Age-related changes in gonadotropin-releasing hormone (GnRH) splice variants in mouse brain.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of the mammalian reproductive axis. We investigated the spatiotemporal expression of GnRH splice variants (V1, V2, and V3) and splicing factors (Srsf7, Srsf9, and Tra-2) in the male mice brain. Further, using in silico tools, we predicted protein structure and the reason for the low translational efficiency of V2 and V3. Messenger RNA levels of GnRH variants and splicing factors were quantified using real-time reverse transcription-polymerase chain reaction at different age groups. Our data show that expression of almost all the variants alters with aging in all the brain regions studied; even in comparison to the hypothalamus, several brain areas were found to have higher expression of these variants. Hypothalamic expression of splicing factors such as Srsf7, Srsf9, and Tra-2 also change with aging. Computational studies have translation repressors site on the V3, which probably reduces its translation efficiency. Also, V2 is an intrinsically disordered protein that might have a regulatory or signaling function. In conclusion, this study provides novel crucial information and multiple starting points for future analysis of GnRH splice variants in the brain.

Alternative splicing analysis showed the splicing factor polypyrimidine tract-binding protein 1 as a potential target in acute myeloid leukemia therapy.

Alternative splicing (AS) is a universal post-transcriptional regulation process in cells, and increasing evidences have validated its crucial role in tumors. We collected AS event, gene expression, and clinical data of 178 AML patients from The Cancer Genome Atlas (TCGA) project. More than 1,000 AS events were found associated with overall survival (OS), and alternate promoter (AP) events were the most significant. The expression of the KIAA0930 transcript was the most significantly different AS event selected from AP events and significantly correlated with the expression of the splicing factor (SF) polypyrimidine tract-binding protein 1 (PTBP1). Then, the roles of PTBP1 on AS of the KIAA0930 and the proliferation of AML cells were confirmed. KIAA0930 variant 1 (KIAA0930-1) was upregulated and variant 2 (KIAA0930-2) downregulated with knockdown PTBP1 expression of AML cells by specific shRNA. A low level of PTBP1 can decrease the proliferation ability of AML cells. In conclusion, the results showed that PTBP1 might be a potential target for AML therapy.

YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the treatment of advanced/metastatic renal cancer. However, resistance to TKIs is common in the clinic, particularly after long-term treatment. YTHDC1 is the main nuclear reader protein that binds with m6A to regulate the splicing, export and stability of mRNA. However, the specific role and corresponding mechanism of YTHDC1 in renal cancer cells are still unclear. METHODS: The Cancer Genome Atlas (TCGA) dataset was used to study the expression of YTHDC1 in ccRCC. Cell counting kit-8 (CCK-8), wound healing, Transwell and xenograft assays were applied to explore the biological function of YTHDC1 in ccRCC. Western blot, quantitative real time PCR (RT‒qPCR), RNA immunoprecipitation PCR (RIP-qPCR), methylated RIP-qPCR (MeRIP-qPCR) and RNA sequencing (RNA-seq) analyses were applied to study the YY1/HDAC2/YTHDC1/ANXA1 axis in renal cancer cells. The CCK-8 assay and xenograft assay were used to study the role of YTHDC1 in determining the sensitivity of ccRCC to sunitinib. RESULTS: Our results demonstrated that YTHDC1 is downregulated in ccRCC tissues compared with normal tissues. Low expression of YTHDC1 is associated with a poor prognosis in patients with ccRCC. Subsequently, we showed that YTHDC1 inhibits the progression of renal cancer cells via downregulation of the ANXA1/MAPK pathways. Moreover, we also showed that the YTHDC1/ANXA1 axis modulates the sensitivity of tyrosine kinase inhibitors. We then revealed that HDAC2 inhibitors resensitize ccRCC to tyrosine kinase inhibitors through the YY1/HDAC2 complex. We have identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC. CONCLUSION: We identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.

OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice.

KEY MESSAGE: P-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3. In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.

Sex-dimorphic Rostro-caudal Patterns of 5'-AMP-activated Protein Kinase Activation and Glucoregulatory Transmitter Marker Protein Expression in the Ventrolateral Ventromedial Hypothalamic Nucleus (VMNvl) in Hypoglycemic Male and Female Rats: Impact of Estradiol.

The ventromedial hypothalamic nucleus-ventrolateral part (VMNvl) is an estradiol-sensitive structure that controls sex-specific behavior. Electrical reactivity of VMNvl neurons to hypoglycemia infers that cellular energy stability is monitored there. Current research investigated the hypothesis that estradiol elicits sex-dimorphic patterns of VMNvl metabolic sensor activation and gluco-regulatory neurotransmission during hypoglycemia. Rostral-, middle-, and caudal-VMNvl tissue was separately micropunch-dissected from letrozole (Lz)- or vehicle-injected male and estradiol- or vehicle-implanted ovariectomized (OVX) female rats for Western blot analysis of total and phosphorylated 5'-AMP-activated protein kinase (AMPK) protein expression and gluco-stimulatory [neuronal nitric oxide synthase (nNOS); steroidogenic factor-1 (SF1) or -inhibitory (glutamate decarboxylase65/67 (GAD)] transmitter marker proteins after sc insulin (INS) or vehicle injection. In both sexes, hypoglycemic up-regulation of phosphoAMPK was estradiol-dependent in rostral and middle, but not caudal VMNvl. AMPK activity remained elevated after recovery from hypoglycemia over the rostro-caudal VMNvl in female, but only in the rostral segment in male. In each sex, hypoglycemia correspondingly augmented or suppressed nNOS profiles in rostral and middle versus caudal VMNvl; these segmental responses persisted longer in female. Rostral and middle segment SF1 protein was inhibited by estradiol-independent mechanisms in hypoglycemic males, but increased by estradiol-reliant mechanisms in female. After INS injection, GAD expression was inhibited in the male rostral VMNvl without estradiol involvement, but this hormone was required for broader suppression of this profile in the female. Neuroanatomical variability of VMNvl metabolic transmitter reactivity to hypoglycemia underscores the existence of functionally different subgroups in that structure. The regional distribution and estradiol sensitivity of hypoglycemia-sensitive VMNvl neurons of each neurochemical phenotype evidently vary between sexes.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

Therapeutic Applications of Targeted Alternative Splicing to Cancer Treatment.

A growing body of studies has documented the pathological influence of impaired alternative splicing (AS) events on numerous diseases, including cancer. In addition, the generation of alternatively spliced isoforms is frequently noted to result in drug resistance in many cancer therapies. To gain comprehensive insights into the impacts of AS events on cancer biology and therapeutic developments, this paper highlights recent findings regarding the therapeutic routes of targeting alternative-spliced isoforms and splicing regulators to treatment strategies for distinct cancers.

Modulation of SF1 Neuron Activity Coordinately Regulates Both Feeding Behavior and Associated Emotional States.

Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans.

Regulation of Energy Balance via BDNF Expressed in Nonparaventricular Hypothalamic Neurons.

Brain-derived neurotrophic factor (BDNF) expressed in the paraventricular hypothalamus (PVH) has been shown to play a key role in regulating energy intake and energy expenditure. BDNF is also expressed in other hypothalamic nuclei; however, the role in the control of energy balance for BDNF produced in these structures remains largely unknown. We found that deleting the Bdnf gene in the ventromedial hypothalamus (VMH) during embryogenesis using the Sf1-Cre transgene had no effect on body weight in mice. In contrast, deleting the Bdnf gene in the adult VMH using Cre-expressing virus led to significant hyperphagia and obesity. These observations indicate that the lack of a hyperphagia phenotype in the Sf1-Cre/Bdnf mutant mice is likely due to developmental compensation. To investigate the role of BDNF expressed in other hypothalamic areas, we employed the hypothalamus-specific Nkx2.1-Cre transgene to delete the Bdnf gene. We found that the Nkx2.1-Cre transgene could abolish BDNF expression in many hypothalamic nuclei, but not in the PVH, and that the resulting mutant mice developed modest obesity due to reduced energy expenditure. Thus, BDNF produced in the VMH plays a role in regulating energy intake. Furthermore, BDNF expressed in hypothalamic areas other than PVH and VMH is also involved in the control of energy expenditure.

SNEV(Prp19/PSO4) deficiency increases PUVA-induced senescence in mouse skin.

Senescent cells accumulate during ageing in various tissues and contribute to organismal ageing. However, factors that are involved in the induction of senescence in vivo are still not well understood. SNEV(P) (rp19/) (PSO) (4) is a multifaceted protein, known to be involved in DNA damage repair and senescence, albeit only in vitro. In this study, we used heterozygous SNEV(+/-) mice (SNEV-knockout results in early embryonic lethality) and wild-type littermate controls as a model to elucidate the role of SNEV(P) (rp19/) (PSO) (4) in DNA damage repair and senescence in vivo. We performed PUVA treatment as model system for potently inducing cellular senescence, consisting of 8-methoxypsoralen in combination with UVA on mouse skin to induce DNA damage and premature skin ageing. We show that SNEV(P) (rp19/) (PSO) (4) expression decreases during organismal ageing, while p16, a marker of ageing in vivo, increases. In response to PUVA treatment, we observed in the skin of both SNEV(P) (rp19/) (PSO) (4) and wild-type mice an increase in γ-H2AX levels, a DNA damage marker. In old SNEV(P) (rp19/) (PSO) (4) mice, this increase is accompanied by reduced epidermis thickening and increase in p16 and collagenase levels. Thus, the DNA damage response occurring in the mouse skin upon PUVA treatment is dependent on SNEV(P) (rp19/) (PSO) (4) expression and lower levels of SNEV(P) (rp19/) (PSO) (4) , as in old SNEV(+/-) mice, result in increase in cellular senescence and acceleration of premature skin ageing.

Effects and mechanisms of acupuncture and moxibustion on reproductive endocrine function in male rats with partial androgen deficiency.

OBJECTIVES: Partial androgen deficiency of the aging male (PADAM) is characterised by a deficiency in serum androgen levels. Both electroacupuncture (EA) and mild moxibustion (MM) can raise serum testosterone levels in PADAM. We investigated the mechanisms underlying the use of EA and MM in a rodent model of PADAM. METHODS: Fifty rats received cyclophosphamide injection over 5 consecutive days to induce PADAM, which was verified by comparing total testosterone (TT) and free testosterone (FT) levels with 10 non-PADAM healthy control rats (CON). Successful modelling was confirmed in 43 of 50 rats, 40 of which were randomly divided into untreated (PADAM), EA-treated (PADAM+EA), MM-treated (PADAM+MM), and androlin (AD)-treated (PADAM+AD) groups (n=10 each). EA and MM were administered at BL23 and CV4 acupuncture points for 8 weeks, and no treatment was given to rats in the PADAM and CON groups. Serum levels of luteinising hormone (LH) and follicle-stimulating hormone (FSH), mRNA expression of cytochrome P450c17 (P450c17) and 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), and protein levels of cytochrome P450 side chain cleavage (P450scc), 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3) and steroidogenic factor 1 (SF-1) were evaluated after 8 weeks. RESULTS: Both EA and mild MM significantly increased serum TT and FT levels with MM displaying superiority. P450scc, 17ß-HSD3 and SF-1 protein expression, and P450c17 and 3ß-HSD1 mRNA expression, were significantly increased and serum LH and FSH levels were significantly decreased in PADAM+EA and PADAM+MM relative to PADAM rats. Moreover, serum LH and FSH levels were significantly lower and 17ß-HSD3 protein expression significantly higher in PADAM+MM relative to PADAM+EA rats. CONCLUSIONS: EA and MM at the BL23 and CV4 acupuncture points appear to be effective treatments for PADAM, and MM displays superior efficacy to EA.