# S‐Propargyl‐Cysteine Attenuates Stroke Heterogeneity via Promoting Protective Autophagy Across Multiple Neural Cell Types: Insights From Single‐Cell Sequencing

## TL;DR

This study shows that S-propargyl-cysteine (SPRC) protects the brain after stroke by boosting protective autophagy, even without key hydrogen sulfide enzymes.

## Contribution

SPRC's neuroprotective effects are revealed to be independent of CBS/3-MST enzymes, offering a novel therapeutic mechanism for stroke.

## Key findings

- SPRC significantly reduces brain injury and improves neurological outcomes in stroke-affected rats.
- SPRC upregulates protective autophagy and preserves endogenous H2S levels post-stroke.
- SPRC provides neuroprotection even when CBS and 3-MST are knocked down, indicating a non-enzymatic mechanism.

## Abstract

Stroke, predominantly ischemic, is a leading cause of mortality and disability worldwide. Despite advances in intervention strategies, effective treatments to mitigate neurological injury post‐ischemic stroke remain limited. Hydrogen sulfide (H2S), a gas signaling molecule, has been implicated in neuroprotection, but its role in stroke is controversial. S‐propargyl‐cysteine (SPRC), an H2S donor, has shown great potential in protecting against neurological injuries, but its mechanisms in ischemic stroke are not fully understood. This study investigates the neuroprotective potential of SPRC and its mechanisms, focusing on the interplay between H2S and autophagy in modulating the cerebral microenvironment post‐stroke.

We conducted a comprehensive single‐cell RNA sequencing analysis on ischemic brain tissue to elucidate the cellular heterogeneity and specific responses related to H2S synthesis and autophagy. We utilized the GEO repository dataset GSE174574, applying stringent filtering and batch effect correction using the Harmony R package. Cellular subpopulations were identified using established markers, and H2S and autophagy scores were calculated using the JASMINE package. We also measured serum H2S levels, evaluated the pharmacodynamics of SPRC in rats, and constructed a cerebral ischemia–reperfusion (I/R) injury model to assess the neuroprotective effects of SPRC. Additionally, we examined the role of SPRC in CBS and 3‐MST knockout mice to determine the dependency on these H2S synthetases.

Our findings revealed a dysregulation in the expression of H2S and autophagy‐related genes in central nervous system cells, particularly in neurons, following stroke. SPRC administration significantly improved neurological behavior, metabolic activity, reduced brain infarction size, and ameliorated ultrastructure changes in stroke‐affected rats. Interestingly, SPRC continued to provide neuroprotection even after the knockdown of CBS and 3‐MST, indicating a CBS/3‐MST‐independent mechanism. Furthermore, SPRC preserved the endogenous H2S level and strongly upregulated protective autophagy.

This study is the first to reveal the neuroprotection of SPRC in cerebral I/R injury in a classical enzymatic CBS/3‐MST independent manner. The potential cellular and molecular mechanisms may rely on the promotion of SPRC to activated protective autophagy. Our results suggest that SPRC could be a promising therapeutic candidate for enhancing neuroprotection and modulating autophagy in ischemic stroke.

Stroke is one of the major contributors to mortality and long‐term disability. Treatments aiming at regulating endogenous protective mechanisms to reduce neurological injury after ischemic stroke are still urgently needed. Herein, we investigated the neuroprotective potential of S‐propargyl‐cysteine (SPRC), a novel water‐soluble donor of H2S, and its possible mechanisms. Histologically, it was observed that the percentage of cells expressing hydrogen sulfide‐related genes in stroke‐affected brain tissues was lower compared to those in healthy brain samples. Autophagy, an emerging area of research in stroke studies, has been shown to modulate the cerebral microenvironment. Nevertheless, the relationship of hydrogen sulfide and autophagy levels remains elusive, which reason is partly due to the inherent heterogeneity in cellular populations present in stroke lesions. This investigation provides a thorough examination of the single‐cell transcriptomic profiles pertaining to hydrogen sulfide synthesis and autophagy across a spectrum of cellular phenotypes within ischemic brain tissue. Our findings reveal dysregulation in the expression of hydrogen sulfide and autophagy‐related genes, particularly in the central nervous system, such as microglial cells, astrocytes, glial cells, and neurons. Moreover, the significant increase in correlation between hydrogen sulfide and autophagy levels was greater in neurons of stroke compared to the control group. Experimental results showed that neurological behavior assays, metabolic activity, brain infarction size, and ultrastructure changes were significantly ameliorated by SPRC administration. After knockdown of cystathionine‐β‐synthase (CBS) and 3‐mercaptopyruvat sulfurtransferase (3‐MST), SPRC could still alleviate neurological injury. Meanwhile, SPRC could preserve the endogenous balance of H2S levels. Furthermore, protective autophagy was strongly upregulated by SPRC administration. Our results first revealed the neuroprotection of SPRC in cerebral I/R injury in a classical enzymatic CBS/3‐MST‐independent manner, and the potential cellular and molecular mechanisms may rely on the promotion of SPRC to the activated protective autophagy.