Aim/Introduction: In the central nervous system, glutamate acts both as excitatory neurotransmitter and metabolic substrate channeled to the Krebs cycle after its conversion to alpha-ketoglutarate. Due to this double role, its extracellular concentration is tightly regulated. In particular, glutamate released by neurons in the synaptic cleft is removed by astrocytes and converted into glutamine to be transferred back to neurons for the conversion to glutamate configuring the so-called glutamate-glutamine cycle (GGC). Accordingly, the catalytic function of glutamine synthetase (GS) highly expressed in astrocytes plays a pivotal role in preventing glutamate toxicity. Although several studies reported an impaired GGC in amyotrophic lateral sclerosis (ALS), methods able to estimate glutamate processing rates by astrocytes are not available so far. Since the main fate of NH3 taken up by cells is the production of glutamine by GS, we aimed to verify whether uptake kinetics of 13N-NH3 is altered in primary astrocytes harvested from SOD1G93A mice as an experimental model of ALS. Materials and Methods: Astrocytes were isolated from brain harvested from neonatal (p2) wild-type (WT) and SOD1G93A mice and cultured under standard conditions. Time-activity curves of cultured cells were defined using a dedicated instrument (LigandTracer, Ridgeview, SE) that consists of a beta-emission detector and a rotating platform harboring a Petri dish inclined at 30 degrees as to alternate cell position from nadir (for incubation) to zenith (for counting) every minute. For the experiments, astrocytes were incubated in saline enriched with 6 MBq/mL 13N-NH3 with or without glutamate 70 microM. Counting rate (counts per second, cps) of cultured cells was monitored for 30 minutes and normalized for both administered dose and cell number (C-Rate). Results: WT and SOD1G93A astrocytes showed a similar 13N-NH3 uptake kinetics in the absence of glutamate and reached similar C-Rate values at 30 minutes (2.8±0.5 vs 2.9 ±0.6 cps, respectively, ns). By contrast, the presence of glutamate increased C-Rate to a greater degree in WT than in SOD1G93A astrocytes (4.1±0.8 vs 2.7±0.8 cps, respectively, p<0.01). Conclusion: The present data suggest that monitoring NH3 uptake might detect an abnormal GGC during the early disease stage of a mouse ALS model. This tool might complement the study of glutamate transporters and GS activity to clarify astrocyte role in ALS progression. References: None.
In vitro Evidence of Abnormal Glutamate to Glutamine Conversion in Astrocytes harvested from a Mouse Model of Amyotrophic Lateral Sclerosis: a potential Experimental Application of 13N-ammonia
C Marini
2019
Abstract
Aim/Introduction: In the central nervous system, glutamate acts both as excitatory neurotransmitter and metabolic substrate channeled to the Krebs cycle after its conversion to alpha-ketoglutarate. Due to this double role, its extracellular concentration is tightly regulated. In particular, glutamate released by neurons in the synaptic cleft is removed by astrocytes and converted into glutamine to be transferred back to neurons for the conversion to glutamate configuring the so-called glutamate-glutamine cycle (GGC). Accordingly, the catalytic function of glutamine synthetase (GS) highly expressed in astrocytes plays a pivotal role in preventing glutamate toxicity. Although several studies reported an impaired GGC in amyotrophic lateral sclerosis (ALS), methods able to estimate glutamate processing rates by astrocytes are not available so far. Since the main fate of NH3 taken up by cells is the production of glutamine by GS, we aimed to verify whether uptake kinetics of 13N-NH3 is altered in primary astrocytes harvested from SOD1G93A mice as an experimental model of ALS. Materials and Methods: Astrocytes were isolated from brain harvested from neonatal (p2) wild-type (WT) and SOD1G93A mice and cultured under standard conditions. Time-activity curves of cultured cells were defined using a dedicated instrument (LigandTracer, Ridgeview, SE) that consists of a beta-emission detector and a rotating platform harboring a Petri dish inclined at 30 degrees as to alternate cell position from nadir (for incubation) to zenith (for counting) every minute. For the experiments, astrocytes were incubated in saline enriched with 6 MBq/mL 13N-NH3 with or without glutamate 70 microM. Counting rate (counts per second, cps) of cultured cells was monitored for 30 minutes and normalized for both administered dose and cell number (C-Rate). Results: WT and SOD1G93A astrocytes showed a similar 13N-NH3 uptake kinetics in the absence of glutamate and reached similar C-Rate values at 30 minutes (2.8±0.5 vs 2.9 ±0.6 cps, respectively, ns). By contrast, the presence of glutamate increased C-Rate to a greater degree in WT than in SOD1G93A astrocytes (4.1±0.8 vs 2.7±0.8 cps, respectively, p<0.01). Conclusion: The present data suggest that monitoring NH3 uptake might detect an abnormal GGC during the early disease stage of a mouse ALS model. This tool might complement the study of glutamate transporters and GS activity to clarify astrocyte role in ALS progression. References: None.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
