Elsevier

Neurobiology of Aging

Volume 36, Issue 2, February 2015, Pages 1130-1139
Neurobiology of Aging

Regular article
Astrocytes show reduced support of motor neurons with aging that is accelerated in a rodent model of ALS

https://doi.org/10.1016/j.neurobiolaging.2014.09.020Get rights and content

Abstract

Astrocytes play a crucial role in supporting motor neurons in health and disease. However, there have been few attempts to understand how aging may influence this effect. Here, we report that rat astrocytes show an age-dependent senescence phenotype and a significant reduction in their ability to support motor neurons. In a rodent model of familial amyotrophic lateral sclerosis (ALS) overexpressing mutant superoxide dismutase 1 (SOD1), the rate of astrocytes acquiring a senescent phenotype is accelerated and they subsequently provide less support to motor neurons. This can be partially reversed by glial cell line–derived neurotrophic factor (GDNF). Replacing aging astrocytes with young ones producing GDNF may therefore have a significant survival promoting affect on aging motor neurons and those lost through diseases such as ALS.

Introduction

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS) are superimposed onto the normal process of aging. Age-related changes in the brain and spinal cord may bring out clinical symptoms of these diseases. Even when born with a causal mutation, neurodegenerative disease onset does not typically occur until an older age. To what extent the aging process influences neuronal death has yet to be fully understood.

One important facet of aging is the accumulation of senescent cells that lose the ability to proliferate. Cells are pushed to the senescent state, primarily to prevent expansion of damaged cells that could potentially cause tumor formation. To ameliorate a damaged tissue environment, senescent cells also release cytokines and proteases, collectively known as the senescence-associated secretory phenotype (SASP) (Campisi and d'Adda di Fagagna, 2007). Senescent cells accumulating in tissues over time result in increased levels of SASP and could contribute to the chronic inflammatory environment seen in old age. Interestingly, removal of senescent cells in an accelerated aging mouse model resulted in the rejuvenation of aged tissue, implicating a driving role for senescent cells in causing age-related tissue damage (Baker et al., 2011). Although the identity and role of senescent cells in aging and disease in the central nervous system have not been thoroughly investigated, it has been suggested that the presence of senescent astrocytes plays a role in aging and disease in the brain (Chinta et al., 2013, Salminen et al., 2011). A study looking at aged and Alzheimer's disease human brains showed that the senescent marker p16 was expressed in a subpopulation of astrocytes in degenerating tissue. Furthermore, the data showed that human astrocyte lines expressing the toxic form of amyloid β rapidly reached a senescent state in vitro (Bhat et al., 2012).

ALS is a progressive neurodegenerative disease characterized primarily by the death of upper and lower motor neurons resulting in muscle atrophy leading to limb and bulbar paralysis. Once the clinical symptoms are present, the disease usually advances rapidly, with 3–5 years for the mean survival time. There is no known genetic link in 90%–95% of ALS patients, and the remainder of patients carry an inherited dominant mutation (Gordon, 2013). The well-characterized superoxide dismutase 1 (SOD1) mutation is a point mutation in a single copy of SOD1G93A that accounts for 20% of familial cases (Rosen, 1993). SOD1G93A is likely a gain-of-function mutation as the SOD1 knockout mouse shows no significant disease-related pathology (Jaarsma et al., 2000). In contrast, overexpression of human SOD1G93A results in widely used transgenic rodent models of ALS that have a phenotype closely mimicking the human disease, including massive loss of motor neurons followed by limb paralysis (Gurney et al., 1994).

Although motor neuron death is prevalent in ALS, other cell types such as glia are likely involved in disease progression. A landmark study using chimeric mice comprised of motor neurons expressing SOD1G93A alongside wild-type astrocytes or chimeric mice comprised of wild-type motor neurons alongside astrocytes expressing SOD1G93A showed a profound role of the glial microenvironment on modulating motor neuron survival in ALS (Clement et al., 2003). The finding that wild-type astrocytes could protect SOD1G93A motor neurons, whereas mutant astrocytes caused wild-type motor neurons to degenerate suggested a direct role of astrocytes in ALS (Yamanaka et al., 2008). This was substantiated by experiments that showed astrocytes derived from SOD1G93A mice or human embryonic stem cells genetically modified to express mutant SOD1 induced more motor neuron death compared with wild-type controls in coculture (Di Giorgio et al., 2007, Marchetto et al., 2008, Nagai et al., 2007, Papadeas et al., 2011). More recently, motor neurons cocultured with astrocytes from adult symptomatic SOD1G93A rats showed significantly high and rapid levels of cell death (Díaz-Amarilla et al., 2011). Similarly, motor neurons cocultured with astrocytes derived from neural progenitors or fibroblasts harvested from postmortem sporadic and familial ALS human tissue showed massive cell death (Haidet-Phillips et al., 2011, Re et al., 2014). However, no study has directly compared the effects of aging on the ability of astrocytes to support motor neurons, either in health or disease.

Astrocytes during neural development express high levels of glial cell line–derived neurotrophic factor (GDNF) (Lin et al., 1993, Schaar et al., 1993, Strömberg et al., 1993) that reduces during aging. During adulthood, exogenous delivery of this growth factor has been shown to have a protective effect on neuronal cells, including motor neurons, through activation of the GDNF receptor, GDNF family receptor alpha-1, and the coreceptor RET (Bresjanac and Antauer, 2000, Rémy et al., 2001). Indeed, neural progenitor cells genetically engineered to secrete GDNF can protect motor neurons and improve survival of SOD1G93A rats after transplantation into the spinal cord (Suzuki et al., 2007). Like motor neurons, adult astrocytes retain expression of the GDNF receptor and the coreceptors and, therefore, also retain the ability to be modulated by GDNF.

As astrocytes clearly play a role in modulating neuronal function and survival in health and disease, we set out to investigate how an aging astrocyte environment could influence ALS. In addition, we hypothesized that there may be a direct role of GDNF that can alter astrocyte function, specifically in terms of their ability to support motor neurons. Our study suggests that even during normal aging, astrocytes become less supportive to motor neurons and that this underlies the significant motor neuron death related to astrocytes in a rodent model of familial ALS. Importantly, we found that priming aged wild-type and SOD1G93A astrocytes with GDNF in the media resulted in increased levels of motor neuron survival in the coculture and decreased levels of certain SASP factors. This gives support for a multifaceted therapeutic approach of transplanting young astrocytes genetically modified to produce GDNF to promote the survival of motor neurons that are prone to death in old age and in diseases such as ALS.

Section snippets

Animals

Male hemizygous SOD1G93A rat breeders (Taconic, Hudson, NY, USA) were crossed with female Sprague-Dawley rats (Harlan, Indianapolis, IN, USA). SOD1G93A rats in our colony displayed onset of symptoms at ∼130 days and reached end stage (inability to right itself within 30 seconds) at ∼150 days. Aged rats were wild-type Sprague-Dawleys that were bred in house and maintained in our colony. All laboratory animal procedures were approved by the Institutional Animal Care and Use Committee of

Astrocyte cultures can be generated from adult rats

Previous studies examining the effect of SOD1 mutations on motor neurons have primarily used neonatal rodent astrocyte cultures (Di Giorgio et al., 2007, Nagai et al., 2007) despite the fact that the disease in rats and humans typically begins in adulthood. Furthermore, the single study using adult rat spinal cord astrocytes could generate them only from adult SOD1G93A animals and thus had no adult wild-type control (Díaz-Amarilla et al., 2011). To fill this void, we first attempted to generate

Discussion

In this study, we conclude that age has a strong influence on dictating astrocyte support to motor neurons, both in wild-type animals and those carrying the SOD1G93A mutation. This confirms the previous studies showing a significant decrease in motor neuron survival after only a few days of exposure to rat or human-derived adult mutant SOD1G93A astrocytes (Díaz-Amarilla et al., 2011, Haidet-Phillips et al., 2011, Meyer et al., 2014) and extends this finding to aged wild-type astrocytes. This

Disclosure statement

All authors declare no biomedical financial interests or conflicts of interest in this study.

Acknowledgements

We thank Genevieve Gowing, Jessica Latter, and Maximus Chen for helpful discussion and technical assistance. We thank Soshana Svendsen for critical editing and review. Author contributions: CNS and MMD conceived and planned the experiments, MMD executed the experiments, CNS and MMD analyzed the data, and CNS and MMD wrote the manuscript.

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