Selva Baltan

White Matter Vulnerability to Ischemic Injury Increases with Age Because of Enhanced Excitotoxicity

Stroke incidence increases with age and this has been attributed to vascular factors. We show here that CNS white matter (WM) is intrinsically more vulnerable to ischemic injury in older animals and that the mechanisms of WM injury change as a function of age. The mouse optic nerve was used to study WM function. WM function in older animals (12 months) was not protected from ischemic injury by removal of extracellular Ca2+ or by blockade of reverse Na+/Ca2+ exchange, as is the case with young adults. Ischemic WM injury in older mice is predominately mediated by glutamate release and activation of AMPA/kainate-type glutamate receptors. Glutamate release, attributable to reverse glutamate transport, occurs earlier and is more robust in older mice that show greater expression of the glutamate transporter. The observation that WM vulnerability to ischemic injury is age dependent has possible implications for the pathogenesis of other age-related CNS conditions.

Metabolic Vulnerability Disposes Retinal Ganglion Cell Axons to Dysfunction in a Model of Glaucomatous Degeneration

We tested the hypothesis that glaucoma disrupts electrophysiological conduction properties and axon function in optic nerve as a function of intraocular pressure (IOP) levels and age in the DBA/2J mouse model of glaucoma. The amplitude and the integral of electrical signals evoked along the axons decreased considerably by 6 months of age as a function of increasing IOP levels. At young ages, raised IOP was directly associated with increased vulnerability to metabolic challenge. Changes in the physiological function of the optic nerves were accentuated with aging, leading to loss of compound action potential in an entire population of fibers: small, slow conducting axons. This loss was accompanied with loss of small fiber axon counts and declining metabolic reserve by demonstrating IOP-dependent ATP decrease in mouse optic nerves. These data shed light on a novel potential mechanism of glaucoma pathology whereby increased IOP and declining metabolic capacity lead to axon liability and eventually dysfunction and loss.

Expression of Histone Deacetylases in Cellular Compartments of the Mouse Brain and the Effects of Ischemia

Drugs that inhibit specific histone deacetylase (HDAC) activities have enormous potential in preventing the consequences of acute injury to the nervous system and in allaying neurodegeneration. However, very little is known about the expression pattern of the HDACs in the central nervous system (CNS). Identifying the cell types that express HDACs in the CNS is important for determining therapeutic targets for HDAC inhibitors and evaluating potential side effects. We characterized the cellular expression of HDACs 1–3, and HDACs 4 and 6, in the adult mouse brain in the cingulate cortex, parietal cortex, dentate gyrus, and CA1 regions of the hippocampus and subcortical white matter. Expression of class I HDACs showed a cell- and region-specific pattern. Transient focal ischemia induced by temporary middle cerebral artery occlusion, or global ischemia induced by in vitro oxygen–glucose deprivation, altered the extent of HDAC expression in a region- and cell-specific manner. The pan-HDAC inhibitor, SAHA, reduced ischemia-induced alterations in HDACs. The results suggest that in addition to promoting epigenetic changes in transcriptional activity in the nucleus of neurons and glia, HDACs may also have non-transcriptional actions in axons and the distant processes of glial cells and may significantly modulate the response to injury in a cell- and region-specific manner.

Novel Protective Effects of Histone Deacetylase Inhibition on Stroke and White Matter Ischemic Injury

Understanding how epigenetics influences the process and progress of a stroke could yield new targets and therapeutics for use in the clinic. Experimental evidence suggests that inhibitors of zinc-dependent histone deacetylases can protect neurons, axons, and associated glia from the devastating effects of oxygen and glucose deprivation. While the specific enzymes involved have yet to be clearly identified, there are hints from somewhat selective chemical inhibitors and also from the use of specific small hairpin RNAs to transiently knockdown protein expression. Neuroprotective mechanisms implicated thus far include the upregulation of extracellular glutamate clearance, inhibition of p53-mediated cell death, and maintenance of mitochondrial integrity. The histone deacetylases have distinct cellular and subcellular localizations, and discrete substrates. As a number of chemical inhibitors are already in clinical use for the treatment of cancer, repurposing for the stroke clinic should be expedited.

Can lactate serve as an energy substrate for axons in good times and in bad, in sickness and in health?

In the mammalian white matter, glycogen-derived lactate from astrocytes plays a critical role in supporting axon function using the astrocyte-neuron lactate transfer shuttle (ANLTS) system with specialized monocarboxylate transporters (MCTs). A rapid breakdown of glycogen to lactate during increased neuronal activity or low glucose conditions becomes essential to maintain axon function. Therefore astrocytes actively regulate their glycogen stores with respect to ambient glucose levels such that high ambient glucose upregulates glycogen and low levels of glucose depletes glycogen stores. Although lactate fully supports axon function in the absence of glucose and becomes a preferred energy metabolite when axons discharge at high frequency, it fails to benefit axon function during an ischemic episode in white matter. Emerging evidence implies a similar lactate transport system between oligodendrocytes and the axons they myelinate, suggesting another metabolic coupling pathway in white matter. Therefore the conditions that activate this lactate shuttle system and the signaling mechanisms that mediate activation of this system are of great interest. Future studies are expected to unravel the details of oligodendrocyte-axon lactate metabolic coupling to establish how white matter components metabolically cooperate and that lactate may be the universal metabolite to sustain CNS function.

Histone deacetylase inhibitors preserve function in aging axons

Aging increases the vulnerability of aging white matter to ischemic injury. Histone deacetylase (HDAC) inhibitors preserve young adult white matter structure and function during ischemia by conserving ATP and reducing excitotoxicity. In isolated optic nerve from 12‐month‐old mice, deprived of oxygen and glucose, we show that pan‐ and Class I‐specific HDAC inhibitors promote functional recovery of axons. This protection correlates with preservation of axonal mitochondria. The cellular expression of HDAC 3 in the central nervous system (CNS), and HDAC 2 in optic nerve considerably changed with age, expanding to more cytoplasmic domains from nuclear compartments, suggesting that changes in glial cell protein acetylation may confer protection to aging axons. Our results indicate that manipulation of HDAC activities in glial cells may have a universal potential for stroke therapy across age groups.

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.

Age-related changes in axonal and mitochondrial ultrastructure and function in white matter

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, Alzheimers 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 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.

MS-275, a Class I histone deacetylase inhibitor, protects the p53-deficient mouse against ischemic injury

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.

Proteolipid protein–deficient myelin promotes axonal mitochondrial dysfunction via altered metabolic coupling

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.