Castro et al. divided the genus Tenuipalpus into two groups, i.e., Tenuipalpus sensu stricto and Tenuipalpus sensu lato. Four new species groups of the Tenuipalpus sensu lato group are proposed in this study, considering the total number of dorsal opisthosomal setae. Additionally, diagnostic keys to new species groups and the world species of Tenuipalpus sensu lato are developed.Tenuipalpus sensu lato group are proposed in the present study based on the total number of dorsal opisthosomal setae, namely, carolinensis with ten pairs of setae (214 species), dubinini with nine pairs of setae (33 species), granati with eight pairs of setae (29 species), and barticanus with seven pairs of setae (7 species) Additionally, diagnostic keys to species groups and 273 species of the Tenuipalpus sensu lato are provided. Three species, T. lustrabilis Chaudhri, T. guptai Sadana and Gupta, and T. solanensis Sadana and Gupta, are synonymized with T. punicae Pritchard and Baker. One species, T. rodionovi Chalilova, is suggested as a junior synonym of T. granati Sayed, and eight species, T. chiococcae De Leon, T. costarricensis Salas and Ochoa, T. ephedrae Livschitz and Mitrofanov, T. molinai Evans, T. santae Manson, T. simplychus Cromroy, T. tetrazygiae De Leon, and T. oxalis (Flechtmann), belonging to the carolinensis species group, are not included in the key. Furthermore, a new species of Tenuipalpus sensu lato, T. jazanensis sp. nov., is described and illustrated based on females collected from the Chamaerops spp. (Arecaceae).Four new species groups of the  Tenuipalpus Donnadieu is the largest genus in the family Tenuipalpidae Berlese (Acari: Prostigmata: Tetranychoidea) and consists of more than 320 species, distributed worldwide, especially in the tropical and subtropical regions [T. granati Sayed, T. punicae Pritchard and Baker, and T. eriophyoides Baker, are serious pests of fruit trees worldwide [ regions ,2,3. Theorldwide .Tenuipalpus was erected by Donnadieu in 1875 [Tenuipalpus species, Extenuipalpus Reck [Aegyptopalpus Mitrofanov [Deleonipalpus Mitrofanov [Gnathopalpus Mitrofanov [Tuttlepalpus Mitrofanov [Ultratenuipalpus Mitrofanov [Tenuipalpus by using the same morphological characters and proposed six species groups, namely, albae, caudatus, elegans, granati, quadrisetosus, and trisetosus. Later, Baker and Tuttle [caudatus and proteae, based on the presence and absence of setae f2 , respectively. Further, they divided the caudatus group into three species subgroups based on the number of intercoxal setae (3a and 4a). Meyer [proteae into three species subgroups and added two more species subgroups to the caudatus group.The genus  in 1875  and Mitrpus Reck , Aegyptotrofanov , Tuttlepd Tuttle  recogniz). Meyer  followed). Meyer  and diviTenuipalpus species. Additionally, some local keys were developed over time by Meyer [Collyer  made theby Meyer , which iby Meyer  includedby Meyer  includedby Meyer  includedby Meyer  includedby Meyer  includedTenuipalpus species groups, as proposed by Baker and Tuttle [Tenuipalpus sensu stricto (36 species), with a pair of lateral projections associated with setae c3 and only one pair of setae 4a, and Tenuipalpus sensu lato (287 species), without a lateral projection associated with setae c3 and one to four pairs of setae 4a. Also, a world key to the species of Tenuipalpus sensu stricto was provided [Tenuipalpus sensu lato has been developed yet.For more than three decades, d Tuttle  and Meyed Tuttle , were cod Tuttle  divided provided ,16. HoweTenuipalpus sensu lato into new species groups based on distinct morphological characters; (ii) develop diagnostic keys to species groups and 273 species of the Tenuipalpus sensu lato; and (iii) examine specimens of Tenuipalpus species collected from different regions of Saudi Arabia.The aims of the present study were to (i) classify all species of Tenuipalpus sensu lato group was collected using different resources: the Acarology Laboratory King Saud University, Google Scholar, ResearchGate, and different acarological journals, and through personal communication . Diagnostic keys to species groups and 273 species are provided. All specimens of the genus that were collected by the Acarology lab have been examined. The mounted specimens of Tenuipalpus species were examined and identified under a phase contrast microscope . Different mite body parts were pictured using an Auto-Montage software system v4.0.1.1  and drawn with Adobe Illustrator v27.7 . All measurements are in micrometers. The terminology used in the research follows that of Mesa et al. [The published taxonomic literature of all known 287 species belonging to the Tenuipalpus sensu lato group was divided into four species groups based on the total number of dorsal opisthosomal setae, namely, the carolinensis group—with ten pairs of setae (214 species), the dubinini group—with nine pairs of setae (33 species), the granati group—with eight pairs of setae (29 species), and the barticanus group—with seven pairs of setae (seven species). This proposed division did not consider whether a specific setae was absent or not . The diagnostic keys to those four new species groups and 273 species of Tenuipalpus sensu lato were also developed.The carolinensis group, eight species were not included in the key: T. chiococcae De Leon, T. costarricensis Salas and Ochoa, T. ephedrae Livschitz and Mitrofanov, T. molinai Evans, T. santae Manson, T. simplychus Cromroy, T. tetrazygiae De Leon, and T. oxalis (Flechtmann)  . Three sTenuipalpus jazanensis sp. nov., belonging to the Tenuipalpus sensu lato group, resulted. The new species is hereby fully described and illustrated based on females collected from the European fan palm, Chamaerops spp. (Arecaceae) from the Jazan region  [s, 1834) .Diagnosis:  except the species T. elegans (Collyer) with two pairs of prodorsum setae , setae v2 absent, opisthosoma with 7–10 pairs of setae; , setae h2 elongate, flagellate; palp one- to three-segmented; venter with one to two pairs of setae 3a  and one to five pairs of setae 4a ; two pairs of pseudanal setae ps1-2 .o et al. ): ProdorTenuipalpus sensu lato Castro et al. [o et al. Diagnosis: . OpisthDiagnosis: . Opisthosoma with ten pairs of dorsal setae. This group consists of 214 species.Diagnosis: . Opisthosoma with nine pairs of dorsal setae. This group consists of 33 species.Diagnosis: . Opisthosoma with eight pairs of dorsal setae. This group consists of 29 species.Diagnosis: . Opisthosoma with seven pairs of dorsal setae. This group consists of seven species.Tenuipalpus T. carolinensis groupTenuipalpus jazanensis sp. nov.urn:lsid:zoobank.org:act:ACDFDCC0-A7D3-4B3B-8618-27E9F14A6A44Diagnosis: . Propodosoma without lateral lobes anterior marginally; propodosoma with transverse striate medially and laterally, reticulated sublaterally. Hysterosoma medially with few reticulations in the area between setae c1-c1 and sublaterally; one pair of seta 3a and five pairs of setae 4a (4a1−5) present; rostrum reaching to the middle of femur I; palp three-segmented; legs setal counts on coxae, 2–2–1–1 trochanters, 1–1–2–1; femora 4–4–2–0; genua 3–2–1–1; tibiae 5–5–3–3 and tarsus 8 (1)–8 (1)–5–5.Description Female (n = 5)c1 and sublaterally; dorsal body setae lanceolate serrate, almost equal in length, except setae h2; opisthosomal pores present; distance between setae v2–h1 339 (335–355), sc2–sc2 180 (160–240). Prodorsum slightly wider than proximal section of opisthosoma; anterior central of prodorsum with transverse striations; lateral regions with reticulations. All dorsal setae short (except h2), not more than 23 μm long; setae v2, sc1, and sc2 serrate, sc2 longer than v2 and subequal in length with sc1; opisthonotum with reticulations; opisthosomal setae serrate; setae e3 shorter than f2 and f3, h2 flagelliform and elongate serrate. Setal lengths: v2 16 (13–16), sc1 23 (20–23), sc2 23 (19–23), c1 13 (13–16), c3 13 (12–15), d1 12 (12–13), d3 14 (12–16), e1 13 (10–15), e3 12 (12–15), 2f 14 (14–16), 3f 14 (14–16), h1 14 (14–20), h2 233 (230–239). Distance between dorsal setae: v2–v2 43 (41–43), sc1–sc1 107 (95–107), c1–c1 57 (52–59), c3–c3 195 (192–197), d1–d1 32 (30–39), d3–d3 156 (160–169), e1–e1 25 (12–25), e3–e3 82 (82–90), f2–f2 71 (66–72), f3–f3 56 (52–57), h2–h2 41 (36–43), h1–h1 20 (20–23), c1–d1 45 (40–45), d1–e1 60 (60–62), c1–c3 66 (66–69), d1–d3 64 (59–65) e1–e3 41 (40–44), c3–d3 38 (30–38), d3–e3 121 (120–127), e3–f3 30 (28–36), f3–f2 10 (12–15), h1–h2 7 (7–10).Dorsum : Anterio4a with broken transverse striate. All ventral setae smooth, setae 1a, 4a flagelliform, and elongate. Setal lengths: 1a 82 (82–92), 1b 30 (22–31), 1c 30 (26–30), 2b 33 (23–33), 2c 33 (23–33), 3a 25 (25–31), 3b 30 (21–31), 4a1 90 (90–100), 4a2 83 (82–91), 4a3 83 (80–86), 4a4 76 (76–84), 4a5 73 (73–102), 4b 33 (30–33), ag 36 (26–36), g1 33 (26–34), g2 33 (25–33), ps1 23 (18–25), ps2 23 (18–23). Distance between ventral setae: 1a–1a 23 (19–24), 3a–3a 30 (30–32), 4a1–4a1 7 (5–7), 4a2–4a2 21 (19–22), 4a3–4a3 35 (31–36), 4a4–4a4 49 (42–49), 4a5–4a5 60 (57–62), 1a–3a 108 (102–110), 3a–4a1 57 (57–60), 1b–1c 14 (13–19), 2b–2c 20 (19–25).Venter : Ventralm 25 (14–25); distance between setae m–m 15 (10–15) Palps 3-segmented (d 15 (12–20); distal segment short, with eupathidium ul′ 4 (4–7) ul″ 15 (12–15).Gnathosoma : Ventralegmented , median –8 (1ω)–5–5; femur IV without seta. Leg I–IV setal count as follows (solenidia in parenthesis).Legs : Setae oMales and immature. UnknownType Materials. Holotype female, four paratype females, from Chamaerops sp. (Arecaceae) Wadi Baydh, Jazan, 17°37.559’ N, 42°22.196’ E, 10 October 2020, coll. J. H. Mirza, H.M.S. Mushtaq and E.M. Khan.Etymology: The specific epithet (jazanensis) is derived from the type region, Jazan.Remarks: The new species, Tenuipalpus jazanensis sp. nov., belongs to the carolinensis species group. This species group is distinguished from other species groups of Tenuipalpus sensu lato by having ten pairs of opisthosomal setae. Tenuipalpus jazanensis sp. nov resembles T. pareriophyoides Meyer and Gerson and T. eriophyoides Baker by having more than three pairs of intercoxal setae 4a, one pair of setae 3a, dorsal setae lanceolate serrate and dorsum with irregular striate laterally. The new species differs from T. pareriophyoides and T. eriophyoides by the following characters; prodorsum medially with transverse striations; area in between setae c1, d1 and d3 with few reticulations ; genu I with three setae (genu I with two setae); setae g and ag smooth .Tenuipalpus, e.g., Aegyptopalpus Mitrofanov [Deleonipalpus Mirofanov [Gnathopalpus Mitrofanov [Tuttlepalpus Mitrofanov [Tenuipalpus [The number of setae  has been used by different authors to erect tenuipalpid genera by transferring species from the genus trofanov , Deleoniirofanov , Gnathoptrofanov , and Tuttrofanov . Later, uipalpus .caudatus and proteae, based on the presence and absence of setae f2 , respectively. Later, these two species groups were divided into species subgroups based on the number intercoxal setae 3a and 4a were recognized [Tenuipalpus species  could not be helpful, because some species  have six of dorsolateral setae (f2 present), but the dorsolateral setae (d3) is absent. Hence, those species can not be placed in any species groups that proposed Baker and Tuttle [caudatus and proteae is based on presence and absense of setae f2. However, some previous works are considering the number of dorsolateral setae to distinguish these two species groups, regardless of which dorsolateral seta is absent. For example, Mahdavi and Asadi [T. clematidos Wang and T. salicis Al-Gboory) in the proteae species group, which have setae f2 present. Therefore, we found that using the total number of opisthosomal setae is proper for dividing species groups, while the absence or presence of certain opisthosomal setae may be used for species differentiation in diagnostic keys. Hence, in this study, the four new species groups were proposed based on the number of opisthosomal setae.Different species groups were proposed based on the number of dorsocentral setae or dorsolateral setae ,9,10. Acl-Gboory ; Khanjanl-Gboory ; Castro l-Gboory ; Xu et al-Gboory ). The lind Asadi  includedcarolinensis, which were not included in the key due to poor/incomplete descriptions and illustrations. Their related species and a possible place in the diagnostic key are provided as follows:The following remarks and additional notes are about those eight species belonging to the species group Tenuipalpus chiococcae De LeonT. chiococcae was originally described as close to T. pigrus Pritchard and Baker [T. pigrus in Tenuipalpus sensu stricto since this species bears a pair of lateral body projections associated with setae c3. The result of our study placed T. chiococcae among five species in the diagnostic key, i.e., T. aurantiacus Wang, T. angolensis Meyer, T. nigerianus Meyer, T. spatulatus Wang, and T. obvelatus Wan.The species nd Baker . The recnd Baker  placed TTenuipalpus costarricensis Salas and OchoaT. costarricencis was considered close to T. granati Sayed [T. granati is placed in the group granati, while T. costarricensis came closer to three species; T. eucleae Meyer, T. garciniae Meyer and Bolland, and T. indicus Maninder and Ghai in the species group carolinensis.Previously, ti Sayed . HoweverTenuipalpus ephedrae Livschitz and MitrofanovT. ephedrae, the morphological characters were obtained from a poor redescription [carolinensis.For the species cription , and theTenuipalpus molinai EvansT. molinai was originally described as close to T. pedrus Manson [T. pedrus Manson had been transferred to the genus Colopalpus Pritchard and Baker, previously [T. molinai belongs only to the species group carolinensis, but could not be assigned in the key.The species s Manson . The cloeviously . HoweverTenuipalpus oxalis FlechtmannT. oxalis is poorly described and illustrated, and the related species was not provided [carolinensis.The species provided . The avaTenuipalpus santae MansonT. santae is morphologically close to T. celtidis Pritchard and Baker in the original description [Tenuipalpus santae could not be placed due to missing leg cheatotaxy and other diagnostic characters.The species cription . TenuipaTenuipalpus simplychus CromroyT. simplychus was described as closely related to T. knorri Baker and Pritchard in the original description [T. simplychus could not be assigned a certain place in the diagnostic key.The species cription . Due to Tenuipalpus tetrazygiae De LeonT. tetrazygiae, it was distinguished by the author from other described species of that time by the shape of dorsocentral setae and dorsum covered with irregular ridges [caroliensis group, no certain place could be identified in the diagnostic key.The species r ridges . AlthougT. lustrabilis was previously reported as a suspected junior synonym of T. punicae Pritchard and Baker [T. lustrabilis in the published key by Meyer [T. lustrabilis is hereby synonymized with T. punicae based on the number of shared characteristics, i.e., leg chaetotaxy, palp segmentation, shape and number of dorsal setae, pattern of dorsal reticulations, as well as its geographic distribution (Pakistan) and host plant (Punica granatum).The species nd Baker ,13. We rnd Baker  as well by Meyer . T. lustT. guptai Sadana and Gupta was also suggested as a junior synonym of T. solanensis Sadana and Gupta [T. guptai vs 7-7 in T. solanensis). This character has been commented to be a miscalculation [T. guptai was described based on a single holotype female while T. solanensis only from three females. Moreover, both species were described based on specimens collected from the same host plant (P. granatum), the same type locality (India), on the same collection date (22-VI-1981), and have been mounted on the same slide (slide#91) [Similarly, the species nd Gupta ,13. We flide#91) .T. solanesis is T. lustrabilis, which is declared as junior synonym of T. punicae. A detailed comparison of available description for both of these species show they are very similar; sharing leg chaetotaxy, palp segmentation, shape and number dorsal setae, pattern of dorsal reticulations, and host plant. Hence, the two species (T. guptai and T. solanensis) are also synonymized with T. punicae.Interestingly, the related species of Tenuipalpus rodionovi Chalilova was described poorly without illustrations [T. granati Sayed, T. zhizhilashviliac Reck, and T. kobachidzei Reck. The latter two species belong to the species group carolinensis, while the former one belongs to the granati new species group. Pritchard and Baker [T. granati. There is a need to check the type specimens of this species to validate its status. Hence, T. rodionovi is considered as a suggested synonym of T. granati until further studies are made.trations . Therefond Baker  and Wainnd Baker  suspecteTenuipalpus is complicated due to taxonomic and classification-based modifications. This research indicates that the genus Tenuipalpus needs more taxonomical studies to raise the level of groups and species groups to higher taxonomical ranking, by using persistent and strong morphological characters. This may even direct future research in the family Tenuipalpidae to study the other closely related genera, in order to validate their status.The history of the genus


Animals that consume fermenting fruit and nectar are at risk of exposure to ethanol and the detrimental effects of inebriation. In this report, we show that the hormone FGF21, which is strongly induced by ethanol in murine and human liver, stimulates arousal from intoxication without changing ethanol catabolism. Mice lacking FGF21 take longer than wild-type littermates to recover their righting reflex and balance following ethanol exposure. Conversely, pharmacologic FGF21 administration reduces the time needed for mice to recover from ethanol-induced unconsciousness and ataxia. FGF21 did not counteract sedation caused by ketamine, diazepam or pentobarbital, indicating specificity for ethanol. FGF21 mediates its anti-intoxicant effects by directly activating noradrenergic neurons in the locus coeruleus region, which regulates arousal and alertness. These results suggest that this FGF21 liver-brain pathway evolved to protect against ethanol-induced intoxication and that it might be targeted pharmaceutically for treating acute alcohol poisoning.  Choi et al. show that the alcohol-induced hormone FGF21 signals from liver to brain to counteract loss of consciousness and coordination caused by ethanol consumption. Pharmacologic FGF21 administration further accelerates recovery from inebriation. FGF21 exerts its sobering effect by activating the noradrenergic nervous system, which regulates arousal and alertness. Accordingly, animals that consume fructose and other simple sugars have evolved liver enzymes to catabolize ethanol. Comparative genetic analyses of alcohol dehydrogenases across species reveal that many strict herbivores and carnivores that are not exposed to ethanol appear to have lost the ability to catabolize it, underscoring the importance of ethanol as an evolutionary driver2.Simple sugars in ripening fruits and nectars are a rich source of calories for many animals. However, consumption of ethanol produced by the natural fermentation of these sugars can cause intoxication, thus impairing mobility and judgement4. In humans, ethanol is by far the most potent inducer of FGF21 described to date7. Previous studies showed that FGF21 suppresses ethanol preference10, induces water drinking to prevent dehydration7, and protects against alcohol-induced liver injury12. Thus, FGF21 plays a broad role in defending against the harmful consequences of ethanol exposure.FGF21 is a hormone that is induced in liver by a variety of metabolic stresses including starvation, protein deficiency, simple sugars and ethanol13. FGF21 binds directly to both FGFR1c and KLB, with FGFR1c serving as the downstream signaling effector. Human genome-wide association studies have linked SNPs in and around both the FGF21 and KLB genes to alcohol consumption15, further highlighting the important relationship between FGF21 and ethanol. In mice, FGF21 crosses the blood-brain-barrier through an unknown mechanism16 and acts on its receptor complex in the nervous system both to suppress ethanol preference and to induce water consumption8.FGF21 acts on a heteromeric cell surface receptor composed of a conventional FGF receptor tyrosine kinase (FGFR1c) in complex with the single-pass transmembrane protein, β-Klotho (KLB)18. In this report, we show that FGF21 directly activates noradrenergic neurons in the LC region. We further show that this FGF21-NE pathway accelerates recovery of righting reflex and balance following ethanol intoxication. Thus, FGF21 is an endogenous anti-intoxicant or “amethystic” agent .Norepinephrine (NE) is an abundant neuromodulator in the CNS. Most central NE is synthesized in the locus coeruleus (LC), a small nucleus in the pons of the brainstem. LC neurons project extensively throughout the brain to regulate diverse biological processes, including arousal and alertnessFgf21–/– mice. Consistent with prior results, plasma FGF21 was induced by ethanol in WT mice, peaking at 2 hours  to wild-type (WT) and global  2 hours . We nextl gavage , Fgf21–/ WT mice . WT and ame rate , and braame rate . Thus, FFgf21-knockout (Fgf21Alb) mice (Klb-knockout (KlbCamk2a) mice (We obtained similar results for both time to loss of righting reflex and its duration with hepatocyte-specific lb) mice  and 2B a2a) mice  and 2E. 2a) mice  and 2F. 19. Thus, circulating FGF21 concentrations are super-physiologic for the duration of the experiments.We next examined whether pharmacologic FGF21 treatment of WT mice stimulates righting reflex recovery following ethanol administration. WT mice were administered a binge ethanol dose  followed one hour later, when the mice were already unconscious, by i.p. injection of recombinant FGF21. Remarkably, FGF21 administration reduced the time required for both male and female mice to recover their righting reflex by ~1.5 hours  and 3B, Fgf21/−− and KlbCamk2a mice. In Fgf21/−− mice, FGF21 administration reduced the righting reflex recovery time to that seen in WT mice  . Convers WT mice . Thus, pWe next tested whether FGF21 counteracts other sedatives, including the glutamatergic receptor antagonist ketamine and the GABA receptor agonists diazepam and pentobarbital. Because these sedatives act more quickly and for a shorter duration than ethanol, we compressed the experimental timeline: mice were i.p. injected with either ethanol or each of the other sedatives followed by i.p. injection of FGF21 or vehicle 30 minutes later. FGF21 retained its ability to accelerate righting reflex recovery in ethanol-treated mice under these modified conditions . In cont22. Moreover, dopamine β-hydroxylase (Dbh)-KO mice, which are unable to synthesize NE, have a prolonged righting reflex recovery time in response to ethanol without any change in ethanol catabolism23, similar to the responses we observed in Fgf21/−− mice. These findings led us to investigate whether physiologic FGF21 is responsible for ethanol-induced activation of noradrenergic neurons in the LC. WT and Fgf21/−− mice were administered ethanol (5 g/kg) by oral gavage and sacrificed 2.5 hours later. Immunostaining of LC sections was performed for c-Fos and the NE transporter (NET), which are markers of neuronal activity and noradrenergic neurons, respectively. As previously reported, ethanol induced c-Fos expression in NET+ LC neurons of WT mice  expressing either Cre recombinase fused with green fluorescent protein (GFP) or GFP alone were bilaterally injected into the LC region. Immunostaining of representative brain sections with a GFP antibody showed successful targeting of the LC region in KlbCreAAV- mice .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Steven A. Kliewer . Fgf21−/− 45, Fgf21Alb7, and KLB-T mice26 were on a C57BL/6J background and KlbCamk2a30 and KlbDbh mice were on mixed C57BL/6J;129/Sv backgrounds.All use of mice and related procedures were approved by the University of Texas Southwestern Medical Center’s Institutional Animal Care and Use Committee. Mice were housed in a temperature-controlled environment (23 ± 1° C) with 12 hour light/dark cycles and fed standard rodent chow ad libitum. Mice were randomly assigned to experimental groups. Experiments were performed with male mice unless indicated otherwise. Dbhfl/fl mice, in which Exon 3 of the Dbh gene is flanked by loxP sites, were generated using a CRISPR/Cas-9 gene editing strategy46. Briefly, two crRNAs (5’-ACTCACCATTGAACCTATGC-3’ for 5’ LoxP and 5’-ACCTGGGTCCCAGAGTTGCA-3’ for 3’ LoxP) and one tracrRNA were used. Two ssODNs, 5’-caggcagagagtgttatggtcttctcatttgctaagcggacagcgaggagcttcacttggtaggaggtcatgtg acatgattctcttcactcaccattgaacctgaattcgttgcgtgaATAACTTCGTATAATGTATGCTATACGAAGT TATatgctggctctgagcgggcaatcaactggttctgtctggctacagg-3’ and 5’-cactgagctacccccacgcttcccccgacaccacatcatcatggtaaacgggggtagagctctgctttccacctgggtcccagagtATA ACTTCGTATAATGTATGCTATACGAAGTTATgttgcgtgaggattctgcatggatcgaggtgctaccctggctccttagaagtagcacatatg-3’ were used as a transgene. We used 5’-gtgcttaacggtgaggacagg-3’ and 5’-ctgtatgcaggcctgaggtg-3’ primers for detecting 5’ LoxP insertion, 5’-cgacaatgagaccacgtactgg-3’ and 5’- ggaaatattcatctcaggggccc-3’ primers for detecting 3’ LoxP insertion, and 5’-ggcagagagtgttatggtc-3’ and 5’-gttctgttacctcctggctctg-3’ primers for sequence confirmation. DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies, Inc. . Two crRNAs, one tracrRNA, two ssODNs, and Cas9 protein were co-injected into fertilized oocytes from C57BL/6N female mice by the University of Texas Southwestern Transgenic Core. Founder mice were bred with C57BL/6J mice and progeny screened for the correct mutation by PCR and sequencing. After back-crossing founder mice with C57BL/6J mice for three generations, Dbhfl/+ mice were mated to generate homozygous Dbhfl/fl mice.Klbfl/fl mice were anesthetized using isoflurane anesthesia  and positioned in a stereotaxic instrument (David Kopf Instruments) with a temperature controller to maintain body temperature. The skull was exposed, bregma was determined, and two small holes were drilled for bilateral injection into the LC . Dorso-ventral coordinates are relative to pia. A volume of 300 nl (1 × 1012 genomic particles/μl) of AAV8-GFP or AAV8-GFP-Cre virus (UNC Vector Core) was injected bilaterally into the LC. The virus was infused at 50 nl/minute using a microinjection syringe pump and Micro2T controller system with a 34G Nanofil needle . After each injection, the needle was maintained in position for 10 minutes to prevent backflow and then slowly removed over 5 minutes. The skin was closed using sutures. Mice were allowed to recover for two weeks before use in experiments.Time to LORR was defined as the time between ethanol administration to mice and LORR. Unless indicated otherwise, ethanol was administered by oral gavage in a volume of 0.02 ml/g to a final concentration of 5 g/kg. Once ataxic, mice were placed in a supine position in V-shaped plastic troughs and the time measured until they were able to right themselves three times within 30 seconds, which was defined as LORR duration. LORR studies were performed in 2–8-month-old mice. For studying FGF21’s pharmacologic effects on ethanol-induced LORR, mice were administered ethanol followed 1 hour later by injection of either FGF21 or vehicle. Unless indicated otherwise, FGF21 was i.p. injected in a volume of 0.01 ml/g to a final concentration of 1 mg/kg. For the sedatives study, ketamine (200 mg/kg), diazepam (30 mg/kg), pentobarbital (55 mg/kg) or ethanol (4.3 g/kg) were i.p. injected followed 30 minutes later by injection of FGF21 or vehicle. Diazepam and ketamine were diluted in 0.9% saline, and pentobarbital was dissolved in 0.9% saline containing 10% ethanol. Ketamine, diazepam and pentobarbital were injected in a volume of 0.01 ml/g, and ethanol was injected in a volume of 0.017 ml/g.For the DSP-4 studies, 2-month-old C57BL/6J mice were i.p. injected with either DSP-4 (50 mg/kg in a volume of 0.01 ml/g) or vehicle. Two days later, mice were administered ethanol followed 1 hour later by injection of either FGF21 or vehicle. For the prazosin and propranolol studies, 2–3-month-old mice were administered ethanol followed 1 hour later by injection of FGF21 or vehicle in the presence or absence of prazosin (0.8 mg/kg in a volume of 0.01 ml/g) or propranolol (10 mg/kg in a volume of 0.01 ml/g). DSP-4, prazosin and propranolol were all dissolved in 0.9% saline just prior to use.Three-to-six-month-old mice were trained for three consecutive days on a Rotamex-5 rotarod (Columbus Instruments) spinning at 5 rpm, with training complete when mice were able to stay on the rotarod without falling completely off for 60 seconds. On the fourth day, mice were i.p. injected with ethanol (2 g/kg in a volume of 0.014 ml/g) followed 30 minutes later by injection of either FGF21 or vehicle. Time on the rotarod spinning at 5 rpm until falling completely off, with a maximum of 60 seconds, was measured at regular intervals.Rectal temperature was measured using a BAT-12 Microprobe digital thermometer with a RET-3 mouse rectal probe . Mice (2–4-month-old) were gently restrained, and the lubricated probe was inserted 1.5 cm into the rectal cavity to determine body temperature.48. c-Fos counts were performed blinded.For the c-Fos induction studies, 2–7-month-old mice were habituated for four days by either i.p. injection of 0.9% saline or oral gavage with water. On the fifth day, mice were i.p. injected with vehicle or FGF21 (2 hour treatment), or orally gavaged with water or ethanol as indicated in the figure legends. For all immunohistochemistry experiments, mice were anesthetized with isoflurane and transcardially perfused first with PBS followed by 10% neutral buffered formalin (NBF). Brains or adrenals were fixed for 24 hours in 10% NBF at 4°C and 50 μm slices were prepared using a Leica VT1000S vibratome. Slices were incubated for 1 hour in blocking buffer  at room temperature with shaking followed by incubation in primary antibodies, including antibodies against NET , cFos , red fluorescent protein , GFP  and DBH  for 48 hours at 4°C. Free-floating slices were washed 3 times in PBS for 10 minutes followed by incubation for 1 hour at room temperature with Alexa Fluor-conjugated secondary antibodies, including goat anti-mouse, goat anti-chicken, goat anti-rabbit IgGs , and DAPI  in blocking buffer. Slices were washed 3 times for 10 minutes in PBS and mounted with Aqua-Poly/Mount (Polysciences). Images were taken using a Zeiss LSM780 confocal microscope and images were processed using Fiji software47 and dissected using a 16G tissue punch. Tissue was homogenized by passage through a 26.5G syringe in RNA-STAT60. Total RNA was isolated from tissue using RNA-STAT60 reagent, and RNA was reverse-transcribed into cDNA (Invitrogen). Gene expression was measured with an Applied Biosystems 7900HT Sequence Detection System using the ddCT assay and normalized to cyclophilin49.Brains from 2–5-month-old mice were extracted from the skull and kept under dry ice vapor for all dissections. Coronal sections (0.5 mm thickness) were cut using a brain-slicing matrix (Braintree Scientific). SCN and LC regions were identified by gross anatomical landmarksKlb (cat# 415221 Mm-Klb) and Fgfr1c (cat# 454941-C2 Mm-Fgfr1-O1-C2) probes purchased from Advanced Cell Diagnostics. Hybridized slides were incubated with amplification reagents and Opal 570 and 690 dyes  followed by sequential incubation with tyrosine hydroxylase , biotinylated anti-chicken secondary  and streptavidin AlexaFluor488  antibodies. Slides were rinsed, dehydrated, cleared, and mounted with EcoMount . Images were taken using a Zeiss LSM880 confocal microscope.C57BL/6J, 5-month-old mice were anesthetized with isoflurane and transcardially perfused first with PBS followed by 10% NBF. Brains were fixed for 24 hours at 4°C in 10% NBF and then switched to 30% sucrose for 24 hours. Brain slices (25 μm) were cut using a freezing microtome (Leica), collected in PBS and treated with hydrogen peroxide for 10 minutes. After a PBS rinse, slices were mounted and desiccated overnight at room temperature. In situ hybridization was performed using RNAScope multiplex fluorescence kits (cat# 323110) and For measuring FGF21 and ethanol concentrations in murine plasma, blood from 4–6-month-old mice was centrifuged at 3,000 rpm for 15 minutes immediately after collection and plasma was stored at −80°C until analysis. Plasma FGF21 concentrations were measured using an FGF21 mouse/rat ELISA kit (BioVendor) according to the manufacturer’s instructions. Plasma ethanol concentrations were measured using an EnzyChrom ethanol assay kit (BioAssay Systems) according to the manufacturer’s instructions. For measuring brain ethanol concentrations, brains were removed, frozen immediately in liquid nitrogen and stored at −80°C. Frozen whole brains were homogenized in 0.1N HCl and centrifuged at 13,000 rpm for 30 minutes at 4°C. Ethanol concentrations in the supernatants were measured using an EnzyChrom ethanol assay kit.P values derived using a likelihood ratio test. In all analyses, a P value < 0.05 was considered significant. All data are expressed as the mean ± SEM. For 123


The same was true for BP values at peak exercise: 190.0 ± 9.5/96.8 ± 3.7 versus 173.3 ± 11.0/88.5 ± 1.8 mmHg . The likelihood of having an EBPR was significantly greater in MH than in their normotensive counterparts . Conclusions: Our meta-analysis suggests that EBPR reflects an increased risk of MH and that BP measurement during physical exercise aimed to assess cardiovascular health may unmask the presence of MH. This underscores the importance of BP measured in the medical setting at rest and in dynamic conditions in order to identify individuals at high cardiovascular risk due to unrecognized hypertension.Aim: Whether exaggerated blood pressure response (EBPR) to exercise represents a marker of masked hypertension (MH) in individuals with no prior history of hypertension is still unclear. We investigated this issue through a review and a meta-analysis of studies providing data on this association in normotensive individuals undergone both to dynamic or static exercise and to 24 h blood pressure monitoring (ABPM). Design: A systematic search was performed using Pub-Med, OVID, EMBASE, and Cochrane library databases from inception up to 31 December 2022. Studies were identified by using the following search terms: “masked hypertension”, “out-of-office hypertension”, “exercise blood pressure”, “exaggerated blood pressure exercise”, “exercise hypertension”. Results: Nine studies including a total of 387 participants with MH and 406 true normotensive controls were considered. Systolic BP (SBP) and diastolic BP (DBP) at rest were significantly higher in MH individuals than in sustained normotensives: 126.4 ± 1.4/78.5 ± 1.8 versus 124.0 ± 1.4/76.3 ± 1.3 mmHg (SMD: 0.21 ± 0.08, CI: 0.06–0.37,  In the As the systematic measurement of out-of-office BP in all members of the general population is unfeasible, the optimal screening strategy for MH remains uncertain, so far. Current hypertension guidelines state that the presence of out-of-office hypertension should be investigated by home or ambulatory BP monitoring (ABPM) in both untreated and treated individuals with office BP classified as high-normal  .In fact, high-normal office BP has been shown to entail an increased risk of MH especially when associated with comorbidities or conditions such as obesity, diabetes, obstructive sleep apnea, active smoking, and job stress ,15,16. FThe review was performed according to the key recommendations provided by Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) statement 2020. Medical literature was reviewed in order to identify all articles evaluating the relationship between MH, as defined by normal BP pre-exercise with elevated ambulatory BP, and EBPR. To this end, a systematic search was performed using Pub-Med, OVID, EMBASE, and Cochrane library databases from inception up to 31 December 2022. Studies were identified by using the following search terms: “masked hypertension”, “out-of- office hypertension”, “exercise blood pressure”, “exaggerated blood pressure exercise”, “hypertensive response to exercise”, “exercise hypertension”. Checks of the reference lists of selected papers and pertinent reviews complemented the electronic search. Data were examined and extracted by three independent investigators . In case of no agreement on a specific record, the full text of the study was analyzed by all reviewers in order to establish its eligibility according to the inclusion criteria mentioned below.http://www.ohrica/programs/clinical_epidemiologyoxford.htm, accessed on 5 January 2023).Main inclusion criteria were: (I) English articles published in peer-reviewed journals; (II) studies providing data on MH and EBPR; (III) minimum set of clinical/demographic data . Specific exclusion criteria were: (I) studies conducted in children and adolescents (age < 18 years); (II) case reports, reviews, and editorials. The Newcastle–Ottawa Scale (NOS) was used to measure the study quality (2 > 75). To assess the effect of individual studies on the pooled result, we conducted a sensitivity analysis by excluding each study one by one and recalculating the combined estimates on remaining studies. Publication bias was assessed by the Begg’s and Egger’s test. Statistical significance was set at p < 0.05.A pooled analysis of rest, exercise, and ambulatory BP values was performed using fixed or random effects meta-analysis by Comprehensive Meta-Analysis Version 2, Biostat, Englewood, NJ. Standard means difference (SMD) with 95% confidence interval (CI) was used to calculate the statistical difference between MH and normotensive individuals . Odds ratios (ORs) and 95% CI were calculated to assess differences in EBPR between the pooled groups. Demographic and clinical data provided by selected studies were expressed as absolute numbers, percentage, mean ± SD, mean ± SE or mean with CI. Heterogeneity was estimated using the I-squared test; random effect models were applied when heterogeneity across studies was high  performed in five countries .2. Seven studies included untreated healthy individuals. Two out of eight studies enrolled clinically normotensive patients with type 2 diabetes mellitus. An exercise test was performed in all studies but one with a treadmill. EBPR definition differed among studies, diagnostic thresholds for SBP ranging from 180 to 210 mmHg. In addition, gender-specific criteria and DBP values were considered by some but not all studies.p = 0.25; men prevalence 66 vs. 61%, p = 0.58; BMI 27.7 ± 1.2 vs. 27.7 ± 1.3 kg/m2, SMD: 0.10 ± 0.13, CI: −0.15/0.35, p = 0.44, respectively). The pooled group of 387 individuals meeting the diagnostic criteria for MH (48.8%) exhibited age, sex distribution, and BMI similar to normotensive controls’ values  and DBP  between groups.Pooled average systolic BP (SBP) and diastolic BP (DBP) at rest were higher in individuals with MH than in sustained normotensives (126.4 ± 1.4/78.5 ± 1.8 versus 124.0 ± 1.4/76.3 ± 1.3 mmHg). As shown by the forest plot in p = 0.002 for SBP and 0.97 ± 0.25, CI: 0.47–1.46, p < 0.0001 for DBP, respectively)  and exercise DBP (96.8 ± 3.7 versus 88.5 ± 1.8 mmHg data from five studies) were higher in MH than in normotensive participants with statistically significant SMDs  .p < 0.0001 for SBP and 1.58 ± 0.11: 1.38–1.80, p < 0.0001, for DBP). This was also the case for night-time SBP and DBP: 129.1 ± 6.2/77.1 ± 3.8 vs. 108.1 ± 1.8/64.5 ± 0.9 mmHg, .Findings provided by five studies showed that pooled daytime SBP and DBP values were greater in MH than in normotensive counterparts:142.9 ± 3.3/85.4 ± 1.3 vs. 120.6 ± 2.3/76.0 ± 1.7 mmHg   .p < 0.0001), but not in the non-diabetic subgroup . A publication bias was present for the following parameters: systolic BP at peak exercise and diastolic BP at rest and peak exercise.The presence of a single study effect was excluded at sensitivity analysis. An analysis by subgroups was performed by comparing studies including diabetic (two studies) vs. non-diabetic (n = 6 studies) patients. By this analysis, the odds ratio for EBPR was still significant in the diabetic subgroup  were both significantly higher in the pooled MH group than in the normotensive group (approximately 2 mmHg for both SBP and DBP). Furthermore, differences between groups were even greater for SBP and DBP at peak exercise (about 17 mmHg higher for SBP and 8 mmHg for DBP in the MH group). Finally, higher ambulatory BP values in individuals with MH were not limited to daytime period but persisted during the night-time. This indicates that EBPR in MH does not exclusively reflect an increased BP reactivity to physical and psychic stimuli during daytime activities but also an altered nocturnal BP profile. Before addressing the details of our study, available evidence on this issue and related topics deserves be considered.In healthy individuals, dynamic exercise during a stress electrocardiography test is associated to BP changes characterized by rapid, large increases in SBP and variable modifications in DBP . Unfortunately, no consensus exists on EBPR metric, given the different diagnostic criteria recommended by major guidelines ,31,32. FThese inconsistencies about the EBPR definition are widely witnessed by the heterogeneous criteria adopted in studies addressing this topic. A meta-analysis based on 12 studies including 53,264 participants revealed that SBP thresholds defining EBPR varied over a 90 mmHg wide range  . In lineAlthough the value of home BP monitoring (HBPM) as a reliable, cost-effective alternative to ABPM in the assessment of out-of-office BP has been universally recognized, MH screening should be preferentially based on ABPM, by using diagnostic criteria for both day- and night-time periods. When MH is defined according to HBPM (only based on daytime measurements) and ABPM daytime criteria, BP status may be misclassified, as nocturnal BP elevations may be missed. In this regard, it should be noted that six out of the eight studies included in the present meta-analysis did not consider average nocturnal BP separately from daytime or 24 h BP and this may have underestimated MH true prevalence, thus leaving isolated nocturnal hypertension to be unmasked.Proposed pathophysiological mechanisms and clinical correlates of EBPR include familial predisposition to hypertension, endothelial dysfunction, impaired arterial baroreflex sensitivity, abnormal neurohormonal response to exercise, large artery stiffness, male gender, pre-hypertension, comorbidities such as obesity, diabetes, obstructive sleep apnea, and subclinical HMOD ,36,37,38It should be underlined, however, that the pooled group with MH had significantly higher resting office BP values  than the normotensive group. The significance of office BP at rest as a predictor of MH deserves a more in-depth comment. In the Masked Hypertension Study , a populA further important result of our meta-analysis was that BP recorded at peak exercise was markedly higher in individuals with MH than in true normotensives, the difference between groups being 17 mmHg for SBP and 8 mmHg for DBP, resulting in a three times higher risk of EBPR in the MH group. Increased adrenergic activity, in addition to abnormal neurohormonal response to exercise, subclinical organ damage such as increased arterial stiffness, and cardiac remodeling are thought to be key factors associated with EBPR.2.7) and relative wall thickness (0.42 ± 0.1 vs. 0.37 ± 0.1) than their normotensive counterparts [Although a discussion about the possible mechanisms of EBPR in patients with MH is beyond the scope of this meta-analysis, some aspects of this topic deserve to be briefly highlighted. Four of the studies included in our meta-analysis provided data on cardiac HMOD ,27,28,29terparts . Similarterparts  and Koleterparts .The value of resting heart rate as an indicator of sympathetic overactive state and as a predictor of adverse CV prognosis has been widely recognized for many decades . In thisA further point should be made. MH may present different clinical patterns such as persistent out-of-office hypertension, isolated daytime, or isolated nocturnal hypertension . InformaSome limitations of our meta-analysis deserve to be reported. Our search was restricted to studies published in English due to the problems in identifying and interpreting papers in other languages. This linguistic approach may have affected our results. The present findings are based on few studies conducted in different settings including a few hundred cases and controls. As a consequence of this, the power of various tests to evaluate publication bias is too low to provide reliable results on this issue. Diagnostic criteria used to define EBPR and MH were heterogeneous and this may have influenced the results. The observational nature of the studies included in the meta-analysis does not allow to draw any conclusion on the causal relationship between MH and EBPR. It should be emphasized that both MH and EBPR are not seen as reproducible clinical traits over time ,51,52. TOur meta-analysis suggests that EBPR portends the likely presence of MH, a BP phenotype which, if not identified, carries a CV risk similar to that of sustained hypertension. This implies that measuring BP in the medical setting during stress testing offers a unique opportunity to identify individuals at high risk of MH. In a clinical perspective, the occurrence of EBPR during an ECG stress test should direct the clinician to plan investigations to unmask the presence of MH and subclinical HMOD in order to reduce the risk of CV complications in this non-marginal fraction of the general population.


SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.Intracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.Human neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.We demonstrate that HKL can successfully reduce αsyn protein levels and SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.This study supports a HKL-mediated downregulation of  SNCA gene locus develop aggressive forms of PD with dementia  accumulation is a key feature in the pathogenesis of Parkinson’s disease (PD) and related synucleinopathies . These ddementia . Experimdementia . Additiodementia  or knockdementia . Diseasedementia . Downregdementia , antisendementia , and virdementia  has demoPhytochemical compounds can contribute to the brain’s chemical balance and current evidence supports the applicability of natural compounds to treat neurodegenerative disorders . The medSNCA expression levels and interrogate the molecular mechanism(s) whereby HKL has its effect on SNCA.A previous study demonstrated that chronic HKL treatment prevented dopaminergic neuronal loss and motor impairments in a hemi-parkinsonian mouse model . Here, wA stable H4 neuroglioma cell line expressing human wt-αsyn was generated and previously described . Cells wPregnant adult CD-1 mice were ordered from Jackson Laboratory . Cell culture dishes were freshly prepared for each litter and were coated with poly-D-lysine (PDL) diluted in DPBS at a final concentration of 0.1 mg/mL. All media were made fresh for each litter and used within 1–2 weeks. Dissection buffer was prepared containing 1X HBSS without phenol red, calcium, or magnesium  and HEPES  at final concentration of 10 mM. Primary culture FBS medium comprised NeuroBasal Medium without L-Glutamine , 10% FBS , 1% GlutaMAX , and 1% Penstrep . Neuronal media comprised NeuroBasal medium without L-Glutamine, 2% B-27 , 1% GlutaMAX, and 1% Penstrep.o C. For treatment of cells, HKL was further diluted in the appropriate cell culture media at a final concentration of 10 μM. DMSO controls were similarly prepared with a final concentration of 0.01% DMSO. Wt-αsyn and primary cortical neurons were treated with 0.01% DMSO or 10 μM HKL for 72 h before being processed.A 100 mM stock solution of HKL  was dissolved in 100% DMSO and stored at -30SNCA gene, reprogrammed into induced pluripotent stem cells (iPSCs) called line ‘3x-1’, and characterized previously  from a patient carrying a triplication in the eviously . iPSCs weviously . Brieflyeviously . After dg for 10 min at 4°C. The protein concentration was determined with Bradford reagent . 5–10 μg of total proteins were separated on bis-tris polyacrylamide gradient gels  or TGX stain-free gels  and transferred to PVDF membranes. Membranes were then blocked for 1 h at room temperature (RT) in TBS-T  supplemented with 5% non-fat dried milk. Subsequently membranes were incubated overnight at 4°C with primary antibodies  lysis buffer  containing 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail. Collected cells were sonicated on ice and centrifuged at 10,000 × Cell toxicity was assessed using the Toxilight bioassay kit  in both primary cortical neurons and in wt-αsyn cells to determine the viability of cells after treatment with HKL. In both cases, cells were treated with 0.01% DMSO or 10 μM HKL for 72 h. The culture plates were removed from the 37°C incubator and left at room temperature for 5 min. 20 μl of conditioned media was transferred from each well to a 96-well luminescence compatible plate. Fresh adenylate kinase (AK) detection reagent was used for each experiment, in which 100μl was added to each conditioned media containing well and incubated at room temperature for 5 min. Luminescence was then read for 1 second in a microplate reader .Cell proliferation was determined using WST-1  assay according to manufacturer instructions. Briefly, WT-αsyn cells were plated in a 96-well plate with 10,000 cells per well in 100μl of media containing tetracycline. The following day, tetracycline was removed, and the cells were treated with either 0.01% DMSO or 10 μM HKL in 100μl of media. After 24, 48, and 72 h, 10 μl/well of WST-1 reagent was added to each well and the plate was incubated at 37°C for 4 h. WST-1 absorbance was read at 450 nm on an EnVision microplate reader . Three biological replicates of the cell proliferation assay were performed each with three technical replicates for each group in each set.SNCA mRNA degradation.Wt-αsyn cells were grown in the absence of tetracycline for 24 h. Then, tetracycline was added to suppress further expression of αsyn, and cells were treated with 10 μM HKL or 0.01% DMSO. Cells were then harvested at 0, 6, 12, 24, 48, and 72 h for western blot and qPCR analysis to evaluate rate of αsyn and SNCA), Mm01188700_m1(mouse Snca), Mm00497442 _m1 , and Hs02800695_m1 probes  followed by DNase RNA cleanup using RNeasy . The quantity and quality of RNA samples were determined by the Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Chip. Complementary DNA (cDNA) synthesized with Applied Biosystems High-Capacity cDNA Archive Kit was used as a template for relative quantitative PCR using ABI TaqMan chemistry . mRNA expression was quantified using Hs00240906_m1 (human in situ hybridization) used to visualize RNA transcripts within cells. Kits and probes were purchased from ACD  and used according to manufacturer’s instructions. Briefly, cells were fixed with 4% PFA, SNCA mRNA was amplified, and the secondary fluorescent detection probe  was added. Cells then underwent ICC to examine αsyn protein levels. Cells were permeabilized using 0.1% Triton-X in 1X PBS and incubated at RT. Following additional washes, bovine serum albumin (BSA) was used to block non-specific antigens. Primary antibody, 4B12  was diluted in blocking buffer and incubated at RT. Secondary fluorescent antibody Alexa Fluor 568 , were incubated after washes in the dark. Hoechst staining  was completed prior to imaging and quantification on a Perkin Elmer Operetta CLS High Content Imager .RNAscope is a variation of FISH (fluorescent Magnolol (#M0125) and 4-0-Methylhonokiol (#M184770) were ordered from LKT Labs . Derivatives arrived in powered form and were prepared in the same manner as HKL .The mRNA samples were sequenced by the Mayo Clinic Genome Analysis Core  using Illumina HiSeq 4000 . Reads were mapped to the mouse genome mm10. The raw gene read counts, along with sequencing quality control, were generated using the Mayo Clinic RNA sequencing (RNA-seq) analytic pipeline: MAP-RSeq version 3.0.1. Conditional quantile normalization (CQN) was performed on raw gene counts to remove biases created by GC content and technical variation, to adjust for gene length and library size differences, and to obtain similar quantile-by-quantile distributions of gene expression across samples. Based on the bimodal distribution of the CQN-normalized and log2-transformed reads per kilobase per million (RPKM) gene expression values, genes with average log2 RPKM ≥ 2 in at least one group were considered to have expression above the detection threshold. Using this selection threshold, 19,005 genes were included in downstream analyses.5 cells/well with tetracycline. The following day, tetracycline was removed and 0.5 μM rotenone  was added for 2 h at 37°C. After the 2-h incubation, cells were washed, fresh media was added, and wells were treated with 0.01% DMSO or 10 μM HKL for 72 h then harvested for protein and mRNA analysis.Wt-αsyn cells were plated in 6-well plates at 1 × 10SNCA mRNA was conducted with paired Student t-test. Differences were considered statistically significant when p < 0.05. Results are presented as mean ± standard error of the mean (SEM).All data were assessed using Graph Pad Prism 9 software  and analyzed by one-way ANOVA with Dunnett’s multiple comparisons test or unpaired Student t-test where appropriate. Proliferation rate of wt-αsyn cells was analyzed with repeated measures two-way ANOVA, followed by Dunnett’s multiple comparisons test. Statistical analysis of degradation rate of αsyn and F  = 21.84, p < 0.0001] (t (14) = 9.18, p < 0.0001] (in vitro.Because HKL has previously been reported to inhibit αsyn amyloid fibril formation in a cell-free aggregation assay  we sough 0.0001] . Therefo 0.0001] . Because 0.0001] . Importa 0.0001] . Togethet (5) = 0.068, p = 0.95] compared to vehicle treated cells.One mechanism by which αsyn expression may be decreased by HKL is via an increase in the rate of protein degradation. Next, we conducted a degradation assay in wt-αsyn cells to determine if increased degradation was the primary mechanism by which HKL regulated αsyn levels . Here weSNCA mRNA levels using quantitative PCR. We observed a significant decrease (51%) in SNCA transcripts levels in cells treated with HKL compared to vehicle   and normalized the average SNCA mRNA spots and the average αsyn protein fluorescence intensity to the average number of nuclei for each treatment. Taken together, qPCR, RNAscope, and ICC confirm that HKL treatment significantly decreases SNCA transcript levels by 73%  and αsyn expression by 45%  while not affecting expression of αTubulin   = 1.38, p = 0.22].Our data so far indicate that HKL can modulate αsyn expression and that the modulation is via a transcription related mechanism. The nature of the H4 overexpressing cells (wt-αsyn) excludes the possibility that regulation is at the level of the promoter; thus, we examined whether HKL modulates  control  . To be consistent, however, we continued to use 10 μM HKL in subsequent experiments. Consistent with our previous data, 10 μM HKL treatment resulted in a 44% decrease in αsyn protein expression (t (10) = 9.05, p < 0.0001] and a 25% decrease in Snca mRNA levels (t (16) = 7.16, p < 0.0001] compared to vehicle (DMSO) treatment.Because our stable cell lines expressing wt-αsyn are under the control of a constitutively active promoter yet our data support HKL reducing expression by altering levels of transcription, we examined the effect of HKL on endogenous levels of αsyn where expression is controlled by the endogenous promoter. To evaluate the effect of HKL on endogenous αsyn expression, mouse primary cortical neurons were treated at 7 days-w as 6μM   and a non-significant 33.4% reduction in SNCA mRNA levels (t (4) = 2.43, p = 0.07].To confirm the effect of HKL on endogenous human d by HKL   and SNCA mRNA levels   targets between groups , and cell signaling and transmembrane transport . To further validate these findings, we selected 4 DEGs and validated the effects of HKL on their expression using quantitative RT-PCR. Consistent with our bulk RNA sequencing data Angptl4 and Neat1 were significantly upregulated by HKL and Snca, Cav1, and Kcnq3 were all significantly downregulated by HKL  for 72 h. As expected, transcriptomic analysis confirmed n groups . Combinid by HKL . FurtherSNCA/Snca and αsyn levels in multiple cellular models, we next asked if HKL can modulate αsyn levels under pathological conditions. Abundant data supports the fact that exposure to rotenone, a worldwide-used pesticide, is associated with human parkinsonism and in vitro treatment with rotenone has been widely used to model synucleinopathy while in vivo rotenone treatment can induce a parkinsonian-like phenotype with nigrostriatal degeneration  = 21.08, p < 0.0001] and SNCA mRNA expression (SNCA expression levels indicating that HKL can prevent a rotenone-induced cellular response   exerting antioxidant activity and improving mitochondrial function . The mecSNCA gene was downregulated in cultures from two mouse litters subjected to sequencing (Angptl4 is an up-regulated gene that encodes angiopoietin-like 4, a secreted protein that modulates triacylglycerol homeostasis (Neat1, an essential structural component of nuclear paraspeckles that has been found increased in the brains and leukocytes of PD patients (Neat1 suppresses the expression of hyper edited Snca transcripts through nuclear retention, and/or inhibition of nuclear-cytoplasm transport (Our RNAseq results are in line with this hypothesis by demonstrating that the quencing . Furthereostasis . Indeed,eostasis . Consideeostasis , it is ppatients . It is pransport . Of noteransport . Howeverin vivo PD models and to clarify pathways through which HKL reduces αsyn.In summary, we demonstrate that the natural small molecule HKL can modulate levels of αsyn protein and transcript in multiple cell models of synucleinopathies. We also provide initial evidence of mechanisms by which HKL regulates αsyn, which suggests both direct gene regulation and indirect metabolism regulation. Additional studies will need to validate these findings in https://www.synapse.org/#!Synapse:syn51376668.Original datasets are available in a publicly accessible repository: MD and PJM contributed to the conception and design of the study. SJF, JDB, MJL, DA, ZBK, AHF, IS, DJQ, PP, and NND conducted the experiments, collected, and analyzed the data. SJF wrote the first draft of the manuscript. SLB contributed to data analysis and the first draft of the manuscript. SJF, SLB, PJM, JDB, and NND contributed to the manuscript revision. All authors contributed to the manuscript revision, read, and approved the submitted version.


The expression and modification levels of key mRNAs in the most enriched pathway were evaluated and compared using western blot and methylated RNA immunoprecipitation-quantitative PCR (MeRIP-qPCR).The mouse model of PCOS were induced by injecting dehydroepiandrosterone (DHEA), and confirmed by observing the morphological structures of ovarian follicles. Subsequently, mSkip mRNAs were hypermethylated in PCOS group.Compared with the control group, 415 hypermethylated and downregulated mRNAs, 8 hypomethylated and upregulated mRNAs, and 14 hypermethylated and upregulated mRNAs were identified in the PCOS group (Fold change ≥ 1.5). Those mRNAs were mainly involved in insulin signaling pathway, type II diabetes mellitus, Fc epsilon RI signaling pathway, inositol phosphate metabolism, and GnRH secretion. In insulin signaling pathway, the expression levels of phosphorylated protein kinase B (p-AKT) were decreased, whereas that of upstream phosphorylated phosphatidylinositol 3-kinase (p-PI3K) were increased in PCOS group. Moreover, skeletal muscle and kidney-enriched inositol polyphosphate 5-phosphatease (SKIP), one of PIP3 phosphatases, was verified to be overexpressed, and 6A modification of mRNAs might play a critical role in PCOS process. The PI3K/AKT pathway is inhibited in the mouse model of PCOS. Whether it is caused by the m6A modification of Skip mRNAs is worthy of further exploration.The altered mThe online version contains supplementary material available at 10.1186/s13048-023-01246-7. Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic dysfunction condition in the women of reproductive age, resulting in irregular menstruation, hyperandrogenism, infertility, and insulin resistance(IR) . PatientFSHR, rs2268361, rs2349415), insulin receptor , it is still difficult to explain the complexity of PCOS etiology and clinical manifestations [The etiology of PCOS is not yet entirely understood. It is widely assumed that PCOS is caused by a combination of environmental and genetic factors. PCOS is associated with hypothalamic-pituitary-ovarian axis (HPOA) neuroendocrine abnormalities, hyperandrogenemia, IR, chronic inflammation, and circadian rhythm disorder , 8. At tstations . Recentlstations , 13. Thestations . Perhapsstations . And the6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs, which is reversible and dynamically regulated by methyltransferase complexes (writers) and demethylases (erasers), and recognized by m6A binding proteins (readers) [6A modification regulates post-transcriptional expression of m6A-tagged genes by participating in RNA metabolism such as pre-mRNA splicing, mRNA translation, nuclear export, mRNA decay, and non-coding RNA biogenesis [6A modifications and their protein machines in oogenesis and in female reproductive tumors, as well as in other female reproductive diseases [6A modification level was increased, and several m6A modulators were dysfunction in PCOS patients [6A proteins are also involved in ovulation, and the m6A writers play an important role in oogenesis, but whether they can be used as therapeutic targets for abnormal ovulation remains to be further investigated. Nevertheless, little attention has been paid to the molecular mechanisms of m6A modification in PCOS.Nreaders) . m6A modogenesis . Recentldiseases . It has patients . In oogepatients , 25. The6A modification landscape of mRNAs in the ovaries of PCOS mice using the epitranscriptomic microarray, and to preliminarily explore the potential signal pathways involved in the PCOS process though KEGG analysis.In this study, we aim to investigate the altered mThe study was approved by the Institutional Review Board (No.2021-S087), and animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animal by International Committees of The Third Xiangya Hospital of Central South University.Female C57BL/6J mice aged 7 weeks were acquired from SJA Laboratory Animal Co. Ltd (Hunan China), and adaptively fed for 1 week on 12 h light/ 12 h dark cycle at room temperature (24 ± 3 °C) with a humidity of 45 ± 2%. During the feeding period, vaginal smears were performed daily at 8:00 a.m. to observe the estrous cycle of mice. Ten mice with normal estrous cycle were selected and randomly divided in two groups: the control and PCOS group. In the PCOS group, 6 mg/(100 g·d) dehydroepiandrosterone (DHEA) and 0.2 ml injectable soybean oil were injected subcutaneously into mice daily for 20 consecutive days. Similarly, mice in the control group were injected with 0.2 ml injectable soybean oil daily for 20 days.6-methyladenosine detection, the right ovaries were preserved at -80 °C for following western blot.Subsequently, five mice with continuous keratosis of vaginal epithelial cells in PCOS group and five mice in control group were randomly sacrificed on the first day. The two ovaries randomly selected mice from each group were embedded in paraffin and sliced for hematoxylin and eosin (HE) staining to observe the morphological changes and confirm the induction of PCOS. And the three remaining mice in each group, the left ovaries were preserved at -80 °C for following NTo isolate total RNA, ovary tissues were cut into small pieces and homogenized in TRIzol reagent before being quantified using a NanoDrop ND-1000 . Supplementary Table S6A spike-in control was immunoprecipitated with anti-m6A rabbit polyclonal antibody  at 4 °C for 2 h. Dynabeads™ M-280 Sheep Anti-Rabbit IgG suspension (20 μL per sample) (Ivitrogen) was blocked with 0.5% bovine serum albumin (BSA) at 4 °C for 2 h, washed three times with IP buffer (300 μL) for 5 min, and resuspended in the prepared RNA-antibody mixture. The RNA was bound to the m6A-antibody beads for 2 h at 4 °C, then the beads were washed with IP buffer , followed by Wash buffer . In this case, the adsorbed RNA was eluted with Elution buffer (200 μL) at 50 °C for 1 h. The immunoprecipitated (IP) RNA and supernatant (Sup) RNA were extracted by acid phenol–chloroform and ethanol precipitation.Total RNA (1-3ug) mixed with m6A-mRNA epitranscriptome microarray slide. The slides were incubated at 65 °C for 17 h in an Agilent Hybridization Oven . The hybridized arrays were washed, fixed, and scanned with an Agilent scanner G2505C (Agilent).The IP RNAs and Sup RNAs were mixed with an equal amount of calibration spike-in control RNA, amplified separately and labeled with Cy3 (for Sup) and Cy5 (for IP) using Arraystar Super RNA Labeling Kit (Arraystar). The synthesized cRNAs was purified by the RNeasy Mini Kit (QIAGEN), and the concentration and specific activity of cRNAs were detected by the NanoDrop ND-1000 (Thermo Fisher Scientific)  was used. The raw IP and Sup intensities were normalized to the log2-scaled Spike-in RNA intensity average. Following Spike-in normalization, probe signals with Present (P) or Marginal (M) QC flags were retained in at least three of six samples for further m6A epitranscriptomic microarray data was validated using IP . MeRIP-qPCR was then used to quantify the RNA enrichment via 2−ΔΔct analysis. Supplementary Table S6A sites were predicted using the sequence-based RNA adenosine methylation site predictor (SRAMP) program (https://www.cuilab.cn/sramp) [The mn/sramp) .The protein concentration of right ovary tissue was determined using the Bradford method after protein extraction (M&C Gene Technology Ltd.). SDS–polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the protein samples, which were then transferred to a polyvinylidene difluoride membrane. Antibodies of interest were used to probe the membranes. The antibodies used were as follows: phosphatidylinositol 3-kinase (PI3K) , phosphorylated PI3K (p-PI3K) , protein kinase B (AKT) , phosphorylated AKT (ser473) (p-AKT) , skeletal muscle and kidney-enriched inositol polyphosphate 5-phosphatase (SKIP) , and β-actin .P < 0.05) revealed differentially m6A-methylated or differentially expressed RNAs between two comparison groups. Hierarchical clustering was carried out using the R software (version 4.02). To perform GO analysis, the topGO package in the R environment for statistical computing and graphics was used, and the Fisher's exact test was used to perform pathway analysis. The western blot data were presented as mean ± SD and compared between groups using the Student's t test or the Mann–Whitney U test. P < 0.05 was regarded as significant. SPSS version 21.0 was used for all statistical analysis .Filtering with the fold change ≥ 1.5 and statistical significance thresholds  between the PCOS and control groups. Among them, 226 and 81 mRNAs were upregulated and downregulated in the PCOS group, respectively  in the PCOS group. Surprisingly, of these 143 mRNAs, 134 mRNAs were found to have higher m6A methylation levels and only 9 mRNAs had lower levels of m6A methylation  , heterogeneous nuclear ribonucleoprotein A2/B1 (Hnrnpa2b1) .Moreover, we found several “writers” and “readers” of mWhen we integrated the mRNAs methylation and expression data, 8 hypomethylated and upregulated mRNAs, 415 hypermethylated and downregulated mRNAs, 14 hypermethylated and upregulated mRNAs, and 0 hypomethylated and downregulated mRNAs (Fold change ≥ 1.5) were identified Fig. A. Then, 6A methylation levels of the skeletal muscle and kidney-enriched inositol polyphosphate 5-phosphatase (Skip) mRNA were verified via MeRIP-PCR (P < 0.01)   Fig. . MoreoveTR) Fig. .Fig. 4ThP < 0.001), whereas the expression of PI3K and p-PI3K were significantly increased (P < 0.05) in PCOS group than that in control group   were significantly decreased  has numerous adverse effects on women. However, the pathogenesis of PCOS is currently unclear, and there is no specific therapy for PCOS. Given the continuously increasing incidence of PCOS in recent years, it is important to explore the mechanisms of PCOS development and to develop effective treatments for patients with PCOS. PCOS is commonly associated with aberrant DNA methylation, and several genes are epigenetically dysregulated, and are associated with the pathological consequences of PCOS and metabolic comorbidities . However6A modification levels of mRNAs were increased, and the m6A ‘readers’  are overexpressed in PCOS mice ovaries. A similar methylation trend was confirmed using MeRIP sequencing (MeRIP-seq) in luteinized granulosa cells (GCs) of PCOS patients [6A modification may mainly participate in the insulin signaling pathway and type II diabetes mellitus pathway, which were closely related to IR and secondary hyperinsulinemia [According to our microarray results, the mpatients . In contpatients . As a reulinemia . The PI3ulinemia , 31. In ulinemia . The dysulinemia , 34. Sim6A modification in PCOS mice. The m6A sites in humans and mice are highly conserved, and mainly enriched in the 3’untranslated region (UTR) and around stop codons [6A peaks in the coding sequence and transcription start regions, but less prominent enrichment near stop codons in PCOS patients' GCs compared to controls [6A sites of Skip mRNA was also mainly predicted in the coding sequence by SRAMP program in this study [6A modification in the coding sequence may alleviate ribosome stalling and thus improve mRNA translation in acute myeloid leukemia [6A modification of Skip mRNA may explain its translation upregulation. Therefore, it is possible that the higher m6A modification of Skip mRNA result in the overexpression of SKIP protein, then reversed the activation of p-PI3K on the downstream AKT.Notably, the activity of upstream and downstream factors in the PI3K/AKT pathway were opposite. In contrast to p-AKT, p-PI3K expression levels were elevated in the PCOS group, which may be related to inositol phosphate metabolism pathway, another enriched pathway. SKIP, a PIP3 5-phosphatase, was localized at endoplasmic reticulum under resting conditions. Insulin stimulation induced its translocation to the plasma membrane, and binding with activated p21-activated protein kinase 1 (PAK1), thereby activating SKIP’s PIP3 phosphatase activity. The rapid and efficient hydrolysis of PIP3, resulting in decreased AKT2 phosphorylation, inhibits membrane ruffle and GLUT4 translocation, thus negatively regulates insulin signaling in skeletal muscle , 36. As p codons . Howevercontrols . Similaris study . In addileukemia . And in leukemia . Therefo6A modification also participant in the etiological mechanism of chronic inflammation and neuroendocrine in PCOS. Increasing number of studies demonstrated that low-grade chronic inflammation can induce IR, obesity, and hyperandrogenemia through related pathways, leading to ovulation disorders in PCOS [In addition, Fc epsilon RI signaling pathway and GnRH secretion pathway, another two enriched pathways, may indicated that the m in PCOS –42. The  in PCOS . The GnR in PCOS , 45.Inevitably, there were several limitations of this study. First, several acknowledged inhibiting factors for PI3K/AKT pathway were not investigated, such as Phosphatase and tensin homolog (PTEN) and c-Jun N-terminal kinase (JNK). Then, we did not further validate our hypothesis that the hypermethylation of SKIP mRNAs enhances its expression, then inhibits the PI3K/AKT signaling pathway.6A modification of mRNAs might play a critical role in PCOS process. And we emphasized the changes in the activity of upstream and downstream factors in the PI3K/AKT signaling pathway. Moreover, the role of m6A modification of Skip mRNA in the pathogenesis of PCOS warrants further studies.In conclusion, our study demonstrates that the altered mAdditional file 1: Supplementary Table S1. the quantification and quality of RNA in ovary tissue of a mouse model of PCOS*. Supplementary Table S2. The specific activity (pmol dyes per μg cRNA) of the labeled RNA. Supplementary Table S3. Primers used in MeRIP-qPCR. Supplementary Table S4. Predicted m6A sites in Skip mRNA by SRAMP program.Additional file 2: Supplementary Figure S1. Characteristics of vaginal smears during various stages of estrus cycle  A. Proestrous, which was characterized by the presence of mostly nucleated and some cornified epithelial cells; B. Estrous, which was characterized by the presence of mostly cornified epithelial cells; C. Metestrus, which was characterized by the presence of cornified epithelial cells and leukocytes; D. Diestrus, which was characterized by the presence of primarily leukocytes.