Tuning Apoptosis and Neuroinflammation: TBK1 Restrains RIPK1.

Partial loss of TANK-binding kinase 1 (TBK1) causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Xu et al. identify the role of TBK1 in suppressing neuroinflammation and apoptosis by its inhibition of the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and elucidate how aging and genetic susceptibility together cause neuroinflammation.

Rethinking Unconventional Translation in Neurodegeneration.

Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.

Scrambling the genome in cancer: causes and consequences of complex chromosome rearrangements.

Complex chromosome rearrangements, known as chromoanagenesis, are widespread in cancer. Based on large-scale DNA sequencing of human tumours, the most frequent type of complex chromosome rearrangement is chromothripsis, a massive, localized and clustered rearrangement of one (or a few) chromosomes seemingly acquired in a single event. Chromothripsis can be initiated by mitotic errors that produce a micronucleus encapsulating a single chromosome or chromosomal fragment. Rupture of the unstable micronuclear envelope exposes its chromatin to cytosolic nucleases and induces chromothriptic shattering. Found in up to half of tumours included in pan-cancer genomic analyses, chromothriptic rearrangements can contribute to tumorigenesis through inactivation of tumour suppressor genes, activation of proto-oncogenes, or gene amplification through the production of self-propagating extrachromosomal circular DNAs encoding oncogenes or genes conferring anticancer drug resistance. Here, we discuss what has been learned about the mechanisms that enable these complex genomic rearrangements and their consequences in cancer.

Mitotic tethering enables inheritance of shattered micronuclear chromosomes.

Chromothripsis, the shattering and imperfect reassembly of one (or a few) chromosome(s)1, is an ubiquitous2 mutational process generating localized and complex chromosomal rearrangements that drive genome evolution in cancer. Chromothripsis can be initiated by mis-segregation errors in mitosis3,4 or DNA metabolism5-7 that lead to entrapment of chromosomes within micronuclei and their subsequent fragmentation in the next interphase or following mitotic entry6,8-10. Here we use inducible degrons to demonstrate that chromothriptically produced pieces of a micronucleated chromosome are tethered together in mitosis by a protein complex consisting of mediator of DNA damage checkpoint 1 (MDC1), DNA topoisomerase II-binding protein 1 (TOPBP1) and cellular inhibitor of PP2A (CIP2A), thereby enabling en masse segregation to the same daughter cell. Such tethering is shown to be crucial for the viability of cells undergoing chromosome mis-segregation and shattering after transient inactivation of the spindle assembly checkpoint. Transient, degron-induced reduction in CIP2A following chromosome micronucleation-dependent chromosome shattering is shown to drive acquisition of segmental deletions and inversions. Analyses of pancancer tumour genomes showed that expression of CIP2A and TOPBP1 was increased overall in cancers with genomic rearrangements, including copy number-neutral chromothripsis with minimal deletions, but comparatively reduced in cancers with canonical chromothripsis in which deletions were frequent. Thus, chromatin-bound tethers maintain the proximity of fragments of a shattered chromosome enabling their re-encapsulation into, and religation within, a daughter cell nucleus to form heritable, chromothriptically rearranged chromosomes found in the majority of human cancers.

Chromosomal fragile site breakage by EBV-encoded EBNA1 at clustered repeats.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with several cancers of lymphocytic and epithelial origin1-3. EBV encodes EBNA1, which binds to a cluster of 20 copies of an 18-base-pair palindromic sequence in the EBV genome4-6. EBNA1 also associates with host chromosomes at non-sequence-specific sites7, thereby enabling viral persistence. Here we show that the sequence-specific DNA-binding domain of EBNA1 binds to a cluster of tandemly repeated copies of an EBV-like, 18-base-pair imperfect palindromic sequence encompassing a region of about 21 kilobases at human chromosome 11q23. In situ visualization of the repetitive EBNA1-binding site reveals aberrant structures on mitotic chromosomes characteristic of inherently fragile DNA. We demonstrate that increasing levels of EBNA1 binding trigger dose-dependent breakage at 11q23, producing a fusogenic centromere-containing fragment and an acentric distal fragment, with both mis-segregated into micronuclei in the next cell cycles. In cells latently infected with EBV, elevating EBNA1 abundance by as little as twofold was sufficient to trigger breakage at 11q23. Examination of whole-genome sequencing of EBV-associated nasopharyngeal carcinomas revealed that structural variants are highly enriched on chromosome 11. Presence of EBV is also shown to be associated with an enrichment of chromosome 11 rearrangements across 2,439 tumours from 38 cancer types. Our results identify a previously unappreciated link between EBV and genomic instability, wherein EBNA1-induced breakage at 11q23 triggers acquisition of structural variations in chromosome 11.

Transient genomic instability drives tumorigenesis through accelerated clonal evolution.

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.

Antisense Oligonucleotide Therapies for Neurodegenerative Diseases.

Antisense oligonucleotides represent a novel therapeutic platform for the discovery of medicines that have the potential to treat most neurodegenerative diseases. Antisense drugs are currently in development for the treatment of amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease, and multiple research programs are underway for additional neurodegenerative diseases. One antisense drug, nusinersen, has been approved for the treatment of spinal muscular atrophy. Importantly, nusinersen improves disease symptoms when administered to symptomatic patients rather than just slowing the progression of the disease. In addition to the benefit to spinal muscular atrophy patients, there are discoveries from nusinersen that can be applied to other neurological diseases, including method of delivery, doses, tolerability of intrathecally delivered antisense drugs, and the biodistribution of intrathecal dosed antisense drugs. Based in part on the early success of nusinersen, antisense drugs hold great promise as a therapeutic platform for the treatment of neurological diseases.

Single-stranded RNAs use RNAi to potently and allele-selectively inhibit mutant huntingtin expression.

Mutant huntingtin (HTT) protein causes Huntington disease (HD), an incurable neurological disorder. Silencing mutant HTT using nucleic acids would eliminate the root cause of HD. Developing nucleic acid drugs is challenging, and an ideal clinical approach to gene silencing would combine the simplicity of single-stranded antisense oligonucleotides with the efficiency of RNAi. Here, we describe RNAi by single-stranded siRNAs (ss-siRNAs). ss-siRNAs are potent (>100-fold more than unmodified RNA) and allele-selective (>30-fold) inhibitors of mutant HTT expression in cells derived from HD patients. Strategic placement of mismatched bases mimics micro-RNA recognition and optimizes discrimination between mutant and wild-type alleles. ss-siRNAs require Argonaute protein and function through the RNAi pathway. Intraventricular infusion of ss-siRNA produced selective silencing of the mutant HTT allele throughout the brain in a mouse HD model. These data demonstrate that chemically modified ss-siRNAs function through the RNAi pathway and provide allele-selective compounds for clinical development.

Chromothripsis drives the evolution of gene amplification in cancer.

Focal chromosomal amplification contributes to the initiation of cancer by mediating overexpression of oncogenes1-3, and to the development of cancer therapy resistance by increasing the expression of genes whose action diminishes the efficacy of anti-cancer drugs. Here we used whole-genome sequencing of clonal cell isolates that developed chemotherapeutic resistance to show that chromothripsis is a major driver of circular extrachromosomal DNA (ecDNA) amplification (also known as double minutes) through mechanisms that depend on poly(ADP-ribose) polymerases (PARP) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Longitudinal analyses revealed that a further increase in drug tolerance is achieved by structural evolution of ecDNAs through additional rounds of chromothripsis. In situ Hi-C sequencing showed that ecDNAs preferentially tether near chromosome ends, where they re-integrate when DNA damage is present. Intrachromosomal amplifications that formed initially under low-level drug selection underwent continuing breakage-fusion-bridge cycles, generating amplicons more than 100 megabases in length that became trapped within interphase bridges and then shattered, thereby producing micronuclei whose encapsulated ecDNAs are substrates for chromothripsis. We identified similar genome rearrangement profiles linked to localized gene amplification in human cancers with acquired drug resistance or oncogene amplifications. We propose that chromothripsis is a primary mechanism that accelerates genomic DNA rearrangement and amplification into ecDNA and enables rapid acquisition of tolerance to altered growth conditions.

Epigenetic centromere propagation and the nature of CENP-a nucleosomes.

Centromeres direct chromosome inheritance, but in multicellular organisms their positions on chromosomes are primarily specified epigenetically rather than by a DNA sequence. The major candidate for the epigenetic mark is chromatin assembled with the histone H3 variant CENP-A. Recent studies offer conflicting evidence for the structure of CENP-A-containing chromatin, including the histone composition and handedness of the DNA wrapped around the histones. We present a model for the assembly and deposition of centromeric nucleosomes that couples these processes to the cell cycle. This model reconciles divergent data for CENP-A-containing nucleosomes and provides a basis for how centromere identity is stably inherited.

The seeds of neurodegeneration: prion-like spreading in ALS.

Misfolded proteins accumulating in several neurodegenerative diseases (including Alzheimer, Parkinson, and Huntington diseases) can cause aggregation of their native counterparts through a mechanism similar to the infectious prion protein's induction of a pathogenic conformation onto its cellular isoform. Evidence for such a prion-like mechanism has now spread to the main misfolded proteins, SOD1 and TDP-43, implicated in amyotrophic lateral sclerosis (ALS). The major neurodegenerative diseases may therefore have mechanistic parallels for non-cell-autonomous spread of disease within the nervous system.

Author Correction: Reversing a model of Parkinson's disease with in situ converted nigral neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reversing a model of Parkinson's disease with in situ converted nigral neurons.

Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2. Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson's disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

How to survive aneuploidy.

Aneuploidy, or an abnormal number of chromosomes, adversely affects cell growth, but it is also linked with cancer and tumorigenesis. Now, Torres et al. (2010) help to resolve this paradox by demonstrating that aneuploid yeast cells can evolve mutations in the proteasome protein degradation pathway that alleviate imbalances in protein production and increase the cell's proliferative capacities.

Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E.

Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here, we demonstrate that Aurora kinases A and B phosphorylate a conserved residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-E's affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation.

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

Antisense Drugs Make Sense for Neurological Diseases.

The genetic basis for most inherited neurodegenerative diseases has been identified, yet there are limited disease-modifying therapies for these patients. A new class of drugs-antisense oligonucleotides (ASOs)-show promise as a therapeutic platform for treating neurological diseases. ASOs are designed to bind to the RNAs either by promoting degradation of the targeted RNA or by elevating expression by RNA splicing. Intrathecal injection into the cerebral spinal fluid results in broad distribution of antisense drugs and long-term effects. Approval of nusinersen in 2016 demonstrated that effective treatments for neurodegenerative diseases can be identified and that treatments not only slow disease progression but also improve some symptoms. Antisense drugs are currently in development for amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, and Angelman syndrome, and several drugs are in late-stage research for additional neurological diseases. This review highlights the advances in antisense technology as potential treatments for neurological diseases.

Cryptic splicing of stathmin-2 and UNC13A mRNAs is a pathological hallmark of TDP-43-associated Alzheimer's disease.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimer's disease. In Alzheimer's disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimer's disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-ß or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimer's disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimer's disease.