Lysosomes are essential degradation organelles and signaling centers in cells, and play a vital role in regulating macroautophagy/autophagy.Lysosomal dysfunction and impaired autophagy are closely related to the development of various neurodegenerative diseases.Enhancing lysosomal activity and increasing the level of autophagy are effective strategies for the treatment of these diseases and have great prospects.However, methods for dynamically modulating lysosomal activity and levels of autophagy in living cells or animals are still lacking.
On August 15, 2024, Cang Chunlei and Qu Lili of China University of Science and Technology jointly communicated inAutophagy Published online entitled“Optogenetic manipulation of lysosomal physiology and autophagic activity”of research papers.The study applied optogenetics to manipulate the physiology and function of lysosomes and developed three optogenetic tools for lysosomes: lyso-NpHR3.0, lyso-ArchT and lyso-ChR2.
These new actuators were able to control lysosomal membrane potential, luminal pH, hydrolytic enzyme activity, degradation processes and Ca in living cells.2 Key aspects such as kinetics are light-dependent regulated.Notably, in a cellular model of Alzheimer's disease, lyso-ChR2 activation induces autophagy through the MTOR pathway, while promoting Aβ clearance through autophagy induction.In addition, lyso-ChR2 activation reduces Aβ deposition and relieves Aβ-induced paralysis in a Caenorhabditis elegans model of Alzheimer's disease.The authors' lysosomal optogenetic actuator provides a way to dynamically regulate lysosomal physiology andautophagy activityThe new method.
Lysosomes are the major degradation organelles in eukaryotic cells, responsible for the degradation and recovery of biological macromolecules from endocytosis, phagocytosis and autophagy.In addition to being the ultimate destination for autophagic degradation, lysosomes exert regulatory control over the upstream process of autophagy through interaction with the MTORC1 complex. In addition, lysosomal-mediated Ca2 + release and regulation of lysosomal membrane potential have been reported to contribute to the regulation of autophagy.Lysosomal dysfunction and impaired autophagy are often manifested in neurodegenerative diseases; therefore, enhancing lysosomal activity and increasing autophagy levels are expected to be effective therapeutic strategies for these diseases.However, there is currently a lack of methods to dynamically regulate lysosomal activity and autophagy levels.
Optogenetics is a method of usinggene codingThe technique of light-sensitive proteins to regulate cellular activity by altering ion homeostasis and membrane potential in response to specific light stimuli.In the authors' recent study [citation 1], the authors utilized lysosome-targeted optogenetic actuators to achieve dynamic modulation of lysosomal physiology and autophagy activity to improve the Alzheimer's disease (AD) phenotype in cells and Caenorhabditis elegans models by enhancing autophagy.
In order to minimize the influence of non-optogenetic factors, the authors used lysosomal targeting signals to achieve precise positioning of optogenetic actuators on lysosomes.After evaluating various lysosomal targeting signals, the authors successfully exploited the EREPLL signal for halophilic rhodopsin -3.0(NpHR3.0) and archaeorhodopsin-T(ArchT) and the GYEVM signal for channelrhodopsin -2(ChR2) to specifically target these proteins to lysosomes. These engineered constructs are designated lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, respectively. Although there is significant lyso-ChR2 residue on the plasma membrane, subsequent control experiments confirmed that changes in lysosomal physiology, autophagy activity, and other related processes are indeed caused by ChR2 specifically located on the lysosomal membrane rather than on the plasma membrane.In addition, total lysosomal patch clamp recordings showed that all three engineered tools were functioning normally on the lysosomal membrane; they responded efficiently to specific light stimuli by eliciting changes in lysosomal membrane potential and current dynamics-thus validating the authors' successful modification.
A series of experiments proved that the three optogenetic tools developed by the authors can effectively regulate various aspects of lysosomal physiology, including lysosomal membrane potential, pH, hydrolytic enzyme activity, degradation ability and Ca2 kinetics (Figure 1).These versatile tools enable researchers to dynamically manipulate lysosomal physiology and function according to their specific research goals.It needs to be emphasized that when using these tools to study specific aspects of lysosomal physiology, researchers should pay attention to their multi-faceted effects and consider all dimensions to fully understand.
Notably, the authors observed that activation of lyso-ChR2 alters the LC3-II:LC3-I ratio, an important indicator of autophagy activity.Further analysis of autophagy flux indicated that lyso-ChR2 activation promoted the initiation of autophagy. Mechanism analysis showed that the activation of lyso-ChR2 promoted the dissociation of MTOR from the lysosomal membrane, resulting in the inactivation of MTOR. This inactivation will release the inhibition of TFEB, leading to its nuclear translocation, thereby activating autophagy (Figure 1). However, the exact mechanism by which lysosomal ChR2 activation induces MTOR dissociation remains unclear. Previous studies have shown that lysosomal Ca mediated by the MCOLN1/TRPML1 channel2 Release-promoting MTORC1 is activated by recruitment of MTOR to lysosomes through CALM (calmodulin) binding. The authors suggest that activation of lysosomal ChR2 may regulate MTOR activity through a similar mechanism. Specifically, lysosomal ChR2 activation induces lysosomal membrane potential depolarization, resulting in reduced MCOLN1 channel activity because of its hyperpolarization-activating properties. The decrease in MCOLN1 activity resulted in a decrease in lysosomal calcium release, which is consistent with the increase in lysosomal calcium content observed by the authors.Subsequently, Ca2 Reduced release attenuates CALM-mediated recruitment of MTOR to lysosomes and promotes autophagy.
Compared with traditional methods of inducing autophagy (such as drugs or genetic intervention), the authors' optogenetics-based method has multiple advantages, including excellent spatial resolution, reversibility, and precise control.By using cell-specific promoters, autophagy induction can be restricted to specific cell types or tissues. Refining the photostimulation region may also improve spatial accuracy. Furthermore, the method is reversible, as removal of the light stimulus is sufficient to restore the original level of autophagy to the cell.In addition, by adjusting the parameters of the light stimulation (such as duration, intensity, and pulse width), precise control of the level of autophagy can be achieved.
Given that various neurodegenerative diseases, including AD, existautophagy disorder, the authors investigated whether the authors' optogenetic tool lyso-ChR2 could improve the AD phenotype by inducing autophagy.In a cellular model of AD, the authors found that several hours of light stimulation significantly promoted Aβ clearance. When using drugs or knocking out keyautophagy geneWhen autophagy is inhibited, this effect is eliminated, suggesting that lyso-ChR2 promotes Aβ clearance in an autophagy-dependent manner. In addition, in the AD C. elegans model that exhibits a paralytic phenotype after Aβ induction, lyso-ChR2 activation significantly reduces Aβ deposition and delays the onset of paralysis.The authors' optogenetics-based approach to induced autophagy may provide new insights into the treatment of AD and other underlying neurodegenerative diseases.
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