NOS3 Inhibition Confers Post-Ischemic Protection to Young and Aging White Matter Integrity by Conserving Mitochondrial Dynamics and Miro-2 Levels.

White matter (WM) damage following a stroke underlies a majority of the neurological disability that is subsequently observed. Although ischemic injury mechanisms are age-dependent, conserving axonal mitochondria provides consistent post-ischemic protection to young and aging WM. Nitric oxide synthase (NOS) activation is a major cause of oxidative and mitochondrial injury in gray matter during ischemia; therefore, we used a pure WM tract, isolated male mouse optic nerve, to investigate whether NOS inhibition provides post-ischemic functional recovery by preserving mitochondria. We show that pan-NOS inhibition applied before oxygen-glucose deprivation (OGD) promotes functional recovery of young and aging axons and preserves WM cellular architecture. This protection correlates with reduced nitric oxide (NO) generation, restored glutathione production, preserved axonal mitochondria and oligodendrocytes, and preserved ATP levels. Pan-NOS inhibition provided post-ischemic protection to only young axons, whereas selective inhibition of NOS3 conferred post-ischemic protection to both young and aging axons. Concurrently, genetic deletion of NOS3 conferred long-lasting protection to young axons against ischemia. OGD upregulated NOS3 levels in astrocytes, and we show for the first time that inhibition of NOS3 generation in glial cells prevents axonal mitochondrial fission and restores mitochondrial motility to confer protection to axons by preserving Miro-2 levels. Interestingly, NOS1 inhibition exerted post-ischemic protection selectively to aging axons, which feature age-dependent mechanisms of oxidative injury in WM. Our study provides the first evidence that inhibition of glial NOS activity confers long-lasting benefits to WM function and structure and suggests caution in defining the role of NO in cerebral ischemia at vascular and cellular levels.SIGNIFICANCE STATEMENT White matter (WM) injury during stroke is manifested as the subsequent neurological disability in surviving patients. Aging primarily impacts CNS WM and mechanisms of ischemic WM injury change with age. Nitric oxide is involved in various mitochondrial functions and we propose that inhibition of glia-specific nitric oxide synthase (NOS) isoforms promotes axon function recovery by preserving mitochondrial structure, function, integrity, and motility. Using electrophysiology and three-dimensional electron microscopy, we show that NOS3 inhibition provides a common target to improve young and aging axon function, whereas NOS1 inhibition selectively protects aging axons when applied after injury. This study provides the first evidence that inhibition of glial cell NOS activity confers long-lasting benefits to WM structure and function.

CK2 inhibition confers functional protection to young and aging axons against ischemia by differentially regulating the CDK5 and AKT signaling pathways.

White matter (WM) is injured in most strokes, which contributes to functional deficits during recovery. Casein kinase 2 (CK2) is a protein kinase that is expressed in brain, including WM. To assess the impact of CK2 inhibition on axon recovery following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), which are pure WM tracts, were subjected to OGD with or without the selective CK2 inhibitor CX-4945. CX-4945 application preserved axon function during OGD and promoted axon function recovery when applied before or after OGD. This protective effect of CK2 inhibition correlated with preservation of oligodendrocytes and conservation of axon structure and axonal mitochondria. To investigate the pertinent downstream signaling pathways, siRNA targeting the CK2α subunit identified CDK5 and AKT as downstream molecules. Consequently, MK-2206 and roscovitine, which are selective AKT and CDK5 inhibitors, respectively, protected young and aging WM function only when applied before OGD. However, a novel pan-AKT allosteric inhibitor, ARQ-092, which targets both the inactive and active conformations of AKT, conferred protection to young and aging axons when applied before or after OGD. These results suggest that AKT and CDK5 signaling contribute to the WM functional protection conferred by CK2 inhibition during ischemia, while inhibition of activated AKT signaling plays the primary role in post-ischemic protection conferred by CK2 inhibition in WM independent of age. CK2 inhibitors are currently being used in clinical trials for cancer patients; therefore, our results will provide rationale for repurposing these drugs as therapeutic options for stroke patients by adding novel targets.

Age-Related Changes in Axonal and Mitochondrial Ultrastructure and Function in White Matter.

UNLABELLED: The impact of aging on CNS white matter (WM) is of general interest because the global effects of aging on myelinated nerve fibers are more complex and profound than those in cortical gray matter. It is important to distinguish between axonal changes created by normal aging and those caused by neurodegenerative diseases, including multiple sclerosis, stroke, glaucoma, Alzheimer's disease, and traumatic brain injury. Using three-dimensional electron microscopy, we show that in mouse optic nerve, which is a pure and fully myelinated WM tract, aging axons are larger, have thicker myelin, and are characterized by longer and thicker mitochondria, which are associated with altered levels of mitochondrial shaping proteins. These structural alterations in aging mitochondria correlate with lower ATP levels and increased generation of nitric oxide, protein nitration, and lipid peroxidation. Moreover, mitochondria-smooth endoplasmic reticulum interactions are compromised due to decreased associations and decreased levels of calnexin and calreticulin, suggesting a disruption in Ca(2+) homeostasis and defective unfolded protein responses in aging axons. Despite these age-related modifications, axon function is sustained in aging WM, which suggests that age-dependent changes do not lead to irreversible functional decline under normal conditions, as is observed in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Aging is a common risk factor for a number of neurodegenerative diseases, including stroke. Mitochondrial dysfunction and oxidative damage with age are hypothesized to increase risk for stroke. We compared axon-myelin-node-mitochondrion-smooth endoplasmic reticulum (SER) interactions in white matter obtained at 1 and 12 months. We show that aging axons have enlarged volume, thicker myelin, and elongated and thicker mitochondria. Furthermore, there are reduced SER connections to mitochondria that correlate with lower calnexin and calreticulin levels. Despite a prominent decrease in number, elongated aging mitochondria produce excessive stress markers with reduced ATP production. Because axons maintain function under these conditions, our study suggests that it is important to understand the process of normal brain aging to identify neurodegenerative changes.

BACE1 regulates the proliferation and cellular functions of Schwann cells.

BACE1 is an indispensable enzyme for generating ß-amyloid peptides, which are excessively accumulated in brains of Alzheimer's patients. However, BACE1 is also required for proper myelination of peripheral nerves, as BACE1-null mice display hypomyelination. To determine the precise effects of BACE1 on myelination, here we have uncovered a role of BACE1 in the control of Schwann cell proliferation during development. We demonstrate that BACE1 regulates the cleavage of Jagged-1 and Delta-1, two membrane-bound ligands of Notch. BACE1 deficiency induces elevated Jag-Notch signaling activity, which in turn facilitates proliferation of Schwann cells. This increase in proliferation leads to shortened internodes and decreased Schmidt-Lanterman incisures. Functionally, evoked compound action potentials in BACE1-null nerves were significantly smaller and slower, with a clear decrease in excitability. BACE1-null nerves failed to effectively use lactate as an alternative energy source under conditions of increased physiological activity. Correlatively, BACE1-null mice showed reduced performance on rotarod tests. Collectively, our data suggest that BACE1 deficiency enhances proliferation of Schwann cell due to the elevated Jag1/Delta1-Notch signaling, but fails to myelinate axons efficiently due to impaired the neuregulin1-ErbB signaling, which has been documented.

Glutamate and ATP at the Interface Between Signaling and Metabolism in Astroglia: Examples from Pathology.

Glutamate is the main excitatory transmitter in the brain, while ATP represents the most important energy currency in any living cell. Yet, these chemicals play an important role in both processes, enabling them with dual-acting functions in metabolic and intercellular signaling pathways. Glutamate can fuel ATP production, while ATP can act as a transmitter in intercellular signaling. We discuss the interface between glutamate and ATP in signaling and metabolism of astrocytes. Not only do glutamate and ATP cross each other's paths in physiology of the brain, but they also do so in its pathology. We present the fabric of this process in (patho)physiology through the discussion of synthesis and metabolism of ATP and glutamate in astrocytes as well as by providing a general description of astroglial receptors for these molecules along with the downstream signaling pathways that may be activated. It is astroglial receptors for these dual-acting molecules that could hold a key for medical intervention in pathological conditions. We focus on two examples disclosing the role of activation of astroglial ATP and glutamate receptors in pathology of two kinds of brain tissue, gray matter and white matter, respectively. Interventions at the interface of metabolism and signaling show promise for translational medicine.

Neutrophil depletion after subarachnoid hemorrhage improves memory via NMDA receptors.

Cognitive deficits after aneurysmal subarachnoid hemorrhage (SAH) are common and disabling. Patients who experience delayed deterioration associated with vasospasm are likely to have cognitive deficits, particularly problems with executive function, verbal and spatial memory. Here, we report neurophysiological and pathological mechanisms underlying behavioral deficits in a murine model of SAH. On tests of spatial memory, animals with SAH performed worse than sham animals in the first week and one month after SAH suggesting a prolonged injury. Between three and six days after experimental hemorrhage, mice demonstrated loss of late long-term potentiation (L-LTP) due to dysfunction of the NMDA receptor. Suppression of innate immune cell activation prevents delayed vasospasm after murine SAH. We therefore explored the role of neutrophil-mediated innate inflammation on memory deficits after SAH. Depletion of neutrophils three days after SAH mitigates tissue inflammation, reverses cerebral vasoconstriction in the middle cerebral artery, and rescues L-LTP dysfunction at day 6. Spatial memory deficits in both the short and long-term are improved and associated with a shift of NMDA receptor subunit composition toward a memory sparing phenotype. This work supports further investigating suppression of innate immunity after SAH as a target for preventative therapies in SAH.

Phosphorylation sites in the Hook domain of CaVß subunits differentially modulate CaV1.2 channel function.

Regulation of L-type calcium current is critical for the development, function, and regulation of many cell types. Ca(V)1.2 channels that conduct L-type calcium currents are regulated by many protein kinases, but the sites of action of these kinases remain unknown in most cases. We combined mass spectrometry (LC-MS/MS) and whole-cell patch clamp techniques in order to identify sites of phosphorylation of Ca(V)ß subunits in vivo and test the impact of mutations of those sites on Ca(V)1.2 channel function in vitro. Using the Ca(V)1.1 channel purified from rabbit skeletal muscle as a substrate for phosphoproteomic analysis, we found that Ser(193) and Thr(205) in the HOOK domain of Ca(V)ß1a subunits were both phosphorylated in vivo. Ser(193) is located in a potential consensus sequence for casein kinase II, but it was not phosphorylated in vitro by that kinase. In contrast, Thr(205) is located in a consensus sequence for cAMP-dependent phosphorylation, and it was robustly phosphorylated in vitro by PKA. These two sites are conserved in multiple Ca(V)ß subunit isoforms, including the principal Ca(V)ß subunit of cardiac Ca(V)1.2 channels, Ca(V)ß2b. In order to assess potential modulatory effects of phosphorylation at these sites separately from the effects of phosphorylation of the α11.2 subunit, we inserted phosphomimetic or phosphoinhibitory mutations in Ca(V)ß2b and analyzed their effects on Ca(V)1.2 channel function in transfected nonmuscle cells. The phosphomimetic mutation Ca(V)ß2b(S152E) decreased peak channel currents and shifted the voltage dependence of both activation and inactivation to more positive membrane potentials. The phosphoinhibitory mutation Ca(V)ß2b(S152A) had opposite effects. There were no differences in peak Ca(V)1.2 currents or voltage dependence between the phosphomimetic mutation Ca(V)ß2b(T164D) and the phosphoinhibitory mutation Ca(V)ß2b(T164A). However, calcium-dependent inactivation was significantly increased for the phosphomimetic mutation Ca(V)ß2b(T164D). This effect was subunit-specific, as the corresponding mutation in the palmitoylated isoform, Ca(V)ß2a, had no effect. Overall, our data identify two conserved sites of phosphorylation of the Hook domain of Ca(V)ß subunits in vivo and reveal differential modulatory effects of phosphomimetic mutations in these sites. These results reveal a new dimension of regulation of Ca(V)1.2 channels through phosphorylation of the Hook domains of their ß subunits.

Cardiomyocytes from AKAP7 knockout mice respond normally to adrenergic stimulation.

Protein kinase A (PKA) is activated during sympathetic stimulation of the heart and phosphorylates key proteins involved in cardiac Ca(2+) handling, including the L-type Ca(2+) channel (Ca(V)1.2) and phospholamban (PLN). This results in acceleration and amplification of the beat-to-beat changes in cytosolic Ca(2+) in cardiomyocytes and, in turn, an increased rate and force of contraction. PKA is held in proximity to its substrates by protein scaffolds called A kinase anchoring proteins (AKAPs). It has been suggested that the short and long isoforms of AKAP7 (also called AKAP15/18) localize PKA in complexes with Ca(V)1.2 and PLN, respectively. We generated an AKAP7 KO mouse in which all isoforms were deleted and tested whether Ca(2+) current, intracellular Ca(2+) concentration, or Ca(2+) reuptake were impaired in isolated adult ventricular cardiomyocytes following stimulation with the ß-adrenergic agonist isoproterenol. KO cardiomyocytes responded normally to adrenergic stimulation, as measured by whole-cell patch clamp or a fluorescent intracellular Ca(2+) indicator. Phosphorylation of Ca(V)1.2 and PLN were also unaffected by genetic deletion of AKAP7. Immunoblot and RT-PCR revealed that only the long isoforms of AKAP7 were detectable in ventricular cardiomyocytes. The results indicate that AKAP7 is not required for regulation of Ca(2+) handling in mouse cardiomyocytes.

Identification of miRNAs That Mediate Protective Functions of Anti-Cancer Drugs During White Matter Ischemic Injury.

We have previously shown that two anti-cancer drugs, CX-4945 and MS-275, protect and preserve white matter (WM) architecture and improve functional recovery in a model of WM ischemic injury. While both compounds promote recovery, CX-4945 is a selective Casein kinase 2 (CK2) inhibitor and MS-275 is a selective Class I histone deacetylase (HDAC) inhibitor. Alterations in microRNAs (miRNAs) mediate some of the protective actions of these drugs. In this study, we aimed to (1) identify miRNAs expressed in mouse optic nerves (MONs); (2) determine which miRNAs are regulated by oxygen glucose deprivation (OGD); and (3) determine the effects of CX-4945 and MS-275 treatment on miRNA expression. RNA isolated from MONs from control and OGD-treated animals with and without CX-4945 or MS-275 treatment were quantified using NanoString nCounter® miRNA expression profiling. Comparative analysis of experimental groups revealed that 12 miRNAs were expressed at high levels in MONs. OGD upregulated five miRNAs (miR-1959, miR-501-3p, miR-146b, miR-201, and miR-335-3p) and downregulated two miRNAs (miR-1937a and miR-1937b) compared to controls. OGD with CX-4945 upregulated miR-1937a and miR-1937b, and downregulated miR-501-3p, miR-200a, miR-1959, and miR-654-3p compared to OGD alone. OGD with MS-275 upregulated miR-2134, miR-2141, miR-2133, miR-34b-5p, miR-153, miR-487b, miR-376b, and downregulated miR-717, miR-190, miR-27a, miR-1959, miR-200a, miR-501-3p, and miR-200c compared to OGD alone. Interestingly, miR-501-3p and miR-1959 were the only miRNAs upregulated by OGD, and downregulated by OGD plus CX-4945 and MS-275. Therefore, we suggest that protective functions of CX-4945 or MS-275 against WM injury maybe mediated, in part, through miRNA expression.

Distinct cellular and molecular mechanisms underlie functional remodeling of repolarizing K+ currents with left ventricular hypertrophy.

Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk, although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and inwardly rectifying (Kir) K(+) channel remodeling was examined in a mouse model of pressure overload-induced LVH, produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV myocytes and QT(c) intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (C(m)) was higher, and I(to,f), I(K,slow), I(ss), and I(K1) densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of the reduced current densities is the increase in C(m), I(K,slow) amplitudes were decreased and I(ss) amplitudes were increased in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and increased I(ss) amplitudes were more pronounced in TAC endocardial LV cells, whereas I(K,slow) amplitudes were selectively reduced in TAC epicardial LV cells. Consistent with the similarities in I(to,f) and I(K1) amplitudes, Kv4.2, Kv4.3, and KChIP2 (I(to,f)), as well as Kir2.1 and Kir2.2 (I(K1)), transcript and protein expression levels were similar in TAC and sham LV. Unexpectedly, expression of I(K,slow) channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was increased in TAC LV. Functional changes in repolarizing K(+) currents with LVH, therefore, result from distinct cellular (cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms.

Phagosomes are competent organelles for antigen cross-presentation.

The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.

Ex Vivo Studies of Optic Nerve Axon Electrophysiology.

The use of ex vivo compound action potential (CAP) recordings from intact optic nerves is an ideal model to study white matter function without the influence of gray matter. Here, we describe how freshly dissected optic nerves are placed in a humidified recording chamber and how evoked CAPs are recorded and monitored in real time for up to 10 h. Evoked CAP recordings allow for white matter to be studied under acute challenges such as anoxia, hypoxia, aglycemia, and ischemia.

Organelle proteomics: looking at less to see more.

The recent finding that the human genome comprises between 21000 and 39000 genes, a number much lower than expected, has in no way simplified the complexity associated with the understanding of how cells perform their functions. Elucidation of the molecular mechanisms underlying cell functions will require a global knowledge of the expressed proteins, including splice variant products, their post-translational modifications, their subcellular localizations and their assembly into molecular machines as deduced from protein-protein interactions, at any given time during the life of the cell or under any cellular conditions. Current and expected advances in mass spectrometry and bioinformatics might help the realization of these goals in a shorter time than is currently predicted.

Role of Brain Glycogen During Ischemia, Aging and Cell-to-Cell Interactions.

The astrocyte-neuron lactate transfer shuttle (ANLS) is one of the important metabolic systems that provides a physiological infrastructure for glia-neuronal interactions where specialized architectural organization supports the function. Perivascular astrocyte end-feet take up glucose via glucose transporter 1 to actively regulate glycogen stores, such that high ambient glucose upregulates glycogen and low levels of glucose deplete glycogen stores. A rapid breakdown of glycogen into lactate during increased neuronal activity or low glucose conditions becomes essential for maintaining axon function. However, it fails to benefit axon function during an ischemic episode in white matter (WM). Aging causes a remarkable change in astrocyte architecture characterized by thicker, larger processes oriented parallel to axons, as opposed to vertically-transposing processes. Subsequently, aging axons become more vulnerable to depleted glycogen, although aging axons can use lactate as efficiently as young axons. Lactate equally supports function during aglycemia in corpus callosum (CC), which consists of a mixture of myelinated and unmyelinated axons. Moreover, axon function in CC shows greater resilience to a lack of glucose compared to optic nerve, although both WM tracts show identical recovery after aglycemic injury. Interestingly, emerging evidence implies that a lactate transport system is not exclusive to astrocytes, as oligodendrocytes support the axons they myelinate, suggesting another metabolic coupling pathway in WM. Future studies are expected to unravel the details of oligodendrocyte-axon lactate metabolic coupling to establish that all WM components metabolically cooperate and that lactate may be the universal metabolite to sustain central nervous system function.

Preserving Mitochondrial Structure and Motility Promotes Recovery of White Matter After Ischemia.

Stroke significantly affects white matter in the brain by impairing axon function, which results in clinical deficits. Axonal mitochondria are highly dynamic and are transported via microtubules in the anterograde or retrograde direction, depending upon axonal energy demands. Recently, we reported that mitochondrial division inhibitor 1 (Mdivi-1) promotes axon function recovery by preventing mitochondrial fission only when applied during ischemia. Application of Mdivi-1 after injury failed to protect axon function. Interestingly, L-NIO, which is a NOS3 inhibitor, confers post-ischemic protection to axon function by attenuating mitochondrial fission and preserving mitochondrial motility via conserving levels of the microtubular adaptor protein Miro-2. We propose that preventing mitochondrial fission protects axon function during injury, but that restoration of mitochondrial motility is more important to promote axon function recovery after injury. Thus, Miro-2 may be a therapeutic molecular target for recovery following a stroke.

PPARalpha-mediated remodeling of repolarizing voltage-gated K+ (Kv) channels in a mouse model of metabolic cardiomyopathy.

Diabetes is associated with increased risk of diastolic dysfunction, heart failure, QT prolongation and rhythm disturbances independent of age, hypertension or coronary artery disease. Although these observations suggest electrical remodeling in the heart with diabetes, the relationship between the metabolic and the functional derangements is poorly understood. Exploiting a mouse model (MHC-PPARalpha) with cardiac-specific overexpression of the peroxisome proliferator-activated receptor alpha (PPARalpha), a key driver of diabetes-related lipid metabolic dysregulation, the experiments here were aimed at examining directly the link(s) between alterations in cardiac fatty acid metabolism and the functioning of repolarizing, voltage-gated K(+) (Kv) channels. Electrophysiological experiments on left (LV) and right (RV) ventricular myocytes isolated from young (5-6 week) MHC-PPARalpha mice revealed marked K(+) current remodeling: I(to,f) densities are significantly (P<0.01) lower, whereas I(ss) densities are significantly (P<0.001) higher in MHC-PPARalpha, compared with age-matched wild type (WT), LV and RV myocytes. Consistent with the observed reductions in I(to,f) density, expression of the KCND2 (Kv4.2) transcript is significantly (P<0.001) lower in MHC-PPARalpha, compared with WT, ventricles. Western blot analyses revealed that expression of the Kv accessory protein, KChIP2, is also reduced in MHC-PPARalpha ventricles in parallel with the decrease in Kv4.2. Although the properties of the endogenous and the "augmented" I(ss) suggest a role(s) for two pore domain K(+) channel (K2P) pore-forming subunits, the expression levels of KCNK2 (TREK1), KCNK3 (TASK1) and KCNK5 (TASK2) in MHC-PPARalpha and WT ventricles are not significantly different. The molecular mechanisms underlying I(to,f) and I(ss) remodeling in MHC-PPARalpha ventricular myocytes, therefore, are distinct.

CK2 inhibition protects white matter from ischemic injury.

Strokes occur predominantly in the elderly and white matter (WM) is injured in most strokes, contributing to the disability associated with clinical deficits. Casein kinase 2 (CK2) is expressed in neuronal cells and was reported to be neuroprotective during cerebral ischemia. Recently, we reported that CK2 is abundantly expressed by glial cells and myelin. However, in contrast to its role in cerebral (gray matter) ischemia, CK2 activation during ischemia mediated WM injury via the CDK5 and AKT/GSK3ß signaling pathways (Bastian et al., 2018). Subsequently, CK2 inhibition using the small molecule inhibitor CX-4945 correlated with preservation of oligodendrocytes as well as conservation of axon structure and axonal mitochondria, leading to improved functional recovery. Notably, CK2 inhibition promoted WM function when applied before or after ischemic injury by differentially regulating the CDK5 and AKT/GSK3ß pathways. Specifically, blockade of the active conformation of AKT conferred post-ischemic protection to young, aging, and old WM, suggesting a common therapeutic target across age groups. CK2 inhibitors are currently being used in clinical trials for cancer patients; therefore, it is important to consider the potential benefits of CK2 inhibitors during an ischemic attack.

Mitochondrial dynamics and preconditioning in white matter.

Mechanisms of ischemic preconditioning have been extensively studied in gray matter. However, an ischemic episode affects both the gray matter (GM) and white matter (WM) portions of the brain. Inhibition of mitochondrial fission is one of the mechanisms of preconditioning neuronal cell bodies against ischemia. Although axons are anatomical extensions of neuronal cell bodies, injury mechanisms differ between GM and WM. Indeed, axonal dysfunction is responsible for much of the disability associated with clinical deficits observed after stroke; however, the signaling process underlying preconditioning remains unexplored in axons. Using mouse optic nerve, which is a pure isolated WM tract, we show that mitochondria in myelinated axons undergo rapid and profuse fission during oxygen glucose deprivation (OGD) that is mediated by translocation of cytoplasmic Dynamin Related Protein-1 (Drp-1) to mitochondria. OGD-induced mitochondrial fission correlates with reduced mitochondrial motility and loss of axon function. Mitochondrial fragmentation and loss of motility become permanent during the recovery period. Inhibiting mitochondrial fission by administering mitochondrial division inhibitor-1 (Mdivi-1) during OGD preserves mitochondrial shape and motility and promotes axon function recovery. In contrast, preconditioning WM by applying Mdivi-1 only before OGD fails to conserve mitochondrial shape or motility and fails to benefit axon function. Our findings suggest that inhibition of mitochondrial fission during ischemia promotes axon function recovery, but is not sufficient to precondition WM against ischemia. These results raise caution in that approaches to preconditioning neuronal cell bodies may not successfully translate into functional improvement following ischemia.

Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy.

Evidence is emerging that systemic metabolic disturbances contribute to cardiac myocyte dysfunction and clinically apparent heart failure, independent of associated coronary artery disease. To test the hypothesis that perturbation of lipid homeostasis in cardiomyocytes contributes to cardiac dysfunction, we engineered transgenic mice with cardiac-specific overexpression of fatty acid transport protein 1 (FATP1) using the alpha-myosin heavy chain gene promoter. Two independent transgenic lines demonstrate 4-fold increased myocardial free fatty acid (FFA) uptake that is consistent with the known function of FATP1. Increased FFA uptake in this model likely contributes to early cardiomyocyte FFA accumulation (2-fold increased) and subsequent increased cardiac FFA metabolism (2-fold). By 3 months of age, transgenic mice have echocardiographic evidence of impaired left ventricular filling and biatrial enlargement, but preserved systolic function. Doppler tissue imaging and hemodynamic studies confirm that these mice have predominantly diastolic dysfunction. Furthermore, ambulatory ECG monitoring reveals prolonged QT(c) intervals, reflecting reductions in the densities of repolarizing, voltage-gated K+ currents in ventricular myocytes. Our results show that in the absence of systemic metabolic disturbances, such as diabetes or hyperlipidemia, perturbation of cardiomyocyte lipid homeostasis leads to cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy. Moreover, the MHC-FATP model supports a role for FATPs in FFA import into the heart in vivo.

Abnormal hepatobiliary and circulating lipid metabolism in the Long-Evans Cinnamon rat model of Wilson's disease.

Long-Evans Cinnamon (LEC) rats exhibit a genetic defect in Atp7b gene, which is homologous to the human Wilson's disease gene, resulting in an inability to mobilize copper from the liver. This study was undertaken to gain insight into the relationship between liver copper accumulation and plasma lipid profile, circulating lipoprotein composition, hepatic sterol metabolism and biliary lipid secretion rates in 12-week-old LEC rats compared to control Long-Evans rats. Concomitant with hepatic copper deposition, LEC rats displayed increased content of triglycerides (TGs), free cholesterol (FC) and cholesteryl ester (CE) in the liver. Hepatic concentrations of malondialdehyde (MDA), an index of lipid peroxidation were also significantly elevated in LEC rats (50%). This steatosis was associated with aberrant microsomal apolipoprotein (apo) B-100 and microsomal triglyceride transfer protein (MTP) content, hypotriglyceridemia, hypocholesterolemia and abnormalities in both circulating lipoprotein composition and size. Atypical hepatobiliary sterol metabolism was established by the assessment of the activity of key intracellular enzymes for cholesterol homeostasis, which demonstrated, with respect to controls, a 40% reduction in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a 30% reduction in cholesterol 7alpha-hydroxylase, and a 54% reduction in acyl CoA:cholesterol acyltransferase. During a 6-h biliary drainage, a decline in the bile acid output was recorded and might be linked to the low protein expression of the bile salt export pump (BSEP or ABCB11). Our data emphasize the crucial role of copper balance in hepatic sterol homeostasis and lipoprotein metabolism in LEC rats. Additional studies are needed to delineate the mechanisms of these disorders.