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Biomedical subjects

Xandra O Breakefield

Publications and source records attributed to Xandra O Breakefield.

At least 37 records · Page 2Linked to original sources
PubMed2004

The early onset dystonia protein torsinA interacts with kinesin light chain 1.

Early onset dystonia is a movement disorder caused by loss of a glutamic acid residue (Glu(302/303)) in the carboxyl-terminal portion of the AAA+ protein, torsinA. We identified the light chain subunit (KLC1) of kinesin-I as an interacting partner for torsinA, with binding occurring between the tetratricopeptide repeat domain of KLC1 and the carboxyl-terminal region of torsinA. Coimmunoprecipitation analysis demonstrated that wild-type torsinA and kinesin-I form a complex in vivo. In cultured cortical neurons, both proteins co-localized along processes with enrichment at growth cones. Wild-type torsinA expressed in CAD cells co-localized with endogenous KLC1 at the distal end of processes, whereas mutant torsinA remained confined to the cell body. Subcellular fractionation of adult rat brain revealed torsinA and KLC associated with cofractionating membranes, and both proteins were co-immunoprecipitated after cross-linking cytoplasmically oriented proteins on isolated rat brain membranes. These studies suggest that wild-type torsinA undergoes anterograde transport along microtubules mediated by kinesin and may act as a molecular chaperone regulating kinesin activity and/or cargo binding.

Adenine↗
PubMed2004

In vivo imaging of HIV protease activity in amplicon vector-transduced gliomas.

In vivo imaging of endogenously expressed mammalian proteases has been useful for the detection of cancer and preneoplastic lesions, for staging of inflammatory and autoimmune diseases, and for testing the efficacy of novel protease inhibitors. Here we report on the synthesis of a novel imaging probe that is specific for HIV-1 protease (PR). The probe was designed to be biocompatible, i.v. injectable, and detectable by fluorescence imaging. Human Gli36 glioblastoma cells infected with an human simplex virus amplicon vector expressing HIV-1PR showed specific fluorescence activation, an effect that could be inhibited by the HIV-1PR inhibitor, indinavir. The transfer of the HIV-1PR marker gene could be detected in vivo after intratumoral delivery of the human simplex virus-amplicon vector. These results are the first proof of principle that viral proteases can directly be imaged in vivo. These findings may be directly applicable in using viral protease expression as a transgene marker in tumor therapy and may have implications in testing the efficacy of HIV-1PR inhibitors in vivo.

Animals↗
PubMed2004

Inhibition of N-linked glycosylation prevents inclusion formation by the dystonia-related mutant form of torsinA.

Most cases of early-onset torsion dystonia are associated with a mutation in the DYT1 gene that results in the loss of a glutamic acid residue in the carboxy terminus of the encoded protein, torsinA. When overexpressed in cultured cells, wild-type torsinA distributes diffusely throughout the endoplasmic reticulum (ER), while the dystonia-related mutant, torsinADeltaE, accumulates within multilamellar membrane inclusions. Here we show that inclusion formation requires the addition of an N-linked oligosaccharide to one of two asparagine residues within the ATP-binding domain of the mutant protein. In the absence of this modification, overexpressed torsinADeltaE was localized diffusely throughout the cell in a reticular pattern resembling that of wild-type torsinA. In contrast, the localization of wild-type torsinA did not appear to vary with its glycosylation state. These results thus indicate that torsinADeltaE must achieve a specific conformation to induce formation of intracellular membrane inclusions.

Amino Acid Sequence↗
PubMed2004

Targeting HSV amplicon vectors.

Several techniques have been developed to deliver DNA directly into mammalian cells, spanning in difficulty from simple mixing procedures to complex systems requiring expensive equipment. Viral vectors have proven able to deliver genes into mammalian cells with high efficiency and low toxicity. In particular, herpes simplex virus type-1 (HSV-1) amplicon vectors are well suited for gene transfer studies as they can infect many cell types, both non-dividing and dividing, have a large transgene capacity and are easy to manipulate. For some applications, it may be desirable to target gene delivery to specific cell populations or to transduce normally non-susceptible cells. This can be achieved by modifying one or more of the glycoproteins found in the viral envelope. Glycoprotein C (gC) has a well-characterized heparan sulfate binding domain (HSBD) necessary for HSV binding to cells. Replacing this region with unique ligands can result in less efficient binding to natural target cells and increase binding to cells which express receptors for these ligands. A method to retarget amplicon vectors by replacing gC HSBD with a model ligand, the hexameric histidine-tag, is described, as well as means to evaluate the binding of modified vector as compared to wild-type virus to cells with or without the appropriate receptor, in this case, a his-tag pseudo-receptor. This protocol demonstrates increased binding of modified virus to receptor-positive cells (at levels greater than wild-type) with no loss of infectivity. Retargeted vectors can provide an additional tool for increasing the efficiency of gene delivery to specific cell types.

Animals↗
PubMed2004

HSV-1 virions engineered for specific binding to cell surface receptors.

Expression of specific peptide epitopes on the surface of virions has significant potential for studying viral biology and designing vectors for targeted gene therapy. In this study, an HSV-1 amplicon plasmid expressing a modified glycoprotein C (gC), in which the heparan sulfate binding domain was replaced with a His-tag, was used in generating HSV-1 virions. Western blot analysis demonstrated the presence of modified gC in the purified virions. The amplicon vectors were packaged using a gC-, lacZ+ helper virus to generate a mixture of high-titer helper virus (lacZ+) and amplicon vectors (GFP+), which expressed modified gC in the virion envelope. His-tagged virions bound to 293 6H cells expressing a cell surface pseudo-His-tag receptor four-fold more efficiently than to parental 293 cells and also proved more effective than wild-type virus in binding to both cell types. Binding resulted in productive infection by the modified virions with expression of reporter genes and cytopathic effect comparable to those of wild-type virions. Thus, not only can HSV-1 tropism be manipulated to recognize a non-herpes simplex binding receptor, but it is also possible to increase the infective capacity of the vectors beyond that of the wild-type virus via specific ligand receptor combinations.

Animals↗
PubMed2004

TorsinB--perinuclear location and association with torsinA.

The torsins comprise a four-member family of AAA+ chaperone proteins, including torsinA, torsinB, torp2A and torp3A in humans. Mutations in torsinA underlie early onset torsion dystonia, an autosomal dominant, neurologically based movement disorder. TorsinB is highly homologous to torsinA with its gene adjacent to that for torsinA on human chromosome 9q34. Antibodies have been generated which can distinguish torsinA and torsinB from each other, and from the torps in human and rodent cells. TorsinB (approximately MW 38 kDa), like torsinA ( approximately MW 37 kDa), is an N-glycosylated protein and both reside primarily in the endoplasmic reticulum (ER) and nuclear envelope in cultured cells. Immunoprecipitation studies in cultured cells and human brain tissue indicate that torsinA and torsinB are associated with each other in cells. Overexpression of both wild-type torsinB and mutant torsinA lead to enrichment of the protein in the nuclear envelope and formation of large cytoplasmic inclusions. We conclude that torsinB and torsinA are localized in overlapping cell compartments within the same protein complex, and thus may carry out related functions in vivo.

Animals↗
PubMed2004

Herpes simplex virus type 1 amplicons and their hybrid virus partners, EBV, AAV, and retrovirus.

HSV-1 amplicons can accommodate foreign DNA of any size up to 150 kbp. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, or multiple transgenes can be inserted in a modular fashion. HSV-1 amplicon vectors deliver DNA efficiently into the cell nucleus as an extrachromosomal, non-replicating circular concatenate, which is rapidly diluted, at least in dividing cells. Consequently, transgene expression is lost within days to weeks in dividing cells, but may be retained for months in non-dividing cells. In contrast, vectors based on Epstein-Barr virus, adeno-associated virus, or retroviruses can mediate long-term transgene expression, as vector DNA is retained by episomal replication or chromosomal integration. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, thereby conserving the large transgene capacity of HSV-1, and genetic elements from other viruses that confer genetic stability to the vector DNA within transduced cells. Additional strategies to sustain genetic material in infected cells include the incorporation of recombinases from different bacteriophages or transposable elements of the Tc1/mariner family in the amplicon vector. Moreover, modification of the HSV-1 virion itself offers a myriad of possibilities to improve gene delivery by targeting specific cell populations or transporting foreign proteins, such as Cre recombinase or the adeno-associated virus Rep protein, which can control the fate and expression of the therapeutic transgene.

Animals↗
PubMed2003

Intravascular delivery of neural stem cell lines to target intracranial and extracranial tumors of neural and non-neural origin.

The remarkable migratory and tumor-tropic capacities of neural stem cells (NSCs and/or neuroprogenitor cells) represent a potentially powerful approach to the treatment of invasive brain tumors, such as malignant gliomas. We have previously shown that whether implanted directly into or at distant sites from an experimental intracranial glioma, NSCs distributed efficiently throughout the main tumor mass and also tracked advancing tumor cells, while stably expressing a reporter transgene. As therapeutic proof-of-concept, NSCs genetically modified to produce the prodrug activating enzyme cytosine deaminase (CD), effected an 80% reduction in the resultant tumor mass, when tumor animals were treated with the systemic prodrug, 5-fluorocytosine. We now extend our findings of the tumor-tropic properties of NSCs (using a well-characterized, clonal NSC line C17.2), by investigating their capacity to target both intracranial and extracranial tumors, when administered into the peripheral vasculature. We furthermore demonstrate their capacity to target extracranial non-neural tumors such as prostate cancer and malignant melanoma. Well-characterized NSC lines (lacZ and/or CD-positive) were injected into the tail vein of adult nude mice with established experimental intracranial and/or subcutaneous flank tumors of neural and non-neural origin. The time course and distribution of NSCs within the tumor and internal organs was assessed in various models. Resulting data suggest that NSCs can localize to various tumor sites when injected via the peripheral vasculature, with little accumulation in normal tissues. Our findings suggest the novel use of intravascularly administered NSCs as an effective delivery vehicle to target and disseminate therapeutic agents to invasive tumors of neural and nonneural origin, both within and outside of the brain.

Animals↗
PubMed2003

Distribution and ultrastructural localization of torsinA immunoreactivity in the human brain.

We have examined the distribution and ultrastructural localization of torsinA, the protein product of the TOR1A gene, in the normal adult human and Macaque brain. TorsinA immunoreactivity was visualized using a monoclonal antibody raised against a fusion protein encoding exon 4 of human torsinA. Western blot analysis of brain homogenates revealed a major species of about 39 kDa, consistent with the predicted size of glycosylated torsinA protein. By light microscopy, torsinA like-immunoreactivity was enriched in gray matter in all brain regions examined. Immunoreactivity was concentrated in the neuropil and immunopositive cell bodies were not observed. Structures particularly enriched in torsinA like-immunoreactivity included the cerebral cortex, the caudate-putamen, globus pallidus, the hippocampal formation, the thalamus, the substantia nigra and molecular cell layer of the cerebellar cortex. Cell bodies of pigmented dopamine neurons in the substantia nigra pars compacta were immunonegative. Biochemical fractionation of the human striata revealed a concentration of torsinA immunoreactivity in particulate fractions. Ultrastructural studies of the human and Macaque striata further revealed an association of torsinA immunostaining with small vesicles within axons and presynaptic terminals forming symmetric synapses. These ultrastructural studies are consistent with a pre-synaptic localization of torsinA protein in the adult striatum and are consistent with a role of torsinA in modulating striatal signaling, although the widespread localization of the protein suggests it probably also participates in signaling in other regions.

Animals↗
PubMed2003

A heteroplasmic mitochondrial complex I gene mutation in adult-onset dystonia.

Mitochondrial DNA (mtDNA) mutations can cause rare forms of dystonia, but the role of mtDNA mutations in other types of dystonia is not well understood. We now report identification by sequencing, restriction endonuclease analyses, and clonal analyses of a heteroplasmic missense A to G base pair substitution at nucleotide position 3796 (A3796G) in the gene encoding the ND1 subunit of mitochondrial complex I in a patient with adult-onset dystonia, spasticity, and core-type myopathy. The mutation converts a highly conserved threonine to an alanine. The same mutation subsequently was identified in 2 of 74 additional unrelated adult-onset dystonia patients. A muscle biopsy was obtained from 1 of these 2 subjects and this revealed abnormalities of electron transport chain (ETC) activities. The mutation was absent in 64 subjects with early onset dystonia, 82 normal controls, and 65 subjects with Parkinson's disease or multiple system atrophy. The A3796G mutation previously has been reported in 3 of 226 subjects from mitochondrial haplogroup H. Each of the 3 subjects in our study harboring the A3796G mutation was also from haplogroup H. However, a subgroup analysis of haplogroup H subjects from our study indicates that the A3796G mutation is significantly overrepresented among haplogroup H adult-onset dystonia subjects compared with haplogroup H controls (P<0.01). This difference remains significant even after excluding the index patient (P=0.04). These data suggest that, among haplogroup H subjects, the presence of the A3796G mutation increases the risk of developing adult-onset dystonia.

Adolescent↗
PubMed2003

Potentiated gene delivery to tumors using herpes simplex virus/Epstein-Barr virus/RV tribrid amplicon vectors.

The development and use of gene transfer techniques creates an opportunity to achieve better treatment modalities for numerous disease entities. Promising results for treatment in tumor cells in culture and in small animal models have been reported. Nevertheless, the lack of widespread vector distribution throughout tumor tissue is one of the current limitations for successful clinical application of gene therapy paradigms. The use of migratory tumor cells themselves as vector delivery vehicles may allow wider vector distribution in tumors. In addition, continuous release of retrovirus vectors on-site could generate a high local virion concentration over an extended time period with consequent increases in transduction efficiency. In this paper, we present in culture and in vivo data of a herpes simplex virus-Epstein-Barr virus hybrid amplicon vector containing retrovirus vector components (tribrid vector) that allows conversion of tumor cells into retroviral producer cells. With this method, we were able to achieve a local fourfold amplification of stable transgene expression in tumors. The application of this system, which can integrate a transgene cassette into tumors with therapeutic bystander effects, could increase the local amplification effect to a level of clinical relevance.

3T3 Cells↗
PubMed2003

TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress.

Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.

Animals↗
PubMed2003

HSV-1 amplicon peptide display vector.

There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33-176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCdelta2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH amplicons carry a GFP transgene and the gCdelta2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1 x 10(5) Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2 x 10(5) amplicon vector transducing units (t.u.)/ml and 1 x 10(7) virus t.u./ml. As the amplicon plasmids also contain the neomycin resistance gene (neo(r)), long term vector producer cell lines were created using G418 selection. This amplicon system provides means to rapidly and efficiently generate HSV-1 amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neo(r) transgenes for marker and selection functions.

Animals↗
PubMed2003

TorsinA protein and neuropathology in early onset generalized dystonia with GAG deletion.

Familial, early onset, generalized torsion dystonia is the most common and severe primary dystonia. Most cases are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A (DYT1) gene, which is widely expressed in human brain and encodes the protein torsinA. This study compares neuropathology and torsinA expression in the normal human brain with that in dystonia cases with and without the GAG deletion. TorsinA-like protein was expressed in neuronal cytoplasm throughout the human brain, including cerebellum, substantia nigra, hippocampus, and neostriatum, with higher levels in specific neurons. This immunostaining pattern was not discernibly different in dystonia and normal brains in midbrain and neostriatal regions. However, nigral dopaminergic neurons appeared to be larger in both GAG-deletion and non-GAG-deletion dystonia brains compared to normal, and may be more closely spaced in GAG-deletion brains. Beyond these apparent changes in neuronal size and spacing in dystonia brains, there was no indication of neuron loss, inflammation, DNA strand breaks, or altered distribution of torsin-like immunoreactivity, supporting a functional rather than degenerative etiology of early onset torsion dystonia.

Adolescent↗