Using CEM screening, we identified a candidate ASO that shows >70% cholesterol-lowering action in monkeys. An N-acetylgalactosamine  ligand then was appended to the candidate ASO to further broaden the therapeutic margin by altering the molecule’s pharmacokinetics. The GalNAc conjugate, HsPCSK9-1811-LNA, was found to be at least ten times more potent in non-human primates (compared with the unconjugated counterpart), with reduced nephrotoxicity in rats. Overall, we successfully showed that our drug development scheme is better suited for selecting clinically relevant BNA-based ASOs, especially for the treatment of liver-associated diseases.Antisense oligonucleotides (ASOs) containing bridged nucleic acids (BNAs) have been proven to be very powerful. However, ensuring a reliable discovery and translational development scheme for this class of ASOs with wider therapeutic windows remains a fundamental challenge. We here demonstrate the robustness of our scheme in the context of the selection of ASOs having two different BNA chemistries  targeting human proprotein convertase subtilisin/kexin type 9 (PCSK9). The scheme features a two-step process, including (1) a unique and sensitive   in vitro screening approach called CEM and (2) a ligand-targeted drug delivery approach to better reach target tissues, averting unintended side effects.A robust discovery and translational development scheme for selection of clinically relevant BNA-based gapmer ASOs was developed. The scheme features a two-step process, including (1) a unique and sensitive  Nucleic acid drugs such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are emerging as pragmatic options for therapeutic modalities, especially for genetic and rare diseases, as the clinical performance of ASOs has been largely improved with innovative chemistry over the past several decades. We have devoted our efforts to developing unique artificial nucleic acids, called bridged nucleic acids or BNAs.in vitro screening methodology that better predicts the in vivo efficacy of ASOs; prior to the implementation of this screening approach, we often failed to obtain in vivo-active ASOs when using conventional lipofection-based in vitro screening.2+ enrichment of medium). Our previous report validated CEM as an alternative in vitro transfection method better suited for selecting in vivo-active ASOs.In this context, we previously devised a unique N-acetylgalactosamine  ligand has proven to remarkably improve the targeting of ASOs to the liver by reinforcing cellular uptake via receptor-mediated endocytosis while reducing renal accumulation of ASOs. The trimeric form of GalNAc-conjugated ligands is known to serve as an excellent ligand for the asialoglycoprotein receptor that is expressed selectively on hepatocytes.,trans-4-hydroxy-l-prolinol-linked GalNAc by showing (in mouse) that this modification reduces the accumulation of ASOs in kidneys while enhancing that in the liver.One reliable approach to avoiding nephrotoxicity is to minimize exposure of the drug to the kidney, given that short oligonucleotides such as high-affinity gapmer ASOs have been shown to preferentially accumulate in the kidney, especially in the proximal tubules.,PCSK9 have been described, including ours .PCSK9 developed by Santaris Pharma (currently Roche),,Our group has focused on the development of BNA-modified gapmer ASOs for the treatment of dyslipidemia. The proprotein convertase subtilisin/kexin type 9 (PCSK9) is widely accepted as a drug target to reduce the residual risk of cardiovascular disease (CVD).in vitro screening step to obtain more potent in vivo activity and (2) a ligand-targeted drug delivery approach to enhance delivery to target tissues, averting unintended accumulation of ASOs, an outcome that often is associated with side effects. Here, we demonstrate our representative discovery/development scheme for high-affinity gapmer ASOs, using PCSK9 as an exemplary target.Our drug discovery scheme has taken the form of a two-step process, including (1) a unique and accurate in vitro screening using the CEM method, we first designed 180 gapmer-type ASOs using two different BNA chemistries . Each of these ASOs was 14 nucleotides long . The library of ASOs corresponded to sequences that ranged across the target transcript’s coding region. The in vitro screen was performed with the CEM method on Huh-7, a human hepatoma cell line. The final concentrations of ASOs and CaCl2 in the screening cultures were 200 nM and 9 mM, respectively. The initial screen revealed that the level of knockdown activity varied considerably among the 180 ASOs tested here  were identified. Both the 311 and the 1811 series are apparently very potent; however, the 311 series lacks homology with rat sequences, which would make the 1811 series more attractive as candidates for clinical drug development. The melting temperatures (Tms) against their complementary RNAs and 50% inhibitory concentration (IC50) values of these selected candidates, along with those of SPC5001, a known PCSK9 inhibitor, were determined and are provided in Tm values of ∼73°C and IC50 values of ∼10–11 nM. Although the Tm values of the HsPCSK9-1811 series are ∼10°C lower than those of the HsPCSK9-311 series, the HsPCSK9-1811 series showed higher potency. In this series, HsPCSK9-1811-LNA(14) has a Tm that is slightly (+3°C) higher than that of HsPCSK9-1811-AM(14), and the former also exhibited slightly higher potency. Santaris’s SPC5001 14-mer showed the lowest Tm; nevertheless, as expected, SPC5001 exhibited excellent potency (IC50 = 8.9 nM). The 2-9-2-1 gapmer system was selected based on previous studies by ourselves and others showing that 14-mer gapmers generally provide affinities sufficient for binding to their target RNAs and exhibit activity both in vitro and in vivo.,in vitro screening successfully identified excellent candidates with activities that were at least comparable to that of SPC5001; we therefore decided to proceed to further in vivo validation.To perform an des long A. All ASted here B. The scted here C. We selted here D and 1E.in vitro screen were evaluated in a dose-escalation study with cynomolgus monkeys. Each ASO was administered subcutaneously once weekly for 3 weeks at doses of 1, 3, and 10 mg/kg in individual male and female monkeys. Serum low-density lipoprotein cholesterol (LDL-C) levels were measured over time through 38 days post-injection. Among the three candidates, HsPCSK9-1811-LNA provided the largest attenuation of cholesterol levels, achieving a nadir of an ∼60% decrease in LDL-C on day 24, after the third injection at 10 mg/kg  selected through the 10 mg/kg A. As wit10 mg/kg B.Figure Next, we tested the dose responsiveness and the possible recovery of LDL-C levels following dosing with the most potent candidate, HsPCSK9-1811-LNA. Additional doses of 3 and 1 mg/kg of HsPCSK9-1811-LNA were administered on days 42 and 61, respectively, to animals originally dosed at 1, 3, or 10 mg/kg. As seen in HsPCSK9-1811-LNA was shown to have a strong LDL-C-lowering effect. To further validate the molecule as an appropriate clinical candidate, we performed 2-week safety studies for HsPCSK9-1811-LNA in rat and monkey. First, groups of two monkeys each were injected subcutaneously, once weekly for 2 weeks, with 10 or 30 mg/kg HsPCSK9-1811-LNA. The monkeys were euthanized 1 week after the final injection. Serum alanine aminotransferase (ALT) levels were elevated in one of the two monkeys treated with 30 mg/kg of the ASO A. As we In rat experiments, groups of 5 animals each were injected subcutaneously with 10, 30, or 100 mg/kg HsPCSK9-1811-LNA. Four of five male rats and one of five female rats administered 100 mg/kg ASO were found dead within 1 week; the remaining rats were euthanized for humane reasons. The rats of the 10- and 30-mg/kg dose groups were administered a second dose after 1 week and observed for another week before euthanasia, necropsy, and collection of tissues for histopathological evaluation. As in the monkey study, an enlargement of the kidneys and an increase of serum creatinine levels were observed F and 3G.,As we anticipated, based on the previous results with SPC5001, the LNA-based ASO we selected also displayed a relatively narrow therapeutic margin, despite exhibiting a strong LDL-C-lowering effect. To avert the renal burden of this ASO, we next incorporated a GalNAc ligand into the 5′ terminus of HsPCSK9-1811-LNA A, yieldi,The potency of HsPCSK9-1811-LNA-GN was evaluated in a single-dose study using monkeys, dosing one monkey each at 0.3 and 1 mg/kg. The dose level of the GalNAc-conjugated ASO was rationalized based on our previous study, in which GalNAc-ASO conjugates were shown to be at least 10 times more potent than their unconjugated counterparts.nd dose), rats dosed with HsPCSK9-1811-LNA exhibited attenuation of body weight gain compared to animals dosed with the other two test compounds. Serum ALT levels were elevated 2- and 4-fold after dosing with 30 mg/kg HsPCSK9-1811-LNA or 3 mg/kg HsPCSK9-1811-LNA-GN, respectively, compared to the level in the saline-treated control ; the 3-mg/kg dose was selected as the higher dosage for HsPCSK9-1811-LNA-GN because we considered a 10-fold margin a reasonably wide therapeutic window in terms of the renal toxicity, based on our previous data and the example of SPC5001. At 2 weeks after the first dose , and tissues were collected and submitted for histopathological evaluation . Macroscin vivo-active ASOs with alleviated “class side effects.” CEM is a simple, highly versatile, and high-throughput in vitro screening system that expedites an otherwise painful in vitro screening step to facilitate the identification of ASOs with in vivo activity. It is known that most cell lines have lost their nucleic acid uptake capacity, impeding our ability to reproduce faithfully the in vivo environment, i.e., the situation whereby ASOs are taken up by cells, in the in vitro cell culture system. Until now, methods using primary cultured cells or attempts to construct new strains based on primary cells have been used, but these specialized methods are difficult to implement in human cell lines, limiting the development of translational nucleic acid drugs. In the CEM method, on the other hand, calcium ions stimulate intracellular uptake by facilitating aggregation of serum albumin, which in turn acts on the cultured cells.in vivo activity of ASOs, enabling the direct exploration of ASOs corresponding to the human PCSK9 sequence and bypassing experiments in small rodents. Notably, CEM does not limit the chemistry or physicochemical properties of ASOs of interest. We used 2′,4′-BNA/LNA- and AmNA-based ASOs, the chemistry of which helps to fortify binding affinity, Of several potent inhibitors selected using CEM, all of the tested candidates demonstrated attenuation of PCSK9 expression in non-human primates. The selected 14-mer gapmers (using the 2-9-2-1 system) have been shown to have affinities sufficient to provide knockdown of PCSK9 activity both in vitro and in vivo owing to the high-affinity BNA modification . In a previous study, Uehara et al. demonstrated the promising effect of AmNA-based gapmer ASOs in a neurodegenerative disease context.,ASOs are eliminated primarily in the kidneys via proximal tubular epithelial cells.in vitro method, the measurement of cellular caspase 3/7 activity, to evaluate the hepatotoxicity of BNA gapmer ASOs in mice. If used in combination with our CEM screening strategy, such a method may permit estimation not only of hepatotoxicity in mice but also of other toxicities in non-human primates and humans.Although there have been many reports on the hepatotoxicity issues of ASOs in early drug development, the pre-clinically observed nephrotoxicity of 2′,4′-BNA/LNA gapmers such as SPC5001 was our primary target in this study. Notably, we have shown an effective approach to avoid nephrotoxicity using GalNAc conjugation, which enables more drug in liver and less in kidney. At the same time, we are aware that it is indeed necessary to be able to select ASO drugs with a wider safety margin for the treatment of chronic diseases such as dyslipidemia. Recently, Dieckmann et al.,in vitro experiments, as assessed with renal proximal tubule epithelial cells.Additionally, it has been reported that ASOs conjugated with triantennary-type GalNAc have lower nephrotoxic potential than non-conjugates in ,,A moderate increase of serum ALT was observed for the highest dose of HsPCSK9-1811-LNA-GN in rats. Hepatotoxicity associated with ASOs is observed more frequently in rodents, and recent studies suggest that its onset can be ascribed to an interaction with RNase H or intracellular proteins.PCSK9 loss-of-function mutations have a much lower rate of CVD than non-carriers.PCSK9 loss-of-function mutations, the ASOs obtained in the present study are expected to exhibit increased potency for reducing the risk of CVD compared with other types of inhibitors (like antibodies) that are in current use.Patients with PCSK9 mRNA while possessing a liver-targeting mechanism. In particular, our HsPCSK9-1811-LNA-GN did show comparable potency and efficacy in non-human primates to an anti-PCSK9 cEt gapmer with a triantennary GalNAc unit, AZD8233, which showed great potential in human trials.ASOs with chemical modifications hold great promise for therapeutic application if such molecules are effective and safe. Here, we established a robust drug discovery scheme and successfully identified highly potent and safe ASOs that showed activity against human One limitation of this study is that the bare minimum number of animals was used in the monkey studies because of the limited number of animals that were available. Although it is reasonable from the perspective of animal protection, and a clear dose dependency was confirmed in the present study, additional follow-up studies with certain control ASOs will be needed to support the alleged efficacy and safety of the ASOs; such studies currently are being conducted as part of pre-clinical studies. Nevertheless, we believe that this approach may be generalizable to the isolation of clinically relevant ASOs, regardless of the target.,PCSK9 mRNA are shown in PCSK9 mRNA from both rat and monkey.The ASOs used in this work were synthesized by Gene Design . The phosphoramidite monomers of AmNA and the monovalent GalNAc units were obtained as previously described.2 in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum  and 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific). Huh-7 cells were seeded at 5,000 cells/well in 96-well plates . Twenty-four hours after seeding, ASOs were added in 10% FBS-DMEM containing 9 mM CaCl2. After another 24 h, cDNA was synthesized directly from cell lysates with a SuperPrep Cell Lysis & RT Kit for qPCR . Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed with a TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and analyzed with a StepOnePlus system . For the IC50 experiments, HsPCSK9-311-AM(14), HsPCSK9-311-LNA(14), HsPCSK9-1811-AM(14), HsPCSK9-1811-LNA(14), and SPC5001 were each transfected into Huh-7 cells at 0.8–200 nM (8–9 points) with the above CaCl2-containing medium. The experiments were performed in technical quadruplicate and were repeated three times. IC50s were calculated by the log(inhibitor) versus response (three parameters) method in Prism 9.2.0 (GraphPad). The TaqMan probes (Applied Biosystems) used in this assay were as follows:Human PCSK9: Hs00545399_m1 (FAM)Human GAPDH: Hs02758991_g1 (VIC)Huh-7 cells, a human hepatoma cell line, were maintained at 37°C and 5% COTm analysis system . Two complementary single-stranded oligonucleotides were mixed in a 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a 1.0 μM oligonucleotide solution. The mixture was annealed by heating at 95°C for 3 min and then cooled at −1°C/min to 20°C. Tm was estimated with the software provided with the TMSPC-8 and defined as the temperature at which the formed duplexes were half-dissociated. The temperature-dependent change in the optical absorption was recorded at 260 nm, and the scan rate was 0.5°C/min from 4°C to 94°C. Each averaged Tm value was determined as the intersection point between the absorption on temperature and the median between the two baselines from three independent measurements.UV melting experiments were performed on a Shimadzu UV-1850 spectrophotometer equipped with the TMSPC-8 ad libitum access to a standard chow  and water.The animal studies using rats were conducted at a certified contract organization  that is fully accredited by AAALAC International. These studies were approved by an Institutional Animal Care and Use Committee  and were performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories Drug Safety Research Laboratories. Rats were maintained on a 12-h light/12-h dark cycle (lights on from 7:00 AM to 7:00 PM) and provided with The non-human primate studies were also conducted at Shin Nippon Biomedical Laboratories. These studies were approved by an Institutional Animal Care and Use Committee  and were performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories Drug Safety Research Laboratories. The studies were performed as non-Good Laboratory Practice (GLP) experiments in male or female cynomolgus monkeys. The monkey age at initiation of treatment was 2–4 years. The monkeys were housed under a 12-h light/12-h dark cycle (lights on from 7:00 AM to 7:00 PM) and fed once daily with ∼100 g of solid food ; for enrichment, animals were provided with apple slices 5 or more times per week. After dosing or collection of blood, the monkeys were provided with raisins or peanuts as treats.A weekly dose-escalation study of HsPCSK9-311-AM, HsPCSK9-1811-AM, and HsPCSK9-1811-LNA was used to evaluate the efficacies of these ASOs. Monkeys that had relatively high LDL-C levels were selected from the monkeys at Shin Nippon Biomedical Laboratories. The candidate ASOs were administered subcutaneously to the monkeys  at a starting dose of 1 mg/kg (on day 0), followed by 3 mg/kg on day 7 and 10 mg/kg on day 14. Blood samples were collected on days 0 (before dose administration), 1, 4, 6, 7 (before dose administration), 8, 11, 13, 14 (before dose administration), 15, 18, 20, 22, 24, 28, 31, 35, and 38. Monkeys treated with HsPCSK9-1811-LNA received an additional dose of 3 mg/kg on day 42  and a final dose of 1 mg/kg on day 61. For animals administered the additional doses, additional blood samples were collected on days 42 (before dose administration), 46, 48, 52, 56, 59, 61 (before dose administration), 65, 67, 71, 75, 78, 82, 85, 92, and 99.g for 10 min, and the resulting serum supernatants were stored at −80°C until analysis. Liver and kidney tissues were fixed in 10% neutral buffered formalin.For all rat studies, animals were 6 weeks old at the initiation of dosing. Each ASO was administered to male or female rats (n = 5) by subcutaneous injection at 0.3, 1, 3, 10, 30, or 100 mg/kg or an equivalent volume of 0.9% saline for a control group (n = 5); doses were administered once weekly for 2 weeks . Urine was collected on days 5 and 12. Fourteen days after the first administration, blood samples were collected via the inferior vena cava, animals were euthanized, and liver and kidney tissues were harvested. After clotting, the blood samples were centrifuged at 1,700 × To evaluate the safety of HsPCSK9-1811-LNA in monkey, a 2-week study was conducted in the same manner as the safety test using rats. HsPCSK9-1811-LNA was administered subcutaneously in monkeys (n = 2) at 10 or 30 mg/kg, once weekly for 2 weeks . Urine was collected on days 2, 6, 9, 13, and 14. Blood samples were collected on days −2, 3, 7, 10, and 14; serum was isolated and stored as described above. Fourteen days after the first administration, animals were euthanized, and liver and kidney tissues were harvested and fixed in 10% neutral buffered formalin.To evaluate the efficacy of HsPCSK9-1811-LNA-GN in monkey, a single-dose study was conducted. One monkey was administered subcutaneously with HsPCSK9-1811-LNA-GN at a single dose of 0.3 or 1 mg/kg. After the first administration, blood samples were collected on days 1, 4, 6, 8, 11, 13, 18, 21, 25, 28, 32, 35, 39, 46, and 53; serum was isolated and stored as described above.Serum ALT, aspartate aminotransferase (AST), creatinine, urea nitrogen, total cholesterol, LDL-C, urinary GGT, and protein levels were measured with an automated analyzer . Serum PCSK9 protein levels were determined with the Human PCSK9 Quantikine ELISA Kit . Urinary Kim-1 levels were determined with xMAP technology using a Bio-Plex 200 Systems (Bio-Rad) kit according to the manufacturer’s instructions.The formalin-fixed tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The histopathological analyses were conducted at Shin Nippon Biomedical Laboratories. The scores were classified into five grades according to the histopathological findings.In vitro dose-dependent test and rat study data are presented as mean ± standard deviation (SD). To compare the efficacy of LNA- and AmNA-modified ASOs, Pearson’s correlation coefficients  were calculated, and single linear regression analysis was performed. Statistical significance (p < 0.05) was determined by Dunnett’s multiple comparison tests. Pearson’s correlation coefficients and statistical significance were analyzed with the statistical programming language R.Data supporting the findings of this study are available from the corresponding author upon reasonable request.


C) is composed of a large intrinsically disordered N-terminal and a structured C-terminal domain, containing three alpha-helical regions and a short, two-stranded beta-sheet. Traditionally, the activity of a protein was linked to the ability of the polypeptide chain to adopt a stable secondary/tertiary structure. This concept has been extended when it became evident that intrinsically disordered domains (IDDs) can participate in a broad range of defined physiological activities and play a major functional role in several protein classes including transcription factors, scaffold proteins, and signaling molecules. This ability of IDDs to engage in a variety of supramolecular complexes may explain the large number of PrPC-interacting proteins described. Here, we summarize diverse physiological and pathophysiological activities that have been described for the unstructured N-terminal domain of PrPC. In particular, we focus on subdomains that have been conserved in evolution.The mammalian prion protein (PrP C) into PrPSc, an aberrantly folded conformer with neurotoxic and infectious properties [C in the formation of PrPSc and infectious prions is highlighted in PrPC-deficient mice and goats, which are resistant to prion infection [C is characterized by a series of co- and posttranslational modifications  was first identified as a protease-resistant protein in brain extracts, which co-purified with the infectious scrapie agent . The ideoperties . The cennfection ,6. Biogeons (see ). After ons see ,9,10,11 ,10,11 C ons  121–230), chicken (aa 121–225), turtle (aa 121–225), and xenopus (aa 90–222) PrPC show extensive structural similarities [C. However, the N-terminal domain is not only important in the context of full-length PrPC. A considerable fraction of mature PrPC is proteolytically processed in vivo, resulting in the release of two distinct soluble, unstructured N-terminal fragments. One fragment, designated N1, is formed by α-cleavage of PrPC under physiological conditions approximately at amino acid position 110. A second cleavage around amino acid position 90 (β-cleavage) is mainly observed under pathological conditions and liberates the fragment N2 [C processing plays a role in generating soluble N-terminal fragments with distinct biological activities .,30.C revC-deficient mice and goats is their resistance to prion diseases [C, such as modulation of synaptic transmission and neuronal excitability; protection against oxidative stress; neuroprotective and neurotoxic signaling; and a role in cell differentiation, neuronal adhesion, and neuro-immune crosstalk. In the context of this review, we focus specifically on biological activities of PrP that are dependent on, or are modulated by, the N-terminal domain or conserved regions therein. For an overview of activities that have been ascribed to PrP thus far, we would like to refer the reader to previous comprehensive reviews [The major phenotype of PrPdiseases ,6. Apart reviews ,41,42,43Sc that lacks aa 23–90. PrP27–30 transmits prion disease, indicating that the infectious properties of prions are not dependent on the N-terminal part of PrP comprising N2 [C into PrPSc, since propagation of infectious prions is supported in transgenic mice expressing only a truncated variant of PrPC devoid of aa 32–93 [C modulates propagation of prions. Notably, mice expressing a variant of PrPC deleted for only aa 23–31 already display a dramatically reduced susceptibility to prion infection and accumulate significantly reduced levels of PrPSc. This study highlights an important role of the PB1 domain of PrPC in the conversion into PrPSc [C(Δ23–111) remain healthy after inoculation with scrapie prions and do not accumulate protease-resistant PrPSc [The prion protein identified in proteinase K-treated brain extracts of scrapie-infected hamsters was PrP27–30, the protease K-resistant core of PrPising N2 ,44. Thisaa 32–93  or aa 23aa 32–93 . Howeverto PrPSc , a findito PrPSc ,49. On tnt PrPSc  . Excluding the octapeptide repeat insertions, all pathogenic mutations found in the N-terminal unstructured domain of PrPC are distal to amino acid 90 [C, contains two beta-strands upon conversion into PrPSc [Inherited prion diseases in humans are caused by mutations in the PrP gene  or PrPC (Δ32–134) [C suppresses the neurotoxic activity of PrPΔHD [C (Δ23–134) instead of PrPC (Δ32–134) display no clinical symptoms or neuropathology, indicating that PB1 is required for the toxic phenotype of PrPC (Δ32–134) [From a study designed to identify regions of PrPΔ32–134) . NotablyΔ32–134) , while aΔ32–134) . Althougf PrPΔHD ,54,55. TΔ32–134)  . Con. ConC coll death . Finallynstrated ,64,65. IC variants devoid of the N-terminal domain or subdomains thereof, it became apparent that some of the biological activities of full-length GPI-anchored PrPC are dependent on its N-terminal regions. We now turn to intriguing findings showing that soluble N-terminal fragments of PrPC have biological activities independently of the globular C-terminal domain. Notably, analyzing the brain interactome of soluble N1 revealed that the intrinsically disordered N-terminal domain is a major mediator of PrP interactions [From the analysis of PrPractions .It was previously shown that the stress protective activity of full-length PrP requires the N-terminal domain ,67,68. IC in mice leads to a chronic demyelinating polyneuropathy affecting Schwann cells [C-deficient goats [C in activating the G protein-coupled receptor Adgrg6. Intriguingly, this activity was dependent on PB1: substitution of the cationic residues in PB1 by alanines abolished the activity of the N-terminal fragment to activate Adgrg6 [Another interesting example for soluble N-terminal fragments showing an activity initially ascribed to GPI-anchored full-length PrP emerged from the observation that the loss of PrPnn cells , a phenont goats . In a foe Adgrg6 . 2) fusion protein. After inoculation with prions, PrP-Fc2 was not converted into PrPSc, and the onset of prion disease was delayed [2 interacts with PrPSc and thereby interferes with its toxic signaling via GPI-anchored PrPC.The first observation that a soluble PrP fragment can protect against neurotoxicity induced by pathogenic protein assemblies was made in transgenic mice expressing a secreted full-length PrP-immunoglobulin Fc  (see ,87,88,89Indeed, LLPS of full-length PrP has been described recently ,96,97,98In a recent study, we provided insight into the mechanism underlying the propensity of the mammalian prion protein to undergo LLPS . Our stuC may be associated with the activity of the PrP/Aβ complex to induce neurotoxic signaling [C and its proteolytic fragments in a cellular context and, in particular, to address the role of membrane anchoring via the GPI-anchor. Although there are only few publications to date that have examined LLPS of PrP in detail ,97,98,99ignaling ,67 faileignaling ,99. Viceignaling ,71. It w


GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.The Connexin43 gap junction gene  Ischemia-Reperfusion (I/R) injury is known to induce excessive reactive oxygen species (ROS) in mitochondria, which results in cellular dysfunction and organ damage. Interestingly, the phenomenon of ischemic preconditioning, first described 35 years ago , is a poRelated to preconditioning is the perplexing dynamic regulation of mitochondria itself. Mitochondria undergo both fission and fusion in an adaptive equilibrium which directly affects cellular activity and response to stress . It is nGJA1, which encodes Cx43, has a single coding exon and thus is not subject to splicing  and mitochondria are associated with preconditioning protection . Little splicing . Howeverisoforms . GJA1-20isoforms  by recruisoforms . In addiisoforms . GJA1-20isoforms , resultiisoforms . It is nIn the present study, we found an inverse relationship between the presence of GJA1-20k and mitochondria size in cultured cells and mice. This relationship is not affected by typical mediators of mitochondrial dynamics by Dynamin Related Protein 1 (DRP1), but rather is strongly dependent on actin dynamics. With the generation of smaller actin-associated mitochondria, we observed decreased oxygen consumption, decreased ROS generation, and profound protection against ischemia-reperfusion damage. Our data demonstrate a novel non-canonical mechanism of protective mitochondrial fission which is dependent on GJA1-20k and cytoskeletal dynamics. The findings also identify GJA1-20k as a pharmaceutical candidate for protection of organs undergoing anticipated ischemia.GJA1 expression  at residue 213 of Gja1 to UUA (Leucine). This M213L mutation removes the internal translation start site, resulting in expression of full length Cx43 protein but not the internally translated GJA1-20k isoform (M213L/M213LGja1 mice have an increase in their average area of mitochondria (Since GJA1-20k is abundantly expressed in cardiomyocytes (CMs) suggesting an important function in heart , and GJA isoform . Neonatachondria , in contchondria . Thus, bM213L/M213LGja1 mice, which lack the expression of GJA1-20k but not full-length Cx43, die from poor gap junction trafficking and arrhythmogenic sudden cardiac death two to four weeks after birth . The mitochondria of 2-week-old cardiomyocytes deficient of GJA1-20k are enlarged (M213L/M213L) had decreased copy number. However, exogenous GJA1-20k did not alter the mitochondrial biogenesis in HEK293 cells. Nor did exogenous GJA1-20k affect membrane potential or baseline ATP production  injury . We meas3L/M213L . In addimutation .M213L/WTGja1 mice to ischemia-reperfusion (I/R) injury. Since homozygous M213L/M213LGja1 mice die in 2–4 weeks after birth, we used heterozygous M213L/WTGja1 mice to be able to work with adult hearts. At baseline, adult M213L/WTGja1 mice with an approximately 50% reduction of GJA1-20k expression, have no change in overall phenotype, or cardiac chamber dimensions or cardiac functional characteristics compared to WT mice, as measured by echocardiography  of GJA1-20k induced fission, it is of interest how fission activity can be limited in stable cells, especially terminally differentiated cells such as adult cardiomyocytes. We have not yet evaluated whether secondary regulators or post-translational modification of either primary actin or GJA1-20k affects GJA1-20k mediated mitochondrial dynamics. One simple form of regulation is in the abundance of GJA1-20k. The paucity of GJA1-20k at baseline, and its large and rapid increase during the onset of ischemic stress , supportWhile smaller mitochondrial size has been traditionally associated with mitochondrial fragmentation and poor cellular health , it is n+ channels  vectors containing GFP-tagged glutathione S-transferase (GST-GFP) or GJA1-20k (GJA1-20k-GFP) driven by the cytomegalovirus (CMV) promoter through retro-orbital injection. Eight weeks post-injection, the heart dissection was performed under anesthesia by isoflurane. Heparin  was injected 20–30 min before dissection. We perfused the heart with cold HEPES buffer to wash out the blood and immediately freeze and proceed DNA extraction. The details of Gja1M213L/M213L mouse generation has been described previously (Gja1M213L/M213L and Wildtype (WT) mice using Pierce Primary Cardiomyocyte Isolation Kit  following manufacturer protocol. The neonatal cardiomyocyte was seeded into gelatin/fibronectin coated 35 mm glass-bottomed dish and subjected to imaging as described below. Adenovirus encoding GJA1-20k-V5  was transduced as previously described Gja1M213L/M213L and WT mice under anesthesia described above for the following experiments. All animal care and study protocols were approved by University of Utah Institutional Animal Care and Use Committee.All mice were maintained under sterile barrier conditions. For the exogenous gene delivery, we used C57BL/6 male mice under same conditions at the age of 8 weeks to start the study procedure according to previous study . We injeeviously . We isol2, 4.8 % Sucrose, at 4°C. Following overnight fixation, the specimens were rinsed two times in buffer and were postfixed in 2% Osmium tetroxide for 1 hr at room temperature. The specimens were rinsed in dH2O and pre-stained with uranyl acetate for 1 hr ate room temperature. Then the samples were dehydrated in graded ethanol series and three times in pure acetone then infiltrated and embedded in epoxy resin, Embed 812 . The blocks were cut at 70 nm thickness using an ultramicrotome  and poststained with uranyl acetate for 10 min, and lead citrate for 5 min. Sections were examined at an accelerating voltage of 120 kV in a JEM-1400 plus or JEM1200-EX  transmission electron microscope with CCD Gatan camera. The number and the total area of mitochondria in each image were measured using imageJ and the average area was calculated by dividing the total area by the number. Electron microscope imaging was performed by the core facility at Electron Microscopy Laboratory at University of Utah and at the Electron Imaging Center of The California NanoSystems Institute at University of California, Los Angeles.The mouse hearts were prepared as described previously . Briefly2 incubator. The cell line was tested negative for mycoplasma contamination. For imaging analysis, we coated 35 mm glass-bottomed dish with 0.1% gelatin  and human fibronectin  incubating 37°C for 2 hr or 4 °C overnight before cell seeding. We seeded the cells (2.0 × 105 cells/dish) into the coated dishes and harvested in the incubator. Next day, we transfected GST- or GJA1-20k-GFP, mCherry-DRP1, or Drp1K38A (0.5 μg/dish) with or without LifeAct-mCherry (1.0 μg/dish) plasmids as described previously (2). To knock-down Gja1, DRP1, or DNM2, we used Gja1 siRNA , DRP1 siRNA , DNM2 siRNA , and Stealth RNAiTM (Thermo Fisher Scientific) as negative control. We transfected 25 pmol of siRNA by LipofectamineTM RNAiMAX (Thermo Fisher Scientific) following manufacturer protocol. After overnight incubation, GST- or GJA1-20k-GFP plasmid was transfected as described above and the samples were subjected to subsequent experiments. To confirm the knock-down, we used six-well culture plate and proceed knock-down exactly same way and same time followed by protein extraction described below. For pharmacological DRP1 inhibition, a mitochondrial division inhibitor 1  or the equal amount of Dimethyl sulfoxide (DMSO) diluted in culture medium was added to the cells at the same time as the plasmid transfection. After overnight incubation, the samples were subjected to imaging. To disrupt the actin polymerization, the cells after the transfection were incubated for 1 hour with Latrunculin A  or the equal amount of DMSO diluted in culture medium. After the incubation, the samples were subjected to imaging.HEK293 cells were purchased from Thermo Fisher Scientific (R70007) and cultured with DMEM containing 10 % fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate (Thermo Fisher Scientific), and Mycozap-CL (Lonza) in 37°C, 5% COeviously , using Feviously . After o2. Mitochondria were labeled by incubating 37 °C for 20 minutes with Mitotracker  before imaging. To measure the mitochondrial membrane potential, we used TMRE  incubating for 20 min at 37°C before imaging. For fixed cell imaging, the cells were fixed by 4% paraformaldehyde for 30 min at room temperature. After fixation, the samples were permeabilized in 0.1% TritonX-100 in PBS for 10 min, washed with PBS 2 × 5 min, and blocked in 5% normal goat serum (NGS) in PBS for 2 hr at room temperature. The following primary antibodies were diluted in 1% NGS in PBS and incubated in dark moisture chamber at 4°C overnight; anti-GFP , anti-TOM20 , ant-DRP1 . Next day, the samples were washed with PBS 3 × 10 min and incubated with host-matched immunoglobin cross-adsorbed secondary antibodies conjugated with Alexa Fluor 488, 555, 647  in 1 % NGS in PBS for 1 hr at room temperature. After washing with PBS 3 × 10 min, the samples were mounted with ProLong Gold antifade regent with DAPI (Thermo Fisher Scientific). For image analysis, we used ImageJ with the mito-morphology plugin ) to extract total protein. The lysate was sonicated on ice, rotated for 1 hr at 4°C, and centrifuged 16,000× g for 20 min at 4°C. The supernatant was collected as the protein sample. To extract mitochondrial fraction, we used Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific) following manufacturer protocol. The protein concentration was measured using DC Protein Assay . The protein samples with sample buffer (NuPAGE LDS sample buffer (NP0007) containing 100 mM DTT) were separated using NuPAGE Bis-Tris gels (4%–12%) with 3-(N-morpholino)propanesulfonic acid (MOPS) or 2-(N-morpholino)ethanesulfonic acid (MES) running buffer (Thermo Fisher Scientific) followed by transferring to polyvinylidene difluoride (PVDF) membrane . After the transferring, the membrane was fixed by soaking in methanol, air drying, and rewetting in methanol followed by blocking with 5% non-fat milk or bovine serum albumin (for phosphorylated protein detection) in Tris-NaCl-Tween (TNT) buffer , and 0.1% Tween20) for 1 hour at room temperature. We used following primary antibodies diluted in TNT buffer; anti-Cx43 C-terminus , anti-DRP1 , anti-phospho-DRP1 at S616 , anti-phospho-DRP1 at S637 , anti-MFN1 , anti-MFN2 , anti-TOM20 , anti-Tubulin , anti-GFP , anti-PGC-1α , anti-mtTFA , anti-LC3B , anti-DNM2 , anti-actin , anti-COX IV , and anti-MEK1/2 . After overnight primary antibody incubation at 4°C, the membrane was washed with TNT buffer 3 × 10 min and incubated with host-matched immunoglobin cross-adsorbed secondary antibodies conjugated with Alexa Fluor 488, 555, 647  diluted in TNT buffer for 1 hour at room temperature. The membrane was washed with TNT buffer 3 × 10 min, soaked in methanol, and air dried followed by the band detection using Chemidoc MP imaging system (Bio-Rad). The band intensity was quantified using Image Lab software (Bio-Rad).We performed Western blot as previously described . We harvGGGGACAAGTTTGTACAAAAAAGCAGGCTTCAGGAGGTATACATATGCATCATCATCATCATCACGGTGGTGGCGGTTCAGGCGGAGGTGGCTCTGTTAAGGATCGGGTTAAGGGAAAG 3'. Reverse primer: 5' GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACTAATCGTCATCATCGTCATCATCGTCATCATCACTTCCACCACTTCCACCGATCTCCAGGTCATCAGGCCG 3'. The fusion protein was expressed in the E. coli expression strain LOBSTR , and we used an elution buffer containing 300 mM NaCl, 50 mM NaH2PO4, 150 mM Imidazole, pH 8.0, 10% glycerol. Before use, the purified protein was concentrated and subjected to a buffer exchange into a final buffer containing 50 mM NaH2PO4, 150 mM NaCl (pH 8.0) with 10% glycerol.GJA1-20k without the putative transmembrane region  was fused at the N-terminus with a 6 × His tag and a linker, and at the C-terminus with a linker and 10 Aspartic Acids. The fusion construct was cloned into the pET301/CT-DEST vector via Gateway cloning (Thermo Fisher Scientific). The primers to amplify the sequence are: Forward primer: 5' n LOBSTR   and polymerized with 0.25× Actin Polymerization Buffer  for 1 hr at room temperature. F-actin was incubated with either 15 nM LatA or an equal volume of DMSO, and/or 1 mM GJA1-20k protein purified as described above. For the polymerization assay, actin was incubated with 1 mM GJA1-20k protein before polymerization. The changes in fluorescence were measured in microplate reader  at excitation wavelength 365 nm and emission wavelength 407 nm. The data was obtained from duplicate or triplicate experimental repeats.The pyrene-actin polymerization and depolymerization assays were performed using the Actin Polymerization Biochem Kit  following the manufacturer protocol. Briefly, lyophilized pyrene-conjugated skeletal muscle actin was reconstituted and diluted to a concentration of 1 mg/ml in G-Buffer  or GST- or GJA1-20k-transfected HEK293 cells (1.0 × 104 cells/well) were seeded into Seahorse XF96 plate. Before seeding neonatal CM, the plate was coated by laminin (20 μg/ml). After 1 (HEK293) or 2 days  incubation at 37°C, OCR was measured in three time points after injection of oligomycin , arbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone , and rotenone/antimycin A . The data was normalized by protein concentration in each well. The maximum respiration was calculated by subtracting the third measurement point of R/A treatment from that of FCCP treatment. For basal respiration, we calculated the difference between the third measurement during the pre-oligomycin phase and the third measurement post-R/A. For proton leak, we calculated the difference between the third measurement during the post-oligomycin and the third measurement post-R/A. For maximum respiration, we calculated the difference between the third measurement during the post-FCCP and the third measurement post-R/A. For ATP-linked respiration, we calculated the difference between the third measurement during the pre-oligomycin phase and the third measurement post-oligomycin. For reserve capacity, we calculated the difference between the third measurement during the post-FCCP and the third measurement the pre-oligomycin phase. For non-mitochondrial respiration, we used the value at the third measurement post-R/A.The mitochondrial oxygen consumption rate (OCR) was measured using Seahorse XF96 analyzer  following the manufacturer protocol and previous study . Isolate2O2  or PBS as control diluted in culture medium for 30 min at 37°C followed by MitoSOX Red Mitochondrial Superoxide Indicator  for 10 min at 37°C or CellROX Deep Red  for 30 min at 37°C. After the incubation, the samples were subjected to live cell imaging. The intensity was calculated using the formula; Total cell intensity – (Area of the cell× Mean intensity of background) by Image J.The cells were harvested in 35 mm glass-bottomed dish and transfected plasmid as described above. The samples were treated with HM213L/WT mice were used for I/R study, as previously described  followed by 60 minutes of reperfusion. During ischemia, the hearts were immersed in warm K-H solution. After reperfusion, the hearts were sliced in 1 mm thickness and immersed in freshly made 1 % triphenyl tetrazolium chloride (TTC) solution for 20 minutes at 37 °C followed by fixation with 4 % paraformaldehyde for 30 min. Scanned image was analyzed using Image J (NIH). The area of the infarcted region of each slice was measured by semiautomatic threshold color setting, and expressed as a percentage of the total slice area. Infarct size was corrected to the weight of each slice as previously described (Adult (14- to 16-week-old) male WT or Gja1escribed  with somescribed . For dihTotal DNA was extracted using NucleoSpin Tissue  from the mouse hearts or the cells cultured in six-well plate following manufacturer protocol. The DNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific) and 5 ng/μl of DNA was subjected to Real-Time PCR analysis (Bio-Rad) using Mouse Mitochondrial DNA Copy Number Assay kit  for the samples from the mouse heart or Human Mitochondrial DNA Monitoring Primer Set (TaKaRa Bio) for the cells following manufacturer protocol.4 cells/well) were seeded into white 96-well microplate . ATP measurement was performed using ATPlite Luminescence Assay System (PerkinElmer) following manufacturer protocol and the luminescence was measured in microplate reader . Note that the cells were washed with PBS before adding lysis buffer and 10 μl of the lysate was used for protein concentration measurement as described above. The result of ATP amount was normalized by total protein amount in each well.GST- or GJA1-20k-transfected HEK293 cells . For two groups comparison, unpaired two-tail Mann-Whitney U-test was performed. For among multiple group comparison, Kruskal-Wallis test with Dunn’s post-hoc test or two-way ANOVA with Bonferroni’s post-hoc test was performed. p values of less than 0.05 was considered significant. All data points, exact p values, and statistical data are provided in the source data. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Acceptance summary:This study identifies a cardioprotective factor, GJA1-20k (a truncated form of Cx43), which appears to confer protection against ischemia/reperfusion (I/R) injury via promotion of mitochondrial fission. This finding is particularly interesting given that hyperfission is generally thought of as an index of toxicity in I/R or hypoxic injury. I/R lesion size in a GJA1 heterozygous mutant mouse is strikingly exacerbated compared to control animals, providing strong in vivo evidence supporting a role for this factor in protection from I/R.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Matt Kaeberlein as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Jason A Mears (Reviewer #3).Thank you for submitting your article "Protective mitochondrial fission induced by stress responsive protein GJA1-20k" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential RevisionsMdivi-1 is has been shown to inhibit ETC CI as well as Drp1 . Changes to mitochondrial dynamics/structure may result from effects on ETC CI. Given the importance of Drp1 independence in the conclusions, the authors need to validate their findings using another method, such as overexpression of the dominant negative Drp1 mutant K38A.K38A (or a similar approach) should also be used for the kinetic experiments.The relative change in GJA1-20k expression in heterozygous (M213L/WT) animals should be shown, and the relative changes to ETC function (Seahorse) and mitochondrial size should be shown for the heterozygous animals. Some of this is already in supplemental, it should be presented in the main figures. This is important for interpreting the data in figure 5. The specificity of the phenotype to the stressed condition is striking, but because the phenotype is only shown in the heterozygotes  the descriptive data in prior figures needs to show this setting.The authors should show mitochondrial size post-I/R in control and heterozygous animals.The H2O2 induced oxidative stress assay is not particularly informative or easy to interpret. I recommend moving this data to the supplement and focusing on additional endpoints from the mouse model in figure 5. The robustness of the phenotype in the I/R assay is much more compelling.Mechanism of Drp1 independent fission is unclear – due to other GTPases such as dynamin-2?The role of increased fission in protecting against ROS production thought to be a particularly interesting finding, but reviewers believe that mitochondria fragmented by Drp1 overexpression and suppression of Mfn1/2 or Opa1 is needed as an additional control.To begin to address whether the fission/ROS findings from HEK293 cells are relevant to the in vivo results, the authors should be repeated in a more relevant cell type, such as in neonatal cardiomyocyte cells.The authors should address the impact of GJA1-20k on mitochondrial membrane potential, ATP generating capacity, and rates of mitophagy.GJA1-20k induces a clear difference in mitochondrial size, while total mitochondrial content appears unaltered (by Tom20). Previously it was suggested that mitochondrial biogenesis occurs with increased levels of GJA1-20k. Is this a difference in the cellular model, and do the changes in cell culture accurately recapitulate the changes seen in animals?Please comment on the role of GJA1-20k in pathologic mitochondrial division during hypoxia/reoxygenation.Please discuss the any perceived therapeutic implications of the findings.Please discuss how altered mitochondrial size may alter cardiomyocyte and cardiac function. Is it possible that the benefits of GJA1-20K to infarct size come at a cost of cardiac function and reserve? Additional data to address this possibility would be helpful, but at minimum a discussion is needed.Reviewer #1 (Recommendations for the authors):This group originally found that cardiomyocyte-specific overexpression of the Cx43 truncated isoform (GJA1-20k) protects the heart against ischemia/reperfusion injury in mice, and this study support their previous study by demonstrating the cardioprotective effect of GJA1-20k peptide administration. The authors suggest that Drp1-independent mitochondrial fission and ROS production precondition the heart against severe ischemic stress. This concept is very interesting and novel, but there are several concerns that should be revised.1. The mechanism of how GJA1-20k induces Drp1-independent fission via actin accumulation to mitochondria is unclear. Please identify what drives the mitochondrial fission. Is it due to other GTPases such as dynamin-2 Nature 540, 139-143 (2016). https://doi.org/10.1038/nature20555?2. The most important point is that GJA1-20k causes mitochondrial fission with few ROS production, and attenuates mitochondrial ROS production caused by hydrogen peroxide. To avoid the possibility of an artifact imaging, they need to carry out comparative experiments with cells that have fragmented mitochondria due to overexpression of Drp1 and suppression of Mfn1/2 or Opa1.3. Most cell-level experiments use HEK293 cells, and it remains questionable whether they reflect in vivo cardiac results. Since the adenoviral gene transfer system into neonatal cardiomyocytes is performed in Figure 1, it is better to see the production of mitochondrial ROS in the same system .4. Does the GJA1-20k overexpression have impact on mitochondrial membrane potential and ATP-producing ability? Is mitochondria that are divided by GJA1-20k removed through mitophagy?5. I would like you to clarify how much this GJA1-20k-dependent mitochondrial division contributes to pathological mitochondrial division under hypoxia /reoxygenation, which can be separated from Drp1-dependent mitochondrial fission. Figure 5—figure supplement 1 compares the mitochondria of WT and Gja1M213L / WT in the physiological state.Reviewer #2 (Recommendations for the authors):The following manuscript from Shimura et al., seeks to investigate the role of a 20kD protein isoform of Cx43 (GJA1-20k) in ischemic preconditioning. The authors discovered that GJA1-20K may provide protection in ischemic hearts through polymerizing actin around mitochondria and inducing mitochondrial fission. Through a series of elegant genetic, chemical, biochemical and cell biology studies including the use of the Gja1 M213L mouse line which was generated by this group, the authors determined that the salutatory effects of GJA1-20K was due to decreased reactive oxygen species (ROS) generation from smaller mitochondria. In general, the studies are a nice extension of published work from this group who initially discovered that a wide range of Cx43 protein isoforms can be generated from internal translation initiation sites within this protein. In particular, this study now further explores the specific role of GJA1-20K in the heart and discovered that it plays a critical role in ischemic preconditioning through affecting mitochondrial size and ROS production. Although the work is well done, there are some outstanding issues that the authors should address. It would be helpful if the authors discuss more the implication of the GJA1-20K isoform as therapy as the AAV9 work suggests the possibility of gene therapy in the future. It remains unclear as to how the authors envision such a scenario. Additional discussion would be helpful as to how smaller mitochondria from GJA1-20K over expression might impact cardiomyocyte and overall cardiac function, especially after ischemia or infarction. Although GJA1-20K may reduce infarct size, could this be at the expense of overall cardiac function or reserve?Reviewer #3 (Recommendations for the authors):1. What are the levels of GJA1-20k over-expression in expts? Is it comparable to what has been described for endogenous increase observed with cardiomyocytes under stress?2. Is there any difference when comparing mitochondria in the SSM and IFM? Are both populations affected?3. The 20 kDa peptide contains some TM sequence, so why is it not targeted to the PM? Is there a MTS? A schematic earlier in the text would help orient the reader – similar to what has been shown in previous articles.4. No change in S616 phosphorylation, but why not check S637? This site is thought to be the major driver of Drp1 activation/inactivation. So changes in the phosphorylation status at 637 is critical to provide a complete evaluation of Drp1 PTM regulation.5. Is Drp1 localization altered when GJA1-20k is overexpressed? Hard to tell from Supplemental Figures No quantification.6. In Figure 5, the Seahorse data show a higher level of non-mitochondrial respiration in the animal samples. This difference also mutes the differences in Basal Respiration, Leak and Maximal Respiration. There is definitely a trend in the data, but the differences are not as convincing if the non-mitochondrial respiration is taken into account (unless I am missing something in the way that the data is presented).7. IMO, the discussion is a little long and winding. Some of it is necessary, but I think it can be more efficiently organized to better highlight the impact of your findings.8. Typo in Line 45… "stress sFriedman and". Reviewer #1 (Recommendations for the authors):This group originally found that cardiomyocyte-specific overexpression of the Cx43 truncated isoform (GJA1-20k) protects the heart against ischemia/reperfusion injury in mice, and this study support their previous study by demonstrating the cardioprotective effect of GJA1-20k peptide administration. The authors suggest that Drp1-independent mitochondrial fission and ROS production precondition the heart against severe ischemic stress. This concept is very interesting and novel, but there are several concerns that should be revised.1. The mechanism of how GJA1-20k induces Drp1-independent fission via actin accumulation to mitochondria is unclear. Please identify what drives the mitochondrial fission. Is it due to other GTPases such as dynamin-2 Nature 540, 139-143 (2016). https://doi.org/10.1038/nature20555?et al., JCI Insight, 2018). While we should not rule out other mediators, we expect GJA1-20k is a direct binding partner and regulator of actin dynamics, as suggested by Figures 3D and 3E, and is therefore responsible for actin organization around mitochondrial bodies, inducing an actin mediated scission event .We thank the Reviewer for raising this point and appreciate the novelty of introducing a type of mitochondrial fission that is independent of known mediators of fission. To address the Reviewer’s question and explore whether dynamin-2 (DNM2) is involved in the mitochondrial fission with GJA1-20k, for the revised manuscript we knocked-down DNM2 and assessed mitochondrial morphology with or without GJA1-20k. As seen in revised Figure 2—figure supplement 1 panels G–I, DNM2 knock-down does not significantly affect overall average area of mitochondria, with or without the presence of GJA1-20k. The failure of DNM2 knockdown to affect GJA1-20k mediated fission in the HEK cells we used, suggests that GJA1-20k activity on mitochondria can be independent of DNM2. Clearly there is still much to learn about GJA1-20k mediated fission. GJA1-20k is associated with the mitochondrial outer membrane  and discussion:— Results section“In addition to DRP1, Dynamin-2 (DNM2) is also a GTPase which can potentially regulate mitochondrial fission . To explore possible DNM2 involvement, we knocked-down DNM2 by siRNA and analyzed mitochondrial morphology. In contrast to siDRP1, siDNM2 alone did not affect mitochondrial morphology , with or without GJA1-20k, suggesting no involvement of DNM2 in mitochondrial dynamics of HEK293 cell lines. It has been reported that DNM2 is not necessary to be responsible for mitochondrial fission in other cell lines as well . The fact that DNM2 is not needed for GJA1-20k activity to occur, indicates it is not an essential co-factor in GJA1-20k mediated mitochondrial fission.”—Discussion sections“We have also investigated the involvement of DNM2 as another GTPase for the mitochondrial fission, but DNM2 knock-down failed to induce sufficient mitochondrial fusion or alter the effects of GJA1-20k . Therefore, it does not appear that DNM2 is essential for GJA1-20k activity on mitochondrial fission. In addition to actin, the endoplasmic reticulum (ER) membrane can be involved in mitochondrial scission . Future studies should be considered whether GJA1-20k induced actin cytoskeleton arrangements involves ER membrane as well. Given the rapidity (less than one minute) of GJA1-20k induced fission, it is of interest how fission activity can be limited in stable cells, especially terminally differentiated cells such as adult cardiomyocytes.”2. The most important point is that GJA1-20k causes mitochondrial fission with few ROS production, and attenuates mitochondrial ROS production caused by hydrogen peroxide. To avoid the possibility of an artifact imaging, they need to carry out comparative experiments with cells that have fragmented mitochondria due to overexpression of Drp1 and suppression of Mfn1/2 or Opa1.We appreciate the Reviewer’s excellent suggestion. To address this concern, we overexpressed DRP1 and measured ROS production under H2O2 stress. As expected (and predicted by the Reviewer), DRP1 overexpression caused fragmented mitochondria . In addition, while H2O2 did not induce an increase in ROS production in the presence of GJA1-20k , H2O2 did induce an increase in ROS in the presence of DRP1 overexpression . Therefore, mitochondrial fragmentation caused by DRP1 overexpression is more sensitive to oxidative stress and produces higher amount of ROS than in the presence of GJA1-20k. These data are supportive to our conclusion that GJA1-20k induces protective (rather than harmful) mitochondrial fission.In addition, to add to the rigor of the study and address concerns about cardiac specificity, we performed new intact heart experiments revealing that there are larger mitochondria, more ROS, and larger areas of infarction in GJA1-20k insufficient mice subjected to I/R injury .The revised manuscript has been modified to include the above new data  and discussion:—Results section“Given that GJA1-20k is a stress-responsive protein which can mediate ischemic preconditioning  and to explore the protective effects of GJA1-20k against oxidative stress, we measured mitochondrial generation of reactive oxygen species (ROS) in HEK293 cells under oxidative stress induced by exogenous H2O2 treatment. In unstressed cells, the presence of GJA1-20k does not affect mitochondrial ROS generation  whereas in the presence of exogenous H2O2, GJA1-20k prevents an increase in mitochondrial ROS . Thus, GJA1-20k, which depresses mitochondrial respiration in unstressed cells, does not affect homeostatic ROS generation, yet has the beneficial effect of decreasing toxic ROS generation during stress . To clarify whether the protective effects come from GJA1-20k expression or mitochondrial size, we measured ROS from HEK293 cells with DRP1 overexpression instead of GJA1-20k overexpression. DRP1 overexpression decreased in mitochondrial size similar to GJA1-20k . However, ROS generation induced by H2O2 was significantly increased and higher than GST control under H2O2 . Taken together, GJA1-20k itself is necessary for the protective mitochondrial fission.”—Discussion section“DRP1-induced fission and smaller mitochondria are associated with excess ROS production, apoptosis, and organ injury , which is also seen in Figure 5 —figure supplement 1F–I, whereas DRP1-independent mitochondrial fission has been observed in cells undergoing protective mitophagy .”—Discussion sectionM213L/WT mouse heart post-I/R . Therefore, GJA1-20k induced decreases in ROS production could limit the amount of I/R injury induced by myocardial infarction.”“Because GJA1-20k-induced fission is associated with less ROS production with oxidative stress , the endogenous generation of GJA1-20k and subsequent decreased ROS production could explain a major benefit of pre-conditioning. Of note, genetic GJA1-20k reduction increases infarct size and ROS production post-I/R injury . In addition, the population of damaged mitochondria is significantly increased in heterozygous Gja13. Most cell-level experiments use HEK293 cells, and it remains questionable whether they reflect in vivo cardiac results. Since the adenoviral gene transfer system into neonatal cardiomyocytes is performed in Figure 1, it is better to see the production of mitochondrial ROS in the same system .et al., Circ Res., 2000). Therefore, to address the Reviewer’s concern about physiological relevance, we used adult mouse heart tissue post-I/R injury and measured ROS production. As seen in new Figure 5 Panels G and H, adult heterozygous (Gja1M213L/WT) mouse hearts experience an increase in ROS production post-I/R injury compared to hearts from WT animals. Moreover, our previous study  AAV9-transduced GJA1-20k overexpressed adult cardiomyocytes decreased ROS production. Taken together, the ROS measurements using HEK293 cells in the present manuscript are consistent with the new ex vivo adult heart results.This is an excellent comment from the Reviewer although we should emphasize, in the original Figure 1, we identified a consistent phenomenon of GJA1-20k induced mitochondrial reduction or GJA1-20k removal induced mitochondrial size increase in not just HEK293 cells, but neonatal cardiomyocytes, cardiomyocytes from the hearts of young adult mice as well as from the hearts of adult mice. With regard to mitochondrial ROS generation, isolated neonatal cardiomyocytes are notoriously difficult to induce mitochondrial ROS  and discussion:—Results sectionM213L/WT mice compared to WT post-I/R . There was no significant difference in mitochondria size at the basal condition between WT and Gja1M213L/WT mice adult CMs as with neonatal CMs , whereas the mitochondria size was significantly increased after I/R injury and the heterozygous Gja1M213L/WT mice had larger mitochondria compared to WT mice post-I/R . Interestingly, the area of mitochondrial matrix was also increased, suggesting loss of cristae in Gja1M213L/WT mice heart . These data indicate that even partial deletion of GJA1-20k results in a profoundly impaired response to ischemic stress.”“However, remarkably, reduced GJA1-20k expression results in an almost complete cardiac infarction after I/R injury . Moreover, ROS production after I/R injury was increased in Gja1—Discussion sectionM213L/WT mouse heart post-I/R . Therefore, GJA1-20k induced decreases in ROS production could limit the amount of I/R injury induced by myocardial infarction.”“Because GJA1-20k-induced fission is associated with less ROS production with oxidative stress , the endogenous generation of GJA1-20k and subsequent decreased ROS production could explain a major benefit of pre-conditioning. Of note, genetic GJA1-20k reduction increases infarct size and ROS production post-I/R injury . In addition, the population of damaged mitochondria is significantly increased in heterozygous Gja14. Does the GJA1-20k overexpression have impact on mitochondrial membrane potential and ATP-producing ability? Is mitochondria that are divided by GJA1-20k removed through mitophagy?We appreciate the Reviewer’s excellent suggestions. To address these concerns, we analyzed mitochondrial membrane potential (measured by TMRE intensity), ATP production, and mitophagy (measured by LC3 intensity). As seen new Figure 1—figure supplement 2, GJA1-20k expression, or its homozygous removal, does not alter membrane potential, ATP production, or mitophagy.The revised manuscript has been modified to include the above new data  and discussion:—Results sectionM213L/M213L mouse hearts .“Previously we reported that GJA1-20k is involved in mitochondrial biogenesis . Consistent with our previous study, AAV9-transduced adult cardiomyocytes showed increased mitochondrial DNA copy number and GJA1-20k deficient mice (Gja1M213L/M213L) had decreased copy number. However, exogenous GJA1-20k did not alter the mitochondrial biogenesis in HEK293 cells. Nor did exogenous GJA1-20k affect membrane potential or baseline ATP production . In addition to mitochondrial DNA copy number, neither biogenesis nor mitophagy protein markers were altered in either GJA1-20k transfected HEK293 cells or Gja1—Discussion section“Yet the presence of GJA1-20k, while inducing mitochondrial fission and smaller mitochondria , does not either reduce MFN1 or MFN2, activate DRP1, change membrane potential, ATP production, mitochondrial biogenesis, or mitophagy .”5. I would like you to clarify how much this GJA1-20k-dependent mitochondrial division contributes to pathological mitochondrial division under hypoxia /reoxygenation, which can be separated from Drp1-dependent mitochondrial fission. Figure 5—figure supplement 1 compares the mitochondria of WT and Gja1M213L / WT in the physiological state.M213L/WT hearts to ex-vivo I/R injury and then used electron microscopy to assess mitochondrial differences. Consistent with previous studies , acute I/R injury caused mitochondrial swelling in both WT and GjaM213L/WT hearts. However, Gja1M213L/WT hearts had a greater increase in mitochondrial swelling compared to WT post-I/R . Furthermore, Gja1M213L/WT hearts had increased mitochondria matrix area (i.e. less cristae), indicating additional fragility of mitochondria from Gja1M231L/WT hearts . These data are supportive of the previous TTC staining results , a necessity of GJA1-20k for cardioprotection against acute I/R injury.We appreciate the Reviewer’s excellent suggestion. In the same protocol for response to point #3 above, we subjected adult WT and Gja1The revised manuscript has been modified to include the above new data  and discussion:—Results sectionM213L/WT mice adult CMs as with neonatal CMs , whereas the mitochondria size was significantly increased after I/R injury and the heterozygous Gja1M213L/WT mice had larger mitochondria compared to WT mice post-I/R . Interestingly, the area of mitochondrial matrix was also increased, suggesting loss of cristae in Gja1M213L/WT mice heart . These data indicate that even partial deletion of GJA1-20k results in a profoundly impaired response to ischemic stress.”“There was no significant difference in mitochondria size at the basal condition between WT and Gja1—Discussion sectionM213L/WT mouse heart post-I/R . Therefore, GJA1-20k induced decreases in ROS production could limit the amount of I/R injury induced by myocardial infarction.”“Because GJA1-20k-induced fission is associated with less ROS production with oxidative stress , the endogenous generation of GJA1-20k and subsequent decreased ROS production could explain a major benefit of pre-conditioning. Of note, genetic GJA1-20k reduction increases infarct size and ROS production post-I/R injury . In addition, the population of damaged mitochondria is significantly increased in heterozygous Gja1Reviewer #2 (Recommendations for the authors):The following manuscript from Shimura et al., seeks to investigate the role of a 20kD protein isoform of Cx43 (GJA1-20k) in ischemic preconditioning. The authors discovered that GJA1-20K may provide protection in ischemic hearts through polymerizing actin around mitochondria and inducing mitochondrial fission. Through a series of elegant genetic, chemical, biochemical and cell biology studies including the use of the Gja1 M213L mouse line which was generated by this group, the authors determined that the salutatory effects of GJA1-20K was due to decreased reactive oxygen species (ROS) generation from smaller mitochondria. In general, the studies are a nice extension of published work from this group who initially discovered that a wide range of Cx43 protein isoforms can be generated from internal translation initiation sites within this protein. In particular, this study now further explores the specific role of GJA1-20K in the heart and discovered that it plays a critical role in ischemic preconditioning through affecting mitochondrial size and ROS production. Although the work is well done, there are some outstanding issues that the authors should address. It would be helpful if the authors discuss more the implication of the GJA1-20K isoform as therapy as the AAV9 work suggests the possibility of gene therapy in the future. It remains unclear as to how the authors envision such a scenario.We agree with the Reviewer that AAV9 mediated cardioprotection of anticipated ischemia would be the natural extension of our work. In addition, gene delivery to other ischemia sensitive organs such as the kidneys and brain should be considered. There is also the possibility of delivery of GJA1-20k peptide acutely. In unpublished preliminary studies, we have found that GJA1-20k peptide in culture is able to cross the cell membrane, enter cells and increase the cells’ resistance to oxidative and fluorescent damage. Furthermore, in a large animal (porcine) model of hypovolemic shock, we have found that intravenous injection of GJA1-20k protects against experimentally induced cardiac ischemia. The revised manuscript has been modified to include this discussion:—Discussion section“Major cardiovascular procedures are associated with low flow and ischemia related damage to end-organs such as the heart, kidneys, and brain. A priori gene therapy mediated delivery of GJA1-20k to these organs could be beneficial. For instance, percutaneous coronary interventions are associated with both cardiac and renal damage  and could benefit from GJA1-20k induced protection. Similarly, coronary bypass surgery with cardiopulmonary bypass is associated with low flow damage to the brain and kidneys , and abdominal aorta surgery is associated with renal damage, all of which may benefit from viral mediated GJA1-20k pretreatment.It is also possible, as suggested by unpublished preliminary studies by our group, that GJA1-20k peptide administration intravenously can gain access to the intracellular environments of the heart and other organs. In this scenario, GJA1-20k administration could occur minutes to hours ahead of an ischemic insult rather than the presumably days to weeks which would be needed for viral mediated introduction. Future studies will focus on acute intravenous administration of GJA1-20k peptide in subjects about to undergo anticipated ischemia.”Additional discussion would be helpful as to how smaller mitochondria from GJA1-20K over expression might impact cardiomyocyte and overall cardiac function, especially after ischemia or infarction. Although GJA1-20K may reduce infarct size, could this be at the expense of overall cardiac function or reserve?et al., JCI insight, 2018; Xiao et al., J Clin Invest, 2020). However, as the Reviewer suggests, acute administration of higher dose of GJA1-20k could still possibly result in decreased function. In fact, as we have found earlier , GJA1-20k is stress responsive and may mediate post-ischemic cardiac stunning which is typified by viable but poorly functioning myocardium post-ischemic insult. The revised manuscript has been modified to include this discussion:Neither viral mediated overexpression of GJA1-20k or development of a mouse line that reduces internal translation initiation of GJA1-20k results in a significant change in cardiac function . However acute administration of GJA1-20k or using GJA1-20k in the context of stressed myocardium could conceivably result in diminished cardiac function. As GJA1-20k is stress responsive, an increase in GJA1-20k may be a mediator of post-ischemic protection , and even responsible for the phenomenon of myocardial stunning which happens to be associated with small mitochondria . On the other hand, GJA1-20k induced functional hibernation of stressed myocardium, while increasing survival, could conceivably result in diminished cardiac function. If indeed GJA1-20k induces an acute cardiac depressant response, appropriate steps would be needed to compensate for lower cardiac output during the myocardial protection period.”Reviewer #3 (Recommendations for the authors):1. What are the levels of GJA1-20k over-expression in expts? Is it comparable to what has been described for endogenous increase observed with cardiomyocytes under stress?et al., JCI insight, 2018). Therefore, our cell line data is applicable for the GJA1-20k overexpression study, especially for acute effects.We appreciate Reviewer #3’s valuable question. In the present study, the exogenous plasmid transfection expresses approximately 5.6-fold increased GJA1-20k compared to endogenous See  . Our pr2. Is there any difference when comparing mitochondria in the SSM and IFM? Are both populations affected?We thank the Reviewer for this excellent question. On reviewing our EM data, we do not have the resolution or immunogold imaging of the older highly organized mouse hearts to permit accurate quantitative comparison between SSM and IFM mitochondria. This is an important question that we should follow-up for a future careful study dedicated to this issue, and appreciate the Reviewer’s insight in pointing out this direction.3. The 20 kDa peptide contains some TM sequence, so why is it not targeted to the PM? Is there a MTS? A schematic earlier in the text would help orient the reader – similar to what has been shown in previous articles.et al., Circ Res., 2006; and Rodriguez-Sinovas et al., Biochim Biophys Acta Biomem., 2018), which would be consistent with GJA-20k localizing to mitochondria during stress. As we have emphasized earlier, prior reports of Cx43 localization by immunocytochemistry may actually have identified GJA1-20k rather than full length Cx43 since the epitope of most anti-Cx43 antibodies is on the C-terminus and will identify GJA1-20k as well. Because GJA1-20k is hydrophilic and does not exist as membrane bound hexamers like Cx43, GJA1-20k does not require vesicle based delivery to the plasma membrane, and therefore is more available for attachment to intracellular organelles such as mitochondria.We appreciate this important question from the Reviewer. By an in silico search, we could not identify a MTS in GJA1-20k. There are reports that Hsp90 helps target Cx43 to mitochondrial membrane  appear earlier or does the Reviewer suggest a GJA1-20k specific explanatory schematic and/or sequence that is introduced earlier on so readers can differentiate GJA1-20k from full length Cx43 hemichannels?4. No change in S616 phosphorylation, but why not check S637? This site is thought to be the major driver of Drp1 activation/inactivation. So changes in the phosphorylation status at 637 is critical to provide a complete evaluation of Drp1 PTM regulation.We appreciate Reviewer #3’s thoughtful comment. We additionally analyzed phospho-DRP1 (S637) expression and found no change in the expression .The revised manuscript has been modified to include the above new data :—Results section“DRP1 is potentiated by phosphorylation of its Serine 616 and 637 (Sabouny and Shutt 2020). We tested abundance of total DRP1 and phosphorylated DRP1 in GJA1-20k transfected HEK293 cells and found no significant difference in either the protein levels of total DRP1, DRP1-pS616, -pS637, or in the ratio  . The levels of MFN1 and MFN2, and mitochondrial marker protein TOM20, a marker of cellular mitochondrial content, were also unchanged .”5. Is Drp1 localization altered when GJA1-20k is overexpressed? Hard to tell from Supplemental Figures No quantification.We appreciate Reviewer #3’s valuable question. To address this issue, we quantified amount of mitochondria associated with DRP1. As shown in Figure 2—figure supplement 1C, there was no significant difference in DRP1 localization to mitochondria between GST control and GJA1-20k transfected group.The revised manuscript has been modified to include the above new data .6. In Figure 5, the Seahorse data show a higher level of non-mitochondrial respiration in the animal samples. This difference also mutes the differences in Basal Respiration, Leak and Maximal Respiration. There is definitely a trend in the data, but the differences are not as convincing if the non-mitochondrial respiration is taken into account (unless I am missing something in the way that the data is presented).M213L/M213L mice.We would like to thank Reviewer #3 for pointing this out. As the reviewer indicates, non-mitochondrial respiration is increased in GJA1 mutant mouse hearts. In the revised manuscript we provide bar graphs indicating each OCR parameter . To calculate these parameters, non-mitochondrial respiration (third measurement point of R/A treatment) was subtracted. The data confirm maximal respiration is increased in the Gja1To clarify this point, we have updated the Methods section:—Methods section“For basal respiration, we calculated the difference between the third measurement during the pre-oligomycin phase and the third measurement post-R/A. For proton leak, we calculated the difference between the third measurement during the post-oligomycin and the third measurement post-R/A. For maximum respiration, we calculated the difference between the third measurement during the post-FCCP and the third measurement post-R/A. For ATP-linked respiration, we calculated the difference between the third measurement during the pre-oligomycin phase and the third measurement post-oligomycin. For reserve capacity, we calculated the difference between the third measurement during the post-FCCP and the third measurement the pre-oligomycin phase. For non-mitochondrial respiration, we used the value at the third measurement post-R/A.”Also, the revised manuscript has been modified to include the above new data :—Results sectionM213L/M213L mice and maximal respiration for heterozygous Gja1M213L/WT mice is between that of WT and Gja1M213L/M213L . In addition, observing other OCR parameters, we found a decrease in ATP-linked respiration and reserve capacity in Gja1M213L/WT cardiomyocytes, and an increase in proton leak and non-mitochondrial respiration in Gja1M213L/M213L suggesting that there can be compensatory long-term effects of the Gja1 mutation .”“Similarly, maximal respiration is increased in neonatal CMs derived from GJA1-20k deficient Gja17. IMO, the discussion is a little long and winding. Some of it is necessary, but I think it can be more efficiently organized to better highlight the impact of your findings.We appreciate Reviewer #3’s thoughtful comment. In the original manuscript and especially in the revised manuscript we have included text to address potential questions from a diversity of readers, from cytoskeleton dynamics to ischemic preconditioning. We have attempted to still make the Discussion coherent but do not was to be too pithy at the expense of addressing an issue important to a particular Reviewer or reader.8. Typo in Line 45… "stress sFriedman and".We thank the reviewer for pointing this out. We corrected the typo.


In many paratuberculosis control programs, test-negative herds are assigned a preferred herd status. This may induce the false belief that these herds are free of paratuberculosis. Hence, farmers may refrain from control measures that could prevent the spread of any undetected infections. The aim of the present study was to develop a predictive model to alert farmers with test-negative herds and a preferred status in the Dutch paratuberculosis control program if they are at an increased risk of positive ELISA results in a subsequent 30-month period. On the basis of the results of this study, we conclude that discrimination of herds with high (52%) and low (17%) risks of positive ELISA results is feasible. This might help farmers with the highest risks of future positive results to make better informed decisions regarding the need to take additional control measures to prevent the spread of any undetected Map infections.c = 0.40, 25% of Status A herds would be alerted with positive and negative predictive values of 0.52 and 0.83, respectively. The model predicting ≥2 positive samples had lower positive, but higher negative, predictive values. This study indicates that discrimination of Status A herds with high and low risks of future positive ELISA results is feasible. This might stimulate farmers with the highest risks to take additional measures to control any undetected Map infections.Dairy herds participating in the Dutch milk quality assurance program for paratuberculosis are assigned a herd status on the basis of herd examinations by ELISA of individual serum or milk samples, followed by an optional confirmatory fecal PCR. Test-negative herds are assigned Status A; the surveillance of these herds consists of biennial herd examinations. Farmers falsely believing that their Status A herds are Map-free may inadvertently refrain from preventive measures. Therefore, we aimed to develop a predictive model to alert Status A farmers at increased risk of future positive ELISA results. Using data of 8566 dairy herds with Status A in January 2016, two logistic regression models were built, with the probabilities of ≥1 or ≥2 positive samples from January 2017–June 2019 as dependent variables, and province, soil type, herd size, proportion of cattle born elsewhere, time since previous positive ELISA results, and the 95th percentile of the S/P ratios in 2015–2016, as explanatory variables. As internal validation, both models were applied to predict positive ELISA results from January 2019–June 2021, in 8026 herds with Status A in January 2019. The model predicting ≥1 positive sample had an area under the receiver operating characteristics curve of 0.76 . At a cut-off predicted probability π Mycobacterium avium subsp. paratuberculosis (MAP). Paratuberculosis is of concern to dairy cattle industries worldwide because of its economic impact on dairy production [Paratuberculosis is an inflammatory bowel disease, primarily affecting ruminants, that is caused by oduction ,2 and unoduction ,4. Controduction . In seveoduction ,6.In 2006, a milk quality assurance program (MQAP) for paratuberculosis in Dutch dairy herds was initiated. The aim of this MQAP is to reduce the concentration of Map in milk delivered to the milk processing industry by controlling Map in the participating herds ,8. Herds3 organisms per liter) delivered to the milk processors [The test scheme of the MQAP was developed to provide assurance that herds with Status A have a high probability of either the absence, or a low concentration, of Map in the milk  could be adequately predicted, a logistic regression analysis was performed on data of herds that had Status A on 1 January 2016. The primary outcome of interest was whether the herd had at least one ELISA result with an S/P ≥1.0 of a sample submitted in a 30-month period between 1 January 2016 and 30 June 2018. This 30-month frame was chosen to ensure that at least one herd examination was performed in each herd, given that the interval between the herd examinations in Status A herds is approximately 24 months. The secondary outcome of interest was whether the herd had at least two samples with an S/P ≥1.0 submitted in the same 30-month period. These outcome variables were chosen because, other than the loss of Status A, they are independent of farmers’ decisions on confirmatory fecal testing of cattle with positive ELISA results.The retrospective data collection and analyses to develop the model were restricted to all herds that matched the following selection criteria: (a) The initial assessment of the herd was prior to 1 January 2011; (b) The herd had a bulk milk tank number and was participating in the MQAP on 1 January 2014, 1 January 2016, and 1 July 2018; (c) The herd was assigned Status A by 1 January 2016; (d) ≥50 ELISA results of samples submitted from the herd between 1 January 2014 and 31 December 2015; (e) On 1 January 2016, the herd had at least 50 adult cattle (≥2 years of age); (f) The S/P could be retrieved for at least one ELISA result of samples submitted from the herd between 1 January 2016 and 30 June 2018; (g) The predominant soil type of the postal code area of the herd could be retrieved.Putative explanatory variables that were included in the full logistic regression model were: the province of the Netherlands; the predominant soil type in the upper 80-cm layer of soil in the terrestrial surface area of the 4-digit postal code of the herd ; the herd size (number of cattle ≥2 years of age on 1 January 2016); the proportion of cattle present in the herd on 1 January 2016 that were born in another herd; the time elapsed since the last ELISA result with an S/P ≥1.0; and the 95th percentile of the distribution of the S/P ratios between 1 January 2014 and 31 December 2015. The 95th percentile of the S/P ratios was chosen because, in comparison with the 1st, 5th, 10th, 25th, 50th, 75th, and 90th percentiles, preliminary univariable receiver-operating characteristics (ROC) analyses indicated a higher area under the curve for prediction of at least one future positive ELISA result based on the 95th percentile. Furthermore, the 95th percentile had fewer missing values than the 99th percentile, as the 99th percentile is not meaningful in herds with less than 100 samples tested. To avoid overfitting, none of the interactions between the explanatory variables were tested. Preliminary analyses indicated nonlinearity of the effects of the time elapsed since the last ELISA result with an S/P ≥1.0 and the proportion of cattle that were born in another herd. Therefore, these variables were categorized . Multicoi to have at least one or two samples with an S/P ≥1.0 was calculated on the basis of the point estimates of the model coefficients. If the logistic regression model reads:i is calculated as:i. The performances of the predictive models were evaluated in ROC analyses on the predicted probabilities of at least one (primary outcome of interest) or two (secondary outcome of interest) samples with an S/P ≥1.0, in comparison with the observed outcome in each herd. In the ROC analyses, the proportion of herds considered to be at an increased risk of positive ELISA results at various cut-off values For both outcomes of interest, a final model was obtained in a backward elimination procedure, with a threshold probability for stepwise removal of 0.10 in the likelihood ratio test. Confounding was monitored by the changes in the regression coefficients. If elimination of a variable resulted in a change in the estimated regression coefficient of any other variable exceeding 25% or 0.1, in case of an estimate between −0.4 and 0.4, the eliminated variable was considered a potential confounder and was re-entered into the model. The model fit was evaluated with the Hosmer–Lemeshow goodness-of-fit test. To correct for overfitting, the model parameters were multiplied with a heuristic shrinkage factor :(1)SFheuAs an internal validation, the predictive models were applied to a second dataset of herds with Status A on 1 January 2019, predicting whether the herd had at least one (primary outcome of interest) or two (secondary outcome of interest) ELISA results with an S/P ≥1.0 of a sample submitted in a 30-month period between 1 January 2019 and 30 June 2021.The dataset used for this internal validation consisted of all herds that matched the following selection criteria: (a) The initial assessment of the herd was prior to 1 January 2013; (b) The herd had a bulk milk tank number and was participating in the MQAP on 1 January 2017, 1 January 2019, and 1 July 2021; (c) The herd was assigned Status A by 1 January 2019; (d) ≥50 ELISA results of the samples submitted between 1 January 2017 and 31 December 2018; (e) On 1 January 2019, the herd had at least 50 adult cattle (≥2 years of age); (f) The S/P could be retrieved for at least one ELISA result of samples submitted from the herd between 1 January 2019 and 30 June 2021; (g) The predominant soil type of the postal code area of the herd could be retrieved.The data on the explanatory variables that were included in the predictive models were retrieved in a similar manner as in the development phase, with 1 January 2019 as a reference date. The province of the Netherlands, the predominant soil type, the herd size (number of cattle ≥2 years of age), the proportion of cattle present in the herd that were born in another herd, and the time elapsed since the last ELISA result with an S/P ≥1.0 were determined on 1 January 2019. The 95th percentile of the S/P ratios was calculated using ELISA results of samples submitted between 1 January 2017 and 31 December 2018.Finally, the performance of the predictive models was evaluated in ROC analyses on the predicted probabilities of at least one (primary outcome of interest) or two (secondary outcome of interest) samples with an S/P ≥1.0, in comparison with the observed outcome in each herd.A total of 8566 dairy herds with Status A on 1 January 2016 matched all the selection criteria for the training dataset. Between 1 January 2016 and 30 June 2018, 1,230,699 samples submitted from these herds were tested by ELISA. For 271 of these samples, the S/P could not be retrieved. The distribution of the S/P of the remaining 1,230,428 samples is shown in heuristicSF equalled 0.98 and, consequently, the adjusted beta values in the predictive models only marginally differed from the estimated beta values in the final logistic regression models  predicted probability of at least one ELISA result with an S/P ≥1.0 . HoweverA total of 8026 dairy herds with Status A on 1 January 2019 matched all selection criteria for the validation dataset. Between 1 January 2019 and 30 June 2021, 1,068,046 samples submitted from these herds were tested by ELISA. For 288 of these samples, the S/P ratio could not be retrieved. The distribution of the S/P ratios of the remaining 1,067,758 samples is shown in The numbers of herds in the various levels of the predictive variables in the predictive model are shown in cπ of at least one ELISA result with an S/P ≥1.0 can be chosen that corresponds to a data point on the ROC curves  to 83%  would receive no ELISA result with an S/P ≥1.0 in the next 30 months, while the remaining 17 to 19% would receive at least one ELISA result with an S/P ≥1.0 , the model predicting ≥2 samples with an S/P ≥1.0 resulted in a lower positive predictive value and a higher negative predictive value than the model predicting ≥1 sample with an S/P ≥1.0 .The aim of the present study was to develop a predictive model to alert farmers with Status A if they are at an increased risk of positive (S/P ≥1.0) ELISA results. The resulting predictive models were able to discriminate between herds with an increased risk , and date of submission of the sample. However, few samples had an S/P between 0.9 and 1.1 . TherefoThe median values of the 95th percentile of the S/P ratios in the training and validation datasets were 0.13 and 0.14, respectively. At the low range of S/P ratios, some batch-to-batch variation may be expected, as verification of new batches of an ELISA kit is mainly focused on test performance around the cut-off values. However, the observed stability of the performance of our predictive models over time indicates that the model performance was rather insensitive to such batch-to-batch variation.c, to discriminate between herds with high and low risks of future positive results. The choice of this cut-off value needs to be based on careful judgement of the relationship between the cut-off value, the proportion of herds that will receive an alert, and the positive and negative predictive values of the alert . Given that results of herd examinations of participating herds become available at varying time points throughout the year, development of a software tool for the automated calculation of the model predictions shortly after each herd examination would seem to be a prerequisite for implementing such an alternative approach in a cost-efficient manner.External validation of predictive models is generally preferred above internal validation . In thisAt present, the prediction models developed in this study are not yet operational. Available options will be discussed with the stakeholders and decision-makers involved in the control of paratuberculosis. The practical applications of our models include management of the expectations of farmers, veterinarians, and other stakeholders. Farmers with an increased risk of positive ELISA results may use the model predictions to make informed decisions on additional control measures, such as preventive management measures, to reduce the potential spread of Map in their herds. It remains to be seen whether farmers do consider a risk in the range of 31 to 67% of positive ELISA results  and low (17%) risks of positive ELISA results is feasible. This might help Status A farmers with the highest risks of positive results to make better informed decisions regarding the need to take additional control measures to prevent the spread of any undetected Map infections.