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1.  TDP-43 toxicity: the usefulness of “junk” 
Nature genetics  2012;44(12):1289-1291.
Loss of the lariat debranching enzyme Dbr1 is found to repress TDP-43 toxicity. The accumulated intronic lariat RNAs, which are normally degraded after splicing, likely act as decoys to sequester TDP-43 away from binding to and disrupting function of other RNAs.
PMCID: PMC3612973  PMID: 23192178
2.  Centrosomes, chromosome instability (CIN) and aneuploidy 
Current opinion in cell biology  2012;24(6):809-815.
Each time a cell divides its chromosome content must be equally segregated into the two daughter cells. This critical process is mediated by a complex microtubule based apparatus, the mitotic spindle. In most animal cells the centrosomes contribute to the formation and the proper function of the mitotic spindle by anchoring and nucleating microtubules and by establishing its functional bipolar organization. Aberrant expression of proteins involved in centrosome biogenesis can drive centrosome dysfunction or abnormal centrosome number, leading ultimately to improper mitotic spindle formation and chromosome missegregation. Here we review recent work focusing on the importance of the centrosome for mitotic spindle formation and the relation between the centrosome status and the mechanisms controlling faithful chromosome inheritance.
PMCID: PMC3621708  PMID: 23127609
3.  Neurodegeneration 
Nature  2008;454(7202):284-285.
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.
PMCID: PMC3625042  PMID: 18633404
4.  Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips 
Nature cell biology  2013;15(9):1079-1088.
During vertebrate mitosis, the centromere-associated kinesin CENP-E transports misaligned chromosomes to the plus ends of spindle microtubules. Subsequently, the kinetochores that form at the centromeres establish stable associations with microtubule ends, which assemble and disassemble dynamically. Here we provide evidence that after chromosomes have congressed and bi-oriented, the CENP-E motor continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends. Using a combination of single molecule approaches and laser trapping in vitro we demonstrate that once reaching microtubule ends, CENP-E converts from a lateral transporter into a microtubule tip-tracker which maintains association with both assembling and disassembling microtubule tips. Computational modeling of this behavior supports our proposal that CENP-E tip-tracks bi-directionally via a “tethered motor” mechanism, which relies on both the motor and tail domains of CENP-E. Our results provide a molecular framework for CENP-E's contribution to the stability of attachments between kinetochores and dynamic microtubule ends.
PMCID: PMC3919686  PMID: 23955301
5.  How to survive aneuploidy 
Cell  2010;143(1):27-29.
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.
PMCID: PMC2955074  PMID: 20887888
6.  Multi-classifier proteomics to define complexes yields new chromosomal proteins 
Developmental cell  2010;19(3):356-359.
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.
PMCID: PMC2943763  PMID: 20833356
7.  Centriole duplication 
Cell Cycle  2010;9(14):2731-2736.
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.
PMCID: PMC3040958  PMID: 20647763
centrosome; centriole; polo-like kinase 4; Plk4; SAK; SCF; phosphodegron; β-TrCP; aneuploidy
8.  NuMA after 30 years: the Matrix Revisited 
Trends in cell biology  2010;20(4):214-222.
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.
PMCID: PMC3137513  PMID: 20137953
9.  Glial cells as intrinsic components of non-cell autonomous neurodegenerative disease 
Nature neuroscience  2007;10(11):1355-1360.
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.
PMCID: PMC3110080  PMID: 17965655
10.  A chemical tool box defines mitotic and interphase roles for Mps1 kinase 
The Journal of Cell Biology  2010;190(1):21-24.
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.
PMCID: PMC2911672  PMID: 20624898
11.  Mechanisms and consequences of localized, complex chromosomal rearrangements in cancer and developmental diseases 
Nature medicine  2012;18(11):1630-1638.
Next-generation DNA sequencing of human tumors has led to discovery of chromoanagenesis, in which large numbers of complex rearrangements occur at one or a few chromosomal loci in a single catastrophic event. Two mechanisms underlie these rearrangements, both of which can be facilitated by a mitotic chromosome segregation error to produce a micronucleus containing the chromosome to undergo rearrangement. In the first, chromosome shattering (called chromothripsis) is produced by mitotic entry before completion of DNA replication within the micronucleus, with failure to disassemble the micronuclear envelope encapsulating the chromosomal fragments for random reassembly in the subsequent interphase. Alternatively, locally defective DNA replication (also potentially within a micronucleus) initiates serial, microhomology-mediated template switching (called chromoanasynthesis) that produces local rearrangements with altered gene copy numbers. Complex, localized rearrangements are present in a broad spectrum of tumors and in individuals with congenital or developmental defects, highlighting the impact of chromoanagenesis in human disease.
PMCID: PMC3616639  PMID: 23135524
12.  Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis 
Nature neuroscience  2013;16(5):571-579.
Oligodendrocytes associate with axons to establish myelin and provide metabolic support to neurons. In the spinal cord of ALS mice, oligodendrocytes downregulate transporters that transfer glycolytic substrates to neurons and oligodendrocyte progenitors (NG2+ cells) exhibit enhanced proliferation and differentiation, although the cause of these changes in oligodendroglia is unknown. Here we report that there is extensive degeneration of gray matter oligodendrocytes in the spinal cord of ALS mice before disease onset. Although new oligodendrocytes were formed, they failed to mature, resulting in progressive demyelination. Oligodendrocyte dysfunction also is prevalent in human ALS, as gray matter demyelination and reactive changes in NG2+ cells were observed in motor cortex and spinal cord of ALS patients. Selective removal of mutant SOD1 from oligodendroglia substantially delayed disease onset and prolonged survival in ALS mice, suggesting that ALS-linked genes enhance the vulnerability of motor neurons and accelerate disease by directly impairing the function of oligodendrocytes.
PMCID: PMC3637847  PMID: 23542689
13.  Aneuploidy Drives A Mutator Phenotype in Cancer 
Science (New York, N.Y.)  2011;333(6045):942-943.
PMCID: PMC3806716  PMID: 21852477
14.  Requirements for NuMA in maintenance and establishment of mammalian spindle poles 
The Journal of Cell Biology  2009;184(5):677-690.
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.
PMCID: PMC2686415  PMID: 19255246
15.  Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited ALS 
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.
PMCID: PMC3711648  PMID: 23486940
16.  Single-Stranded RNAs Use RNAi to Potently and Allele-Selectively Inhibit Mutant Huntingtin Expression 
Cell  2012;150(5):895-908.
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.
PMCID: PMC3444165  PMID: 22939619
17.  Revisiting oxidative damage in ALS: microglia, Nox, and mutant SOD1 
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.
PMCID: PMC2213376  PMID: 18219386
18.  Propagation of centromeric chromatin requires exit from mitosis 
The Journal of Cell Biology  2007;176(6):795-805.
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.
PMCID: PMC2064054  PMID: 17339380
19.  Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling 
The Journal of Cell Biology  2005;170(6):873-880.
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.
PMCID: PMC2171436  PMID: 16144904
20.  Replicating centromeric chromatin: spatial and temporal control of CENP-A assembly 
Experimental cell research  2012;318(12):1353-1360.
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.
PMCID: PMC3616609  PMID: 22561213
21.  Misregulated RNA processing in amyotrophic lateral sclerosis 
Brain research  2012;1462:3-15.
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.
PMCID: PMC3707312  PMID: 22444279
Amyotrophic lateral sclerosis; Frontotemporal dementia; TDP-43; FUS/TLS; C9orf72; RNA processing
22.  Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis 
Neuron  2012;74(6):1031-1044.
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.
PMCID: PMC3383626  PMID: 22726834
23.  Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss 
The Journal of Cell Biology  2003;162(4):551-563.
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.
PMCID: PMC2173788  PMID: 12925705
kinetochore; mitosis; cell cycle; LENP-E; BubR1
24.  Modest loss of peripheral axons and formation of brain inclusions in mice with targeted deletion of gigaxonin exon 1 
Journal of neurochemistry  2008;107(1):253-264.
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.
PMCID: PMC3657508  PMID: 18680552
intermediate filaments; inclusion bodies; neuropathy
25.  Elevated PGC-1α Activity Sustains Mitochondrial Biogenesis and Muscle Function without Extending Survival in a Mouse Model of Inherited ALS 
Cell metabolism  2012;15(5):778-786.
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.
PMCID: PMC3565468  PMID: 22560226

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