The impact of neurodegenerative disorders is profound. An estimated 1 million people in the U.S. endure Parkinson’s disease1, and another 5.3 million live with Alzheimer’s disease2. Over 69,000 ischemic strokes occur each year in the U.S.3, and those lucky enough to survive them are at risk for neurodegeneration.
There is tremendous need for a better arsenal of medications to treat neurodegenerative disorders. While a limited number of pharmaceutical agents are available to lessen symptoms, there are currently no drugs that slow or halt the progression of neuronal cell death. Two recently published research articles have investigated the neuroprotective activity of compounds that affect protein kinase A (PKA), a cAMP-dependent protein kinase that regulates glycogen, sugar, and lipid metabolism. In neuronal cells, PKA plays a key role in promoting cell survival by suppressing apoptosis. Curiously, each of the two compounds used in these studies protects neurons from cell death – even though one is a PKA activator, and the other is a PKA inhibitor.
An article published by Chong et al. in Free Radical Biology and Medicine focused on a compound named BHDPC (benzyl 7(4-hydroxy3methoxyphenyl)5-methyl-4,7-dihydrotetrazolo-[1,5a]-pyrimidine6carboxylate), which was originally identified in a screen for agents preventing atorvastatin-induced cerebral hemorrhage injury in zebrafish. The researchers tested BHDPC for neuroprotective activity in a zebrafish model of Parkinson’s disease, and conducted assays on brain slices and the human neuroblastoma cell line SH-SY5Y.
In animal models, administering the lipid-soluble neurotoxic chemical MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) induces a Parkinson’s disease-mimicking process. MPTP is converted to an active metabolite called MPP+ that is taken up by neurons via dopamine transporters, where it reacts with mitochondria complex I of the electron transport chain and causes irreversible oxidative damage and activation of the apoptotic cascade. Chong et al. found that BHDPC ameliorates MPTP-induced dopaminergic neuron loss at both the whole-organism level (zebrafish) and the tissue level (rat cerebellar slices, where treatment with 30 μM BHDPC attenuated MPP+-induced cell death from 100% to 35%). Importantly, they also demonstrated that 30 μM BHDPC restores normal locomotive behavior in MPTP-treated zebrafish.
To pinpoint the cell signaling pathways involved in neuroprotection by BHDPC, researchers exposed human neuroblastoma SH-SY5Y to MPTP. The resulting MPP+ induced loss of mitochondrial membrane potential and activated pro-apoptotic caspase 3, as expected. Pre-treatment with BHDPC prevented mitochondrial membrane potential loss and caspase 3 activation, but did not affect release of reactive oxygen species (ROS), suggesting that the drug is not simply an
BHDPC did, however, increase expression of the apoptosis regulator Bcl2. And it also increased phosphorylation of CREB, a nuclear transcription factor that regulates Bcl2. Furthermore, BHDPC caused phosphorylation of PKA, an effect that could be abolished by co-administration of the PKA inhibitor H89. Altogether, the authors suggested that BHDPC acts through PKA, and that further evaluation of BHDPC as a neuroprotective agent is warranted.
While Chong et al. used H89 as a tool to verify that BHDPC activates PKA, H89 also played a key role in the
second study, as well—but in an entirely different light. An article appearing in Neural Plasticity investigated the possibility that H89 might attenuate synaptic dysfunction and neuronal cell death after ischemic injury. However, in doing so, the authors focused on effects of H89 that are unrelated to its inhibition of PKA.
Neuronal cell death and synaptic dysfunction following cerebral ischemia can lead to memory impairment and other severe pathogenic events. Alterations in neuronal cell morphology and neurite extension are key steps in this pathogenesis. After noting that H89 affects ROCK II—a serine/threonine-specific protein kinase that plays a crucial role in organizing the actin cytoskeleton—Song et al. focused on how H89 affects expression of important proteins involved in neurodegeneration after ischemic and reperfusion injury. These include brain-derived neurotrophic factor (BNDF), postsynaptic density protein 95 (PSD95), microtubule-associated protein 2 (MAP2), and the apoptosis regulators Bcl2 and cleaved caspase 3. They also assessed the effect of H89 on neurite outgrowth.
In mice, Song et al. showed that hypoxia and reperfusion injury caused an inhibition of neurite outgrowth, but that H89 administration could reverse this process. MAP2 was suggested to be involved, since H89 restored MAP2 expression at the RNA and protein levels. (Although described as a neuronal-specific microtubule-associated protein, homologs of MAP2 are evolutionarily conserved in many species including Arabidopsis4.)
Not only did H89 up-regulate MAP2 if administered after hypoxia and reperfusion injury, it also increased cell survival, increased expression of anti-apoptotic Bcl2, and increased expression of cleaved caspase-3, and the neurotrophic factor BDNF. PSD-95, which increases synaptic plasticity, was similarly up-regulated by H89. Interestingly, these anti-apoptotic, pro-survival, and pro-synaptic plasticity responses elicited by H89 may be occurring through activation of AKT. That observation could explain why these H89 effects are occurring due to activity that is unrelated to its
inhibition of PKA.
While much more study is needed, preclinical studies such as Chong et al. and Song et al. exemplify the importance of deeply investigating the potential for known compounds to slow or halt disease progression. In some cases—such as H89—this may even require a fresh look at a “classic” inhibitor.
For more information on how Transcreener® technology can be used as a robust Protein Kinase A assay, we suggest “Determination of Km for ATP and Kemptide with Protein Kinase A Enzyme using Transcreener® ADP2 FP Assay,” an application note published by researchers at Bellbrook Labs.
1 Source: Parkinson’s Disease Foundation
2 Source: Alzheimer’s Association
3 Source: Centers for Disease Control
4 Perrin et al. 2007. WVD2 is a novel microtubule-associated protein in Arabidopsis thaliana. Plant J 49:961–971.