That mutations in the SOD1 enzyme underlie familial form of the motor neuron disease ALS is clear. But there seems to be more than one answer to the question of what are the consequences of such mutations.
Mitochondria have been proposed as targets for toxicity in amyotrophic lateral sclerosis (ALS), a progressive, fatal adult-onset neurodegenerative disorder characterized by the selective loss of motor neurons. A decrease in the capacity of spinal cord mitochondria to buffer calcium (Ca2+) has been observed in mice expressing ALS linked mutants of SOD1 that develop motor neuron disease with many of the key pathological hallmarks seen in ALS patients. In mice expressing three different ALS-causing SOD1 mutants, we now test the contribution of the loss of mitochondrial Ca2+ buffering capacity to disease mechanism(s) by eliminating ubiquitous expression of cyclophilin D, a critical regulator of Ca2+ mediated opening of the mitochondrial permeability transition pore (mPTP) that determines mitochondrial calcium content. A chronic increase in mitochondrial buffering of Ca2+ in the absence of cyclophilin D was maintained throughout disease course and was associated with improved mitochondrial ATP synthesis, reduced mitochondrial swelling, and retention of normal morphology. This was accompanied by an attenuation of glial activation, reduction in levels of misfolded SOD1 aggregates in the spinal cord, and a significant suppression of motor neuron death throughout disease. Despite this, muscle denervation, motor axon degeneration, and disease progression and survival were unaffected, thereby eliminating mutant SOD1-mediated loss of mitochondrial Ca2+ buffering capacity, altered mitochondrial morphology, motor neuron death, and misfolded SOD1 aggregates, as primary contributors to disease mechanism for fatal paralysis in these models of familial ALS.
Mutant huntingtin (HTT) protein causes Huntington’s 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 silencing RNAs (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.
Aneuploidy, or the abnormal number of chromosomes, adversely effects cell growth, but it is also linked with cancer and tumorigenesis. Now Torres et al. (2010) help 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.
The centromere is the fundamental unit for insuring chromosome inheritance. This complex region has a distinct type of chromatin in which histone H3 is replaced by a structurally different homologue identified in humans as CENP-A. In metazoans, specific DNA sequences are neither required nor sufficient for centromere identity. Rather, an epigenetic mark comprised of CENP-A containing chromatin is thought to be the major determinant of centromere identity. In this view, CENP-A deposition and chromatin assembly are fundamental processes for the maintenance of centromeric identity across mitotic and meiotic divisions. Several lines of evidence support CENP-A deposition in metazoans occurring at only one time in the cell cycle. Such cell cycle-dependent loading of CENP-A is found in divergent species from human to fission yeast, albeit with differences in the cell cycle point at which CENP-A is assembled. Cell cycle dependent CENP-A deposition requires multiple assembly factors for its deposition and maintenance. This review discusses the regulation of new CENP-A deposition and its relevance to centromere identity and inheritance.
In a recent issue of Cell, Ohta et al. (2010) report a method of quantitative proteomics coupled with bioinformatic analysis for the identification of associated components in complex mixtures. Using this approach, they assayed the protein composition of mitotic chromosomes, identifying 4029 associated proteins, 562 of which are previously uncharacterized.
Amyotrophic lateral sclerosis (ALS) research is undergoing an era of unprecedented discoveries with the identification of new genes as major genetic causes of this disease. These discoveries reinforce the genetic, clinical and pathological overlap between ALS and frontotemporal lobar degeneration (FTLD). Common causes of these diseases include mutations in the RNA/DNA-binding proteins, TDP-43 and FUS/TLS and most recently, hexanucleotide expansions in the C9orf72 gene, discoveries that highlight the overlapping pathogenic mechanisms that trigger ALS and FTLD. TDP-43 and FUS/TLS, both of which participate in several steps of RNA processing, are abnormally aggregated and mislocalized in ALS and FTLD, while the expansion in the C9orf72 pre-mRNA strongly suggests sequestration of one or more RNA binding proteins in pathologic RNA foci. Hence, ALS and FTLD converge in pathogenic pathways disrupting the regulation of RNA processing. This article is part of a Special Issue entitled RNA-Binding Proteins.
Amyotrophic lateral sclerosis; Frontotemporal dementia; TDP-43; FUS/TLS; C9orf72; RNA processing
In interphase and mitosis, centrosomes play a major role in the spatial organization of the microtubule network. Alterations in centrosome number and structure are associated with genomic instability and occur in many cancers. Centrosome duplication is controlled by centriole replication. In most dividing animal cells, centrioles duplicate only once per cell cycle at a site adjacent to existing centrioles. The conserved protein kinase Polo-like kinase 4 (Plk4) has a key role in controlling centriole biogenesis. Overexpression of Plk4 drives centrosome amplification and is associated with tumorigenesis in flies. By contrast, haploinsufficiency of Plk4 promotes cytokinesis failure, leading to an increased incidence of tumors in mice. Recent studies have shown that Plk4 is a low abundance protein whose stability is linked to the activity of the enzyme. We discuss how this autoregulatory feedback loop acts to limit the damaging effects caused by too much or too little Plk4.
centrosome; centriole; polo-like kinase 4; Plk4; SAK; SCF; phosphodegron; β-TrCP; aneuploidy
The large Nuclear Mitotic Apparatus (NuMA) protein is an abundant component of interphase nuclei and an essential player in mitotic spindle assembly and maintenance. With its partner, cytoplasmic dynein, NuMA uses its cross-linking properties to tether microtubules to spindle poles. NuMA and its invertebrate homologues play a similar tethering role at the cell cortex, thereby mediating essential asymmetric divisions during development. Despite its maintenance as a nuclear component for decades after the final mitosis of many cell types (including neurons), an interphase role for NuMA remains to be established, although its structural properties implicate it as a component of a nuclear scaffold, perhaps as a central constituent of the proposed nuclear matrix.
A lesson from dominantly inherited forms of diverse neurodegenerative diseases, including amyotrophic lateral sclerosis, spinocerebellar ataxia and Huntington’s disease, is that the selective dysfunction or death of the neuronal population most at risk in each disease is not mediated solely by mutant derived damage within the target neurons. The disease-causing toxic process, which in each case is caused by mutation in a gene that is widely or ubiquitously expressed, involves mutant damage within the non-neuronal glial cells of the central nervous system - especially astrocytes and microglia. Disease mechanism is non-cell autonomous, with toxicity derived from glia as a prominent contributor to driving disease progression and in some instances even disease initiation.
The primary cause of Huntington’s disease (HD) is expression of huntingtin with a polyglutamine expansion. Despite an absence of consensus on the mechanism(s) of toxicity, diminishing the synthesis of mutant huntingtin will abate toxicity if delivered to the key affected cells. With antisense oligonucleotides (ASOs) that catalyze RNase H-mediated degradation of huntingtin mRNA, we demonstrate that transient infusion into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal. Similar ASO infusion into non-human primates is shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, our findings establish a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.
In this issue, three groups (Hewitt et al. 2010. J. Cell Biol. doi:10.1083/jcb.201002133; Maciejowski et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001050; Santaguida et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001036) use chemical inhibitors to analyze the function of the mitotic checkpoint kinase Mps1. These studies demonstrate that Mps1 kinase activity ensures accurate chromosome segregation through its recruitment to kinetochores of mitotic checkpoint proteins, formation of interphase and mitotic inhibitors of Cdc20, and correction of faulty microtubule attachments.
Mutations in gigaxonin are responsible for Giant Axonal Neuropathy (GAN), a progressive neurodegenerative disorder associated with abnormal accumulations of Intermediate Filaments (IFs). Gigaxonin is the substrate-specific adaptor for a new Cul3-E3-ubiquitin ligase family that promotes the proteasome dependent degradation of its partners MAP1B, MAP8 and TBCB. Here, we report the generation of a mouse model with targeted deletion of Gan exon 1 (GanΔexon1;Δexon1). Analyses of the GanΔexon1;Δexon1 mice revealed increased levels of various IFs proteins in nervous system and the presence of IFs inclusion bodies in the brain. Despite deficiency of full length gigaxonin, the GanΔexon1;Δexon1 mice do not develop overt neurological phenotypes and giant axons reminiscent of the human GAN disease. We propose that the existence of a short gigaxonin isoform expressed in the spinal cord could underlie the mitigation of GAN-phenotypes in GanΔexon1;Δexon1 mice. Nonetheless, the GanΔexon1;Δexon1 mice exhibited modest increase in axon calibers and 27% axonal loss in the L5 ventral roots. This new mouse model should provide a useful tool for testing potential therapeutic approaches for GAN disease.
intermediate filaments; inclusion bodies; neuropathy
The transcriptional coactivator PGC-1α induces multiple effects on muscle, including increased mitochondrial mass and activity. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, adult-onset neurodegenerative disorder characterized by selective loss of motor neurons and skeletal muscle degeneration. An early event is thought to be denervation-induced muscle atrophy accompanied by alterations in mitochondrial activity and morphology within muscle. We now report that elevation of PGC-1α levels in muscles of mice that develop fatal paralysis from an ALS-causing SOD1 mutant elevates PGC-1α-dependent pathways throughout disease course. Mitochondrial biogenesis and activity are maintained through end-stage disease, accompanied by retention of muscle function, delayed muscle atrophy, and significantly improved muscle endurance even at late disease stages. However, survival was not extended. Therefore, muscle is not a primary target of mutant SOD1-mediated toxicity, but drugs increasing PGC-1α activity in muscle represent an attractive therapy for maintaining muscle function during progression of ALS.
Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.
Mutation in superoxide dismutase–1 (SOD1) causes the inherited degenerative neurological disease familial amyotrophic lateral sclerosis (ALS), a non–cell-autonomous disease: mutant SOD1 synthesis in motor neurons and microglia drives disease onset and progression, respectively. In this issue of the JCI, Harraz and colleagues demonstrate that SOD1 mutants expressed in human cell lines directly stimulate NADPH oxidase (Nox) by binding to Rac1, resulting in overproduction of damaging ROS (see the related article beginning on page 659). Diminishing ROS by treatment with the microglial Nox inhibitor apocynin or by elimination of Nox extends survival in ALS mice, reviving the proposal that ROS mediate ALS pathogenesis, but with a new twist: it’s ROS produced by microglia.
Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.
The mitotic checkpoint plays an important role in preventing chromosome segregation errors and the production of aneuploid progeny. In this issue, Zhang et al. examine mice and cells lacking the deubiquitinating enzyme USP44. Surprisingly, they find that USP44 prevents chromosome segregation errors through a function independent of its previously identified role in the mitotic checkpoint. Usp44-null animals develop aneuploidy and experience increased rates of tumorigenesis, implicating USP44 as novel tumor suppressor.
The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible “stop anaphase” inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1–CENP-E–microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.
Dominant mutation in two DNA/RNA binding proteins, TDP-43 and FUS/TLS, are causes of inherited Amyotrophic Lateral Sclerosis (ALS). TDP-43 and FUS/TLS have striking structural and functional similarities, implicating alterations in RNA processing as central in ALS. TDP-43 has binding sites within a third of all mouse and human mRNAs in brain and this binding influences the levels and splicing patterns of at least 20% of those mRNAs. Disease modeling in rodents of the first known cause of inherited ALS – mutation in the ubiquitously expressed superoxide dismutase (SOD1) – has yielded non-cell autonomous fatal motor neuron disease caused by one or more toxic properties acquired by the mutant proteins. In contrast, initial disease modeling for TDP-43 and FUS/TLS has produced highly varied phenotypes. It remains unsettled whether TDP-43 and FUS/TLS mutants provoke disease from a loss of function or gain of toxicity or both. TDP-43 or FUS/TLS misaccumulation seems central not just to ALS (where it is found in almost all instances of disease), but more broadly in neurodegenerative disease, including frontal temporal lobular dementia (FTLD-U) and many examples of Alzheimer’s or Huntington’s disease. (182 words)
Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.
kinetochore; mitosis; cell cycle; LENP-E; BubR1
A growing body of evidence suggests that cell-to-cell spread of misfolded protein aggregates represents a mechanism underlying the pathogenesis of several neurodegenerative diseases.
Protein misfolding is common to most neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Recent work using animal models with intracellular α-synuclein and tau inclusions adds decisively to a growing body of evidence that misfolded protein aggregates can induce a self-perpetuating process that leads to amplification and spreading of pathological protein assemblies. When coupled with the progressive nature of neurodegeneration, recognition of such cell-to-cell aggregate spread suggests a unifying mechanism underlying the pathogenesis of these disorders.
Misfolded proteins accumulating in several neurodegenerative diseases (including Alzheimer’s, Parkinson’s and Huntington’s 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 that provide a molecular pathway for non-cell autonomous disease spread within the nervous system.
Polo-like kinase 4 (Plk4) plays an essential role in centriole duplication, but recent work led to the conclusion that Plk4 also directly regulates cytokinesis. The consequence of reduced Plk4 levels in human and mouse cells is studied. It is shown that Plk4 controls centriole duplication but does not directly regulate cytokinesis.
Centrioles organize the centrosome, and accurate control of their number is critical for the maintenance of genomic integrity. Centrioles duplicate once per cell cycle, and duplication is coordinated by Polo-like kinase 4 (Plk4). We previously demonstrated that Plk4 accumulation is autoregulated by its own kinase activity. However, loss of heterozygosity of Plk4 in mouse embryonic fibroblasts has been proposed to cause cytokinesis failure as a primary event, leading to centrosome amplification and gross chromosomal abnormalities. Using targeted gene disruption, we show that human epithelial cells with one inactivated Plk4 allele undergo neither cytokinesis failure nor increase in centrosome amplification. Plk4 is shown to localize exclusively at the centrosome, with none in the spindle midbody. Substantial depletion of Plk4 by small interfering RNA leads to loss of centrioles and subsequent spindle defects that lead to a modest increase in the rate of cytokinesis failure. Therefore, Plk4 is a centriole-localized kinase that does not directly regulate cytokinesis.