Å with a switching distance of 10.0 Å. The long-range electrostatic interactions were computed with the particle mesh Ewald method. Periodic boundary conditions were imposed in all directions and coordinates were stored every 2 ps.The system studied comprises a single graphene layer with a nanopore of radius 33 corresponding to potential differences ranging from −0.5 to −4 volts at 0.5 V intervals. The constant field was computed as Ez = V/Lz where V is the desired potential difference, while Lz is the length of the simulation box along the z-axis. Following where the sum runs over all the ions of a given type, Lz is the length of the simulation box in the direction of the channel axis, α is a conversion factor to express the current in amperes, qi and zi are the charge and position of ion i and the displacement is computed over two sampling intervals δt. In order to perform a statistical analysis through block-averages, the 40 ns long trajectories, run in the NVT ensemble at the temperature of 300 K, were split in 4 10 ns-blocks. Currents are computed as averages, and errors as standard deviations over the four blocks.After equilibration the system was simulated in the presence of a uniform electric field25 was employed. In these simulations the axial coordinate z of a single K+ was harmonically restrained  while the radial position was subject to a semi-harmonic wall at 4.5 Å from the pore axis. We considered 81 axial windows from z = −20.0 Å to 20.0 Å of width 0.5 Å. In the calculation of the K+ PMF each window was run for 6.0 ns with the first 1.0 ns considered as equilibration and excluded from the analysis. In the calculation of the Cl− PMF, due to slower convergence, each window was run for 16 ns with the first nanosecond discarded as equilibration. Using the implementation by Grossfield35 the Weighted Histogram Analysis Method (WHAM)36 was used to calculate the PMF with a tolerance of 10−7.In order to obtain the potential of mean force (PMF) of the permeating potassium ion the umbrella sampling method+ permeation, we ran MD simulations with an external applied potential in the range from −0.5 V to −4.0 V at 0.5 V intervals  the force exerted by the other K+ ions; (ii) the force exerted by Cl− ions; (iii) the force exerted by water molecules; (iv) the force exerted by graphene. + pushing it away from the bulk and towards graphene. By contrast, the negatively charged chlorides attract the reference K+ ion pulling it into the bulk. Due to the 1 : 1 stoichiometry of KCl it is not surprizing that the forces exerted by potassium and chloride ions are almost symmetrical and tend to balance each other. However, at high values of the pore charge  there is the appearance of a high local density of potassium ions on either side of the graphene layer. This local layer of potassium ions repels the reference K+ ion, pushing it away from graphene and reinforcing the effect of chloride ions. The force exerted by K+ and Cl− ions is however, comparatively small with respect to that exerted by graphene and water. When the pore is neutral, graphene exerts a zero electrostatic force. However, as the pore charge is increased, graphene exerts a stronger and stronger attracting force on the reference K+ ion. Finally, the force exerted by water pulls the K+ ion strongly into the bulk. In summary, the reversal in the sign of the total electrostatic force occurs when the graphene force becomes dominant compared to the water force and the potassium force starts pushing the reference K+ ion into the bulk.Both the van der Waals and electrostatic forces acting on the K+ ion is pulled closer and closer to the charged pore entering into the repulsive branch of the Lennard-Jones potential of the carbon atoms. Another notable feature of the van der Waals forces is that the force exerted on the reference K+ by the other potassium ions is always negligible. This is due to the fact that the repulsive K+–K+ electrostatic force keeps the potassium ions far away from the reference K+ so that the short-range van der Waals force is vanishing. Other trends can easily be identified, but they are more difficult to interpret. For instance, the van der Waals force exerted by Cl− ions always pushes the reference K+ ion towards the graphene pore. Moreover, the van der Waals force exerted by water molecules, at intermediate distance from graphene pulls the reference K+ ion into the bulk, but it pushes it towards the pore at closer distances from the graphene layer. The pattern exhibited by the chloride and water van der Waals force is discussed in more detail in the next section. As a final note, we can now explain the behaviour of the total van der Waals force. In particular, the reversal in the direction of this force occurs when the repulsive force exerted by graphene becomes dominant with respect to the force exerted by water that tends to push the reference K+ ion towards the pore.The van der Waals force also shows some interesting trends. First of all, note that graphene exerts a repulsive force, increasing with the pore charge. This is due to the fact that, as the pore charge is increased, the reference K+ has negligible Lennard-Jones interactions with the hydrogen atoms belonging to water molecules of both the first and second shell. This is because the peak of the first two shells (determined from the RDF profiles) corresponds to the flat tail of the Lennard-Jones curve. For the same reason K+ experiences vanishing Lennard-Jones forces from the oxygen atoms belonging to the water molecules of the second shell. Conversely, potassium is subject to repulsive forces from the oxygen atoms of the first water shell because the peak of this shell is located in the repulsive branch of the LJ potential.The pattern exhibited by the van der Waals force of water can be explained in terms of the several factors. First of all, the analysis of the Lennard-Jones (LJ) potential of the potassium/water-oxygen and potassium/water-hydrogen interactions  and close to the pore but not so close as to exit from the bulk phase and lose their hydration shells. On the other hand, when the pore charge becomes very high, the coulombic attraction is so strong that a close contact with the charged pore becomes more energetically convenient than remaining in the bulk solution to maintain the hydration energy.In order to explain this behaviour we plot the number density of potassium and chloride ions in axial bins with height 0.5 Å and radius 4.5 Å  choosing as an example the simulation at −4.0 V. The potassium profile in ESI Fig. SF6(a)+ ions. When Qp = 0 there is a single chloride density peak at z = −4.0 Å. This is due to the fact that the electric field is oriented towards the negative z-axis. This means that the negatively charged chlorides are pushed towards the positive z-axis. However, since graphene is not very permeable to chloride, they tend to accumulate just below the pore. As the rim charge is increased up to Qp = −4, the chloride peak decreases and shifts further away from the pore at z = −5.0 Å due to the increasing electrostatic repulsion exerted by the pore. However, when the charge is increased to Qp = −6 and −7 there will be the appearance of three high density peaks at z = −7.0 Å, z = −5.0 Å and z = −2.0 Å. This behaviour is clearly a consequence of the build-up of two high potassium density peaks close to the pore. The high local potassium concentration screens the negative charge of the pore and the K+/Cl− attraction allows the appearance of a high Cl− concentration. Moreover, the shielding of the pore charge allows an increase of the Cl− current as shown in the current plot ent plot  at Qp = ESI Fig. SF7+ PMF except that each window was run for 16 ns instead of 6 ns to reach convergence. The first nanosecond of the simulations was discarded as equilibration and the analysis was performed on the remaining 15 ns. The PMF profiles appearing in Qp = 0 to Qp = −3 and then drops from Qp = −4 to Qp = −7. This behaviour is seemingly at odds with the current/charge plots shown in Qp = 0 to Qp = −1 and Qp = −2 that does not seem to be justified by the very similar peaks of the PMF profiles. A clue to help in disentangling this contradiction is provided by the current plot in Qp = 0 to Qp > 0 becomes more and more pronounced as the applied voltage increases. This suggests that the high current observed at Qp = 0 could be due to some peculiar ion distribution induced by the external electric field. To test this hypothesis in ESI Fig. SF8+ and Cl− ions in the simulation with an applied potential of −4.0 V. It can be noticed that at Qp = 0 the ion distribution around the pore is highly asymmetrical. Specifically, the Cl− concentration below the pore is much higher than the concentration of the same ion above the graphene layer, while the potassium concentration above the pore is only slightly higher than the K+ concentration below the pore. This distribution determines that, in the neighborhood of the pore, there is a large availability of chloride ions that could be involved in permeation. This results in a high chloride current even if the free energy barrier of the PMF is high. It must be stressed explicitly that, since the PMF was computed in absence of external field, this concentration polarization effect could not be accounted for by the PMF profiles. As a comparison, we also computed the ion density profiles for the system with pore charge Qp = −2 in the presence of an external potential of −4.0 V. In this case it can be observed that the chloride density below the pore is only moderately higher than that above the pore. Thus, the gradient of concentration of Cl− is smaller than in the Qp = 0 case explaining why the current at Qp = −2 is much smaller than that at Qp = 0 even though the peaks of the PMFs are almost identical. As a final note, it can be observed that the shallow free energy wells appearing on either side of the barrier at Qp = −6 and Qp = −7 are also due to the high local density of K+ ions that attract the incoming chloride.More insight into the permeation mechanism of chloride can be attained from the analysis of the PMF profiles. The PMF was again computed through umbrella sampling simulations using a protocol identical to the one employed for the calculation of the Ket al.16 showed that even large graphene nanopores, if negatively charged, can enhance K+ transport while completely rejecting Cl−. Even if this work explores a wide range of values of pore charge and radius, no attempt was made to study the influence of the charge on potassium and chloride PMFs. Similarly, Li et al.37 recently showed that a charged graphene surface can induce selectivity of Li+ and Na+ over K+. However, these authors, instead of analyzing classical pores generated by removal of carbon atoms, considered ion flow through the stretched aromatic rings of a graphene membrane under mechanical strain.One of the many potential applications of graphene concerns water desalination and purification. While the sieving capability of small graphene pores depends on steric effects, the selectivity of larger pores critically depends on the charge. Yet, only a couple of studies systematically addressed the issue of selectivity/charge relationship. Zhao I/V curves and the PMF profiles fills a gap in the scientific literature. As a cautionary note, it must be observed that we chose to use a simplified pore charge distribution . In realistic functionalized graphene nanopores the charge distribution can be different. Remarkable examples include the bio-inspired nanopores studied by Kang et al.21 and the graphene-based crown ethers analysed by Guo et al.38 and by Smolyanitsky et al.39 where the electrostatic potential maps critically depend on the placement and orientation of the ether dipoles. Despite its simplicity however, our approach can qualitatively reproduce the properties of realistic systems. This is due to the fact that many patterns are significantly robust with respect to the fine details of the charge distribution. As an example, Zhao et al.16 who used a charge model similar to ours, showed that, as long as the total charge is kept constant, charging all pore atoms or only alternating ones led to comparable ion fluxes. Similarly to what is reported in +, we found that, as the pore charge is increased, the barrier of K+ PMF decreases until it turns into a well that partially traps potassium, reducing the current. This trend can be explained in terms of an increasing electrostatic compensation of a roughly constant desolvation cost. This is in agreement with the seminal theory developed by Eisenman26,27 that identifies two driving forces for ion permeation: (i) the ion–pore electrostatic interactions and (ii) the desolvation cost the ion incurs when crossing the pore. More formally, an ion with charge q and radius r has a hydration free energy represented by the Born approximation: Ghydr = q2/8πεwr where εw is the dielectric constant of water and its electrostatic interaction with a site within the channel of charge qs and radius rs is Gint = qqs/4πεw(r + rs). Ion permeation can only occur if Gint ≥ Ghydr. It is noteworthy that use of the Born formula to express the dehydration cost, amounts to assuming that the ion is completely desolvated upon entering the pore. Eisenman's ideas were further developed by Zwolak and coworkers28,29 who expressed the desolvation cost as a function of the pore radius. This approach accounts for partially desolvated states since dehydration occurs in a quantized way, a shell at a time. According to this model desolvation only depends on the geometric properties of the pore, namely its radius. Our results however, show that this picture is oversimplified. Even if the large number of water molecules lost by the ion upon entering the neutral pore confirms the importance of geometry, + but also for Na+ and Li+.Our systematic exploration of the influence of the pore charge on both the 40 on the grounds of theoretical modelling and Brownian dynamics simulations suggested that knock-on barrierless conduction can be considered as a general mechanism of transport in ion channels with multiple occupancy. Similar conclusions have been reached by the Ionic Coulomb Blockade model41 predicting that conduction bands occur when the charge of the selectivity filter balances the charge of the ions already inside the channel plus the image charge of a further potentially incoming ion. Our simulations show that barrierless conduction can be tuned using pore charge as an adjustable parameter. In fact, when the rim charge is high enough, the ion/pore interaction almost perfectly balances the desolvation cost flattening the permeation barrier. Recent computational studies have shown that barrierless conduction can be induced using other control parameters. For instance Fang et al.42 showed that a modest mechanical strain on graphene-embedded crown ether pores reduces the K+ release barrier, significantly increasing the current. Interestingly, in this case also, the barrier arises from both the desolvation cost and ion–pore electrostatics. The mechanical pore expansion flattens the barrier simultaneously weakening the ion/pore interactions and strengthening the ion/water ones.The decrease of the free energy barrier as a function of increasing pore charge leads to the issue of near-barrierless permeation. In the literature it is widely debated whether barrierless conduction must be considered a universal mechanism of ion channels or a peculiar property of KcsA-like channels where two- and three-ion occupation states are isoenergetic. Yesylevskyy and Kharkyanen+ ion towards the pore while electrostatic forces tend to keep it in the bulk. However, as the pore charge is increased, the directions of both forces are reversed. The reversal of the van der Waals force is due to the build-up of an increasing repulsive force exerted by graphene that becomes dominant over the LJ forces exerted by chloride and water. The reversal of the electrostatic force depends on two main factors. First of all, as pore charge increases, the attractive electrostatic force exerted by graphene overcomes the repulsive force exerted by water. The interplay between these two forces is the one expected based on simplified models like Eisenman's and Zwolak's. The simulations however, also reveal an important but unexpected role of potassium and chloride ions. For small values of the pore charge these ions are evenly spread in the bulk phase so that their forces (the reference K+ ion is attracted to the bulk by chloride and pushed to graphene by the other potassium ions) tend to balance each other. At high pore charges however, the formation of a concentration polarization layer of K+ ions switches the sign of the electrostatic force that potassium ions exert on the reference K+ ion. The formation of concentration polarization layers is even more significant in the case of Cl− permeation reconciling the seeming mismatch between the modest decrease of the barrier of the chloride PMF (when Qp varies from 0 to −3) and the significant drop of the current.The atomistic level insight provided by molecular dynamics not only sheds light on the details of the desolvation process, but it also reveals a rich picture not foreseen by simplified physical models. Our simulations show that van der Waals and electrostatic forces exert opposite effects on the permeation process . For smaet al.43 observed a surprisingly large K+/Cl− selectivity in pores even as large as 20 nm in diameter. A careful examination of the results attributed this counter-intuitive result to the elevated concentration of mobile cations near the electronegative graphene surface. More recently, Hu et al.24 showed through MD simulations that sodium and potassium form concentration polarization layers on either side of a graphene sheet. In their work with a neutral pore, Na+ forms symmetrical density peaks on either side of the graphene pore similarly to what happens with K+ in our simulation with Qp = 0. Chloride, on the other hand, forms a single very high density peak just above the membrane. This mismatch with our simulations where the Cl− density peak is below the pore, is simply due to the different orientation of the electric field in their work.Concentration polarization, the non-uniform ion distribution close to a sufficiently small pore is a widespread phenomenon also highlighted in other studies. For instance Rollings In summary, we have characterized the influence of pore charge on the permeation of potassium and chloride across a graphene nanopore. While the simulations basically confirm the validity of simplified physical models, they also reveal a much richer phenomenology where the asymmetry of ion distributions and the orientational effects induced by the applied potential and pore charge significantly affect conduction, thus calling for more detailed and comprehensive modelling.There are no conflicts to declare.RA-009-C9RA03025E-s001


Drosophila mdg4-ERV (gypsy) or the human ERV, HERV-K (HML-2) are each sufficient to stimulate cytoplasmic aggregation of human TDP-43. Viral ERV transmission also triggers TDP-43 pathology in recipient cells that express physiological levels of TDP-43, whether they are in contact or at a distance. This mechanism potentially underlies the TDP-43 proteinopathy-caused neurodegenerative propagation through neuronal tissue.Inter-cellular movement of “prion-like” proteins is thought to explain propagation of neurodegeneration between cells. For example, propagation of abnormally phosphorylated cytoplasmic inclusions of TAR-DNA-Binding protein (TDP-43) is proposed to underlie progression of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). But unlike transmissible prion diseases, ALS and FTD are not infectious and injection of aggregated TDP-43 is not sufficient to cause disease. This suggests a missing component of a positive feedback necessary to sustain disease progression. We demonstrate that endogenous retrovirus (ERV) expression and TDP-43 proteinopathy are mutually reinforcing. Expression of either  Drosophila or human endogenous retroviruses (ERVs) is sufficient to cause TDP-43 protein aggregation, and viral transmission of the ERVs triggers TDP-43 pathology in recipient cells. This mechanism may underly spread of neurodegenerative effects in a Drosophila model.Expression of  Mutations in TDP-43 are responsible for a fraction of the inherited forms of ALS, providing a direct causal link between this gene and neurodegenerative cascades. But the vast majority of cases involve the mis-localization and aggregation of TDP-43 of normal amino acid sequence1. Thus, initiation of TDP-43 protein pathology is triggered by upstream factors, likely ones that accumulate with age. On the other hand, disease progression after diagnosis is relatively rapid, and is thought to involve inter-cellular propagation of disease pathology from a point of stochastic focal origin. This inflection in rate of disease progression after decades of apparently normal physiology is consistent with involvement of a self-sustaining biological processes, a positive feedback loop6.Cytoplasmic aggregation of abnormally phosphorylated TDP-43 (pTDP-43) is seen in both neurons and glia in the affected brain regions of 97% of patients with ALS, ~40% of cases with FTD, in many cases of Alzheimer’s disease (AD), and in patients with an AD-like presentation 6. In this model, pathological forms of misfolded TDP-43 are able to template misfolding in trans of normal wild-type TDP-43, much as has been demonstrated for the prion protein (PrP) that underlies infectious prion diseases8. Such misfolded TDP-43 is proposed to be released from neurons and/or glial cells and then to be taken up by recipient cells, where the prion-like templating process is repeated. Indeed, numerous biochemical experiments support the idea that TDP-43 protein misfolding can template in trans, leading to propagation of pathological forms of TDP-43 in vitro, and there are elegant experiments that demonstrate inter-cellular spread, both in cell culture and by injection into cortex of mice that also over-express a sub pathological level of transgenic human TDP-43 variants11. These experiments reveal that pathological cytoplasmic hyperphosphorylated TDP-43 appears in cells far from the site of injection. But unlike the case of PrP, injections of pathological TDP-43 does not cause robust propagation of proteinopathy in recipient animals, and does not cause neurodegenerative cellular effects, unless those animals also over-express transgenic human TDP-43 in addition to the endogenous mouse gene11. We and others have documented that TDP-43 protein pathology or knockout of TDP-43 are each sufficient to cause dysfunctional expression of ERVs and retrotransposable elements (RTEs). This has been shown in Drosophila, human neuroblastoma cells, and cortical tissue from human subjects21. Here, we provide evidence that ERVs provide a hitherto unknown component of a positive feedback loop to sustain propagation of TDP-43 pathology and to spread neurodegenerative effects through neuronal tissue.One model that has been proposed to explain such a self-sustaining inter-cellular propagation is the prion hypothesis24. Indeed, enteroviral infection can cause pathological accumulation of TDP-4324. We therefore tested whether expression of ERVs could impact the localization and phosphorylation of TDP-43. We first tested the impact of expressing HERV-K, a human ERV whose expression has been observed in cortical tissue of patients that exhibit TDP-43 pathology16. We generated a HERV-K reporter construct that was tagged with a nuclear localized H2B-mCherry (HERV-K-H2B-mCherry)  Fig. . To accory) Fig. . In contry) Fig. .Fig. 1Hu26. We also observe cytoplasmic mislocalization of TDP43 using a pan-TDP-43 antibody  is sufficient to trigger cytoplasmic accumulation of pathologically phosphorylated human TDP-43 . Indeed, by 24 h after induced expression of a V5-tagged human TDP-43, 64.31% of cells that were transfected with an mCherry tagged mdg4-ERV exhibited redistribution of TDP-43-V5 to the cytoplasm compared with just 35% of cells transfected with a control mCherry  has been replaced by a human TDP-43 cDNA with a 3xFlag tag fused at the N-terminal (abbreviated as TDP-43WT-KI)28. In this strain, only the human TDP-43 gene is expressed, at physiological levels via the endogenous fly ortholog’s promoter28. Although knockout of the endogenous fly ortholog is lethal, this humanized TDP-43WT-KI fly strain exhibits normal longevity that is indistinguishable from that of the laboratory wild-type strain28  only in a spatially restricted glial subtype called the subperineurial glia (SPG)  Fig. . It is wset Fig. . This pets), so that we could induce spatially restricted TDP-43 expression after allowing normal development (induction relies on shifting the growth temperature from 21 °C to 29 °C). The use of an inducible expression system also provides the means for observing effects over a time-course after initiating expression. We found that Gal4 mediated induction of hTDP-43 in SPG (SPGts > hTDP-43) greatly reduces median survival of animals that contain the TDP-43WT-KI replacement of the fly ortholog. Median survival of this strain was just 18 days compared 31 days for animals containing just the TDP-43WT-KI and 34 days for animals that contain the SPGts and TDP-43WT-KI, but no UAS-hTDP-43 , we observed only nuclear Flag-labeled TDP-43 in neurons, with no evidence of pTDP-43 accumulation. This was true over a 15 day time course after shifting to induction temperature  Fig. . This efure Fig. . By conture Fig. . This efure Fig. . For exaure Fig. , and theure Fig. . We alsoure Fig. . We alsoure Fig. . This in36. DNA damage has been observed in post mortem brain tissue from patients with TDP-43 related disorders36, in iPSC derived neurons from C9orf72 familial patients that exhibit TDP-43 pathology37, and in animal models including Drosophila when TDP-43 is over-expressed34. We therefore wondered whether we would detect DNA damage foci in neurons that exhibited pTDP-43 pathology due to focal induction of hTDP-43 over-expression in SPG. Importantly, such neurons exhibit pTDP-43 due to induced hTDP-43 over-expression in a neighboring cell type while only expressing human TDP-43 at physiological levels themselves  rather than the humanized TDP-43WT-KI knock in 39 in SPG using the same Gal80ts approach. This allowed us to monitor both pTDP-43 signal and the RFP signal independently. We also co-expressed a membrane tethered mCD8-GFP in the SPG in order to mark the membrane, yielding an outline of SPG morphology. As with induction of the untagged TDP-43, induction in SPG of the TDP-43-RFP (SPGts > hTDP-43-RFP in TDP-43WT-KI) shortened the animal’s lifespan to a median survival of 15 days  resulted in a strong cell autonomous effect on TDP-43 pathology, resulting in robust pTDP-43 signal in all SPG at D7, and this persisted to D15    Fig. . Such ecWT-KI instead of the fly gene  and in a higher fraction of cells at (29.02% vs 12.12%) at D15 than at D7 , we significantly attenuated these non-cell autonomous effects on pTDP-43 in neurons , we greatly reduced the appearance of DNA damage in the nearby neurons both at D7 and D15  was sufficient to greatly ameliorate the loss of neurons  that are capable of transmitting between cells grown in culture. Indeed, both of these ERVs contain an enveloped glycoprotein that is required to mediate such intercellular transmission. We previously established Drosophila S2 cell culture assays to detect transmission of mdg4-ERV between cells grown in contact, or between cells that were separated by a 0.4 µm filter in a transwell system40. We used these assays to test whether viral transmission of mdg4-ERV was sufficient to mediate propagation of TDP-43 pathology to recipient cells.Both the 40. In contrast, the H2B-mCherry control was never seen to transfer to the hTDP-43-V5 expressing population of cells that are grown in contact  were co-transfected into S2 cells 59 and in Aicardi-Goutieres syndrome61. RTE and ERV expression has the potential to contribute to many of the cellular impacts that are thought to be at play across these disorders, including inflammatory signaling, DNA damage and defects in RNA metabolism and proteostasis65. But our findings raise the possibility that RTE and ERV expression provides a feedback amplification to sustain the proteinopathy in other disorders as well. This suggests the potential of clinical interventions to block RTE/ERV expression or function, as a means to interrupt a positive feedback mechanism driving disease progression.Normal aging is associated with a progressive rise in RTE and ERV expressionNo statistical methods were used to predetermine sample size. Animal age for each experiment was noted throughout the text. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment.Drosophila Stock Center. The hTDP-43-KI28 and UAS-hTDP-43-RFP39 were generous gifts from Professor David Morton  and Professor Jane Y. Wu (Northwestern University School of Medicine). The following stocks, UAS-GFP-IR, UAS-mdg4-IR , UAS-hTDP-43, tub-Gal80ts and UAS-mCD8-GFP were used in our previous studies43. To prevent artifacts from genetic variation between groups, all strains used in this study were backcrossed to our laboratory wild-type strain, Canton-S derivative w1118 (isoCJ1), for at least five generations. Male flies were chosen as the experimental subjects throughout the study. All flies for TARGET66 temperature-shift experiments  were raised in a 21 °C incubator from the embryonic stage66. All flies for TARGET experiments were immediately shifted to the permissive temperature at 29 °C after eclosion, which was designated as day 0. These TARGET flies were then incubated at 29 °C for lifespan assays or dissected at desired time points for analyzing the patterns of molecular markers dependent on the experimental designs.We obtained SPG-Gal4  and UAS-myr-RFP (#7119) from Bloomington 40 and moxBFP  constructs and then fused by PCR to generate the H2B-BFP fragment. The P2A was separately added to the C-terminal of H2B-BFP and N-terminal of hTDP-43 by PCR. The final H2B-BFP-P2A-hTDP-43 was then created by PCR fusing H2B-BFP-P2A and P2A-hTDP-43 fragments and then cloned into the inducible pMT-V5-6xHis plasmid. The pAc-H2B-mCherry-HA, pAc-mdg4-H2B-mCherry-HA and pAc-mdg4-H2B-mCherry-HAΔEnv constructs were generated described previously40.To generate the inducible hTDP-43-V5 construct, the full length of hTDP-43 was PCR amplified but the stop codon was removed in order to generate an in frame fusion protein with the V5 tag provided by the pMT-V5-6xHis plasmid . To generate the pMT-H2B-BFP-P2A-hTDP-43-V5 construct, the H2B and BFP were separately PCR amplified from pAc-H2B-mCherry13 and used as the template to generate the constitutive pcDNA3.1-HERV-K-P2A-H2B-mCherry-HA plasmid. In short, the P2A-H2B-mCherry-HA fragment was PCR amplified and integrated downstream of Env of pcDNA3.1-HERV-K while removing stop codon of Env.To generate the constitutively expressing pcDNA3.1-H2B-mCherry-HA, the H2B-mCherry-HA was moved from pAc-H2B-mCherry-HA by restriction enzyme NotI and reinserted the H2B-mCherry-HA DNA fragment into the NotI site of pcDNA3.1 . The backbone of HERV-K (pcDNA3.1-HERV-K) was obtained from Professor Nath’s laboratory14. In short, ten adult flies of a given genotype were housed within one vial, and >100 flies in total for experimental group were used for each experiment. The surviving flies from each vial were flipped into fresh vials with fly food every other day, and dead flies at specific time points were recorded for the final survival curve analysis. The Log-Rank (Mantel-Cox) test and the Gehan-Breslow-Wilcoxon test were used to compare the significance of survival curves.Adult male flies with desired genotypes raised at 21 °C were collected immediately after eclosion and incubated in a 29 °C incubator for survival assay as addressed in previous studies12. Adult brains were dissected in ice-cold phosphate-buffered-saline (PBS) and then transferred into 4% paraformaldehyde (Electron Microscopy Sciences #15713) PBS solution ) and incubated for 30 min under vacuum twice. After fixation, dissected brains were washed three times for 10 min with 1XPBST wash solution (1XPBS with 1% Triton-X-100 and 3% NaCl). Brain samples were then incubated in blocking solution  overnight at 4 °C on a nutator. After blocking, dissected brains were transferred into primary antibodies and incubated overnight at 4 °C with the following dilutions: mouse anti-Repo , rat anti-Elav , mouse anti-Elav , mouse anti-Flag , rabbit anti-pTDP-43 , rabbit anti-pTDP-43  and rabbit anti-γH2Av  in 1XPBST washing solution with 10% normal horse serum. Samples were then washed with 1XPBST four times, 15 min each. Fly brains were incubated in secondary antibody solution  at 4 °C overnight. Secondary antibodies were combined from the following lists: donkey anti-mouse Alexa Fluor 488 , donkey anti-rabbit Alexa Fluor 488 , donkey anti-rat Alexa Fluor 594 , donkey anti-mouse Alexa Fluor 647 , donkey anti-rat DyLight 405 . After washing with 1XPBST for 4 × 15 min, fly brains were mounted in FocusClear , imaged using a Zeiss LSM 800 with Airyscan mode, and acquired images were processed by the Zeiss Zen software package.Adult brains at specific time points for each experiment were dissected, and immunofluorescent staining was performed as previously described2 (77.99 µm x 71.75 µm) area  were maintained in Schneider’s Drosophila medium  supplemented with 10% fetal bovine serum (FBS)  and 100U/ml Penicillin-Streptomycin  in a 25 °C incubator. Human neuroblastoma cells, SH-SY5Y , were grown in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12)  with 10% FBS and 100U/ml Penicillin-Streptomycin in a 37 °C incubator with 5% CO2 supplementation.42. 5 × 105 cells were seeded onto coverslips coated with 0.5 mg/ml concanavalin A (ConA)  and placed in the 6 well culture plate overnight prior to the transfection. Single or co-transfections with desired construct combinations  were performed with 1 µg of DNA each and Effectene transfection reagents . Post 24 h transfection, transfection complexes were washed away and supplied with the fresh complete medium containing 700 µM CuSO4 to induce hTDP-43-V5 expression.Conditions for S2 cells transfection were modified from our previous studies5 cells were seeded onto coverslips coated with 0.1 mg/ml Poly-D-Lysine and placed in 6 well culture plates overnight prior to transfection. Single transfection was performed under the manufacturer’s guideline with a mixture of 4 µg pcDNA3.1-H2B-mCherry or pcDNA3.1-HERV-K-P2A-H2B-mCherry with 12 µl TransIT-2020 reagent in a 1:3 ratio . After 24 h of incubation, transfection complexes were washed away, and fresh complete medium was supplied daily until the desired time points for analysis.Transfection conditions of SH-SY5Y were as follows: 8 × 105 of donor (pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry) and recipient (pMT-hTDP-43-V5) cells were seeded at a 1:1 ratio onto the ConA-coated coverslip and maintained for 72 h to enhance the entrance of VLPs into recipient cells. At 20 or 24 h prior to the experimental time, the culture medium was replaced with a medium containing 700 µM CuSO4 to induce hTDP-43-V5 expression in recipient cells. For transwell assays, 5 × 105 of recipient (pMT-hTDP-43-V5) cells were plated onto ConA-coated coverslips and placed in the bottom well. At the same time, 1 × 106 of donor (pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry) cells were placed into the upper well. 20 or 24 h prior to analysis, CuSO4 was added to induce hTDP-43-V5 expression in recipient cells.In order to expand the donor cell population and enhance the production of virus-like particles (VLPs), S2 cells were transfected with pAc-H2B-mCherry or pAc-mdg4-P2A-H2B-mCherry 48 h before co-culture or transwell assays were performed  PBS solution ) for 10 min on a nutator. After fixation, cells were washed three times with 1XPBST wash solution (1XPBS with 1% Triton-X-100 and 3% NaCl) and then incubated for 1 h in blocking solution  at room temperature. Cells were transferred into primary antibodies in 1XPBST washing solution with 10% normal horse serum after blocking and incubated for 1 h at room temperature with the following dilutions: mouse anti-V5 , rabbit anti-TDP-43  and rabbit anti-pTDP-43 , rabbit anti-pTDP-43 , mouse anti-HERV-K-Env , rat anti-mCherry  and rabbit anti-STMN2 . Samples were then washed with 1XPBST three times for 10 min each. Cells were incubated within secondary antibody solution  with DAPI at room temperature for 1 h. After washing three times with 1XPBST for 10 min each, cells were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific), imaged using a Zeiss LSM 800 with Airyscan mode, and acquired images were processed by Zeiss Zen software package.25. To obtain the relative fold changes of POLDIP3α, values from each group were further normalized to the levels in pcDNA3.1-H2B-mCherry transfection at Day 2 in order to obtain the relative levels.Total RNA was extracted from pcDNA3.1-H2B-mCherry or pcDNA3.1-HERV-K-P2A-H2B-mCherry transfected SH-SY5Y cells using Trizol reagent  at different time points, and the total RNA extractions were treated with RQ1 DNase  before performing reverse transcription. 1 µg of total RNA from each sample was used to synthesize cDNA with SuperScript IV VILO Master Mix . qPCR was then performed using PowerUp SYBR Green master mix  on an Applied Biosystems QuantStudio 3 Real Time PCR System. The primer sets against POLDIP3α (Forward: 5'-CCAAAACCATCCAGGTTCCACAGCAG-3' and Reverse: 5'-GTGGTGGAGAAAGCCGCCTGAG-3') and GAPDH (Forward: 5'-GTTCGACAGTCAGCCGCATC-3' and Reverse: 5'-GGAATTTGCCATGGGTGGA-3') used in this study were as previously described27. Transfected S2 cells were washed, collected with 1XPBS, and then homogenized in ice-cold RIPA buffer  with protease inhibitors  and phosphatase inhibitors . Cell lysates were centrifuged at 16000 g for 30 min at 4 °C, and the supernatant was saved as the RIPA-soluble fraction. The RIPA-insoluble pellets were washed with RIPA buffer by centrifuging at 16000 g for 15 min at 4 °C. Supernatants were discarded and the pellets were extracted and homogenized with urea buffer . The lysates were centrifuged at 16000 g for 30 min at 4 °C. The supernatant was kept as the RIPA-insoluble and urea-soluble fraction.The soluble and insoluble TDP-43 fractions within the S2 cells were sequentially extracted dependent on the solubilities in the extraction buffers as previously descripted30 µg protein samples from each cellular fraction were separated by 10% polyacrylamide gel  and followed by a canonical Western Blot protocol. In short, membranes were blocked in 1XTris Buffered Saline-Tween-20 (TBST) (1XTBS + 0.2% Tween-20) with 5% SlimFast (chocolate flavor) at room temperature for 30 min. After blocking, membranes were incubated with primary antibodies mouse anti-GFP , and rabbit anti-pTDP-43  in 5% SlimFast -TBST for 1 h at room temperature or overnight at 4 °C. After washing three times with 1XTBST, membranes were incubated with secondary antibodies  or goat anti-rabbit-HRP  at 1:1000 or 1:2000 dilutions) for 1 h at room temperature. Signals were developed in chemiluminescent HRP substrate  and imaged with Sapphire Biomolecular Imager (Azure Biosystems).p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Statistical analyses for different experimental setups were performed using GraphPad Prism 9. Specific statistical analyses used for comparing the significance between experimental groups were addressed in the corresponding figure legends and the experimental reproducibility was also listed in the figure legends. The significant levels were indicated as star numbers in the following format: *Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The antagonism between strength and corrosion resistance in graphene‐reinforced aluminum matrix composites is an inherent challenge to designing reliable structural components. Heteroatom microstructural modification is highly appreciated to conquer the obstacle. Here, a bottom‐up strategy to exploit the heterogeneous phase interface to enable high corrosion durability is proposed. Deformation‐driven metallurgy derived from severe plastic deformation is developed to produce Mg‐alloyed fluorinated graphene structures with homogeneous dispersion. These structures allow for absorbing corrosion products, forming a dense protective layer against corrosion, and local micro‐tuning of the suppression of charge transfer. This results in superior corrosion resistance with an outstanding strength‐ductility balance of the composites via ultrafine‐grained and precipitation strengthening. The anti‐corrosion polarization resistance remains 89% of the initial state after 2‐month immersion in chloride‐containing environment, while the ultra‐tensile strength and elongation of 532 ± 39 MPa and 17.3 ± 1.2% are obtained. The economical strategy of heteroatom modification broadens the horizon for anti‐corrosion engineering in aluminum matrix composites, which is critical for the design of carbonaceous nanomaterial‐reinforced composites to realize desired performances for practical applications. A bottom‐up strategy is proposed to exploit the heterogeneous phase interface to enable high corrosion durability via deformation‐driven metallurgy. The anti‐corrosion polarization resistance remains 89% of the initial state after 2‐month immersion in chloride‐containing environment, which is critical for the design of carbonaceous nanomaterial‐reinforced composites to realize desired performances for practical applications. This barrier characteristic and chemical stability make it not surprising to shield the metals underneath it from unexpected reactions and corrosion. However, when these nanomaterials are applied to prepare aluminum matrix composites in practical applications, especially for high specific‐strength functions, fatal acceleration of the corrosion rate is often to be obtained instead of corrosion improvement. The undesirable anti‐corrosion weakening of graphene reinforced aluminum matrix composites can be attributed to three major inherent issues: (a) Graphene can trigger corrosion‐promotion activity (CPA) to enhance the localized galvanic corrosion due to the heterogeneous corrosion potential, such as graphene  and aluminum matrix (−1.1 V vs SCE). When coupling with a relatively active metal, the electrochemical stable and high electrically conductive graphene tends to behave like noble cathode, contributing to the formation of micro‐galvanic corrosion that initiates localized corrosion and boosts the anodic dissolution; (b) Small molecules such as O2 and H2O may infiltrate from the edge defects of graphene over the long‐term application, which consequently accelerated the corrosion rate; (c) Graphene is easy to agglomerate and exhibits poor compatibility due to its large specific surface area and strong van der Waals interaction, which severely reduces their dispersibility in the aluminum matrices and deteriorates the corrosion resistance performances.Sparked by its in‐plane impermeability to nearly all molecules,1 grap It can reduce the electrical conductivity by increasing the electron scattering in graphene lattice, as well as modifying the local electron density to inhibit the electrochemical activity of graphene. Fluorinated graphene is promising to enhance the anti‐corrosion performance for the insulating nature induced by F doping, as well as the molecule impermeability inherited from graphene. An alternative to suppressing the corrosion rate is to design microstructures that are intrinsically electrochemically homogeneous . However, the combination of these two approaches loses effectiveness since the composites reinforced by fluorinated graphene are susceptible to weak interfacial bonding and low load‐bearing capacity due to the chemical inertness of this modified graphene. The desirable interfacial bonding cannot be simply obtained by the high‐value plastic strain induced by severe plastic deformation. Developing cheap but effective microstructure solutions that enable both high corrosion resistance and mechanical performances thus remains a fundamental challenge.In light of these, several tailoring strategies were proposed to suppress the CPA behavior toward highly applicable aluminum matrix composites. Heteroatom tailoring of graphene shows great potential for changing the electronic density to tailor its electronic and electrochemical properties.9 It cHere we propose a bottom‐up strategy based on the principle of exploiting great interfacial bonding between fluorinated graphene nanoplatelets (F‐GNPs) and aluminum matrices via Mg alloying and severe plastic deformation. A severe plastic deformation technique, deformation‐driven metallurgy (DDM), was applied to obtain the homogeneous dispersion of F‐GNPs to realize extraordinary corrosion resistance. Corrosion‐suppression activity (CSA) induced by the synergistic strategy of active metal and heteroatomic modification was studied. This work aims to examine the effectiveness of synergistic modification and DDM techniques in terms of corrosion behavior in an aggressive chloride‐containing environment. Microstructural factors, interfacial characteristics, reinforcement dispersion, and electrochemical response were evaluated and discussed in detail.22.1Figure Based on the principle of heteroatom modification toward high corrosion durability, we designed a type of carbonaceous nanomaterial‐reinforced aluminum matrix composites with great interfacial bonding via Mg‐alloyed F‐GNPs and severe plastic deformation. Figure ID/IG A higher ID/IG value refers to the more broken graphene, indicating that there are more active open edges to provide necessary conditions for subsequent metallurgical bonding. Compared with the original (F)‐GNPs, an increase in this value can be seen in all the ball milling routes. Due to the more significant lubricating effect of the fluorinated graphene, the crushing effect caused by ball milling is weakened in the AM+F and MF+A routes. On the one hand, the integrity of F‐GNPs themselves is better preserved, resulting in a more efficient load transfer effect for mechanical performances and surficial absorbing integrity for corrosion resistance. On the other hand, it also means the reduction of the active reaction sites. It is also worth noting that the characteristic peak of MgF2 is observed in Figure 2 has high chemical stability and bond energy, the existence of this substance implies the strong chemical bonding between the carbonaceous phases and the matrices. This phenomenon compensates for the adverse effects of fewer active sites during the ball milling process. In addition, the ball milling routes of preferential mixing of (F)‐GNPs in the first step have a low ID/IG value, indicating that the physical isolation effect between Al and Mg brought by (F)‐GNPs can reduce the overreaction of the ball‐milled powders. Figure ID/IG values have decreased correspondingly. Obviously, graphene cannot be re‐spliced into a larger piece via severe plastic deformation. The decrease in this value indicates that the decrease in the thickness and layers of the (F)‐GNPs, and finally results in the increase of the specific surface area of graphene. This provides better prerequisites for the load transfer effect. In addition, the results of X‐ray diffraction (XRD) tests ‐GNPs and the matrices.To access the influence of different bottom‐up preparation routes on the microstructural factors of the (F)‐GNPs and the matrices, we conducted Raman spectroscopy, as shown in s Figure  show tha3 to sp2 ratio, which is in line with the bonding form of F‐GNPs, that is, one C atom forms a bond with two in‐plane C atoms and one out‐of‐plane F atom. Besides, there are basically no C‐Al bonds in the F‐GNPs DDM composites, which proves that the existence of F atoms isolates the direct reaction between Al matrix and C atoms, thereby avoiding the formation of the easily hydrolyzable compounds Al4C3. Since this substance is detrimental to the structural integrity of materials exposed to the aqueous environment, this phenomenon provides the possibility to improve corrosion resistance. Figure ). This is consistent with our vision properly, which is to achieve indirect bonding between aluminum and (F)‐GNPs through Mg alloys, thereby avoiding the CPA effect and improving the comprehensive corrosion resistance of the DDM composites.X‐ray photoelectron spectroscopy (XPS) was also applied to analyze the microstructural evolution and further illustrate the bonding behavior of the DDM composites. Figure e F atom.17 BesFigure  which significantly promotes the flow intensity of the composites during the severe plastic deformation process. More obvious dynamic recovery and recrystallization are triggered by the enhanced lattice distortion and defect energy; (2) by adding (F)‐GNPs in the first step of the ball milling process, the excessive reaction of Al and Mg can be avoided, thereby reducing the dynamic viscosity of the thermo‐plasticized composites during DDM process. This makes the material more prone to plastic deformation and further improves the grain refinement effect. From the above two explanations, it can be found that the DDM composites obtained via the MF+A route has achieved the finest microstructures with an average grain diameter of 1.14 ± 0.45 µm. We further observed the grain characteristics of the MF+A DDM composites, as shown in Figure  From the energy spectrum results, the distribution area of Mg and F‐GNPs overlaps, indicating that a self‐assembled nanoscale structure is formed between Mg and graphene. As shown in Figure 4C3, and enhances the bonding strength between the Al matrix and the reinforcements, since F‐GNPs are relatively chemically inert to aluminum. In addition, due to the poor electronic conductivity of the fluoride, it also provides a necessary condition for reducing the CPA effect on the composites.Figure 22 Fro2.2Figure  the precipitation/solid solution strengthening produced by the Mg element, the sound interfacial bonding between the matrix and the reinforcements, and the Hall‐Petch strengthening brought by the severe plastic deformation, the DDM composites we prepared has obtained the extraordinary mechanical performances. We further studied the comprehensive mechanical responses with different Mg addition. One can see that the tensile strength performance with all the compositions is several times that of pure aluminum, which confirms the good mechanical properties. According to our previous research, the comprehensive mechanical performance has reached the highest known level in related carbonaceous nanomaterial‐reinforced aluminum matrix composites. In the two preferred preparation routes, the mechanical properties of the composites basically increase gradually with the increase in the Mg addition. Regrettably, the elongation has shown a downward trend. Since ductility is also the primary component of the comprehensive mechanical properties, we need to strictly consider the balance of strength and ductility. As such, a 5.5% Mg addition can achieve the relatively ideal mechanical responses.Since our strategy is to conquer the antagonism between strength and corrosion resistance in graphene‐reinforced aluminum matrix composites, we tested the mechanical performances of the DDM composites with four different preparation routes. Figure 25 the2.33+ at the electrical double layer formed at the corrosion surface, while the second is the relaxation and exfoliation of the corrosion products at the exposed surface. The passive oxide film of pure aluminum is porous in the chlorine‐containing solution, resulting in low electrical resistance. The two response loops of pure aluminum are prone to be separated. In contrast, due to the dense protective film formed by the (F)‐GNPs and heteroatom‐doped oxide film according to our previous research, the electrochemical responses in this paper cannot be distinguished, indicating that the impedance value of the doped oxide film is relatively high and shows great protection against corrosion. Besides, the larger diameter of the capacitive loop means a bigger electrochemical impedance. The corrosion resistance of MF+A DDM composites gradually increases and decreases over time. It is worth noting that even when the exposure time reaches 1440 h, that is, 2 months later, the diameter of the capacitive loop still does not show a sharp weakening trend, which shows that our composites can show good anti‐corrosion performance under long‐term service conditions. We further conducted EIS tests on various preparation routes, as well as pure aluminum, and fitted them according to the equivalent circuit in Figure Figure  surface.30 The), the corrosion current densities are significantly lower than that of pure aluminum. The Tafel curves only show a slight change over time, indicating that the corrosion rate is not sensitive to time. Figure Figure 33), t2.4 undesirable anti‐corrosion weakening is easily triggered due to the CPA effect, high‐density graphene defects, and agglomeration. The problem of agglomeration can be well solved by the severe plastic deformation process. Our bottom‐up DDM process can realize homogeneous distribution of (F)‐GNPs in the matrix and even further reduce the thickness of graphene through interlayer shear to improve the service performance of graphene. Nevertheless, how the corrosion acceleration caused by the CPA effect and high‐density defects can be avoided or even achieved the CSA effect is still puzzling. We therefore performed elemental distribution analysis and characterization of the electrochemical corroded surface, as shown in Figure  At the junction between F‐GNPs, due to the existence of the F‐doped oxide film, these F‐GNPs form a tight bond with the oxide film, thereby avoiding the problem of the decrease in the diffusion distance of the corrosive medium caused by the microscopic defects. In a word, this layer composed of F‐GNPs and F‐doped oxide film and coated by Mg alloying finally realizes the CSA effect with a low corrosion rate better than pure aluminum over long‐term service.Electrochemical results and hydrogen evolution rate measurements seem to prove that the corrosion resistance of the DDM composites is higher than that of pure aluminum. However, according to Sun et al.,34 und2O3 (0001) slab, the F‐GNP@Al2O3 (0001) slab, and the F‐GNP+Mg@Al2O3 (0001) slab, respectively. The existence of F‐GNP significantly improves the work function of the composites, indicating that the difficulty of electrons removed from the matrix is increased. Moreover, although F‐GNP without Mg alloying seems to have a higher work function, we can see from the electron density difference image  calculation to clarify the electron transfer characteristics of the protective layer containing Mg‐alloyed F‐GNPs. The detailed calculation method can be found in Supplementary Information ncreased.37 More Figure  that the3We designed a novel bottom‐up strategy to achieve charming carbonaceous nanomaterial‐reinforced aluminum matrix composites with a better balance between corrosion resistance and mechanical performances. This strategy is to conquer the corrosion promotion activity caused by heterogeneous corrosion potential, graphene defects, and agglomeration. Mg‐alloyed fluorinated graphene nanoplatelets were introduced via severe plastic deformation and designed preparation routes. Homogeneously dispersed Mg‐alloyed fluorinated graphene nanoplatelets and ultrafine‐grained microstructures contributed to the formation of dense and strong protective film again chloride‐containing aqueous environment. Corrosion suppression activity was discovered in the designed composites. The anti‐corrosion polarization resistance remained 89% of the initial state after 2‐month immersion in chloride‐containing environment, while the ultra‐tensile strength and elongation of 532 ± 39 MPa and 17.3 ± 1.2% were achieved. The microstructural characterization and density function theory calculation confirmed the existence and rationality of the dense anti‐corrosion layer composed of fluorinated graphene nanoplatelets and F‐doped oxide film and coated by Mg alloying. Moreover, this work is significant for conquering the antagonism between mechanical performances and corrosion resistance, which is indeed critical for the design of carbonaceous nanomaterial‐reinforced aluminum matrix composites to realize some desired performance for practical applications.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.