Anales de la RANM

82 A N A L E S R A N M R E V I S T A F U N D A D A E N 1 8 7 9 AMYOTROPHIC LATERAL SCLEROSIS IS NOT ONLY A MOTOR NEURON DISEASE Fernández A, et al. An RANM. 2022;139(01): 78 - 87 These various disturbances in neurosecretory systems could have in common a synaptopathy that drives a disturbed exocytotic machinery and the last steps of exocytosis. To explore these questions the sympathetic neuronal-like CC has been used during the last decade or so, to inquire about the alterations of ion channels, cell excitability and exocytosis in transgenic mouse models of neurodegenerative diseases (25). Thus, altered exocytosis either in its kinetics or in the fusion pore formation and expansion, has been investigated in CCs of the APP/PS1 mouse model of Alzheimer’s disease (AD) (34), in the knockout mouse model of huntingtin-associated protein 1 (HAP1), a model of Huntington’s disease (HD) (35), in mice carrying specific mutations in proteins of the exocytotic machinery, in the SOD1 G93A mouse model of ALS (36), and in the R6/1 mouse model of HD (37). In a thorough study done in CCs of SOD1 G93A mice, we investigated various ion currents and cell excitability using the patch-clamp technique (38), as well as the quantal release of catecholamines at the single-cell level using the online amperometric recording of secretory spike events with a carbon fibre microelec- trode (39). Upon stimulation with acetylcholine (ACh) an initial spike burst of secretion occurred in wildtype (WT) as well as in SOD1 G93A cells; this was followed by a period of slower secretion rates in both cell types. Although the number of spikes was similar, the cumulative secretion was however substantially higher in SOD1 G93A cells. Also, the tau of activation of the exocytotic rate was faster in the SOD1 G93A CCs (Fig. 2). The kinetic analysis of single spikes (equivalent to the quantal release of single catecholamine-storing vesicles) indicated differ- ences in SOD1 G93A CCs versus WT CCs, namely: 35.9 lower rise rate, 60.7 longer decay time, 55.3 longer half-width, 16.6 lower amplitude, and 52.2 % higher quantal size. This indicated a slower but higher exocytotic release of catecholamines per single vesicle in SOD1 G93A cells, with respect to WT cells. Of interest was the comparison of the kinetics of single exocytotic spikes obtained from CCs of the APP/PS1 mouse model of AD and the SOD1 G93A mouse model of ALS. The averaged AD spike has higher Imax and faster decay time with decreased quantal size, suggesting a lower catecholamine content per vesicle, compared with WT cells. On the other hand, the averaged amperometric spikes of SOD1 G93A CCs has lower amplitude and slower activa- tion and decay rates, indicating a slower but higher total secretion. The drastic differences between AD and SOD1 G93A spikes can be observed in Fig. 3; much faster and higher amplitude of the AD spike, with respect the SOD1 G93A spike. This suggests that the different kinetics of exocytosis could potentially serve as a biomarker of AD and ALS, respectively. D I S T U R B E D E X O C Y T O S I S I N C H R OMA F F I N C E L L S F R OM T H E S OD1G93A MOU S E MOD E L O F A L S Figure 2. Higher total catecholamine release evoked by ACh stimulation in SOD1G93A CCs, compared with WT CCs. Cumu- lative secretion (summation of secretory spikes areas) calculated at 5-s intervals in the 60-s recording of amperometric single spikes. (Adapted from Calvo-Gallardo et al., 2015).