David A. Rempe

Neurological diseases as primary gliopathies: a reassessment of neurocentrism

Diseases of the human brain are almost universally attributed to malfunction or loss of nerve cells. However, a considerable amount of work has, during the last decade, expanded our view on the role of astrocytes in CNS (central nervous system), and this analysis suggests that astrocytes contribute to both initiation and propagation of many (if not all) neurological diseases. Astrocytes provide metabolic and trophic support to neurons and oligodendrocytes. Here, we shall endeavour a broad overviewing of the progress in the field and forward the idea that loss of homoeostatic astroglial function leads to an acute loss of neurons in the setting of acute insults such as ischaemia, whereas more subtle dysfunction of astrocytes over periods of months to years contributes to epilepsy and to progressive loss of neurons in neurodegenerative diseases. The majority of therapeutic drugs currently in clinical use target neuronal receptors, channels or transporters. Future therapeutic efforts may benefit by a stronger focus on the supportive homoeostatic functions of astrocytes.

The Good, the Bad, and the Cell Type-Specific Roles of Hypoxia Inducible Factor-1α in Neurons and Astrocytes

Hypoxia inducible factor-1α (HIF-1α) is a key regulator of oxygen homeostasis, because it is responsible for the regulation of genes involved in glycolysis, erythropoiesis, angiogenesis, and apoptosis. In the CNS, HIF-1α is stabilized by insults associated with hypoxia and ischemia. Because its many target genes mediate both adaptive and pathological processes, the role of HIF-1α in neuronal survival is debated. Although neuronal HIF-1α function has been the topic of several studies, the role of HIF-1α function in astrocytes has received much less attention. To characterize the role of HIF-1α in neurons and astrocytes, we induced loss of HIF-1α function specifically in neurons, astrocytes, or both cell types in neuron/astrocyte cocultures exposed to hypoxia. Although loss of HIF-1α function in neurons reduced neuronal viability during hypoxia, selective loss of HIF-1 function in astrocytes markedly protected neurons from hypoxic-induced neuronal death. Although the pathological processes induced by HIF-1α in astrocytes remain to be defined, induction of inducible nitric oxide synthase likely contributes to the pathological process. This study delineates, for the first time, a cell type-specific action for HIF-1α within astrocytes and neurons.

Blockade of Gap Junctions In Vivo Provides Neuroprotection After Perinatal Global Ischemia

Background and Purpose— We investigated the contribution of gap junctions to brain damage and delayed neuronal death produced by oxygen-glucose deprivation (OGD). Methods— Histopathology, molecular biology, and electrophysiological and fluorescence cell death assays in slice cultures after OGD and in developing rats after intrauterine hypoxia-ischemia (HI). Results— OGD persistently increased gap junction coupling and strongly activated the apoptosis marker caspase-3 in slice cultures. The gap junction blocker carbenoxolone applied to hippocampal slice cultures before, during, or 60 minutes after OGD markedly reduced delayed neuronal death. Administration of carbenoxolone to ischemic pups immediately after intrauterine HI prevented caspase-3 activation and dramatically reduced long-term neuronal damage. Conclusions— Gap junction blockade may be a useful therapeutic tool to minimize brain damage produced by perinatal and early postnatal HI.

Targeting astrocytes for stroke therapy

SummaryStroke remains a major health problem and is a leading cause of death and disability. Past research and neurotherapeutic clinical trials have targeted the molecular mechanisms of neuronal cell death during stroke, but this approach has uniformly failed to reduce stroke-induced damage or to improve functional recovery. Beyond the intrinsic molecular mechanisms inducing neuronal death during ischemia, survival and function of astrocytes is absolutely required for neuronal survival and for functional recovery after stroke. Many functions of astrocytes likely improve neuronal viability during stroke. For example, uptake of glutamate and release of neurotrophins enhances neuronal viability during ischemia. Under certain conditions, however, astrocyte function may compromise neuronal viability. For example, astrocytes may produce inflammatory cytokines or toxic mediators, or may release glutamate. The only clinical neurotherapeutic trial for stroke that specifically targeted astrocyte function focused on reducing release of S-100β from astrocytes, which becomes a neurotoxin when present at high levels. Recent work also suggests that astrocytes, beyond their influence on cell survival, also contribute to angiogenesis, neuronal plasticity, and functional recovery in the several days to weeks after stroke. If these delayed functions of astrocytes could be targeted for enhancing stroke recovery, it could contribute importantly to improving stroke recovery. This review focuses on both the positive and the negative influences of astrocytes during stroke, especially as they may be targeted for translation to human trials.

HUMMR, a hypoxia- and HIF-1α–inducible protein, alters mitochondrial distribution and transport

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 α (HIF-1α). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states.

The Janus-faced effects of hypoxia on astrocyte function

Astrocytes are increasingly recognized for their impact on neuronal function and viability in health and disease. Hypoxia has Janus-faced influences on astrocytes and their ability to support neuronal viability. For example, hypoxia induces astrocyte-dependent protection of neurons following hypoxia preconditioning. Yet, hypoxia induces processes in astrocytes that augment neuronal death in other situations, such as the coincidence of hypoxia with inflammatory signaling. A complex array of gene expression is induced by hypoxia within astrocytes and neurons through multiple transcription factors and intracellular molecular pathways. The hypoxia inducible factors (HIFs) are transcription factors that are likely instrumental in orchestrating adaptive and pathological functions of astrocytes. As such, the HIFs are postulated to mediate both adaptive and pathological functions during hypoxia/ ischemia. Identifying the conditions under which hypoxia induces signaling in astrocytes that alters autonomous or neuronal survival will undoubtedly have important implications regarding the development of new strategies for stroke treatment.

Loss of c/EBP-β activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons

Understanding the mechanisms governing the switch between hypoxia-induced adaptive and pathological transcription may reveal novel therapeutic targets for stroke. Using an in vitro hypoxia model that temporally separates these divergent responses, we found apoptotic signaling was preceded by a decline in c/EBP-beta activity and was associated with markers of ER-stress including transient eIF2alpha phosphorylation, and the delayed induction of the bZIP proteins ATF4 and CHOP-10. Pretreatment with the eIF2alpha phosphatase inhibitor salubrinal blocked the activation of caspase-3, indicating that ER-related stress responses are integral to this transition. Delivery of either full-length, or a transcriptionally inactive form of c/EBP-beta protected cultures from hypoxic challenge, in part by inducing levels of the anti-apoptotic protein Bcl-2. These data indicate that the pathologic response in cortical neurons induced by hypoxia involves both the loss of c/EBP-beta-mediated survival signals and activation of pro-death pathways originating from the endoplasmic reticulum.

Prophylactic neuroprotection against stroke: low-dose, prolonged treatment with deferoxamine or deferasirox establishes prolonged neuroprotection independent of HIF-1 function.

Prophylactic neuroprotection against stroke could reduce stroke burden in thousands of patients at high risk of stroke, including those with recent transient ischemic attacks (TIAs). Prolyl hydroxylase inhibitors (PHIs), such as deferoxamine (DFO), reduce stroke volume when administered at high doses in the peristroke period, which is largely mediated by the hypoxia-inducible transcription factor (HIF-1). Yet, in vitro experiments suggest that PHIs may also induce neuroprotection independent of HIF-1. In this study, we examine chronic, prophylactic, low-dose treatment with DFO, or another iron chelator deferasirox (DFR), to determine whether they are neuroprotective with this paradigm and mediate their effects through a HIF-1-dependent mechanism. In fact, prophylactic administration of low-dose DFO or DFR significantly reduces stroke volume. Surprisingly, DFO remained neuroprotective in mice haploinsufficient for HIF-1 (HIF-1 +/ –) and transgenic mice with conditional loss of HIF-1 function in neurons and astrocytes. Similarly, DFR was neuroprotective in HIF-1 +/ mice. Neither DFO nor DFR induced expression of HIF-1 targets. Thus, low-dose chronic administration of DFO or DFR induced a prolonged neuroprotective state independent of HIF-1 function. As DFR is an orally administered and well-tolerated medication in clinical use, it has promise for prophylaxis against stroke in patients at high risk of stroke.

In Cultured Astrocytes, p53 and MDM2 Do Not Alter Hypoxia-inducible Factor-1α Function Regardless of the Presence of DNA Damage

A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α). Several studies describe a binding of p53 to HIF-1α in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1α. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1α plasmid constructs and/or p53 and HIF-1α reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1α-mediated transactivation of endogenous HIF-1α targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1α targets that were most dependent on HIF-1α function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1α-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1α target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1α target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1α targets, regardless of p53 activation, in primary astrocytes.

Targeting Glia for Treatment of Neurological Disease

Translational research in neurological disease has focused on neuronal disorders. Yet, the function of glia is indispensable for proper neuronal survival and CNS function. Some of the most prominent supportive functions of astrocytes include glutamate uptake, water transport, control of cerebral blood flow, buffering of ions, and neurotrophic support (see Kimelberg and Nedergaard, page 338). In recent years, astrocytes have also been shown to play an integral role in complex cognitive functions, including sensory processing and sleep (see Kimelberg and Nedergaard, page 338). Similarly, microglia play critical roles for regulating immune responses in the brain and provide neurotrophic support. Given these pleiotropic functions of astrocytes and microglia, it is not surprising that their dysfunction is an important contributor to multiple neurological diseases. In fact, in some cases, astrocyte dysfunction is the primary basis of the disease (see Messing, LaPash Daniels, and Hagemann, page 507). Although initially neglected, the examination of glia is now flourishing and providing valuable new insight into the function and dysfunction of glia during disease. As such, opportunities are emerging by which astrocyte and microglial function might be targeted to suppress progression of the disease. Following a detailed discussion of normal physiological functions of astrocytes (see Kimelberg and Nedergaard, page 338), this issue of Neurotherapeutics will explore the role of astrocytes and microglia in specific disease states and highlight potential therapeutic opportunities. It is our hope that these articles will help to spark interest, research, and funding directed at targeting glia for future treatment of neurological disease. The neurotoxic factors released by activated microglia are important contributors to neuronal death and dysfunction in neurological disease. In this issue, the contributions of microglia to neurodegenerative disease (see Lull and Block, page 354), traumatic brain injury (see Loane and Byrnes, page 366), stroke (see Yenari, Kauppinen, and Swanson, page 378), and HIV-associated neurocognitive disease (HAND) (see Gelbard et al., page 392) are explored. In the case of neurodegenerative diseases, the pathological processes that contribute to neuronal death and dysfunction extend over multiple years. Mirroring this time course, chronic activation of microglia is present in neurodegenerative diseases (see Lull and Block, page 354). Interestingly, this chronic activation may be induced by either multiple stimuli or a single stimulus. Thus, the effects of an injurious stimulus may propagate through the actions of microglia, contributing to neuronal death over extended time periods. Of course, microglia are not simply harbingers of neuropathological processes. In contrast, during acute injury, their function as the resident immunological cell in the brain may be an important contribution to neuronal survival. This paradox of the “good and bad” functions of microglia in traumatic brain injury and spinal cord injury presents an important paradox to consider when targeting microglial function (see Loane and Byrnes, page 366). Similarly, the role of microglia in stroke, the third leading cause of death in the U.S., is complex. In the acute phase of stroke, microglia contribute importantly to cell death. The various stimuli that activate microglia, the signal transduction pathways that contribute to microglial activation, and the mechanisms of microglial cytotoxicity in stroke are discussed in detail in the review by Yenari, Kauppinen, and Swanson (page 378). Interestingly, therapeutic agents that reduce microglial activation during stroke, such as minocycline, are being entered into clinical trials for stroke. However, as the authors stress, it will be important to develop specific therapies that diminish the pathological functions of microglia while maintaining their salutary effects. HAND contributes significantly to the morbidity of HIV, and its impact remains significant even in patients treated with retroviral therapy (see Gelbard et al., page 392). Interestingly, in patients with HIV, the mixed lineage kinase type 3 (MLK3) is pathologically activated in microglia, as well as other cell types in the brain. This activation of MLK3 leads to neuronal dysfunction. Translational research is identifying small molecule inhibitors of MLK3 as a potential treatment for HAND. Astrocytes significantly contribute to neuronal health in the neurodegenerative diseases, including Alzheimer’s disease (AD) (see Verkhratsky et al., page 399), Parkinson’s disease (PD) (see Rappold and Tieu, page 413), and amyotrophic lateral sclerosis (ALS) (see Vargas and Johnson, page 471). In AD, astrocytes influence -amyloid abundance in the extracellular space, are involved in the neuroinflammatory response to -amyloid, and likely impact the neurovascular dysfunction observed in AD. In the case of PD, astrocytes play dual roles, which may be helpful or harmful (see Rappold and Tieu, page 413). Astrocytes likely improve dopaminergic neuronal survival by uptake of toxic molecules, release of trophic Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics