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Exp Neurobiol 2016; 25(5): 233-240
Published online October 31, 2016
© The Korean Society for Brain and Neural Sciences
Junghee Lee1,2*, Seung Jae Hyeon3, Hyeonjoo Im3, Hyun Ryu2, Yunha Kim3 and Hoon Ryu1,2,3*
1Veterans Affairs Boston Healthcare System, Boston, MA 02130, 2Department of Neurology, Boston University School of Medicine, Boston, MA 02118, USA, 3Center for Neuromedicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul 04535, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 1-857-364-6034, 5910, FAX: 1-857-364-4540
Junghee Lee e-mail: Junghee@bu.edu, Hoon Ryu firstname.lastname@example.org
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that leads to a progressive muscle wasting and paralysis. The pathological phenotypes are featured by severe motor neuron death and glial activation in the lumbar spinal cord. Proposed ALS pathogenic mechanisms include glutamate cytotoxicity, inflammatory pathway, oxidative stress, and protein aggregation. However, the exact mechanisms of ALS pathogenesis are not fully understood yet. Recently, a growing body of evidence provides a novel insight on the importance of glial cells in relation to the motor neuronal damage via the non-cell autonomous pathway. Accordingly, the aim of the current paper is to overview the role of astrocytes and microglia in the pathogenesis of ALS and to better understand the disease mechanism of ALS.
Keywords: amyotrophic lateral sclerosis, astrocyte, microglia, motor neuron, non-cell autonomous toxicity
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with a prevalence of 2~3 per 100,000 people and is generally fatal within a few years of disease onset. Affected motor neurons in the brain stem, spinal cord, and motor cortex undergo significant loss, and it eventually causes progressive muscle wasting and paralysis in ALS patients. ALS was initially reported by Dr. Jean-Martin Charcot, a French neurologist, in 1869 . Since Charcot's initial reporting, ALS received international attention when Lou Gehrig, a baseball player of the New York Yankees (Bronx, NY, USA), retired from baseball after being diagnosed with ALS in 1939. For this reason ALS has also been referred as 'Lou Gehrig's disease'. Interestingly, Gulf War veterans have a significantly increased risk (above two fold) of developing ALS . Evidence has shown that the incidence of ALS has risen in recent years and it is reasonable to expect that it will continue to rise in the future. Most cases of ALS occur sporadically, but about 5~10% of ALS cases are familial ALS (FALS). In FALS, more than 90 mutations are found in
In the past when scientists had focused on the study of neuronal function and activity, the events related to neuronal damage and cell death were only investigated from a narrow viewpoint. This view was based on the notion that neurons are damaged due to the dysfunction and deregulation by themselves (so called cell autonomous pathway), and this damage was not related to the dysfunction of any other cell types. As time went by, the view and knowledge of scientists on the mechanisms of neuronal damage have more evolved and advanced. Importantly, a growing body of evidence have proven that non-neuronal cells such as astrocytes, microglia, and oligodendrocytes directly contribute to the motor neuronal damage and cell death (so called non-cell autonomous pathway) in ALS including other neurodegenerative diseases. Indeed, the disease onset and progression is modulated via non-cell autonomous pathway in transgenic ALS [mutant SOD1 (G93A)] mice . The mutant SOD1 expression within motor neurons initiates a damage process and drives the disease onset. In parallel, activation of astrocytes and microglia by mutant SOD1 markedly exacerbates the disease progression while motor neuronal mutant SOD1 has little influence on the progression of ALS. Thus, the paradigm of the non-cell autonomous toxicity has been determined and proven in several experimental conditions of ALS [22,23].
A major pathological feature of ALS is the generation and migration of new cells, specifically astrocytes, within and around damaged regions of the spinal cord . Astrocytes respond to cellular stresses by proliferating and adopting a reactive phenotype characterized by the development of long and thick processes with an increased content of glial fibrillary acidic protein (GFAP). Interestingly, a similar increase in GFAP immunoreactivity was found when cultured primary spinal cord astrocytes were exposed to oxidative stress, suggesting that such morphological changes may be triggered by stress signals . It seems likely that epigenetic alterations induced by mutant SOD1 (mtSOD1) and other pathological stresses are involved in the transformation of astrocytes to a neurotoxic reactive phenotype. In this scenario, non-cell autonomous cell death of motor neurons in ALS could result from either a loss of normal astrocytic support and/or the secretion of neurotoxic cytokines. Several studies have proven this idea as following: co-culture of astrocytes expressing mtSOD1 (G93A) or exposure to conditioned medium derived from astrocytes expressing mtSOD1 (G93A) damages both primary motor neurons and embryonic stem cell-derived motor neurons [25,26]. Previous studies have suggested that cytokines and other toxic factors released from SOD1(G93A) astrocytes may trigger motor neuronal damage [27,28,29,30]. For example,
Excitatory amino acid transporter-2 (EAAT2) is known as a typical glial glutamate transporter that uptakes neurotransmitters glutamate and aspartate from the synaptic cleft . It is believed that EAAT2 uptakes more than 90% of glutamate into glia. In normal condition, astrocytes uptake glutamate and turn it into glutamine, and nourish motor neurons by supplying them as energy source. However, when astrocytes become reactive, the expression of
In comparison to the astrocytic phenotype in ALS, different astrocytic behaviors in relation to the excitotoxicity may be derived due to either the different damage region of CNS (brain versus spinal cord) or the different stress stimuli (bolus excitotoxicity versus chronic oxidative stress). For instance, GFAP-positive astrocytes appear extensively around the damage sites 7 days after injection of N-ethyl-D-aspartic acid (NMDA) while EAAT2- and GFAP-positive astrocytes disappear in a kainic acid (KA)-injected cortical region of the brain . This study shows that two excitotoxic injury models exhibit quite different pattern of astrocyte behaviors such as astrogliogenesis versus astrocyte loss that are distinguished from the pathology of ALS. Accordingly, it will be challenging to pursue how the difference of region or stress stimuli concerts and affects astrocyte behaviors in future studies.
Our group has previously addressed this question using primary astrocytes from the spinal cord of wild type (WT) and ALS transgenic [mutant SOD1 (G93A)] mice. Our study shows that astrocyte survival is correlated with the elevation of Ets-2 transcription factor and with
Oxidative stress due to the mutation of SOD1 is highly implicated in the pathogenesis of ALS. Not only does superoxide anion (O2-) lead to cellular damage including oxidation of DNA and protein and lipid peroxidation but nitric oxide (NO) is also thought to play a key pathogenic role in ALS . Motor neurons are particularly vulnerable to oxidative stress in ALS which is a phenomena attributed to a low level of antioxidant enzymes and a high content of easily oxidized substrates [5,24,40]. NO is synthesized by NO synthases (NOSs) from arginine, which is a rate-limiting factor for NO production. We have reported that neuronal NOS (nNOS)-positive motor neurons are depleted while inducible NOS (iNOS)-positive reactive astrocytes are increased in ALS transgenic [mutant SOD1 (G93A)] mice . The expression of
Despite its controversy, microglia are also known to be linked to motor neuronal damage and the pathogenesis of ALS via the non-cell autonomous pathway [22,47]. Interestingly, deletion of NF-κB signaling in microglia rescues motor neurons from microglial-mediated death
On the other hand, in order to examine whether proliferating microglia leads to motor neuron degeneration in ALS mice, Gowing et al. (2008) generated double transgenic mice with CD11b-TK(mut-30) and mutant SOD1(G93A) in which a 50% reactive microglia is specifically reduced in the lumbar spinal cord . Unexpectedly, reduction of reactive microglia had no effect on the degeneration of motor neuron. This study implies that proliferating microglia-expressing mutant SOD1 (G93A) does not play a pivotal role in triggering neuronal damage in an animal model of ALS. This study raises a question regarding whether different stages of microglia are involved in different modes of action for protecting versus being involved in the damaging of motor neurons through yet unidentified mechanisms. We suggest that future studies are necessary to uncover the precise action mechanism behind the obscure role of microglia in ALS.
Is microglia activation beneficial or disadvantageous to motor neurons? Microglia function is necessary for surveilancing the condition of motor neurons and for restoring tissue injury in response to acute and reversible stress: microglia are beneficial before the threshold limit reached. However, constitutive activation of microglia by a chronic and irreversible stress such as ALS stress may transform them as a non-cell autonomous player to be toxic to motor neurons: microglia are disadvantageous after they become fully activated.
We have previously found that the expression of c-Ret is altered in motor neurons of the lumbar spinal cord in ALS transgenic [mutant SOD1 (G93A)] mice and ALS [mutant SOD1 (G85R) and (G93A)] motor neuronal cell lines . c-Ret oncoprotein is a protein kinase receptor and responds to glial cell line-derived neurotrophic factor (GDNF). c-Ret-mediated signal transduction is important to maintain cellular activity and survival function. Notably, the levels of non-phosphorylated and phosphorylated c-Ret were markedly elevated in active microglia of the lumbar spinal cord of ALS mice in an age-dependent manner. Our findings suggest that ALS stress-induced expression of c-Ret in microglia may trigger non-cell autonomous toxic signals and exacerbate damage responses in motor neurons by disturbing the GDNF signaling pathway in motor neurons . Our previous study does not provide a direct evidence that microglia contribute to non-cell autonomous motor neuronal damage in ALS. However, based on our findings, we suggest an indirect contribution of microglia to motor neuronal damage. For instance, the increased level of c-Ret in microglia elevates interaction with GDNF. As a result, the c-Ret and GDNF interaction promotes the survival of microglia whereas the subsequent deprivation of NFs by activated microglia in the niche of spinal cord may lead to motor neuronal damage (Fig. 2).
In the pathogenesis of ALS, non-motor neuronal cells such as astrocytes and microglia undergo a series of molecular and cellular changes in that these cells become unprofitable to motor neurons, leading to irrecoverable neurodegeneration. The mechanism of non-cell autonomous motor neuron death is closely associated with the pathophysiological change in ALS that is apparently distinguished from cell autonomous pathway.
Neuroinflammation is now identified as a key contributor to motor neuron damage in ALS [52,53,54]. Reactive astrocytes and microglia are triggers of neuroinflammation that accelerate disease progression [55,56] which is further exacerbated by ongoing neuronal injury . Inflammatory cytokines released by astrocytes and microglia may facilitate glutamate excitotoxicity thereby linking neuroinflammation and excitotoxic death [18,57,58].
Taken together, previous findings suggest that the molecular and cellular adaptation between astrocytes, microglia, and motor neurons may be differently modulated by epigenetic components upon ALS stresses. In this paradigm, due to chronic oxidative stress or other irreversible mechanisms, a critical threshold limit is reached and that reactive astrocytes and microglia trigger the pathological processes that subsequently lead to a non-cell autonomous death of motor neurons in ALS. This idea suggests that future therapeutic strategy for the treatment of ALS should be aimed at specific interception of pro-oxidant and pro-death signals in a cell-type specific manner [59,60,61,62].