Direct haplotype-resolved 5-base HiFi sequencing for genome-wide profiling of hypermethylation outliers in a rare disease cohort.

Long-read HiFi genome sequencing allows for accurate detection and direct phasing of single nucleotide variants, indels, and structural variants. Recent algorithmic development enables simultaneous detection of CpG methylation for analysis of regulatory element activity directly in HiFi reads. We present a comprehensive haplotype resolved 5-base HiFi genome sequencing dataset from a rare disease cohort of 276 samples in 152 families to identify rare (~0.5%) hypermethylation events. We find that 80% of these events are allele-specific and predicted to cause loss of regulatory element activity. We demonstrate heritability of extreme hypermethylation including rare cis variants associated with short (~200 bp) and large hypermethylation events (>1 kb), respectively. We identify repeat expansions in proximal promoters predicting allelic gene silencing via hypermethylation and demonstrate allelic transcriptional events downstream. On average 30-40 rare hypermethylation tiles overlap rare disease genes per patient, providing indications for variation prioritization including a previously undiagnosed pathogenic allele in DIP2B causing global developmental delay. We propose that use of HiFi genome sequencing in unsolved rare disease cases will allow detection of unconventional diseases alleles due to loss of regulatory element activity.

Massively parallel identification of functionally consequential noncoding genetic variants in undiagnosed rare disease patients.

Clinical whole genome sequencing has enabled the discovery of potentially pathogenic noncoding variants in the genomes of rare disease patients with a prior history of negative genetic testing. However, interpreting the functional consequences of noncoding variants and distinguishing those that contribute to disease etiology remains a challenge. Here we address this challenge by experimentally profiling the functional consequences of rare noncoding variants detected in a cohort of undiagnosed rare disease patients at scale using a massively parallel reporter assay. We demonstrate that this approach successfully identifies rare noncoding variants that alter the regulatory capacity of genomic sequences. In addition, we describe an integrative analysis that utilizes genomic features alongside patient clinical data to further prioritize candidate variants with an increased likelihood of pathogenicity. This work represents an important step towards establishing a framework for the functional interpretation of clinically detected noncoding variants.

Estrogen Protects Optic Nerve Head Astrocytes Against Oxidative Stress by Preventing Caspase-3 Activation, Tau Dephosphorylation at Ser422 and the Formation of Tau Protein Aggregates.

Glaucoma is a neurodegenerative disorder that leads to the slow degeneration of retinal ganglion cells, and results in damage to the optic nerve and concomitant vision loss. As in other disorders affecting the viability of central nervous system neurons, neurons affected by glaucoma do not have the ability to regenerate after injury. Recent studies indicate a critical role for optic nerve head astrocytes (ONHAs) in this process of retinal ganglion cell degeneration. Cleavage of tau, a microtubule stabilizing protein and constituent of neurofibrillary tangles (NFT), plays a major part in the mechanisms that lead to toxicity in CNS neurons and astrocytes. Here, we tested the hypothesis that estrogen, a pleiotropic neuro- and cytoprotectant with high efficacy in the CNS, prevents tau cleavage, and hence, protects ONHAs against cell damage caused by oxidative stress. Our results indicate that estrogen prevents caspase-3 mediated tau cleavage, and thereby decreases the levels of the resulting form of proteolytically cleaved tau protein, which leads to a decrease in NFT formation, which requires proteolytically cleaved tau protein. Overall, our data propose that by stopping the reduction of estrogen levels involved with aging the sensitivity of the optic nerve to glaucomatous damage might be reduced. Furthermore, our data suggest that therapeutic use of estrogen may be beneficial in slowing or preventing the onset or severity of neurodegenerative diseases such as glaucoma and potentially also other degenerative diseases of the CNS through direct control of posttranslational modifications of tau protein.

Expediting rare disease diagnosis: a call to bridge the gap between clinical and functional genomics.

Approximately 400 million people throughout the world suffer from a rare disease. Although advances in whole exome and whole genome sequencing have greatly facilitated rare disease diagnosis, overall diagnostic rates remain below 50%. Furthermore, in cases where accurate diagnosis is achieved the process requires an average of 4.8 years. Reducing the time required for disease diagnosis is among the most critical needs of patients impacted by a rare disease. In this perspective we describe current challenges associated with rare disease diagnosis and discuss several cutting-edge functional genomic screening technologies that have the potential to rapidly accelerate the process of distinguishing pathogenic variants that lead to disease.

Resveratrol Protects Optic Nerve Head Astrocytes from Oxidative Stress-Induced Cell Death by Preventing Caspase-3 Activation, Tau Dephosphorylation at Ser422 and Formation of Misfolded Protein Aggregates.

Optic nerve head astrocytes (ONHAs) are the major cell type within the optic nerve head, providing both structural and nutrient support to the optic nerve. Astrocytes are necessary for the survival of neurons with controlled activation of astrocytes being beneficial to neurons. However, overactive astrocytes can be harmful and the loss of normal astrocyte function can be a primary contributor to neurodegeneration. The neuroprotective properties of reactive astrocytes can be lost or they might gain neurotoxic properties in neurodegenerative diseases. The activated astrocytes are crucial in the development of glaucoma, where they serve as a source for cytotoxic substances that participate in ganglion apoptosis. There is increasing evidence indicating that neuroinflammation is an important process in glaucoma. Under pathological conditions, astrocytes can induce an inflammatory response. Extensive evidence shows that inflammatory responses mediated by astrocytes can also influence pathology development, synapse health, and neurodegeneration. The elimination of activated astrocytes by apoptosis is also expected in unfavorable conditions. In neurodegenerative diseases, a common feature is the presence of aggregates found in astrocytes, which can disrupt astrocyte function in such a way as to be detrimental to the viability of neurons. The biological processes involved in vision loss in glaucoma are not well understood. Despite the rapid advances in our understanding of optic nerve head (ONH) structure and function, numerous potential contributions of the ONHAs to optic nerve damage remain unanswered. The present study investigated the role of ONHAs during oxidative stress in order to determine novel cell biological processes underlying glaucoma pathogenesis. ONHAs were exposed to chemically induced oxidative stress using tert-butyl hydroperoxide (tBHP) in order to model extracellular oxidative stress as it occurs in the glaucomatous retina and ONH. In order to determine the impact of an intervention approach employing potential glioprotective treatments for central nervous system tissue we pretreated cells with the polyphenolic phytostilbene and antioxidant trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene). ONHAs exposed to tBHP-mediated oxidative stress displayed decreased viability and underwent apoptosis. In addition, increased levels of activated caspases, dephosphorylation of Tau protein at Ser422, an important site adjacent to the caspase cleavage site controlling Tau cleavage, caspase-mediated Tau cleavage, and cytoskeletal changes, specifically formation of neurofibrillary tangles (NFTs) were detected in ONHAs undergoing oxidative stress. When cells were pretreated with resveratrol cell viability increased along with a significant decrease in activated caspases, cleaved Tau, and NFT formation. Taken together, ONHAs appear to act similar to neurons when undergoing oxidative stress, where proteolytic cleavage of Tau by caspases leads to NFT formation. In addition, resveratrol appears to have promise as a potential protective treatment preventing ONHA dysfunction and degeneration. There is currently no cure for glaucoma or a neuro- and glioprotective treatment that directly targets the pathogenic mechanisms in the glaucomatous retina and optic nerve. The present study identified a potential mechanism underlying degeneration of astrocytes that is susceptible to pharmaco-therapeutic intervention in the eye and potentially elsewhere in the central nervous system. Identification of such mechanisms involved in glaucoma and other disorders of the eye and brain is critical to determine novel targets for effective therapies.

Distinct Mechanisms Underlying Resveratrol-Mediated Protection from Types of Cellular Stress in C6 Glioma Cells.

The polyphenolic phytostilbene, trans-resveratrol, is found in high amounts in several types and tissues of plants, including grapes, and has been proposed to have beneficial effects in the central nervous system due to its activity as an antioxidant. The objective of the present study was to identify the mechanisms underlying the protective effects of resveratrol under conditions of oxidative stress or DNA damage, induced by the extracellularly applied oxidant, tert-butyl hydrogen peroxide, or UV-irradiation, respectively. In C6 glioma cells, a model system for glial cell biology and pharmacology, resveratrol was protective against both types of insult. Prevention of tau protein cleavage and of the formation of neurofibrillary tangles were identified as mechanisms of action of resveratrol-mediated protection in both paradigms of cellular damage. However, depending on the type of insult, resveratrol exerted its protective activity differentially: under conditions of chemically induced oxidative stress, inhibition of caspase activity, while with DNA damage, resveratrol regulated tau phosphorylation at Ser422. Results advance our understanding of resveratrol's complex impact on cellular signaling pathway and contribute to the notion of resveratrol's role as a pleiotropic therapeutic agent.

Caspase-3-Dependent Proteolytic Cleavage of Tau Causes Neurofibrillary Tangles and Results in Cognitive Impairment During Normal Aging.

Mouse models of neurodegenerative diseases such as Alzheimer's disease (AD) are important for understanding how pathological signaling cascades change neural circuitry and with time interrupt cognitive function. Here, we introduce a non-genetic preclinical model for aging and show that it exhibits cleaved tau protein, active caspases and neurofibrillary tangles, hallmarks of AD, causing behavioral deficits measuring cognitive impairment. To our knowledge this is the first report of a non-transgenic, non-interventional mouse model displaying structural, functional and molecular aging deficits associated with AD and other tauopathies in humans with potentially high impact on both new basic research into pathogenic mechanisms and new translational research efforts. Tau aggregation is a hallmark of tauopathies, including AD. Recent studies have indicated that cleavage of tau plays an important role in both tau aggregation and disease. In this study we use wild type mice as a model for normal aging and resulting age-related cognitive impairment. We provide evidence that aged mice have increased levels of activated caspases, which significantly correlates with increased levels of truncated tau and formation of neurofibrillary tangles. In addition, cognitive decline was significantly correlated with increased levels of caspase activity and tau truncated by caspase-3. Experimentally induced inhibition of caspases prevented this proteolytic cleavage of tau and the associated formation of neurofibrillary tangles. Our study shows the strength of using a non-transgenic model to study structure, function and molecular mechanisms in aging and age related diseases of the brain.

Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis.

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT(C/ala)) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT(C/ala) did not affect circadian behavior differently from wild-type DBT (DBT(WT)), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT(WT) protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT(C/ala) did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

Drosophila spaghetti and doubletime link the circadian clock and light to caspases, apoptosis and tauopathy.

While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. It is not known if age-dependent circadian dysfunction leads to neurodegeneration or vice-versa, and the proteins that mediate the effect remain unidentified. Here, we show that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthens circadian period and causes expression of activated Dronc, and a loss-of-function mutation in Clk also leads to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments place Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt are shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurs in cells that do not express the spag RNAi or dominant negative Dbt and requires PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity leads to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function show markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibit Dbt modification and activated caspase at particular times of day. These results suggest that Dbt suppresses expression of activated Dronc to prevent Tau cleavage, and that the circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. They establish a link between the circadian clock factors, light, cell death pathways and Tau toxicity, potentially via dysregulation of circadian neuronal remodeling in the optic lobes.

The role of casein kinase I in the Drosophila circadian clock.

The circadian clock mechanism in organisms as diverse as cyanobacteria and humans involves both transcriptional and posttranslational regulation of key clock components. One of the roles for the posttranslational regulation is to time the degradation of the targeted clock proteins, so that their oscillation profiles are out of phase with respect to those of the mRNAs from which they are translated. In Drosophila, the circadian transcriptional regulator PERIOD (PER) is targeted for degradation by a kinase (DOUBLETIME or DBT) orthologous to mammalian kinases (CKIɛ and CKIδ) that also target mammalian PER. Since these kinases are not regulated by second messengers, the mechanism (if any) for their regulation is not known. We are investigating the possibility that regulation of DBT is conferred by other proteins that associate with DBT and PER. In this chapter, the methods we are employing to identify and analyze these factors are discussed. These methods include expression of wild type and mutant proteins with the GAL4/UAS binary expression approach, analysis of DBT in Drosophila S2 cells, in vitro kinase assays with DBT isolated from S2 cells, and proteomic analysis of DBT-containing complexes and of DBT phosphorylation with mass spectrometry. The work has led to the discovery of a previously unrecognized circadian rhythm component (Bride of DBT, a noncanonical FK506-binding protein) and the mapping of autophosphorylation sites within the DBT C-terminal domain with potential regulatory roles.

Intrahaemocoelic infection of Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus does not induce tracheal cell basal lamina remodelling.

Infection of the lepidopteran insect Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by the oral route stimulates activation of host matrix metalloproteases (MMP) and effector caspases, a process dependent on expression of the viral fibroblast growth factor (vFGF). This pathway leads to tracheal cell basal lamina remodelling, enabling virus escape from the primary site of infection, the midgut epithelium, and establishment of efficient systemic infection. In this study, we asked whether the MMP-caspase pathway was also activated following infection by intrahaemocoelic injection. We found that intrahaemocoelic infection did not lead to any observable tracheal cell or midgut epithelium basal lamina remodelling. MMP and caspase activities were not significantly stimulated. We conclude that the main role of the AcMNPV vFGF is in facilitating virus midgut escape.

Viral fibroblast growth factor, matrix metalloproteases, and caspases are associated with enhancing systemic infection by baculoviruses.

Most arthropod-borne and invertebrate viruses are orally ingested and commence infection in cells of the invertebrate intestine. Infection of secondary sites and eventual transmission to other hosts is hindered by basal lamina, a tightly interwoven and virus-impenetrable noncellular layer, lining the intestine and other organ cell layers. The mechanisms for viral escape across basal laminae are unknown. We describe an elegant mechanism mediated by a baculovirus-encoded fibroblast growth factor (vFGF) that signals a previously undescribed stepwise cascade of protease activation wherein matrix metalloproteases activate effector caspases, leading to remodeling of basal lamina lining tracheal cells associated with the intestine and culminating in the establishment of efficient systemic infections. Because FGFs coordinate diverse functions during development, metabolic processes, and tissue repair, it is plausible that the vFGF-mediated pathway described here is widely used during developmental and pathogenic processes that involve basal lamina remodeling.

Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus.

Members of the baculovirus p35 gene family encode proteins that specifically inhibit caspases, cysteine proteases that are involved in apoptosis. To date, p35 homologs have only been found in baculoviruses. We have identified AMVp33, a gene from Amsacta moorei entomopoxvirus with low but significant homology to baculovirus p35 genes. Expression of AMVp33 blocked apoptosis in several different insect and human cell lines. Purified recombinant P33 protein was an efficient inhibitor of insect and human effector caspases, but not initiator caspases. P33 was cleaved by effector caspases, and the resulting cleavage fragments stably associated with the caspases. Mutation of the predicted caspase cleavage site in P33 eliminated cleavage, caspase inhibition and anti-apoptotic function. Thus, AMVp33 encodes a caspase inhibitor similar to baculovirus P35 with a preference for effector caspases. This is the first report of a p35 homolog from any viral or cellular genome outside of the baculovirus family.

The baculovirus anti-apoptotic protein Op-IAP does not inhibit Drosophila caspases or apoptosis in Drosophila S2 cells and instead sensitizes S2 cells to virus-induced apoptosis.

The Op-IAP protein from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) is highly effective at inhibiting apoptosis triggered by a variety of different stimuli in lepidopteran cells as well as in several different mammalian cell types, suggesting that it functions at a highly conserved step in the apoptotic pathway. However, the mechanism by which Op-IAP inhibits apoptosis is unclear. Since some IAP proteins can bind and inhibit caspases, we tested whether Op-IAP could inhibit the activity of caspases from Drosophila melanogaster. We found that recombinant Op-IAP protein was not able to bind or directly inhibit the activity of the Drosophila caspases DRONC, DrICE, or DCP-1 in vitro. In addition, expression of Op-IAP was unable to inhibit apoptosis triggered by either actinomycin D or UV light in D. melanogaster S2 cells. Surprisingly, Op-IAP expression in S2 cells enhanced apoptosis caused by baculovirus infection, but did not cause increased sensitivity to either actinomycin D or UV damage-induced apoptosis. The observation that Op-IAP cannot inhibit these insect caspases suggests that it functions by a mechanism that does not involve direct caspase inhibition.

Cleavage of the apoptosis inhibitor DIAP1 by the apical caspase DRONC in both normal and apoptotic Drosophila cells.

In Drosophila S2 cells, the apical caspase DRONC undergoes a low level of spontaneous autoprocessing. Unintended apoptosis is prevented by the inhibitor of apoptosis DIAP1, which targets the processed form of DRONC for degradation through its E3 ubiquitin protein ligase activity. Recent reports have demonstrated that shortly after the initiation of apoptosis in S2 cells, DIAP1 is cleaved following aspartate residue Asp-20 by the effector caspase DrICE. Here we report a novel caspase-mediated cleavage of DIAP1 in S2 cells. In both living and dying S2 cells, DIAP1 is cleaved by DRONC after glutamate residue Glu-205, located between the first and second BIR domains. The mutation of Glu-205 prevented the interaction of DIAP1 and processed DRONC but had no effect on the interaction with full-length DRONC. The mutation of Glu-205 also had a negative effect on the ability of overexpressed DIAP1 to prevent apoptosis stimulated by the proapoptotic protein Reaper or by UV light. These results expand our knowledge of the events that occur in the Drosophila apoptosome prior to and after receiving an apoptotic signal.

Silencing of the baculovirus Op-iap3 gene by RNA interference reveals that it is required for prevention of apoptosis during Orgyia pseudotsugata M nucleopolyhedrovirus infection of Ld652Y cells.

The Op-iap3 gene from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) inhibits apoptosis induced by a mutant of Autographa californica MNPV (AcMNPV) that lacks the antiapoptotic gene p35, as well as apoptosis induced by a wide range of other stimuli in both mammalian and insect cells. However, the role of Op-iap3 during OpMNPV infection has not been previously examined. To determine the function of the Op-IAP3 protein during OpMNPV infection, we used RNA interference (RNAi) to silence Op-iap3 expression during OpMNPV infection of Ld652Y cells. Infected cells treated with Op-iap3 double-stranded RNA (dsRNA) did not accumulate detectable Op-iap3 mRNA, confirming that the Op-iap3 gene was effectively silenced. Op-IAP3 protein was found to be a component of the budded virion; however, in OpMNPV-infected cells treated with Op-iap3 dsRNA, the Op-IAP3 protein that was introduced by the inoculum virus decreased to almost undetectable levels by 12 h after dsRNA addition. Apoptosis was observed in infected cells treated with Op-iap3 dsRNA beginning at 12 h, and by 48 h, almost all of the cells had undergone apoptosis. These results show for the first time that Op-IAP3 is necessary to prevent apoptosis during OpMNPV infection. In addition, our results demonstrate that the RNAi technique can be an effective tool for studying baculovirus gene function.