Baker M (2011) Nat Methods "More options for gene editing."

Baker M

[Nat Methods, 2011]

Engineering precise genetic changes in a genome is powerful way to study gene function, and several recent papers describe new applications of gene-editing tools. Working with researchers at Sangamo BioSciences, Howard Hughes Medical Institute investigator Barbara Meyer and her colleagues at the University of California, Berkeley, described the first systems for making targeted genomic modifications in the roundworm Caenorhabditis elegans, a valuable model organism (Wood et al., 2011).

Baker M et al. (2006) Nature "Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17."

Neary D, Robinson T, Mackenzie IR, Dickson D, Snowden J, Rollinson S, Pickering-Brown SM, Cannon A, Crook R, Boeve B, Melquist S, Berger Z, Dickey CA, Adamson J, Mann D, Rademakers R, Gass J, Dwosh E, Sadovnick AD, Zehr C, Baker M, Hutton M, Richardson A, Eriksen J, McGowan E, Feldman H, Lindholm C

[Nature, 2006]

Frontotemporal dementia (FTD) is the second most common cause of dementia in people under the age of 65 years. A large proportion of FTD patients (35-50%) have a family history of dementia, consistent with a strong genetic component to the disease. In 1998, mutations in the gene encoding the microtubule-associated protein tau (MAPT) were shown to cause familial FTD with parkinsonism linked to chromosome 17q21 (FTDP-17). The neuropathology of patients with defined MAPT mutations is characterized by cytoplasmic neurofibrillary inclusions composed of hyperphosphorylated tau. However, in multiple FTD families with significant evidence for linkage to the same region on chromosome 17q21 (D17S1787-D17S806), mutations in MAPT have not been found and the patients consistently lack tau-immunoreactive inclusion pathology. In contrast, these patients have ubiquitin (ub)-immunoreactive neuronal cytoplasmic inclusions and characteristic lentiform ub-immunoreactive neuronal intranuclear inclusions. Here we demonstrate that in these families, FTD is caused by mutations in progranulin (PGRN) that are likely to create null alleles. PGRN is located 1.7 Mb centromeric of MAPT on chromosome 17q21.31 and encodes a 68.5-kDa secreted growth factor involved in the regulation of multiple processes including development, wound repair and inflammation. PGRN has also been strongly linked to tumorigenesis. Moreover, PGRN expression is increased in activated microglia in many neurodegenerative diseases including Creutzfeldt-Jakob disease, motor neuron disease and Alzheimer''s disease. Our results identify mutations in PGRN as a cause of neurodegenerative disease and indicate the importance of PGRN function for neuronal survival.

Baker B M et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "The Mitochondrial UPR, a Stress Response to Maintain Organelle Protein Homeostasis and Function"

Baker B M, Haynes C M

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Protein folding in eukaryotic cells is compartmentalized. The load of unfolded proteins confronting the compartment specific machinery of the cytosol, ER and mitochondria varies with cell type, age and physiological state. Signaling pathways that adjust the folding capacity of each organelle to its load of unfolded proteins are collectively referred to as unfolded protein responses (UPR). Dysfunction of these pathways compromises the ability of cells to cope with compartment-specific stress and may contribute to diseases of aging and protein misfolding. Compartment-specific protein folding sensors responsive to chaperone occupancy signal a transcriptional response that selectively upregulates compartment-specific machinery to fold or degrade unfolded/misfolded proteins. Through a genetic screen we have identified several components that make up a signal transduction pathway that regulates expression of mitochondrial chaperone genes. The most upstream component of the UPRmt is a matrix-localized orthologue of the bacterial protease ClpXP, a protease known to play a role in protein quality control. UPRmt signaling occurs as unfolded proteins accumulate beyond the mitochondrial chaperone capacity. The excess unfolded proteins are degraded by ClpXP to prevent the accumulation of deleterious proteins or aggregation. Genetic and biochemical evidence indicate that HAF-1, a mitochondrial-localized ABC transporter contributes to signaling the UPRmt by pumping ClpXP-derived peptides across the mitochondrial inner membrane. We have recently identified a novel bZip transcription factor, ZC376.7, required for mitochondrial chaperone expression, the downstream effector of ClpP-mediated peptide efflux in signaling the UPRmt. These observations are consistent with unfolded protein degradation and peptide efflux in signaling changes in the mitochondrial matrix protein folding state or chaperone occupancy to activate the UPRmt and reestablish homeostasis within the organelle. Furthermore, we have established a role for this pathway in stress resistance to conditions that perturb mitochondrial function and will discuss further its involvement in development and aging. Haynes et al., 2007. ClpP mediates activation of a mitochondrial unfolded protein response in C.elegans. Developmental Cell 13, 467-80. Haynes et al., 2010. The matrix peptide exporter HAF-1 signals a mitochondrial UPR by activating the transcription factor ZC376.7 in C. elegans. Molecular Cell 26, 529-40.

Kimberly M Baker et al. (2005) International Worm Meeting "Molecular and genetic interactions of SAX-7 with the dystrophin-associated proteins, gamma-syntrophin and KIN-4, in the germline and gonad."

Lihsia Chen, Kimberly M Baker, Karla Opperman

[International Worm Meeting, 2005]

The L1 family of cell adhesion molecules (L1CAMs) has been implicated in neuronal development. Non-neuronal L1CAM expression and function have been identified recently. However, their roles and mechanisms are poorly understood. We have identified requirement for the C. elegans L1CAM homologue, SAX-7, in multiple non-neuronal tissues, including the gonad and germline. To better determine SAX-7 functions and mechanisms, we performed a directed yeast two hybrid screen for PDZ proteins that interact with the SAX-7 consensus PDZ binding motif. We identified two proteins, gamma-syntrophin and KIN-4, both of which are associated with the dystrophin-glycoprotein complex (DGC) (1, 2). Reduced levels of gamma-syntrophin or KIN-4 accomplished by RNAi in sax-7 mutant animals but not in wild-type animals resulted in obvious germline and gonad defects. This genetic interaction suggests that the molecular interaction between SAX-7 and gamma-syntrophin and KIN-4 is functionally significant, particularly with respect to germline and gonad development. 1. Piluso et al. (2000) J. Biol. Chem. 275: 15851-15860; 2. Lumeng et al. (1999) Nat. Neurosci. 2: 611-617.

Hodgkin J (2002) Genes Dev "The remarkable ubiquity of DM domain factors as regulators of sexual phenotype: ancestry or aptitude?"

Hodgkin J

[Genes Dev, 2002]

The CM domain is a cysteine-rich DNA-binding motif first recognized in proteins encoded by the Drosophila set determination gene doublesex (Erdman and Burtis 1993; Zhu et al. 2000). As the name doublesex (dsx) suggests, this gene has functions in both sexes: Its transcripts undergo sex-specific alternative splicing, so that it can encode either a male-specific isoform, DSX(M), or a female-specific isoform, DSX(F) (Baker and Wolfner 1988; Burtis and Baker 1989). These proteins have the same N-terminal DNA-binding domain, but different C termini that confer different regulatory properties on the two forms. The expression of DSX(M) directs male development, and the expression of DSX(F) directs female development, throughout most of the somatic tissues of the fruit fly.

Kao AW et al. (2011) Proc Natl Acad Sci U S A "A neurodegenerative disease mutation that accelerates the clearance of apoptotic cells."

Cabello J, Kao AW, Neukomm LJ, Farese RV, Eisenhut RJ, Huang A, Bagley JA, Martens LH, Nakamura A, Zhou P, de Luis A, Kenyon C

[Proc Natl Acad Sci U S A, 2011]

Frontotemporal lobar degeneration is a progressive neurodegenerative syndrome that is the second most common cause of early-onset dementia. Mutations in the progranulin gene are a major cause of familial frontotemporal lobar degeneration [Baker M, et al. (2006) Nature 442:916-919 and Cruts M, et al. (2006) Nature 442:920-924]. Although progranulin is involved in wound healing, inflammation, and tumor growth, its role in the nervous system and the mechanism by which insufficient levels result in neurodegeneration are poorly understood [Eriksen and Mackenzie (2008) J Neurochem 104:287-297]. We have characterized the normal function of progranulin in the nematode Caenorhabditis elegans. We found that mutants lacking pgrn-1 appear grossly normal, but exhibit fewer apoptotic cell corpses during development. This reduction in corpse number is not caused by reduced apoptosis, but instead by more rapid clearance of dying cells. Likewise, we found that macrophages cultured from progranulin KO mice displayed enhanced rates of apoptotic-cell phagocytosis. Although most neurodegenerative diseases are thought to be caused by the toxic effects of aggregated proteins, our findings suggest that susceptibility to neurodegeneration may be increased by a change in the kinetics of programmed cell death. We propose that cells that might otherwise recover from damage or injury are destroyed in progranulin mutants, which in turn facilitates disease progression.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.