Science Note: Lysosome

Recent research shows that lysosomal dysfunction in cells leads to toxic accumulation, causing stress responses, cell senescence and cell death. Here are some of the papers showing that lysosomal homeostasis is important for disease prevention.

Lysosomal dysfunction disrupts essential cellular processes and leads to the promotion of several diseases, including neurodegenerative diseases and senescence. In neurodegenerative diseases such as Alzheimer's and Parkinson's, impaired lysosomal activity leads to the accumulation of toxic proteins that contribute to neuronal death and disease progression. Lysosomal dysfunction is also associated with cellular senescence, where it impairs the degradation of damaged cellular components, leading to the accumulation of senescent cells and promoting age-related diseases. Therapeutic strategies targeting lysosomal function are being explored to alleviate these conditions and improve overall cellular health.

PLD3 and PLD4 synthesize S,S-BMP, a key phospholipid enabling lipid degradation in lysosomes
Click here for the original article: Shubham Singh, et. al., Cell, 2024.

Modeling Parkinson’s disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines
Click here for the original article: Armin Bayati, et. al., Nature Neuroscience, 2024.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence
Click here for the original article: Mengying Cui, et. al., PNAS, 2024.

Point of Interest

- PLD3 and PLD4 synthesize lysosomal S,S-bis(monoacylglycero)phosphate (BMP), which is critical for lipid degradation and brain health.

- Lysosomal S,S-BMP is essential for lipid degradation; its synthesis by PLD3/4 prevents gangliosidosis and supports cellular health.

- Mutations in PLD3 associated with neurodegenerative diseases reduce its activity, affecting S,S-BMP levels and contributing to lysosomal dysfunction.

Point of Interest

- Immune-induced lysosomal dysfunction promotes Lewy body (LB) formation in dopaminergic neurons, mimicking Parkinson's disease (PD) pathology.

- α-Synuclein fibrils and immune challenges (e.g., interferon-γ treatment) induce LD-like inclusions in iPSC-derived dopaminergic neurons.

- LB formation in PD may result from lysosomal-autophagic dysfunction, exacerbated by immune challenge and α-synuclein accumulation.

Point of Interest

- Mitochondrial and lysosomal functions are linked, with transcription factor EB (TFEB) and hexokinase domain containing 1 (HKDC1) playing critical roles in their regulation and maintenance.

- HKDC1, regulated by TFEB, is essential for mitophagy and lysosomal repair, independent of its glycolysis function.

- Loss of HKDC1 accelerates cellular senescence by causing mitochondrial hyperfusion and lysosomal damage.

Lysosomal Function Analysis 

With existing reagents, it was difficult to determine whether lysosomal mass or their function (pH) fluctuated because the discussion was based on changes in the fluorescence brightness of a single dye. This kit contains pHLys Green, which is highly specific to lysosomes and shows pH-dependent changes in fluorescence, and pH-resistant LysoPrime Deep Red. Using these two dyes, lysosomal pH and volume of the same sample can be measured for a detailed analysis of lysosomal function.

Accurate Measurement for Lysosomal pH changes 

Existing lysosomal pH detection reagents have issues with dye localization, pH sensitivity, and retention. pHLys Green is a dye that solves these issues. The improved dye retention and localization enable detection of normal lysosomes, and the improved pH sensitivity enables detection of slight pH changes.

	1. High sensitive pH detection Comparison of pH response of cells treated with low concentrations of lysosomal acidification inhibitor Bafilomycin A1	2. High specificity for lysosomes Comparison of specificity for lysosomes using lysosomal marker protein LAMP1-GFP expressing cells	3. High retention in lysosomes Comparison of intracellular retention

Product in Use:
   - Lysosomal Acidic pH Detection Kit-Green/Deep Red

Related Product:
   - pHLys Red- Lysosomal Acidic pH Detection

Some research on Alzheimer's disease (AD) shows that lysosomal dysfunction is observed in the early stages of AD and affects the progression of AD. Here are some of the studies that show one of the mechanisms of lysosomal dysfunction in AD and ideas for therapy.

Lysosomes play a critical role in the removal and degradation of cellular waste, including the degradation of proteins and damaged organelles by autophagy. Lysosomal dysfunction is commonly observed in Alzheimer's disease (AD), contributing to the accumulation of amyloid beta plaques and tau tangles that are hallmarks of the disease. Impaired lysosomal activity disrupts cellular homeostasis, leading to the accumulation of toxic proteins and organelle damage, further exacerbating neurodegeneration. Restoring lysosomal function has emerged as a potential therapeutic target for slowing or ameliorating the progression of Alzheimer's disease.

Accumulation of APP C-terminal fragments causes endolysosomal dysfunction through the dysregulation of late endosome to lysosome-ER contact sites
Click here for the original article:Marine Bretou, et. al.,Developmental Cell, 2024.

Urolithin A improves Alzheimer's disease cognition and restores mitophagy and lysosomal functions
Click here for the original article:Yujun Hou, et. al., Alzheimers Dement., 2024.

Rescue of ApoE4-related lysosomal autophagic failure in Alzheimer’s disease by targeted small molecules
Click here for the original article:Meenakshisundaram Balasubramaniam, et. al., Communications Biology, 2024.

Point of Interest

- Endolysosomal collapse in Alzheimer's disease (AD), initiated by decreased lysosomal calcium and increased cholesterol, is triggered by γ-secretase inhibition, with APP depletion rescuing these dysfunctions.

- APP C-terminal fragments (CTFs) are localized to late endosome-lysosome-ER contacts, and excess APP CTFs cause lysosomal Ca²⁺ deficits, leading to cholesterol accumulation at endosome-lysosome-ER contacts.

- Failure to maintain balanced APP-CTF levels perpetuates signaling, causing lysosomal dysfunction and contributing to early AD pathology.

Point of Interest

- Long-term treatment with urolithin A (UA) improves learning, memory and olfactory function in Alzheimer's disease mice.

- UA reduces amyloid beta and tau pathology and improves mitophagy by restoring lysosomal function through cathepsin Z regulation.

- UA shows therapeutic potential for Alzheimer's disease by improving lysosomal function and modulating immune responses and Alzheimer's disease-related pathways.

Point of Interest

- The APOE ε4 allele increases the risk of Alzheimer's disease by impairing autophagy by blocking the transcription of autophagy genes.

- Small molecules have been identified that bind ApoE4 and restore autophagy gene transcription, thereby reducing amyloid aggregation in models.

- These findings suggest that ApoE4-targeted drugs could be used to rescue lysosomal autophagy in neurodegenerative diseases such as Alzheimer's.

Analysis of autophagic flux without transfection 

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.

Experimental Conditions
CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

Products in Use
Autophagic Flux Assay Kit

Lysosomal dysfunction is increasingly recognized as a critical factor in the development and progression of several neurological diseases. In neurodegenerative diseases such as Parkinson's and Alzheimer's, impaired lysosomal function leads to the accumulation of misfolded proteins and neuronal toxicity, contributing to cell death and disease progression. Although rare, lysosomal storage diseases often have significant neurological manifestations due to the accumulation of undigested substances in neurons, impairing their function and survival. Therefore, understanding and targeting lysosomal pathways is emerging as a promising therapeutic approach to treat and potentially prevent a range of neurological disorders. 

Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation
Click here for the original article: Terytty Yang Li, et. al., Cell Discovery, 2023.

ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization
Click here for the original article: Dilip Menon, et. al., Cell Reports, 2023.

Nutrient-regulated control of lysosome function by signaling lipid conversion
Click here for the original article: Michael Ebner, et. al., Cell, 2023.

Point of Interest
- Mitochondrial stress activates mTORC1 through v-ATPase-mediated lysosomal stimulation, leading to phosphorylation of ATF4.

- This phosphorylation of ATF4 triggers the mitochondrial unfolded protein response (UPRmt).

- Interfering with the ability of mTORC1 to phosphorylate ATF4 inhibits the UPRmt.

- The phosphorylation of ATF4 prevents ROS-induced cell death under mitochondrial stress.

Point of Interest
- ARL8B enhances the lysosomal lipolysis of triacylglycerol stored in lipid droplets (LDs).

- ARL8B in its GDP-bound state associates with LDs, whereas ARL8B-GTP is predominantly associated with lysosomes.

- ARL8B facilitates the contact between LDs and lysosomes, with both its GDP- and GTP-bound states forming a complex.

- The ARL8B-mediated lysosomal lipolysis of LDs represents a pathway for the turnover of neutral lipids in macrophages.

Point of Interest
- Starvation enhances lysosomal catabolism through the localized synthesis of PI(4)P on the lysosomal membrane.

- Cells possess diverse lysosome populations, distinguished by the presence of either PI(3)P or PI(4)P.

- A lipid switch, regulated by nutrient availability, facilitates the interconversion between these distinct lysosome populations.

- mTORC1 signaling and the synthesis of lysosomal PI(4)P are mutually inhibitory, each suppressing the other's activity.

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe²⁺

Through a combination of various scientific methods, this research reveals the vital role of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in maintaining lysosomal pH balance. Despite being mainly recognized as lysosomal markers, LAMP-1 and LAMP-2 directly influence the lysosomal cation channel TMEM175, aiding the acidification of lysosomes to a pH level that optimizes hydrolase activity. Interruptions to the interaction between LAMP and TMEM175 can increase lysosomal pH, impairing their hydrolytic function.
We offer small fluorescent probes for detecting lysosomal mass and pH with several color options: Lysosomal Acidic pH Detection kit-Green/Red and Green/Deep Red. 

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel    
Click here for the original article: Jiyuan Zhang, et. al., Mol. Cell. (2023)

Point of Interest
- The lysosomal proteins LAMP-1 and LAMP-2 have a direct interaction with the TMEM175 channel.
- This complex formation between TMEM175 and LAMPs is facilitated by their transmembrane (TM) domains.  
- The binding of LAMP proteins inhibits the channel activity of TMEM175.
- This inhibition of TMEM175 promotes lysosomal acidification, thereby enhancing the efficiency of hydrolase activity.

Accurate Measurement for Lysosomal pH changes

Existing lysosomal pH detection reagents have issues with dye localization, pH sensitivity, and retention. pHLys Green is a dye that solves these issues. The improved dye retention and localization enable detection of normal lysosomes, and the improved pH sensitivity enables detection of slight pH changes.

Product in Use:
   - Lysosomal Acidic pH Detection Kit-Green/Deep Red

Related Product:
   - pHLys Red- Lysosomal Acidic pH Detection

Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis    Ruilin Tian, et. al., Nature Neuroscience (2022)

Point of Interest
- The study reveals pathways that govern neuronal response to chronic oxidative stress, a factor in neurodegenerative diseases.
- Suppression of the lysosomal protein prosaposin makes neurons highly susceptible to oxidative stress. 
- This happens through the induction of lipofuscin formation which sequesters iron.
- Iron accumulation contributes to creating reactive oxygen species and lipid peroxidation, triggering ferroptosis, exclusively in neurons.

The simultaneous detection of lysosomal function with Mitochondrial ROS and intracellular Fe²⁺

Induction of Ferroptosis by Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe²⁺, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe²⁺ , ROS, and lipid peroxides.

• - Pathogenic species of αSyn accumulate within neuronal lysosomes in mouse brains and primary neurons.
• - Neurons release these pathogenic αSyn species via SNARE-dependent lysosomal exocytosis
• - The released aggregates are non-membrane enveloped and seeding-competent
• - This release is dependent on neuronal activity and cytosolic Ca²⁺

• - PEN2 is a binding partner of metformin with a dissociation constant at micromolar levels
• - Metformin-bound PEN2 forms a complex with ATP6AP1 which leads to the inhibition of v-ATPase and the activation of lysosomal AMPK
• -  In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects.
• -  Knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan

• - TMEM175 is the proton “leak” channel of lysosomes and endosomes
• - TMEM175 is a highly proton-selective channel that is gated by luminal protons
• - An endogenous lipid can also activate TMEM175 to trigger lysosomal proton release
• - TMEM175 sets the lysosomal pH optimum via a classic negative feedback mechanism