Pamela J. Skinner

Ataxin-1 Nuclear Localization and Aggregation: Role in Polyglutamine-Induced Disease in SCA1 Transgenic Mice

Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1[82] with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1[77] containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.

Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures ∼0.5 µm across, whereas the expanded ataxin-1 localizes to a single ∼2-µm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.

Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection

In the early stages of viral infection, outcomes depend on a race between expansion of infection and the immune response generated to contain it. We combined in situ tetramer staining with in situ hybridization to visualize, map, and quantify relationships between immune effector cells and their targets in tissues. In simian immunodeficiency virus infections in macaques and lymphocytic choriomeningitis virus infections in mice, the magnitude and timing of the establishment of an excess of effector cells versus targets were found to correlate with the extent of control and the infection outcome (i.e., control and clearance versus partial or poor control and persistent infection). This method highlights the importance of the location, timing, and magnitude of the immune response needed for a vaccine to be effective against agents of persistent infection, such as HIV-1.

CD8+ T-Lymphocyte Response to Major Immunodominant Epitopes after Vaginal Exposure to Simian Immunodeficiency Virus: Too Late and Too Little

ABSTRACT In the acute stage of infection following sexual transmission of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), virus-specific CD8+ T-lymphocyte responses partially control but do not eradicate infection from the lymphatic tissues (LTs) or prevent the particularly massive depletion of CD4+ T lymphocytes in gut-associated lymphatic tissue (GALT). We explored hypothetical explanations for this failure to clear infection and prevent CD4+ T-lymphocyte loss in the SIV/rhesus macaque model of intravaginal transmission. We examined the relationship between the timing and magnitude of the CD8+ T-lymphocyte response to immunodominant SIV epitopes and viral replication, and we show first that the failure to contain infection is not because the female reproductive tract is a poor inductive site. We documented robust responses in cervicovaginal tissues and uterus, but only several days after the peak of virus production. Second, while we also documented a modest response in the draining genital and peripheral lymph nodes, the response at these sites also lagged behind peak virus production in these LT compartments. Third, we found that the response in GALT was surprisingly low or undetectable, possibly contributing to the severe and sustained depletion of CD4+ T lymphocytes in the GALT. Thus, the virus-specific CD8+ T-lymphocyte response is “too late and too little” to clear infection and prevent CD4+ T-lymphocyte loss. However, the robust response in female reproductive tissues may be an encouraging sign that vaccines that rapidly induce high-frequency CD8+ T-lymphocyte responses might be able to prevent acquisition of HIV-1 infection by the most common route of transmission.

Cutting Edge: In Situ Tetramer Staining of Antigen-Specific T Cells in Tissues

Staining Ag-specific T cells with fluorescently labeled tetrameric MHC/peptide complexes has provided a powerful experimental approach to characterizing the immune response. In this report, we describe an extension of this method to directly visualize Ag-specific T cells in tissues. We successfully stained transgenic T cells with MHC tetramers in spleen sections from both 2C and OT-1 TCR transgenic mice. In addition, with the in situ tetramer staining technique, we detected a very small population of Ag-specific T cells in tissue after adoptive transfer of transgenic TCR T cells to a syngeneic nontransgenic mouse. We also show that the in situ tetramer technique can be applied to lightly fixed as well as frozen tissue, thus extending the method to archived tissue collections. This in situ tetramer staining technique offers a general approach to tracking the Ag-specific T cells in tissues.

Dominance of CD8 Responses Specific for Epitopes Bound by a Single Major Histocompatibility Complex Class I Molecule during the Acute Phase of Viral Infection

ABSTRACT Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8+ T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8+ responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIVmac239 and determined all of the simian immunodeficiency virus-specific CD8+ T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8+ T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8+ immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8+ responses against Mamu-A*01-restricted epitopes Tat28-35SL8 and Gag181-189CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag181-189CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8+ cellular immune response.

CTL fail to accumulate at sites of HIV-1 replication in lymphoid tissue

The inability of HIV-1-specific CTL to fully suppress virus replication as well as the failure of administration of exogenous CTL to lower viral loads are not understood. To evaluate the hypothesis that these phenomena are due to a failure of CTL to localize at sites of HIV-1 replication, we assessed the distribution of HIV-1 RNA and HIV-1-specific CTL identified by HIV-1 peptide/HLA class I tetrameric complexes (tetramers) within lymph nodes of 14 HIV-1-infected individuals who were not receiving antiretroviral therapy. A median of 0.04% of follicular compared with 0.001% of extrafollicular CD4+ cells were estimated to be producing HIV-1 RNA, a 40-fold difference (p = 0.0001). Tetramer-stained cells were detected by flow cytometry in disaggregated lymph node cells from 11 subjects and constituted a significantly higher fraction of CD8+ cells in lymph node (mean, 2.15%) than in PBMC (mean, 1.52%; p = 0.02). In situ tetramer staining in three subjects’ lymph nodes, in which high frequencies of tetramer-stained cells were detected, revealed that tetramer-stained cells were primarily concentrated in extrafollicular regions of lymph node and were largely absent within lymphoid follicles. These data confirm that HIV-1-specific CTL are abundant within lymphoid tissues, but fail to accumulate within lymphoid follicles where HIV-1 replication is concentrated, suggesting that lymphoid follicles may be immune-privileged sites. Mechanisms underlying the exclusion of CTL from lymphoid follicles as well as the role of lymphoid follicles in perpetuating other chronic pathogens merit further investigation.

Altered Trafficking of Membrane Proteins in Purkinje Cells of SCA1 Transgenic Mice

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expression of mutant ataxin-1 that contains an expanded polyglutamine tract. Overexpression of mutant ataxin-1 in Purkinje cells of transgenic mice results in a progressive ataxia and Purkinje cell pathology that are very similar to those seen in SCA1 patients. Two prominent aspects of pathology in the SCA1 mice are the presence of cytoplasmic vacuoles and dendritic atrophy. We found that the vacuoles in Purkinje cells seem to originate as large invaginations of the outer cell membrane. The cytoplasmic vacuoles contained proteins from the somatodendritic membrane, including mGluR1, GluRDelta1/Delta2, GluR2/3, and protein kinase C (PKC) gamma. Further examination of PKCgamma revealed that its sequestration into cytoplasmic vacuoles was accompanied by concurrent loss of PKCgamma localization at the Purkinje cell dendritic membrane and decreased detection of PKCgamma by Western blot analysis. In addition, the vacuoles were immunoreactive for components of the ubiquitin/proteasome degradative pathway. These findings present a link between vacuole formation and loss of dendrites in Purkinje cells of SCA1 mice and indicate that altered somatodendritic membrane trafficking and loss of proteins including PKCgamma, are a part of the neuronal dysfunction in SCA1 transgenic mice.

Gene expression alterations in brains of mice infected with three strains of scrapie

BackgroundTransmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders which occur in humans and various animal species. Examples include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep, and experimental mice. To gain insights into TSE pathogenesis, we made and used cDNA microarrays to identify disease-associated alterations in gene expression. Brain gene expression in scrapie-infected mice was compared to mock-infected mice at pre-symptomatic and symptomatic time points. Three strains of mouse scrapie that show striking differences in neuropathology were studied: ME7, 22L, and Chandler/RML.ResultsIn symptomatic mice, over 400 significant gene expression alterations were identified. In contrast, only 22 genes showed significant alteration in the pre-symptomatic animals. We also identified genes that showed significant differences in alterations in gene expression between strains. Genes identified in this study encode proteins that are involved in many cellular processes including protein folding, endosome/lysosome function, immunity, synapse function, metal ion binding, calcium regulation and cytoskeletal function.ConclusionThese studies shed light on the complex molecular events that occur during prion disease, and identify genes whose further study may yield new insights into strain specific neuropathogenesis and ante-mortem tests for TSEs.

Compartmentalization of Simian Immunodeficiency Virus Replication within Secondary Lymphoid Tissues of Rhesus Macaques Is Linked to Disease Stage and Inversely Related to Localization of Virus-Specific CTL

We previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA+ cells and SIV-specific CTL in situ in spleen, lymph nodes, and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS, SIV-producing cells were more concentrated in follicular (F) compared with extrafollicular (EF) regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with simian AIDS, which often have low-frequency CTL responses. SIV-specific CTL were consistently more concentrated within EF regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within F and EF compartments predicted SIV RNA+ cells within these compartments in a mixed model. Few SIV-specific CTL expressed the F homing molecule CXCR5 in the absence of the EF retention molecule CCR7, possibly accounting for the paucity of F CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection.