Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub.

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers.

Lithium rescues dendritic abnormalities in Ank3 deficiency models through the synergic effects of GSK3ß and cyclic AMP signaling pathways.

Bipolar disorder (BD) is a highly heritable mood disorder with intermittent episodes of mania and depression. Lithium is the first-in-line medication to treat BD, but it is only effective in a subset of individuals. Large-scale human genomic studies have repeatedly linked the ANK3 gene (encoding ankyrin-G, AnkG) to BD. Ank3 knockout mouse models mimic BD behavioral features and respond positively to lithium treatment. We investigated cellular phenotypes associated with BD, including dendritic arborization of pyramidal neurons and spine morphology in two models: (1) a conditional knockout mouse model which disrupts Ank3 expression in adult forebrain pyramidal neurons, and (2) an AnkG knockdown model in cortical neuron cultures. We observed a decrease in dendrite complexity and a reduction of dendritic spine number in both models, reminiscent of reports in BD. We showed that lithium treatment corrected dendrite and spine deficits in vitro and in vivo. We targeted two signaling pathways known to be affected by lithium using a highly selective GSK3ß inhibitor (CHIR99021) and an adenylate cyclase activator (forskolin). In our cortical neuron culture model, CHIR99021 rescues the spine morphology defects caused by AnkG knockdown, whereas forskolin rescued the dendrite complexity deficit. Interestingly, a synergistic action of both drugs was required to rescue dendrite and spine density defects in AnkG knockdown neurons. Altogether, our results suggest that dendritic abnormalities observed in loss of function ANK3 variants and BD patients may be rescued by lithium treatment. Additionally, drugs selectively targeting GSK3ß and cAMP pathways could be beneficial in BD.

Cancer immunotherapy based on image-guided STING activation by nucleotide nanocomplex-decorated ultrasound microbubbles.

The cytosolic innate immune sensor cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is crucial for priming adaptive antitumour immunity through antigen-presenting cells (APCs). Natural agonists, such as cyclic dinucleotides (CDNs), activate the cGAS-STING pathway, but their clinical translation is impeded by poor cytosolic entry and serum stability, low specificity and rapid tissue clearance. Here we developed an ultrasound (US)-guided cancer immunotherapy platform using nanocomplexes composed of 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) electrostatically bound to biocompatible branched cationic biopolymers that are conjugated onto APC-targeting microbubbles (MBs). The nanocomplex-conjugated MBs engaged with APCs and efficiently delivered cGAMP into the cytosol via sonoporation, resulting in activation of cGAS-STING and downstream proinflammatory pathways that efficiently prime antigen-specific T cells. This bridging of innate and adaptive immunity inhibited tumour growth in both localized and metastatic murine cancer models. Our findings demonstrate that targeted local activation of STING in APCs under spatiotemporal US stimulation results in systemic antitumour immunity and improves the therapeutic efficacy of checkpoint blockade, thus paving the way towards novel image-guided strategies for targeted immunotherapy of cancer.

Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca2+ homeostasis and network synchrony via PMCA2/ATP2B2.

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony by a shed ectodomain.

Noble-Metal Nanoframes and Their Catalytic Applications.

Noble-metal nanoframes consisting of interconnected, ultrathin ridges have received considerable attention in the field of heterogeneous catalysis. The enthusiasm arises from the high utilization efficiency of atoms for significantly reducing the material loading while enhancing the catalytic performance. In this review article, we offer a comprehensive assessment of research endeavors in the design and rational synthesis of noble-metal nanoframes for applications in catalysis. We start with a brief introduction to the unique characteristics of nanoframes, followed by a discussion of the synthetic strategies and their controls in terms of structure and composition. We then present case studies to elucidate mechanistic details behind the synthesis of mono-, bi-, and multimetallic nanoframes, as well as heterostructured and hybrid systems. We discuss their performance in electrocatalysis, thermal catalysis, and photocatalysis. Finally, we highlight recent progress in addressing the structural and compositional stability issues of nanoframes for the assurance of robustness in catalytic applications.

Preserving the shape of silver nanocubes under corrosive environment by covering their edges and corners with iridium.

Silver nanocubes have found use in an array of applications but their performance has been plagued by the shape instability arising from the oxidation and dissolution of Ag atoms from the edges and corners. Here we demonstrate that the shape of Ag nanocubes can be well preserved by covering their edges and corners with a corrosion-resistant metal such as Ir. In a typical process, we titrate a Na3IrCl6 solution in ethylene glycol (EG) into a suspension of Ag nanocubes in an EG solution in the presence of poly(vinylpyrrolidone) (PVP) held at 110 °C. The Ir atoms derived from the reduction of Na3IrCl6 by EG and Ag are deposited onto the edges and then corners for the generation of Ag-Ir core-frame nanocubes. Remarkably, our results indicate that a small amount of Ir atoms on the edges and corners is adequate to prevent the Ag nanocubes from transforming into nanospheres when heated in a PVP/EG solution up to 110 °C. We further demonstrate that these Ag-Ir nanocubes embrace plasmonic properties comparable to those of the original Ag nanocubes, making them immediately useful in a variety of applications. This strategy for stabilizing the shape of Ag nanocubes should be extendible to Ag nanocrystals with other shapes or nanocrystals comprised of other metals.

Toxicity and Survival of Hepatocellular Carcinoma Patients with Hepatitis B Infection Treated with Yttrium-90 Radioembolization: An Updated 15-Year Study.

PURPOSE: To evaluate the toxicity and survival of hepatocellular carcinoma (HCC) secondary to hepatitis B virus (HBV) infection treated with yttrium-90 transarterial radioembolization (TARE) over a 15-year period. MATERIALS AND METHODS: This study retrospectively analyzed 93 consecutive patients with HBV HCC-all derived from an original cohort of 1,000 patients-who were treated with TARE via standard radiation segmentectomy/lobectomy between December 2003 and December 2018. This group comprised 80 males and 13 females, with 79 having only HBV and 14 having additional liver comorbidities. Toxicity grades were determined by Common Terminology Criteria for Adverse Events, version 5.0. Overall survival (OS) was reported using intention-to-treat (ITT), censored, or competing risk. Univariate/multivariate analyses were used to evaluate predictors of OS. RESULTS: Posttreatment grade 3/4 toxicities included albumin (1.1%), bilirubin (4.3%), aspartate transaminase (6.5%), and alanine transaminase (3.2%). Median censored OS was 16.9 months (95% confidence interval [CI], 11.8-23.5): 17.5 months (95% CI, 11.5-86.9) for Child-Pugh (CP) A and 14.5 months (95% CI, 5.2-22.5) for CP B; not reached, 16.9 months (95% CI, 11.2-68.7), and 11.5 months (95% CI, 8.6-17.5) for Barcelona Clinic Liver Cancer (BCLC) A, B, and C, respectively. Multivariate analysis revealed albumin, alpha-fetoprotein, and portal vein thrombosis as independent predictors of ITT OS and albumin and tumor size as predictors when curative therapy was assigned as a competing risk. CONCLUSIONS: This retrospective study showed that TARE therapy resulted in minimal toxicity in patients with HBV-derived HCC. Patients with CP A or BCLC A disease had superior survival outcomes compared to patients with CP B and BCLC B/C disease. These findings suggest that TARE is a viable treatment option for certain patient groups with HCC tumors secondary to HBV infection.

A novel role for the late-onset Alzheimer's disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling.

Postsynaptic trafficking plays a key role in regulating synapse structure and function. While spiny excitatory synapses can be stable throughout adult life, their morphology and function is impaired in Alzheimer's disease (AD). However, little is known about how AD risk genes impact synaptic function. Here we used structured superresolution illumination microscopy (SIM) to study the late-onset Alzheimer's disease (LOAD) risk factor BIN1, and show that this protein is abundant in postsynaptic compartments, including spines. While postsynaptic Bin1 shows colocalization with clathrin, a major endocytic protein, it also colocalizes with the small GTPases Rab11 and Arf6, components of the exocytic pathway. Bin1 participates in protein complexes with Arf6 and GluA1, and manipulations of Bin1 lead to changes in spine morphology, AMPA receptor surface expression and trafficking, and AMPA receptor-mediated synaptic transmission. Our data provide new insights into the mesoscale architecture of postsynaptic trafficking compartments and their regulation by a major LOAD risk factor.

CNTNAP2 is targeted to endosomes by the polarity protein PAR3.

A decade of genetic studies has established contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2's molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and partitioning-defective 3 (PAR3)-a polarity molecule isolated in a yeast two-hybrid screen with CNTNAP2's C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+ /PAR3+ complexes are largely associated with clathrin-coated endocytic vesicles in heterologous cells and that PAR3 causes an enlargement of CNTNAP2 puncta size. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that PAR3 limits the mobility of CNTNAP2. Finally, overexpression of PAR3 but not a PAR3 mutant lacking all PDZ domains (PAR3∆PDZall) can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that PAR3 regulates CNTNAP2 spatial localization.

The CNTNAP2-CASK complex modulates GluA1 subcellular distribution in interneurons.

GABAergic interneurons are emerging as prominent substrates in the pathophysiology of multiple neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, and epilepsy. Interneuron excitatory activity is influenced by 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs), which in turn affects excitatory transmission in the central nervous system. Yet how dysregulation of interneuronal AMPARs distinctly contributes to the molecular underpinning of neurobiological disease is drastically underexplored. Contactin-associated protein-like 2 (CNTNAP2) is a neurexin-related adhesion molecule shown to mediate AMPAR subcellular distribution while calcium/calmodulin-dependent serine protein kinase (CASK) is a multi-functional scaffold involved with glutamate receptor trafficking. Mutations in both genes have overlapping disease associations, including autism spectrum disorders, intellectual disability, and epilepsy, thus suggesting converging perturbations of excitatory/inhibitory balance. Our lab has previously shown that CNTNAP2 stabilizes interneuron dendritic arbors through CASK and that CNTNAP2 regulates AMPAR subunit GluA1 trafficking in excitatory neurons. The interaction between these three proteins, however, has not been studied in interneurons. Using biochemical techniques, structured illumination microscopy (SIM) and shRNA technology, we first confirm that these three proteins interact in mouse brain, and then examined relationship between CNTNAP2, CASK and GluA1 in mature interneurons. Using SIM, we ascertain that a large fraction of endogenous CNTNAP2, CASK, and GluA1 molecules collectively colocalize together in a tripartite manner. Finally, individual knockdown of either CNTNAP2 or CASK similarly alter GluA1 levels and localization. These findings offer insight to molecular mechanisms underlying GluA1 regulation in interneurons.

CNTNAP2 stabilizes interneuron dendritic arbors through CASK.

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2's C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

A Schizophrenia-Linked KALRN Coding Variant Alters Neuron Morphology, Protein Function, and Transcript Stability.

BACKGROUND: Large-scale genetic studies have revealed that rare sequence variants, including single nucleotide variants (SNVs), in glutamatergic synaptic genes are enriched in schizophrenia patients. However, the majority are too rare to show any association with disease and have not been examined functionally. One such SNV, KALRN-P2255T, displays a penetrance that greatly exceeds that of previously identified schizophrenia-associated SNVs. Therefore, we sought to characterize its effects on the function of kalirin (Kal)-9, a dual Ras-related C3 botulinum toxin substrate 1 and Ras homologue gene family, member A (RhoA) guanine nucleotide exchange factor, upregulated in human schizophrenia brain tissue. METHODS: Kal9 was overexpressed in primary rat cortical neurons or human embryonic kidney 293 (HEK293) cells. The effects of the P2255T variant on dendritic branching, dendritic spine morphology, protein and messenger RNA stability, and catalytic activity were examined. RESULTS: Kal9-P2255T leads to diminished basal dendritic branching and dendritic spine size, compared with wild-type Kal9. The P2255T SNV directly affected Kal9 protein function, causing increased RhoA activation in HEK293 cells, but had no effect on Ras-related C3 botulinum toxin substrate 1 activation. Consistent with human postmortem findings, we found that Kal9-P2255T protein levels were higher than those of wild-type Kal9 in neurons. Increased messenger RNA stability was detected in HEK293 cells, indicating that this was the cause of the higher protein levels. When analyzed together, increased intrinsic RhoA guanine nucleotide exchange factor catalytic activity combined with increased messenger RNA expression led to net enhancement of RhoA activation, known to negatively impact neuronal morphology. CONCLUSIONS: Taken together, our data reveal a novel mechanism for disease-associated SNVs and provide a platform for modeling morphological changes in mental disorders.

Psychiatric risk factor ANK3/ankyrin-G nanodomains regulate the structure and function of glutamatergic synapses.

Recent evidence implicates glutamatergic synapses as key pathogenic sites in psychiatric disorders. Common and rare variants in the ANK3 gene, encoding ankyrin-G, have been associated with bipolar disorder, schizophrenia, and autism. Here we demonstrate that ankyrin-G is integral to AMPAR-mediated synaptic transmission and maintenance of spine morphology. Using superresolution microscopy we find that ankyrin-G forms distinct nanodomain structures within the spine head and neck. At these sites, it modulates mushroom spine structure and function, probably as a perisynaptic scaffold and barrier within the spine neck. Neuronal activity promotes ankyrin-G accumulation in distinct spine subdomains, where it differentially regulates NMDA receptor-dependent plasticity. These data implicate subsynaptic nanodomains containing a major psychiatric risk molecule, ankyrin-G, as having location-specific functions and open directions for basic and translational investigation of psychiatric risk molecules.

Increased expression of Kalirin-9 in the auditory cortex of schizophrenia subjects: its role in dendritic pathology.

Reductions in dendritic arbor length and complexity are among the most consistently replicated changes in neuronal structure in post mortem studies of cerebral cortical samples from subjects with schizophrenia, however, the underlying molecular mechanisms have not been identified. This study is the first to identify an alteration in a regulatory protein which is known to promote both dendritic length and arborization in developing neurons, Kalirin-9. We found Kalirin-9 expression to be paradoxically increased in schizophrenia. We followed up this observation by overexpressing Kalirin-9 in mature primary neuronal cultures, causing reduced dendritic length and complexity. Kalirin-9 overexpression represents a potential mechanism for dendritic changes seen in schizophrenia.