To determine the ability of antibodies to provide protection from Ebola viruses, monoclonal antibodies (mAbs) to the Ebola glycoprotein were generated and evaluated for efficacy. We identified several protective mAbs directed toward five unique epitopes on Ebola glycoprotein. One of the epitopes is conserved among all Ebola viruses that are known to be pathogenic for humans. Some protective mAbs were also effective therapeutically when administered to mice 2 days after exposure to lethal Ebola virus. The identification of protective mAbs has important implications for developing vaccines and therapies for Ebola virus.

The ataxia (ax J ) mutation is a spontaneous recessive mutation that results in reduced expression of ubiquitinspecific protease 14, Usp14. Mice homozygous for the ax J mutation are retarded for growth and exhibit several behavioral disorders, including a resting tremor and hindlimb paralysis. Although pathological defects appear to be limited to the central nervous system, reduction of Usp14 expression was widespread in the ax J mice. Usp14 co-fractionated with proteasomes isolated from livers and brains of wildtype mice. Proteasomes isolated from the ax J brains still possessed deubiquitinating activity and were functionally competent to hydrolyze 20S proteasomal substrates in vitro. However, the levels of monomeric ubiquitin were reduced approximately 35% in most of the ax J tissues examined.These results indicate that Usp14 functions to maintain the cellular levels of monomeric ubiquitin in mammalian cells, and that alterations in the levels of ubiquitin may contribute to neurological disease. Keywords: ataxia, mutation, neurological, proteasome, ubiquitin, ubiquitin-specific protease 14. . These disorders are associated with changes in protein degradation that are manifested through the production of protein aggregates. However, it is unclear whether these alterations in the UPS are the primary cause or a secondary consequence of these disorders. Our recent studies on ataxia (ax J ) mice indicate that primary alterations in the UPS can lead to neurological dysfunction (Wilson et al. 2002). The ax J mutation is a spontaneously arising recessive neurological mutation (D'Amato and Hicks 1965). The ax J mice suffer from progressive motor system abnormalities that first appear as a resting tremor when the mice are 2-3 weeks old. At 4 weeks of age, ax J mice exhibit severe hind muscle wasting and ataxia. These mice are completely immobile by 6 weeks of age and death occurs at about 8 weeks of age. The neurological phenotypes observed in the ax J mice are the result of a genetic lesion in the gene encoding the deubiquitinating enzyme Usp14 (Wilson et al. 2002). The insertion of an intracisternal A-particle into intron 5 of Usp14 reduces the expression of Usp14 in the ax J mice to only 5-10% of the levels found in wild-type mice, indicating that the ax J mutation represents a hypomorphic allele of Usp14. A better understanding of the deficiencies in the ax J mice, and in other mice that harbor mutations in the UPS, should therefore provide valuable insights into how the UPS functions in the nervous system and how alterations in these pathways lead to disease.

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Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, includingAlzheimer's, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient ax J mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the ax J mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.

The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (ax J ) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of ax J mice with neuronally expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resultingfromthelossofUsp14,demonstratingthatubiquitindeficiencyisamajorcauseoftheneurologicaldefectsobservedinthe ax J mice.We also show that proteasome components are normally induced during the first 2 weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.

The ataxia mutation (ax J ) is a recessive neurological mutation that results in reduced growth, ataxia, and hindlimb muscle wasting in mice. The ax J gene encodes ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme (DUB) that associates with the proteasome via its ubiquitin-like (Ubl) domain and is involved in processing ubiquitin chains. Analysis of Usp14 gene products demonstrated that Usp14 undergoes alternative pre-mRNA splicing to produce a full-length form of Usp14 that is capable of binding proteasomes and a form that contains a deletion in the Ubl domain. The full-length form of Usp14 is the only form that appears to be reduced in the ax J mice. Transgenic rescue of the ax J mice with neuronal-specific expression of Usp14 demonstrated that the full-length form of Usp14 was sufficient to restore viability and motor system function to the ax J mice. Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of ax J mice. However, the ax J -rescued mice still displayed the Purkinje cell axonal swellings that are seen in the ax J mice, indicating that this cerebellar alteration is not the primary cause of the ax J movement disorders. These results show that the motor defects observed in the ax J mice are attributable to a neuropathic disease rather than to a muscular disorder and suggest that changes in proteasomal function may contribute to neurological dysfunction in the ax J mice.

The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and Usp14, result in neurological phenotypes in mice. We identified a new mutation in Uch-L1 and compared the roles of Uch-L1 and Usp14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or Usp14 result in decreased levels of ubiquitin, suggesting that they both regulate ubiquitin stability in the nervous system. However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the Usp14-deficient mice, and changes in hippocampal synaptic transmission were only observed in Usp14-deficient mice. In addition, while Usp14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and Usp14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.

Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8 ؉ T-cell responses to six Ebola virus proteins were examined. CD8 ؉ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8 ؉ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8؉ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.