Calpain-Dependent Degradation of Nucleoporins Contributes to Motor Neuron Death in a Mouse Model of Chronic Excitotoxicity

Abstract

Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as a common pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence to suggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1 and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunction in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deleting astroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST(+/-)/GLT1-cKO). GLAST(+/-)/GLT1-cKO mice (both sexes) exhibited nuclear irregularity and calpain-mediated degradation of nuclear pore complexes (NPCs), which are responsible for nucleocytoplasmic transport. These abnormalities were associated with progressive motor neuron loss, severe paralysis, and shortened lifespan. The nuclear export inhibitor KPT-350 slowed but did not prevent motor neuron death, whereas long-term treatment of the AMPA receptor antagonist perampanel and the calpain inhibitor SNJ-1945 had more persistent beneficial effects. Thus, NPC degradation contributes to AMPA receptor-mediated excitotoxic motor neuronal death, and preventing NPC degradation has robust protective effects. Normalization of NPC function could be a novel therapeutic strategy for neurodegenerative disorders in which AMPA receptor-mediated excitotoxicity is a contributory factor.SIGNIFICANCE STATEMENT Despite glial glutamate transporter dysfunction leading to excitotoxicity has been documented in many neurological diseases, it remains unclear whether its dysfunction is a primary cause or secondary outcome of neuronal death at disease state. Here we show the combined loss of glial glutamate transporters GLT1 and GLAST in spinal cord caused motor neuronal death and hindlimb paralysis. Further, our novel mutant exhibits the nuclear irregularities and calpain-mediated progressive nuclear pore complex degradation. Our study reveals that glial glutamate transporter dysfunction is sufficient to cause motor neuronal death in vivo. Glutamate-mediated excitotoxicity induces neuronal death by altering various intracellular signaling pathways and is implicated as a common pathogenic pathway in many neurodegenerative diseases. In the case of motor neuron disease, there is significant evidence to suggest that the overactivation of AMPA receptors due to deficiencies in the expression and function of glial glutamate transporters GLT1 and GLAST plays an important role in the mechanisms of neuronal death. However, a causal role for glial glutamate transporter dysfunction in motor neuron death remains unknown. Here, we developed a new animal model of excitotoxicity by conditionally deleting astroglial glutamate transporters GLT1 and GLAST in the spinal cords of mice (GLAST ϩ/Ϫ /GLT1-cKO). GLAST ϩ/Ϫ /G...

Region‐specific deletions of the glutamate transporter GLT1 differentially affect seizure activity and neurodegeneration in mice

“…Cell counting was previously described (Sugiyama et al, 2017). NeuNpositive cells were counted in the bilateral region of interest using a cell counter plug-in of ImageJ software (NIH).…”

“…Emx1-Cre knock-in mice (Emx1 cre/1 mice, Iwasato et al, 2004) and Foxb1 cre/1 mice (Zhao, Zhou, Szab o, Leitges, & Alvarez-Bolado, 2007) were obtained from the RIKEN BioResource Center. Floxed-GLT1 mice, harboring GLT1 gene exon 4 flanked by loxP sequences (GLT1 F/F ), were previously described (Aida et al, 2015;Cui et al, 2014;Sugiyama et al, 2017). We crossed GLT1 F/F mice with Emx1 cre/1 and Foxb1 cre/1 mice to obtain Emx1 cre/1 ;GLT1 F/1 and Foxb1 cre/1 ;GLT1 F/1 mice, respectively.…”

“…Immunohistostaining was previously described (Aida et al, 2015;Cui et al, 2014;Sugiyama et al, 2017). Mice were deeply anesthetized and transcardially perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS.…”

“…Western blotting was performed as previously described (Aida et al, 2015;Sugiyama et al, 2017). Mouse brains dissected in each region were homogenized in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF and 1 mM Na 3 VO 4 ) containing protease inhibitor cocktail (Roche).…”

Potential Alzheimer’s Disease Therapeutics Among Weak Cysteine Protease Inhibitors Exhibit Mechanistic Differences Regarding Extent of Cathepsin B Up-Regulation and Ability to Block Calpain

“…For over 30 years, research programs for developing neurotherapeutics and other disease treatments have included cysteine protease inhibitors (see Wang & Yuen, 1994; Nixon, 2000; Vanderklish & Bahr, 2000; Trinchese et al, 2008; Saatman et al, 2010; Ono et al, 2016; Sugiyama et al, 2017). For protein accumulation disorders (e.g., AD, Parkinson’s disease, and Huntington’s disease), it is counterintuitive to use protease inhibitors that would block the same protein clearance pathways whose dysfunctions are part of pathogenic cascades (see Torres et al, 2012; Burbulla et al, 2017; Farizatto et al, 2017).…”