A novel disease connection for TRPM2 channels

Alzheimer's Disease (AD) is a progressive neurodegenerative condition responsible for dementia in older adults and for which no effective treatment is available. Amyloid-β (Aβ), a proteolytic product of amyloid precursor protein (APP), is recognized as a primary pathogenic molecule in AD, but the exact mechanism(s) by which this protein induces brain dysfunction and cognitive impairment has not been fully resolved. In the present study, the authors have examined the cellular pathways by which Aβ induces vasoconstriction of cerebral arteries and vascular dysfunction, leading to compromised cerebral blood flow (CBF) and glucose/oxygen delivery to the brain. As now reported by Park and coworkers, Aβ can directly impair brain endothelial cells via a cascade of events culminating in the opening of transient receptor potential melastatin-2 (TRPM2) channels and Ca²⁺-related cellular dysfunction. Given its important role in maintaining adequate blood flow, impairment of vascular endothelium would have negative consequences on neuronal viability and contribute to the pathogenesis of AD.

In anesthetized mice with cranial windows, neocortical superfusion of 5 μM Aβ protein 1–40 reduced basal CBF, as measured by a laser-Doppler flow probe, and blunted CBF responses induced by sensory stimulation (i.e. whisker movement) and local application of the endothelium-dependent vasodilator acetylcholine. However, Aβ did not impair CBF responses to adenosine, a smooth muscle relaxant. Aβ mediated reductions in CBF correlated with the presence of peroxynitrite in cerebral arteries, as detected by 3-nitrotyrosine fluorescence imaging. Peroxynitrite (ONOO⁻) is an oxidative chemical generated by the reaction of ROS with nitric oxide (NO), a potent vasodilator synthesized in vascular endothelial cells. Functionally, Aβ appears to interact with innate immunity receptors (i.e. CD36) on endothelial cells to stimulate ROS production via NADPH oxidase and overproduction of ROS would be expected to promote peroxynitrite formation and reduce NO levels. Data demonstrating that chemical scavengers of peroxynitrite, genetic inactivation of NADPH oxidase or inhibition of endothelial NO synthase (eNOS) could prevent the Aβ mediated decreases in blood flow confirmed the importance of peroxynitrite in the cerebrovascular effects of Aβ.

Peroxynitrite is known to cause DNA damage and the authors observed that neocortical superfusion of Aβ activated the DNA repair enzyme poly-ADP-ribose polymerase (PARP) in penetrating cortical arterioles. Interestingly, pharmacologic inhibition of PARP prevented Aβ evoked decreases in both basal and stimulated CBF, whereas Aβ had no effects on CBF in PARP1 knockout mice. Inhibition of poly-ADR-ribose glycohydrolase (PARG), which degrades poly-ADP-ribose to generate ADP-ribose monomers, also prevented the negative effects of Aβ on resting and stimulated blood flow. So, how would the activities of DNA repair enzymes contribute to the vasoconstrictor actions of Aβ? The authors hypothesized that PARP/PARG activities may lead to elevated levels of ADP-ribose in endothelial cells, leading to stimulation of ADP-ribose-sensitive targets, such as the TRPM2 channel. In cultured murine brain endothelial cells, patch clamp recordings revealed that 300 nM Aβ protein 1–40 stimulated TRPM2-like whole cell currents that were sensitive to the pharmacologic TRPM2 blockers 2-APB (30 μM) and ACA (20 μM). Knock down of TRPM2 protein expression using siRNA treatment (confirmed by western blot) also prevented the Aβ evoked increase in whole cell current magnitude. Importantly, stimulation of TRPM2-like currents by Aβ was also blocked by a chemical scavenger of ROS, inhibition of NO synthase and NADPH oxidase or inhibitors of PARP and PARG; these agents, however, did not prevent enhancement of TRPM2 currents by intracellular application of ADP-ribose. This latter observation indicates that ADP-ribose mediated TRPM2 activation is downstream of peroxynitrite formation and PARP/PARG activities. As TRPM2 channels are Ca²⁺ permeable, it was further observed that Aβ evoked a significant elevation of cytosolic Ca²⁺ in cultured endothelial cells loaded with the Ca²⁺-sensitive fluorescent dye Fura-2. These elevations were blocked by siRNA-mediated knock down of TRPM2 protein and pharmacologic inhibitors of TRPM2 channels. TRPM2 blockers also prevented the depression of CBF by Aβ under stimulatory conditions (i.e. whisker movement or acetylcholine superfusion) and restored normal CBF responses in a transgenic mouse model of AD (i.e. tg2576) overexpressing a mutant form of APP. Finally, Aβ had no effects on CBF responses in TRPM2 knockout mice, demonstrating the critical role of this channel in the Aβ mediated impairment of CBF regulation.

Based on their observations, the authors suggest that the binding of Aβ to cerebral endothelial cells stimulates NADPH oxidase activity to generate ROS, which combines with NO to form peroxynitrite and cause DNA damage. The ensuing activation of PARP and PARG activities results in the generation of free ADP-ribose and the sustained activation of TRPM2 channels, leading to excessive Ca²⁺ influx and endothelial dysfunction. The subsequent disruption of vascular function would thus be expected to compromise CBF regulation and promote ischemic brain injury. Collectively, these observations suggest that suppression of Aβ induced oxidative stress and TRPM2 channel activity in cerebral arteries may counteract the pathogenesis of AD. As extracellular Aβ can accumulate in the walls of cerebral blood vessels (i.e. cerebral amyloid angiopathy) of AD patients, it will be of interest to elucidate whether the acute effects of Aβ observed in this study may also help explain the progressive brain deterioration and cognitive decline that occurs over several years in AD patients.