A Smart Window with Passive Radiative Cooling and Switchable Near-Infrared Light Transmittance via Molecular Engineering.

Smart windows with synergetic light modulation have heightened demands for applications in smart cars and novel buildings. However, improving the on-demand energy-saving efficiency is quite challenging due to the difficulty of modulating sunlight with a broad bandwidth in an energy-saving way. Herein, a smart window with switchable near-infrared light transmittance and passive radiative cooling is prepared via a monomer design strategy and photoinduced polymerization. The effects of hydrogen bonds and fluorine groups in acrylate monomers on the electro-optical properties as well as microstructures of polymer-dispersed liquid crystal films have been systematically studied. Some films show a high contrast ratio of 90.4 or a low threshold voltage (Vth) of 2.0 V, which can be roll-to-roll processed in a large area. Besides, the film has a superior indoor temperature regulation ability due to its passive radiative cooling and controllable near-infrared light transmittance properties. Its radiative cooling efficiency is calculated to be 142.69 W/m2 and NIR transmittance could be switched to below 10%. The introduction of a carboxylic monomer and fluorinated monomer into the system endows the film with a highly efficient temperature management capability. The film has great potential for applications in fields such as flexible smart windows, camouflage materials, and so on.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Heterodimerization of apelin and opioid receptor-like 1 receptors mediates apelin-13-induced G protein biased signaling.

The apelin receptor (APJ) and the opioid-related nociceptin receptor 1 (ORL1) are family A G protein-coupled receptors that participate in a variety of physiological processes. The distribution and function of APJ and ORL1 in the nervous system and peripheral tissues are similar; however, the detailed mechanism of how these two receptors modulate signaling and physiological effects remains unclear. Here, we examined whether APJ and ORL1 form dimers, and investigated signal transduction pathways. The endogenous co-expression of APJ and ORL1 in SH-SY5Y cells was confirmed by western blotting and RT-PCR. Bioluminescence and fluorescence resonance energy transfer assays, as well as a proximity ligation assay and co-immunoprecipitation experiments, demonstrated that APJ and ORL1 heterodimerize in HEK293 cells. We found that the APJ-ORL1 heterodimer is selectively activated by apelin-13, which causes the dimer to couple to Gαi proteins and reduce the recruitment of GRKs and ß-arrestins to the dimer. We showed that the APJ-ORL1 dimer exhibits biased signaling, in which G protein-dependent signaling pathways override ß-arrestin-dependent signaling pathways. Our results demonstrate that the structural interface of the APJ-ORL1 dimer switches from transmembrane domain TM1/TM2 in the inactive state to TM5 in the active state. We used mutational analysis and BRET assays to identify key residues in TM5 (APJ L2185.55, APJ I2245.61, and ORL1 L2295.52) responsible for the receptor-receptor interaction. These results provide important information on the APJ-ORL1 heterodimer and may assist the design of new drugs targeting biased signaling pathways for treatment of pain and cardiovascular and metabolic diseases.

Laforin targets malin to glycogen in Lafora progressive myoclonus epilepsy.

Glycogen is the largest cytosolic macromolecule and is kept in solution through a regular system of short branches allowing hydration. This structure was thought to solely require balanced glycogen synthase and branching enzyme activities. Deposition of overlong branched glycogen in the fatal epilepsy Lafora disease (LD) indicated involvement of the LD gene products laforin and the E3 ubiquitin ligase malin in regulating glycogen structure. Laforin binds glycogen, and LD-causing mutations disrupt this binding, laforin-malin interactions and malin's ligase activity, all indicating a critical role for malin. Neither malin's endogenous function nor location had previously been studied due to lack of suitable antibodies. Here, we generated a mouse in which the native malin gene is tagged with the FLAG sequence. We show that the tagged gene expresses physiologically, malin localizes to glycogen, laforin and malin indeed interact, at glycogen, and malin's presence at glycogen depends on laforin. These results, and mice, open the way to understanding unknown mechanisms of glycogen synthesis critical to LD and potentially other much more common diseases due to incompletely understood defects in glycogen metabolism.

Glycogen synthase downregulation rescues the amylopectinosis of murine RBCK1 deficiency.

Longer glucan chains tend to precipitate. Glycogen, by far the largest mammalian glucan and the largest molecule in the cytosol with up to 55 000 glucoses, does not, due to a highly regularly branched spherical structure that allows it to be perfused with cytosol. Aberrant construction of glycogen leads it to precipitate, accumulate into polyglucosan bodies that resemble plant starch amylopectin and cause disease. This pathology, amylopectinosis, is caused by mutations in a series of single genes whose functions are under active study toward understanding the mechanisms of proper glycogen construction. Concurrently, we are characterizing the physicochemical particularities of glycogen and polyglucosans associated with each gene. These genes include GBE1, EPM2A and EPM2B, which respectively encode the glycogen branching enzyme, the glycogen phosphatase laforin and the laforin-interacting E3 ubiquitin ligase malin, for which an unequivocal function is not yet known. Mutations in GBE1 cause a motor neuron disease (adult polyglucosan body disease), and mutations in EPM2A or EPM2B a fatal progressive myoclonus epilepsy (Lafora disease). RBCK1 deficiency causes an amylopectinosis with fatal skeletal and cardiac myopathy (polyglucosan body myopathy 1, OMIM# 615895). RBCK1 is a component of the linear ubiquitin chain assembly complex, with unique functions including generating linear ubiquitin chains and ubiquitinating hydroxyl (versus canonical amine) residues, including of glycogen. In a mouse model we now show (i) that the amylopectinosis of RBCK1 deficiency, like in adult polyglucosan body disease and Lafora disease, affects the brain; (ii) that RBCK1 deficiency glycogen, like in adult polyglucosan body disease and Lafora disease, has overlong branches; (iii) that unlike adult polyglucosan body disease but like Lafora disease, RBCK1 deficiency glycogen is hyperphosphorylated; and finally (iv) that unlike laforin-deficient Lafora disease but like malin-deficient Lafora disease, RBCK1 deficiency's glycogen hyperphosphorylation is limited to precipitated polyglucosans. In summary, the fundamental glycogen pathology of RBCK1 deficiency recapitulates that of malin-deficient Lafora disease. Additionally, we uncover sex and genetic background effects in RBCK1 deficiency on organ- and brain-region specific amylopectinoses, and in the brain on consequent neuroinflammation and behavioural deficits. Finally, we exploit the portion of the basic glycogen pathology that is common to adult polyglucosan body disease, both forms of Lafora disease and RBCK1 deficiency, namely overlong branches, to show that a unified approach based on downregulating glycogen synthase, the enzyme that elongates glycogen branches, can rescue all four diseases.

FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability.

Ongoing inchworm-like CAG and CGG repeat expansions in brains, arising by aberrant processing of slipped DNAs, may drive Huntington's disease, fragile X syndrome, and autism. FAN1 nuclease modifies hyper-expansion rates by unknown means. We show that FAN1, through iterative cycles, binds, dimerizes, and cleaves slipped DNAs, yielding striking exo-nuclease pauses along slip-outs: 5'-C↓A↓GC↓A↓G-3' and 5'-C↓T↓G↓C↓T↓G-3'. CAG excision is slower than CTG and requires intra-strand A·A and T·T mismatches. Fully paired hairpins arrested excision, whereas disease-delaying CAA interruptions further slowed excision. Endo-nucleolytic cleavage is insensitive to slip-outs. Rare FAN1 variants are found in individuals with autism with CGG/CCG expansions, and CGG/CCG slip-outs show exo-nuclease pauses. The slip-out-specific ligand, naphthyridine-azaquinolone, which induces contractions of expanded repeats in vivo, requires FAN1 for its effect, and protects slip-outs from FAN1 exo-, but not endo-, nucleolytic digestion. FAN1's inchworm pausing of slip-out excision rates is well suited to modify inchworm expansion rates, which modify disease onset and progression.

Canine Lafora Disease: An Unstable Repeat Expansion Disorder.

Canine Lafora disease is a recessively inherited, rapidly progressing neurodegenerative disease caused by the accumulation of abnormally constructed insoluble glycogen Lafora bodies in the brain and other tissues due to the loss of NHL repeat containing E3 ubiquitin protein ligase 1 (NHLRC1). Dogs have a dodecamer repeat sequence within the NHLRC1 gene, which is prone to unstable (dynamic) expansion and loss of function. Progressive signs of Lafora disease include hypnic jerks, reflex and spontaneous myoclonus, seizures, vision loss, ataxia and decreased cognitive function. We studied five dogs (one Chihuahua, two French Bulldogs, one Griffon Bruxellois, one mixed breed) with clinical signs associated with canine Lafora disease. Identification of polyglucosan bodies (Lafora bodies) in myocytes supported diagnosis in the French Bulldogs; muscle areas close to the myotendinous junction and the myofascial union segment had the highest yield of inclusions. Postmortem examination of one of the French Bulldogs revealed brain Lafora bodies. Genetic testing for the known canine NHLRC1 mutation confirmed the presence of a homozygous mutation associated with canine Lafora disease. Our results show that Lafora disease extends beyond previous known breeds to the French Bulldog, Griffon Bruxellois and even mixed-breed dogs, emphasizing the likely species-wide nature of this genetic problem. It also establishes these breeds as animal models for the devastating human disease. Genetic testing should be used when designing breeding strategies to determine the frequency of the NHLRC1 mutation in affected breeds. Lafora diseases should be suspected in any older dog presenting with myoclonus, hypnic jerks or photoconvulsions.

Gys1 antisense therapy rescues neuropathological bases of murine Lafora disease.

Lafora disease is a fatal progressive myoclonus epilepsy. At root, it is due to constant acquisition of branches that are too long in a subgroup of glycogen molecules, leading them to precipitate and accumulate into Lafora bodies, which drive a neuroinflammatory response and neurodegeneration. As a potential therapy, we aimed to downregulate glycogen synthase, the enzyme responsible for glycogen branch elongation, in mouse models of the disease. We synthesized an antisense oligonucleotide (Gys1-ASO) that targets the mRNA of the brain-expressed glycogen synthase 1 gene (Gys1). We administered Gys1-ASO by intracerebroventricular injection and analysed the pathological hallmarks of Lafora disease, namely glycogen accumulation, Lafora body formation, and neuroinflammation. Gys1-ASO prevented Lafora body formation in young mice that had not yet formed them. In older mice that already exhibited Lafora bodies, Gys1-ASO inhibited further accumulation, markedly preventing large Lafora bodies characteristic of advanced disease. Inhibition of Lafora body formation was associated with prevention of astrogliosis and strong trends towards correction of dysregulated expression of disease immune and neuroinflammatory markers. Lafora disease manifests gradually in previously healthy teenagers. Our work provides proof of principle that an antisense oligonucleotide targeting the GYS1 mRNA could prevent, and halt progression of, this catastrophic epilepsy.

Ppp1r3d deficiency preferentially inhibits neuronal and cardiac Lafora body formation in a mouse model of the fatal epilepsy Lafora disease.

Mammalian glycogen chain lengths are subject to complex regulation, including by seven proteins (protein phosphatase-1 regulatory subunit 3, PPP1R3A through PPP1R3G) that target protein phosphatase-1 (PP1) to glycogen to activate the glycogen chain-elongating enzyme glycogen synthase and inactivate the chain-shortening glycogen phosphorylase. Lafora disease is a fatal neurodegenerative epilepsy caused by aggregates of long-chained, and as a result insoluble, glycogen, termed Lafora bodies (LBs). We previously eliminated PPP1R3C from a Lafora disease mouse model and studied the effect on LB formation. In the present work, we eliminate and study the effect of absent PPP1R3D. In the interim, brain cell type levels of all PPP1R3 genes have been published, and brain cell type localization of LBs clarified. Integrating these data we find that PPP1R3C is the major isoform in most tissues including brain. In the brain, PPP1R3C is expressed at 15-fold higher levels than PPP1R3D in astrocytes, the cell type where most LBs form. PPP1R3C deficiency eliminates ~90% of brain LBs. PPP1R3D is quantitatively a minor isoform, but possesses unique MAPK, CaMK2 and 14-3-3 binding domains and appears to have an important functional niche in murine neurons and cardiomyocytes. In neurons, it is expressed equally to PPP1R3C, and its deficiency eliminates ~50% of neuronal LBs. In heart, it is expressed at 25% of PPP1R3C where its deficiency eliminates ~90% of LBs. This work studies the role of a second (PPP1R3D) of seven PP1 subunits that regulate the structure of glycogen, toward better understanding of brain glycogen metabolism generally, and in Lafora disease.

An inducible glycogen synthase-1 knockout halts but does not reverse Lafora disease progression in mice.

Malstructured glycogen accumulates over time in Lafora disease (LD) and precipitates into Lafora bodies (LBs), leading to neurodegeneration and intractable fatal epilepsy. Constitutive reduction of glycogen synthase-1 (GYS1) activity prevents murine LD, but the effect of GYS1 reduction later in disease course is unknown. Our goal was to knock out Gys1 in laforin (Epm2a)-deficient LD mice after disease onset to determine whether LD can be halted in midcourse, or even reversed. We generated Epm2a-deficient LD mice with tamoxifen-inducible Cre-mediated Gys1 knockout. Tamoxifen was administered at 4 months and disease progression assessed at 12 months. We verified successful knockout at mRNA and protein levels using droplet digital PCR and Western blots. Glycogen determination and periodic acid-Schiff-diastase staining were used to analyze glycogen and LB accumulation. Immunohistochemistry using astrocytic (glial fibrillary acidic protein) and microglial (ionized calcium-binding adapter molecule 1) markers was performed to investigate neuroinflammation. In the disease-relevant organ, the brain, Gys1 mRNA levels were reduced by 85% and GYS1 protein depleted. Glycogen accumulation was halted at the 4-month level, while LB formation and neuroinflammation were significantly, though incompletely, prevented. Skeletal muscle analysis confirmed that Gys1 knockout inhibits glycogen and LB accumulation. However, tamoxifen-independent Cre recombination precluded determination of disease halting or reversal in this tissue. Our study shows that Gys1 knockdown is a powerful means to prevent LD progression, but this approach did not reduce brain glycogen or LBs to levels below those at the time of intervention. These data suggest that endogenous mechanisms to clear brain LBs are absent or, possibly, compromised in laforin-deficient murine LD.

GYS1 or PPP1R3C deficiency rescues murine adult polyglucosan body disease.

OBJECTIVE: Adult polyglucosan body disease (APBD) is an adult-onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease. METHODS: We characterized the effects of knocking out two pro-glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified. RESULTS: APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro- and micro-gliosis in the brains of GYS1- and PPP1R3C-deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies. INTERPRETATION: GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.

An EHBP-1-SID-3-DYN-1 axis promotes membranous tubule fission during endocytic recycling.

The ACK family tyrosine kinase SID-3 is involved in the endocytic uptake of double-stranded RNA. Here we identified SID-3 as a previously unappreciated recycling regulator in the Caenorhabditis elegans intestine. The RAB-10 effector EHBP-1 is required for the endosomal localization of SID-3. Accordingly, animals with loss of SID-3 phenocopied the recycling defects observed in ehbp-1 and rab-10 single mutants. Moreover, we detected sequential protein interactions between EHBP-1, SID-3, NCK-1, and DYN-1. In the absence of SID-3, DYN-1 failed to localize at tubular recycling endosomes, and membrane tubules breaking away from endosomes were mostly absent, suggesting that SID-3 acts synergistically with the downstream DYN-1 to promote endosomal tubule fission. In agreement with these observations, overexpression of DYN-1 significantly increased recycling transport in SID-3-deficient cells. Finally, we noticed that loss of RAB-10 or EHBP-1 compromised feeding RNAi efficiency in multiple tissues, implicating basolateral recycling in the transport of RNA silencing signals. Taken together, our study demonstrated that in C. elegans intestinal epithelia, SID-3 acts downstream of EHBP-1 to direct fission of recycling endosomal tubules in concert with NCK-1 and DYN-1.

Transmembrane peptide 4 and 5 of APJ are essential for its heterodimerization with OX1R.

Increasing evidence indicates some G protein-coupled receptors function as a heterodimer, which provide a novel target for therapeutics investigation. However, study on the receptor-receptor interaction interface, a potent target on interfering dimer formation, are still limited. Here, using bioluminescence resonance energy transfer (BRET) combined with co-immunoprecipitation (Co-IP), we found a new constitutive GPCR heterodimer, apelin receptor (APJ)-orexin receptor type 1 (OX1R). Both APJ and OX1R co-internalized when constantly subjected to cognate agonist (apelin-13 or orexin-A) specific to either protomer. Combined with BRET and immunostaining, the in vitro synthesized transmembrane peptides (TMs) interfering experiments suggests that TM4 and 5 of APJ act as the interaction interface of the APJ-OX1R heterodimer, and co-internalization could be disrupted by these peptides as well. Our study not only provide new evidence on GPCR heterodimerization, but address a novel heterodimerization interface, which can be severed as a potential pharmacological target.

Skeletal Muscle Glycogen Chain Length Correlates with Insolubility in Mouse Models of Polyglucosan-Associated Neurodegenerative Diseases.

Lafora disease (LD) and adult polyglucosan body disease (APBD) are glycogen storage diseases characterized by a pathogenic buildup of insoluble glycogen. Mechanisms causing glycogen insolubility are poorly understood. Here, in two mouse models of LD (Epm2a-/- and Epm2b-/-) and one of APBD (Gbe1ys/ys), the separation of soluble and insoluble muscle glycogen is described, enabling separate analysis of each fraction. Total glycogen is increased in LD and APBD mice, which, together with abnormal chain length and molecule size distributions, is largely if not fully attributed to insoluble glycogen. Soluble glycogen consists of molecules with distinct chain length distributions and differential corresponding solubility, providing a mechanistic link between soluble and insoluble glycogen in vivo. Phosphorylation states differ across glycogen fractions and mouse models, demonstrating that hyperphosphorylation is not a basic feature of insoluble glycogen. Lastly, model-specific variances in protein and activity levels of key glycogen synthesis enzymes suggest uninvestigated regulatory mechanisms.

Dominant LMAN2L mutation causes intellectual disability with remitting epilepsy.

Mis-secreted glycoproteins (LGI1, reelin) are emerging causes of epilepsy. LMAN2L belongs to a glycoprotein secretion chaperone family. One recessive LMAN2L missense mutation predicted to impair the chaperone's interaction with glycoproteins was reported in a family with intellectual disability (ID) and remitting epilepsy. We describe four members of a family with autosomal dominant inheritance of a similar phenotype. We show that they segregate a NM_001142292.1:c.1073delT mutation that eliminates LMAN2L's endoplasmic reticulum retention signal and mislocalizes the protein from that compartment to the plasma membrane. LMAN2L mislocalization, like impaired glycoprotein interaction, disturbs brain development, including generation of developmentally restricted epilepsy.

Ghrelin Through GHSR1a and OX1R Heterodimers Reveals a Gαs-cAMP-cAMP Response Element Binding Protein Signaling Pathway in Vitro.

Growth hormone secretagogue receptor 1α (GHSR1a) and Orexin 1 receptor (OX1R) are involved in various important physiological processes, and have many similar characteristics in function and distribution in peripheral tissues and the central nervous system. We explored the possibility of heterodimerization between GHSR1a and OX1R and revealed a signal transduction pathway mechanism. In this study, bioluminescence and fluorescence resonance energy transfer and co-immunoprecipitation (Co-IP) analyses were performed to demonstrate the formation of functional GHSR1a/OX1R heterodimers. This showed that a peptide corresponding to the 5-transmembrane domain of OX1R impaired heterodimer construction. We found that ghrelin stimulated GHSR1a/OX1R heterodimer cells to increase the activation of Gαs protein, compared to the cells that express GHSR1a. Stimulation of GHSR1a/OX1R heterodimers with orexin-A did not alter GPCR interactions with Gα protein subunits. GHSR1a/OX1R heterodimers induced Gαs and downstream signaling pathway activity, including increase of cAMP-response element luciferase reporter activity and cAMP levels. In addition, ghrelin induced a higher proliferation rate in SH-SY5Y cells than in controls. This suggests that ghrelin GHSR1a/OX1R heterodimers promotes an upregulation of a Gαs-cAMP-cAMP-responsive element signaling pathway in vitro and an increase in neuroblastoma cell proliferation.

Nationwide genetic testing towards eliminating Lafora disease from Miniature Wirehaired Dachshunds in the United Kingdom.

BACKGROUND: Canine DNA-testing has become an important tool in purebred dog breeding and many breeders use genetic testing results when planning their breeding strategies. In addition, information obtained from testing of hundreds dogs in one breed gives valuable information about the breed-wide genotype frequency of disease associated allele. Lafora disease is a late onset, recessively inherited genetic disease which is diagnosed in Miniature Wirehaired Dachshunds (MWHD). It is one of the most severe forms of canine epilepsy leading to neurodegeneration and, frequently euthanasia within a few years of diagnosis. Canine Lafora disease is caused by a dodecamer repeat expansion mutation in the NHLRC1 gene and a DNA test is available to identify homozygous dogs at risk, carriers and dogs free of the mutation. RESULTS: Blood samples were collected from 733 MWHDs worldwide, mostly of UK origin, for canine Lafora disease testing. Among the tested MWHD population 7.0% were homozygous for the mutation and at risk for Lafora disease. In addition, 234 dogs were heterozygous, indicating a carrier frequency of 31.9% in the tested population. Among the tested MWHDs, the mutant allele frequency was 0.2. In addition, data from the tested dogs over 6 years (2012-2017) indicated that the frequency of the homozygous and carrier dogs has decreased from 10.4% to 2.7% and 41.5% to 25.7%, respectively among MWHDs tested. As a consequence, the frequency of dogs free of the mutation has increased from 48.1% to 71.6%. CONCLUSIONS: This study provides valuable data for the MWHD community and shows that the DNA test is a useful tool for the breeders to prevent occurrence of Lafora disease in MWHDs. DNA testing has, over 6 years, helped to decrease the frequency of carriers and dogs at risk. Additionally, the DNA test can continue to be used to slowly eradicate the disease-causing mutation in the breed. However, this should be done carefully, over time, to avoid further compromising the genetic diversity of the breed. The DNA test also provides a diagnostic tool for veterinarians if they are presented with a dog that shows clinical signs associated with canine Lafora disease.