Loss of Katnal2 leads to ependymal ciliary hyperfunction and autism-related phenotypes in mice.

Autism spectrum disorders (ASD) frequently accompany macrocephaly, which often involves hydrocephalic enlargement of brain ventricles. Katnal2 is a microtubule-regulatory protein strongly linked to ASD, but it remains unclear whether Katnal2 knockout (KO) in mice leads to microtubule- and ASD-related molecular, synaptic, brain, and behavioral phenotypes. We found that Katnal2-KO mice display ASD-like social communication deficits and age-dependent progressive ventricular enlargements. The latter involves increased length and beating frequency of motile cilia on ependymal cells lining ventricles. Katnal2-KO hippocampal neurons surrounded by enlarged lateral ventricles show progressive synaptic deficits that correlate with ASD-like transcriptomic changes involving synaptic gene down-regulation. Importantly, early postnatal Katnal2 re-expression prevents ciliary, ventricular, and behavioral phenotypes in Katnal2-KO adults, suggesting a causal relationship and a potential treatment. Therefore, Katnal2 negatively regulates ependymal ciliary function and its deletion in mice leads to ependymal ciliary hyperfunction and hydrocephalus accompanying ASD-related behavioral, synaptic, and transcriptomic changes.

Increased Pro-Inflammatory T Cells, Senescent T Cells, and Immune-Check Point Molecules in the Placentas of Patients With Gestational Diabetes Mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is the most common metabolic complication of pregnancy. To define the altered pathway in GDM placenta, we investigated the transcriptomic profiles from human placenta between GDM and controls. METHODS: Clinical parameters and postpartum complications were reviewed in all participants. Differentially expressed canonical pathways were analyzed between the GDM and control groups based on transcriptomic analysis. CD4+ T, CD8+ T, and senescent T cell subsets were determined by flow cytometry based on staining for specific intracellular cytokines. RESULTS: Gene ontology analysis revealed that the placenta of GDM revealed upregulation of diverse mitochondria or DNA replication related pathways and downregulation of T-cell immunity related pathways. The maternal placenta of the GDM group had a higher proportion of CD4+ T and CD8+ T cells than the control group. Interestingly, senescent CD4+ T cells tended to increase and CD8+ T cells were significantly increased in GDM compared to controls, along with increased programmed cell death-1 (CD274+) expression. Programmed death-ligand 1 expression in syncytotrophoblasts was also significantly increased in patients with GDM. CONCLUSION: This study demonstrated increased proinflammatory T cells, senescent T cells and immune-check point molecules in GDM placentas, suggesting that changes in senescent T cells and immune-escape signaling might be related to the pathophysiology of GDM.

Dietary zinc supplementation rescues fear-based learning and synaptic function in the Tbr1+/- mouse model of autism spectrum disorders.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by a dyad of behavioural symptoms-social and communication deficits and repetitive behaviours. Multiple aetiological genetic and environmental factors have been identified as causing or increasing the likelihood of ASD, including serum zinc deficiency. Our previous studies revealed that dietary zinc supplementation can normalise impaired social behaviours, excessive grooming, and heightened anxiety in a Shank3 mouse model of ASD, as well as the amelioration of synapse dysfunction. Here, we have examined the efficacy and breadth of dietary zinc supplementation as an effective therapeutic strategy utilising a non-Shank-related mouse model of ASD-mice with Tbr1 haploinsufficiency. METHODS: We performed behavioural assays, amygdalar slice whole-cell patch-clamp electrophysiology, and immunohistochemistry to characterise the synaptic mechanisms underlying the ASD-associated behavioural deficits observed in Tbr1+/- mice and the therapeutic potential of dietary zinc supplementation. Two-way analysis of variance (ANOVA) with Sídák's post hoc test and one-way ANOVA with Tukey's post hoc multiple comparisons were performed for statistical analysis. RESULTS: Our data show that dietary zinc supplementation prevents impairments in auditory fear memory and social interaction, but not social novelty, in the Tbr1+/- mice. Tbr1 haploinsufficiency did not induce excessive grooming nor elevate anxiety in mice. At the synaptic level, dietary zinc supplementation reversed α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) hypofunction and normalised presynaptic function at thalamic-lateral amygdala (LA) synapses that are crucial for auditory fear memory. In addition, the zinc supplemented diet significantly restored the synaptic puncta density of the GluN1 subunit essential for functional NMDARs as well as SHANK3 expression in both the basal and lateral amygdala (BLA) of Tbr1+/- mice. LIMITATIONS: The therapeutic effect of dietary zinc supplementation observed in rodent models may not reproduce the same effects in human patients. The effect of dietary zinc supplementation on synaptic function in other brain structures affected by Tbr1 haploinsufficiency including olfactory bulb and anterior commissure will also need to be examined. CONCLUSIONS: Our data further the understanding of the molecular mechanisms underlying the effect of dietary zinc supplementation and verify the efficacy and breadth of its application as a potential treatment strategy for ASD.

Fetal cardiac parameters for predicting postnatal operation type of fetuses with tetralogy of Fallot.

BACKGROUND: To assess fetal cardiac parameters predictive of postnatal operation type in fetuses with tetralogy of Fallot (TOF). METHODS: Echocardiographic data obtained in the second and third trimesters were retrospectively reviewed for fetuses diagnosed with TOF between 2014 and 2018 at Asan Medical Center. The following fetal cardiac parameters were analyzed: 1) pulmonary valve annulus (PVA) z-score, 2) right pulmonary artery (RPA) z-score, 3) aortic valve annulus (AVA) z-score, 4) pulmonary valve peak systolic velocity (PV-PSV), 5) PVA/AVA ratio, and 6) RPA/descending aorta (DAo) ratio. These cardiac parameters were compared between a primary corrective surgery group and a palliative shunt operation followed by complete repair group. RESULTS: A total of 100 fetuses with TOF were included. Only one neonatal death occurred. Ninety patients underwent primary corrective surgery and 10 neonates underwent a multistage surgery. The PVA z-score, RPA z-score, and RPA/DAo ratio measured in the second trimester and the PVA z-score, RPA z-score, and PVA/AVA raio measured in the third trimester were significantly lower in the multistage surgery group, while the PV-PSV as measured in both trimesters were significantly higher in the multistage surgery group. CONCLUSION: Fetal cardiac parameters are useful for predicting the operation type necessary for neonates with TOF.

In vitro zinc supplementation alters synaptic deficits caused by autism spectrum disorder-associated Shank2 point mutations in hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders characterised by deficits in social interactions and repetitive behaviours. ASDs have a strong genetic basis with mutations involved in the development and function of neural circuitry. Shank proteins act as master regulators of excitatory glutamatergic synapses, and Shank mutations have been identified in people with ASD. Here, we have investigated the impact of ASD-associated Shank2 single nucleotide variants (SNVs) at the synaptic level, and the potential of in vitro zinc supplementation to prevent synaptic deficits. Dissociated rat hippocampal cultures expressing enhanced green fluorescent protein (EGFP) tagged Shank2-Wildtype (WT), and ASD-associated Shank2 single nucleotide variants (SNVs: S557N, V717F, and L1722P), were cultured in the absence or presence of 10 µM zinc. In comparison to Shank2-WT, ASD-associated Shank2 SNVs induced significant decreases in synaptic density and reduced the frequency of miniature excitatory postsynaptic currents. These structural and functional ASD-associated synaptic deficits were prevented by chronic zinc supplementation and further support zinc supplementation as a therapeutic target in ASD.

Isolation of adult mouse microglia using their in vitro adherent properties.

Microglia are the primary innate immune effectors of the central nervous system. Although numerous protocols have been developed to isolate fetal mouse microglia, the isolation of adult mouse microglia has proven more difficult. Here, we present a simple, widely accessible protocol to isolate pure microglia cultures from 4- to 14-month-old mouse brains using their adherent properties in vitro. These isolated microglia recapitulate the adherent properties of adult human microglia and present a more suitable model for studying age-related diseases. For complete details on the use and execution of this protocol in adult human microglia, please refer to Rustenhoven et al. (2016).

Shankopathies in the Developing Brain in Autism Spectrum Disorders.

The SHANK family of proteins play critical structural and functional roles in the postsynaptic density (PSD) at excitatory glutamatergic synapses. Through their multidomain structure they form a structural platform across the PSD for protein-protein interactions, as well as recruiting protein complexes to strengthen excitatory synaptic transmission. Mutations in SHANKs reflect their importance to synapse development and plasticity. This is evident in autism spectrum disorder (ASD), a neurodevelopmental disorder resulting in behavioural changes including repetitive behaviours, lack of sociability, sensory issues, learning, and language impairments. Human genetic studies have revealed ASD mutations commonly occur in SHANKs. Rodent models expressing these mutations display ASD behavioural impairments, and a subset of these deficits are rescued by reintroduction of Shank in adult animals, suggesting that lack of SHANK during key developmental periods can lead to permanent changes in the brain's wiring. Here we explore the differences in synaptic function and plasticity from development onward in rodent Shank ASD models. To date the most explored brain regions, relate to the behavioural changes observed, e.g., the striatum, hippocampus, sensory, and prefrontal cortex. In addition, less-studied regions including the hypothalamus, cerebellum, and peripheral nervous system are also affected. Synaptic phenotypes include weakened but also strengthened synaptic function, with NMDA receptors commonly affected, as well as changes in the balance of excitation and inhibition especially in cortical brain circuits. The effects of shankopathies in activity-dependent brain wiring is an important target for therapeutic intervention. We therefore highlight areas of research consensus and identify remaining questions and challenges.

Isolation and culture of functional adult human neurons from neurosurgical brain specimens.

The ability to characterize and study primary neurons isolated directly from the adult human brain would greatly advance neuroscience research. However, significant challenges such as accessibility of human brain tissue and the lack of a robust neuronal cell culture protocol have hampered its progress. Here, we describe a simple and reproducible method for the isolation and culture of functional adult human neurons from neurosurgical brain specimens. In vitro, adult human neurons form a dense network and express a plethora of mature neuronal and synaptic markers. Most importantly, for the first time, we demonstrate the re-establishment of mature neurophysiological properties in vitro, such as repetitive fast-spiking action potentials, and spontaneous and evoked synaptic activity. Together, our dissociated and slice culture systems enable studies of adult human neurophysiology and gene expression under normal and pathological conditions and provide a high-throughput platform for drug testing on brain cells directly isolated from the adult human brain.

Influence of maternal zinc supplementation on the development of autism-associated behavioural and synaptic deficits in offspring Shank3-knockout mice.

Autism Spectrum Disorders (ASD) are characterised by deficits in social interactions and repetitive behaviours. Multiple ASD-associated mutations have been identified in the Shank family of proteins that play a critical role in the structure and plasticity of glutamatergic synapses, leading to impaired synapse function and the presentation of ASD-associated behavioural deficits in mice. Shank proteins are highly regulated by zinc, where zinc binds the Shank SAM domain to drive synaptic protein recruitment and synaptic maturation. Here we have examined the influence of maternal dietary zinc supplementation during pregnancy and lactation on the development of ASD-associated behavioural and synaptic changes in the offspring Shank3 knockout (Shank3-/-) mice. Behavioural and electrophysiological experiments were performed in juvenile and adult Shank3-/- and wildtype littermate control mice born from mothers fed control (30 ppm, ppm) or supplemented (150 ppm) dietary zinc. We observed that the supplemented maternal zinc diet prevented ASD-associated deficits in social interaction and normalised anxiety behaviours in Shank3-/- offspring mice. These effects were maintained into adulthood. Repetitive grooming was also prevented in adult Shank3-/- offspring mice. At the synaptic level, maternal zinc supplementation altered postsynaptic NMDA receptor-mediated currents and presynaptic function at glutamatergic synapses onto medium spiny neurons in the cortico-striatal pathway of the Shank3-/- offspring mice. These data show that increased maternal dietary zinc during pregnancy and lactation can alter the development of ASD-associated changes at the synaptic and the behavioural levels, and that zinc supplementation from the beginning of brain development can prevent ASD-associated deficits in Shank3-/- mice long term.

Presynaptic PTPσ regulates postsynaptic NMDA receptor function through direct adhesion-independent mechanisms.

Synaptic adhesion molecules regulate synapse development and function. However, whether and how presynaptic adhesion molecules regulate postsynaptic NMDAR function remains largely unclear. Presynaptic LAR family receptor tyrosine phosphatases (LAR-RPTPs) regulate synapse development through mechanisms that include trans-synaptic adhesion; however, whether they regulate postsynaptic receptor functions remains unknown. Here we report that presynaptic PTPσ, a LAR-RPTP, enhances postsynaptic NMDA receptor (NMDAR) currents and NMDAR-dependent synaptic plasticity in the hippocampus. This regulation does not involve trans-synaptic adhesions of PTPσ, suggesting that the cytoplasmic domains of PTPσ, known to have tyrosine phosphatase activity and mediate protein-protein interactions, are important. In line with this, phosphotyrosine levels of presynaptic proteins, including neurexin-1, are strongly increased in PTPσ-mutant mice. Behaviorally, PTPσ-dependent NMDAR regulation is important for social and reward-related novelty recognition. These results suggest that presynaptic PTPσ regulates postsynaptic NMDAR function through trans-synaptic and direct adhesion-independent mechanisms and novelty recognition in social and reward contexts.

Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Shank3 Exon 13-16 Mutant Mice.

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in SHANK1-3 are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of SHANK3 causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength. As ASD-associated SHANK3 mutations retain responsiveness to zinc, here we investigated how increasing levels of dietary zinc could alter behavioral and synaptic deficits that occur with ASD. We performed behavioral testing together with cortico-striatal slice electrophysiology on a Shank3 -/- mouse model of ASD (Shank3 ex13-1616-/-), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in Shank3 ex13-16-/- mice prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. Dietary zinc supplementation also increased the recruitment of zinc sensitive SHANK2 to synapses, reduced synaptic transmission specifically through N-methyl-D-aspartate (NMDA)-type glutamate receptors, reversed the slowed decay tau of NMDA receptor (NMDAR)-mediated currents and occluded long term potentiation (LTP) at cortico-striatal synapses. These data suggest that alterations in NMDAR function underlie the lack of NMDAR-dependent cortico-striatal LTP and contribute to the reversal of ASD-related behaviors such as compulsive grooming. Our data reveal that dietary zinc alters neurological function from synapses to behavior, and identifies dietary zinc as a potential therapeutic agent in ASD.

Lipopolysaccharide-Induced Preconditioning Attenuates Apoptosis and Differentially Regulates TLR4 and TLR7 Gene Expression after Ischemia in the Preterm Ovine Fetal Brain.

Acute exposure to subclinical infection modulates subsequent hypoxia-ischemia (HI) injury in a time-dependent manner, likely by cross-talk through Toll-like receptors (TLRs), but the specific pathways are unclear in the preterm-equivalent brain. In the present study, we tested the hypothesis that repeated low-dose exposure to lipopolysaccharide (LPS) before acute ischemia would be associated with induction of specific TLRs that are potentially neuroprotective. Fetal sheep at 0.65 gestation (term is ∼145 days) received intravenous boluses of low-dose LPS for 5 days (day 1, 50 ng/kg; days 2-5, 100 ng/kg) or the same volume of saline. Either 4 or 24 h after the last bolus of LPS, complete carotid occlusion was induced for 22 min. Five days after LPS, brains were collected. Pretreatment with LPS for 5 days decreased cellular apoptosis, microglial activation and reactive astrogliosis in response to HI injury induced 24 but not 4 h after the last dose of LPS. This was associated with upregulation of TLR4, TLR7 and IFN-ß mRNA, and increased fetal plasma IFN-ß concentrations. The association of reduced white matter apoptosis and astrogliosis after repeated low-dose LPS finishing 24 h but not 4 h before cerebral ischemia, with central and peripheral induction of IFN-ß, suggests the possibility that IFN-ß may be an important mediator of endogenous neuroprotection in the developing brain.

SHON is a novel estrogen-regulated oncogene in mammary carcinoma that predicts patient response to endocrine therapy.

Endocrine therapies are the primary systemic intervention for patients with estrogen receptor-positive (ER(+)) breast cancer. However, a significant proportion of initially responsive ER(+) tumors develop resistance, with relapses occurring in up to 50% of patients. Lack of reliable predictive biomarkers remains an unfilled need for enhanced clinical management of this disease. In this study, we address this need in identifying a novel estrogen-regulated gene called SHON (secreted hominoid-specific oncogene). Enforced expression of SHON in breast cancer cells increased their proliferation, survival, migration, and invasion in vitro. Furthermore, SHON enhanced the oncogenicity of these cells in xenograft models of human breast cancer and was also sufficient to oncogenically transform MCF10A human mammary epithelial cells. Conversely, SHON attenuation mediated by RNA interference- or antibody-based methods reduced the oncogenicity of breast cancer cells. Mechanistic investigations indicated that the oncogenic transforming properties of SHON were mediated by BCL-2 and NF-κB. In primary clinical specimens, SHON was immunohistochemically detected in 62% of breast cancers, in which its expression was positively correlated with ER expression. In this setting, SHON expression predicted a favorable response to endocrine therapy in high-risk patients with ER(+) breast cancer. Taken together, our findings identify SHON as a novel human oncogene with predictive utility in ER(+) breast cancer, perhaps offering a simple biomarker to predict the therapeutic efficacy of antiestrogen therapy in patients with breast cancer.

Artemin Reduces Sensitivity to Doxorubicin and Paclitaxel in Endometrial Carcinoma Cells through Specific Regulation of CD24.

We have previously reported that artemin (ARTN) stimulates the oncogenicity and invasiveness of endometrial carcinoma cells. Herein, we demonstrate that ARTN modulates the sensitivity of endometrial carcinoma cells to agents used to treat late-stage endometrial carcinoma. Forced expression of ARTN in endometrial carcinoma cells decreased sensitivity to doxorubicin and paclitaxel. Accordingly, depletion of ARTN by small interfering RNA or functional inhibition of ARTN with antibodies significantly increased sensitivity of endometrial carcinoma cells to doxorubicin and paclitaxel. Forced expression of ARTN in endometrial carcinoma cells abrogated doxorubicin-induced G(2)-M arrest and paclitaxel-induced apoptosis. ARTN increased CD24 expression in endometrial carcinoma cells by transcriptional up-regulation, and CD24 was partially correlated to ARTN expression in endometrial carcinoma. Forced expression of CD24 in endometrial carcinoma cells stimulated cell proliferation and oncogenicity, enhanced cell invasion, and decreased sensitivity to doxorubicin and paclitaxel. Depletion of CD24 in endometrial carcinoma cells abrogated ARTN-stimulated resistance to doxorubicin and paclitaxel. ARTN-stimulated resistance to doxorubicin and paclitaxel in endometrial carcinoma cells is therefore mediated by the specific regulation of CD24. Functional inhibition of ARTN may therefore be considered as an adjuvant therapeutic approach to improve the response of endometrial carcinoma to specific chemotherapeutic agents.