Antisense nucleic acids are single-stranded oligonucleotides that have been specially chemically modified, which can bind to RNA expressed by target genes through base complementary pairing and affect protein synthesis at the level of posttranscriptional processing or protein translation. In recent years, the application of antisense nucleic acid technology in the treatment of neuromuscular diseases has made remarkable progress. In 2016, the US FDA approved two antisense nucleic acid drugs for the treatment of Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA), and the development to treat other neurodegenerative diseases has also entered the clinical stage. Therefore, ASO represents a treatment with great potential. The article will summarize ASO therapies in terms of mechanism of action, chemical modification, and administration methods and analyze their role in several common neurodegenerative diseases, such as SMA, DMD, and amyotrophic lateral sclerosis (ALS). This article systematically summarizes the great potential of antisense nucleic acid technology in the treatment of hereditary neurodegenerative diseases. Neurodegenerative diseases are a range of conditions that are characterized by the progressive functional and structural degeneration of the central or peripheral nervous system, and the patient's cognitive ability and athletic ability are severely impaired, placing a huge burden on the individual's family and society . NeurodeAntisense oligonucleotides (ASOs) are single-stranded oligonucleotides of 8 to 50 bases in length that specifically bind to selected RNA sequences and regulate the expression of genes by several mechanisms depending on their chemical properties and targets, which will affect protein synthesis at posttranscriptional processing or protein translation levels . In 1978Antisense oligonucleotides can regulate gene expression by recruiting RNase H to degrade target RNA or prevent splicing factors from binding through steric hindrance to treat neurodegenerative diseases.It has been found that the DNA-RNA hybrid is a substrate of the RNase H enzyme, while the main function of RNase H is to degrade RNA-DNA hybrids synthesized by lagging strands in the nucleus. Meanwhile, RNase H is also present in the cytoplasm and can degrade mature mRNA , 15. OliSplicing is a process of removing introns from the initial DNA transcription product and joining the exons to form a contiguous RNA molecule. The eukaryotic mRNA precursor is spliced in the nucleus to produce mature mRNA, which is transported to the cytoplasm and translated into protein. Alternative splicing can produce protein variants with different functions and can produce beneficial proteins or reduce harmful proteins with splicing regulation. Splicing is a complex process regulated by multiple mechanisms involving multiple proteins, such as the mRNA precursor with splicing signals and some splicing factors bind to these signal sequences to perform specific functions. The mRNA precursor has basic splicing signals such as 5′ splice site, 3′ splice site, branch point sequence, and polypyrimidine sequence, as well as regulatory signals such as silencer and enhancer , 22.ASO regulates splicing by binding to the splicing signal on mRNA precursor through base complementary pairing, thereby blocking the binding of splicing factors, which will change the original splicing pattern or lead to the activation of a new splicing site and significant changes in the inclusion of exons and ultimately obtain the desired protein product  23]. Th. Th23]. The rapid degradation of antisense oligonucleotides by endo- and exonuclease is one of the main factors affecting the effectiveness of antisense oligonucleotides. 3′-5′ exonuclease can degrade serum unprotected antisense oligonucleotides in half an hour . IncreasThe first-generation oligonucleotide modification refers to the phosphorothioate (P = S) backbone, which is achieved by replacing the nonbridging oxygen atoms in the phosphate group of the nucleotide with a sulfur atom. The addition of sulfur atoms increases the backbone negative density of the antisense nucleic acid, which can enhance the absorption efficiency of ASO by various types of cells . In addiThe glycosyl-modified ASO is called the second-generation ASO. The most important feature is that the cholesterol at the 2′position on the glycosyl is replaced by other high-stability groups and was usually used in combination with P = S in application. The second-generation ASO has three advantages: firstly, the 2′modification blocks the nucleophilic properties of the original 2′hydroxyl group and further increases the resistance of ASO to nucleases; secondly, this modification improves the thermal stability of complementary hybridization, and the increased specificity of binding makes it possible to use shorter oligonucleotide; and thirdly and finally, it can reduce the cytotoxicity caused by P = S modification –38. The The third-generation ASO combines phosphate, ribose, and nucleoside modifications, which mainly includes peptide nucleic acids (PNA) and phosphorodiamidate morpholine oligomer (PMO). In brief, PNA modification is the connection of chemically modified oligonucleotides to short peptides, where the oligonucleotides perform base complementary pairing to provide targeting, and the short peptides provide functionality such as splicing regulation or translation inhibition. PNA is uncharged, so it also has the advantages of stability and binding specificity and cannot activate RNase H , 41. HowIn neurodegenerative diseases, the destruction of the blood-brain barrier is common. It has been shown in animal models that the damaged blood-brain barrier itself can cause neurodegeneration , 47. AltFor systemic administration, ASO must be selectively transported at an appropriate concentration to cells of the brain and spinal cord tissue through the blood circulation. The general way for small molecules to pass through the blood vessel barrier is simple diffusion. Although chemical modification gives ASOs the ability to bind to hemoglobin, the molecular weight of antisense nucleic acid is relatively large that it cannot reach an effective concentration in the nervous system by simple diffusion through the blood vessel barrier. Early studies have shown that only about one percent of peripherally injected ASOs can be detected in the brain , 49. ASOMost ASOs currently used to treat neurodegenerative diseases must be administered by intraventricular injection or intrathecal injection. In these ways, it is unnecessary for ASOs to pass through the blood-brain barrier and can be transported to the cerebrospinal fluid, thereby distributing throughout the central nervous system. This method of administration has certain advantages over peripheral administration. Because of the material exchange between the cerebrospinal fluid and the brain parenchyma, the drug can be directly and quickly delivered to the nervous system and produce a higher drug concentration, which means that a smaller drug dose can be used to achieve the therapeutic effect and minimize the toxicity. Over time, ASOs gradually shift from the cerebrospinal fluid to the blood circulation and can also enter the peripheral tissues. Central nervous system administration of antisense oligonucleotides has been widely used in rodent models and nonhuman primates of neurodegenerative diseases . In clinNasal administration is an emerging mode of administration. After administration, the drug can enter the nerve center through the olfactory nerve route and the olfactory mucosal epithelial route. Although a few studies have shown that antisense oligonucleotides can be delivered to the brain by intranasal administration so far, this is still a very promising alternative delivery route. Clinical trials have shown that nasal administration of insulin can significantly improve the cognitive ability of patients with diabetes and AD . In addiSMN1) gene mutation or deletion which will lead to the lack of SMN protein. Its pathological characteristics are degeneration of motor neurons in the anterior horn of the spinal cord, neuromuscular junction necrosis, while the clinical manifestations are of myasthenia and amyotrophy. The parallel homologous gene of gene SMN1 in humans, which is called SMN2, can express a small amount of SMN protein. SMN2 gene is very similar to SMN1 gene, with only a few bases different. One of the key base differences is that the sixth base in exon 7 of SMN1 is C, while it is T in SMN2 gene. This does not affect protein coding, but it will severely affect RNA splicing. The result is that about 90% of exon 7 of the SMN2 gene is skipped, while only about 5% of SMN1 is skipped. Therefore, SMN2 gene can only produce about 10–20% of the functional SMN protein, which is insufficient to compensate for the decrease in SMN protein expression caused by SMN1 mutations or deletion. Therefore, activating SMN2 expression can be used as an important strategy for the treatment of SMA [SMA is a common autosomal recessive genetic disease. It is caused by survival of motor neuron 1 element on intron 7 to prevent the binding of the splicing inhibitor HNRNPA1/2, thereby increasing the inclusion of exon 7 MOE P = S modified ASO10-27 (nusinersen once used its name), improved motor function in SMA mouse models, and extended the life span of SMA mice by 25 times [A breakthrough in the use of antisense nucleic acids to treat SMA is that Hua and colleagues working in Cold Spring Harbor used the ASO walk method to systematically and comprehensively screen 25 times –64. Prec25 times , 66.ALS is a fatal devastating neurodegenerative disorder which predominantly affects the motor neurons in the brain and spinal cord. The death of motor neurons in ALS causes subsequent muscle atrophy, paralysis, and eventually death. The pathological mechanism of ALS has not been clarified, which is mainly caused by genetic and environmental factors; there is no effective treatment method yet , 68. AccAntisense nucleic acid drugs for the treatment of ALS mainly target the two genes C9ORF72 and SOD1. It is not completely clear how SOD1 gene mutation causes ALS. It may be due to the aggregation of the mutant protein that causes proteins to become toxic. Intrathecal administration of 2′-MOE P = S gapmer ASO targeting SOD1 mutant mRNA significantly reduces the levels of SOD1 protein, mRNA in the brain and spinal cord of rats. Preonset administration can delay disease progression and prolong the survival period . The firDMD is a rare and incurable disease due to mutations in the DMD gene located at the Xp21 locus. DMD gene mutation is the main reason for the pathology and progression of neuromuscular diseases that cause neuroatrophy. Most of the mutations found in DMD genes are deletions/duplications and are nonrandomly distributed. Due to the lack of dystrophin, there is progressive muscle weakness, which usually leads to a decrease in muscle membrane elasticity in their twenties and eventually death due to respiratory and heart failure –81.The DMD gene is one of the largest genes in the human genome, and many mutations in this gene have been reported in DMD patients. The latest research showed that, in patients with DMD, 69% have large deletions, 11% have large duplications, 10% have nonsense mutations, 7% have missense mutations, and 3% have introns or other mutations . Some reTwo ASOs with splicing regulation effects modified by 2′oMeP = S (drisapersen) and PMO (eteplirsen) have been successfully applied to induce exon 51 skipping. Yokota et al. found that intramuscular injection of drisapersen and eteplirsen in a dog model of muscular dystrophy can effectively increase exon skipping, inducing dystrophin expression and thus improving its clinical phenotype , 87. DriThe latest progress of antisense oligonucleotides in the treatment of neurodegenerative diseases is encouraging. This series of progress is the result of joint efforts of chemists, biologists, and clinicians driven by the lack of effective drugs for decades. Antisense nucleic acid technology is actually a targeted therapy, which relies on clear pathogenesis. The promotion of this technology will surely promote research in basic medicine. The success of Nusinersen et al. confirmed the broad prospects of ASOs in the treatment of neurodegenerative diseases, which also paved the way for the use of ASO strategies to treat a wider range of diseases with known pathogenesis. It is believed that this technology not only has the potential to overcome more neurological diseases in the near future but also shines in other diseases such as cancer.


The domain is then likely cleaved away immediately before, during, or immediately after the incorporation of a new CAF subunit into the nascent amyloid. We had previously identified a 21 amino acid long region, which mediates the regulatory activity of the N-terminus towards the CAF. However, many mutations in the respective segment caused misfolding and/or blocked PMEL export from the endoplasmic reticulum, leaving their phenotype hard to interpret. Here, we employ a saturating mutagenesis approach targeting the motif at single amino acid resolution. Our results confirm the critical nature of the PMEL N-terminal region and identify several residues essential for PMEL amyloidogenesis.The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Excluded from the aggregate is the regulatory N-terminus, which nevertheless must be linked  In these cells, it forms a functional amyloid matrix in melanosomes2, which are lysosome-related organelles3. The amyloid matrix serves for the deposition of the pigment melanin2. Sequestering highly oxidative reaction intermediates derived from the melanin synthesis pathway4 on PMEL fibrillar aggregates likely prevents their toxic leakage out of melanosomes and may accelerate melanin formation5. Mutations in PMEL are associated with pigmentation disorders and/or impairments of eye development in various species including fish6, chickens7, mice9, horses10, cattle11, and dogs12. In humans, mutations in PMEL were recently linked to pigment dispersion syndrome and pigmentary glaucoma13. Gain-of-function mutations in PMEL, perhaps driving toxic misaggregation, have a tendency to lead to stronger pigmentation phenotypes than loss-of-function mutations14. Besides its well-established role in pigmentation biology, PMEL is also an abundant tumor antigen highly expressed in human melanoma15. Epitopes derived from the protein are promising targets for cancer immunotherapy17. Recent reports implicate PMEL aggregation in melanoma progression18.PMEL is a pigment cell protein expressed in melanocytes and retinal pigment epithelium20. This gives rise to a ~ 120 kDa form of PMEL named P2, which is subsequently cleaved by proprotein convertases21 during passage through secretory compartments23. This cleavage results in a ~ 90 kDa N-terminal Mα fragment and a ~ 28 kDa C-terminal, membrane-tethered Mβ fragment. These fragments remain linked to each other by a disulfide bond involving cysteine-301 in a PMEL cross-dimer24. After arrival at the cell surface, the protein is endocytosed via a di-leucine-based endocytic motif and delivered into stage I melanosomes25, which represent a specialized early endosomal compartment26. Via a lumenal determinant, PMEL is then sorted into domains that invaginate inwards from the limiting melanosomal membrane in a CD63-dependent manner. These invaginations eventually pinch off, and form intralumenal vesicles (ILVs)28. Subsequently, the protease BACE2 cleaves within Mβ, which releases the Mα fragment, probably still disulfide-linked to a small piece of Mβ29, from the ILV surface and into the lumen of the developing melanosome30. We provide a detailed overview of the PMEL processing pathway in Suppl. Fig. As a type I transmembrane protein, newly synthesized PMEL is inserted into the endoplasmic reticulum (ER) membrane as a ~ 100 kDa species called P1. P1 is rapidly exported from the ER and undergoes extensive O-glycosylation, particularly in the repeat (RPT) domain, as well as maturation of various N-glycans in the Golgi31  (amino acid (aa) 26 to ~ 147), the core amyloid fragment (CAF) (aa ~ 148 to ~ 223), the polycystic kidney disease-like (PKD) domain (aa 255 to 297), and the RPT domain (302–457)34. Because bacterially produced, unglycosylated RPT domain was shown to aggregate in vitro, it was originally discussed as the leading candidate for forming the amyloid core35. However, there exists no evidence that the RPT domain forms amyloid in vivo, and the dense O-glycosylation of this fragment in vertebrate cells36, would likely shield necessary protein–protein interactions. Moreover, amyloid formed in vitro by artificial unglycosylated RPT domain fragments dissolves at neutral pH37. This is non-physiological, because pH neutralization during melanosome development40 is required for tyrosinase to be enzymatically active and produce melanin42. As a consequence, acidic pH-addicted RPT fibrils would be predicted to fall apart in vivo in the melanosome right in the moment when they are most needed, at the time when the pigment is beginning to be synthesized.It is also long known that the highly O-glycosylated RPT domain is associated with melanosomal amyloid43. Moreover, the RPT domain is not required for PMEL amyloid formation in vitro33. Recently, we demonstrated that the RPT domain is also dispensable for amyloid formation in vivo in living human cells44. Our laboratory extensively characterized PMEL amyloid formed in the absence of the RPT domain in situ in melanosomes by electron tomography36. This work demonstrated that if the RPT domain is lacking, the characteristic sheet morphology45 of the aggregated matrix collapses36. This phenomenon is dependent on RPT domain-associated O-glycans, the only conserved feature of this domain, but it is not dependent on the RPT domain amino acid sequence36. Taken together, the RPT domain is almost certainly not the amyloid-forming domain of the PMEL protein. Instead, it represents an accessory module that likely associates peripherally with the fibrillar matrix and shapes its morphology.The RPT domain is the only major PMEL domain whose sequence is not conserved in evolution, although RPT domains from all vertebrates are predicted to be highly O-glycosylated33. Moreover, fibrillar fractions from melanoma cells contain high levels of this fragment46. Alanine-scanning mutagenesis revealed numerous residues that when mutated fully suppressed or substantially reduced amyloid formation in living human cells, while the precursors of these mutants often behaved normally with respect to trafficking and processing up until arriving in melanosomes31. Various amyloid prediction algorithms identified amyloidogenic regions within the fragment31. The CAF is highly conserved in evolution, and many of the residues identified as most critical for amyloidogenesis display 100% conservation in species as diverse as mammals, birds, reptiles, amphibians, and fish31. Across these species, structure modeling predicts a β-solenoid fold for the fragment, and β-solenoids can be building blocks of amyloids47. Strikingly, the prediction of the β-solenoid structure breaks down when mutations which disrupt amyloid formation in vivo are introduced into the sequence31. Thus, we conclude that the CAF, not the RPT domain, is the major driver of amyloid formation in melanosomes.In search of the PMEL domain driving amyloidogenesis in melanosomes another candidate has emerged: the ~ 8 kDa CAF. The first evidence pointing in this direction was the demonstration by mass spectrometry that amyloid cores of in vitro-aggregated PMEL fragments contain sequences derived from the CAF31, which gives rise to the N-terminal MαN fragment (harboring the NTF and the CAF) and the C-terminal MαC fragment (harboring the PKD domain and the RPT domain)48. Although the NTF is absent from the fibrils33, it is essential for their formation44. Its function is of a regulatory nature and appears to govern the activation and/or incorporation of the CAF into nascent fibrils. To achieve this, the NTF must be linked in cis to the CAF via an intact protein backbone44. This requirement distinguishes it from the accessory RPT domain, which can associate with the amyloid in trans44. A wildtype CAF subunit linked in cis to a mutant non-functional NTF (e.g. D73K) fails to be incorporated into nascent amyloid even in the presence of functional seeds44. Thus, the NTF is not merely required for amyloid seeding, but controls the insertion of new CAF subunits into growing fibrils.First mechanistic insights have emerged into how PMEL aggregation may be regulated in vivo. Once it is proteolytically released from the ILV membrane, the Mα fragment undergoes a further cleavage between the CAF and the PKD domain44. In the respective study, seven triplets of PMEL residues were exchanged against corresponding triplets of alanine (or sometimes glycine), which resulted in loss-of-function phenotypes in all cases except for mutant 85SIA87 → AAG44. The remaining triplet mutants fell into two categories. The first category folded sufficiently well to pass ER quality control and accumulated in post-ER compartments without forming fibrils 44. Of these triplet mutants, only one was further analyzed by substituting individual amino acids, which revealed that residues Asp-73 and Pro-75 are essential for PMEL amyloid formation44. The second category of mutants misfolded more severely, causing their full retention in the ER (82ASF84 → GAA and 88LNF90 → AAA)44, where they only marginally interacted with conformation-sensitive antibodies44. Because these triplet mutants did not reach stage I melanosomes, the site of amyloid formation  substitutions on the level of individual amino acids. This allowed all constructs to be released from the ER and to vigorously react with conformation-sensitive antibodies, indicating that misfolding was largely resolved or at least significantly reduced when compared to the previously generated set of triplet mutants. Our analysis based on well-established quantitative electron microscopy, immunofluorescence, and Western blotting assays identifies several critical residues within the NTF, some of which are absolutely essential for fibril formation.44. In order to reduce broad structural effects on PMEL folding as much as possible and to identify individual amino acids that are functionally critical for fibril formation, we constructed a set of point mutants, in which individual residues were substituted with alanine or glycine. Because Asp-73, Gly-74, and Pro-75 mutants have been described elsewhere44, we did not re-include them in this analysis. Hence, our set of mutants is comprised of 18 individual alanine- (or glycine-) scanning mutants.Prior work has demonstrated that the 21 amino acid long regulatory NTF region within PMEL is structurally sensitive to mutations49. In this experimental system, we had previously determined that PMEL expression is somewhat higher (about two-fold) than endogenous PMEL levels in the human melanoma cell line buf128046. We nevertheless note that even mild overexpression may to some extent affect PMEL trafficking, processing, and/or amyloidogenesis, which should be taken into account when interpreting the data. To characterize PMEL maturation, Western blots were employed using antibodies recognizing the C-terminus of the protein . PMEL mutants mostly displayed EP4863(2) labeling consistent with this distribution  indicating largely normal trafficking. Only mutants F84A and F90A were an exception, as they did not show significant levels of newly synthesized PMEL beyond the reticular pattern likely corresponding to the ER . This is in line with our Western blotting data  analysis using antibody EP48632), which recognizes the PMEL N-terminus, which r19. However, likely because of the extremely high epitope density on PMEL fibrils, IF generally finds HMB50 labeling depleted from early secretory and stage I melanosomal compartments. Instead, practically all observable signal accumulates in compartments that contain mature fibrils, which in Mel220 cells are stage II melanosomes46. This is why the EP4863(2)-labeled and the HMB50-labeled populations of wildtype PMEL do not normally overlap in IF applications46  and HMB50 labeling by immunofluorescence was observed for PMEL mutants L77A  and F84A , indicating that Leu-77 and Phe-84 are essential for PMEL fibril formation. This overlap was also reflected in significantly higher Pearson’s coefficients for the EP4863(2) / HMB50 colocalization in the case of these mutants relative to wt-PMEL  but not all cells . These results suggest that Asn-72 and Ala-82 are important, but not essential for NTF function. We note that heterogeneous phenotypes that are stronger in some cells and weaker in other cells of the same bulk transfectant cell line are frequently observed in our experimental system and have been described previously44.In fact, overlap of EP48632) labeling and HMB50 labeling in IF applications is a well-characterized signature of nonfunctional PMEL that fails to aggregate into amyloid form labeling46. We have described previously that in Mel220 cells, lysosomes concentrate in the perinuclear area, while melanosomes distribute into a peripheral horseshoe pattern. Thus, H4A3 labeling and HMB50 labeling are typically segregated in wildtype PMEL-expressing cells . However, in previous studies we have reported many PMEL mutants displaying aberrant misaggregation of PMEL. A shared signature of these mutants is the sometimes partial and sometimes complete accumulation of PMEL aggregates in lysosomes instead of melanosomes46. In the respective cases, full overlap of H4A3 and HMB50 labeling is seen in the perinuclear region46. Interestingly, this signature of misaggregation was strongly observed for mutants V70A and G79A , indicating that Val-70 and Gly-79 are required for proper PMEL amyloid formation. In addition, N72A and A82G cells that did not display the “lack of aggregation phenotype” as discussed above, invariably showed the misaggregation signature . Thus, it seems that mutating Asn-72 and Ala-82 not only strongly reduces PMEL fibril formation overall, but that in those cells in which aggregation does occur at a low level, the process is abnormal and aggregates are largely deposited in lysosomal LAMP1high compartments. Loss-of-function mutants L77A and F84A displayed reduced colocalization with LAMP1, likely reflecting their lack of accumulation in either melanosomes or lysosomes  and HMB50, we also labeled the cells with the LAMP1-specific antibody H4A3. LAMP1 is present at high levels in lysosomes, but only at low levels in melanosomes and HMB544. Accordingly, no CAF was detected for loss-of-function mutants L77A and F84A  Fig. A. The quing Fig. B/C and Iing Fig.  and 3, ming Fig. A. This c54. The discovery that amyloids can also represent physiological structures that serve crucial cellular functions surprised many in the field, and an increasing number of examples has been found in recent years58. PMEL is one of these functional amyloids, playing a key role in vertebrate pigmentation2.The formation of amyloid was long regarded as a strictly pathological process. Amyloid fibrils and prefibrillar oligomers are well-known to damage membranes, to be toxic to cells, and to either directly cause or at least to contribute to neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s disease as well as to various other amyloidoses61, which is likely not a coincidence. Changes in pH can drive conformational rearrangements favoring aggregation, for instance by local structural unfolding leading to the exposure of new interaction interfaces that were previously buried in the protein core62. It is therefore not surprising that low pH as found in endosomes and lysosomes has been reported to support amyloid formation in both physiological as well as pathological systems70. In pH-sensitive proteins, the pKa value of the imidazole side chain of histidine, which is close to 6.1, is frequently exploited to drive pH-responsive conformational rearrangements73. The pKa value of the negatively charged carboxylate group of aspartic and glutamic acid is typically lower 74, but can shift significantly depending on the molecular environment in which these amino acids are located within a protein75. Hence, aspartic and glutamic acid residues also frequently act as pH sensors responding to moderately acidic conditions79.It has been noted that amyloid formation is often efficient in acidic organelles, such as lysosomes or early melanosomes44  Fig. A. Underlrow 5, and Fig. row 3, and Suppl. Fig. rows 2, 4 and 9, and Fig. row 2). In different cells of the same mutant cell line either the degradation pathway or the misaggregation pathway might dominate, depending on how rapidly fibrils can be cleared, which could explain some of the heterogeneity we observe in our immunofluorescence samples . Without convincing support from structural data it is hard to provide a strong hypothesis on the role that these residues and also the essential residues Leu-77 and Phe-84 are playing mechanistically during PMEL fibril formation. Nevertheless, one possibility is that they could be involved in releasing the CAF from an inhibitory interaction in response to Asp-73 protonation. This could, for instance, occur by breaking a hypothetical NTF-CAF domain interface, which one could assume is keeping the CAF in an inactive state until the final cascade of maturation and processing begins /10% FCS (HyClone) containing non-essential amino acids (Gibco), GlutaMax (Gibco), and penicillin/streptomycin (Gibco). PMEL transfectants were cultured in medium additionally containing 2 mg/ml G418 (Gibco). PMEL expression, subcellular localization, and reactivity with various specific antibodies in Mel220 cells has been extensively characterized in previous work46.LG2-MEL-220 (Mel220) is a human PMEL-deficient melanoma cell line88 employing human PMEL in pBMN-IRES-neo46 as a template in combination with primer pairs listed in Suppl. Table Human PMEL mutants were cloned using a standard QuikChange mutagenesis approach44 into Mel220 cells and selection with 2 mg/ml G418 (Gibco).All vectors were sequenced before retroviral transduction33. Pep13h is a rabbit polyclonal antiserum raised against the carboxyterminal 15 residues of PMEL59. HMB50 is a conformation-sensitive mouse monoclonal antibody recognizing an epitope in PMEL that is contained within residues 224–254, likely located in the PKD domain31. HMB45 (NeoMarkers) is a mouse monoclonal antibody recognizing residues 328–344 in the sialylated RPT domain48. EP4863(2) (Abcam) and EPR4864 (Abcam) are rabbit monoclonal antibodies recognizing the PMEL N-terminus (NTF)44 and the PMEL C-terminus36, respectively. We had previously mapped the EP4863(2) epitope to PMEL residues 28–7531. H4A3 (Abcam) is a mouse monoclonal antibody recognizing LAMP1. HRP- and fluorophore-labeled secondary antibodies were purchased from Jackson ImmunoResearch and Molecular Probes.I51 is a rabbit polyclonal antiserum raised against residues 206–220 in the PMEL CAF89. Briefly, we resuspended 5 × 106 cells in 1 ml ice-cold 10 mM Tris–HCl pH 7.4 containing protease inhibitor  and incubated on ice for 10 min. Lysed cells were Dounce-homogenized with 30 strokes using the “tight” pestle and centrifuged at 800g and 4 °C for 10 min. The resulting supernatant was spun at 45,000 rpm and 4 °C using the TLA-55 rotor in a Beckman Optima TL ultracentrifuge. The pellet was subsequently lysed in PBS/1% SDS/1% β-mercaptoethanol for 10 min at RT followed by 10 min at 95 °C and subjected to SDS-PAGE. Western blotting was carried out as described90. For compliance with journal guidelines, extended images of the films corresponding to the experiments shown in Fig. Total membranes were prepared as described91. Briefly, we fixed and permeabilized Mel220 transfectants before staining with primary antibodies in PBS/0.5% BSA/0.5% saponin for 1 h followed by staining with Alexa647-, Alexa546-, or Alexa488-conjugated secondary antibodies in the same buffer at 1:100. Primary antibodies H4A3 (0.100 mg/ml), EP4863(2) (0.357 mg/ml), and HMB50 (hybridoma supernatant) were used at 1:25, 1:100, and 1:100, respectively. Cells were mounted in ProLong Gold reagent (Invitrogen). Images were acquired using a Leica TCS SP8 confocal microscope (Leica Microsystems) at 63 × magnification.Immunofluorescence was performed as described46. Samples were viewed using an FEI Tecnai Biotwin TEM at 80 kV. Images were collected using Morada CCD and iTEM (Olympus) software.For Epon embedding, we fixed the cells in 2.5% glutaraldehyde / 2% sucrose in 0.1 M sodium cacodylate buffer pH 7.4 (NaCaCo buffer) for 30 min at RT followed by 30 min at 4 °C. Subsequently, cells were rinsed with NaCaCo buffer and further processed as describedEpon-embedded EM samples were first inspected to qualitatively assess if cells formed fibril-containing melanosomes. To quantify fibril formation, we then counted in one view field fibril-containing organelles in 15 arbitrarily chosen cells. Each count was performed once and the respective means are shown in Suppl. Fig. Supplementary Information


N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide‐antimicrobial‐Xenorhabdus” (PAX) peptide‐producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems.Non‐ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N‐ and C‐terminal docking domains ( CDD) cooperate to bind to the N‐terminal docking domain (NDD) with nanomolar affinity—the highest docking domain affinity yet observed in such megasynthases.The interaction between two non‐ribosomal peptide synthetase (NRPS) proteins mediates the biosynthesis of PAX peptides and involves an extended docking interface, where a part of the thiolation (T) domain and a minimal C‐terminal docking domain ( The reactive thiol group of the Ppant arm reacts with the aminoacyl intermediate activated by the preceding A domain and forms a thioester with the amino acid while releasing AMP. The subsequent condensation domain (C) then catalyzes the formation of a peptide bond between the amino acid bound to its preceding T domain and an amino acid bound to the T domain of the downstream module. Additional domains with specific tailoring functions such as methyltransferases or epimerization domains can be included in a functional module. The final product is cleaved off from the Ppant arm of the T domain in the last elongation module by a thioesterase (TE) domain localized at the C‐terminus of a termination module, which releases either linear, cyclic or branched cyclic peptides. Multiple modules can be located on a single protein. An extreme example in this regard is the NRPS synthesizing kolossin A which consists of 15 modules on a single polypeptide chain.Non‐ribosomal peptide synthetases (NRPSs) manufacture a very diverse range of natural products starting from amino acids. Many of these products such as daptomycin, vancomycin or bleomycin are highly relevant for clinical applications.In single‐protein NRPSs the linear order of modules from the N‐ to the C‐terminus directly determines the linear order of amino acid building blocks in the final product. This relationship is known as the collinearity rule.m.[The structural analysis of docking domains and their complexes revealed a large structural diversity.14, 15Xenorhabdus and produces “peptide‐antimicrobial‐Xenorhabdus (PAX)” peptides.1 and 2 . PaxB consists of three canonical elongation modules with a preference for arginine/lysine or lysine. PaxC contains three additional canonical elongation modules and a C‐terminal thioesterase (TE) domain which also catalyzes the cyclization between a lysine side chain amino group and the TE‐bound C‐terminus of the peptide. Based on the structure of the PAX peptides and the collinearity rule PaxA, PaxB and PaxC form a unidirectional assembly line where the C‐terminus of PaxA supposedly interacts with the N‐terminus of PaxB and the C‐terminus of PaxB with the N‐terminus of PaxC.Here, we focus on the multiprotein NRPS PaxS consisting of the three proteins PaxA, PaxB and PaxC Figure . PaxS oc2 Figure  are cyclCDD) and the PaxC N‐terminal docking domain (NDD) in the PaxS NRPS of Xenorhabdus bovienii SS‐2004.CDD/PaxC NDD complex defined a novel type of docking domain interaction.CDD/PaxC NDD pair can be grafted onto unrelated multiprotein NRPS systems to induce functional and specific interchain interactions in these systems.We recently established the structural basis for the specific interaction between the PaxB C‐terminal docking domain ‐3‐hydroxytetradecanoyl fatty acid moiety as expected. Two additional side products ‐3‐hydroxytetradec‐7‐enoyl‐SRkK (5)) and ((3R)‐3‐hydroxytetradecanoyl‐SK (6))) were detected  and the comparison of the MS2 fragmentation patterns with those of synthetic standards  . In this truncated NRPS product formation should only rely on a productive non‐covalent interaction between PaxA and PaxB. Deletion of either the putative PaxB NDD or of both the PaxB NDD and the PaxA CDD almost completely abolished product formation in vivo while a deletion of the PaxA CDD alone significantly reduced product formation to 25 % for 3 and to 50 % for 4  and 4  and a PaxB1–36 (PaxB NDD) peptide . Surprisingly, the interaction between the PaxA CDD and the PaxB NDD was too weak to be reliably quantified by isothermal titration calorimetry . However, when we titrated PaxA986–1089, a di‐domain construct containing both the entire T1 domain in its apo state and the putative CDD of PaxA, with the PaxB NDD peptide in ITC experiments we measured a KD of 201±20 nm  for this interaction with a 1:1 stoichiometry (n: 0.74±0.08). In contrast, in a titration of the isolated T1 domain of PaxA (PaxA986–1076) with the PaxB NDD peptide the interaction was again too weak to be quantified . Thus, in the PaxS NRPS from X. bovienii SS‐2004 the non‐covalent interaction between the PaxA and PaxB proteins is dependent on the presence of both the T1 and the CDD of PaxA which apparently have to cooperate to bind the PaxB NDD with an affinity that is remarkably high in comparison to the affinities for other characterized docking domain pairs.[To test the interaction of the putative PaxA 1‐CDD di‐domain construct and the PaxB NDD peptide from the PaxS NRPS from X. bovienii SS‐2004 was not suitable for a full structure determination. The closely related species Xenorhabdus cabanillasii JM26 also contains the PaxS NRPS with PaxA and PaxB . More importantly, the X. cabanillasii PaxA T1‐CDD di‐domain (PaxA981–1084) construct bound a slightly length‐optimized X. cabanillasii PaxB NDD construct (PaxB1–30) with a similarly high affinity (248±18 nm) and a 1:1 stoichiometry (n: 0.88±0.01) according to ITC . No quantifiable interactions were observed by ITC between the isolated PaxA CDD (PaxA1066–1084) and the PaxB NDD as well as between the isolated PaxA T1 domain (PaxA981–1071) and the PaxB NDD, respectively . Apparently, the T domain features the characteristic four α‐helices (α1–α4) typical of the canonical carrier protein fold.CDD are unstructured and flexible both in the framework of the di‐domain construct based on the NMR data as well as in the absence of the T1 domain according to CD spectroscopy .The secondary structure of the PaxA TNDD showed a surprisingly high α‐helical content  in contrast to the intrinsically disordered protein (IDP) character typically observed for many other docking domains in the absence of their binding partner.[The free PaxB  partner.15, 17trans peptide bond conformation based on its 13Cγ and 13Cβ chemical shifts.1H},15N‐hetNOE data for the free PaxB NDD the helix α2 from L11 to K22 is stable while the helical turn from L6 to S8 forms only transiently . The stability of α2 can be attributed to the presence of two pairs of residues with oppositely charged side chains—K15/E19 and E13/R16—spaced in a manner to enable favorable intra‐helical electrostatic interactions. The structure of the free PaxB NDD reveals a very uneven charge distribution for α2. One side of the helix is highly positively charged while the other surface is mostly hydrophobic with interspersed negative charges  which is separated from α2 by a sharp kink (ca. 83°). The kink is induced by P10 which adopts a s Figure .1‐CDD di‐domain construct and the PaxB NDD was determined based on a large number of intra‐ and intermolecular NOEs (Table S2) and its dynamics was characterized . The structural bundle representing the NMR solution structure of this complex (PDB ID: 7B2B) is shown in Figure NDD interacts extensively with the C‐terminal half of helix α4 from the T1 domain of PaxA as well as with the CDD of PaxA. Importantly, in the complex the CDD now forms a stable three‐turn α‐helix (α5) involving residues A1073–S1082. The PaxB NDD now features an extended stable N‐terminal α‐helix spanning residues N3–S8 (α1) while the sharp kink centered around P10 and helix α2 (L11–A24) are preserved. The pairs of interacting helices from PaxA (α4 and α5) and PaxB (α1 and α2) each form a V shape and the two V shapes interlock in an antiparallel orientation .The solution structure of the 1:1 complex between the PaxA T1 domain in the bound state closely corresponds to the classical right‐handed up‐and‐down four‐helical bundle fold observed in many other T domain structures in particular and carrier protein structures in general.[NDD. Importantly, the docking interface between PaxA and PaxB is located on the site of the T domain that is opposite to the site for the attachment of the Ppant arm to residue S1027  show hydrophobic interactions with amino acids in the C‐terminal part of helix α4 and of helix α5 in the PaxA T1 domain . Residues L1076, I1079 and Y1080 from the PaxA CDD (α5) add further hydrophobic interactions with both helices of the PaxB NDD  to the docking interface  suggesting that the full‐length PaxB NDD is required for a high‐affinity docking interaction. The equivalent deletion of helix α1 in our truncated X. bovienii SS‐2004 PaxS NRPS  overexpressed in E. coli strongly reduced product formation in vivo to about 12 % for 3 and about 14 % for 4 in comparison to the wild type (WT) .Our structure of the complex suggested that the extensive intermolecular contacts contributed by residues in helix α2 in the PaxB NDD, which is induced by the presence of P10 in the sequence and already preformed in the structure of the free PaxB NDD, is important for the binding affinity. Therefore, we replaced P10 by a leucine which promotes and stabilizes α‐helical conformations. According to the chemical shift index  the P10L variant adopts a conformation with a continuous straight α‐helix spanning residues L6–K25 . Accordingly, the CD spectrum of the P10L mutant showed an increased ellipticity at circa 222 nm compared to the WT  and the bound PaxB NDD  despite the presence of the mutation. This suggests that the conformation of both binding partners in the WT and the mutant complex are very similar. In particular, the continuous α‐helix in the free mutant NDD P10L apparently breaks and the kinked conformation is adopted upon binding. In agreement with such a scenario, particularly large chemical shift changes are observed for residues T7–E13 upon binding of the PaxB NDD P10L mutant to the PaxA T1‐CDD di‐domain . Thus, P10 preorganizes the conformation of the PaxB NDD in a binding‐competent conformation. The conformational preorganization in the PaxB NDD is apparently also important in vivo for efficient product formation. The introduction of the equivalent P10L mutation in our truncated X. bovienii SS‐2004 PaxS NRPS  lead to a clear reduction in product formation in vivo to about 35 % for 3 and about 20 % for 4 in comparison to the WT . Overall, the pronounced effects of the salt bridge abrogating mutations on binding demonstrated that these interactions are important for establishing the high affinity interaction between PaxA and PaxB in vitro. The introduction of the PaxB NDD R14E/K15E double mutant also strongly inhibited product formation for the truncated X. bovienii SS‐2004 PaxS NRPS  in vivo. Only approximately 5 %/10 % of the amount of 3/4 formed by the WT were detected for this mutant  and its affinity therefore higher than for many other previously described functional docking domain interactions.[Importantly, all PaxB t Figure . The effT Figure . HoweverCDD must cooperate to bind with high affinity to the PaxB NDD is not limited to PaxS‐related NRPS systems from Xenorhabdus species. A simple BlastP search of NCBI's non‐redundant protein sequence database1‐CDD and the PaxB NDD from X. cabanillasii and X. bovienii as input revealed a number of hits to putative multiprotein NRPS systems in different classes of the proteobacteria as well as in cyanobacteria . In these hits, the predicted C‐terminal T domains feature long α4 helices (ca. 4‐turns) in one protein chain of the NRPS gene cluster which precede short CDDs while a circa 35 amino acids long potential NDD with α‐helical secondary structure propensity in another protein chain precedes a C domain. Furthermore, the α4 helix regions of the T domains and the putative CDD regions are in general rich in negatively charged residues whereas the predicted NDDs contain a number of positively charged residues . Unfortunately, none of the putative NRPS systems found in this search were functionally characterized or have known peptide products . For a hit from the β‐proteobacterium Chromobacterium violaceum Bergonzini we expressed and purified the PaxA T1‐CDD di‐domain analog and the PaxB CDD analog. In an NMR titration experiment the addition of the unlabeled PaxB NDD analog to the 15N‐labeled PaxA T1‐CDD di‐domain analog caused widespread chemical shift perturbations indicative of binding . In contrast, NMR titration experiments of an isolated 15N‐labeled PaxA T1 domain analog  or an isolated PaxA CDD analog  with the PaxB NDD analog showed no obvious evidence for an interaction. Thus, also in this system the T domain and the CDD must cooperate to bind to their cognate NDD suggesting that this type of composite docking interface is widespread in NRPS and NRPS‐PKS systems from different classes of proteobacteria and also occurs in additional bacterial phyla such as the cyanobacteria .The extended docking interface observed here for the PaxS NRPS between PaxA and PaxB where the T domain and a minimal KD values in the mid‐ and low‐micromolar range. Here, we have identified a novel type of docking interaction in a multiprotein NRPS system where the C‐terminal T domain and a short C‐terminal docking domain of one NRPS protein cooperate to create a binding interface for the N‐terminal docking domain of another NRPS protein to non‐covalently assemble a productive NRPS complex. In comparison to previously described docking interactions the affinity of this interaction is significantly higher with a KD in the nanomolar range. The T domain takes part in the docking interface based on the presence of an elongated helix α4 where the C‐terminal part of this extended helix contributes to the docking interaction. Since this helix is part of the canonical carrier protein fold it is structurally pre‐organized already prior to docking. The NDD also shows a high degree of structural preorganization in its free form and only the rather short CDD behaves as an IDP in contrast to previous observations in 4 α‐helix bundle (4HB) docking domains.[NDD.Previous structural and functional studies of docking domains in megasynthases have not only uncovered their rich structural diversity but also established them as interesting building blocks for megasynthase engineering since they are able to mediate non‐covalent protein–protein interactions in multiprotein NRPS or PKS systems independently from other domains and can be transplanted between systems without loss of function.10, 11Xenorhabdus. Due to the high affinities observed for these systems they might be attractive tools for mediating non‐native intermolecular interactions in “designer” NRPS systems. However, their functional introduction into non‐native contexts will be more challenging compared to free‐standing docking domains since it will involve the re‐engineering of the sequence and structure of helix α4 of the T domain while avoiding interference with the T domain fold and its other functions. Fortunately, the contacts between helix α4 of the T domain and the NDD are limited to its C‐terminal extension, which does not interact with the core of the T domain. Thus, it might be possible to extend helix α4 and add the CDD in more conventional T domains to create a suitable docking interface for the NDD in artificial NRPS. Most likely, the high affinity provided by this type of docking interface will be beneficial for the efficiency of the assembled NRPS systems.Docking interactions involving T domains such as the one described here are apparently not limited to the PaxS NRPS but also occur in NRPS and NRPS‐PKS systems from organisms not related to The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Mycoplasma hyopneumoniae (Mhyo) is the causative agent of enzootic pneumonia in pigs which adversely affects animal health and welfare, in addition to causing considerable economical losses. This paper presents the implementation of the national Mhyo eradication program in Norway, the subsequent population wide surveillance and documentation on the current freedom from Mhyo in the Norwegian pig population.In 1994, the Board of The Norwegian Pig Health Service decided on conducting a national surveillance and eradication program for Mhyo. The program aimed for population wide freedom from Mhyo, based on serological surveillance. A partial depopulation program was initiated in all Mhyo positive farrow-to-feed and farrow-to-finish herds. Total depopulation was performed in all positive finisher herds.From 1994 to 2009, a total of 138,635 pigs in 3211 herds were serologically tested for the presence of antibodies against Mhyo. Of these, 5538 (4%) individual samples and 398 (12.4%) of the herds were defined as positive. In 2009, the Norwegian pig population was declared free from Mhyo, and has been so since then. From 2009 through 2019, a total of 44,228 individual serum samples have been analyzed for the presence of antibodies against Mhyo and found negative in the National surveillance program.Eradication of Mhyo infections has resulted in improved health and welfare of the Norwegian pig population. The success of the strategy is based on numerous factors, such as moderate to low prevalence of the agent, well documented and effective eradication protocols, accurate diagnostic tests, relatively small herds, low herd density in most parts of the country and negligible import of live pigs. In addition, economic benefit due to a premium on pigs marketed from herds free from Mhyo, a well-structured commercial pig population, and finally, the loyalty and significant effort of farmers, abattoir employees and veterinarians were crucial factors. To maintain the infection-free status at national level, a continuous alertness is required in the future to discover possible Mhyo infections and ensure rapid sampling and diagnostics. Any findings of Mhyo positive pig herds in Norway will result in immediate eradication. Mycoplasma hyopneumoniae (Mhyo) is the causative agent of enzootic pneumonia (EP) in pigs and is prevalent in pig populations worldwide [Pasteurella multocida (PMT), Actinobacillus pleuropneumoniae (APP), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine circovirus type 2 (PCV2) [orldwide . EP is aorldwide , 35. Mhy2 (PCV2) .The main clinical sign associated with Mhyo infections is a chronic non-productive cough appearing 10 to 16 days post infection , and is In order to improve animal welfare and reduce the economic losses, several control measures, including optimizing management and housing, vaccination and strategic antimicrobial medication are utilized . WhIn the 1990s, lung lesions indicative of Mhyo infections were found in 10 to 20% of Norwegian slaughter pigs scored at the abattoir , 17, 18,In 1994, the Norwegian pig production sector  and the abattoir organizations) agreed on a long-term strategy for surveillance and control of EP. The strategy included the ambition of implementing a national eradication program for Mhyo with the goal of population-wide freedom. The intermediate objectives were to maintain the Mhyo-free status of all nucleus and multiplier breeding herds and to perform a serological screening for antibodies against Mhyo in all sow herds. In addition, the strategy aimed for strictly regulated trade and thereby preventing pigs from infected herds being mixed with pigs from non-infected herds. The final objectives were to eradicate Mhyo from all pig herds in defined regions in 2–3 years, and from all herds in the country within 5 to 8 years.This paper presents the implementation of the national eradication program in Norway, the subsequent population wide surveillance and documentation on the current freedom from Mhyo in the Norwegian pig population.Between 1994 and 2009, serum or colostrum samples from a total of 138,635 pigs in 3211 herds were serologically tested for the presence of antibodies against Mhyo (Table n = 216) were tested for antibodies against Mhyo. A total of 12.5% of these (n = 27) were found positive and were obliged to perform an eradication program. In 1998 the annual serological screening for antibodies to Mhyo detected no seropositive nucleus herds and only one seropositive multiplier herd. During the years 1999 to 2003 all nucleus and multiplier herds were documented free from Mhyo. One multiplier herd in Rogaland county was reinfected by Mhyo both in 2004 and 2007, most probably from contaminated animal transport vehicles or infected neighboring herds. All nucleus and multiplier herds have been free from Mhyo since 2007.Between 1994 and 1998 all nucleus and multiplier herds  was performed. Seven of the 390 sow herds (1.8%) were found positive. In addition, in October, the multiplier herd, mentioned above, tested positive again. The infection had spread to five commercial herds through trade of live pigs from the multiplier herd. The cause of the reinfection was never discovered. Based on this finding, it was decided to test all pig herds in Rogaland during 2008 and 2009, both sow herds and finisher herds. Due to high herd density and a history of several reinfections in this area, commercial Mhyo vaccines (Respisure™ or Suvaxyn Mhyo™) were used in Mhyo positive sow herds (n = 8) in Rogaland in 2006 to 2008.In 2007 an intensive serological screening of all sow herds in Rogaland  was in 2009 roughly estimated to 7–18 million NOK [The benefits of disease-free populations of pigs include improved animal welfare, increased production at reduced cost, reduced need for antibiotics and increased job satisfaction. Several countries have established programs to create pig herds free from defined respiratory agents, such as Mhyo , but regIn Switzerland, trade of subclinically infected live animals was found to cause most outbreaks of respiratory disease during the national control program , 34. ThiTotal depopulation and repopulation (depop-repop) have long been recognized as the most successful and reliable means to eradicate disease . In addiStärk et al.  found thManagement factors such as production system, purchase of animals, animal stocking density, biosecurity measures and housing conditions are important factors in the control of respiratory infections . All-in-The commercial Norwegian pig population is organized in a pyramidal structure with unidirectional animal flow from closed pure-breed genetic nucleus herds at the apex, through multiplier herds producing hybrid sows for productions herds (farrow-to-feed or farrow-to-finish) and specialized fattening pig producers. Hence, by annual serological testing for Mhyo of all nucleus and multiplier herds and sow pool systems, the potential risk of spreading a possible infection is limited. The risk of reintroduction of Mhyo in the Norwegian pig population is relatively small as there are mandatory requirements of documented Mhyo freedom for all import of pigs from abroad, and the import itself is negligible. From 1994 to 2018 a total of 254 pigs in 11 batches were imported, only 36 of them (in two imports) after 2008 [The growing interest for outdoor pig production represents an increased concern of introducing Mhyo through direct contact between potentially infected wild boar and domesticated pigs kept outdoor. Therefore, in 2018 the Norwegian Veterinary Institute, in cooperation with the Norwegian pig production sector, initiated a surveillance program for Mhyo in wild boar. During 2018 and 2019, blood samples from a total of 92 wild boars were tested for the occurrence of antibodies against Mhyo. All samples were negative  concludeAnother potential risk factor is the use of mini pigs as companion animals. Mini pigs are not part of the surveillance program for Mhyo in pigs in Norway. Even though mini pigs are normally kept as pets, and as such away from pigs in commercial farms, there is still a risk of spreading pathogens between the two. This is exemplified by some farms having used mini pigs as teaser boars for stimulating the sows before artificial insemination. After entering the breeding herd, these teaser boars usually come in close nose-to-nose contact with a considerable portion of the sow herd within a short time period. The use of mini pigs as teaser boars is not in accordance with the Norwegian pig productions sector’s recommendations.When tracking swine respiratory disease outbreaks in endemically Mhyo infected populations, various diagnostic methods are available , but serThe success of the Norwegian eradication strategy is based on ambitious decisions made by the board of the Norwegian Pig Health Service, but several factors have been crucial to obtain and are still crucial to maintain the national status of Mhyo freedom. This includes the initial moderate to low incidence of the agent, well documented and effective eradication protocols, accurate diagnostic tests, small commercial herds in an international perspective, low herd density and negligible import of live pigs with mandatory requirements of documented Mhyo freedom for those imported. In addition, a well-structured commercial pig population with stringent adherence to unidirectional live animal flow, the economic benefit due to a premium on pigs marketed from herds free from Mhyo, and finally, the loyalty and significant effort of farmers, abattoir employees and local veterinarians were crucial factors for success.The prolonged active serosurveillance, in combination with the low prevalence of EP-like lesions in abattoirs, provides a solid basis for concluding that the commercial pig population in Norway is free from Mhyo. To maintain the infection-free status at national level, a continuous alertness from veterinarians, the food safety authorities performing clinical registrations at the abattoir, farmers and other advisors is required in the future to discover possible Mhyo infections and ensure rapid sampling and diagnostics. Any findings of Mhyo positive pig herds in Norway will result in immediate eradication.Already in 1992, four farrow-to-finish herds (two nucleus herds and two farrow-to-finish herds), with an average size of 25 sows, were selected for an Mhyo eradication attempt . The diaTo control the Mhyo status of each herd, fatteners born after completing the eradication were serologically tested at slaughter (30–113 samples per herd). In addition, lungs from all pigs slaughtered were examined macroscopically for lesions 18–33 months after the eradication program was completed. Lungs with lesions suggestive of Mhyo infection were subject to histopathological examination.All sampled fatteners in participating herds were seronegative after the eradications, and the average prevalence of gross lesions indicative of pneumonia and pleurisy recorded at slaughter in the four herds was reduced from 34.3 to 2.5 and 7.4 to 2.4%, respectively .Loss of production was minimal in all 4 units, as young animals  were transferred to other units. In two herds (the nucleus herds) the average daily weight gain (ADG) from birth to 138 days of age increased by 92 and 33 g respectively after eradication compared to before. In the two other herds the period from birth to slaughter was reduced by 1 to 1.5 months.Pasteurella multocida (PMT)), sarcoptic mange and swine dysentery (caused by Brachyspira hyodysenteriae) [Several important events led to the implementation of a screening and eradication program for Mhyo on a national level Table . Based onteriae) . In 1999The Norwegian Pig Health Service was responsible for the planning and implementation of the project, but the eradication program was primarily based on voluntary efforts from farmers and was to be a joint effort of private and farmer owned abattoirs, and Norsvin. All costs, including laboratory analyses, were paid by the pig production sector, shared in part by the affected farmers individually and in part through collective funds from a pork levy all farmers are required to pay based on kilograms delivered to the abattoir.Due to higher herd density and a higher prevalence of Mhyo positive herds in Rogaland county compared to the rest of the country, additional efforts were necessary to succeed with the eradication program here. Therefore, in 2002, a separate project group for Mhyo eradication in Rogaland was established. The project group was comprised by representatives from the Norwegian Pig Health Service, the local/regional abattoirs and the pig breeder’s organization (Norsvin SA).Mycoplasma hyopneumoniae ELISA, DAKO®, Denmark) according to the manufacturers’ instructions [In nucleus and multiplier breeding herds, blood samples from 60 and 40 conveniently sampled fatteners or sows from each herd, respectively were collected . In otheructions . All anaIf all samples were found negative, the herd was classified as free from Mhyo. All herds with three or more positive samples were defined as positive. In herds with one or two positive samples, new samples were collected, and the herd was retested before the conclusion on Mhyo status was made.All herds defined as positive for Mhyo, were contacted by an advisor or veterinarian from their abattoir to plan the eradication, at least 6 months before scheduled startup. Each eradication was thoroughly planned to minimize the production losses during the eradication period. Before eradication was initiated, the risk of reinfection from neighboring farms or transport vehicles was considered. The inseminations were planned to avoid farrowing in a period of at least 4, and preferably 6 weeks.A partial depopulation program according to the Swiss method , was iniAll units and pens were cleaned and disinfected, using sodium hypochlorite or potassium peroxymonosulfate (Virkon S®) as disinfectants. Cleaning and disinfection were preferably performed in empty units. The herds were restocked by the medicated sows and piglets born after completed eradication. In the great majority of positive herds, the eradication was performed according to this protocol, but in a few farms suckling piglets younger than 3 weeks of age were present during the eradication period. Also, in some of the Mhyo positive herds the farmer decided to discontinue the pig production. In Mhyo positive finisher herds, total depopulation by slaughtering finished pigs was performed, followed by cleaning and disinfection as described above.To confirm freedom from Mhyo after eradication, all herds were retested by analyzing blood or colostrum samples from 10 pigs and/or sows twice a year, in total 20 samples per herd. Additional sampling was performed in herds with clinical symptoms of respiratory disease and in herds where the prevalence of gross lesions indicative of pneumonia and pleurisy recorded at slaughter was high.Since 1997, all nucleus and multiplier herds have been tested for the occurrence of antibodies against Mhyo, based on blood or colostrum samples from at least ten pigs in each herd twice a year [Since 2009, all sow pool systems and samples collected from other herd categories (integrated herds and piglet-producing herds) have also been analyzed for antibodies against Mhyo. The testing performed until 2011 was organized by the Norwegian Pig Health Service. From 2012, the samples have been collected through the Surveillance Program for Specific Viral Infections in Swine Herds in Norway, implemented by the Norwegian Food Safety Authority (NFSA) . Plucks Serosurveillance for Mhyo is financed by the Norwegian Pig Health Service and analyses are performed by the Norwegian Veterinary Institute by a monoclonal blocking ELISA, as described above.


Further results showed that GPR30/G1 activation decreased MMP-9 expression via ERK1/2 inhibition, which further reduced TGF-β1 expression. Inhibition of the ERK1/2 signaling pathway by its inhibitor PD98059 suppressed the induction of the cardiomyocyte MMP-9 level caused by the GRP30 antagonist G15 and inhibited TGF-β1 expression in cardiac fibroblast in vitro. In summary, our results from in vivo and in vitro studies indicated that GPR30 activation inhibited myocardial fibrosis and preserved cardiac function via inhibiting ERK-mediated MMP-9 expression. Thus, the present study may provide the novel drug targets for prevention and treatment of cardiac pathological hypertrophy in postmenopausal women.The incidence of cardiovascular diseases was significantly increased in postmenopausal women. The protection of estrogen in the cardiovascular system has been further reported for decades. Although menopausal hormone therapy has been used in many clinical trials, the debatable results indicate that the studies for elucidating the precise molecular mechanism are urgently required. G protein–coupled estrogen receptor 30 (GPR30) is a membrane receptor of estrogen and displays protective roles in diverse cardiovascular diseases. Previous studies have revealed that ERK1/2-mediated MMP-9 signaling was involved in ischemic heart diseases. However, the role of ERK1/2-mediated MMP-9 signaling in the protection of GPR30 against cardiac hypertrophy in aged female mice has not been investigated. Our present study demonstrated that GPR30 overexpression and its agonist G1 co-administration reduced transverse aortic constriction–induced myocardial fibrosis and preserved cardiac function in aged female mice. MMP-9 expression was markedly increased  Cardiovascular disease is the leading cause of death worldwide, which accounts for 17.3 million death each year . ClinicaG protein–coupled receptor 30 (GPR30), also designated as G protein–coupled estrogen receptor (GPER), is a seven-transmembrane protein and a novel estrogen receptor, besides the two classic nuclear receptors ERα and ERβ. Previous studies have demonstrated that GPR30 is widely expressed and participates in multiple biological functions including glucose tolerance, bone growth, and blood pressure regulation . It was via matrix metalloprotein degradation  were purchased from Cell Signaling Technology. GAPDH was obtained from Santa Cruz Biotechnology . The rabbit anti-goat, goat anti-rabbit, and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company . The MMP-9 ELISA kit was purchased from Elabscience Bioengineering Institute . GPR30 agonist G1  and GPR30 antagonist G15 14,673) were purchased from Cayman Chemical . The ERK inhibitor PD98059 (HY-12028) and DOX (HY-15142) were purchased from MCE. AAV-GPR30 was purchased from the GeneChem Company . DAPI was obtained from Roche Molecular Biochemicals . The ECL reagent was purchased from Millipore .GPR30 (ab37742), vimentin (ab92547), cardiac troponin I (ab47003), TGF-β1 (ab27969), and MMP-9 38,898) were purchased from Abcam. ERK 4,695) and ad libitum. The subjects in this study were mice which were raised and used in experiments in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee.Female C57BL/6 mice  were housed in individual cages under a 12:12-h light/dark cycle (light on 06:00) at 22–24 °C and fed with regular pellet diet in vivo experiments were designed to investigate the influence of the estrogen receptor GPR30 on the cardiac function and myocardial fibrosis in hypertrophied aged female mice. Mice were randomly divided into the following four experimental groups: 1) aged female mice (AG group), 2) the AG + GPR30/G1 group, 3) the AG/TAC group, and 4) the AG/TAC + GPR30/G1 group. Mice were subjected to a tail intravenous injection of 5× 1010 adeno-associated virus genome particles carrying GPR30 in the AG + GPR30/G1 group and the AG/TAC + GPR30/G1 group, while the equal amount of empty adeno-associated virus was injected in the AG group and the AG/TAC group. Briefly, awake mice were placed in a special device to avoid the movement of mice for tail vein injection. Then the tail of the mouse was wiped with a 75% ethanol cotton ball, followed by a tail vein injection with the volume of 50 μl. Mice in the AG/TAC group and the AG/TAC + GPR30/G1 group underwent TAC operation after 3 weeks, while those in the AG group and the AG + GPR30/G1 group were underwent sham operation. Following the surgery, the AG + GPR30/G1 group and the AG/TAC + GPR30/G1 group were injected intraperitoneally with G1 each day with a dose of 35 μg/kg body weight. The cardiac function of the mouse from each group was assessed at the time of fourth and eighth weeks following the operation.All animal experiments were approved by the Institutional Animal Care and Use Committee of Fourth Military Medical University. The TAC surgery was performed to establish cardiac hypertrophy in the mouse model, as previously described . BrieflyThe ultrasound technicians were not informed of the protocol of the study and the details of the animal groups to ensure unbiased reporting. After the mouse was anesthetized with 1–2% isoflurane, the left front chest hair of the mouse was removed. Transthoracic ultrasonography was performed using a VisualSonics 2,100 echocardiograph , and a 30-MHz transducer was used to obtain the images in both parasternal long axis and short axis of the left ventricle. The indexes that can be detected through echocardiography included ejection fraction (EF)% and fraction shortening (FS)% (indicating the cardiac function), interventricular septal thickness at end diastole (IVSd), and left ventricular posterior wall thickness at end diastole (LVPWd) . The aforementioned parameters were calculated by Vevo Lab 3.1.0 software .After completion of the experiments, mice were euthanized, and their hearts were harvested. The heart weight (HW) and body weight (BW) were recorded. From these data, the HW/BW ratios were calculated.Hearts of mice used for morphological observation were fixed in 4% paraformaldehyde for 72 h, embedded in paraffin, and cut into 5-μm sections. Histomorphology was evaluated by hematoxylin and eosin (H&E) staining. Myocardial fibrosis was evaluated by Masson Staining, and the cell cross-sectional area was evaluated by wheat germ agglutinin (WGA) staining.2 incubator. The non-adherent NRCMs were transferred to another culture flask or confocal dish, and the remaining adherent cells are mostly NRCFs. NRCMs were plated at a density of 5× 105 cells per ml and were cultured in the serum containing culture medium DME/F-12 , 10% new bovine serum , penicillin (100 U/ml), streptomycin (100 U/ml), and bromodeoxyuridine (BrdU) (0.1 mM) for 48 h at 37 °C with 5% CO2. After culturing for 24 h, NRCFs were plated at a density of 5×105 cells/ml and cultured in the same medium as NRCMs.Newborn Sprague–Dawley (SD) rats were obtained from the Experimental Animal Center of the Fourth Military Medical University. Isolation and culture of neonatal rat cardiomyocytes (NRCMs) and neonatal rat cardiac fibroblasts (NRCFs) were performed, as described previously . BrieflyIn vitro experiments are designed to investigate the expression of p-ERK1/2, ERK1/2, and MMP-9 in primary cultured cardiomyocytes in the Dox + Ang Ⅱ state (simulating the aging pressure-overload stress) by the GPR30 agonist G1/antagonist G15 and the changes in the content of MMP-9 in the culture medium of each group. After the intervention of G1 or G15, the effects of the culture supernatant on cardiomyocytes and the subsequent different treatments on the expression of TGF-β1 of cardiac fibroblast were examined. The neonatal rat cardiomyocytes were isolated and divided into five groups. The cell without any treatment was considered as the control group (CON group). The cells in the Dox group were treated with 0.1 μM Dox for 24 h to mimic cell senescence, while the cells in the Dox + Ang II group were treated with 0.1 μM Dox combined with 0.1 μM Ang II for 24 h (for 24 h . Cardiomp-ERK (1:800), TGF-β1 (1:200), vimentin (1:250), or troponin Ⅰ (1:100) overnight at 4°C. Then cells were incubated with secondary antibodies for 2 h at room temperature. Nuclear staining was performed with DAPI. Tissue slides and cell slides were examined with an Olympus FV1000 laser confocal microscope . ImageJ software was used to quantify the fluorescence of tissue slides and cell slides in each group.The immunofluorescence staining experiments were performed with myocardium or isolated cells from neonatal hearts. NRCM- or NRCF-covered confocal dishes were washed with PBS following diverse treatments and then fixed with 4% paraformaldehyde at 4°C for 30 min. The tissues or cells were then treated with 0.05% Triton X-100 for permeabilization and were incubated with 1% bovine serum albumin, followed by incubation with primary antibodies targeting GPR30 (1:250), MMP-9 (1:800), p-ERK , or TGF-β1  overnight at 4°C. Then the membranes were incubated with the corresponding secondary horseradish peroxidase–conjugated antibodies  for 2 h at room temperature. The blots were visualized using enhanced chemiluminescence reagent (Millipore). GAPDH was determined as an internal loading control. The density of each band was quantified using Image Lab software .As previously described, protein samples from myocardium and cells were extracted using a RIPA buffer with a protease inhibitor and a phosphatase inhibitor, separated by 8–12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane (Millipore). Membranes were blocked in Tris-buffered saline with 0.1% Tween containing 5% non-fat milk for 2 h at room temperature, followed by incubation with primary antibodies targeting GPR30 , MMP-9 , The levels of MMP-9 in the culture supernatant of NRCMs in different treatment groups were determined with ELISA kits , following the manufacture’s instruction.p < 0.05 was considered statistically significant.All values were presented as mean ± S.E.M, and all statistical tests were performed using GraphPad Prism software . The normality of the distribution was assessed using the Shapiro–Wilk test. The statistical analysis among multiple groups was performed using one-way ANOVA followed by Bonferroni multiple comparisons test. A value of in vivo study tried to elucidate the effects of GPR30 activation on cardiac hypertrophy in aged female mice. To fully activate GPR30, the GPR30-specific agonist G1 and GPR30 adeno-associated virus were used. M-mode echocardiography results of different groups are shown in This The hallmarks of cardiac remodeling caused by pressure overload are changes in the myocardial cell size, ventricular wall thickness, and myocardial fibrosis. HE staining, Masson staining, and WAG staining were used to observe the effect of GPR30 activation on myocardial remodeling caused by pressure overload at the fourth week after TAC surgery. HE staining showed that the ventricular wall thickness was increased in the AG/TAC group compared to the AG group, while the reduction trend of ventricular wall thickness was observed in the AG/TAC + GPR30/G1 group, which was consistent with our echocardiographic data and the HW/BW ratio. The results of Masson staining  showed tTo further assess the long-term effects of GPR30 activation on cardiac hypertrophy in aged female mice, cardiac function and fibrosis were examined in 8 weeks post TAC surgery. The results showed that cardiac LVEF and LVFS in the AG/TAC group further deteriorated compared to those in the AG group, whereas those indices in the AG/TAC + GPR30/G1 group were significantly recovered compared to those in the AG/TAC group . The IVSHE staining, Masson staining, and WAG staining were employed to investigate the effects of GPR30 on cardiac remodeling caused by pressure load after 8 weeks of TAC surgery. The results of Masson staining  showed tp-ERK, ERK, MMP-9, and TGF-β1 proteins in the myocardium of aged TAC mice (p-ERK1/2 to ERK1/2 in the myocardial tissue of the AG/TAC group increased significantly, while GPR30/G1 can significantly reduce the ratio of p-ERK1/2 to ERK1/2 in the myocardial tissue compared to the AG/TAC group (Western blot results revealed that continuous intraperitoneal injection of GPR30 agonist G1 for 8 weeks combined with GPR30 overexpression affected the expressions of GPR30, TAC mice . GPR30/GTAC mice . FollowiAC group . The expAC group . MeanwhiAC group .The results of immunofluorescent staining revealed that GPR30/G1 significantly elevated the expressions of GPR30 in the TAC-injured myocardium of aged female mice . Furtherin vivo results showed that GPR30/G1 activation may be associated with the reduction of TGF-β1 and myocardial fibrosis, which may be related with ERK-regulated MMP-9 expression.As shown in p-ERK1/2 to ERK1/2 in the Dox group and the Dox + Ang II group (Primary cultured cardiomyocytes express troponin but not vimentin, and fibroblasts express vimentin but not troponin, which is consistent with the description of cardiomyocytes and cardiac fibroblasts . Dox andII group  and C. FII group  and G.The results from Western blot showed that G15 treatment upregulated phosphorylated ERK1/2 and MMP-9 expression compared to the Dox + Ang II group, while PD98059 could reverse the effects of G15 addition, as evidenced by the reduction of phosphorylated ERK1/2 and MMP-9 expression . The datWith the use of ovariectomized rodent models, we have previously explored the function of GPR30 in pathological condition such as myocardial infarction and diabetes-induced myocardial injury . Here, wSince the incidence of cardiovascular diseases differs significantly between men and women, the protection of estrogen in the cardiovascular system has been proposed and reported for decades . EspeciaEstrogen receptors play important roles to mediate multiple biological functions of estrogen. Till date, GPR30 is the only membrane receptor of estrogen that protects the heart from diverse pathological injuries . A previin vivo. These results indicated that this reduction of cardiac fibrosis was associated with suppressed ERK1/2 signaling. Meanwhile, the reduction of MMP-9 in myocardium was observed following GPR30/G1 treatments. The components of the myocardial extracellular matrix were regulated by the balance of numerous matrix metalloproteinases including MMP-9, and the disruption of this balance can cause cardiac fibrosis (via ERK1/2 signaling in H9c2 cells challenged by lipopolysaccharides (in vitro study showed that G1 treatment decreased angiotensin II and doxorubicin-induced MMP-9 expression in neonatal cardiomyocytes. The MMP-9 level in the supernatant of the cardiomyocyte culture was increased following angiotensin II and doxorubicin treatment, which was reduced by G1 treatment. Furthermore, addition of GPR30 antagonist G15 could elevate ERK1/2 phosphorylation and MMP-9 expression, which can be reversed by ERK1/2 inhibitor PD98059. By adding the culture medium from cardiomyocytes to fibroblasts, the TGF-β1 expression of fibroblasts was increased in the medium, which was treated by angiotensin II and doxorubicin, while the TGF-β1 expression of fibroblast was inhibited in the medium which was treated by G1. Taken together, these results favored the notion that GRP30 activation inhibited MMP-9 expression via ERK1/2 signaling to at least partially preserve cardiac function and inhibited myocardial fibrosis in aged female hypertrophied hearts.Furthermore, the underlying molecular mechanism of GPR30 activation on cardiac fibrosis was further clarified. The present data showed that cardiac fibrotic areas at both myocardial interstitial tissue and the perivascular space were decreased following GPR30 overexpression and G1 administration evidenced by Masson staining. The phosphorylated ERK1/2 was upregulated in response to pressure overload induced by TAC, which as a member of MAPK superfamily has been shown to contribute to pathogenesis of cardiac hypertrophy . Our datfibrosis . It was charides . Mice lacharides , which iAlthough the ovariectomized female mouse is a powerful model for studying the effects of estrogen use, aging is a more complex pathological process, instead of bilateral oophorectomy. Thus, in order to fully mimic the pathological condition of postmenopausal women, mice at the age of 18 months were used in this study. Second, despite overexpression of GPR30 by adeno-associated virus delivery in our study, we further intraperitoneally injected the agonist G1 to thoroughly activate GPR30. While our present study clarified the vital role of GPR30 activation in aged hypertrophied female hearts, there are some limitations, which can be improved in further studies. In this study, we used GPR30 adeno-associated virus to overexpress GPR30. But GPR30 knockout or transgenic mice should be used in future studies, the result of which may provide valid evidence. Second, we only observed cardiac hypertrophy and cardiac function till 8 weeks post-TAC surgery. The long-term results may indicate the potential beneficial or detrimental effects of GPR30 activation.in vitro model, the importance of ERK1/2 in mediating GPR30 protection against TAC-induced cardiac fibrosis was verified by ERK1/2 signaling inhibitor PD98059. Collectively, our results presented the new potential drug for the treatment of cardiac pathological hypertrophy in postmenopausal women.In summary, this study demonstrated that GPR30 and G1 co-administration reduced TAC-induced cardiac fibrosis and preserved cardiac contractile function in aged female hearts. These effects were attributed to GPR30 activation and the subsequent inhibition of ERK1/2-mediated MMP-9 expression. By using an