The Endocannabinoid System and Oligodendrocytes in Health and Disease.

Cannabinoid-based interventions are being explored for central nervous system (CNS) pathologies such as neurodegeneration, demyelination, epilepsy, stroke, and trauma. As these disease states involve dysregulation of myelin integrity and/or remyelination, it is important to consider effects of the endocannabinoid system on oligodendrocytes and their precursors. In this review, we examine research reports on the effects of the endocannabinoid system (ECS) components on oligodendrocytes and their precursors, with a focus on therapeutic implications. Cannabinoid ligands and modulators of the endocannabinoid system promote cell signaling in oligodendrocyte precursor survival, proliferation, migration and differentiation, and mature oligodendrocyte survival and myelination. Agonist stimulation of oligodendrocyte precursor cells (OPCs) at both CB1 and CB2 receptors counter apoptotic processes via Akt/PI3K, and promote proliferation via Akt/mTOR and ERK pathways. CB1 receptors in radial glia promote proliferation and conversion to progenitors fated to become oligodendroglia, whereas CB2 receptors promote OPC migration in neonatal development. OPCs produce 2-arachidonoylglycerol (2-AG), stimulating cannabinoid receptor-mediated ERK pathways responsible for differentiation to arborized, myelin basic protein (MBP)-producing oligodendrocytes. In cell culture models of excitotoxicity, increased reactive oxygen species, and depolarization-dependent calcium influx, CB1 agonists improved viability of oligodendrocytes. In transient and permanent middle cerebral artery occlusion models of anoxic stroke, WIN55212-2 increased OPC proliferation and maturation to oligodendroglia, thereby reducing cerebral tissue damage. In several models of rodent encephalomyelitis, chronic treatment with cannabinoid agonists ameliorated the damage by promoting OPC survival and oligodendrocyte function. Pharmacotherapeutic strategies based upon ECS and oligodendrocyte production and survival should be considered.

Motoneuron programmed cell death in response to proBDNF.

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.

Exogenous Hsc70, but not thermal preconditioning, confers protection to motoneurons subjected to oxidative stress.

Proper sensing of stress and the initiation of the stress response are critical to maintaining cell viability in response to noxious stimuli. Induction of the stress response prior to the exposure of a lethal stress (preconditioning) can be protective. Heat shock proteins (Hsps), the main products of the stress response, are considered to be responsible for this protective effect. Most cells readily initiate a stress response, but some neuronal phenotypes, including motoneurons (MNs), have a diminished capacity to do so. We have found that, given a proper stimulus, MNs can execute a heat stress response; but, it does not protect them from death caused by hydrogen peroxide (H(2)O(2)) induced oxidative stress, despite inhibiting H(2)O(2)-induced caspase activation. Conversely, we demonstrate that incubation with the heat shock cognate 70 (Hsc70) protein prior to oxidative insult can protect MNs from oxidative stress. This survival promoting effect may be mediated through the substrate binding domain (SBD) of Hsc70. Our data suggest that stress preconditioning may not be beneficial to MNs, but that pharmacological interventions and alternative means of acquiring components of the stress response are an effective means of ameliorating lethal stress in MNs and may be potentially useful therapeutically in preventing pathological MN loss.

Exogenous delivery of heat shock protein 70 increases lifespan in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder that results in the progressive loss of motoneurons (MNs) in the CNS. Several survival and death mechanisms of MNs have been characterized and it has been determined that MNs do not appear to mount a complete stress response, as determined by the lack of heat shock protein 70 (Hsp70) upregulation after several stress paradigms. Hsp70 has been shown to confer neuroprotection and the insufficient availability of Hsp70 may contribute to MNs' susceptibility to death in ALS mice. In this study, recombinant human Hsp70 (rhHsp70) was intraperitoneally injected three times weekly, beginning at postnatal day 50 until endstage, to G93A mutant SOD1 (G93A SOD1) mice. The administration of rhHsp70 was effective at increasing lifespan, delaying symptom onset, preserving motor function and prolonging MN survival. Interestingly, injected rhHsp70 localized to skeletal muscle and was not readily detected in the CNS. Treatment with rhHsp70 also resulted in an increased number of innervated neuromuscular junctions compared with control tissue. Together these results suggest rhHsp70 may delay disease progression in the G93A SOD1 mouse via a yet to be identified peripheral mechanism.

Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases.

The ability to mount a successful stress response in the face of injury is critical to the long-term viability of individual cells and to the organism in general. The stress response, characterized in part by the upregulation of heat shock proteins, is compromised in several neurodegenerative disorders and in some neuronal populations, including motoneurons (MNs). Because astrocytes have a greater capacity than neurons to survive metabolic stress, and because they are intimately associated with the regulation of neuronal function, it is important to understand their stress response, so that we may to better appreciate the impact of stress on neuronal viability during injury or disease. We show that astrocytes subjected to hyperthermia upregulate Hsp/c70 in addition to intracellular signaling components including activated forms of extracellular-signal-regulated kinase (ERK1/2), Akt, and c-jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). Furthermore, astrocytes release increasing amounts of Hsp/c70 into the extracellular environment following stress, an event that is abrogated when signaling through the ERK1/2 and phosphatidylinositol-3 kinase (PI3K) pathways is compromised and enhanced by inhibition of the JNK pathway. Last, we show that the Hsp/c70 is released from astrocytes in exosomes. Together, these data illustrate the diverse regulation of stress-induced Hsp/c70 release in exosomes, and the way in which the balance of activated signal transduction pathways affects this release. These data highlight how stressful insults can alter the microenvironment of an astrocyte, which may ultimately have implications for the survival of neighboring neurons.

Astrocyte and muscle-derived secreted factors differentially regulate motoneuron survival.

During development, motoneurons (MNs) undergo a highly stereotyped, temporally and spatially defined period of programmed cell death (PCD), the result of which is the loss of 40-50% of the original neuronal population. Those MNs that survive are thought to reflect the successful acquisition of limiting amounts of trophic factors from the target. In contrast, maturation of MNs limits the need for target-derived trophic factors, because axotomy of these neurons in adulthood results in minimal neuronal loss. It is unclear whether MNs lose their need for trophic factors altogether or whether, instead, they come to rely on other cell types for nourishment. Astrocytes are known to supply trophic factors to a variety of neuronal populations and thus may nourish MNs in the absence of target-derived factors. We investigated the survival-promoting activities of muscle- and astrocyte-derived secreted factors and found that astrocyte-conditioned media (ACM) was able to save substantially more motoneurons in vitro than muscle-conditioned media (MCM). Our results indicate that both ACM and MCM are significant sources of MN trophic support in vitro and in ovo, but only ACM can rescue MNs after unilateral limb bud removal. Furthermore, we provide evidence suggesting that MCM facilitates the death of a subpopulation of MNs in a p75(NTR) - and caspase-dependent manner; however, maturation in ACM results in MN trophic independence and reduced vulnerability to this negative, pro-apoptotic influence from the target.

Cell cycle events distinguish sensory neuronal death from motoneuron death as a result of trophic factor deprivation.

The clarification of mechanisms of developmental cell death may provide hints in the prevention of pathological neuronal death. The execution phase of cell death has been extensively characterized; however, events that occur prior to this phase are less well understood. Previous studies have suggested that terminally differentiated neurons induced to die in various experimental paradigms may be making an abortive attempt to reenter the cell cycle. We have examined this process in postmitotic motoneurons and dorsal root ganglia sensory neurons in the developing chick embryo in vitro and in vivo. An examination of the programmed cell death of postmitotic motoneurons does not implicate a role for cell cycle-related proteins. We did, however, observe a decrease in the amount of cell death in dorsal root ganglion cells of embryos treated with cell cycle inhibitors. These results indicate that upstream initiators of the neuronal cell death pathway vary between phenotypes.

Bcl-2 rescues motoneurons from early cell death in the cervical spinal cord of the chicken embryo.

Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.