Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis
Introduction
Astrocytes constitute one of the major building blocks of the blood-brain barrier (BBB), which tightly regulates the brain microenvironment to maintain proper neuronal functioning. Brain microvessels are wrapped by the basal lamina and astrocyte endfeet, which are known to serve as physical barriers and mutual functional bridges between neurons and endothelial cells/pericytes; this anatomical/physiological multi-cellular complex is termed the “neurovascular unit” (Abbott et al., 2006, Zhao et al., 2015, Zlokovic, 2005). Astrocyte proliferation (astrogliosis) and BBB breakdown have been implicated in the pathogenesis of many neurological or mental disorders (Abbott et al., 2006, Najjar et al., 2017, Zlokovic, 2005).
Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease affecting the upper and lower motor neurons in the brain and spinal cord (van Es et al., 2017). Many studies have reported that astrogliosis is one of the main pathological hallmarks observed in the spinal cords of patients with ALS, as well as in animal models of the disease (Schiffer et al., 1996, Hall et al., 1998). Recent studies have shown that reactive microglial cells and astrocytes actively contribute to the death of motor neurons in ALS mouse models and in patients with ALS (Turner et al., 2004, Philips and Robberecht, 2011). Astrocytes were shown to contribute to microglial activation, leading to further damage to motor neurons and accelerated disease progression via a non-cell autonomous mechanism (Yamanaka et al., 2008, Ilieva et al., 2009). Moreover, BBB disruption has been observed at an early, presymptomatic stage in mouse models of ALS (Garbuzova-Davis et al., 2007, Zhong et al., 2008) and in patients with ALS (Garbuzova-Davis et al., 2012), suggesting that vascular disease can occur simultaneously in ALS.
Aquaporin-4 (AQP4), the most abundantly expressed AQP isoform in the brain, is specifically expressed in astrocytes. AQP4 shows highly polarized localization (Nielsen et al., 1997) and serves as a marker for astrocyte endfeet, which suggests a possible role in maintaining BBB properties (Nicchia et al., 2004, Tomás-Camardiel et al., 2005, Wolburg et al., 2009). In a rat model of ALS in which mutant human superoxide dismutase 1 (SOD1) is expressed, the overexpression and mislocalization of AQP4 was observed in the affected motor nuclei in the brainstem and spinal cord (Nicaise et al., 2009, Bataveljić et al., 2012). However, it is not known whether the observed AQP4 abnormalities are a cause or consequence of ALS. Recent studies showed that AQP4 upregulation induced BBB disruption and brain edema, while inhibition of AQP4 attenuated BBB disruption in mouse models of ischemia (Tang et al., 2014, Wang et al., 2015).
In the present study, we investigated the expression of AQP4 in a mouse model of ALS, as well as in tissue from patients with ALS. In both cases, AQP4 was upregulated, which is consistent with previous studies in a rat model of ALS (Garbuzova-Davis et al., 2007, Zhong et al., 2008). We then utilized AQP4 knockout mice to create an AQP4-deficient ALS mouse model, and examined the role of AQP4 in BBB permeability and ALS disease progression.
Section snippets
Animals
Transgenic mice carrying the human mutant SOD1G93A gene (B6.Cg-Tg(SOD1*G93A)1Gur/J) were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained on a C57BL/6 background. Mice carrying the human SOD1G85R gene (line 148) or the wild-type human SOD1 gene (B6.Cg-Tg(SOD1)76Dpr) were kind gifts from Dr. Don Cleveland (University of California, San Diego). LoxSOD1G37R mice were previously described (Yamanaka et al., 2008). The AQP4 knockout mice (Ikeshima-Kataoka et al., 2013, Acc. No.
Overexpression of AQP4 in a mouse model of ALS and in tissue from patients with ALS
Immunohistochemical analysis revealed that AQP4 was specifically observed in astrocyte endfeet surrounding blood vessels in the lumbar spinal cord of wild-type (WT) control mice. AQP4 was shown to be mislocalized in age-matched SOD1G93A mice during the progression of ALS symptoms (Fig. 1). Numerous studies have reported the presence of activated astrocytes in ALS animal models and in patients with ALS (Bataveljić et al., 2012, Hall et al., 1998, Nicaise et al., 2009). In the present study,
Discussion
The overexpression of astrocytic AQP4 in the SOD1G93A rat model of ALS has been reported in previous studies (Bataveljić et al., 2012, Nicaise et al., 2009). In this study, we also showed that astrocytic AQP4 is overexpressed in mouse models of ALS, as well as in tissue from patients with ALS. In the spinal cord, abnormal AQP4 localization was more conspicuous in the ventral horn than in the dorsal horn. Moreover, we showed that AQP4 overexpression is uniquely associated with ALS disease state,
Author contributions
S.W-M., Y.M., T.O., and H.M. designed the experiments. S.W-M. and H.M. performed the experiments. K.Y., Y.A., and M.Y. contributed critical analytic tools and input. S.O. provided clinical and pathological data and tissue samples of human cases. S.W-M. and H.M. wrote the manuscript, and all authors discussed the data and commented on the manuscript.
Acknowledgments
We thank Airi Iwasaki, Yumiko Date, Akihiro Kondo, Yuki Sudo for help with the experiments. This work was supported by Grants-in-Aid for Scientific Research on Innovative Areas (Grant Number 23111006 to KY and HM) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; JSPS KAKENHI Grant Number 16K15129 (to HM) and 26293208 (to KY).
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