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The astrocyte-neuron lactate shuttle (ANLS) is an essential metabolic support system that uptakes glucose, stores it as glycogen in astrocytes, and provides glycogen-derived lactate for axonal function. Aging intrinsically increases the vulnerability of white matter (WM) to injury. Therefore, we investigated the regulation of this shuttle to understand vascular-glial metabolic coupling to support axonal function during aging in two different WM tracts. Aging astrocytes displayed larger cell bodies and thicker horizontal processes in contrast to thinner vertically oriented processes of young astrocytes. Aging axons recovered less following aglycemia in mouse optic nerves (MONs) compared to young axons, although providing lactate during aglycemia equally supported young and aging axonal function. Incubating MONs in high glucose to upregulate glycogen stores in astrocytes delayed loss of function during aglycemia and improved recovery in both young and aging axons. Providing lactate during recovery from aglycemia unmasked a metabolic switch from glucose to lactate in aging axons. Young and aging corpus callosum consisting of a mixture of myelinated and unmyelinated axons sustained their function fully when lactate was available during aglycemia and surprisingly showed a greater resilience to aglycemia compared to fully myelinated axons of optic nerve. We conclude that lactate is a universal substrate for axons independent of their myelination content and age.

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.

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.

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/GSK3β 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/GSK3β 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.

BACE1 is an indispensable enzyme for generating β-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.

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.

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.

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.

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 Ca2+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 STATEMENTAging 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.

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.

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.

Axonal injury and dysfunction are responsible for much of the disability observed following a stroke. Human brain comprises equal proportions of gray matter and white matter and white matter is injured in most strokes. Casein kinase 2 (CK2) is a protein kinase expressed in brain, including white matter, and is regulated by ischemia. We therefore hypothesized that transient CK2 inhibition would protect white matter from ischemic injury. To assess the impact of CK2 inhibition on axonal electrical activity following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), a pure white matter track, from C57BL/6J were subjected to OGD (1h) while eliciting compound action potentials (CAPs) and exposed to CX-4945, a selective CK2 inhibitor, or control artificial cerebrospinal fluid (ACSF). We observed that CX-4945 preserved CAPs when applied either before or after OGD. Then to determine the impact of CK2 inhibition on glial cell survival following OGD, MONs exposed to OGD that were treated with either CX-4945 or control ACSF were processed for immunohistochemistry. We observed that CX-4945 treatment protected oligodendrocytes from OGD. And finally, to determine if CK2 inhibition protected mitochondrial from OGD, MONs from Thy-1 mito-CFP mice were similarly subjected to OGD in the presence of either CX-4945 or control ACSF. We observed that CX-4945 maintained Thy-1 mito-CFP fluorescence following OGD. In conclusion, our results suggest that CK2 inhibition preserves axonal function by preserving oligodendrocytes and mitochondrial function following ischemic injury. We propose that CK2 inhibitors, which are currently in phase II-III clinical trials for cancer therapy could be repurposed and provide a novel therapeutic target to protect white matter against ischemic injury, reducing mortality and morbidity and improving recovery following stroke.

The authors show that central nervous system myelin lacking proteolipid protein (PLP) induces mitochondrial dysfunction, including altered motility, degeneration, and ectopic smooth endoplasmic reticulum interactions, leading to axonal structural defects and degeneration. Mutated PLP occurs in hereditary spastic paraplegia, and these cellular effects provide potential insight into the pathology of the disease., Hereditary spastic paraplegia (HSP) is a neurological syndrome characterized by degeneration of central nervous system (CNS) axons. Mutated HSP proteins include myelin proteolipid protein (PLP) and axon-enriched proteins involved in mitochondrial function, smooth endoplasmic reticulum (SER) structure, and microtubule (MT) stability/function. We characterized axonal mitochondria, SER, and MTs in rodent optic nerves where PLP is replaced by the peripheral nerve myelin protein, P0 (P0-CNS mice). Mitochondrial pathology and degeneration were prominent in juxtaparanodal axoplasm at 1 mo of age. In wild-type (WT) optic nerve axons, 25% of mitochondria–SER associations occurred on extensions of the mitochondrial outer membrane. Mitochondria–SER associations were reduced by 86% in 1-mo-old P0-CNS juxtaparanodal axoplasm. 1-mo-old P0-CNS optic nerves were more sensitive to oxygen-glucose deprivation and contained less adenosine triphosphate (ATP) than WT nerves. MT pathology and paranodal axonal ovoids were prominent at 6 mo. These data support juxtaparanodal mitochondrial degeneration, reduced mitochondria–SER associations, and reduced ATP production as causes of axonal ovoid formation and axonal degeneration.

White matter (WM) is frequently affected by stroke. Aging WM recovers less following stroke compared to younger WM. Free radicals accumulate with aging resulting in increased oxidative stress. Mitochondria/endoplasmic reticulum (ER) interactions are crucial for protection from oxidative stress. We hypothesized that aging WM is more susceptible to oxidative stress damage mediated by mitochondrial/ER dysfunction, leading to decreased ATP synthesis and impaired Ca2+ homeostasis. Mouse optic nerves (MON) obtained from C57BL/6J and Thy-1 CFP(+) mice at 1 (young) or 12 months (aging) of age were used. Axon function was quantified by recording evoked compound action potentials. Mitochondrial/ER structure and interactions were determined by 3D-electron microscopy, Western blotting, ATP assays, and CFP (+) fluorescent mitochondrial imaging. Oxidative stress was assessed by NOS and glutathione assays and by Western blotting for oxidative stress markers, 3-NT and 4-HNE. Oxygen-glucose deprivation (OGD) for 60 min resulted in decreased axon function recovery of aging WM. Aging WM showed elevated NOS activity and increased levels of by-products of lipid peroxidation (4-HNE) and protein nitration (3-NT), indicating aggravated oxidative stress. Structurally, aging axons were larger, with thicker myelin, and were characterized by longer and thicker mitochondria due to altered levels of mitochondrial shaping proteins. This was further confirmed by 3D-EM and CFP (+) imaging and may underlie the decreased ATP levels detected in aging WM. Moreover, mitochondrial-ER interactions were compromised due to decreased association between the organelles and due to decrease in levels of mitochondrial trafficking protein miro-2, which suggests defective Ca2+ homeostasis in aging axons. Calnexin, an ER stress response chaperone protein, was decreased under baseline conditions in aging axons, but did not show upregulation following OGD when compared to young axons, suggesting a defect in unfolded protein responses. We conclude that aging WM is increasingly vulnerable to stroke because of inherent structural changes in axons and mitochondria/ER interactions, leading to depletion of ATP and defective Ca2+ dynamics, resulting in increased oxidative stress.

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Amyloid precursor protein, which generates amyloid beta peptides, is intimately associated with Alzheimer’s disease (AD) pathogenesis. We previously showed that transgenic mice overexpressing amyloid precursor protein intracellular domain (AICD), a peptide generated simultaneously with amyloid beta, develop AD-like pathologies, including hyperphosphorylated tau, loss of synapses, and memory impairments. AICD is known to bind c-Jun N-terminal kinase (JNK)einteracting protein 1 (JIP1), a scaffold protein that associates with and activates JNK. The aim of this study was to examine the role of JIP1 in AICD-induced AD-like pathologies in vivo, since the JNK pathway is aberrantly activated in AD brains and contributes to AD pathologies. We generated AICD-Tg mice lacking the JIP1 gene (AICD; JIP1 � /� ) and found that although AICD; JIP1 � /� mice exhibit increased AICD, the absence of JIP1 results in decreased levels of hyperphosphorylated tau and activated JNK. AICD; JIP1 � /� mice are also protected from synaptic loss and show improved performance in behavioral tests. These results suggest that JIP1 mediates ADlike pathologies in AICD-Tg mice and that JNK signaling may contribute to amyloid-independent mechanisms of AD pathogenesis.

The administration of pan histone deacetylase (HDAC) inhibitors reduces ischemic damage to the CNS, both in vitro and in animal models of stroke, via mechanisms which we are beginning to understand. The acetylation of p53 is regulated by Class I HDACs and, because p53 appears to play a role in ischemic pathology, the purpose of this study was to discover, using an in vitro white matter ischemia model and an in vivo cerebral ischemia model, if neuroprotection mediated by HDAC inhibition depended on p53 expression. Optic nerves were excised from wild-type and p53-deficient mice, and then subjected to oxygen-glucose deprivation in the presence and absence of a specific inhibitor of Class I HDACs (MS-275, entinostat) while compound action potentials were recorded. Furthermore, transient focal ischemia was imposed on wild-type and p53-deficient mice, which were subsequently treated with MS-275. Interestingly, and in both scenarios, the beneficial effects of MS-275 were most pronounced when p53 was absent. These results suggest that modulation of p53 activity is not responsible for MS-275-mediated neuroprotection, and further illustrate how HDAC inhibitors variably influence p53 and associated apoptotic pathways. Optic nerves from wild-type and p53-deficient mice, engineered to express cyan fluorescent protein (CFP) in neuronal mitochondria, were subjected to oxygen-glucose deprivation (OGD) in the presence and absence of a specific inhibitor of Class I histone deacetylases. The protective effect of MS-275 was evidenced by mitochondrial preservation, and this was most pronounced in the absence of p53.