A perspective on recent findings and future strategies for reactive aldehyde removal in spinal cord injury

Seth A.Herr, Anna J.Prall, Riyi Shi

Acrolein in spinal cord injury:The propensity of reactive aldehydes such as acrolein to both initiate and perpetuate tissue damage after spinal cord injury (SCI) is well established.Formed primarily from lipid peroxidation, acrolein is known to be one of the most reactive aldehydes.Acrolein will quickly overwhelm endogenous clearance mechanisms and antioxidants, and form adducts with lipids, proteins, and DNA.Acrolein is proinflammatory and contributes significantly to cellular stress, eventually leading to cell death of neurons (Figure 1).After a neurotrauma, acrolein forms immediately and remains significantly elevated for at least 2 weeks (Burcham, 2017).When injected at physiologically relevant concentrations, acrolein-induced pathologies mirror those seen in trauma (Burcham, 2017).On the other hand, when acrolein is successfully sequestered, significant benefits including reduction of pain, increased locomotion, and restoration of cord tissue structure are observed.Yet, more complete removal of acrolein proves challenging given the high concentrations of acrolein that are generated after injury.There are currently no Food and Drug Administration(FDA)-approved medications for SCI patients that remove acrolein, and promote recovery, despite the known benefits.One of the main challenges of establishing an effective anti-acrolein based therapy for clinical usage is the need for small molecule aldehyde scavengers to cross the bloodbrain barrier and do so at concentrations that will remove acrolein effectively without harmful side effects.

New strategies to enhance endogenous pathways for acrolein removal:Traditional acrolein scavenging has utilized exogenous small molecules and antioxidants.However, many endogenous mechanisms already exist in the cell,such as glutathione, NRF/Keap2, and aldehyde dehydrogenases, including mitochondrial aldehyde dehydrogenase (ALDH2).Without intervention,reactive aldehydes can propagate for weeks through lipid peroxidation, and overwhelm endogenous systems which are slow to respond and often require the production of new proteins.In contrast with traditional small molecule scavengers, Alda-1, a known ALDH2 activator, does not directly bind acrolein for clearance.Instead, it catalyzes the activity of ALDH2 by approximately 2 times above the physiological baseline, and increases many folds when ALDH2 is depressed.Alda-1 is a small molecule capable of crossing the blood-brain barrier and having a beneficial effect on several animal models of neurological disorders including stroke, alcoholism, and depression.However, the ability to remove acrolein in SCI by enhancement of the endogenous ALDH2 pathway has never been explored before and is the subject of our recent publication,critical role of mitochondrial aldehyde dehydrogenase 2 in acrolein sequestering in rat spinal cord injury(Herr et al., 2022b).After acute administration, we observed significant acrolein reduction in cord tissue in rats given Alda-1.We further found that Alda-1 can protect fromtissue damage determined by measuring the size of cord cysts.Finally, we show that chronic Alda-1 administration at a high daily dose for 2 weeks caused a significant reduction in pain behavior and increased locomotion (Herr et al., 2022b).Such findings reflect those of previously used aldehyde scavengers hydralazine and phenelzine.Interestingly, we found a significant decrease in the expression of ALDH2 after SCI, but no significant decrease after acute Alda-1 administration when compared with control (healthy rats).This suggests that Alda-1 may be important for the preservation of this enzyme (Herr et al., 2022b).

Figure 1｜Cartoon demonstrating various mechanisms of acrolein removal following spinal cord injury.

Our study and others suggest Alda-1 can provide enzymatic protection from reactive aldehydesin vivo.At certain levels, aldehydes overwhelm ALDH and they no longer function (Yoval-Sánchez and Rodríguez-Zavala, 2012).Many different types of ALDH’s exist, including ALDH1A1 and ALDH3A1,although their relevance to spinal cord injury tissue remains unclear due to few publications examining them.While ALDH2 shows the highest catalytic efficiency for acrolein, other ALDH subtypes may still prove effective targets for the removal of acrolein or related reactive aldehydes(Yoval-Sánchez and Rodríguez-Zavala, 2012).Even though it is the most effective, ALDH2 is also the most vulnerable to inactivation, possibly caused by adduct formation at the site of NAD+binding(Yoval-Sánchez and Rodríguez-Zavala, 2012).Alda-1 acts not only as a catalyst but also allows the enzyme to persist through surging aldehyde concentrations, making it an ideal and unique catalyst for ALDH2.The stability of some ALDH’s(some resistant even to mM concentrations of acrolein, while ALDH2 is inactivated in the uM range), but a slower rate of aldehyde clearance,indicates that other ALDH subtypes may benefit from catalysts as well (Yoval-Sánchez and Rodríguez-Zavala, 2012).Already, similar ALDH catalysts are being studied, including Alda-89,AD-9308, and even omeprazole, and could prove effective alone or when combined with Alda-1 post SCI (Calleja et al., 2020; Lee et al., 2021).

Most ALDH’s including ALDH2 are oxidoreductases and require NAD+as a cofactor.Thus, while Alda-1 can catalyze this enzyme, NAD+availability remains a rate-limiting step.Few studies have examined NAD+in SCI; however, some suggest that NAD+levels are significantly lowered after injury.For instance, in a weight drop model of traumatic brain injury, NAD+levels were significantly lower in the injury group, when compared with sham.This could limit the effectiveness of Alda-1.However,it should be considered that variability exists amongst different labs, for instance, standard rat chow may have different levels of NAD+precursor niacin (here, our standard chow is fortified with niacin).In order to effectively study NAD+levels in SCI, diets with more relevant levels of niacin,instead of an excess that’s not needed for healthy survival, should be used.Furthermore, the amount of chow each animal consumes after injury should be measured.Relevantly, NAD+boosting has shown benefits in lowering oxidative stress in spinal cord injury, including lowering reactive aldehydes(Xie et al., 2017).There is also strong evidence that NAD+boosting protects from myelin damage and can even rejuvenate lost myelin (Kobayakawa et al., 2019).Thus boosting NAD+appears to be a logical step in both enhancing ALDH2 and protecting cord tissue from lipid peroxidation and myelin damage.

New strategies through exogenously applied scavengers to remove acrolein:While enhancing endogenous ALDH2 activity to sequester acrolein is novel and promising, using exogenous acrolein scavengers has a relatively long history with much success in promoting functional recovery in SCI (Burcham, 2017).For example, traditional scavengers such as FDA-repurposed drugs hydralazine, phenelzine, and dimercaprol have been administered to remove acrolein (Hill et al.,2019; Shi et al., 2021).While these scavengers show significant benefits, they are unable to fully remove acrolein.Boosting the levels of these drugs will likely increase the power of clearing acrolein, but unfortunately will be accompanied by potentially harmful side effects.

In light of these challenges, we have adopted a strategy of utilizing drug-targeting technology to re-engineer traditional exogenous scavengers,aiming to increase its efficacy and reduce side effects.The primary goal is to modify a known effective exogenous acrolein scavenger to reduce its side effects and enhance its translation ability for clinical usage.For instance, while hydralazine is one of the most examined and best-known aldehyde scavengers, it is an FDA-approved medication for lowering blood pressure.Thus,despite its proven benefits after SCI and safety for human use, lowering blood pressure is a serious side effect that hinders its application for human trauma patients who may have circulatory and blood pressure issues due to SCI (Kish et al., 2021).In our recent publicationTargeted delivery of acrolein scavenger hydralazine in spinal cord injury using folate-linker-drug conjugation, we took advantage of the fact that during instances of high inflammation, dividing cells including microglia and macrophages will express the folate receptors alpha and beta (FOLR) (Herr et al., 2022a).Folate is used in DNA synthesis and therefore selectively upregulated on dividing cells (Ebara,2017).Because SCI is hallmarked by significantly increased levels of inflammation (actively dividing FOLR positive cells) and therefore high levels of folate receptors at the injury site, we aimed to selectively target drugs to those cells through the binding of the folate receptors.Specifically, by chemically conjugating desired payloads to folate,we delivered such payloads through a trojan horse mechanism and receptor-mediated endocytosis.Our results show that the folate targeting strategy delivers drugs directly to the site of injury, (and much less to the adjacent regions of the cord), as indicated by folate conjugated to a near infrared dye.Furthermore, IBA1+macrophage/microglia,which are major producers of oxidative stress and acrolein, were shown uptaking the folatelinked dye.Such selective targeted delivery largely eliminates hydralazine’s effect on blood pressure,yet preserves its acrolein scavenging capabilities(Herr et al., 2022a).

The ability of hydralazine to lower blood pressure involves direct action on vascular smooth muscle.Smooth muscle will cause vasoconstriction when internal Ca2+concentrations increase in the cells.Hydralazine is believed to act directly as an inhibitor of inositol trisphosphate, which reduces intracellular Ca2+release from the sarcoplasmic reticulum, thus lowering cellular calcium levels and promoting vasodilation.However, with the extensive chemical modification involved with folate-hydralazine, it is unlikely that this drug will enter FOLR-negative cells and still act as an inhibitor of inositol trisphosphate.Therefore,while the majority of the folate-hydralazine drugs will enter FOLR-positive cells, the remaining compounds may not be able to enter FOLRnegative cells, further reducing its possible side effect.Interestingly, a few studies have shown that increased folate improved outcomes after SCI.However, in our study, folate was chemically modified and cells would no longer be able to use our drugs in a similar manner as they use unconjugated folate (such as for DNA synthesis).Thus, any observed benefits from our drug targeting will likely be due to the delivery of the payload (hydralazine).

As an additional benefit of drug targeting,concentrations of the drug will increase in the targeted region at much higher levels than in blood, due to the process of continual receptor recycling, which internalizes the drug.Therefore,folate-hydralazine could be effective even if administered in nanomole doses, as done with other folate-conjugated drugs including FDAapproved Cytalux (OTL38).Since hydralazine only lowers blood pressure when at micromolar levels in the blood, folate-hydralazine could be dosed at low enough concentrations, (nanomole level), to avoid interference with blood pressure, but still reach desired concentrations in injured tissue.

Conclusion:SCI represents a great medical challenge.Despite its relevance to injury pathogenesis, aldehydes remain a problem as there is no approved therapy targeting aldehyde accumulation in humans.Traditional aldehyde scavengers show effectiveness in scavenging acrolein, but are often accompanied by undesirable side effects and warrant chemical improvements to mitigate side effects or discover additional novel anti-acrolein treatments.Our recent investigations have examined new strategies for both endogenous and exogenous removal of acrolein, as summarized inFigure 1.Endogenously, we have shown that Alda-1 works to remove acrolein post SCI by potentiating a mitochondria bound acrolein-metabolizing enzyme, ALDH2.Furthermore, many additional opportunities exist in this line of investigation,including a combination of Alda-1 with other novel ALDH catalysts, or supplementation with cofactor NAD+boosting compounds.Exogenously, we have found that traditional acrolein scavengers can be targeted to the injury site, enhancing/preserving function and eliminating side effects.With these initial new and promising results involving both endogenous and exogenous strategies, further studies can be initiated to examine the long-term benefits of these improved therapeutic strategies.

Seth A.Herr, Anna J.Prall, Riyi Shi*Center for Paralysis Research, Purdue University,West Lafayette, IN, USA (Herr SA, Prall AJ, Shi R)Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, West Lafayette, IN, USA (Herr SA, Prall AJ, Shi R)Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA (Shi R)

*Correspondence to:Riyi Shi, MD, PhD,riyi@purdue.edu.https://orcid.org/0000-0002-7297-9428(Riyi Shi)

Date of submission:November 7, 2022

Date of decision:December 21, 2022

Date of acceptance:January 12, 2023

Date of web publication:March 3, 2023

https://doi.org/10.4103/1673-5374.369107

How to cite this article:Herr SA, Prall AJ, Shi R(2023) A perspective on recent findings and future strategies for reactive aldehyde removal in spinal cord injury.Neural Regen Res 18(10):2190-2191.

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