The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/ß-catenin pathway by inhibiting the proteasome.

UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear ß-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

Inhibition of Tau aggregation by three Aspergillus nidulans secondary metabolites: 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde.

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies.

Association of allelic combinations of FSHR gene polymorphisms with ovarian response.

During an IVF protocol, exogenous FSH is administered to women for ovulation induction. The ovarian response to gonadotrophin stimulation is variable and unpredictable in these women. The FSHR is the most studied gene in relation to ovarian response. The association of a FSHR gene polymorphism at position 680 (p.Asn680Ser) with ovarian response has been well documented. Recently, a polymorphism at position -29 in the 5'-untranslated region of FSHR (g.-29G>A) has been reported to be associated with poor ovarian response and reduced FSHR expression. The present study evaluated the combined effect of the polymorphisms at positions -29 and 680 of FSHR with type of ovarian response and receptor expression. The two FSHR gene polymorphisms together formed four discrete haplotypes and nine allelic combinations. Various clinical parameters revealed that 75% of the subjects with A/A-Asn/Asn genotype were poor ovarian responders (odds ratio 7.92; P=0.009). The relative FSHR mRNA expression in granulosa cells indicated that subjects with A/A-Asn/Asn genotype express significantly lower level of FSHR as compared to the subjects with G/G-Asn/Ser genotype (P=0.029). These results indicate that A/A-Asn/Asn genotype could be used as a potential marker to predict poor ovarian response. The action of FSH is mediated by its receptor (FSHR) present on the granulosa cells in the ovary. Any alterations in the hormone or its receptor are likely to disrupt its normal function, thus causing infertility. Several alterations (mutations/polymorphisms) of the FSHR gene have been reported in women with primary or secondary amenorrhoea. It has also been reported that FSHR gene polymorphisms are associated with variable ovarian response to FSH stimulation during IVF. Women may show poor or normal or hyperovarian response to FSH stimulation. It is well documented that the level of FSHR expression has a great effect on FSH action and is associated with ovarian response. In the present study, we screened normally menstruating women undergoing IVF due to tubal/male factor or unexplained infertility. We analysed two polymorphisms of FSHR, g-29G>A and p.Asn680Ser, in these women. In the subjects studied, 75% women with A/A-Asn/Asn genotype were observed to be poor ovarian responders to FSH stimulation. FSHR expression at the transcript level was observed to be significantly lower in women with A/A-Asn/Asn genotype as compared to women with G/G-Asn/Ser genotype. We also observed that women with A/A-Ser/Ser genotype were not present in the study population. These findings indicate the significance of A/A-Asn/Asn genotype as a predictive marker for poor ovarian response to FSH stimulation.

Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

High-throughput screening and classification of chemicals and their effects on neuronal gene expression using RASL-seq.

We previously used RNA-seq to identify chemicals whose effects on neuronal gene expression mimicked transcriptional signatures of autism, aging, and neurodegeneration. However, this approach was costly and time consuming, which limited our study to testing a single chemical concentration on mixed sex cortical neuron cultures. Here, we adapted a targeted transcriptomic method (RASL-seq, similar to TempO-seq) to interrogate changes in expression of a set of 56 signature genes in response to a library of 350 chemicals and chemical mixtures at four concentrations in male and female mouse neuronal cultures. This enabled us to replicate and expand our previous classifications, and show that transcriptional responses were largely equivalent between sexes. Overall, we found that RASL-seq can be used to accelerate the pace at which chemicals and mixtures that transcriptionally mimic autism and other neuropsychiatric diseases can be identified, and provides a cost-effective way to quantify gene expression with a panel of marker genes.

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

Azaphilones inhibit tau aggregation and dissolve tau aggregates in vitro.

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

Human cancer xenografts in outbred nude mice can be confounded by polymorphisms in a modifier of tumorigenesis.

Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.