Disturbances in the secretion of neurotransmitters in IA-2/IA-2beta null mice: changes in behavior, learning and lifespan

Abstract

Islet-associated protein 2 (IA-2) and IA-2beta are major autoantigens in type 1 diabetes and transmembrane proteins in dense core secretory vesicles (DCV) of neuroendocrine cells. The deletion of these genes results in a decrease in insulin secretion. The present study was initiated to test the hypothesis that this deletion not only affects the secretion of insulin, but has a more global effect on neuroendocrine secretion that leads to disturbances in behavior and learning. Measurement of neurotransmitters showed that norepinephrine, dopamine and 5-HT were significantly decreased in the brain of double knockout (DKO) mice (P<0.05 to <0.001). In tests evaluating anxiety-like behavior and conditioned-learning, the DKO mice showed a highly significant increase in anxiety-like behavior (P<0.01 to <0.001) and impairment of conditioned learning (P<0.01) as compared to WT mice. The DKO mice also displayed an increase in spontaneous and induced seizures (P<0.01) and age-related death. Contrary to the generally held view that IA-2 and IA-2beta are expressed exclusively in DCV, subcellular fractionation studies revealed that IA-2beta, but not IA-2, co-purifies with fractions rich in synaptic vesicles (SV), and that the secretion of dopamine, GABA and glutamate from the synaptosomes of the DKO mice was significantly decreased as was the number of SV (P<0.01). Taken together, these findings show that IA-2beta is present in both DCV and SV, and that the deletion of IA-2/IA-2beta has a global effect on the secretion of neurotransmitters. The impairment of secretion leads to behavioral and learning disturbances, seizures and reduced lifespan.

Fig. 1. Decreased neurotransmitters in brain of DKO mice

(A) Western blot of whole brain showing expression of IA-2 and IA-2β in WT, but not DKO mice. (B) Concentration of norepinephrine, dopamine and serotonin in the brain of WT and DKO mice at 12 and 30 weeks of age as determined by HPLC. Each group represents 6 mice. Data are expressed as mean ± SEM. *P < 0.05, **P<0.01, ***P<0.001. WT mice (open bars); DKO mice (closed bars).

Fig. 2. Behavioral changes in open field (A-E) and height-fear (F) tests in DKO mice

(A) The average walking speed of WT and DKO mice was similar. (B) Exploratory activity was significantly delayed as determined by the time required to leave a fixed spot in the center of the cage. (C) DKO mice show reduced frequency of rearings. (D) DKO mice show reduced travel distance. (E) DKO mice show reduced number of visits to the center area of a cage. (F) Latency (seconds) to step down from elevated platforms of different heights in the height-fear test. Open and closed bars represent WT and DKO mice, respectively. Bars show mean ± SEM. 12 to 16 mice per group, *P < 0.05, **P<0.01, ***P<0.001 (DKO mice vs. WT mice).

Fig. 3. Conditioned learning in DKO mice as evaluated by taste aversion, olfactory-place preference and rotarod-latency to fall

(A) Prior to conditioning WT and DKO mice consumed the same amount of water (10 mice per group). (B) Both preferred saccharine-flavored over plain water (10 mice per group). (C) After conditioning (linking the saccharine-flavored water with LiCl which caused abdominal discomfort) the WT mice preferred plain water for the 14 days of the experiment, whereas the DKO mice showed accelerated extinction of learning and rapidly resumed their preference for saccharine-flavored water (10 mice per group). Open and closed circles represent WT and DKO mice, respectively. (D) Behavioral preference for conditioning versus non-conditioning odorant (8 mice per group). WT mice spent significantly more time than DKO mice in the compartment containing the conditioning odorant (stripped bar) as compared to the compartment containing the non-conditioning odorant (dotted bar). (E) Motor learning as evaluated by latency to fall in the rotarod test (20 mice per group). Both the WT and DKO mice improved performance with increase in training, but the response of the DKO mice never reached that of the WT mice. Rotarod data, analyzed with a two-way ANOVA, revealed a significant main effect of genotype (P<0.01) and a significant main effect of trial (P<0.0001), but no genotype effect by trial interaction (P = 0.33). Open and closed circles represent WT and DKO mice, respectively. Data points and bars represent mean ± SEM. **P< 0.01, ***P< 0.001.

Fig. 4. Increase in spontaneous and induced seizures and shorten lifespan in DKO mice

(A) Frequency of handling-induced seizure increases with age in DKO mice (20-30 animals per group). (B) Latency in seconds for the development of PTZ-induced seizures (40 mg/kg body weight) as evaluated by loss of posture (10 mice per group). (C) Severity of kainic acid induced seizures (20mg/kg body weight) (10 mice per group). (D) Survival of WT (28 untreated, open circles) and DKO (31 untreated, closed circles) mice. Bars represent mean ± SEM. **P< 0.01.

Fig. 5. Distribution of IA-2 and IA-2β in SV and DCV

(A) Homogenized brain lysates were fractionated on a 0.6-1.8 M sucrose gradient and analysed by immunoblotting with antibodies to IA-2, IA-2β, synaptophysin and secretogranin II. (B) Biotinylated labeling of cell surface proteins, followed by separation from the non-biotinylated cytosolic proteins by avidin-coated beads. Western blot shows that IA-2β, but not IA-2, is expressed on the surface of the synaptosomes. Surface biotinylation of HSP60 was not observed, indicating that synaptosomal damage and contamination of proteins from the cytoplasm did not occur. (C) Colocalization of IA-2β and synapsin 1 in cultured primary neurons from the hippocampus.

Fig. 6. Decreased secretion of neurotransmitters from synaptosomes of DKO mice

(A) Uptake of [H³]dopamine, [H³]GABA and [H³]glutamate by synaptosomes of DKO mice as percentage of uptake by WT mice. Data are mean ± SEM from three separate experiments performed in triplicate. (B) Basal and K⁺-induced (25 mM) release of [H³]dopamine, [H³]GABA, and [H³]glutamate from synaptosomes of WT and DKO mice. The amount of radiolabelled neurotransmitter released, expressed as percentage of total radiolabelled neurotransmitter, is shown. Values are mean ± SEM from three separate experiments performed in triplicate; ** P<0.01. Open and closed bars represent WT and DKO mice, respectively. (C) Quantification of synaptic vesicles from 10 areas of hippocampus each representing 50 μm². *** P<0.001. (D) Effect of neuropharmacologic agents on reducing the increased anxiety-like behavior in DKO mice (saline) as compared to WT mice, as measured by the decrease in immobility time in the tail suspenstion test (TST) (8-10 mice per group). (§) Untreated (saline) DKO mice differ significantly from untreated (saline) WT mice (P<0.001). (†) Treated (i.e., fluoxetine 20 mg/kg, bupropion 20 mg/kg, pargylene 75 mg/kg) WT mice differ significantly from untreated (saline) WT mice (P<0.001). (#) Fluoxetine and bupropion-treated DKO mice differ significantly from untreated (saline) DKO mice (P<0.001). (&) Treated DKO mice differ significantly from treated WT mice (P<0.001).