Byung Park

Mitochondria-Targeted Antioxidants Protect Against Amyloid-β Toxicity in Alzheimer's Disease Neurons

The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimers disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.

Differential Expression of Oxidative Phosphorylation Genes in Patients with Alzheimer's Disease: Implications for Early Mitochondrial Dysfunction and Oxidative Damage

In Alzheimer’s disease (AD) pathogenesis, increasing evidence implicates mitochondrial dysfunction resulting from molecular defects in oxidative phosphorylation (OXPHOS). The objective of the present study was to determine the role of mRNA expression of mitochondrial genes responsible for OXPHOS in brain specimens from early AD and definite AD patients. In the present article, using quantitative real-time polymerase chain reaction (PCR) techniques, we studied mRNA expression of 11 mitochondrial-encoded genes in early AD patients (n=6), definite AD patients (n=6), and control subjects (n=6). Using immunofluorescence techniques, we determined differentially expressed mitochondrial genes—NADH 15-kDa subunit (complex I), cytochrome oxidase subunit 1 (complex IV), and ATPase δ-subunit (complex V)—in the brain sections of AD patients and control subjects. Our quantitative reverse transcription (RT)-PCR analysis revealed a downregulation of mitochondrial genes in complex I of OXPHOS in both early and definite AD brain specimens. Further, the decrease of mRNA fold changes was higher for subunit 1 compared to all other subunits studied, suggesting that subunit 1 is critical for OXPHOS. Contrary to the downregulation of genes in complex I, complexes III and IV showed increased mRNA expressions in the brain specimens of both early and definite AD patients, suggesting a great demand on energy production. Further, mitochondrial gene expression varied greatly across AD patients, suggesting that mitochondrial DNA defects may be responsible for the heterogeneity of the phenotype in AD patients. Our immunofluorescence analyses of cytochrome oxidase and of the ATPase δ-subunit suggest that only subpopulations of neurons are differentially expressed in AD brains. Our double-labeling immunofluorescence analyses of 8-hydroxyguanosine and of cytochrome oxidase suggest that only selective, over-expressed neurons with cytochrome oxidase undergo oxidative damage in AD brains. Based on these results, we propose that an increase in cytochrome oxidase gene expression might be the result of functional compensation by the surviving neurons or an early mitochondrial alteration related to increased oxidative damage.

Differential loss of synaptic proteins in Alzheimer's disease: Implications for synaptic dysfunction

The objective of our research was to determine synaptic protein levels in brain specimens from AD subjects and age-matched control subjects. Further, to determine whether presynaptic or postsynaptic compartments of neurons are preferentially affected in AD patients, we studied 3 presynaptic vesicle proteins (synaptotagmin, synaptophysin, and Rab 3A), 2 synaptic membrane proteins (Gap 43 and synaptobrevin), and 2 postsynaptic proteins (neurogranin and synaptopodin) in specimens from AD and age-matched control brains. Two brain regions--the frontal and parietal cortices--were assessed for protein levels by immunoblotting analysis. We found a loss of both presynaptic vesicle proteins and postsynaptic proteins in all brain specimens from AD patients compared to those from age-matched control subjects. Further, we found that the loss of synaptic proteins was more severe in the frontal cortex brain specimens than in the parietal cortex brain specimens from the AD subjects compared to those from the control subjects, suggesting that the frontal brain may be critical for synaptic function in AD. Using immunohistochemistry techniques, we also determined the distribution pattern of all synaptic proteins in both the frontal and parietal cortices brain specimens from control subjects. Of the 7 synaptic proteins studied, the presynaptic proteins synaptophysin and rab 3A and the postsynaptic protein synaptopodin were the most down-regulated. Our study suggests that postsynaptic proteins and presynaptic proteins are important for synaptic function and may be related to cognitive impairments in AD.

Time‐course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging

The study of aging is critical for a better understanding of many age‐related diseases. The free radical theory of aging, one of the prominent aging hypotheses, holds that during aging, increasing reactive oxygen species in mitochondria causes mutations in the mitochondrial DNA and damages mitochondrial components, resulting in senescence. Understanding a mitochondrial gene expression profile and its relationship to mitochondrial function becomes an important step in understanding aging. The objective of the present study was to determine mRNA expression of mitochondrial‐encoded genes in brain slices from C57BL6 mice at four ages (2, 12, 18, and 24 months) and to determine how these altered mitochondrial genes influence age‐related changes, including oxidative damage and cytochrome c in apoptosis. Using northern blot analysis, in situ hybridization, and immunofluorescence analyses, we analyzed changes in the expression of mitochondrial RNA encoding the mitochondrial genes, oxidative damage marker, 8‐hydroxyguanosine (8‐OHG), and cytochrome c in brain slices from the cortex of C57BL6 mice at each of the four ages. Our northern blot analysis revealed an increased expression of mitochondrial‐encoded genes in complexes I, III, IV, and V of the respiratory chain in 12‐ and 18‐month‐old C57BL6 mice compared to 2‐month‐old mice, suggesting a compensatory mechanism that allows the production of proteins involved in the electron transport chain. In contrast to the up‐regulation of mitochondrial genes in 12‐ and 18‐month‐old C57BL6 mice, mRNA expression in 24‐month‐old C57BL6 mice was decreased, suggesting that compensation maintained by the up‐regulated genes cannot be sustained and that the down‐regulation of expression results in the later stage of aging. Our in situ hybridization analyses of mitochondrial genes from the hippocampus and the cortex revealed that mitochondrial genes were over‐expressed, suggesting that these brain areas are critical for mitochondrial functions. Our immunofluorescence analysis of 8‐OHG and cytochrome c revealed increased 8‐OHG and cytochrome c in 12‐month‐old C57BL6 mice, suggesting that age‐related mitochondrial oxidative damage and apoptosis are associated with mitochondrial dysfunction. Our double‐labeling analysis of in situ hybridization of ATPase 6 and our immunofluorescence analysis of 8‐OHG suggest that specific neuronal populations undergo oxidative damage. Further, double‐labeling analysis of in situ hybridization of ATPase 6 and immunofluorescence analysis of cytochrome c suggest cytochrome c release is related to mitochondrial dysfunction in the aging C57BL6 mouse brain. This study also suggests that these mitochondrial gene expression changes may relate to the role of mitochondrial dysfunction, oxidative damage, and cytochrome c in aging and in age‐related diseases such as Alzheimers disease and Parkinsons disease.

Dramatic increase in naïve T cell turnover is linked to loss of naïve T cells from old primates

The loss of naïve T cells is a hallmark of immune aging. Although thymic involution is a primary driver of this naïve T cell loss, less is known about the contribution of other mechanisms to the depletion of naïve T cells in aging primates. We examined the role of homeostatic cycling and proliferative expansion in different T cell subsets of aging rhesus macaques (RM). BrdU incorporation and the expression of the G1-M marker Ki-67 were elevated in peripheral naïve CD4 and even more markedly in the naïve CD8 T cells of old, but not young adult, RM. Proliferating naïve cells did not accumulate in old animals. Rather, the relative size of the naïve CD8 T cell compartment correlated inversely to its proliferation rate. Likewise, T cell receptor diversity decreased in individuals with elevated naïve CD8 T cell proliferation. This apparent contradiction was explained by a significant increase in turnover concomitant with the naïve pool loss. The turnover increased exponentially when the naïve CD8 T cell pool decreased below 4% of total blood CD8 cells. These results link the shrinking naïve T cell pool with a dramatic increase in homeostatic turnover, which has the potential to exacerbate the progressive exhaustion of the naïve pool and constrict the T cell repertoire. Thus, homeostatic T cell proliferation exhibits temporal antagonistic pleiotropy, being beneficial to T cell maintenance in adulthood but detrimental to the long-term T cell maintenance in aging individuals.

The prognosis for patients with chronic myeloid leukemia who have clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells

Clonal cytogenetic abnormalities (CCA) were detected in Philadelphia chromosome (Ph)‐negative cells in some patients with chronic myeloid leukemia (CML) who attained a cytogenetic response to imatinib mesylate. In some patients, CCA/Ph‐negative status was associated with myelodysplasia or acute myeloid leukemia. The objective of the current study was to determine the prognostic impact of CCA/Ph‐negative cells.

Loss of Naive T Cells and Repertoire Constriction Predict Poor Response to Vaccination in Old Primates

Aging is usually accompanied by diminished immune protection upon infection or vaccination. Although aging results in well-characterized changes in the T cell compartment of long-lived, outbred, and pathogen-exposed organisms, their relevance for primary Ag responses remain unclear. Therefore, it remains unclear whether and to what extent the loss of naive T cells, their partial replacement by oligoclonal memory populations, and the consequent constriction of TCR repertoire limit the Ag responses in aging primates. We show in this study that aging rhesus monkeys (Macaca mulatta) exhibit poor CD8 T cell and B cell responses in the blood and poor CD8 responses in the lungs upon vaccination with the modified vaccinia strain Ankara. The function of APCs appeared to be maintained in aging monkeys, suggesting that the poor response was likely intrinsic to lymphocytes. We found that the loss of naive CD4 and CD8 T cells, and the appearance of persisting T cell clonal expansions predicted poor CD8 responses in individual monkeys. There was strong correlation between early CD8 responses in the transitory CD28+ CD62L− CD8+ T cell compartment and the peak Ab titers upon boost in individual animals, as well as a correlation of both parameters of immune response to the frequency of naive CD8+ T cells in old but not in adult monkeys. Therefore, our results argue that T cell repertoire constriction and naive cell loss have prognostic value for global immune function in aging primates.

Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques

Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1–specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8+ T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.

Effects of sex and APOE ε4 on object recognition and spatial navigation in the elderly

Abstract To determine effects of APOE e4 (e4) on cognitive performance of healthy elderly, 116 nondemented elders (mean age 81 years) were cognitive tested. The established tests Faces, Family Pictures, Spatial Span Forward and Backward, and the object recognition and spatial navigation tests developed in our laboratory were used as cognitive tests. Salivary samples were collected to determine APOE genotype and salivary testosterone and cortisol levels. Non-e4- and e4-carrying men and women did not differ in age, Mini-Mental State Examination, Wide Range Achievement Test-Reading, Beck Anxiety Inventory, or reaction time scores. There was an effect of e4 on the object recognition and spatial navigation tests, with non-e4 carriers outperforming e4 carriers, but not in the other cognitive tests. No relationship was found for sex and e4 status or sex and performance during the hidden session of Memory Island. In men, salivary cortisol levels correlated with object recognition. These results show that object recognition and spatial navigation tests are useful to assess cognitive function in the elderly.

Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors

Aging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG‐I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro‐inflammatory and antiviral cytokines and chemokines including TNFα, IL‐6, IL‐1β, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up‐regulation of PD‐L1 on monocytes and T cells, and increased expression of PD‐L2 and B7‐H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR‐stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T‐cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age‐associated changes in cytokine, chemokine and interferon production, as well as co‐stimulatory protein expression could contribute to the blunted memory B‐ and T‐cell immune responses to vaccines and infections.