Genetic Inactivation of Two-Pore Channel 1 Impairs Spatial Learning and Memory

Abstract

Two-pore channels (TPCs) constitute a small family of cation channels that are localized in membranes of endosomal and lysosomal compartments. Although their roles for vesicular fusion and endolysosomal trafficking have been investigated, our knowledge on their expression pattern and higher order functions in the murine brain is still limited. Western blot analysis indicated a broad expression of TPC1 in the neocortex, cerebellum and hippocampus. In order to investigate the consequences of the genetic inactivation of TPC1, we performed a set of behavioural studies with TPC1^-/- mice. TPC1^-/- mice were analysed for an altered motor coordination and grip-strength, exploratory drive and anxiety as well as learning and memory. TPC1^-/- mice did not show any differences in their exploratory drive or in their anxiety levels. There were also no differences in spontaneous activity or motor performance. However, the Morris water maze test uncovered a deficit in spatial learning and memory in TPC1^-/- mice.

Keywords: Morris water maze; NAADP; Spatial learning; TPC1; Two-pore channel.

Fig. 1

Strategy for the genetic inactivation of TPC1 and western blot analysis of murine brain regions. a Schematic representation of the wild type, targeted, “floxed” exon 3 and knock out tpc1 gene locus. The numbers indicate the exon number. The TPC1 knock out model was generated by two rounds of recombination. First, the neomycin resistance cassette was removed by Cre-mediated recombination in targeted ES cells. Second, the knock out locus was generated by crossing mice containing a floxed exon 3 allele with ROSA26 Cre mice. A Acc65I, B BamHI, C ClaI, H HindIII, X XhoI. b Schematic representation of the TPC1 transmembrane topology and consequences of deletion of exon 3. Cre-mediated deletion of exon 3 causes a frameshift and a premature stop codon resulting in a 73 amino acid residues N-terminal fragment. The amino acid sequence (aa 774–790) for generating the TPC1 antibody is indicated in blue. c Representative western blot of membrane preparations obtained from WT and TPC1 knock out mouse brains. First line: Expression of TPC1 in neocortex, hippocampus and cerebellum of WT and TPC1 knock out mice. Second line: Corresponding brain regions do not show a compensatory upregulation of TPC2 in TPC1 knock out mice. Third line: Loading control using an antibody for Na⁺/K⁺-ATPase (Color figure online)

Fig. 2

Elevated plus maze and open field test. Activity of 12 to 21 weeks old male wild type (N = 15) and TPC1^−/− mice (N = 17) was recorded over a period of 9 min during an elevated plus maze test. a Percentage of time spent at the open arms is presented for the first 3, 6 and 9 min. b Total covered distance for the same periods. c Spontaneous activity and exploratory behaviour of 10 weeks old male wild type (N = 15) and TPC1 knockout mice (N = 17) was recorded over a period of 20 min in the open field arena and percentage of time spent at central area is shown. d Total covered distance during the open field experiment. Statistical significance was assessed with two-tailed t test. Significance levels are indicated as P values

Fig. 3

Voluntary wheel running. Single caged, adult male mice (12 to 28 weeks old) were allowed for voluntary wheel running during 21 days. At day 14 (indicated by the red dashed line) standard wheels (regular rung configuration) were replaced by “complex wheels”, meaning that particular rungs were removed to create an irregular rung configuration (Mandillo et al. 2014). Wheel running activity was recorded for wildtype (grey, N = 10) and TPC1^−/− mice (white, N = 10) and average running speed (a) and average distance (b) was calculated. Both parameters tested, showed no significantly different motor performances for WT and TPC1^−/− mice. Data are represented as mean ± SEM (Color figure online)

Fig. 4

Morris water maze task. Figures present the results of the acquisition phase (a) and of the retention trial (b) to (d); e shows a scheme of the experimental setup of the Morris water maze (number and age of male mice: WT: N = 16 and TPC1^−/−: N = 17; 12 to 21 weeks old). a Covered distance during day 1 to 5 of the acquisition phase. Data of the acquisition trials were averaged across four trials per day. Differences in path length were analyzed using two-way analysis of variance (ANOVA) with repeated measures and additional Holm–Sidak pairwise comparison procedure (WT vs. TPC1^−/− within each day). b Spatial memory was determined by preference of the platform (target) sector when the platform was removed at day 6 of the experiment (retention trial). Differences for sector occupancy (sector 1 – sector 4 within WT and TPC1^−/−) were analyzed using one way analysis of variance (ANOVA). The Student–Newman–Keuls post-hoc test was used pairwise for multiple comparison. Differences in path length at sector 4 (target sector) between WT and TPC1^−/− mice was analyzed using two-tailed t test. c Average distance to platform position was measured as an independent parameter. Statistical significance was assessed with two-tailed t test. d Average swimming speed of mice during retention trial. Statistical significance was assessed with two-tailed t test. e Experimental setup of Morris water maze task indicating the arrangement of the four sectors. During retention animals were launched at quadrant two (start sector). Sector four (target sector) previously contained the submerged platform (“P” indicates platform position) during acquisition trials. Data are represented as mean + SEM, significance levels are indicated as P values; n.s. not significant (P > 0.05)