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A Squirrely New Stroke Discovery

by Rose Liu art by Luke Estrada

Walking through Morningside Park earlier this fall, you might have seen some squirrels scurrying around. These fluffy critters were preparing for hibernation, and their ability to return from this state of torpor might hold the key for stroke patients a block away at Mt. Sinai Hospital.

Deep in their underground hideout, hibernating ground squirrels undergo a complex process of metabolic depression characterized by decreased body temperature and cerebral blood flow, or ischemia [1]. These mammals show a remarkable capacity to tolerate drastic fluctuations in oxygen availability without significant brain damage [2]. The disruption of blood supply to the brain often leads to serious health consequences in stroke patients, yet these hibernating mammals seem to endure prolonged periods of ischemia without any significant brain injury [2]. Researchers have observed that a biological process called SUMOylation increases in hibernating squirrels, and further investigation of this pathway may lead to the development of novel stroke treatments.


What is an Ischemic Stroke? What is a Stroke? What is…?

Tripping and falling, you keep losing your balance. You try to stay still, but the people around you multiply suddenly in double vision. The world is spinning, so you desperately reach for a wall to lean against, but your arm is numb. Soon, sirens are blaring. You’re rushed to the ER. Doctors and physicians whizz around you in a blur as you wonder—how exactly did I get here?

While you will hopefully never have to experience a stroke, this situation is a life-changing event for over 800,000 Americans annually [3]. As a matter of fact, stroke is one of the leading causes of disability in the United States. If you have high blood pressure, smoke cigarettes, or have heart disease, there’s a good chance that your stroke is what is called an ischemic stroke [4]. Ischemic strokes are the most common in the United States, accounting for 87 percent of reported strokes [5]. An ischemic stroke occurs when blood flow to the brain is interrupted by blood clots [5]. Clots are formed by immune cells called platelets when a fatty plaque in the brain artery ruptures [6]. The body’s immune response sends platelets to mend the opening by forming a clot directly at the damage. These blockages prevent blood from flowing down to critical areas [7].


But how does this blockage cause your brain cells to die?

After a clot, there exists a mismatch between cerebral blood flow and metabolic demands for oxygen and glucose in the brain, as well as insufficient waste clearance [8]. This deficiency is what causes damage to the neurons, leading ion pumps to fail and cellular waste to build up [9]. The failure of these ion pumps lead the neuron to fill with excess sodium and calcium, which are toxic for neurons [10]. The excess calcium results in depolarization of the neurons, and they will thus fire more action potentials, increasing metabolic demands [10]. This process where neurons fire themselves to death is called excitotoxicity [11]. Because some neurons make long, projecting connections, diaschisis can occur, where neurons far from the ischemic core can be damaged [12]. As cells in the core experience progressive damage, further complications like neuroinflammation and oxidative stress occur and generate further damage and neuron death [10, 13]. Even after blood flow is restored, there is often reperfusion injury, in which the suddenly increased blood flow causes increased inflammation and oxidative stress [14].

Neuroinflammation is a form of further cerebral damage after stroke onset, which is instigated by sodium and calcium accumulation in neurons [10]. The increased sodium levels cause the neuron to take in more water, which then causes the neuron to swell and undergo apoptosis, or cell death [15]. The increased calcium levels activate harmful enzymes that damage cell organelles and lead to further cell death [16].

Oxidative stress, another main cause of brain tissue damage, occurs after stroke onset [13]. Oxidative stress is a condition where antioxidant levels are low, and there is an imbalance between reactive oxygen species and antioxidant defenses [17]. A reactive oxygen species is an unstable molecule that contains oxygen and has the capacity to damage biomolecules [18]. The accumulation of toxic reactive oxygen species can lead to cell and tissue damage [17]. Depending on where the initial blockage is, different brain regions could die [19]. Because each area of the brain controls different body functions, cell death in one area could lead to a loss of the motor, cognitive, and behavioral functions with which this area is associated [19].


Save Me! Treatment Options for Strokes

While many of us may know someone who has suffered from a stroke, not all of us understand what treatments they might have undergone. There are currently numerous treatments for ischemic stroke, the most popular being emergency intravenous (IV) medications [20].

The drug recombinant tissue plasminogen activator (rTPA) is the most common IV medication used for ischemic strokes, and is administered through a catheter. It works by dissolving the blood clot, thus restoring blood flow [20]. More specifically, when IV-rTPA interacts with the blood clot, it cleaves the peptide bonds in the proteins and creates plasmin—the primary enzyme in dissolving blood clots [21]. For IV-rTPA to work effectively, it must be administered within three hours after stroke, a significant limitation to this treatment option [20]. With administration after three hours not being FDA approved, doctors must carefully decide whether to administer IV-rTPA after the recommended time frame. Although clinical studies have shown that administering IV-rTPA within three to four hours improves clinical outcomes [22], administering IV-rTPA four-and-a-half hours after stroke onset has been shown to increase the risk of death [23]. This time dependency of treatment can make utilization difficult because before medication can even be administered, doctors must first perform numerous tests to identify the type and location of the stroke [24]. It’s critical for doctors to identify that the stroke is indeed a case of ischemic stroke rather than the almost identical hemorrhagic stroke, for which the treatment would be lethal [25]. This identification process can take around 45 minutes from the time a patient is admitted to a hospital [26], which is a large amount of time during a medical crisis in which every minute counts.

Despite its time restrictions, IV-rTPA proves beneficial. When compared to patients that are not treated with IV-rTPA, patients treated with IV-rTPA between zero and three hours after onset are more often discharged home rather than to a nursing home and report fewer cases of permanent impairment [27, 28]. In a 2013 study, the percentage of patients who recovered successfully from severe stroke when using IV-rTPA was 35 percent, which was comparable to the success rate of more invasive stroke therapies [29]. Among patients with moderate stroke symptoms, the recovery rate was 74 percent [29].

With America’s growing population of aging adults, novel developments in stroke treatments could greatly benefit this population’s health.


Just Sleep it Away: Hibernation

To investigate hibernation, scientists have turned to ground squirrels as an important model for treatment purposes. Although hibernating animals experience the same lack of blood flow as ischemic stroke patients, these animals are able to return to their previous state with no brain damage [2].

Contrary to popular belief, hibernation is not just one continuous sleep. It is actually categorized by periods of “torpor,” in which the animal “sleeps,” and interbout arousal, in which the animal wakes up [30]. During torpor—the “sleep”—oxygen consumption of squirrels drops to just two to three percent of its original rate. Their core body temperatures drop from 37°C to 4-6°C, and their heart rates drop to 3-10 beats/min, compared to its usual 200-400 beats/min when the animal is active. The duration of torpor can vary from one day to many weeks, and afterward, the squirrel wakes up into interbout arousal [30]. Interbout arousal is a period of 24 hours in which the animal’s body temperature and other homeostatic functions return to normal [31]. Then, the cycle repeats.


SUMO & Hibernation

One common trend scientists have observed within ground squirrels’ neural activity is the correlation between hibernation and a specific chemical process that modifies the function of various proteins within the brain—SUMOylation [32]. Increased SUMOylation activity is found in the brains of hibernating ground squirrels [32]. This correlation has been observed and studied since 2007, when research groups found that the increased SUMOylation had neuroprotective mechanisms against oxygen deprivation and hypothermia during the squirrels’ hibernation [33].

Oxygen deprivation is a direct cause of ischemic strokes and leads to cell death in the ischemic core and penumbra of human brains [9]. However, it is also important to note that scientists still cannot say with certainty how SUMO protects against ischemic damage. Hypothermia is the significant cooling of body temperatures below 35℃ [34]. Normally, hypothermia is considered a warning sign for health, and immediate medical attention should be offered [35]. However, it is also a beneficial and common surgical technique that allows surgeons to operate on a patient during cardiac operations by decreasing metabolism and therefore decreasing oxygen and energy demands by the body, which would in turn decrease the burden on the heart to pump large volumes of blood throughout the body [36]. Some surgeons take this beneficial idea of hypothermia for reducing metabolism a step further and induce suspended animation onto the patient—the patient’s blood is replaced with cold, glucose-laced saline solution to reduce energy demands on the brain—through a technique known as emergency preservation and resuscitation [37].

Combating the impacts of oxygen deprivation and hypothermia helps the animal maintain good health. Luckily for us, SUMOylation is a cellular process humans experience as well [32].


Clearing up SUMOylation

But what exactly is SUMOylation? SUMOylation is a post-translational chemical modification in which SUMO, a Small Ubiquitin-like Modifier, is added to a target protein. A post-translational chemical modification is either the cleavage or addition of a functional group to a protein after it has been synthesized [38]. By changing the chemical composition of a protein, the 3-D conformation of the protein changes, allowing it to have different functions [38]. Ubiquitin is a regulatory protein that aids in synthesizing and degrading defective proteins [39]. Despite its name, SUMO does not share the degradative properties of ubiquitin—rather, SUMO’s name derives from the structural similarities between SUMO and ubiquitin [40].

In the case of SUMOylation, SUMO polypeptides attach to the protein after it is synthesized. This attachment between the SUMO polypeptide and the target protein alters the target protein’s shape and function [38]. The enzymes conjugate, or attach, the SUMO molecule to the target protein [33]. By modifying proteins, SUMOylation regulates cell signaling, apoptosis, protein processing, immune response, neuronal maturation, synapse formation, plasticity, and DNA repair [33].

In the brain, SUMOylation also plays an important role in neuronal maturation, synapse formation, and brain plasticity [41]. More specifically, SUMOylation is assumed to provide cytoprotection of brain cells from oxygen-glucose deprivation [33]. Afterward, certain molecules deconjugate, or disconnect, the SUMO polypeptide from the target protein in a process known as deSUMOylation [32].

Considering SUMOylation’s restorative properties, scientists have sought to increase SUMOylation as a potential treatment for stroke. In 2018, Bernstock et al. analyzed numerous candidate molecules in order to increase SUMOylation activity by inhibiting the SUMO-specific protease SENP [42]. Proteases are enzymes that cleave peptide bonds, and a SUMO-specific protease only targets the bond between SUMO and its target protein [42, 43]. Inhibiting this SUMO-specific protease, SENP2, would prevent it from cleaving the bond between SUMO and its target protein [42]. Blocking the enzyme SENP2 from degrading SUMO would prevent the deconjugation of SUMO from SUMOylated proteins, thus increasing SUMOylation levels. Bernstock et al. revealed eight candidate molecules as potential binders to SENP2, which would decrease deSUMOylation by inhibiting SENP2 activity [42]. Among those molecules, ebselen emerged as a viable drug. Six of the eight candidate drugs were, unfortunately, toxic. The last, 6-thioguanine, was not tested: due to its use in chemotherapy, it was determined impractical for treating strokes [42].


Moving Towards a New Stroke Treatment

Ebselen emerged as the most viable drug because it blocks SENP2, which boosts SUMOylation activity [42]. This effect could make ebselen incredibly helpful in increasing SUMOylation in the brains of stroke patients. Bernstock et al. further demonstrated that, in the absence of glucose and oxygen, ebselen sustains rat cells longer than no drug at all, protecting them from oxygen- and glucose-deprivation-induced cell death [42]. In short, ebselen could be used as a stroke treatment in humans and greatly improve stroke outcomes.

This data by Bernstock et al. aligns with past research that reveals ebselen as a promising neuroprotective agent for ischemic strokes [42, 44]. Through trials, ebselen was found to be most effective as a short-term intervention, as it was most useful when administered within 24 hours of the initial stroke—far longer than the three-hour window of emergency IV medications [44]. Ebselen was found to influence key processes involved in ischemic brain damage, including decreasing oxidative stress [44].

Ebselen helps protect neurons, especially those in the ischemic penumbra, from cell death during oxygen deprivation [45]. Ebselen mimics the behavior of antioxidant enzymes, which reduce oxidative stress and free radical damage [46]. Oxidative stress is the accumulation of free radicals: molecules that have at least one unpaired valence electron and are therefore extremely reactive [47]. Together, these radicals cause oxidative stress as they bind to cells and attack their membrane [47]. Ebselen is neuroprotective because it neutralizes free radicals, which prevents radicals from destroying the lipids of the cell membrane [48]. This in turn reduces tissue damage from oxidative stress.

While ebselen was not approved as a clinical drug candidate for stroke treatment in Japan in 2000 due to limited evidence of its efficacy, Japan’s decision was largely based on results from small studies performed by one company [49, 50]. This decision led to the halt of other ongoing trials due to concerns over toxicity. Now that ebselen’s toxicity has been disproven, it is possible that there will be future clinical trials of ebselen in alternate contexts [49].

For example, even though ebselen appears ineffective on its own, it exhibits synergistic effects when administered alongside IV-rTPA in rabbit models, significantly improving behavioral outcomes [51]. Therefore, combination therapies involving ebselen and other stroke therapies may be an avenue of interest for future clinical studies [51].

But even if ebselen may not prove to have a sufficient impact on stroke patients, it doesn't necessarily mean scientists haven't learned a new avenue for intervention. The idea of increasing SUMOylation has incredible potential for influencing the development of new stroke medications, neurodegeneration therapies, and hypothermia interventions. For scientists who constantly synthesize new drugs, it's only a matter of finding the right drug for the pathway.


The Drawbacks of SUMOylation

The drawback of increasing SUMOylation to treat strokes is SUMOylation’s relationship with cancer. While SUMOylation could potentially treat ischemic stroke patients, this post-translational modification is also involved in cell replication [52]. More specifically, high levels of SUMOylation are necessary for the replication of cancer cells in stressful environments [53]. Therefore, increases in SUMOylation could increase the risk of cancer [54, 55]. Doctors would have to weigh the benefits and costs of SUMOylation before administering drugs that influence its expression. This dilemma between doctors choosing to increase or inhibit

SUMOylation could be something we see in our future, as cancer and strokes are both prevalent health concerns for an aging population.


Bright Eyed and Bushy Tailed

Morningside Heights feels chilly this winter: the ski jackets are out and the students walk sleepily to class. What these students now know is that this winter cold has many scientific applications. From hypothermia to hibernation, researchers will still keep uncovering beneficial pathways like SUMOylation. However, while scientists discovered a fascinating application of SUMOylation through observing hibernating animals, scientists also have to be cautious when applying their knowledge in clinical settings due to the delicate nature of the human body. Compounds like those discussed above that can be beneficial to one body’s process may be detrimental to others as the same molecules can impact a number of physiological functions. The nature of the SUMOylation pathway is still instrumental, and most certainly a stroke of good luck for the future of medicine.


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