BEATING ALZHEIMER'S DISEASE: NEW FLUOROGENIC MARKER

BEATING ALZHEIMER'S DISEASE

A NEW FLUOROGENIC MARKER PUTS ROUTINE TESTS FOR ALZHEIMER'S IN REACH

By Jack LeGrow Ashley Chen

Alzheimer’s disease (AD) and other neurodegenerative disorders pose some of the most concerning threats to an aging population. According to the World Health Organization, there are approximately 55 million people worldwide currently living with AD or some related form of dementia. The 7th leading cause of death internationally and the 4th among those aged 70 and older, Alzheimer’s not only induces stress and fatigue among its victims but also weighs a heavy burden on the family members of patients. Caretakers and loved ones report constant physical and emotional stress, feeling on-duty 24/7, and statistically disproportionate frictional unemployment.

Considering the distress placed on patients and their families, the Alzheimer’s Association championed clinical trials and extensive research to treat and understand AD beginning in 1978. Since then, several AD medications have proved efficient at delaying the onset of neuronal damage. Molecular causes for AD have been identified as the buildup of amyloid-beta (Aβ) and tau neuritic plaques in the cerebral cortex. Unfortunately, finding a cure for AD presents a challenge to the medical community despite massive investment and decades of research.

The primary roadblock preventing the development of a comprehensive cure for dementia has been the difficult timeline implicated in AD clinical studies. Alzheimer’s has a long prodromal period. Patients appear asymptomatic while plaques and neurofibrillary tangles accumulate in the gray matter of the brain. By the time the first symptoms appear, AD is often too far advanced to effectively treat in clinical trials. Therefore, early detection of AD is critical for meaningful advances in its treatment.

Conventionally, cerebrospinal and ocular fluid are used to sample Aβ, tau, and other biomarkers associated with the onset of AD; however, these techniques often yield inconclusive concentrations or are too invasive for early diagnoses. Conversely, AD can be effectively diagnosed throughout its early stages using amyloid-positron emission tomography (PET) and magnetic resonance imaging (MRI) to map regions of plaque buildup and neuronal damage. Unfortunately, these techniques implicate other disadvantages, including high cost and exposure to radionucleotides. To better diagnose asymptomatic AD, there is a need for low-cost, non-invasive, and spatially precise imaging to identify amyloid and neurofibrillary tangle buildup in the cerebral cortex. With such leverage, the hope is that frequent AD testing could be integrated into check-ups at the doctor’s office for patients over a certain age—as standard as blood work.

Recently, reactive oxygen species (ROS) have also become promising candidate indicators for AD diagnostics research. These species are critical in cellular and genetic signaling, and their concentration is strictly controlled for homeostatic balance. If not properly regulated, however, excessive ROS can build up and cause cellular damage in a process called oxidative stress. The buildup of Aβ plaques in AD has been directly linked to oxidative stress. Oligomer Aβ can insert itself in the cell membrane of neurons, inducing an effect called lipid peroxidation. This generates highly reactive aldehydes like acrolein, malondialdehyde, and 4-hydroxynonenal (HNE). HNE is of particular importance in AD pathology because it can readily react with cellular components containing thiol (–SH) and amine (–NH2) groups, such as nucleotides and proteins. Oxidative stress generating HNE has been shown to result in multiple deleterious events like RNA and DNA inhibition, the disruption of protein functions, and stagnated protein synthesis. These events interfere with typical neuron signaling and can initiate premature apoptosis, contributing to cognitive impairment in AD.

Oxidative stress is directly correlated with the progression of AD, and therefore, its detection can provide the same confidence as the presence of amyloids and tau in the cerebral cortex. Furthermore, ROS are direct products of plaques interacting with brain tissue, so their detection would offer the same relative spatial mapping as MRI or amyloid-PET. The advantage of ROS is that they are much more reactive than Aβ, tau, and the other biomarkers linked to AD. Thus, indicators can be designed to react with ROS in a manner that makes them detectable in the presence of ROS and undetectable otherwise—an

approach that has not previously been possible.

Considering the role of oxidative stress in Alzheimer’s pathology, researchers have developed a fluorogenic molecular probe that senses O2^˙−, a major ROS produced by oligomer Aβ and HNE reactions. They achieved this by chemically “caging” resorufin—a fluorescent dye used in bioimaging—so that nonfluorescent derivatives have their fluorescence recovered in the presence of O2^˙− through selective nucleophilic cleavage of a chemical cage unit. They appended several benzene-sulfonyl chlorides with various electron-donating and electron-withdrawing substituents to examine which resorufin probes would generate the best sensitivity to inflammatory oxidative stress stimulation in HeLa cancer cells and mice.

Pentafluorobenzenesulfonyl cage units were found to produce ideal resorufin fluorescence sensitivity in the presence of O2^˙−. With a 2.6 × 10^-7 M limit of detection, its sensitivity sits comfortably between the O2^˙− concentration expressed under normal (10^-10 –10^-12 M) and abnormal/inflamed (10^-6 M) conditions. Compellingly, this pentafluorobenzenesulfonyl-caged resorufin derivative was used to identify oxidative stress in an array of demonstrations. Human cervical epithelioid carcinoma cells stained with the molecular probe produced a strong fluorogenic response when intentionally placed under oxidative stress. The probe was given to mice who had simulated inflammatory-induced oxidative stress in their peritoneal cavity where it was capable of spatiotemporally visualizing the affected tissue in-vivo. Justification that this fluorogenic response was oxidative stress-induced was provided when the same conditions were generated and when Tiron, an O2^˙− scavenger, was introduced. These experiments produced no fluorogenic response, indicating the O2^˙−-specific fluorescence.

This new molecular probe shows promise to provide the necessary early imaging for conclusive AD diagnosis. Microglial cells, known to cause neuronal death in dementia through the release of inflammatory cytokines, were tested with the caged resorufin derivative. The molecular probe produced the same fluorogenic response when the microglial cells were placed under oxidative stress from Aβ presence. Indeed, this suggests that the probe is effective under the exact conditions of neurofibrillary tangle and plaque buildup in the early stages of AD.

Noninvasive and cheap molecular probes like pentafluorobenezenesulfonyl-resorufin open doors to a new age in Alzheimer’s treatment and the pursuit for a cure. With the potential to conclusively diagnose AD in its earliest stages by imaging oxidative stress in the cerebral cortex, patients will be able to receive preemptive treatments and participate in new studies that can eventually lead to cures starting from the disease’s asymptomatic, prodromal period. Proper diagnostics serve as the pillar for effective treatments. This cutting-edge research presents a substantial addition to the toolkit in the fight against AD and neurodegenerative disorders.

The future for oxidative stress biomarkers is vast. By demonstrating resorufin’s ability to fluoresce in cells with Aβ-induced oxidative stress, researchers can tap into the studies of cancer, Parkinson’s disease, Lou Gehrig’s disease and heart failure. Identifying localized oxidative stress in regular exams could help to diagnose dormant tumors, increased risk for organ failure, and asymptomatic neurodegenerative disorders alike. Having the benefit of early diagnosis in any disease helps save lives, not only to help delay or prevent symptoms and disease progression but also in more robust clinical trials for future cures.

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