Targeting the autophagy-NAD axis protects against cell death in Niemann-Pick type C1 disease models.

Impairment of autophagy leads to an accumulation of misfolded proteins and damaged organelles and has been implicated in plethora of human diseases. Loss of autophagy in actively respiring cells has also been shown to trigger metabolic collapse mediated by the depletion of nicotinamide adenine dinucleotide (NAD) pools, resulting in cell death. Here we found that the deficit in the autophagy-NAD axis underpins the loss of viability in cell models of a neurodegenerative lysosomal storage disorder, Niemann-Pick type C1 (NPC1) disease. Defective autophagic flux in NPC1 cells resulted in mitochondrial dysfunction due to impairment of mitophagy, leading to the depletion of both the reduced and oxidised forms of NAD as identified via metabolic profiling. Consequently, exhaustion of the NAD pools triggered mitochondrial depolarisation and apoptotic cell death. Our chemical screening identified two FDA-approved drugs, celecoxib and memantine, as autophagy activators which effectively restored autophagic flux, NAD levels, and cell viability of NPC1 cells. Of biomedical relevance, either pharmacological rescue of the autophagy deficiency or NAD precursor supplementation restored NAD levels and improved the viability of NPC1 patient fibroblasts and induced pluripotent stem cell (iPSC)-derived cortical neurons. Together, our findings identify the autophagy-NAD axis as a mechanism of cell death and a target for therapeutic interventions in NPC1 disease, with a potential relevance to other neurodegenerative disorders.

Partial Inhibition of Complex I Restores Mitochondrial Morphology and Mitochondria-ER Communication in Hippocampus of APP/PS1 Mice.

Alzheimer's disease (AD) has no cure. Earlier, we showed that partial inhibition of mitochondrial complex I (MCI) with the small molecule CP2 induces an adaptive stress response, activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, Aß and pTau accumulation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD. Here, using serial block-face scanning electron microscopy (SBFSEM) and three-dimensional (3D) EM reconstructions combined with Western blot analysis and next-generation RNA sequencing, we demonstrate that CP2 treatment also restores mitochondrial morphology and mitochondria-endoplasmic reticulum (ER) communication, reducing ER and unfolded protein response (UPR) stress in the APP/PS1 mouse brain. Using 3D EM volume reconstructions, we show that in the hippocampus of APP/PS1 mice, dendritic mitochondria primarily exist as mitochondria-on-a-string (MOAS). Compared to other morphological phenotypes, MOAS have extensive interaction with the ER membranes, forming multiple mitochondria-ER contact sites (MERCS) known to facilitate abnormal lipid and calcium homeostasis, accumulation of Aß and pTau, abnormal mitochondrial dynamics, and apoptosis. CP2 treatment reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reductions in MERCS, ER/UPR stress, and improved lipid homeostasis. These data provide novel information on the MOAS-ER interaction in AD and additional support for the further development of partial MCI inhibitors as a disease-modifying strategy for AD.

A Comprehensive Approach to Sample Preparation for Electron Microscopy and the Assessment of Mitochondrial Morphology in Tissue and Cultured Cells.

Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high-resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Mitochondrial complex I as a therapeutic target for Alzheimer's disease.

Alzheimer's disease (AD), the most prominent form of dementia in the elderly, has no cure. Strategies focused on the reduction of amyloid beta or hyperphosphorylated Tau protein have largely failed in clinical trials. Novel therapeutic targets and strategies are urgently needed. Emerging data suggest that in response to environmental stress, mitochondria initiate an integrated stress response (ISR) shown to be beneficial for healthy aging and neuroprotection. Here, we review data that implicate mitochondrial electron transport complexes involved in oxidative phosphorylation as a hub for small molecule-targeted therapeutics that could induce beneficial mitochondrial ISR. Specifically, partial inhibition of mitochondrial complex I has been exploited as a novel strategy for multiple human conditions, including AD, with several small molecules being tested in clinical trials. We discuss current understanding of the molecular mechanisms involved in this counterintuitive approach. Since this strategy has also been shown to enhance health and life span, the development of safe and efficacious complex I inhibitors could promote healthy aging, delaying the onset of age-related neurodegenerative diseases.

A genome-wide association study in human lymphoblastoid cells supports safety of mitochondrial complex I inhibitor.

Novel therapeutic strategies for Alzheimer's disease (AD) are of the greatest priority given the consistent failure of recent clinical trials focused on Aß or pTau. Earlier, we demonstrated that mild mitochondrial complex I inhibitor CP2 blocks neurodegeneration and cognitive decline in multiple mouse models of AD. To evaluate the safety of CP2 in humans, we performed a genome-wide association study (GWAS) using 196 lymphoblastoid cell lines and identified 11 SNP loci and 64 mRNA expression probe sets that potentially associate with CP2 susceptibility. Using primary mouse neurons and pharmacokinetic study, we show that CP2 is generally safe at a therapeutic dose.

Partial inhibition of mitochondrial complex I ameliorates Alzheimer's disease pathology and cognition in APP/PS1 female mice.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

The evolution of operative access in lung surgery.

In modern surgical practice, considerable attention is paid to reducing surgical trauma and reducing the incidence of postoperative complications, which has a direct impact on the duration of hospitalization and patient recovery. In chest surgery, this problem is most significant, since to perform small interventions on the lungs and pleura, a wide thoracotomy was required with the transection of the chest muscles and the separation of the ribs. The article describes modern minimally invasive approaches used in lung surgery. Particular attention is paid to the role of video-assisted surgical interventions in the surgical treatment of non-small cell lung cancer. The results of traditional multiport thoracoscopic lung resections were compared with standard open thoracotomy. The advantages and possible disadvantages of various options for video-assisted surgical interventions on the lungs are described.

Differential effect of amyloid beta peptides on mitochondrial axonal trafficking depends on their state of aggregation and binding to the plasma membrane.

Inhibition of mitochondrial axonal trafficking by amyloid beta (Aß) peptides has been implicated in early pathophysiology of Alzheimer's Disease (AD). Yet, it remains unclear whether the loss of motility inevitably induces the loss of mitochondrial function, and whether restoration of axonal trafficking represents a valid therapeutic target. Moreover, while some investigations identify Aß oligomers as the culprit of trafficking inhibition, others propose that fibrils play the detrimental role. We have examined the effect of a panel of Aß peptides with different mutations found in familial AD on mitochondrial motility in primary cortical mouse neurons. Peptides with higher propensity to aggregate inhibit mitochondrial trafficking to a greater extent with fibrils inducing the strongest inhibition. Binding of Aß peptides to the plasma membrane was sufficient to induce trafficking inhibition where peptides with reduced plasma membrane binding and internalization had lesser effect on mitochondrial motility. We also found that Aß peptide with Icelandic mutation A673T affects axonal trafficking of mitochondria but has very low rates of plasma membrane binding and internalization in neurons, which could explain its relatively low toxicity. Inhibition of mitochondrial dynamics caused by Aß peptides or fibrils did not instantly affect mitochondrial bioenergetic and function. Our results support a mechanism where inhibition of axonal trafficking is initiated at the plasma membrane by soluble low molecular weight Aß species and is exacerbated by fibrils. Since trafficking inhibition does not coincide with the loss of mitochondrial function, restoration of axonal transport could be beneficial at early stages of AD progression. However, strategies designed to block Aß aggregation or fibril formation alone without ensuring the efficient clearance of soluble Aß may not be sufficient to alleviate the trafficking phenotype.

Interleukins 6 and 8 and abdominal fat depots are distinct correlates of lipid moieties in healthy pre- and postmenopausal women.

Available data associate lipids concentrations in men with body mass index, anabolic steroids, age, and certain cytokines. Data were less clear in women, especially across the full adult lifespan, and when segmented by premenopausal and postmenopausal status. SUBJECTS: 120 healthy women (60 premenopausal and 60 postmenopausal) in Olmsted County, MN, USA, a stable well studied clinical population. Dependent variables: measurements of 10 h fasting high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglycerides. INDEPENDENT VARIABLES: testosterone, estrone, estradiol, 5-alpha-dihydrotestosterone, and sex-hormone binding globulin (by mass spectrometry); insulin, glucose, and albumin; abdominal visceral, subcutaneous, and total abdominal fat [abdominal visceral fat, subcutaneous fat, total abdominal fat by computerized tomography scan]; and a panel of cytokines (by enzyme-linked immunosorbent assay). Multivariate forward-selection linear-regression analysis was applied constrained to P < 0.01. Lifetime data: High-density lipoprotein cholesterol was correlated jointly with age (P < 0.0001, positively), abdominal visceral fat (P < 0.0001, negatively), and interleukin-6 (0.0063, negatively), together explaining 28.1 % of its variance (P = 2.3 × 10-8). Total cholesterol was associated positively with multivariate age only (P = 6.9 × 10-4, 9.3 % of variance). Triglycerides correlated weakly with sex-hormone binding globulin (P = 0.0115), and strongly with abdominal visceral fat (P < 0.0001), and interleukin-6 (P = 0.0016) all positively (P = 1.6 × 10-12, 38.9 % of variance). Non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol correlated positively with both total abdominal fat and interleukin-8 (P = 2.0 × 10-5, 16.9 % of variance; and P = 0.0031, 9.4 % of variance, respectively). Premenopausal vs. postmenopausal comparisons identified specific relationships that were stronger in premenopausal than postmenopausal individuals, and vice versa. Age was a stronger correlate of low-density lipoprotein cholesterol; interleukin-6 of triglycerides and high-density lipoprotein; and both sex-hormone binding globulin and total abdominal fat of non high-density lipoprotein cholesterol in premenopausal than postmenopausal women. Conversely, sex-hormone binding globulin, abdominal visceral fat, interleukin-8, adiponectin were stronger correlates of triglycerides; abdominal visceral fat, and testosterone of high-density lipoprotein cholesterol; and age of both non high-density lipoprotein and low-density lipoprotein in postmenopausal than premenopausal women. Our data delineate correlations of total abdominal fat and interleukin-8 (both positively) with non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in healthy women across the full age range of 21-79 years along with even more specific associations in premenopausal and postmenopausal individuals. Whether some of these outcomes reflect causal relationships would require longitudinal and interventional or genetic studies.

Altered brain energetics induces mitochondrial fission arrest in Alzheimer's Disease.

Altered brain metabolism is associated with progression of Alzheimer's Disease (AD). Mitochondria respond to bioenergetic changes by continuous fission and fusion. To account for three dimensional architecture of the brain tissue and organelles, we applied 3-dimensional electron microscopy (3D EM) reconstruction to visualize mitochondrial structure in the brain tissue from patients and mouse models of AD. We identified a previously unknown mitochondrial fission arrest phenotype that results in elongated interconnected organelles, "mitochondria-on-a-string" (MOAS). Our data suggest that MOAS formation may occur at the final stages of fission process and was not associated with altered translocation of activated dynamin related protein 1 (Drp1) to mitochondria but with reduced GTPase activity. Since MOAS formation was also observed in the brain tissue of wild-type mice in response to hypoxia or during chronological aging, fission arrest may represent fundamental compensatory adaptation to bioenergetic stress providing protection against mitophagy that may preserve residual mitochondrial function. The discovery of novel mitochondrial phenotype that occurs in the brain tissue in response to energetic stress accurately detected only using 3D EM reconstruction argues for a major role of mitochondrial dynamics in regulating neuronal survival.

Modulation of mitochondrial complex I activity averts cognitive decline in multiple animal models of familial Alzheimer's Disease.

Development of therapeutic strategies to prevent Alzheimer's Disease (AD) is of great importance. We show that mild inhibition of mitochondrial complex I with small molecule CP2 reduces levels of amyloid beta and phospho-Tau and averts cognitive decline in three animal models of familial AD. Low-mass molecular dynamics simulations and biochemical studies confirmed that CP2 competes with flavin mononucleotide for binding to the redox center of complex I leading to elevated AMP/ATP ratio and activation of AMP-activated protein kinase in neurons and mouse brain without inducing oxidative damage or inflammation. Furthermore, modulation of complex I activity augmented mitochondrial bioenergetics increasing coupling efficiency of respiratory chain and neuronal resistance to stress. Concomitant reduction of glycogen synthase kinase 3ß activity and restoration of axonal trafficking resulted in elevated levels of neurotrophic factors and synaptic proteins in adult AD mice. Our results suggest metabolic reprogramming induced by modulation of mitochondrial complex I activity represents promising therapeutic strategy for AD.

Serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of LSEPAELTDAVK peptide.

CONTEXT: Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays. OBJECTIVE: To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays. DESIGN: Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer. RESULTS: The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis. CONCLUSIONS: This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.

Immunologic and mass-spectrometric estimates of SHBG concentrations in healthy women.

OBJECTIVE: Sex-hormone binding globulin (SHBG) concentrations across the adult female lifespan are not well defined. To address this knowledge gap, SHBG was quantified by both immunological and criterion methods, viz, mass spectrometry (MS). SETTING: Center for Translational Science Activities (CTSA). PARTICIPANTS: Healthy nonpregnant women (N=120) ages 21 to 79 years. OUTCOMES: SHBG, testosterone (T), estradiol (E2) and estrone (E1) each determined by MS. Uni- and multivariate regression of SHBG concentrations on age, body mass index (BMI), total and visceral abdominal fat (TAF, AVF), albumin, glucose, insulin, sex steroids, selected cytokines, blood pressure, and lipids. RESULTS: By univariate regression, MS-estimated SHBG correlated negatively with BMI, TAF, AVF, insulin, free T and bioavailable T (bio T) (each P≤10(-4)), but not with blood pressure or lipids. By stepwise multivariate regression analysis, free and total T (both positive) and bio T (negative) were correlated with SHBG in all 4 assays (each P<10(-15), R(2)≥0.481). In addition, TAF and BMI were negatively associated with SHBG (P≤0.0066) in 2 SHBG assays, and estrone and IL-8 with SHBG weakly (P≤0.035) in one SHBG assay each. When nonsignificant cytokines were excluded, SHBG was jointly associated with AVF, total T and HDL (P<10(-9), R(2)=0.358). CONCLUSION: According to MS, three metabolic factors, T, AVF and HDL, together explain more than one-third of the interindividual variation in SHBG levels. We speculate that these measures reflect insulin action.

Mass spectrometry measurements of prostate-specific antigen (PSA) peptides derived from immune-extracted PSA provide a potential strategy for harmonizing immunoassay differences.

OBJECTIVES: Harmonization of prostate-specific antigen (PSA) immunoassays is important for good patient care. The specificity of the antibodies used to detect circulating PSA could cause differences in the PSA measurements. METHODS: We used mass spectrometry (MS) to quantitate the concentration of five peptides cleaved from trypsin digestion of PSA and compared these measurements with six automated immunoassays. Linear regression and a mixed-effects model were used to analyze the results. RESULTS: PSA measurements from the immunoassays and the five MS peptide assays were highly correlated (R(2) > 0.99), but the recovery of the World Health Organization standard and the regression slopes differed across assays. The same relative patterns of immunoassay differences were seen in comparing their results with each of the five MS peptide measurements from different parts of the circulating PSA molecules. CONCLUSIONS: Mass spectrometry quantitation of peptides derived from trypsin digestion of immune-extracted PSA could be used to harmonize PSA immunoassays.

Immunological and mass spectrometric assays of SHBG: consistent and inconsistent metabolic associations in healthy men.

CONTEXT: SHBG concentrations correlate inconsistently with metabolic parameters. HYPOTHESIS: SHBG assay platforms contribute to nonuniformities according to the literature. DESIGN: The design of the study was a noninterventional quantification of SHBG by two immuno- and two mass spectrometric assays and abdominal visceral fat by computed tomography scan. SETTING: The study was conducted at the Center for Translational Science Activities. PARTICIPANTS: Healthy men (n=120) aged 18-80 years with a body mass index of 20-43 kg/m2 participated I the study. OUTCOMES: Outcomes of the study included a correlation of log SHBG with age, metabolic surrogates [body mass index, albumin, glucose, insulin, abdominal (total and visceral) fat, homeostasis model assessment insulin resistance index], sex steroids (estrone, 17ß-estradiol, T, and dihydrotestosterone by mass spectrometry), and adipocytokines (IL-1ß, IL-6, IL-8, IL-10 and IL-12, TNF-α, and adiponectin). RESULTS: By univariate regression, age (P<10(-4)), dihydrotestosterone (P<10(-4)), T (P≤.00022), and adiponectin (P≤.0084) were positive correlates, and insulin and homeostasis model assessment insulin resistance index were negative correlates (P≤.0060) of SHBG in all four assays. Stepwise multivariate analysis unveiled that age and T together could explain 38.1%-52.5% of the statistical variance in SHBG in all assays (P<10(-11)). Multivariate regression without sex steroids unveiled that age (P<10(-5)) and insulin (P<10(-3)) are jointly associated with SHBG levels in the four assays with overall R2=0.215-0.293 and P<10(-6). In one immunological SHBG assay each, abdominal visceral fat and adiponectin were weak multivariates also. CONCLUSION: Immunological and mass spectrometric SHBG assays yield both consistent and inconsistent correlations with key metabolic variables in healthy men, thereby potentially explaining earlier inconsistencies in the literature.

SDF-1α degrades whereas glycoprotein 120 upregulates Bcl-2 interacting mediator of death extralong isoform: implications for the development of T cell memory.

After a primary immune response, T cell memory occurs when a subset of Ag-specific T cells resists peripheral selection by acquiring resistance to TCR-induced death. Recent data have implicated Bcl-2 interacting mediator of death (Bim) as an essential mediator of the contraction phase of T cell immunity. In this article, we describe that stromal-derived factor-1α (SDF-1α) ligation of CXCR4 on activated T cells promotes two parallel processes that favor survival, phospho-inactivation of Foxo3A, as well as Bim extralong isoform (Bim(EL)) degradation, both in an Akt- and Erk-dependent manner. Activated primary CD4 T cells treated with SDF-1α therefore become resistant to the proapoptotic effects of TCR ligation or IL-2 deprivation and accumulate cells of a memory phenotype. Unlike SDF-1α, gp120 ligation of CXCR4 has the opposite effect because it causes p38-dependent Bim(EL) upregulation. However, when activated CD4 T cells are treated with both gp120 and SDF-1α, the SDF-1α-driven effects of Bim(EL) degradation and acquired resistance to TCR-induced death predominate. These results provide a novel causal link between SDF-1α-induced chemotaxis, degradation of Bim(EL), and the development of CD4 T cell memory.