04 Jul 2010

A potential therapeutic strategy for Alzheimer disease (AD) is to inhibit the β-secretase (BACE) enzymes that kickstart processing of amyloid precursor protein (APP) to form Aβ. BACE inhibitors are currently the subject of intense drug development—including analysis of possible serious side effects. In a paper published June 30 in The Journal of Neuroscience, researchers led by Riqiang Yan at the Lerner Research Institute in Cleveland, Ohio, report that BACE1-knockout mice produce more sodium channels in axons, have increased neuronal excitability, and are more susceptible to seizures than are normal mice. The paper is a cautionary note for drug developers, although further evidence will be needed before scientists can determine if these BACE1 effects will be a problem for human trials.

BACE1-null mice have been particularly valuable in determining the normal functions of the enzyme; for example, these mice show hypomyelination of central and peripheral nerves, which has been traced to BACE1’s action on neuregulin-1 (see ARF related news story on Meyer-Luehmann et al., 2006 and Willem et al., 2006). BACE1 also cleaves the β subunits of sodium channel proteins (see Wong et al., 2005).

While characterizing BACE1 knockouts, first authors Xiangyou Hu and Xiangdong Zhou observed that many of the animals experienced epileptic seizures. The same behavior was also seen by another group looking at a separate line of BACE1-negative mice (see Kobayashi et al., 2008), implying it was not just an artifact of this particular line. Yan and colleagues noted that about 11 percent of young BACE1-null mice had seizures, with incidence increasing with age to about 22 percent of elderly mice. To investigate this phenomenon, the authors implanted eight knockout mice and eight wild-type mice with devices that permitted electroencephalogram (EEG) monitoring of brain activity and electromyogram (EMG) monitoring of muscle contractions. They found that all of the knockout mice showed increased electrical discharges in their brains compared to wild-type mice, although only one of the knockouts experienced a visible seizure during the monitoring period. These electrical discharges are referred to as “silent seizures” because there are no behavioral indications. The EEG results revealed that even BACE1-null mice without visible seizures have a pattern of abnormal electrical activity and hyperexcitability in the brain.

Sodium channels are closely involved in neuron excitability, as their opening depolarizes neurons, bringing them to the threshold where an action potential will fire. And since these channels are linked to seizures (see Catterall, 2002; Stafstrom, 2007) and are BACE1 substrates, the authors chose to examine their expression in the brains of wild-type and BACE1-null mice. They stained brain sections with antibodies to the two β and one α subunit that make up the sodium channel, and found a greater abundance of subunits on the surface of hippocampal mossy fibers of the knockouts, consistent with axonal expression of the channels.

To verify these immunostaining results, the authors directly measured the activity of sodium channels in young BACE1-knockout mice by isolating hippocampal neurons and using whole-cell patch-clamping methods. Cells from the knockout mice were more easily depolarized than wild-type neurons, and showed higher current density, implying more sodium channels were available. Hippocampal slices from knockout animals had enhanced neuronal excitability and more robust synchronous neuronal firing.

Their results demonstrate another physiological role for BACE1, Yan said, adding to the evidence that “BACE1 is important not only for generating Aβ peptide, but also for other brain functions.”

It’s not yet clear whether this finding will affect therapeutic development of BACE1 inhibitors. The knockout mouse is not the best model of a human patient taking a BACE1 inhibitor, noted Adam Simon, president of the science consulting firm AJ Simon Enterprises in Yardley, Pennsylvania, who worked on BACE1 inhibitor development at Merck for several years. Most pharmacological inhibitors reduce levels of their target protein by only 30 to 70 percent, not 100 percent as in the knockout mouse. A better genetic model, Simon said, would be the heterozygous BACE1 knockout, which would have about 50 percent gene dosage.

Yan said his group plans to examine the BACE1 heterozygote to see if these mice also show silent seizures. They also want to cross BACE1 heterozygotes with AD model mice, Yan said, a model that would even more closely mimic an AD patient receiving a BACE1 inhibitor. Complicating the issue is the fact that AD mice, as well as human AD patients, have an increased incidence of epileptic seizures, which may be due to high Aβ levels (see ARF related news story on Palop et al., 2007). Lowering BACE1 levels in AD mice might reduce these seizures by reducing Aβ, Yan said, or conversely lowering BACE1 might increase seizures by increasing the surface expression of sodium channels; there’s no way to know until they do the experiment.

Another complication is that BACE1 expression is extremely high during development, Simon pointed out, which means that some problems in the knockout mouse could be due to lack of the enzyme during developmental stages, not due to adult loss of BACE1 function. Since AD patients have had normal levels of BACE1 all their lives, a conditional knockout of BACE1 would be the best model for drug development, Simon said. Without a conditional knockout model to test, he said, scientists can’t rule out a developmental explanation for the observed brain changes in BACE1 knockouts.

Drug discoverers should be mindful of the possible effect of BACE1 inhibitors on sodium channel activity, Simon suggested, and might want to look for sodium channel changes and examine EEGs in their preclinical animal models. But this finding does not ring any alarm bells for the therapeutic viability of BACE1 inhibitors, Simon said. “Many more controls need to be done to tease apart whether [this finding] is relevant to pharmacological inhibition of BACE1 in the elderly.”—Madolyn Bowman Rogers

Comments

No Available Comments

Make a Comment

To make a comment you must login or register.

References

News Citations

1. Double Paper Alert—A Function for BACE, a Basis for Amyloid 22 Sep 2006
2. Do "Silent" Seizures Cause Network Dysfunction in AD? 7 Sep 2007

Paper Citations

1. Meyer-Luehmann M, Coomaraswamy J, Bolmont T, Kaeser S, Schaefer C, Kilger E, Neuenschwander A, Abramowski D, Frey P, Jaton AL, Vigouret JM, Paganetti P, Walsh DM, Mathews PM, Ghiso J, Staufenbiel M, Walker LC, Jucker M. Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science. 2006 Sep 22;313(5794):1781-4. PubMed.
2. Willem M, Garratt AN, Novak B, Citron M, Kaufmann S, Rittger A, Destrooper B, Saftig P, Birchmeier C, Haass C. Control of peripheral nerve myelination by the beta-secretase BACE1. Science. 2006 Oct 27;314(5799):664-6. PubMed.
3. Wong HK, Sakurai T, Oyama F, Kaneko K, Wada K, Miyazaki H, Kurosawa M, De Strooper B, Saftig P, Nukina N. beta Subunits of voltage-gated sodium channels are novel substrates of beta-site amyloid precursor protein-cleaving enzyme (BACE1) and gamma-secretase. J Biol Chem. 2005 Jun 17;280(24):23009-17. PubMed.
4. Kobayashi D, Zeller M, Cole T, Buttini M, McConlogue L, Sinha S, Freedman S, Morris RG, Chen KS. BACE1 gene deletion: impact on behavioral function in a model of Alzheimer's disease. Neurobiol Aging. 2008 Jun;29(6):861-73. PubMed.
5. Catterall WA. Molecular mechanisms of gating and drug block of sodium channels. Novartis Found Symp. 2002;241:206-18; discussion 218-32. PubMed.
6. Stafstrom CE. Persistent sodium current and its role in epilepsy. Epilepsy Curr. 2007 Jan-Feb;7(1):15-22. PubMed.
7. Palop JJ, Chin J, Roberson ED, Wang J, Thwin MT, Bien-Ly N, Yoo J, Ho KO, Yu GQ, Kreitzer A, Finkbeiner S, Noebels JL, Mucke L. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron. 2007 Sep 6;55(5):697-711. PubMed.

Further Reading

News

1. Double Paper Alert—A Function for BACE, a Basis for Amyloid 22 Sep 2006
2. Do "Silent" Seizures Cause Network Dysfunction in AD? 7 Sep 2007