FMRP deficiency leads to multifactorial dysregulation of splicing and mislocalization of MBNL1 to the cytoplasm.

Fragile X syndrome (FXS) is a neurodevelopmental disorder that is often modeled in Fmr1 knockout mice where the RNA-binding protein FMRP is absent. Here, we show that in Fmr1-deficient mice, RNA mis-splicing occurs in several brain regions and peripheral tissues. To assess molecular mechanisms of splicing mis-regulation, we employed N2A cells depleted of Fmr1. In the absence of FMRP, RNA-specific exon skipping events are linked to the splicing factors hnRNPF, PTBP1, and MBNL1. FMRP regulates the translation of Mbnl1 mRNA as well as Mbnl1 RNA auto-splicing. Elevated Mbnl1 auto-splicing in FMRP-deficient cells results in the loss of a nuclear localization signal (NLS)-containing exon. This in turn alters the nucleus-to-cytoplasm ratio of MBNL1. This redistribution of MBNL1 isoforms in Fmr1-deficient cells could result in downstream splicing changes in other RNAs. Indeed, further investigation revealed that splicing disruptions resulting from Fmr1 depletion could be rescued by overexpression of nuclear MBNL1. Altered Mbnl1 auto-splicing also occurs in human FXS postmortem brain. These data suggest that FMRP-controlled translation and RNA processing may cascade into a general dys-regulation of splicing in Fmr1-deficient cells.

The effects of herbal medicine (Jujadokseo-hwan) on quality of life in patients with mild cognitive impairment: Cost-effectiveness analysis alongside randomized controlled trial.

Background: Mild cognitive impairment (MCI), the early stage of dementia, requires effective intervention for symptom management and improving patients' quality of life (QoL). Jujadokseo-hwan (JDH) is a Korean herbal medicine prescription used to improve MCI symptoms, such as memory deficit. This study evaluates the improvement in QoL through JDH. Alongside a clinical trial, it estimates the cost-effectiveness of JDH, compared to placebo, for MCI over 24 weeks. Methods: Changes in QoL were measured using the EuroQol-5 Dimensions (EQ-5D) and Korean version QoL-Alzheimer's Disease (KQOL-AD). Direct medical and non-medical costs were surveyed and incremental cost-effectiveness ratios (ICER) per QALY for JDH were produced. Results: In total, 64 patients were included in the economic evaluation (n = 35 in JDH, n = 29 in placebo). In the JDH group, EQ-5D and KQOL-AD improved by 0.020 (p = .318) and 3.40 (p = .011) over 24 weeks, respectively. In the placebo group, they increased by 0.001 (p=.920) and 1.07 (p=.130), respectively. The ICER was KRW 76,400,000 per QALY and KRW 108,000 per KQOL-AD for JDH, compared to the placebo group. Conclusion: JDH is not considered a cost-effective treatment option compared with placebo; however, it positively affects QoL improvement in patients with MCI.

CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia.

Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.

Oppositional poly(A) tail length regulation by FMRP and CPEB1.

Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3'UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases GLD2 (TENT2, PAPD4, Wispy) and GLD4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor GLD4 to RNA, we expressed double tagged GLD4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that coprecipitated with GLD4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA nanopore sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation.

FMRP links optimal codons to mRNA stability in neurons.

Fragile X syndrome (FXS) is caused by inactivation of the FMR1 gene and loss of encoded FMRP, an RNA binding protein that represses translation of some of its target transcripts. Here we use ribosome profiling and RNA sequencing to investigate the dysregulation of translation in the mouse brain cortex. We find that most changes in ribosome occupancy on hundreds of mRNAs are largely driven by dysregulation in transcript abundance. Many down-regulated mRNAs, which are mostly responsible for neuronal and synaptic functions, are highly enriched for FMRP binding targets. RNA metabolic labeling demonstrates that, in FMRP-deficient cortical neurons, mRNA down-regulation is caused by elevated degradation and is correlated with codon optimality. Moreover, FMRP preferentially binds mRNAs with optimal codons, suggesting that it stabilizes such transcripts through direct interactions via the translational machinery. Finally, we show that the paradigm of genetic rescue of FXS-like phenotypes in FMRP-deficient mice by deletion of the Cpeb1 gene is mediated by restoration of steady-state RNA levels and consequent rebalancing of translational homeostasis. Our data establish an essential role of FMRP in codon optimality-dependent mRNA stability as an important factor in FXS.

HDAC6 regulates thermogenesis of brown adipocytes through activating PKA to induce UCP1 expression.

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). UCP1 increases the conductance of the inner mitochondrial membrane (IMM) for protons to make BAT mitochondria generate heat rather than ATP. HDAC6 is a cytosolic deacetylase for non-histone substrates to regulate various cellular processes, including mitochondrial quality control and dynamics. Here, we showed that the body temperature of HDAC6 knockout mice is slightly decreased in normal hosing condition. Interestingly, UCP1 was downregulated in BAT of HDAC6 knockout mice, which extensively linked mitochondrial thermogenesis. Mechanistically, we showed that cAMP-PKA signaling plays a key role in HDAC6-dependent UCP1 expression. Notably, the size of brown adipocytes and lipid droplets in HDAC6 knockout BAT is increased. Taken together, our findings suggested that HDAC6 contributes to mitochondrial thermogenesis in BAT by increasing UCP1 expression through cAMP-PKA signaling pathway.

HDAC6 deficiency induces apoptosis in mesenchymal stem cells through p53 K120 acetylation.

The acetylation of p53 is critical in modulating its pro-apoptotic roles. However, its regulatory mechanism and physiological significance are unclear. Here, we show HDAC6 negatively regulates pro-apoptotic acetylation of p53 at lysine residue 120 (K120) in mesenchymal stem cells (MSCs). The loss of HDAC6 expression in MSCs increases K120 acetylation of p53, which is successfully reversed by the wild-type but not by catalytically dead HDAC6. Deletion of HDAC6 induces caspase-dependent apoptosis by promoting transactivation of Bax and suppression of Bcl-2. Moreover, HDAC6 deficiency leads to mitochondrial dysfunction characterized by aberrant reactive oxygen species production and defective oxidative phosphorylation, which is reversed by ectopic expression of wild-type or acetylation mimetic p53. This study demonstrates that HDAC6 is a critical regulator of a pro-apoptotic p53 K120 acetylation and mitochondrial function in MSCs, suggesting that the modulation of HDAC6 activity could be a novel approach to improve MSC- based therapies.

The DTL ERG electrode comes in different shapes and sizes: Are they all good?

PURPOSE: Although the DTL fiber electrode has been in use in the ERG field for more than four decades, its composition was never clearly defined. We compared five different types of conductive (DTL type) yarn (differing in terms of mass, number of filaments, and crimping degree) in order to determine whether we could identify one that would be better suited for the recording of ERGs. METHODS: Photopic flash ERGs were recorded from five subjects using the following DTL electrodes: 27/7, 22/1, 11/1, 11/1*2, and 22/1*2. Data analysis included amplitude and peak time measurements of the a- and b-waves in the time domain (TD) as well as measurements of specific frequency descriptors of the ERG waveform in the time-frequency domain using the discrete wavelet transform (DWT) approach. The degree of comfortableness was also assessed in 12 subjects with two surveys (Likert 5-point and the ranking scale). RESULTS: Comparisons of TD and DWT parameters did not permit to identify the best DTL electrode, all yielding comparable measures. There was a slight trend for the largest electrode (22/1*2) to yield the largest response, but this was at the expense of comfort, the 22/1*2 electrode being rated as the least comfortable. CONCLUSIONS: Given the minimal impact the different electrodes had on the amplitude of the signal, we believe that comfort should dictate our choice. It would appear from our results that use of a multifilament electrode is the best choice since one can get an electrode whose size is optimized for the recording of large responses while minimizing the foreign-body sensation due to the small size of each of the filaments that compose this multifilament electrode.

Sildenafil Improves Functional and Structural Outcome of Retinal Injury Following Term Neonatal Hypoxia-Ischemia.

PURPOSE: The purpose of this study was to investigate the effects of sildenafil on retinal injury following neonatal hypoxia-ischemia (HI) at term-equivalent age in rat pups. METHODS: Hypoxia-ischemia was induced in male Long-Evans rat pups at postnatal day 10 (P10) by a left common carotid ligation followed by a 2-hour exposure to 8% oxygen. Sham-operated rats served as the control group. Both groups were administered vehicle or 2, 10, or 50 mg/kg sildenafil, twice daily for 7 consecutive days. Retinal function was assessed by flash electroretinograms (ERGs) at P29, and retinal structure was assessed by retinal histology at P30. RESULTS: Hypoxia-ischemia caused significant functional (i.e., attenuation of the ERG a-wave and b-wave amplitudes and photopic negative response) and structural (i.e., thinning of the total retina, especially the inner retinal layers) retinal damage in the left eyes (i.e., ipsilateral to the carotid ligation). Treatment with the different doses of sildenafil led to a dose-dependent increase in the amplitudes of the ERG a- and b-waves and of the photopic negative response in HI animals, with higher doses associated with greater effect sizes. Similarly, a dose response was observed in terms of improvements in the retinal layer thicknesses. CONCLUSIONS: Hypoxia-ischemia at term-equivalent age induced functional and structural damage mainly to the inner retina. Treatment with sildenafil provided a dose-dependent recovery of retinal function and structure.

Impact of bronchopulmonary dysplasia on brain and retina.

Many premature newborns develop bronchopulmonary dysplasia (BPD), a chronic lung disease resulting from prolonged mechanical ventilation and hyperoxia. BPD survivors typically suffer long-term injuries not only to the lungs, but also to the brain and retina. However, currently it is not clear whether the brain and retinal injuries in these newborns are related only to their prematurity, or also to BPD. We investigated whether the hyperoxia known to cause histologic changes in the lungs similar to BPD in an animal model also causes brain and retinal injuries. Sprague Dawley rat pups were exposed to hyperoxia (95% O2, 'BPD' group) or room air (21% O2, 'control' group) from postnatal day 4-14 (P4-14); the rat pups were housed in room air between P14 and P28. At P28, they were sacrificed, and their lungs, brain, and eyes were extracted. Hematoxylin and eosin staining was performed on lung and brain sections; retinas were stained with Toluidine Blue. Hyperoxia exposure resulted in an increased mean linear intercept in the lungs (P<0.0001). This increase was associated with a decrease in some brain structures [especially the whole-brain surface (P=0.02)], as well as a decrease in the thickness of the retinal layers [especially the total retina (P=0.0008)], compared to the room air control group. In addition, a significant negative relationship was observed between the lung structures and the brain (r=-0.49,P=0.02) and retina (r=-0.70,P=0.0008) structures. In conclusion, hyperoxia exposure impaired lung, brain, and retina structures. More severe lung injuries correlated with more severe brain and retinal injuries. This result suggests that the same animal model of chronic neonatal hyperoxia can be used to simultaneously study lung, brain and retinal injuries related to hyperoxia.

Visual Impairments Following Term Neonatal Encephalopathy: Do Retinal Impairments Also Play a Role?

PURPOSE: We investigated the effects of term neonatal encephalopathy on retinal function and structure. METHODS: A rat model of term neonatal hypoxic-ischemic (HI) encephalopathy (Vannucci model) was used. Hypoxia-ischemia was induced by a left common carotid ligation followed by a 2-hour period of hypoxia (8% oxygen) in Long-Evans rat pups at postnatal day 10 (P10). Sham operated rats served as controls.. Retinal function was assessed at P30 and P60 by electroretinograms (ERGs), after which retinal histology was performed. Retinocortical function was assessed with visual evoked potentials (VEPs) at P60 and subsequently brain histology was performed. RESULTS: The ERGs of the HI animals at P30 and P60 demonstrated a significant reduction in the scotopic and photopic b-wave amplitudes, but a preserved a-wave amplitude. The retinal histology of the HI animals confirmed that the photoreceptor layer remained intact, whereas the inner layers of the retina were damaged. The HI animals also showed reduced VEP P100 amplitudes, which correlated with reduced left cerebral hemisphere surfaces. There was no correlation between the severities of retinal versus cerebral injuries. CONCLUSIONS: Our findings suggest that term neonatal encephalopathy resulting from HI induces functional and structural damages to the inner retina, while relatively sparing the photoreceptors. These findings raise the possibility that retinal injuries may contribute to visual impairments with or without the presence of brain injury in term asphyxiated newborns and, thus, warrant further studies with humans and animals to better understand the disease process.

Targeted delivery of vaccine to dendritic cells by chitosan nanoparticles conjugated with a targeting peptide ligand selected by phage display technique.

The paper presents a novel dendritic cells (DC)-targeting peptide, TPAFRYS (TP) identified by phage display technique and conjugated to chitosan in order to develop an efficient DC-targeting vaccine delivery carrier. TP-conjugated chitosan nanoparticles (TPC-NPs) were prepared with ovalbumin (OVA) as a model vaccine by ionic gelation. Flow cytometry and immunocytochemistry studies demonstrated the higher targeting ability of TPC-NPs to DCs in compared to chitosan NPs. Moreover, TPC-NPs exhibited higher targeting specificity in DCs than macrophage and myoblasts. Furthermore, immunization of mice with OVA-loaded TPC-NPs enhanced OVA-specific serum IgG and IgG isotype antibodies production. Thus, DC-targeting strategy demonstrates a potential approach to enhance the effectiveness of vaccines.

Solid solution cermet: (Ti,Nb)(CN)-Ni cermet.

Solid solution powders without W, (Ti,Nb)(CN) powders with a B1 structure (NaCl like), were synthesized by high energy milling and carbothermal reduction in nitrogen. The range of molar ratios of Ti/Nb for forming complete (Ti,Nb)(CN) phase was broader than that of Ti/W for the (Ti,W)(CN) phase because carbide or carbonitride of Nb had a B1 crystal structure identical to Ti(CN) while WC had a hexagonal crystal structure. The results revealed that the hardness of (Ti,Nb)(CN)-Ni cermets was higher than that of (Ti,W)(CN)-Ni cermets. The lower density of the (Ti,Nb)(CN) powder contributed to the higher hardness compared to (Ti,W)(CN) because the volumetric ratio of (Ti,Nb)(CN) in the (Ti,Nb)(CN)-Ni cermets was higher than that of (Ti,Nb)(CN) in the (Ti,W)(CN)-Ni cermets at the same weight ratio of Ni. Additionally, it was assumed that intrinsic the properties of (Ti,Nb)(CN) could also be the cause for the high hardness of the (Ti,Nb)(CN)-Ni cermets.

Solid-solution nanocrystallite formation by high-energy milling.

Solid-solution nanocrystalline powders were prepared by the high-energy milling of Ti alloys with graphite. The B1 structure (NaCl-like structure) phases, (Ti, Cr)C and (Ti, Al)C, were formed during the milling process of Ti-Cr + graphite and Ti-Al + graphite, and the synthetic procedures were investigated in terms of the phase evolution from XRD data. The (Ti, Al)C phase was obtained after milling for 20 hr at BPR = 40:1 (under a more severe condition), while the (Ti, Cr)C phase formed after milling for 20 hr at BPR = 20:1 (a relatively soft condition). The difference in the tendency to create a solid solution with Ti in the B1 structure caused a difference in the synthetic behavior of (Ti, Al)C and (Ti, Cr)C. In other words, (Ti, Cr)C is formed earlier than (Ti, Al)C during milling because the atomic size of Cr (0.166 nm) is similar to that of Ti (0.176 nm), which leads to the straightforward formation of the solid-solution (Ti, Cr)C as compared to when (Ti, Al)C is used. As a result, the crystallite size of the (Ti, Al)C phase (2-3 nm) synthesized at a later stage becomes smaller than that of the (Ti, Cr)C phase (5 10 nm) formed at an earlier stage during milling.

Leptin and interleukin-6 alter the function of mesolimbic dopamine neurons in a rodent model of prenatal inflammation.

Maternal inflammation during critical stages of gestation is thought to underlie the link between prenatal infection and several neurodevelopmental psychiatric disorders in the offspring, including schizophrenia. Increased activity of mesolimbic dopamine (DA) neurons, a hallmark of psychosis, is found in offspring of rodents exposed to a prenatal inflammatory challenge but it is unclear how this effect is elicited. Using an experimental model of localized aseptic inflammation with turpentine oil (TURP) we sought to establish whether circulating interleukin-6 (IL-6) and leptin play a role in the effects of prenatal inflammation on DA neurons. Both mediators are involved in the systemic inflammatory response to immunogens, with IL-6 mediating the early phase, followed by leptin in the late phase of the response. Maternal treatment with TURP at gestational day (GD) 15 enhanced the locomotor response to the DA indirect agonist, amphetamine (AMPH), increased the expression of tyrosine hydroxylase (TH), an enzyme involved in DA synthesis, DA levels and the expression of the post-synaptic protein spinophilin in the nucleus accumbens (NAcc) in the adult offspring. All of these alterations were totally abolished by co-treating the pregnant dams with a neutralizing IL-6 antiserum. Neutralization of maternal leptin prevented the enhanced behavioral sensitization and elevation of DA and spinophilin in the NAcc but spared other changes regulated by IL-6, such as increased NAcc TH levels and acute locomotor response to AMPH. Our results provide novel evidence to suggest that prenatal surges in both maternal circulating IL-6 and leptin contribute to the appearance of sensitized DA function in the adult offspring.