Hippocampal astrocytes induce sex-dimorphic effects on memory.

Astrocytic receptors influence cognitive function and can promote behavioral deficits in disease. These effects may vary based on variables such as biological sex, but it is not known if the effects of astrocytic receptors are dependent on sex. We leveraged in vivo gene editing and chemogenetics to examine the roles of astrocytic receptors in spatial memory and other processes. We show that reductions in metabotropic glutamate receptor 3 (mGluR3), the main astrocytic glutamate receptor in adults, impair memory in females but enhance memory in males. Similarly, increases in astrocytic mGluR3 levels have sex-dependent effects and enhance memory in females. mGluR3 manipulations also alter spatial search strategies during recall in a sex-specific manner. In addition, acute chemogenetic stimulation of Gi/o-coupled or Gs-coupled receptors in hippocampal astrocytes induces bidirectional and sex-dimorphic effects on memory. Thus, astrocytes are sex-dependent modulators of cognitive function and may promote sex differences in aging and disease.

Astrocytes in selective vulnerability to neurodegenerative disease.

Selective vulnerability of specific brain regions and cell populations is a hallmark of neurodegenerative disorders. Mechanisms of selective vulnerability involve neuronal heterogeneity, functional specializations, and differential sensitivities to stressors and pathogenic factors. In this review we discuss the growing body of literature suggesting that, like neurons, astrocytes are heterogeneous and specialized, respond to and integrate diverse inputs, and induce selective effects on brain function. In disease, astrocytes undergo specific, context-dependent changes that promote different pathogenic trajectories and functional outcomes. We propose that astrocytes contribute to selective vulnerability through maladaptive transitions to context-divergent phenotypes that impair specific brain regions and functions. Further studies on the multifaceted roles of astrocytes in disease may provide new therapeutic approaches to enhance resilience against neurodegenerative disorders.

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS-based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III, and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP, and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Systems-level analyses dissociate genetic regulators of reactive oxygen species and energy production.

Respiratory chain dysfunction can decrease ATP and increase reactive oxygen species (ROS) levels. Despite the importance of these metabolic parameters to a wide range of cellular functions and disease, we lack an integrated understanding of how they are differentially regulated. To address this question, we adapted a CRISPRi- and FACS- based platform to compare the effects of respiratory gene knockdown on ROS to their effects on ATP. Focusing on genes whose knockdown is known to decrease mitochondria-derived ATP, we showed that knockdown of genes in specific respiratory chain complexes (I, III and CoQ10 biosynthesis) increased ROS, whereas knockdown of other low ATP hits either had no impact (mitochondrial ribosomal proteins) or actually decreased ROS (complex IV). Moreover, although shifting metabolic conditions profoundly altered mitochondria-derived ATP levels, it had little impact on mitochondrial or cytosolic ROS. In addition, knockdown of a subset of complex I subunits-including NDUFA8, NDUFB4, and NDUFS8-decreased complex I activity, mitochondria-derived ATP and supercomplex level, but knockdown of these genes had differential effects on ROS. Conversely, we found an essential role for ether lipids in the dynamic regulation of mitochondrial ROS levels independent of ATP. Thus, our results identify specific metabolic regulators of cellular ATP and ROS balance that may help dissect the roles of these processes in disease and identify therapeutic strategies to independently target energy failure and oxidative stress.

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP.

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

Astrocytic TDP-43 dysregulation impairs memory by modulating antiviral pathways and interferon-inducible chemokines.

Transactivating response region DNA binding protein 43 (TDP-43) pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 pathology are not clear, and therapeutic strategies to alleviate TDP-43-linked cognitive decline are lacking. We found that patients with Alzheimer's disease or frontotemporal dementia have aberrant TDP-43 accumulation in hippocampal astrocytes. In mouse models, induction of widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused progressive memory loss and localized changes in antiviral gene expression. These changes were cell-autonomous and correlated with impaired astrocytic defense against infectious viruses. Among the changes, astrocytes had elevated levels of interferon-inducible chemokines, and neurons had elevated levels of the corresponding chemokine receptor CXCR3 in presynaptic terminals. CXCR3 stimulation altered presynaptic function and promoted neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 dysregulation, and blockade of CXCR3 reduced this activity. Ablation of CXCR3 also prevented TDP-43-linked memory loss. Thus, astrocytic TDP-43 dysfunction contributes to cognitive impairment through aberrant chemokine-mediated astrocytic-neuronal interactions.

Rib Fracture Mortality: Are There Clues in the Core?

BACKGROUND: Sarcopenia is associated with increased morbidity and mortality in the trauma patient. The primary objective of this study was to determine the relationship of psoas cross sectional area with hospital mortality in patients with rib fractures over the age of 55 years. MATERIALS AND METHODS: We retrospectively reviewed 1223 patients presenting to a Level 1 Trauma Center between 1/1/2002 and 1/31/2019. Psoas cross sectional area was measured using a polygonal tracing tool. Patients were stratified into four quartiles based on sex-specific values. RESULTS: There was increased in-hospital mortality for patients with a lower psoas cross sectional area (10 %, 8%, 6%, and 4%, Q1-Q4 respectively; P=0.021). The logistic regression model determined for every increase in psoas cross sectional area by 1 cm2 the odds of in-hospital mortality decreased by 4%. CONCLUSIONS: In-hospital mortality is multifactorial; however, psoas cross sectional area may provide a clue in predicting adverse outcomes after traumatic rib fractures.

Pituitary sellar spine in a pediatric patient with diabetes insipidus.

A sellar spine is a midline bony spur arising from the ventral aspect of the dorsum sellae. Representing a remnant of the anterior notochord, it is often an incidental finding and thought to be of no clinical significance. However, it has recently been suggested that a potential association may exist between sellar spine and pediatric pituitary endocrinopathies, possibly caused by deformation of the developing pituitary gland by the sellar spine. To our knowledge, this is the first case report demonstrating an association between sellar spine and clinical diabetes insipidus.

A phylogenomic approach reveals a low somatic mutation rate in a long-lived plant.

Somatic mutations can have important effects on the life history, ecology, and evolution of plants, but the rate at which they accumulate is poorly understood and difficult to measure directly. Here, we develop a method to measure somatic mutations in individual plants and use it to estimate the somatic mutation rate in a large, long-lived, phenotypically mosaic Eucalyptus melliodora tree. Despite being 100 times larger than Arabidopsis, this tree has a per-generation mutation rate only ten times greater, which suggests that this species may have evolved mechanisms to reduce the mutation rate per unit of growth. This adds to a growing body of evidence that illuminates the correlated evolutionary shifts in mutation rate and life history in plants.

The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production.

Reactive oxygen species are important signaling molecules crucial for muscle differentiation and adaptation to exercise. However, their uncontrolled generation is associated with an array of pathological conditions. To identify and quantify the sources of superoxide and hydrogen peroxide in skeletal muscle we used site-specific suppressors (S1QELs, S3QELs and NADPH oxidase inhibitors). We measured the rates of hydrogen peroxide release from isolated rat muscle mitochondria incubated in media mimicking the cytosol of intact muscle. By measuring the extent of inhibition caused by the addition of different site-specific suppressors of mitochondrial superoxide/hydrogen peroxide production (S1QELs for site IQ and S3QELs for site IIIQo), we determined the contributions of these sites to the total signal. In media mimicking resting muscle, their contributions were each 12-18%, consistent with a previous method. In C2C12 myoblasts, site IQ contributed 12% of cellular hydrogen peroxide production and site IIIQo contributed about 30%. When C2C12 myoblasts were differentiated to myotubes, hydrogen peroxide release increased five-fold, and the proportional contribution of site IQ doubled. The use of S1QELs and S3QELs is a powerful new way to measure the relative contributions of different mitochondrial sites to muscle hydrogen peroxide production under different conditions. Our results show that mitochondrial sites IQ and IIIQo make a substantial contribution to superoxide/hydrogen peroxide production in muscle mitochondria and C2C12 myoblasts. The total hydrogen peroxide release rate and the relative contribution of site IQ both increase substantially upon differentiation to myotubes.

To be or not to be pink(1): contradictory findings in an animal model for Parkinson's disease.

The PTEN-induced putative kinase 1 knockout rat (Pink1-/-) is marketed as an established model for Parkinson's disease, characterized by development of motor deficits and progressive degeneration of half the dopaminergic neurons in the substantia nigra pars compacta by 8 months of age. In this study, we address our concerns about the reproducibility of the Pink1-/- rat model. We evaluated behavioural function, number of substantia nigra dopaminergic neurons and extracellular striatal dopamine concentrations by in vivo microdialysis. Strikingly, we and others failed to observe any loss of dopaminergic neurons in 8-month-old male Pink1-/- rats. To understand this variability, we compared key experimental parameters from the different studies and provide explanations for contradictory findings. Although Pink1-/- rats developed behavioural deficits, these could not be attributed to nigrostriatal degeneration as there was no loss of dopaminergic neurons in the substantia nigra and no changes in neurotransmitter levels in the striatum. To maximize the benefit of Parkinson's disease research and limit the unnecessary use of laboratory animals, it is essential that the research community is aware of the limits of this animal model. Additional research is needed to identify reasons for inconsistency between Pink1-/- rat colonies and why degeneration in the substantia nigra is not consistent.

Neuronal Apolipoprotein E4 Expression Results in Proteome-Wide Alterations and Compromises Bioenergetic Capacity by Disrupting Mitochondrial Function.

Apolipoprotein (apo) E4, the major genetic risk factor for Alzheimer's disease (AD), alters mitochondrial function and metabolism early in AD pathogenesis. When injured or stressed, neurons increase apoE synthesis. Because of its structural difference from apoE3, apoE4 undergoes neuron-specific proteolysis, generating fragments that enter the cytosol, interact with mitochondria, and cause neurotoxicity. However, apoE4's effect on mitochondrial respiration and metabolism is not understood in detail. Here we used biochemical assays and proteomic profiling to more completely characterize the effects of apoE4 on mitochondrial function and cellular metabolism in Neuro-2a neuronal cells stably expressing apoE4 or apoE3. Under basal conditions, apoE4 impaired respiration and increased glycolysis, but when challenged or stressed, apoE4-expressing neurons had 50% less reserve capacity to generate ATP to meet energy requirements than apoE3-expressing neurons. ApoE4 expression also decreased the NAD+/NADH ratio and increased the levels of reactive oxygen species and mitochondrial calcium. Global proteomic profiling revealed widespread changes in mitochondrial processes in apoE4 cells, including reduced levels of numerous respiratory complex subunits and major disruptions to all detected subunits in complex V (ATP synthase). Also altered in apoE4 cells were levels of proteins related to mitochondrial endoplasmic reticulum-associated membranes, mitochondrial fusion/fission, mitochondrial protein translocation, proteases, and mitochondrial ribosomal proteins. ApoE4-induced bioenergetic deficits led to extensive metabolic rewiring, but despite numerous cellular adaptations, apoE4-expressing neurons remained vulnerable to metabolic stress. Our results provide insights into potential molecular targets of therapies to correct apoE4-associated mitochondrial dysfunction and altered cellular metabolism.

Plate-Based Measurement of Superoxide and Hydrogen Peroxide Production by Isolated Mitochondria.

Superoxide and hydrogen peroxide produced by mitochondria play important roles in various physiological and pathological processes. This chapter describes a plate-based method to measure rates of superoxide and/or hydrogen peroxide production at specific sites in isolated mitochondria.

Loss of α-Synuclein Does Not Affect Mitochondrial Bioenergetics in Rodent Neurons.

Increased α-synuclein (αsyn) and mitochondrial dysfunction play central roles in the pathogenesis of Parkinson's disease (PD), and lowering αsyn is under intensive investigation as a therapeutic strategy for PD. Increased αsyn levels disrupt mitochondria and impair respiration, while reduced αsyn protects against mitochondrial toxins, suggesting that interactions between αsyn and mitochondria influences the pathologic and physiologic functions of αsyn. However, we do not know if αsyn affects normal mitochondrial function or if lowering αsyn levels impacts bioenergetic function, especially at the nerve terminal where αsyn is enriched. To determine if αsyn is required for normal mitochondrial function in neurons, we comprehensively evaluated how lowering αsyn affects mitochondrial function. We found that αsyn knockout (KO) does not affect the respiration of cultured hippocampal neurons or cortical and dopaminergic synaptosomes, and that neither loss of αsyn nor all three (α, ß and γ) syn isoforms decreased mitochondria-derived ATP levels at the synapse. Similarly, neither αsyn KO nor knockdown altered the capacity of synaptic mitochondria to meet the energy requirements of synaptic vesicle cycling or influenced the localization of mitochondria to dopamine (DA) synapses in vivo. Finally, αsyn KO did not affect overall energy metabolism in mice assessed with a Comprehensive Lab Animal Monitoring System. These studies suggest either that αsyn has little or no significant physiological effect on mitochondrial bioenergetic function, or that any such functions are fully compensated for when lost. These results implicate that αsyn levels can be reduced in neurons without impairing (or improving) mitochondrial bioenergetics or distribution.

Ancient genes establish stress-induced mutation as a hallmark of cancer.

Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts. We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching "protected" genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer.

Long-term oral kinetin does not protect against α-synuclein-induced neurodegeneration in rodent models of Parkinson's disease.

Mutations in the mitochondrial kinase PTEN-induced putative kinase 1 (PINK1) cause Parkinson's disease (PD), likely by disrupting PINK1's kinase activity. Although the mechanism(s) underlying how this loss of activity causes degeneration remains unclear, increasing PINK1 activity may therapeutically benefit some forms of PD. However, we must first learn whether restoring PINK1 function prevents degeneration in patients harboring PINK1 mutations, or whether boosting PINK1 function can offer protection in more common causes of PD. To test these hypotheses in preclinical rodent models of PD, we used kinetin triphosphate, a small-molecule that activates both wild-type and mutant forms of PINK1, which affects mitochondrial function and protects neural cells in culture. We chronically fed kinetin, the precursor of kinetin triphosphate, to PINK1-null rats in which PINK1 was reintroduced into their midbrain, and also to rodent models overexpressing α-synuclein. The highest tolerated dose of oral kinetin increased brain levels of kinetin for up to 6 months, without adversely affecting the survival of nigrostriatal dopamine neurons. However, there was no degeneration of midbrain dopamine neurons lacking PINK1, which precluded an assessment of neuroprotection and raised questions about the robustness of the PINK1 KO rat model of PD. In two rodent models of α-synuclein-induced toxicity, boosting PINK1 activity with oral kinetin provided no protective effects. Our results suggest that oral kinetin is unlikely to protect against α-synuclein toxicity, and thus fail to provide evidence that kinetin will protect in sporadic models of PD. Kinetin may protect in cases of PINK1 deficiency, but this possibility requires a more robust PINK1 KO model that can be validated by proof-of-principle genetic correction in adult animals.

Suppressors of Superoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury.

Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

The NHLBI-sponsored Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR): a new paradigm for rigorous, accurate, and reproducible evaluation of putative infarct-sparing interventions in mice, rabbits, and pigs.

RATIONALE: Despite 4 decades of intense effort and substantial financial investment, the cardioprotection field has failed to deliver a single drug that effectively reduces myocardial infarct size in patients. A major reason is insufficient rigor and reproducibility in preclinical studies. OBJECTIVE: To develop a multicenter, randomized, controlled, clinical trial-like infrastructure to conduct rigorous and reproducible preclinical evaluation of cardioprotective therapies. METHODS AND RESULTS: With support from the National Heart, Lung, and Blood Institute, we established the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR), based on the principles of randomization, investigator blinding, a priori sample size determination and exclusion criteria, appropriate statistical analyses, and assessment of reproducibility. To validate CAESAR, we tested the ability of ischemic preconditioning to reduce infarct size in 3 species (at 2 sites/species): mice (n=22-25 per group), rabbits (n=11-12 per group), and pigs (n=13 per group). During this validation phase, (1) we established protocols that gave similar results between centers and confirmed that ischemic preconditioning significantly reduced infarct size in all species and (2) we successfully established a multicenter structure to support CAESAR's operations, including 2 surgical centers for each species, a Pathology Core (to assess infarct size), a Biomarker Core (to measure plasma cardiac troponin levels), and a Data Coordinating Center-all with the oversight of an external Protocol Review and Monitoring Committee. CONCLUSIONS: CAESAR is operational, generates reproducible results, can detect cardioprotection, and provides a mechanism for assessing potential infarct-sparing therapies with a level of rigor analogous to multicenter, randomized, controlled clinical trials. This is a revolutionary new approach to cardioprotection. Importantly, we provide state-of-the-art, detailed protocols ("CAESAR protocols") for measuring infarct size in mice, rabbits, and pigs in a manner that is rigorous, accurate, and reproducible.

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria.

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.