K145Q resulted in stress-induced nuclear TDP-43 foci and loss of TDP-43 function in primary mouse and human-induced pluripotent stem cell (hiPSC)-derived cortical neurons. Mice harboring the TDP-43K145Q mutation recapitulated key hallmarks of FTLD, including progressive TDP-43 phosphorylation and insolubility, TDP-43 mis-localization, transcriptomic and splicing alterations, and cognitive dysfunction. Our study supports a model in which TDP-43 acetylation drives neuronal dysfunction and cognitive decline through aberrant splicing and transcription of critical genes that regulate synaptic plasticity and stress response signaling. The neurodegenerative cascade initiated by TDP-43 acetylation recapitulates many aspects of human FTLD and provides a new paradigm to further interrogate TDP-43 proteinopathies.TDP-43 proteinopathies including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders characterized by aggregation and mislocalization of the nucleic acid-binding protein TDP-43 and subsequent neuronal dysfunction. Here, we developed endogenous models of sporadic TDP-43 proteinopathy based on the principle that disease-associated TDP-43 acetylation at lysine 145 (K145) alters TDP-43 conformation, impairs RNA-binding capacity, and induces downstream mis-regulation of target genes. Expression of acetylation-mimic TDP-43 TDP-43 proteinopathies are characterized by the dysfunction and aggregation of transactivation response element DNA-binding protein of 43 kDa (TDP-43), with ~95% of all amyotrophic lateral sclerosis (ALS) and ~50–60% of all frontotemporal lobar dementia (FTLD-TDP) cases harboring TDP-43 pathology . There iTARDBP mutations that are causative for FTLD/ALS  and gene transcription . StructuFTLD/ALS . In sporFTLD/ALS . Both TDFTLD/ALS .TARDBP mutations that disrupt RNA binding, and thereby may act in a similar manner to TDP-43 acetylation, have been identified in FTLD-TDP patients  (K145Q) is sufficient to neutralize the positive charge, disrupt RNA binding, and induce several hallmarks of TDP-43 pathology in vitro , such as phosphorylation, acetylation, and ubiquitination may play a role. PTMs modulate TDP-43’s biochemical properties leading to conformational changes, modulation of nucleic acid-binding affinity, regulation of liquid–liquid phase separation, and propensity to form insoluble TDP-43 aggregates, all of which are disease-associated phenomena . Among td K181E) , supportin vitro , supportTardbp gene, thereby generating acetylation-mimic TDP-43K145Q knock-in mice, which enabled us to investigate the pathophysiological impacts of an aberrant TDP-43 PTM while leaving native upstream and downstream genomic elements intact. Using mouse cortical neurons, human hiPSC-derived cortical neurons, and aged cohorts of TDP-43K145Q homozygous mice, we found that acetylation-mimic TDP-43K145Q-induced nuclear TDP-43 foci and cytoplasmic TDP-43 accumulation, which coincided with several disease-associated and loss-of-function measures including widespread transcriptome and splicing alterations. Finally, we observed prominent FTLD-like cognitive and behavioral deficits in acetylation-mimic TDP-43 mice that correlated with biochemical and splicing alterations in affected brain regions. Our study supports lysine acetylation of TDP-43 as a driver of dysfunction in sporadic TDP-43 proteinopathies.Here, we used CRISPR/Cas9 genome editing to introduce a K145Q substitution into the endogenous mouse wt), acetylation-deficient (TDP-43K145R), and acetylation-mimic (TDP-43K145Q) variants to overexpress TDP-43 species in neurons, and then examined their subcellular localization by immunofluorescence microscopy. In the absence of acute cellular stress, most neurons overexpressing TDP-43K145Q showed distinct nuclear foci that were more prominent compared to TDP-43wt or TDP-43K145R constructs  mRNA splice variants, as a more sensitive endogenous indicator of loss of TDP-43-dependent splicing function. Functional nuclear TDP-43 results in the production of a mature spliced Sort1 mRNA transcript, however, in the setting of TDP-43 depletion or loss of function, TDP-43 is unable to repress the inclusion of exon 17b, generating a longer Sort1+ex17b transcript variant , acetylation-deficient TDP-43 (TDP-43K145R.2 and TDP-43K145R.12), or unmodified TDP-43   . We confquencing , verifiequencing , and thequencing . We thenlization . All hiPlization . Followiive foci . In compK145Q cortical neurons show more robust functional deficits compared to the mouse neuron data above. We again employed RT-qPCR to measure levels transcripts associated with TDP-43 loss of function, beginning with TARDBP as an indicator of impaired TDP-43 autoregulation. In contrast to mouse neurons, we observed an approximately twofold increase TARDBP mRNA in TDP-43K145Q.12 and TDP-43K145Q.18 neurons and a slight but significant reduction in TARDBP mRNA in TDP-43K145R.12 neurons  was reduced about 50% in both acetylation-mimic TDP-43K145Q lines but unchanged in TDP-43K145R.2 neurons  failed to detect the abnormal splice product in TDP-43wt and TDP-43K145R.2 lines, with only low levels observed in some TDP-43K145R.12 samples. In contrast, both TDP-43K145Q lines dramatically accumulated the UNC13A cryptic exon containing mRNA (UNC13A WT) were similar across all five lines  buffer-extracted) and insoluble (Urea-extracted) protein fractions. TDP-4310 locus  -TDP-43 -TDP-43  and mode)-TDP-43  in the s animals . We thuswt or TDP-43KQ/KQ mice, representing the regions and timepoints with the most striking biochemical and behavioral and abnormalities and identified nearly 400 differentially expressed genes (DEGs) in each brain region in TDP-43KQ/KQ mice compared to TDP-43wt, after correcting for underlying batch effects. As expected by acetylation-induced loss of TDP-43 function and subsequent autoregulation, the Tardbp transcript was increased , gliogenesis , and myelination . Sema5b was the most dramatically reduced transcript in the hippocampus, and the third most highly reduced in the cortex  were selectively downregulated in the neocortex, while trans-synaptic signaling and ion transport mechanisms  were decreased in the hippocampus.We then clustered the DEGs based on their up- or downregulation and the brain region affected , which re cortex  with a tSesn1, Nrros, Plat, Klf15) and many apoptotic regulators  in both the hippocampus and neocortex, along with an over-representation of GO terms related to metabolism, localization, and cell adhesion . Interestingly, while genes associated with trans-synaptic signaling were decreased in the hippocampus, another set of genes involved in this same pathway was upregulated in the cortex   .KQ/KQ mice, we sought to draw parallels between the TDP-43KQ/KQ mouse and the human FTLD-TDP transcriptome. Comparison of the DEGs in TDP-43KQ/KQ mice to the mouse orthologs of those found in FTLD-TDP frontal or temporal cortex tissue , with 81.7% of loci containing at least one cryptic splice junction, and 29.8% containing two cryptic splice junctions   . Of all identified DSGs, the most substantially altered transcript was Sort1, encoding the Sortilin-1 (Sort1) protein. We noticed that exclusion of a Sort1 3′ exon was reduced by 55.9% in TDP-43KQ/KQ cortex and by 57.0% in the hippocampus  , transcr, Lrrk2) , and alspocampus , such thSORT1 exon 17b inclusion, this leads to the production of a soluble and putatively toxic SORT1 variant that is increased in FTLD-TDP patients , and a primer pair that recognizes all Sort1 variants  on tissues isolated from TDP-43KQ/KQ or WT mice  and regulates endosomal/lysosomal function through a pathway that is genetically linked to FTLD-TDP . Becausepatients . To conf WT mice . We founeocortex  and hippeocortex  of TDP-4with age .Sort1+ex17b transcript in TDP-43KQ/KQ mice leads to the generation of a distinct Sort1 protein product, we immunoblotted hippocampus and cortex homogenates and identified a higher molecular weight Sort1 variant in TDP-43KQ/KQ compared to WT mice . Using a combination of molecular, cellular, and transcriptomic readouts combined with in vivo behavioral assays, we provide evidence that aberrant TDP-43K145Q recapitulates key hallmarks of TDP-43 proteinopathies, including altered TDP-43 solubility and localization, impaired autoregulation, and abnormal splicing function. Taken together, our findings suggest that TDP-43 acetylation-induced dysfunction likely contributes to disease progression.Here, we developed novel neuronal and mouse models of RNA-binding deficient acetylation-mimic TDP-43  and likely other factors. Based on our original description of these TDP-43 foci, and several more recent studies of acetyl-mimic TDP-43 variants  The levels of insoluble phosphorylated TDP-43 in the spinal cord have not crossed a critical threshold required for functional decline. (2) There is a time-dependent delay in the accumulation of TDP-43 and the onset of motor symptoms that could emerge beyond 18 months, which will require additional studies in aged cohorts of mice. (3) Spinal motor neurons in mice may possess some intrinsic resilience that suppresses functional decline. (4) It is possible that TDP-43KQ/KQ mice do indeed show subtle motor deficits that could be detected by more sensitive measures, such as electromyography-based techniques  and altered splicing of TDP-43 targets   . Similar/KQ mice . Hippoca/KQ mice . This al/KQ mice , the K14nockdown . This isutations , and TDPKQ/KQ mouse brains. Many up- and downregulated transcripts identified in acetylation-mimic mice were also differentially expressed in FTLD-TDP human tissue  was significantly altered in TDP-43KQ/KQ mice, and abnormalities in LRP8 can cause learning and memory defects, likely by disrupting Reelin-mediated synaptic plasticity and long-term potentiation (LTP)  protein that regulates LTP and synaptic plasticity via the production of nitric oxide  and other significantly altered splice variants  have been linked to AD and other neurodegenerative diseases  . Similaric oxide , showed diseases . Our traGiven the correlation between TDP-43 acetylation and FTLD-TDP presented in this study, it is perhaps surprising that acetylated TDP-43 (detected with an acetylation-specific ac-K145 antibody) is found within ALS spinal cord inclusions but has not yet been detected in FTLD-TDP cortex . HoweverK145Q mutation results in an age-dependent, dementia-like phenotype characterized by signatures of FTLD, including progressive cognitive deficits, TDP-43 mislocalization and phosphorylation, impaired autoregulation, and prominent RNA dysregulation. TDP-43 acetylation affected many known FTLD-associated pathways, including STMN2, SORT1, and stress response signaling, while also pinpointing new putative pathways as mediators of disease pathogenesis. By developing models of TDP-43 acetylation, we expand our understanding of sporadic TDP-43 proteinopathies and provide new avenues to identify therapeutics that target the pathogenic mechanisms underlying TDP-43 proteinopathies.In conclusion, disrupting RNA binding with an acetylation-mimic TDP-43Mice were housed in ventilated microbarrier cages on racks providing high efficiency particulate air (HEPA)-filtered air supply to each cage. Animals were kept on a 12-hr light–dark cycle with access to food and water ad libitum. All animal husbandry, experiments, and procedures were performed in strict compliance with animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill (Protocol #21.257).wt/KQ breeding pairs. Timed pregnant females at embryonic days 15–16 were lethally anesthetized with isoflurane. The abdominal cavity was opened, and the uterus incised to remove the placentas and embryonic sacs, which were washed briefly in ice-cold 70% ethanol and then placed in cold 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Bank’s balanced salt solution (HBSS). The embryos were transferred into a 10-cm dish containing cold HBSS, the embryos removed from the amniotic sac, and the brains were extracted from the cranium. For TDP-43wt/KQ dissections, process was paused for approximately 2 hr to permit genotyping of the fetuses, during which the brains were stored at 4°C protected from light in a Hibernate-E (BrainBits NC9063748) solution supplemented with B27 (Gibco 17504044) and GlutaMAX (Thermo Fisher 35050061). After genotyping, if applicable, the cerebral cortices from each brain were isolated under a stereomicroscope, pooled by genotype, minced with forceps, and digested for 30 min at 37°C in a filter-sterilized HBSS solution containing 20 U/ml papain , 1 mM EDTA, 0.2 mg/ml l-cysteine, and 5 U/ml DNAse (Promega M6101). The enzyme solution was removed, and the digested tissue was washed twice with sterile HBSS. Warm plating media  containing 5 U/ml DNAse, was added and the tissue was dissociated mechanically using a P1000 pipette. The resulting cell suspension was spun down for 5 min at 1.5 rcf to pellet the cells, resuspended in plating media, and filtered through a 40-mm cell strainer. Cells were counted using a hemocytometer and plated onto poly-D-lysine (PDL)-coated 12-well tissue culture plates (Corning 356470) at 300 K cells/well (for RNA extraction) or 96-well glass bottom black wall plates (Cellvis P96-1.5H-N) at 30 K/well (for immunofluorescence and microscopy). 16–24 hr after plating, all plating media was removed and replaced with neuronal cell media . Cultures were incubated at 37°C, 5% CO2 and 95% humidity with half-media exchanges every 3 days for the duration of all experiments.Murine primary cortical neurons were performed using wild-type C57Bl/6 mice (Charles River) or TDP-43http://n2t.net/addgene:24129; RRID:Addgene_24129)  . To genee_24129)  and a pc plasmid  as the tg for 10 min. Lentiviral particles were purified using a double-sucrose gradient method. Briefly, the supernatants were loaded onto a 70–60−30–20% sucrose gradient and centrifuged at 70,000 × g for 2 hr at 17°C . The 30–60% fraction containing the viral particles was retrieved, resuspended in phosphate-buffered saline (PBS), filtered with a 0.45-μm filter flask before loaded onto a 20% sucrose cushion, and centrifuged a second time at 70,000 × g for 2 hr at 17°C. The supernatants were carefully discarded, and the viral particles present in the pellet were resuspended in PBS, aliquoted and stored at −80°C.Lentiviral production was performed by co-transfecting  37.5 μg lenti-plasmid with 25 μg psPAX2, 12.5 μg VSVG, and 6.25 μg REV for each 15 cm dish of lenti-X 293T cells (Takara 632180) and 3 dishes of cells were used for each lentiviral production. Three days after transfection, culture media were collected and centrifuged at 2000 × Lentivirus aliquots were evaluated for neuronal transduction ability prior to each experiment and diluted to approximately equal concentrations of effective virus. Lentiviral transductions were performed on DIV10 for a DIV14 harvest or DIV24 for a DIV28 harvest. In brief, lentiviruses were diluted to a 2× concentration in neuronal media, and then added to plates via half-media exchange. Twenty-four hours after viral transduction, all virus-containing media was removed and replaced with 50% fresh neuronal media and 50% conditioned media from plates containing WT non-transduced neurons. Neurons were then cultured to DIV14 or DIV28 as the experiment required.The HPC26 iPSCs were maintained on Matrigel-coated dishes (Corning 354480) in StemFlex Medium (Thermo Fisher Scientific A3349401) and passaged every 3–4 days with 0.5 mM EDTA dissociation solution as previously described .5 iPSCs were electroporated on the Neon electroporation system (Thermo Fisher Scientific) with recombinant protein complexes made of Cas9 v2 protein (Thermo Fisher Scientific A36498), 900 ng sgRNA-TARDBP and 2700 ng of single-stranded donor oligonucleotide-K145Q or donor oligonucleotide-K145R  supplemented with 10 µM Y27632 (PeproTech 1293823) and cultured in suspension on an orbital shaker at 37°C and 5% CO2. After 48 hr, cells were dissociated with Accutase and 3 × 105 cells differentiated into NPCs as neutrosphere in StemScale PSC medium supplemented with 10 µM Y27632 (PeproTech 1293823) and 1.5 µM CHIR99021 (PeproTech 2520691), 10 µM SB431542 (PeproTech 3014193) and 50 nM LDN-193189 (Sigma SML0755). CHIR99021 was removed after 24 hr and cells were cultured for 10 days with daily medium changes and neurospheres dissociated at 1:3 ratio twice a week. Then NPCs were expanded for 7 days in the presence of 20 ng/ml FGF (Peprotech 100-18B) in Neuronal Expansion Medium [1:1 Advanced Dulbecco's Modified Eagle Medium (DMEM)/F12 (12634028) and Neural Induction Supplement (A1647801)]. NPCs were plated for maturation on PDL/Laminin-coated plates or coverslips in cortical neuron maturation medium  supplemented with 10 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF), and 10 µM N-[N-- L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Seventy-two hours after plating, cells were treated with 1 µg/ml Mitomycin C for (Sigma M5353) for 1 hr. Cellular identity was assessed specific markers of neuronal progenitor , and mature neurons   . All rea, neurons were treated for 2 hr with 200 µM sodium arsenite (NaAsO2) or vehicle (molecular biology grade water). Following treatment, neurons were fixed with 4% paraformaldehyde (PFA) in 1× PBS and washed with 1× PBS. Neurons were permeabilized with 0.3% Triton X-100 in 1× PBS for 15 min at RT, blocked in an 8% normal goat serum (NGS) 0.2% Triton X-100 in 1xPBS solution for 1–2 hr at RT, and incubated with primary antibodies (On DIV14 (mouse primary neurons) or mature hiPSC-derived cortical neurons (~DIV50)tibodies  diluted tibodies  diluted Mouse primary cortical neurons in 96-well plates were visualized via automated fluorescence microscopy using EVOS M7000 Imaging System (Thermo Fisher AMF7000) equipped with Olympus ×20/0.75 NA UPlanSApo and Olympus ×40/0.95 NA UPlanSApo objectives, and the following filter cubes: DAPI , GFP , Texas Red , and Cy5.5 . Images were acquired in an automated fashion, using DAPI fluorescence as the autofocus substrate, taking 16–32 images per well of 2–3 wells per experimental condition. Automated quantitative image analysis was performed using CellProfiler 4.0  to measuTardbp K145 codon were identified using Benchling software . Three guide RNAs were selected for activity testing. Guide RNAs were cloned into a T7 promoter vector followed by in vitro transcription and spin column purification . Guide RNAs were tested for cleavage activity by in vitro cleavage assay. Each guide RNA was incubated with Cas9 protein (UNC Protein Expression and Purification Core Facility) and PCR-amplified guide RNA target site. The products were run on an agarose gel for analysis of target site cleavage. Based on this assay, the guide RNA selected for genome editing in embryos was Tardbp-g79T  containing (1) 20 ng/µl Cas9 mRNA, 10 ng/µl g79T guide RNA, and 50 ng/µl donor oligonucleotide (founder #9) or (2) 20 ng/µl Cas9 mRNA, 400 nM Cas9 protein, 10 ng/µl g79T guide RNA, and 50 ng/µl donor oligonucleotide . Injected embryos were implanted in recipient pseudopregnant females and resulting pups were screened by PCR and sequencing for the presence of the desired mutant allele. Founder lines were propagated as heterozygotes and regularly sequenced to confirm retention of the K145Q and silent mutations.Tardbp locus.We assessed the likelihood of off-target mutations using prediction algorithms to ensure low likelihood of off-target effects. The Benchling MIT off-target score for Tardbp-g79T is 72 , and theTardbp locus was performed with ApexRed master mix (Genesee Scientific 42-138B) and TDPKQ F′ and TDPKQ R′ primers  crossed by a grid of photobeams . Counts were taken of the number of photobeams broken during the trial in 5-min intervals, with separate measures for locomotor activity  and vertical rearing movements. Time spent in the center region was used as an index of anxiety-like behavior.Training. On day 1, mice were placed in the test chambers, contained in sound-attenuating boxes. The mice were allowed to explore the novel chambers for 2 min before presentation of a 30-s tone (80 dB), which co-terminated with a 2-s scrambled foot shock (0.4 mA). Mice received two additional shock-tone pairings, with 80 s between each pairing, and were removed from the test chambers 80 s following the third shock. Context- and cue-dependent learning. On day 2, mice were placed back into the original conditioning chambers for a test of contextual learning. Levels of freezing (immobility) were determined across a 5-min session. On day 3, mice were evaluated for associative learning to the auditory cue in another 5 min session. The conditioning chambers were modified using a Plexiglas insert to change the wall and floor surface, and a novel odor (dilute vanilla flavoring) was added to the sound-attenuating box. Mice were placed in the modified chamber and allowed to explore. After 2 min, the acoustic stimulus (an 80-dB tone) was presented for a 3-min period. Levels of freezing before and during the stimulus were obtained by the image tracking system.Mice were evaluated for conditioned fear using the near-infrared image tracking system . The procedure had the following phases: training on day 1, a test for context-dependent learning on day 2, and a test for cue-dependent learning on day 3. Grip strength was evaluated using precision force gauges. Measures were based on paw grasp of a metal grid by a mouse gently pulled by the tail. Digital force meters  were mounted on an acrylic platform (San Diego Instruments), with two different grids: a left-hand grid for the front paws, and a right-hand grid for all-four paws. Each grid connected to a force transducer, which provided measures of peak force (newtons). Each mouse was given three trials, with at least 1 min between each trial. Each trial had two components: front-paw measures from the left-hand grid, immediately followed by all-four-paw measures from the right-hand grid.Subjects were tested for motor coordination and motor learning on an accelerating rotarod . For the first test, mice were given three trials, with 45 s between each trial. Two additional trials were given 48 hr later. Rpm (revolutions per minute) was set at an initial value of 3, with a progressive increase to a maximum of 30 rpm across 5 min . Measures were taken for latency to fall from the top of the rotating barrel.The water maze was used to assess spatial and reversal learning, swimming ability, and vision. The water maze consisted of a large circular pool (diameter = 122 cm) partially filled with water , located in a room with numerous visual cues. The procedure involved three separate phases: a visible platform test, acquisition in the hidden platform task, and a test for reversal learning (an index of cognitive flexibility).Each mouse was given 4 trials per day, across 2 days, to swim to an escape platform cued by a patterned cylinder extending above the surface of the water. For each trial, the mouse was placed in the pool at one of four possible locations (randomly ordered), and then given 60 s to find the visible platform. If the mouse found the platform, the trial ended, and the animal was allowed to remain 10 s on the platform before the next trial began. If the platform was not found, the mouse was placed on the platform for 10 s, and then given the next trial. Measures were taken of latency to find the platform and swimming speed via an automated tracking system (Noldus Ethovision).Following the visible platform task, mice were tested for their ability to find a submerged, hidden escape platform (diameter = 12 cm). Each mouse was given 4 trials per day, with 1 min per trial, to swim to the hidden platform. The criterion for learning was an average group latency of 15 s or less to locate the platform. Mice were tested until the group reached criterion, with a maximum of 9 days of testing. When the group reached criterion (on day 5 in the present study), mice were given a 1-min probe trial in the pool with the platform removed. Selective quadrant search was evaluated by measuring the number of crosses over the location where the platform (the target) had been placed during training, versus the corresponding area in the opposite quadrant. Following the acquisition phase, mice were tested for reversal learning, using the same procedure as described above. In this phase, the hidden platform was re-located to the opposite quadrant in the pool. As before, measures were taken of latency to find the platform. On day 5 of testing, the platform was removed from the pool, and the group was given a probe trial to evaluate reversal learning.This procedure was used to assess auditory function, reactivity to environmental stimuli, and sensorimotor gating. The test was based on the reflexive whole-body flinch, or startle response, that follows exposure to a sudden noise. Measures were taken of startle magnitude and PPI, which occurs when a weak prestimulus leads to a reduced startle in response to a subsequent louder noise.Mice were placed into individual small Plexiglas cylinders within larger, sound-attenuating chambers. Each cylinder was seated upon a piezoelectric transducer, which allowed vibrations to be quantified and displayed on a computer (San Diego Instruments SR-Lab system). The chambers included a ceiling light, fan, and a loudspeaker for the acoustic stimuli. Background sound levels (70 dB) and calibration of the acoustic stimuli were confirmed with a digital sound level meter (San Diego Instruments). Each session began with a 5-min habituation period, followed by 42 trials of 7 different types: no-stimulus (NoS) trials, trials with the acoustic startle stimulus  alone, and trials in which a prepulse stimulus  occurred 100 ms before the onset of the startle stimulus. Measures were taken of the startle amplitude for each trial across a 65 ms sampling window, and an overall analysis was performed for each subject’s data for levels of PPI at each prepulse sound level  × 100]).At each end point, mice were anesthetized deeply with isoflurane and euthanized via rapid decapitation. The hippocampus and neocortex were dissected out of the brain on a cold surface using clean surgical tools, placed into cryo-safe nuclease-free microcentrifuge tubes, flash frozen in liquid nitrogen (LN2), and stored at −80°C until processing. Frozen tissue was then pulverized in LN2-cooled stainless steel Cryo-Cups using cold stainless-steel pestles and transferred into cold nuclease-free microcentrifuge tubes and immediately stored at −80°C until use.Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride (TPCK) (Sigma T7254), 1 µg/ml Trypsin inhibitor (Sigma T9003), 1 µg/mL N-p-Tosyl-l-phenylalanine chloromethyl ketone (TLCK) (Sigma T4376), 0.67 µg/ml trichostatin A, 10 mM nicotidamide, 1 mM phenyline thanosulfyl fluoride, 1 mM phenylmethylsulfonyl fluoride]. The solution was homogenized by sonication and centrifuged at 4°C for 45 min at 18,000 × rcf. The supernatant was removed and saved as the RIPA-soluble (soluble) protein fraction. The pellet was resuspended in RIPA buffer with inhibitor mixture, sonicated, and centrifuged as described above, and the supernatant discarded. The resulting pellet of RIPA-insoluble material was resuspended in approximately 1 µl/µg of Urea buffer  with the inhibitor mixture (as above), sonicated to homogenize, and centrifuged at RT for 45 min at 21,000 × rcf. The resulting supernatant of RIPA-insoluble, urea-soluble protein fraction was saved as the ‘insoluble’ protein fraction.All steps of protein fractionation were performed on ice unless otherwise indicated. For solubility fractionation, pulverized tissue was suspended in 5 µl/mg of ice-cold 1× RIPA buffer  containing a mix of protease, phosphatase, and deacetylase inhibitors [1 µg/ml Peptstatin A (Sigma P4265), 1 µg/ml Leupeptin (Sigma L2023), 1 µg/ml Nuclear and cytoplasmic soluble proteins were isolated using the Thermo Fisher NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific 78835) per the manufacturer’s instructions, with the addition of the protease, phosphatase, and deacetylase inhibitors (as above) to each buffer.All soluble protein fractions were analyzed by Bicinchoninic acid (BCA) assay (Thermo Scientific 23225) to determine protein concentration. Equal quantities of protein per sample were run onto 4–20% Tris-Glycine SDS–polyacrylamide gel electrophoresis gels (Bio-Rad 5671095) under reducing conditions and then transferred onto nitrocellulose membranes. Total transferred protein (TTP) was assessed using PonceauS  protein stain on nitrocellulose membranes per the manufacturer’s instructions. Membranes were washed three times in Tris-buffered saline with 0.1% Tween-20 (TBST), once in 1x Tris-buffered saline (TBS), and blocked in 2% nonfat milk in 1× TBS for 1–2 hr at RT. Membranes were incubated with primary antibodies  diluted Mouse brain tissue was isolated, flash frozen, and pulverized as described above. Approximately 20 mg of pulverized brain tissue per sample was used to isolate RNA. 1 ml of TRIzol (Invitrogen 15596018) was added to each nuclease-free Eppendorf tube containing pulverized tissue, and tissues were lysed via trituration with a P1000 pipette tip, followed by trituration with a 21-G and then a 25-G needle on a 1-ml syringe. Samples were centrifuged at 4°C for 5 min at 10,000 rcf to remove tissue debris. The supernatant was removed and added to a new tube containing 200 µL of chloroform, which were mixed by inversion and cooled on ice for 5 min. Samples were centrifuged at 4°C for 15 min at 10,000 rcf, and the upper aqueous phase containing RNA was transferred into a new tube, followed by the addition of 100 µl of isopropanol to precipitate RNA and overnight incubation at −20°C. The next day, samples were centrifuged at top speed  for 20 min at 4°C to pellet the RNA. The pellets were washed twice with 1 ml of ice-cold 70% molecular biology grade ethanol and then air dried for 15 min at RT. The RNA pellets were resuspended in 20 µl of nuclease-free water. On-column DNAse digestion and RNA clean-up were then performed using the QIAGEN RNeasy mini kit  per the manufacturer’s instructions, followed by elution in nuclease-free water.RNA concentration was assessed using Qubit RNA BR Assay Kit (Q10210) and a Qubit 3.0 Fluorometer. RNA integrity was assessed using an Agilent 4150 TapeStation system and associated RNA screen tape reagents (Agilent 5067-5576). Only samples with an estimated RNA integrity number (RIN) ≥7.0 were sent to the New York Genome Center (NYGC) for bulk total RNA sequencing. Upon receipt at NYGC, RNA samples were re-evaluated for quantification and integrity, using Ribogreen and Fragment Analyzer 5300, respectively. Total RNA libraries were prepped using Kapa Total library prep with Ribo-Erase, in accordance with manufacturer recommendations. Briefly, 500 ng of total RNA was used for ribosomal depletion and fragmentation of total RNA. Depleted RNA underwent first- and second-strand cDNA synthesis. cDNA was then adenylated, ligated to Illumina sequencing adapters, and amplified by PCR (using nine cycles). The cDNA libraries were quantified using Fragment Analyzer 5300  kit FA-NGS-HS (Agilent DNF-474-1000) and Spectramax M2 (Molecular Devices) kit Picogreen (Life Technologies P7589). Libraries were sequenced on an Illumina NovaSeq sequencer, using paired end sequencing (2 × 100 bp cycles) to a depth of >75 M read pairs per sample.Raw reads were then trimmed and filtered of adapter sequencing using cutadapt  and filtadj < 0.05) were then separated into 6 groups based on their intersections between the two brain regions using UpSetR v1.4.0  experiments involving mouse primary cortical neurons or human iPSC-derived cortical neurons, cells were washed with 1× PBS, harvested from the culture dish, and RNA was extracted using the QIAGEN RNeasy mini kit  with on-column DNAse digestion per the manufacturer’s instructions. RNA concentration was determined using a NanoDrop 2.0 spectrophotometer, 250 ng to 1 µg of RNA was used to generate cDNA using Applied Biosystems High-capacity RNA-to-cDNA kit (#4387406) per the manufacturer’s instructions. cDNA was generated from 250 to 500 ng of RNA using Applied Biosystems High-capacity RNA-to-cDNA kit (#4387406) per the manufacturer’s instructions. Quantitative PCR was performed on a QuantStudio 6 Real Time Polymerase Chain Reaction (PCR) system  using PowerUp SYBR Green master mix (Applied Biosystems A25776). The PCR phase consisted of 40 cycles of 15 s at 95°C and 1 min at 60°C. Forward and reverse primer sequences for imerBank  and syntMice were transcardially perfused with 1× PBS followed by 4% PFA, and brains were removed and post-fixed for 24 hr in 4% PFA. Brains were cryoprotected in 15% sucrose in 1× PBS for 24 hr followed then 30% sucrose in 1× PBS for 48 hr, and then embedded in Fisher Tissue-Plus OCT compound (4585). Forebrain (~Bregma +1.78 mm), midbrain (~Bregma +0.50 mm), and hippocampal (~Bregma –1.94 mm) 10 mm cryosections were collected onto Superfrost Plus charged slides using a Leica CM1950 cryostat.Slides were acclimated to RT and rinsed in distilled water. They were then incubated in 70% ethanol for ~60 hr followed by 95% ethanol for 30 min. Tissues were stained with 0.1% Luxol Fast Blue Solution overnight at 60°C, rinsed in distilled water, and differentiated as needed by 0.05% Lithium Carbonate followed by 70% ethanol. They were then counterstained with 0.1% Cresyl Echt Violet, rinsed in distilled water, and differentiated again by 95% ethanol. Finally, slides were dehydrated in 100% ethanol, cleared in Xylene, and coverslipped with DPX mountant (Millipore-Sigma 44581).Slides were acclimated to RT and rinsed in distilled water. Heat-induced epitope retrieval was performed at 120°C using pH 6.0 buffer . The tissues were then blocked in 10% NGS for 1 hr. Primary antibodies were applied overnight at 4°C: Mouse Anti-NeuN (clone A60) conjugated to Alexa Fluor 555 , and Rabbit Anti-TDP-43  or Rabbit anti-Iba1  and mouse anti-GFAP conjugated to Alexa Fluor 488  . After rBrain sections stained with Cresyl Violet (CV) and brain sections labeled with Iba1 and GFAP anatibodies were imaged on a Nikon Eclipse Ti2 widefield microscope equipped with a Nikon DS-Fi3 CMOS color camera (for CV imaging) and a pco.edge 4.2Q High QE sCMOS camera  using a ×20/0.5 NA Plan Fluor objective and NIS-Elements software . Immunofluorescently labeled brain sections were imaged on a Leica SP8X Falcon confocal microscope equipped with hybrid GaAsP detectors using a ×40/1.30 NA Plan Apochromatic (oil) objective and Leica Application Suite X Life Sciences software . Cell counting (density of CV- and NeuN-positive cells), as well as TDP-43 fluorescence intensity, TDP-43 localization measurements, GFAP fluorescence intensity and area, and Iba1 fluorescence intensity and area were performed using CellProfiler . Prior thttps://www.graphpad.com. For datasets with n > 10, outliers were identified and removed (if any) using the ROUT method , as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.Comments on the latest version:The authors have addressed most concerns. The additional analysis demonstrating a lack of neuron loss is quite different from the original study -- it is good that the authors pursued this question. In addition, new data focusing on native TDP-43 splice targets, rather than the splicing reporter, are excellent. This paper extends prior work demonstrating the importance of K145 acetylation of TDP-43 as a post-translational modification that impacts its RNA-binding capacity and may contribute to pathology in FTLD-ALS. The main strengths of this paper are the generation of a novel mouse model, using CRISPR gene editing, in which an acetylation-mimetic mutation (K to Q) is introduced at position 145. Behavioral, biochemical, and genetic analyses indicate that these mice display phenotypes relevant to TDP-43-associated disease and will be a valuable contribution to the field. Numerous experimental models are phenotyped in this manuscript including mouse neurons, iPSC-derived human neurons, knock-in mice, and knock-in iPSCs. Expression of acetylation-mimic or acetylation-null TDP-43 protein is achieved either with overexpression or CRISPR-Cas9-based knock-in. A complex phenotype is observed including loss of TDP-43 function  and a gain of TDP-43 (increased insoluble TDP-43 protein). These correlate with downstream neurobehavioral changes which are most consistent with a cortical/hippocampal phenotype without a motor phenotype. Post-translational modifications of disease-associated proteins are thought to contribute to neurodegenerative disease pathogenesis, and this study succeeds in demonstrating that TDP-43 acetylation results in downstream molecular and behavioral phenotypes.TDP-43 acetylation is a post-translational modification that is known to be associated with TDP-43 inclusions that are characteristic of human diseases. An important strength is the rigorous use of multiple different experimental models  with overall consistent results. Moreover, multiple orthogonal endpoints are presented including histology/cytology/immunostaining, biochemistry, molecular biology, and neurobehavioral assays. As TDP-43 acetylation is known to block RNA binding, these novel cellular and mouse models represent interesting albeit complex tools to study the functional consequences of a partial loss of function. As TDP-43 regulates its own expression (i.e. autoregulation), the complexity lies at least in part due to the loss of RNA binding leading to a functional loss of TDP-43 function which includes the increased expression of the TARDBP transcript and TDP-43 protein.Conceptually, there is a disconnect in that the mouse model exhibits primarily a cortical/hippocampal phenotype more akin to frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), while TDP-43 acetylation is only seen in ALS tissues and not in FTLD-TDP tissues because most of the pathologic protein in the latter is N-terminally truncated (i.e. the acetylation site is not present). That being said, there is no mouse model which completely and faithfully recapitulates the human disease, and this mouse model avoids overt overexpression  and avoids the use of synthetic/artificial mutation .This revision addresses most of my prior comments including documenting the lack of neurodegeneration in this model, the use of more appropriate statistical methods, and the use of more robust/quantitative aberrant splicing measures. The one thing which would still be helpful is sequencing the top predicted off target genomic loci for their various CRISPR'd models irrespective of whether these loci are exonic or noncoding. Having actual sequencing verification of the lack of mutations at these loci is preferable over relying only on computational likelihood estimates. The following is the authors’ response to the original reviews.Reviewer #11.1 Fig. 1: A good control for these studies would be a TDP-43 variant with an RRM1 mutation that impairs RNA binding, but not an acetylation mimic In our original paper , we already characterized TDP-43 acetylation and employed a complete RNA-deficient mutant (F147/149L), as the reviewer suggested. In that original study, this mutant showed maximal RNA binding-deficiency, and therefore we proposed that acetylation mimic mutations represent a comparable RNA-binding deficient variant.1.2 Fig. 1: time and expression level can influence nuclear TDP-43 puncta formation. It is important that the authors take these into account when measuring puncta number/frequency.All expression levels and transfection/transduction times were identical across samples. We chose the optimal times to express TDP-43 constructs yet minimize toxicity and found that neuronal transduction at DIV10 and arsenite exposure on DIV14 in mature neurons was optimal.1.3 Fig. 2: to accurately refer to the nuclear foci as anisosomes, the authors will need to conduct higher-resolution imaging.We agree with the reviewer and since anisosomes are not well characterized in terms of their relationship to TDP-43 nuclear foci (and may represent only a subset of foci), we have now omitted any mention of anisosomes but instead refer to them in the discussion, where we suggest that TDP-43 K145Q foci may partially represent anisosomes.1.4 Fig. 2D: it seems as though the splicing reporter should have a fluorescence-based readout . Is this the case, and is the ratio informative?We have now removed the splicing reporter data and replaced this with much more robust data showing RT-qPCR of downstream TDP-43 targets including Sortilin-1 .1.5 Line 145: "Overall, these results indicate that a single endogenously expressed acetylation-mimic TDP-43(K145Q) mutation is sufficient to alter TDP-43 localization, induce TDP-43 phase separation, and impair splicing in a murine primary neuron culture model." The authors did not assess phase separation in this study. Moreover, it would be more convincing to assess native splice targets of TDP-43 in K145Q primary neurons, rather than an exogenous splicing reporter.See comment 1.1 above. We have now avoided mentioning phase separation in the main text but mention this as a potential mechanism in the discussion. In addition, we have now evaluated native TDP-43 splice targeted in primary neurons.1.6 Fig. 4A: is the loss of neurons selective for a specific layer or region of the cortex?Since we did not observe any gliosis, we have gone back and completely re-evaluated neuronal loss since the concept of neurodegeneration is a critical question in the TDP-43KQ/KQ mice. We do not find any significant neuronal loss in the homozygous TDP-43KQ/KQ mice .1.7 Fig. 6: The authors suggest that the large majority of splicing changes are direct results of the TDP43(K145Q) mutation and impaired RNA binding by TDP-43. However, without a direct assessment of TDP43(K145Q) target RNAs in comparison to those of TDP-43(WT), this is only an assumption. Moreover, given the fact that RNA-seq was performed in aged animals, the potential for indirect gene expression changes is very high.In our original study , we showed that the K145Q is severely deficient in RNA binding. In this study, we now show strong evidence that many known targets of direct TDP-43 binding are dysregulated, supporting the expected loss of function if TDP-43 K145Q mutation abrogated RNA binding. Although we have not performed direct RNA binding studies to the Sort1 transcript, for example, other studies have clearly indicated that wild-type TDP-43 binds these targets. We infer that loss of function mutations  impact direct targets of TDP-43. Future studies employing RNA-immunoprecipitation followed by RNA sequencing (RIP-seq) could be useful in this regard and will be required to mechanistically address this point.1.8 Sup Fig. 8 is very interesting and suggests that any TDP-43 variant that is unable to bind RNA may lead to upregulation of TDP43 RNA and phenotypes similar to those observed n K145Q animals. This is alluded to in the discussion but never specifically tested.Yes, we agree with this reviewer’s comment. Loss of RNA binding, whether due to acetylation  or otherwise is expected to cause autoregulatory up-regulation of the TARDBP transcript and impact other targets, potentially yielding phenotypes similar to the TDP-43KQ/KQ mice. However, new in vivo models would be needed to prove this point. For example, in the future, we will consider this possibility by characterizing recently identified RNA-binding deficient familial TARDBP mutants .1.9 The authors should also provide some comment or potential explanation for why TDP43(K145Q) animals show no signs of motor neuron disease.We now show a moderate level of TDP-43 aggregation and hyper-phosphorylation in spinal cord of mutant mice in Figure 6 – Figure Supplement 3. We also speculate in the discussion why we observe aspects of TDP-43 dysfunction in spinal cord without overt motor phenotypes up until 18 months old.1.10 Line 79: "However, TARDBP mutations that disrupt RNA binding, and thereby may act in a similar manner to TDP-43 acetylation, have been identified in FTLD-TDP patients." Evidence suggests that the D169G mutation does not interfere with RNA binding. See Furukawa et al., 2016.We thank the reviewer for pointing this out. We have now removed the D169G mutation from the discussion.1.11 It is unclear why the authors focused solely on homozygous K145Q animals, rather than heterozygous mice.We focused initially on homozygous mutant mice to provide better statistical power to detect small effect sizes. However, we have now included a thorough analysis of heterozygous mice including molecular analysis of brain tissue and mouse behavior, as shown in Figure 4 – Figure Supplements 1-2 and Figure 6 – Figure Supplements 1-3.Reviewer #22.1 A strength of this paper is the generation of a new mouse model to study the effects of K145 acetylation in TDP-43 proteinopathy. While the authors note an absence of a behavioral phenotype on neuromuscular testing in aged animals, it would be appropriate to include some analysis of spinal cord and skeletal muscle in this initial description of their model. At a minimum, I wonder if there is pathology in the cord  or muscle (fiber atrophy) if insoluble p-TDP-43 is detectable in these tissues, and whether dysregulated splicing of TDP-43 target genes  occurs at these sites.See comment 1.9 above. We analyzed TDP-43 aggregation, localization, and splicing in the spinal cord of TDP-43KQ/KQ mice and found mild loss of TDP-43 function that was comparable, though not to same extent, as that seen in hippocampus and cortex. We discussed these findings in the discussion and provide several possibilities for why there are no overt motor phenotypes in these mice. We note that TDP-43 Q331K knock-in mice also have cognitive but no motor deficits, suggesting TDP-43 dysfunction may preferentially  impact cognitive function :552-563).2.2 Fig 2: Differences in the splicing reporter are hard to appreciate from the images shown in panel E. Is the quantification shown in panel F corroborated by an analysis of green vs yellow fluorescence or by another method? Quantification of results shown in panel 2G  should be included.We have now removed the splicing reporter data in lieu of the more robust RT-qPCR data shown in Figure 2E and 3B-I. We have also now included more biological replicates from our iPSC neuron imaging, as shown in Figure 3A. Due to time and resource constraints, we were not able to quantify the images shown in figure 3A, and reinforce in the text that our statements are qualitative. However, we were able to add quantitative analysis of TDP-43 dysfunction, by detecting genotype-dependent splicing changes in hiPSC neurons, as mentioned above, which strengthens our claim that TDP-43 dysfunction is prominent in this culture modee.2.3 Fig 4: Differences in NeuN quantification without changes in cresyl violet staining or gliosis are surprising and a bit difficult to understand. Is there confirmation of neuron loss through another metric? Is it possible that NeuN expression is lower in mutants without frank neuron loss? Also, although no significant differences were seen by IF for TDP-43 staining, did IF for phospho TDP-43 show differences? One might expect this to be the case given the biochemical findings in Fig 5.See comment 1.6 above. After a much more in-depth and rigorous assessment, we find little evidence for neurodegeneration. Given the transcriptome data showing that TDP-43 regulates a subset of synaptic genes, we suggest that synaptic deficits underlie the behavioral phenotype rather than neuronal loss.Regarding phospho-TDP-43 pathology by immunofluorescence (IF) staining, after much effort, we have not been able to detect phospho-TDP-43 pathology by IF in TDP-43KQ/KQ mice. Currently available phospho-TDP-43 antibodies (including those acquired from collaborators) do not work well to detect endogenous mouse TDP-43 by histology or IF staining, and therefore we are somewhat limited technically. Nonetheless, given the increase in phospho-TDP-43 in the insoluble fractions by western blotting combined with the increase in cytoplasmic TDP-43 via biochemical fractionation, our data suggest that phospho-TDP-43 is the relevant species accumulating in the cytoplasm of TDP-43KQ/KQ mice.2.4 Fig 5: Probing the NC fractions for phospho TDP-43 would be an interesting addition to support the conclusion that increased cytoplasmic localization of the KQ mutant occurs prior to its phosphorylation.We agree that this would be an excellent addition to our data. Unfortunately, after rigorous antibody validation experiments, we were not able to find a phospho-TDP-43 antibody that specifically detected phosphorylated TDP-43 and did not cross-react with unphosphorylated TDP-43 in the buffers used for N-C fractionations. We tested phospho-TDP antibodies in RIPA (soluble), Urea (detergent-insoluble), and the N-C fractionation buffers, using samples treated or untreated with lambda phosphatase (to de-phosphorylate TDP-43). Only one antibody reliably detected the phosphorylated TDP-43 and not the lambda phosphatase-treated TDP-43 samples, and only did so in the Urea buffer, which is shown by straight westerns in our manuscript. Because of these technical difficulties with the phospho-TDP-43 antibodies, this was a challenging point to address at the moment. As better phospho-TDP antibodies become available, we hope to be able to address this. We therefore cannot definitively conclude that cytoplasmic phospho-TDP-43 pathology is present in these mice, but nonetheless the total phospho-TDP-43 levels are significantly elevated in urea (insoluble) fractions.2.5 Fig 1: What quantitative criteria were used to distinguish between puncta and foci, as highlighted in panel A? What is the biological significance of this distinction? From the images in panel A, it is difficult to see the TDP-43 foci in wt and K145R expressing cells.Although the size of nuclear TDP-43 foci can be quite variable, and we are certainly interested in the biological significance of this parameter, we did not focus this study on size profiles of K145Q-induced foci, only their accelerated formation and abundance. Therefore, in the revised manuscript we chose not to explicitly state any differences in “foci” vs. “puncta” and now refer to all nuclear TDP-43 structures as “foci” (removed the word “puncta” throughout).2.6 Fig 3: In describing the results of context-dependent fear testing, it is more appropriate to state that significant deficits appeared at 18 months, deleting the word "more" on line 186.We have deleted the work “more”.Reviewer #33.1 Multiple figures  include data with multiple measurements per field of view and multiple fields of view per condition. It appears that each measurement was considered an "n" for ANOVA or t-tests, but the data structure violates the requirement that data points are independent. More rigorous statistical methods such as mixed effect models should be considered (see DOI: 10.1016/j.neuron.2021.10.030) which in many cases provide more statistical power. Mixed effects models are the more appropriate statistical method for much of their data. Should the authors want to reanalyze their data with this method, they can reach out to me for an introduction to this statistical model.We have now re-evaluated the figures mentioned using linear mixed effects models, similar to what the reviewer has mentioned. The new statistical measurements have been incorporated into the revised Figures 1, 2, and 5 . A description of the statistical methods used is now provided in the revised methods section.3.2 In the introduction, the authors write "we avoid both TDP-43 overexpression and disruption of autoregulatory genomic elements of the endogenous Tardbp transcript" but they show that autoregulation is altered. So shouldn't the acetylation sites be considered a genomic element that regulates autoregulation?We agree and have now stated that our knock-in approach avoids disrupting surrounding genomic elements  in order to retain the native Tardbp gene in its unaltered form.3.3 Suggest editing the language regarding potential neurodegeneration/neuron loss as the same results could be obtained with tissue volume and/or developmental effects independent of progressive neurodegeneration.See comments 1.6 and 2.3 above. The language has been edited to reflect no apparent neurodegeneration.3.4 Sequencing the top predicted off-target loci in CRISPR'd mice and iPSC cell lines would help show the absence of off-target mutations.We described in the methods how potential off-target effects were avoided. We assessed the likelihood of off-target mutations using prediction algorithms to ensure low likelihood. All of the predicted exonic off-target sites have 4 mismatches, making them extremely unlikely to be mutated.3.5 The authors describe a subtle shift in electrophoretic mobility of the SORT1 protein band in figure 7d/e, but it is unclear why the entire SORT1 band should be shifted up in mutant mice given that the RNA analysis suggests that WT species  is still the major RNA that is expressed. In addition, others have shown that the WT versus +ex17b bands can be resolved (see DOI: 10.1073/pnas.1211577110). Perhaps knockout/knockdown cells can facilitate by providing a positive control for sizing/separation of Sort1 by immunoblotting.Please refer to our RNA-seq data shown in Figure 8A. In WT mice, nearly 80% of Sort1 transcripts lack exon17b, while this number drops to 23% in the TDP-43KQ/KQ mice. Therefore, the abnormally spliced +ex17b becomes the dominant transcript in TDP-43KQ/KQ mice. Given the prominent +ex17b inclusion that we are observing at the transcript level, it is not surprising that we mostly observe the up-shifted ex17b-containing Sort1 protein band. We have been unable to resolve two distinct bands by immunoblotting in mouse tissues using multiple Sort1 antibodies, including those used in Prudencio et al Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21510-5. Nonetheless, the up-shifted Sort1 protein is clearly the abnormal variant, as it becomes destabilized in our mice. Another possibility is that partial loss of TDP-43 function, as we suspect occurs in the TDP-43KQ/KQ mice, may magnify (or enhance) the effects on Sort1 such that the dominant Sort1 variant observed is the +ex17b containing variant. We suspect this to be true since this phenomenon was also observed in the Prudencio et al study .3.6 The authors may try to corroborate their CFTR splicing results by examining fluorescence as it appears that the construct allows for analysis of splicing differences using GFP vs mCherry expression. This is a minor point as RNA-seq analysis demonstrates abundant splicing changes in acetylation-mimic expression models.We have now removed the CFTR splicing data entirely and replaced it with more robust readouts of endogenous TDP-43 splicing targets both in vitro  and in vivo .3.7 Should the bars in figure 3d for 1 and 2 min be colored in grey/pink? It is unclear why they are clear and only outlined in color.This point is clarified in the revised Figure 4D legend. In our cue-dependent conditioned fear testing, the filled bars beyond 2 min represents the presence of the auditory cue (tone) and the period of statistical analysis.3.8 The statistical test used (Fisher's exact test?) for determining overlap between transcriptome datasets should be stated.We clarified our comment in the results section to reflect the use of over-enrichment analysis. In the methods section, it reads “Previously published differentially expressed genes from Hasan et al95 and Polymenidou et al96 were retrieved from the respective publications; significant over-enrichments as well as human gene symbol mappings to mouse orthologs were performed using gprofiler2 (g:Orth).”


The coupling effects of surface plasmon resonance (SPR) from metamaterials induce variation in both the frequency and intensity of plasmonic modes. Here, we report an angular-dependent THz modulator with hybrid metal–graphene metastructures. The metastructures composed of the period gold split-rod arrays on top of a monolayer graphene, which show redshift modulation in the THz region with an increasing incident angle due to the strong out-of-plane magnetic flux introduced by the clockwise circular current at the oblique incidence. By utilizing graphene-based actively tunable conductor with ion-gel electrical gating, the THz transmission can be significantly modified. The modulation depth of the hybrid metal–graphene metastructure modulator can reach ~37.6% at 0.62 THz with a gate voltage of −3 V. The theoretical modeling of transmitted dependency on frequency and incident angle is demonstrated at different Fermi energies, which fits well with the experimental results. This hybrid device can offer a useful method for THz applications (such as angle sensors or angular-resolved spectroscopy), where angle-dependent modulation is needed. The terahertz (THz) spectral region, serving as a bridge between electronics and optics, has attracted tremendous interest over the past few decades for its potential applications in noninvasive imaging , high-sp2 hybridized carbon atoms, possesses exceptional gapless electronic band structure [fE) and well described by the Drude model [Graphene, a two-dimensional structure arranged in a hexagonal lattice with sptructure , high motructure  and linetructure  for electructure ,14. The tructure . In the tructure , which cde model . Such unMetamaterials composed of discrete sub-wavelength structures have attracted enormous attention for versatile applications, such as perfect lensing , invisibHere, we demonstrate an angular-dependent modulator with hybrid metal–graphene metastructures in the THz region. By applying ion-gel doping, the transmission characteristics of the gated hybrid metal–graphene metastructures can be tuned, which consequently induces modulation of the transmitted wave. We observe that the resonance increases with the incident angle due to the enhanced localized field in the split-rod arrays. The THz modulation enhancement of the hybrid device is verified by both experimental and theoretical results, which shows dependency on the incidence angles and gating voltages. It turns out that the maximum modulation depth can reach −37.6% with a gate voltage of −3 V at an incident angle of 30°. Our results show that the hybrid metal–graphene metastructures can be useful in various active devices for angle-dependent photonic applications.xP and yP) of 120 µm, length (L) of 100 µm, split width (w) of 10 µm and split gap (g) of 6 µm are designed to generate the optical resonance at range of 0–1 THz, which is to fit with the operation wavelength of our THz measurement setup. The configuration of the metamaterials is sketched in 2 and a beam resolution of 0.1 µm. After e-beam lithography, the sample has to be immersed in AZ351  for 10 min to remove Al firstly and developed in a solution made of Methyl Iso Butyl Ketone (MIBK):Isopropanol (IPA) = 1:3 for 1 min. The morphology of the gold metamaterials is depicted in −1) triblock copolymer and 20 mL dichloromethane. By using the mask with apertures, the electrode with proper scale can be obtained by engaging with the e-beam evaporation. Finally, wire-bonding technology is used to apply the electrical connection, and the scheme of the measured device is shown in Experimentally, the gold split-rod metamaterials are fabricated on a sapphire substrate with further covered with chemical-vapor-deposition (CVD) graphene sheet, and the fabrication diagram is displayed in An angular-solved THz-time domain spectroscopy (TDS) setup is used to measure the transmission properties at oblique incidence from the hybrid metal–graphene metastructure. The source laser is a Ti/Sapphire laser  with a pulse width of 200 fs and repetition rate of 75.9 MHz at 800 nm, which further impinge onto a photoconductive switch integrated on low-temperature grown GaAs (LT-GaAs) at an average power of 30 mW. The switch is biased at 40 V and produces the linear polarized THz pulses that propagate in a nitrogen-purged environment through the sample, with probe detection performed via an LT-GaAs-based photoconductive switch as well. The spot size of the THz pulse is approximately 3 mm with a frequency resolution of ~50 GHz. By rotating the sample, the incident angle of the light is typically set to 0–30° for the angular-resolved THz transmission measurement.The morphology of the metastructure is characterized by field emission scanning electron microscopy . Graphene properties are characterized by Raman spectroscopy . The electrical properties of the device are measured using a sourcemeter (Agilent 2400 and 2401).gσ) can be described by altering the Fermi energy (fE) of graphene using the Kubo formula, which is present with both intraband and interband transitions [bk and T is the Boltzmann constant and temperature, Г is the carrier relaxing time, e is the charge of an electron, and ω is angular frequency, respectively.In calculations, the optical conductivity of graphene σgГ defined by μ and the Fermi velocity μ of 10,000 cm2/(V•s) and 6 m/s throughout the entire calculations.In the lower THz region, the optical conductivity of graphene is mainly governed by the intraband transition due to the Fermi level of graphene increasing above half of the photo energy. Here we only consider graphene with the ke model :(4)σg(ω)X- and Y-directions. In simulations, the thickness of the metal and substrate are 50 nm and 2 µm, respectively. The permittivity of gold in the THz region is described by the Drude model with a plasma frequency of pω = 1.37 × 1016 s−1 and damping constant dω = 4.08 × 1013 s−1, respectively [By using the frequency domain solver of CST Microwave Studio, the numerical calculations at oblique incidence can be performed with the unit cell boundary condition only supplying in ectively . The dieectively . The traBy investigating the electromagnetic response of the split-rod arrays, it is obvious that the gold split-rod metamaterials can be viewed as an LC resonance in which the effective inductance arises from the loop formed by the split-rod and effective capacitance due to the gap region between split-rod arms. The degree of asymmetry is increased by increasing the incident angle and further leads to the redshift of the resonance frequency. As a result, the electric field can effectively couple to the metastructures and induces a circular current, which accounts for a magnetic dipole perpendicular to the plane of metastructures.gV), the electrical measurement is employed to study the chemical doping states of the device and helps to define the charge neutral point (CNP) of graphene, as given in dsV = 1.0 V. It is apparent that the leakage current (gsI) (black line) is stabilized around zero, while the source–drain current (dsI) (blue line) varies conspicuously with the applied gate voltage. This behavior reveals that the electric doping of graphene can be efficiently tunable, and the Fermi level of graphene can be adjusted approaching the Dirac point with a gate voltage of 0.5 V , which implies a natural p-type doping of our graphene samples. It is worth noting that the asymmetry response in the hole and electron transportation regions is observed, which is affected by current leakage through the electrical gating [gV, the theoretical relation between fE and gV is given by [a = 1.45 × 1013/m2V is the capacitance per charge of the ion-gel [Due to the fact that carrier doping in graphene can be controlled with the gate voltage /T0, gT and 0T represent the transmission of the device with applied at gV and CNPV) is defined as the relative transmittance normalized to the value near the CNP for a selection of spectra for CNPV = 0.5 V under normal incidence, as shown in gV = −3.0 V under normal incidence. The relative transmittance with incident angles of 10°, 20°and 30° are present in In order to evaluate the modulation of the gated metal-graphene metastructure device more comprehensively, the modulation depth  at around 0.62 THz with gV = −3.0 V.To verify the angular-dependent modulation more intuitively, we investigate the transmission dip as a function of incident angle, as shown in To gain a better understanding of the physical mechanisms of the angular-dependent modulation characteristics, the simulated transmission spectra of hybrid metal–graphene metastructure device are applied by numerical results versus the incident angle and the Fermi energy of graphene layer, as shown in z-component magnetic field and x-component electric field at frequencies centered within resonant modes under normal incidence, respectively. It is evident that the magnetic fields are mainly localized at the edge of individual split-rods with TE mode, which lead to a magnetic dipole perpendicular to the surface plane of metamaterials. Hence the circulating currents can be excited, which naturally induces opposite electrical fields in the x-direction. Similarly, the distribution of magnetic fields at oblique incidence shows further localization at the edge of individual split-rods as portrayed in gV = −3 V at an incident angle of 30°. To further reveal the physical mechanism of the modulation properties, a theoretical analysis for transmitted dependency on the incident angle and Fermi energy of graphene is proposed. It is observed that the LC resonant model is employed to describe the near-field interaction in this hybrid device, which agrees well with the experimental results. The distribution of magnetic and electric fields further illuminates that the modulated behaviors lie in the recombination effect of the conductive graphene. These results prove that the angular-dependent modulation of the gated graphene-metastructure device can potentially provide a platform for strong light–matter interactions. Further tunable modulation of graphene-based metastrctures can be achieved by applying vertical or broadside coupling, which can be effective routes through exploiting the interlayer near-field electromagnetic coupling. Our works indicate that the graphene-based optical modulator can be used under multi-angle incident conditions.In conclusion, in this work we investigate the angle-dependent modulation of electrostatic gating for a hybrid metal–graphene metastructure device. Through an experiment performed on an angle-resolved TDS setup, the maximum angular-dependent modulation depth is obtained as −37.6% with


The competence of ChatGPT (Chat Generative Pre-Trained Transformer) in non-English languages is not well studied.This study compared the performances of GPT-3.5 (Generative Pre-trained Transformer) and GPT-4 on the Japanese Medical Licensing Examination (JMLE) to evaluate the reliability of these models for clinical reasoning and medical knowledge in non-English languages.This study used the default mode of ChatGPT, which is based on GPT-3.5; the GPT-4 model of ChatGPT Plus; and the 117th JMLE in 2023. A total of 254 questions were included in the final analysis, which were categorized into 3 types, namely general, clinical, and clinical sentence questions.The results indicated that GPT-4 outperformed GPT-3.5 in terms of accuracy, particularly for general, clinical, and clinical sentence questions. GPT-4 also performed better on difficult questions and specific disease questions. Furthermore, GPT-4 achieved the passing criteria for the JMLE, indicating its reliability for clinical reasoning and medical knowledge in non-English languages.GPT-4 could become a valuable tool for medical education and clinical support in non–English-speaking regions, such as Japan. ChatGPT  is a state-of-the-art large language model (LLM) that can simulate human-like conversations based on user input . As a coHowever, in a previous study, ChatGPT, based on GPT-3.5 (Generative Pre-trained Transformer), performed poorly for 77 out of 79 medical students on a South Korean parasitology examination, which resulted in questions about its ability to provide medically accurate responses in non-English languages . On MarcTherefore, this study compared the accuracy of GPT-3.5 and GPT-4 on the Japanese Medical Licensing Examination (JMLE) . FurtherWe used the default mode of ChatGPT, which is based on GPT-3.5, and the GPT-4 model of ChatGPT Plus. The latest JMLE, number 117, conducted on February 4 and 5, 2023, was also used for this study. The JMLE comprises 400 questions, which were classified into 3 categories: essential knowledge questions, which test the knowledge and ethics required of a doctor; general clinical questions, which cover numerous diseases; and specific disease questions, which test the knowledge of each disease . FurtherQuestions and their multiple-choice answers from the JMLE were used in their original Japanese form, as was the official national examination rubric. Instructions for using ChatGPT were also provided in Japanese. A typical rubric is as follows:We will present questions for the Japanese National Medical Examination. There will be five options from a to e, and you must choose the appropriate option for the question. If there is no specific limit on the number of options to choose, please select one option only.15The definition of “correct” answers to the questions asked to GPT-3.5 and GPT-4 was based on the answers to the JMLE, which were published on the website of the MHLW . Only thWe evaluated the difficulty level of each question and categorized them as hard (n=82), normal (n=112), and easy (n=60) based on the correct response rate published by medu4, a preparatory school for the JMLE ,17. QuesFinally, we simultaneously collected responses from both GPT-3.5 and GPT-4 between March 16 and 18, 2023, and scored them using the definition of correct answers. P<.05.Standard descriptive statistics were used to calculate the numbers, proportions, and means for each data set. The McNemar test was used to compare correct response rates. All analyses were performed using the Stata statistical software (StataCorp LLC) . All tesThis study only used information that was already published on the internet and did not involve human subjects; rather, an analysis of the JMLE was performed. Therefore, approval by the Institutional Review Board of Shimane University was not required.P<.001). In terms of the correct response rate for individual questions, the examinees’ rate for essential knowledge questions was 89.2% compared to 87.2% for GPT-4. Notably, this represents a considerable 32.1% improvement over GPT-3.5, which had a 55.1% correct response rate. Similarly, a 29.5% increase was observed for general clinical questions, and a 25.4% increase was observed for specific disease questions. In all cases, GPT-4 achieved the passing rates for the JMLE. However, none of these rates exceeded the total percentage of correct answers by examinees.A total of 254 questions from the 117th JMLE were used in the experiment. P<.001) and a 29.6% increase for clinical questions (P<.001) compared to GPT-3.5. Notably, a 36.3% increase was observed in the number of correct responses to clinical sentence questions, with a significant improvement in all question types .P<.001), a 31.5% increase for normal-level questions (P<.001), and an 18.3% increase for easy-level questions (P<.001).P<.05). In particular, for hard-level questions, the correct response rate of GPT-4 was 17% higher than the examinees’ average correct response rate.Finally, GPT-4 significantly outperformed GPT-3.5 in all formats in terms of correct response rates (all We compared the correct response rates of GPT-3.5 and GPT-4 on the 2023 JMLE. GPT-3.5 did not satisfy the passing criteria, whereas GPT-4 achieved the required scores. Furthermore, GPT-4 demonstrated a significantly improved correct response rates compared with GPT-3.5 across various question types and difficulty levels. The correct response rate of GPT-4 was particularly enhanced for the challenging hard-level questions and surpassed the average correct response rate of actual examinees. Based on these results, we discuss 2 factors that explain the significant improvement in the correct response rates of GPT-4 on the JMLE.First, we ascribe this enhancement to the augmented NLP capabilities in non-English languages. A performance disparity between English and other languages in LLMs is ubiquitous in NLP . AdditioSecond, since improving the information processing capabilities in professional and academic domains is imperative, OpenAI’s development of GPT-4 aimed to handle more intricate and nuanced tasks beyond those encountered in many real-world situations . The JMLAs the results of this study and several previous studies indicate, LLMs such as ChatGPT have made remarkable progress ,7,13. HoThis study had several limitations. First, the results reflect the capabilities of ChatGPT as of March 17 and 18, 2023, and different results could be obtained even if the same methods were used. The knowledge and interpretation capabilities of ChatGPT will rapidly improve in the future because of user feedback and deep learning. Second, although GPT-4 is a multimodal artificial intelligence that is inherently capable of inputting images and tables, among other things, this study excluded them for an accurate comparison with GPT-3.5, and only text questions were used. Third, the JMLE has a supplementary assessment that states that if an absolute contraindication answer is selected 2 or more times, the applicant will fail the examination, even if they have achieved the passing scores . BecauseGPT-4 passed the 117th JMLE, whereas GPT-3.5 failed the examination. This phenomenon revealed GPT-4’s rapid evolution in Japanese language processing. Investigations are necessary to evaluate its safety, efficiency, and cost-effectiveness for potential application as an LLM artificial intelligence tool for medical practice support, learning in clinical settings, and medical education.


I-V measurements. An increased electrical conductance is observed for the bulk-NCG-based FET, despite its higher defect density, the channel displaying a transconductance of up to ≊The progress of advanced materials has invoked great interest in promising novel biosensing applications. Field-effect transistors (FETs) are excellent options for biosensing devices due to the variability of the utilized materials and the self-amplifying role of electrical signals. The focus on nanoelectronics and high-performance biosensors has also generated an increasing demand for easy fabrication methods, as well as for economical and revolutionary materials. One of the innovative materials used in biosensing applications is graphene, on account of its remarkable properties, such as high thermal and electrical conductivity, potent mechanical properties, and high surface area to immobilize the receptors in biosensors. Besides graphene, other competing graphene-derived materials (GDMs) have emerged in this field, with comparable properties and improved cost-efficiency and ease of fabrication. In this paper, a comparative experimental study is presented for the first time, for FETs having a channel fabricated from three different graphenic materials: single-layer graphene (SLG), graphene/graphite nanowalls (GNW), and bulk nanocrystalline graphite (bulk-NCG). The devices are investigated by scanning electron microscopy (SEM), Raman spectroscopy, and  Field-effect transistors (FETs) have attracted extensive attention in biosensing applications. Unlike traditional sensing methods, FET-based biosensors do not involve extensive photodetectors or fluorescent labeling parameters. A biosensor has two key components: a bioreceptor and a transducer. The bioreceptor is a biomolecule that generates a physical or chemical reaction upon interaction with a target analyte in a biological media. The transducer primarily converts the identification process into a measurable signal, which can be either electrical, electrochemical, optical, or mechanical. One electric-type biosensor that has gained a significant amount of traction over the last few years is the FET. The FET biosensor is composed of a substrate with source, drain, and gate electrodes, as well as a conductive channel that is functionalized with a bioreceptor  and acts as the transducer. The bioreceptor–analyte reactions will cause a change in the electrical conductivity of the transducer, and the FET’s biosensitivity occurs when the respective change in conductivity is large enough to be measured ,2,3.As the size and performance of silicon transistors have approached their physical limits, it is necessary to look for alternative materials to support emerging technologies. One of these materials is graphene, which is an attractive option for the source-drain channel of field effect transistors due to its excellent electrical, optical, mechanical, and thermal properties . In receAs with traditional silicon-based FETs, the GFET’s gate modulates the flow of electrons or holes in the channel. Due to the nanoscale thickness of the GFET’s channel, the current flows mainly on its surface; therefore, the GFETs possess a high sensitivity. The higher electrical and thermal conductivity of graphene results in reduced losses and enhanced heat dissipation compared to silicon. As a result, graphene-based transistors have the potential to deliver enhanced performance and efficiency ,8,9.graphene is used as a general definition for graphene materials, such as graphene (single or multilayer), graphene oxide, or reduced graphene oxide [In the literature, the term ne oxide ,11. Howene oxide . Single-ne oxide  and was ne oxide . Alternane oxide . Their inditions ,16. Overnditions .I-V curve with respect to the gate voltage [One of the first devices to employ graphene-derived materials for biosensing applications was reported by Mohanty and Berry in 2008. They produced cost-efficient chemically modified graphene-based transistor-like devices for the purpose of bacterial and DNA detection with enhanced sensitivity tunability . Exosome voltage .Gold’s standard potential of 1.52 V makes Au nanoparticles (AuNPs) more stable from an electrical standpoint compared to other noble metal nanoparticles such as AgNPs  . Their hThe feasibility of terahertz operation was also determined in 2012 by Mehr et al., with a transistor based on vertically orientated graphene sheets . Later, The GFETs reported in this paper are four-terminal devices , similarThe greater part of their most noteworthy features resides in their electrical properties. Generally, graphene has a very low resistivity at room temperature, as well as high intrinsic mobility, over 100 times that of Si. Theoretically, graphene can transfer electric current with 100% efficiency, and even the earliest manufactured graphene layers, although prepared using relatively modest methods in limited controlled conditions, presented remarkable qualities with reasonably high quasiparticle mobility for FET-like devices (up to 5000 cmV−1 s−1) . Over thV−1 s−1) , and thu2, h-BN)  and over samples ,28. For  samples . Their dubstrate  and up tnded SLG . As it igraphene . Despitef layers ,32. Grapf layers ,33.The SLG thin films were grown on Cu substrates by Chemical Vapor Deposition (CVD), while the GNW and bulk-NCG films were grown directly on SiOThe SLG thin films are grown via thermal CVD on a catalyst substrate, which consists of 35 rocesses ,35,36.The GNW and bulk-NCG films used for the GFETs’ fabrication are grown via PECVD in a capacitively coupled RF reactor from CH process ,38,39, isorption . The PEC4) ratio . The GNW4) ratio ,16, and 4) ratio ,35.The GFETs’ fabrication process differs from the silicon-based FETs’ process as it entails a more complex series of photolithographic steps revolving around both wet and dry etching. The microfabrication of the graphenic-based FETs involves the deposition of the carbonic materials on a thermally oxidized Si wafer, either directly  or by transfer , followed by the metallic contacts’ deposition  and functionalization of the source-drain channel.All presented GFETs were manufactured on In preparation for the back gate deposition, the back-side SiOone bath . The wafIn the case of the SLG-based GFETs, an additional step is required to properly identify the areas onto which the SLG thin films are to be transferred. This is accomplished by wet chemical etching, using a specifically designed mask a within Next, the source and drain electrodes are deposited through e-beam evaporation and lift-off techniques similar to the back gate deposition, except this time a 365 nm thick Au layer and a 30 nm thick Cr adhesion layer are used. Following the subsequent cleaning in acetone and isopropyl alcohol, the graphenic source-drain channels on all wafers are masked with an AZ 4562 photoresist and the rest of the graphenic material is dry etched in an oxygen plasma. The channel patterning is carried out in a capacitively-coupled RF reactor with the Plasma Etcher—Etchlab 200  equipment, at 250 W RF discharge power, a pressure of 20 Pa, and an OThe functionalization of the graphenic drain-source channel is accomplished by AuNPs decoration. The AuNPs’ purpose is to enhance the electron transfer rate and to provide a larger contact surface, thusly improving the sensor’s response.The AuNPs were chemically synthesized by Au (III) to Au (0) reduction: 10 mL of chlorauric acid  investigations confirm the binding of  channel .The graphenic-based channel is deposited on a dielectric layer  at an acceleration voltage of 15 kV. Top-view SEM micrographs showcasing the graphenic source-drain channel and its respective electrodes, along with the top gate electrode, are presented in Additionally, Raman spectroscopy was used to evaluate the source-drain channel and analyze the structure of the different graphenic materials. The spectra  were acqX, respectively, could be explained by defects induced during the transfer process. The appearance of transfer-induced defects is also corroborated by the presence of a small intensity D peak, which can be observed at ≊1340 cmAll Raman spectra are initially vector normalized, and a Savitzky–Golay filter is applied over an 11-point window with a third-order polynomial to improve the signal-to-noise ratio. The spectra are then background corrected by linear subtraction and deconvoluted into individual Lorentzian peaks, with the exception of the G peak in the case of the GNW and bulk-NCG films, which is described by a Breit–Wigner–Fano shape . The coe phonons . Typical phonons ,42. The an bands ,42,43. MI-V readout. The output characteristics of the GFETs at different gate voltages, presented in The electrical performance of the GFETs was investigated at room temperature using the Keithley 4200-SCS Semiconductor Characterization System  coupled with the Manual Wafer Probing Station EP6 . Initially, several attempts were made with varying back gate voltages but, as depicted in I-V transfer curves can be observed in the case of all three materials (The GFETs are unique in that they can act as both n- or p-type channel transistors, depending on the top gate applied voltage. The critical transition between the two regions of the GFETs’ aterials d–f. At tAnother important distinction between the three graphenic channels that can be ascertained from nfluence , as mentThe measured transconductance values of the GFETs and the charge carrier mobility, calculated using Equation , are shoI-V transfer characteristics of the source-drain channel before and after AuNPs decoration, we notice an improved sensitivity. The Dirac point of the bulk-NCG FET showed a As the bulk-NCG channels showed the best electrical characteristics, further investigations were employed in an attempt to improve their sensitivity by AuNP decoration. When comparing the  ≊746.43 .Graphenic materials have proven to be valuable assets in the fabrication of FETs for sensing applications. In addition to their close relation to atomically thin graphene, in terms of their composition and molecular structure, which relate to similar physico-chemical properties, they provide a more feasible fabrication process for the respective GFETs. The PECVD synthesis of the graphenic materials is accomplished directly on an insulating layer, therefore eliminating the requirement of a transfer step from the technological workflow, a step that is still necessary for SLG. Their shared sp


We find that the magnitude of the Seebeck coefficientis up to nine times larger than that of Au–C60–Ptmolecular junctions. Moreover, the sign of the thermopower can beeither positive or negative depending on the details of the bindinggeometry and on the local value of the Fermi energy. Our results demonstratethe potential of using graphene electrodes for controlling and enhancingthe thermoelectric properties of molecular junctions and confirm theoutstanding performance of [60]fullerene derivatives.The thermoelectric properties of molecular junctionsconsistingof a metal Pt electrode contacting [60]fullerene derivatives covalentlybound to a graphene electrode have been studied by using a conducting-probeatomic force microscope (c-AFM). The [60]fullerene derivatives arecovalently linked to the graphene via two  However, functionalization of only one side has also been accomplishedby using a solid substrate where the graphene layer is deposited.13 In this sense, in most of the practical applications of graphene,it is actually supported on a solid substrate which could be easilyincorporated into an electronic device. Therefore, the chemical functionalizationof graphene on a substrate with [60]fullerene may drastically expandthe material capabilities by fine-tuning its chemical and physicalproperties.Graphene and [60]fullerene aretwo Nobel laureated disruptive materials representing new allotropesof carbon whose properties could be fine-tuned upon chemical covalent14 They also open the possibility of designingnew thermoelectric devices with enhanced thermoelectric propertiesthat could find applications in energy management, on-chip cooling,temperature sensing, or energy harvesting.20 All-carbon hybrid devices show great promise in their ability toexploit desirable thermoelectric properties.25 One such property is the delicate control of thesign and magnitude of the Seebeck coefficient. This control has beendemonstrated, for example, by applying tip pressure to modulate thecoupling of the molecule to the electrode26 and by tuning intermolecular interactions between C60 molecules.18Studies of the thermoelectric properties of molecularjunctionsconsisting of organic molecules connected to electrodes provide insightinto the charge transport mechanisms at the molecular level.27 They found that for C60, PCBM, and C70, the Seebeck coefficient was negative,being largest in magnitude for C70 and for the Ag tip (−33μV/K). These are among the highest measured values for molecularjunctions.14 Evangeli et al.18 found that the thermopower of C60 dimers between gold electrodes reaches values of −33 μV/K,almost double that of C60 (−18 μV/K). Thefact that the thermopower is negative for all these fullerene molecularjunctions shows that electron transport takes place through theirLUMOs . In contrast, it hasbeen shown that, in molecular junctions of endohedral fullerenes,the Seebeck coefficient can be either positive or negative dependingon the orientation of the encapsulated moiety and also in responseto pressure; this behavior was explained by the presence of transmissionresonances close to the Fermi level.17The thermopower ofvarious single fullerene molecules between agold substrate and a metal tip of Au, Ag, and Pt was studied by Yeeet al.1–3) bound covalently to grapheneon SiO2/Si  as shown in 60–Ptjunctions. We use density functional theory (DFT) calculations toobtain a detailed understanding of the origin of these values andthe molecular configurations responsible for these amazing behaviors.Here, we investigate the thermoelectric properties of single-moleculejunctions formed by contacting diphenylmethanofullerene molecules led to derivatives GOS-1, GOS-2, and GOS-3 , affording the final amino or diamino-containing diphenylmethanofullerenes 1–3.28 Thecovalent functionalization of GOS was conducted via aryl diazoniumchemistry in the presence of isoamyl nitrite, following a slightlymodified protocol of that previously reported by Tour and co-workers.29 Thus, the corresponding aryl diazonium saltsare generated in advance by adding isoamyl nitrite to a solution of 1, 2, or 3 and subsequently dropcasted on the GOS. As proof of concept and to validate the covalentfunctionalization over the graphene grafted on top of SiO2/Si, we have also synthesized the analogous all-carbon hybrid materialsover few layers graphene (FLG) to achieve a complete characterizationof the final derivatives. Pristine FLG was obtained through graphiteexfoliation following Colleman’s method with a high degreeof purity.30 A suspension of FLG in o-DCB was immediately reacted with [60]fullerene derivatives 1 and 2 in the presence of isoamyl nitrite providing FLG-1 and FLG-2, respectively  oncopper foils, cut into smaller pieces and transferred onto SiOnd GOS-3 1. The syectively 1.2 carbon atoms to sp3 as a result of the covalent attachment of 1, 2, or 3, respectively. Therefore, the ratio withthe G band ID/IG is a way to quantify the covalent functionalization degree of thosematerials. According to this criterion, in our measurement we found ID/IG ratios of 0.26for GOS-1, 0.54 for GOS-2, and 0.23 for GOS-3 . Moreover,a new peak around 1457 cm–1 is clearly observedfor all fullerene aggregates which can be assigned to the pentagonalpinch mode [Ag(2)] of C60.31 These results indicate a higher functionalization in the case of GOS-2, which is also confirmed by the observed ID/IG ratios for FLG-1 and FLG-2 . Thus,this evidence confirms the covalent attachment of 1, 2, or 3 on the FLG or GOS. A characterizationof FLG-1 and FLG-2 employing ATG, FTIR,XPS, and TEM has been carried out and is available in the SI.Raman spectroscopy under 532 nm laser wavelengthsgives insightsof the surface functionalization of FLG and GOS. All aggregates presentan increase in the D-band with respect to the G-band, which is generallyassociated with a higher defect degree on the nanomaterials, alsoattributed to functionalization. This increase is related with a substantialrehybridization from spGOS-1, GOS-2, or GOS-3 that partially cover the graphene sheet with the substrate and held stationarythrough a feedback loop controller. This allows us to measure current–voltage(I–V) characteristics in which the thermoelectricvoltage shows as a voltage-offset ΔV, fromwhich one can obtain the thermopower or Seebeck coefficient S = −ΔV/ΔT. A zero-calibration of the voltage reading is performed in shorttime intervals by disconnecting the c-AFM from the V source and connecting a resistor R instead, facilitatinga precise voltage reading. Then we can determine S and the conductance G (from the slope of the I–V curves) for a number of givenpoints on the substrate.To characterize the thermoelectricresponse of the [60]fullerenederivatives on graphene, we used a conductive atomic force microscope(c-AFM) with Pt-coated tips (Multi75-G from BudgetSensors). S measured with the tip in contactwith the graphene. We obtain a very small average value of 2 μV/K,corresponding to the thermopower of the Pt-tip/graphene/SiO2 junction. This measurement configuration is totally different tothat of previously reported experiments that determine the in-planethermopower of graphene, which is found to be between −50 and+50 μV/K, depending on doping.32GOS-1 yield twodistinct sets of values centered on S = −56μV/K and S = 74 μV/K , as described in the SI.To further understand this behavior,we used density functionaltheory (DFT) combined with quantum transport theory to compute theelectrical conductance SI, the calculatedSeebeck coefficients were found to be sensitive to the location ofthe metallic tip on the surface of the C60 and to the valueof the Fermi energy, which can vary across the film due to the presenceof adsorbates, such as water. Therefore, values of S for a range of tip-C60 contact locations and a rangeof Fermi energies were calculated. These were then used to createthe histograms of Seebeck coefficients shown in GOS-3 values of S being positive,whereas GOS-1 and GOS-2 histograms possesspeaks at both positive and negative values. This suggests that theexistence of two peaks in the histograms cannot be considered as anindication of the presence of molecules bound by one or two legs andthat the precise shape of Seebeck histograms depends on the rangeof Fermi energies and tip-C60 configurations sampled inthe experiments. The sign of the Seebeck coefficient depends on theenergetic location of the frontier orbitals relative to the Fermienergy. If the LUMO is closer to the Fermi energy, then the Seebeckcoefficient is negative, whereas if the HOMO is closer, the Seebeckcoefficient is positive. Bonding of the tip to the unattached phenylring was not considered, since the unattached ring points toward thesubstrate, making it unavailable to the tip.As discussed in the 60 has been considered in a previous work.33 This study showed that intermolecular interactions causesmall changes in the Seebeck coefficient, but these are negligiblecompared to the sample-to-sample fluctuations shown in In the above simulations, each junction containeda single molecule,whereas in the experiments, many molecules are present. The effectof intermolecular interactions in junctions with more than one C1–3) have been covalently linkedto 2Dgraphene either through two meta-connected phenylrings, two para-connected phenyl rings, or just asingle phenyl ring, resulting in the new systems GOS1–3. All the [60]fullerene/graphene hybrids havebeen characterized by spectroscopic and microscopic techniques, revealingthe efficient covalent linkage between the two carbon allotropes.In summary, suitably functionalized [60]fullerene derivatives(GOS-1, GOS-2, and GOS-3 generateexceptionally high quantum thermopowers when covalently anchored tographene, thus suggesting great promise for molecular/graphene compositematerials in thermoelectric applications. The possibility to changethe sign of the most probable Seebeck coefficient when utilizing twoanchoring groups implies potential for tailoring thermoelectric performancein more complex device architectures in which positive and negativecontributions may be combined to build thermoelectric modules.Our work demonstrates that the [60]fullerene derivatives GOS-1 and GOS-2 is very sensitive tolocal variations of the Fermi level, leading to both positive andnegative values of the Seebeck coefficient. In contrast, the calculationspredict that, for the same local variations in the Fermi energy, thethermopower of GOS-3 is more robust when anchored tographene, since its transmission function exhibits a consistentlynegative slope around the energy range of interest. Fluctuations inenergies of the frontier orbitals relative to the Fermi energy aremodeled by sampling a range of Fermi energies. The precise range sampledexperimentally is unknown, because small changes in the bonding configurationand defects in the graphene will change the Fermi energy relativeto frontier orbital energies. Therefore, we sampled a range, whichwas consistent with experiment. In this sense, although the theoryis not predictive, we are able to show that the experiment can beconsistent with theory. In conclusion, the geometries and Fermi energiesvary so much that a broad distribution of Seebeck coefficients areobserved. The sign of the Seebeck coefficient depends on the energeticlocation of the frontier orbitals relative to the Fermi energy. Theseare shifted relative to their gas-phase values by the real part ofthe self-energy, due to contact with the substrate and tip. This shiftdepends on the strength of the contact, which in turn depends on theorientation of the molecule and details of the linker. Random energylevel shifts also occur due to imperfections in the graphene substrate.DFT calculations show that the sign of the Seebeck coefficientof