Autophagy is an intracellular mechanism by which cells remove damaged or senescent cytoplasmic components. Autophagy helps maintain cellular homeostasis by eliminating dysfunctional organelles, misfolded proteins, and other cellular debris. There is growing evidence that autophagy, observed in a variety of cell types, plays a key role in brain pathology after ischemic stroke. Thus, the regulation of autophagy is a potential target for ischemic stroke therapy.

Ace Therapeutics, a leading company in the field of stroke research, offers comprehensive analytical services to investigate the role of autophagy in stroke, encompassing key signaling pathways and various autophagy subtypes, including mitophagy, pexophagy, aggrephagy, endoplasmic reticulum-phagy, and lipophagy. Leveraging advanced methodologies and strategic partnerships, we empower our clients to develop innovative stroke therapies that selectively target autophagy-related signaling pathways.

Related Services

• Autophagy Mechanism Analysis in Stroke
• Analysis of mTOR-related Signaling Pathways in Stroke
• Analysis of Beclin 1/Bcl-2 Signaling Pathway in Stroke

Overview of Autophagy Process and Signaling in Normal Condition

Autophagy plays a critical role in cellular growth and differentiation and is essential for maintaining cellular homeostasis by ensuring the continuous turnover of dysfunctional proteins and organelles.

Autophagy Process in Normal Condition

(1) Sequestration: A specialized membrane, known as the isolation membrane, engulfs cytoplasmic components, including organelles, forming an autophagosome.

(2) Transportation: The autophagosome transports the sequestered material to the lysosome.

(3) Degradation: The autophagosome fuses with the lysosome, forming a structure referred to as an "autolysosome" or "autophagolysosome." Lysosomal hydrolases then degrade both the inner membrane and the contents of the autophagosome.

(4) Recycling: The degradation products are exported to the cytoplasm for reuse, contributing to cellular renewal and energy efficiency.

Autophagy relies on the coordinated interaction of various complexes composed of autophagy-related (Atg) proteins. These include ubiquitin-like conjugation systems(Atg12-Atg5 and Atg8/LC3-PE conjugation systems), phosphatidylinositol 3-kinase (PI3K) complex, Atg1/Unc-51-like kinase (ULK) complexes, mAtg9, and Atg2-Atg18 complexes.These complexes work together to regulate and execute the autophagy process

Types of Autophagy

There are three types of autophagy, including macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA), each differing in cargo delivery to lysosomes. They are difference in manner and physiological function in which they deliver cargo to the lysosomal cavity.

• Macroautophagy is a catabolic process characterized by sequestering cytoplasmic matter in a double-membrane vacuole, known as an autophagosome, which is then transported to the lysosome for degradation.
• Microautophagy is non-selective and involves lysosomal membrane invagination for degradation of cytoplasmic components and membrane regulation.
• CMA selectively degrades proteins with KFERQ motifs through chaperones like HSC70.

Autophagy Signaling in Normal Condition

Autophagy is regulated by a complex signaling network, with mTORC1 (mechanistic target of rapamycin complex 1) and AMPK (AMP-activated protein kinase) as key upstream regulators. Under nutrient-rich conditions, mTORC1 is hyperactivated, inhibiting the ULK1 kinase complex and reducing autophagy. In contrast, during nutrient depletion, AMPK activates ULK1 by phosphorylating it and suppressing mTORC1, enhancing autophagy.

Selective autophagy enables the degradation of specific cellular components like damaged mitochondria, aggregated proteins, excess peroxisomes, and pathogens, maintaining intracellular homeostasis. This selectivity is mediated by autophagy receptors, which recognize specific cargo through protein modifications in response to proteotoxic stress.

Fig. 1 Schematic overview of the main pathways involved in the regulation of autophagy. (Stanzione, et al., 2024)

The Dual Role of Autophagy in Ischemic Stroke

Protective Role of Autophagy in Ischemic Stroke

After ischemic stroke, Beclin-1, a key regulator of autophagy, is overexpressed, initiating autophagosome formation and autophagy through the Beclin-1-Vps34-Vps15 complex. Overexpression of Beclin-1 causes DNA damage but does not always lead to cell death. Autophagy inhibitors (e.g., 3-MA) reduce Beclin-1 expression and shift cell death from apoptosis to necrosis. Conversely, rapamycin induces autophagy, upregulates Beclin-1, decreases cell death, and reduces brain damage.

Detrimental Role of Autophagy in Ischemic Stroke

Overactivation of autophagy can worsen brain injury while inhibiting autophagy aids in neural tissue recovery after cerebral ischemia/reperfusion. Loss of the autophagy gene Atg7 in a neonatal rat ischemia model reduced autophagic neuronal death and brain damage, indicating the protective effects of autophagy inhibition.

Fig. 2 Dual role of autophagy in ischemic stroke. (Stanzione, et al., 2024)

Autophagy Signaling Pathways in Ischemic Stroke

Cerebral ischemia can activate multiple signaling pathways that subsequently feed into the autophagy pathway.

Fig. 3 Possible autophagy signaling pathways in cerebral ischemia. (Chen, et al., 2014)

Autophagy as Therapeutic Target in Ischemic Stroke

Restoration of autophagy in models of ischemic stroke in most cases exerts beneficial effects and reduces brain injury. To date, several strategies have been developed to enhance autophagy with pharmacological agents.

Autophagy can be inhibited using pharmacologic inhibitors or by knocking down specific proteins such as Beclin 1, LC3, and Atgs. PI3K inhibitors like 3-methyladenine (3-MA), LY294002, and wortmannin are effective tools for suppressing autophagy by inhibiting autophagosome formation. On the other hand, autophagy induction can be achieved with drugs like rapamycin and RAD001, which inhibit mTOR. However, mTOR inhibitors lack specificity, making interpretation of results challenging. Despite the widespread use of autophagy-regulating drugs in research, their low specificity can lead to uncertainties in conclusions, especially regarding ischemic neuronal damage.

1. Stanzione, R., et al. (2024). Role of autophagy in ischemic stroke: insights from animal models and preliminary evidence in the human disease. Frontiers in Cell and Developmental Biology, 12, 1360014.
2. Chen, W., et al. (2014). Autophagy: a double-edged sword for neuronal survival after cerebral ischemia. Neural regeneration research, 9(12), 1210-1216.