p21 Both Attenuates and Drives Senescence and Aging in BubR1 Progeroid Mice

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Nicotinamide Improves Aspects of Healthspan,but Not Lifespan,in Mice

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Mutation of Elfn1 in Mice Causes Seizures and Hyperactivity

A growing number of proteins with extracellular leucine-rich repeats (eLRRs) have been implicated in directing neuronal connectivity. We previously identified a novel family of eLRR proteins in mammals: the Elfns are transmembrane proteins with 6 LRRs, a fibronectin type-3 domain and a long cytoplasmic tail. The recent discovery that Elfn1 protein, expressed postsynaptically, can direct the elaboration of specific electrochemical properties of synapses between particular cell types in the hippocampus strongly reinforces this hypothesis. Here, we present analyses of an Elfn1 mutant mouse line and demonstrate a functional requirement for this gene in vivo. We first carried out detailed expression analysis of Elfn1 using a β-galactosidase reporter gene in the knockout line. Elfn1 is expressed in distinct subsets of interneurons of the hippocampus and cortex, and also in discrete subsets of cells in the habenula, septum, globus pallidus, dorsal subiculum, amygdala and several other regions. Elfn1 is expressed in diverse cell types, including local GABAergic interneurons as well as long-range projecting GABAergic and glutamatergic neurons. Elfn1 protein localises to axons of excitatory neurons in the habenula, and long-range GABAergic neurons of the globus pallidus, suggesting the possibility of additional roles for Elfn1 in axons or presynaptically. While gross anatomical analyses did not reveal any obvious neuroanatomical abnormalities, behavioural analyses clearly illustrate functional effects of Elfn1 mutation. Elfn1 mutant mice exhibit seizures, subtle motor abnormalities, reduced thigmotaxis and hyperactivity. The hyperactivity is paradoxically reversible by treatment with the stimulant amphetamine, consistent with phenotypes observed in animals with habenular lesions. These analyses reveal a requirement for Elfn1 in brain function and are suggestive of possible relevance to the etiology and pathophysiology of epilepsy and attention-deficit hyperactivity disorder.     

Neurofilaments and Orthograde Transport Are Reduced in Ventral Root Axons of Transgenic Mice that Express Human SOD1 with a G93A Mutation

Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly⁹³ → Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained ∼30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By ∼200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.Neurofilaments (NFs)¹ comprise the major class of neuron-specific intermediate filaments and are the most abundant cytoskeletal components found in large myelinated axons (for reviews see Nixon, 1993; Fuchs and Weber, 1994). NFs are heteropolymers formed by three subunits known as the high (NFH; 110 kD), middle (NFM; 95 kD), and low (NFL; 62 kD) molecular weight NF proteins, all of which are synthesized in neuronal perikarya and transported in the SCa phase (i.e., slow component a) of axonal transport at ∼0.2 to 1.2 mm/d (Lasek and Hoffman, 1976). Other cytoskeletal components (e.g., actin, tubulin) are transported three to four times faster in the SCb of slow axonal transport (Hoffman and Lasek, 1980).The carboxy termini or tail domains of NFH and NFM harbor tandem repeats of lysine-serine-proline (KSP) motifs, and the serine in this motif may be phosphorylated under both physiological and pathological conditions (Jones and Williams, 1982; Julien and Mushynski, 1982; Black and Lee, 1988; Lee et al., 1988a ,b; Clark and Lee, 1991; Giasson and Mushynski, 1996). Normally, the serines in these motifs become highly phosphorylated only after NFH and NFM have been transported into axons where this phosphorylation regulates the caliber of axons (de Waegh et al., 1992; Cole et al., 1994; Tu et al., 1995). In a variety of human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson''s disease (PD), as well as in neurotoxin-induced neuropathies, such as those that result from exposure to aluminum and acrylamide (Troncoso et al., 1985; Yase, 1988; Johnson and Jope, 1988; Johnson et al., 1990; Strong, 1994), NF inclusions typically form in the perikarya and processes of neurons, and these inclusions contain highly phosphorylated NFH and NFM (Tu et al., 1997a ,b). The major functions of NFs are to provide mechanical support, especially in large myelinated axons, and to regulate axonal caliber. Thus, disruption of the NF network has been hypothesized to play a mechanistic role in the degeneration of selectively vulnerable neurons that accumulate inclusions in a subset of human neurodegenerative disease (Lee et al., 1994; Julien, 1995). Significantly, this hypothesis has been supported by several recent studies of a number of different lines of transgenic mice that develop an ALS-like phenotype, including prominent NF inclusions in motor neurons that subsequently degenerate (Côté et al., 1993; Xu et al., 1993; Eyer and Peterson, 1994; Lee et al., 1994; Tu et al., 1997a ). Since only variations in the number of KSP motifs in NFH have been observed in some ALS patients (Figlewicz et al., 1994), other factors may lead to disruption of the NF network in neurodegenerative disorders. For example, mutations in the Cu/Zn superoxide dismutase gene (SOD1), which occurs in ∼20% of familial ALS (FALS) kindreds, lead to perturbations of the NF network (for recent review see Tu et al., 1997b ). FALS (as well as sporadic ALS) is a motor neuron disease (MND) characterized by progressive motor weakness due to the selective degeneration of motor neurons, many of which accumulate NF inclusions before their demise (Schmidt et al., 1987; Hill et al., 1991; Hirano, 1991; Tu et al., 1997b ). Transgenic mice that express one of four different mutant forms of the human SOD1 gene recapitulate many of the hallmarks of FALS including a fatal, progressive motor neuron weakness, the selective loss of motor neurons (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995; Bruijn et al., 1997), fragmentation of the Golgi apparatus (Mourelatos et al., 1996), and the accumulation of NF inclusions in motor neurons that are vulnerable to degenerate (Dal Canto and Gurney, 1994, 1995, 1997; Tu et al., 1996). The precise mechanisms that lead to the selective degeneration of neurons in authentic ALS as well as in transgenic mouse models of this disorder remain enigmatic, but there is evidence to suggest that NF inclusions may impede axonal transport and thereby contribute to the degeneration of affected neurons (Collard et al., 1995). Alternatively, other studies suggest that NF inclusions may compromise the viability of affected neurons by sequestering vital organelles (Tu et al., 1997a ). Thus, the present study exploited a classic experimental paradigm to determine if axonal transport in the ventral roots was impaired in transgenic mice that expresses human SOD1 with a Gly⁹³→ Ala mutation (G93A) and develop an ALS-like phenotype.     

Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington's Disease Mutation

Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington's disease (HD) patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24-48 hours) in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as 'foreign' and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as 'abnormal' by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.     

Some Behavioral Features of Drosophila melanogasterStrains Carrying a flamencoGene Mutation

Olfactory sensitivity and locomotor activity was assayed in Drosophila melanogasterstrains carrying a mutation of the flamencogene, which controls transposition of retrotransposon gypsy. A change in olfactory sensitivity was detected. The reaction to the odor of acetic acid was inverted in flies of the mutator strain (MS), which carried the flammutation and active gypsycopies and were characterized by genetic instability. Flies of the genetically unstable strains displayed a lower locomotor activity. The behavioral changes in MS flies can be explained by the pleiotropic effect of the flammutation or by insertion mutations which arise in behavior genes as a result of genome destabilization by gypsy.     

Pandemic 2009 H1N1 Influenza A Virus Carrying a Q136K Mutation in the Neuraminidase Gene Is Resistant to Zanamivir but Exhibits Reduced Fitness in the Guinea Pig Transmission Model

Resistance of influenza A viruses to neuraminidase inhibitors can arise through mutations in the neuraminidase (NA) gene. We show here that a Q136K mutation in the NA of the 2009 pandemic H1N1 virus confers a high degree of resistance to zanamivir. Resistance is accompanied by reduced numbers of NA molecules in viral particles and reduced intrinsic enzymatic activity of mutant NA. Interestingly, the Q136K mutation strongly impairs viral fitness in the guinea pig transmission model.     

Hyperphosphorylation and Accumulation of Neurofilament Proteins in Transgenic Mice with Alzheimer Presenilin 1 Mutation

Neurofilaments (NFs) are hyperphosphorylated and accumulate in Alzheimer’s disease (AD) brains. In this study, employing the transgenic mouse model, we explored the effect of presenilin 1 (PS-1) mutation on the phosphorylation and distribution of NFs. Western blot analysis showed that there was a significant increase in the phosphorylation of NF-H and NF-M subunits with a concomitant increase in phosphorylated c-Jun N-terminal protein kinase 1/2 (JNK1/2) mitogen-activated protein kinase (MAPK) in hippocampus of PS-1 transgenic mice compared to that of wild-type littermates. Immunohistochemical analysis revealed that phosphorylated NFs accumulated throughout the hippocampal neurons of the transgenic mice. These findings suggest that PS-1 mutation may induce hyperphosphorylation and accumulation of NFs via a JNK1/2-involved mechanism.     

A Novel MVA Vectored Chikungunya Virus Vaccine Elicits Protective Immunity in Mice

Background

Chikungunya virus (CHIKV) is a re-emerging arbovirus associated with febrile illness often accompanied by rash and arthralgia that may persist for several years. Outbreaks are associated with high morbidity and create a public health challenge for countries affected. Recent outbreaks have occurred in both Europe and the Americas, suggesting CHIKV may continue to spread. Despite the sustained threat of the virus, there is no approved vaccine or antiviral therapy against CHIKV. Therefore, it is critical to develop a vaccine that is both well tolerated and highly protective.

Methodology/Principal Findings

In this study, we describe the construction and characterization of a modified Vaccinia virus Ankara (MVA) virus expressing CHIKV E3 and E2 proteins (MVA-CHIK) that protected several mouse models from challenge with CHIKV. In particular, BALB/c mice were completely protected against viremia upon challenge with CHIKV after two doses of MVA-CHIK. Additionally, A129 mice (deficient in IFNα/β) were protected from viremia, footpad swelling, and mortality. While high anti-virus antibodies were elicited, low or undetectable levels of neutralizing antibodies were produced in both mouse models. However, passive transfer of MVA-CHIK immune serum to naïve mice did not protect against mortality, suggesting that antibodies may not be the main effectors of protection afforded by MVA-CHIK. Furthermore, depletion of CD4⁺, but not CD8⁺ T-cells from vaccinated mice resulted in 100% mortality, implicating the indispensable role of CD4⁺ T-cells in the protection afforded by MVA-CHIK.

Conclusions/Significance

The results presented herein demonstrate the potential of MVA to effectively express CHIKV E3-E2 proteins and generate protective immune responses. Our findings challenge the assumption that only neutralizing antibodies are effective in providing protection against CHIKV, and provides a framework for the development of novel, more effective vaccine strategies to combat CHIKV.     

Mutation of POLB Causes Lupus in Mice

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Chromosome damage in mitosis induces BubR1 activation and prometaphase arrest

The effect of double-strand DNA breaks (DSBs) on the spindle assembly checkpoint (SAC) has important implications with respect to the relationship between SAC function and chromosome instability of cancer cells. Here, we demonstrate that induction of DSBs in mitosis results in prolonged hyper-phosphorylation of the SAC protein BubR1 and association of BubR1 with kinetochores in mammalian cells. Combining single cell time-lapse microscopy with immunofluorescence, flow cytometry, and Western blot analysis in synchronized cells, we provide evidence that DSBs activate BubR1, leading to prometaphase arrest. Accordingly, elimination of BubR1 expression by siRNA resulted in the abrogation of mitotic delay in response to chromosome damage. These results suggest that BubR1 links DNA damage to kinetochore-associated SAC function.     

Caspase-mediated specific cleavage of BubR1 is a determinant of mitotic progression

The fidelity of chromosomal duplication is monitored by cell cycle checkpoints operational during mitosis. One such cell cycle delay is invoked by microtubule-targeting agents such as nocodazole or paclitaxel (Taxol) and is mediated by mitotic checkpoint proteins that include BubR1. Relatively little is known about the regulation of expression and stability of BubR1 (or other checkpoint proteins) and how these factors dictate the durability of the cell cycle delay. We report here that treatment of HeLa cells with spindle-disrupting agents resulted in caspase activation and precipitated the cleavage of BubR1. This mechanism ultimately leads to reduced levels of full-length protein, which are accompanied by abrogation of the mitotic block; the checkpoint abrogation is substantially accelerated by inhibition of de novo protein synthesis. In contrast, inhibition of caspase activity blocked BubR1 degradation and prolonged mitosis. To confirm a direct link between caspase activity and BubR1 protein expression, we identified by site-directed mutagenesis the specific caspase cleavage sites cleaved after exposure to paclitaxel. Surprisingly, BubR1 has two sites of cleavage: primarily at Asp607/Asp610 and secondarily at Asp576/Asp579. BubR1 mutated at both locations (BubR1Delta579Delta610) was resistant to paclitaxel-induced degradation. Expression of BubR1Delta579Delta610 augmented the mitotic delay induced by spindle disruption in transfected cells as well as in clones engineered to inducibly express the mutant protein upon exposure to doxycycline and ultimately led to increased aneuploidy. Underscoring the importance of these caspase cleavage sites, both tetrapeptide motifs are identified in the amino acid sequences of human, mouse, chicken, and Xenopus BubR1. These results are potentially the first to link the control of the stability of a key mitotic checkpoint protein to caspase activation, a regulatory pathway that may be involved in killing defective cells and that has been evolutionarily conserved.     

Mice Lacking NCF1 Exhibit Reduced Growth of Implanted Melanoma and Carcinoma Tumors

The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 ^m1J mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 ^m1J mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 ^m1J mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.