For all the scrutiny of the amyloid precursor protein (APP) as the source of pathogenic Aβ peptides, there is little understanding of the parent protein’s normal role in neurons. Using APP knockout mice, Hui Zheng and colleagues at Baylor College of Medicine, Houston, Texas, have been studying the need for APP in synapses, and their latest work identifies a new function for the protein in regulating calcium fluxes in some neurons. Specifically, the researchers find that APP controls the levels of an L-type calcium channel, CaV1.2, specifically in GABAergic neurons. Loss of APP results in higher levels of CaV1.2 protein and activity, which leads to altered short-term plasticity at synapses, they show. While the study does not directly link any of these changes to Alzheimer disease, the results suggest that alterations in APP metabolism or function could be linked to failed calcium homeostasis and changes in synaptic plasticity, both events that occur early on in the disease. In other APP news this week, researchers identify new signaling pathways that may mediate the effects of the precursor protein (see ARF related news story).
The work follows on Zheng’s previous studies on the neuromuscular junction in APP knockout mice, where she identified problems in synapse development (see ARF related news story and Wang et al., 2009) and function, including defects in GABA-induced short-term plasticity involving aberrant activation of L-type calcium channels (Yang et al., 2007). In the new study, the investigators extended their work to the CNS by looking at the expression of various classes of calcium channels in different brain regions in APP-null mice by Western blot. When they found a specific upregulation of the CaV1.2 L-type calcium channel in the striatum, first author Li Yang followed up with electrophysiology studies on striatal neuron cultures. Yang found that the increase in CaV1.2 coincided with the presence of exaggerated calcium currents that were reversed by nifedipine, a specific L-type calcium channel blocker. The cells also showed alterations in short-term plasticity (reduced paired-pulse inhibition and increased post-tetanic potentiation), which could be corrected by nifedipine. Re-expression of APP in the neurons using a lentivirus rescued both normal channel expression and calcium currents. Thus, it appears that loss of APP led to increased CaV1.2, which was responsible for increased calcium influx and accompanying functional changes in striatal GABAergic neurons.
What about neurons in the hippocampus, the site of early changes in calcium homeostasis in AD? The researchers did not see a significant difference in overall CaV1.2 expression in the hippocampal neurons between wild-type and APP-knockout mice. At the same time, they knew from previous work (Seabrook et al., 1999) that loss of APP results in a significant reduction in paired-pulse inhibition in GABAergic synapses there. When the researchers used quantitative immunostaining to look specifically at GABAergic neurons (which make up a minority of hippocampal neurons), they did find an elevation of CaV1.2. Along with this, they found altered short-term plasticity in the GABAergic neurons, which was reversed by nifedipine. Thus, it seems that increased CaV1.2 levels and calcium currents are a general result of loss of APP in GABAergic neurons, whether in the hippocampus or striatum.
Other recent data suggest that APP regulates L-type calcium currents and calcium oscillations in rat cortical (glutamatergic) neurons as well (Santos et al., 2009). In that study, though, increased channel function resulted from overexpressing human APP, the opposite to what Zheng and colleagues report. Taken together, the two studies “support the notion that APP may affect L-type calcium channel function in multiple neurons and by multiple mechanisms,” the authors write.
How does APP regulate CaV1.2 levels? The mechanism did not seem to involve transcription, as CaV1.2 mRNA levels were unchanged in APP knockout mice compared to wild-type. That ruled out a role for the APP intracellular domain (AICD), but another possibility was Aβ, which itself modulates calcium influx in neurons. However, treating wild-type cells with a γ-secretase inhibitor had no effect on channel levels, suggesting that Aβ likewise was not involved. The researchers did find a direct and specific physical interaction between the proteins by co-immunoprecipitation, and they speculate that APP may control CaV1.2 trafficking to the membrane.
As yet, Zheng says they have no clue why APP might regulate CaV1.2 specifically in GABAergic neurons. “If you look at the APP expression pattern and the L-type calcium channel expression pattern, we did find a higher level of L-type calcium channels in inhibitory neurons, but they are expressed in all the neurons. We hypothesize that there must be some intermediate molecule that is GABAergic specific, but we have not been able to identify that particular molecule,” she told ARF.
Calcium dysregulation is one of earliest manifestations of pathology in mouse models of AD, but it is not clear how this new function of APP might relate to disease processes. Zheng told ARF that her lab is now studying APP mutants to see if they affect calcium channel levels or function.—Pat McCaffrey
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