Autophagy plays a critical role in ischemic stroke and has been identified as an important target for prevention of ischemic injury during ischemia/reperfusion events. The abnormal initiation of autophagic flux in neurons following ischemic stroke disrupts the autophagy-lysosome system, leading to both a blockage of autophagic flux and the subsequent autophagic death of neurons.

As a leading provider of stroke research services, Ace Therapeutics can help clients investigate the molecular mechanisms that lead to neuronal autophagy-lysosomal dysfunction after ischemic stroke and develop potential therapies for stroke.

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• Autophagy Mechanism Analysis in Stroke

What Is Autophagy?

Autophagy is an evolutionarily conserved process for the degradation and recycling of cellular materials through the lysosomal pathway. The process is typically divided into five stages: nucleation of the phagophore membrane, expansion, closure of the phagophore, fusion between autophagosomes and multivesicular endosomes or lysosomes, and degradation of the autophagosome contents. Disruption at any stage, such as lysosomal dysfunction, can lead to autophagy dysfunction.

Autophagy and Lysosomal Dysfunction

Lysosomes are essential organelles critical for maintaining protein and cellular homeostasis by degrading unwanted cellular components, including damaged or misfolded proteins. Lysosomal function is vital in virtually all cell types, including neurons, and any disruption in lysosomal activity can lead to detrimental effects. Lysosomes are vital for autophagy machinery, and maintenance of lysosomal functions is important for cellular hemostasis.

Autophagy-Lysosome Dysfunction After Ischemic Stroke

Autophagy can be activated in various brain cell types and serves as an endogenous protective mechanism following ischemic stroke. Autophagic flux, which involves the initiation, fusion, and digestion of autophagosomes by lysosomes, is often used to assess autophagic activity.

Disruptions in Autophagic Flux

After ischemic stroke, the hydrolytic capacity and number of lysosomes in neurons decrease, leading to increased membrane permeability. This results in insufficient lysosomal function, causing autophagic flux disorders. These disorders can impair the process of autophagy, triggering neuronal damage.

Fusion Defects

The ischemic stroke also reduces the proteins involved in the fusion of autophagosomes and lysosomes. This dysfunction prevents the formation of autophagolysosomes, further exacerbating autophagic flux disorders and contributing to neuronal autophagic death and neurological damage.

Abnormal Activation of Autophagy

In addition to deficiencies, ischemic stroke can lead to abnormal activation of autophagic flux. This results in excessive autophagosome formation and lysosomal storage disorders, further complicating the injury response.

Fig. 1 Schematic diagram of the pathogenesis of autophagy-lysosome dysfunction in neurons after ischemic stroke. (Shi, et al., 2023)

The Molecular Mechanisms of Neuronal Autophagy-Lysosomal Dysfunction After Ischemic Stroke

Instability of the Acidic Environment in Neuronal Lysosomes Following Ischemic Stroke

The acidic environment of lysosomes in neurons becomes unstable following ischemic stroke. This instability disrupts the normal lysosomal function, which relies on a stable acidic pH (around 4.5–5) for optimal hydrolytic activity. Ischemic stroke leads to a significant reduction in ATP levels, weakening the activity of vacuolar H⁺-ATPases (V-ATPases) that maintain lysosomal acidity. This disturbance results in impaired hydrolysis, causing the accumulation of autophagosomes and disrupting autophagic flux. Additionally, ischemic stroke reduces the expression of TMEM175, a protein crucial for maintaining the acidic environment of lysosomes. The decreased activity of TMEM175 further exacerbates ATP deficiency and lysosomal acidification imbalance, contributing to neuronal damage.

Fig. 2 Schematic representation of the processes involved in regulating the acidic environment of the lysosomes after ischemic stroke. (Shi, et al., 2023)

Decreased Levels of Lysosomal Cathepsins in Neurons Following Ischemic Stroke

Cathepsins, crucial proteolytic enzymes in lysosomes, are responsible for degrading polypeptides and proteins. Among them, CTSB (cathepsin B) and CTSD (cathepsin D) have garnered significant attention after ischemic stroke. The levels of CTSD and CTSB in lysosomes in neurons are signifcantly reduced after ischemic stroke. The decrease in these enzymes impairs the lysosomal hydrolysis function, hindering the cell's ability to remove damaged materials. This dysfunction contributes to the accumulation of damaged proteins and cellular debris, further exacerbating neuronal injury and dysfunction.

Fig. 3 The mechanisms that regulated the maturation of CTSB and CTSD after ischemic stroke. (Shi, et al., 2023)

Ischemic Stroke Induces Increased Lysosomal Permeability in Neurons

Ischemic stroke increases lysosomal membrane permeability in neurons, which plays a critical role in autophagic-lysosomal pathway dysfunction. The elevated permeability leads to the release of protein hydrolases from the lysosomal lumen into the cytosol, impairing autophagic flux and contributing to neuronal death. Studies show that ischemic stroke activates NMDA receptors, increasing intracellular calcium levels and triggering calpain-1 (CAPN-1) activation. CAPN-1 damages lysosome-associated membrane protein 2 (LAMP2), a key factor for maintaining lysosomal membrane stability. This disruption was reversed when CAPN-1 was knocked out. Additionally, receptor interacting protein kinase 1 (RIPK1), which promotes autophagosome formation, also increases lysosomal membrane permeability. RIPK1 levels peak 3–12 hours after ischemic stroke, coinciding with increased permeability. In vitro experiments further show that ischemic conditions significantly increase lysosomal permeability, which is improved by RIPK1 shRNA treatment. Therefore, ischemic stroke enhances lysosomal membrane permeability through the activation of RIPK1 and CAPN-1, contributing to autophagy-lysosome dysfunction.

Fig. 4 The mechanism that regulated the increased lysosomal membrane permeability after ischemic stroke. (Shi, et al., 2023)

Ischemic Stroke Causes a Decrease in the Number of Lysosomes in Neurons

The markers LAMP-1 and LAMP-2, which are commonly used to assess lysosome numbers, show significant reductions in neurons after ischemic events. Further studies demonstrated that the lysosome rupture mediated by CAPN-1 and ROS combined with heat shock protein 70.1 (Hsp70.1) was the main reason for the reduction of lysosomes. The loss of lysosomes prevents their fusion and hydrolysis with autophagosomes, thereby obstructing autophagic flux and contributing to neuronal damage.

Abnormal Initiation of Autophagic Flux Leads to Autophagosome Accumulation and Autophagy-Lysosome Pathway Disorder Following Ischemic Stroke

After ischemic stroke, various signaling pathways contribute to the excessive activation of autophagy, leading to the accumulation of autophagosomes and disruption of the autophagy-lysosome pathway. Elevated homocysteine (Hcy) suppresses mTOR signaling, causing mTORC1 phosphorylation disorder, which leads to the excessive activation of Beclin 1 and autophagy. Additionally, AMPK, a negative upstream regulator of mTORC1, is activated by Ca²⁺ overload and ATP depletion, which inhibits mTORC1 phosphorylation and further promotes Beclin 1 activation, driving excessive autophagosome production.

The Unc-51-like kinase complex dissociates from mTORC1 and assembles with AMPK, indirectly activating Beclin 1 and enhancing autophagy. The ischemic-hypoxic environment further exacerbates autophagy by increasing Beclin 1 level through the activation of the HIF-1α-BNIP3 pathway. Moreover, the inflammatory cytokine IL-17A induces hyperactivation of autophagy by activating the Src kinase, which leads to downstream activation of phospholipase C-γ (PLC-γ) and phosphatase 2B (PP2B). PP2B dephosphorylates mTOR, promoting excessive autophagy and contributing to ALP dysfunction.

Fig. 5 Schematic representation of the accumulation of autophagic vesicles triggered by abnormal activation of autophagic fux after ischemic stroke. (Shi, et al., 2023)

Impaired Autophagosome-Lysosome Fusion in Neurons Following Ischemic Stroke

After ischemic stroke, autophagosome-lysosome fusion, crucial for autophagic flux, was impaired, leading to the accumulation of autophagosomes and worsened neuronal damage. This fusion process is regulated by the SNARE complex, consisting of STX17, SNAP29, and VAMP8. During fusion, LAMP-2 facilitates the colocalization of STX17 to LC3 on the autophagosome and its recruitment to SNAP29, forming the STX17-SNAP29 complex. This complex then binds to VAMP8, forming the SNARE complex that promotes fusion. However, overactivated CAPN-1 cleaves LAMP-2, disrupting fusion. Furthermore, ischemic stroke reduces SNAP29 levels, impairing fusion, while ATG14 and VAMP8 also play critical roles. After ischemia and reperfusion, the interaction of ATG14 with LC3 is damaged, further hindering fusion. The decreased expression of VAMP8 in stroke also contributes to SNARE complex dysfunction, exacerbating autophagy-lysosome fusion impairment.

Fig. 6 Schematic presentation of the mechanism that regulated autophagosome-lysosome fusion after ischemic stroke. (Shi, et al., 2023)

1. Shi, G. S., et al. (2023). The pathological mechanism of neuronal autophagy-lysosome dysfunction after ischemic stroke. Cellular and Molecular Neurobiology, 43(7), 3251-3263.