Decreased levels of ARMS/Kidins220 in vivo lead to enhanced LTP and increased basal synaptic transmission at the Schaffer collateral-CA1 synapse. A, ARMS/Kidins220^+/− mice display enhanced LTP. LTP was induced by a θ-burst stimulus (arrow) in acute hippocampal slices from 3–6-month-old ARMS/Kidins220^+/− mice and wild-type littermates. Each point represents the average of three successive events (WT, black circles, n = 8; ARMS/Kidins220^+/−, white circles, n = 11; data are represented as the means ± S.E.). B, input-output curve shows increased basal synaptic transmission at lower stimulus intensities in ARMS/Kidins220^+/−mice. Input-output curve of field excitatory postsynaptic potential slope (fEPSP) (V s⁻¹) versus stimulus (V) at Schaffer collateral-CA1 synapses in adult hippocampal slices from ARMS/Kidins220^+/− and wild-type mice (WT, black circles, n = 11; ARMS/Kidins220^+/−, white circles, n = 15; *, p < 0.05, t test; data are represented as the means ± S.E.).

ARMS/Kidins220 degradation induced by KCl depolarization requires extracellular calcium. A, hippocampal cultures were depolarized with 50 mm KCl for 3 h in the presence of the calcium chelator EGTA, and ARMS/Kidins220 protein levels were assessed by Western blot. EGTA fully rescued the degradation of full-length ARMS/Kidins220 and the appearance of N-terminal degradation fragments (arrows a, b, and c). Actin is shown as a loading control. 95- and 150-kDa nonspecific bands were detected by the N-terminal antibody. B, quantification of ARMS/Kidins220 protein levels in A, as detected with the C-terminal antibody, normalized to actin levels, and plotted relative to untreated control (n ≥ three independent experiments; **, p < 0.01, t test; data are represented as the means ± S.E.).

Activity-dependent degradation of ARMS/Kidins220 protein is not mediated by proteasomes and lysosomes. A, inhibition of proteasomes does not rescue activity-induced degradation of ARMS/Kidins220. Hippocampal cultures were depolarized with 50 mm KCl for 3 h in the presence of the proteasomal inhibitor epoxomicin or lactacystin, and ARMS/Kidins220 protein levels were assessed by Western blot. Actin is shown as a loading control. B, inhibition of lysosomes does not rescue activity-induced degradation of ARMS/Kidins220. Experiments were performed as in A using the lysosomal inhibitor chloroquine. Actin is shown as a loading control. C, quantification of ARMS/Kidins220 protein levels in A and B normalized to actin levels and plotted relative to untreated control (n ≥ three independent experiments; data are represented as the means ± S.E.).

Inhibition of calpain prevents enhancement of LTP in ARMS/Kidins220^+/− mice. Acute hippocampal slices from 3–7-month-old ARMS/Kidins220^+/− mice and wild-type littermates were incubated with the calpain inhibitor BDA-410 before and during LTP induction by a θ-burst stimulus (arrow). Calpain blockade did not affect LTP expression in wild-type animals, whereas it prevented enhancement of LTP in ARMS/Kidins220^+/− mice. Each point represents the average of three successive events (WT: vehicle, black circles, n = 7; BDA-410, black triangles, n = 8; ARMS/Kidins220^+/−: vehicle, white circles, n = 7; BDA-410, white triangles, n = 9; data are represented as the means ± S.E.). fEPSP, field excitatory postsynaptic potential.

ARMS/Kidins220 is degraded upon KCl activation of hippocampal neurons. A, ARMS/Kidins220 is degraded upon neuronal activation by KCl. Hippocampal cultures (14–21 days in vitro) were depolarized with 50 mm KCl for the indicated times. Cell lysates were immunoblotted for ARMS/Kidins220 using antibodies against the C or N terminus and for actin as a loading control. In response to neuronal activation, 220-kDa full-length ARMS/Kidins220 as detected by both C- and N-terminal antibodies was degraded, leading to the accumulation of smaller sized fragments as detected by the N-terminal antibody (arrows), estimated to be 140 (a), 120 (b), and 85 (c) kDa. 95- and 150-kDa nonspecific bands were detected by the N-terminal antibody. B, quantification of ARMS/Kidins220 protein levels in hippocampal neurons after KCl depolarization, as detected with the C-terminal antibody, normalized to actin levels, and plotted relative to untreated control (n ≥ three independent experiments; data are represented as the means ± S.E.).

Glutamate activation of hippocampal neurons induces ARMS/Kidins220 degradation through a calcium-mediated process. A, ARMS/Kidins220 is degraded upon glutamate treatment of hippocampal neurons. Hippocampal cultures were stimulated with 200 μm glutamate for 1 min, and cell lysates were collected at the indicated times after stimulation and immunoblotted for ARMS/Kidins220. As was the case after KCl depolarization, full-length ARMS/Kidins220 was degraded, leading to the accumulation of similar smaller sized N-terminal fragments (arrows a, b, and c). Actin is shown as a loading control. A 150-kDa nonspecific band was detected by the N-terminal antibody. B, glutamate-mediated ARMS/Kidins220 degradation requires extracellular calcium. Hippocampal cultures were stimulated with 200 μm glutamate for 1 min in the presence of EGTA, and cell lysates were collected 3 h after stimulation. ARMS/Kidins220 protein levels, as detected by the C-terminal antibody, were assessed by Western blot and quantified. ARMS/Kidins220 levels were normalized to actin levels and plotted relative to untreated control (n ≥ three independent experiments; **, p < 0.01, t test; data are represented as the means ± S.E.).

ARMS/Kidins220 is degraded by calpain upon neuronal activation. A, inhibition of the protease calpain rescues KCl-induced degradation of ARMS/Kidins220. Hippocampal cultures were depolarized with 50 mm KCl for 3 h in the presence of the calpain inhibitor MDL28170 or N-acetyl-Leu-Leu-Met (ALLM), and ARMS/Kidins220 protein levels were assessed by Western blot. Both of these inhibitors significantly reduced the degradation of ARMS/Kidins220 and prevented the appearance of N-terminal degradation fragments (arrows a, b, and c). The 145- and 150-kDa breakdown products of spectrin mark calpain activity, and actin is shown as a loading control. 95- and 150-kDa nonspecific bands were detected by the N-terminal antibody. B, quantification of ARMS/Kidins220 protein levels in A, as detected by the C-terminal antibody, normalized to actin levels, and plotted relative to untreated control (n ≥ three independent experiments; **, p < 0.01, t test; data are represented as the means ± S.E.). C, hippocampal cultures were stimulated with 200 μm glutamate for 1 min in the presence of the calpain inhibitor MDL28170 or N-acetyl-Leu-Leu-Met, and cell lysates were collected at the indicated times after stimulation. ARMS/Kidins220 protein levels, as detected by the C-terminal antibody, were assessed by Western blot and quantified. ARMS/Kidins220 levels were normalized to actin levels and plotted relative to untreated control (n ≥ three independent experiments; *, p < 0.05, t test; data are represented as the means ± S.E.). D, hippocampal cultures were depolarized with 50 mm KCl for the indicated times in the presence or absence of the calpain inhibitor MDL28170, and the indicated proteins were probed by Western blot. The degradation of full-length ARMS/Kidins220, as well as the appearance of N-terminal degradation fragments (arrows a, b, and c), was correlated with the degradation of GluA1, and the degradation of both proteins was prevented by calpain inhibition. In contrast, GluA2 was not degraded.