Lysosomal Membrane Permeabilization and Therapeutic Use

Lysosomal Membrane Permeabilization: From Basic Research to Putative Therapeutic Use

  • Loss of lysosome membrane permeability in some conditions like stress conditions.

The Lysosome

  • Organelle that contains many enzymes called acid hydrolases, which are active at an acidic pH.

  • Hydrolyzes macromolecules into small molecules.

  • Approximately 60 different acid hydrolases, including lipases and phosphatases.

  • Acidic environment is maintained by v-ATPase, a proton pump that uses ATP to accumulate protons.

Lysosomal Membrane Proteins

  • v-ATPase: Proton pump.

  • Ion channels and transporters: Important for exporting products of hydrolyzation for reuse by the cell, such as amino acid and glucose transporters.

  • Tethering factors, SNAREs, and Small GTPases: Mediate vesicle fusion, organelle contact, etc.

  • Motor adaptors: Facilitate lysosome motility by binding to microtubules.

  • Signaling complexes: Lysosomes have a role in metabolic signaling.

  • LAMPs (Lysosomal-Associated Membrane Proteins): Most abundant protein in the lysosomal membrane; highly glycosylated and form a protective layer called the lysosomal glycocalyx.

    • Lysosomal proteins are N-glycosylated.

    • LAMPs carry many oligosaccharidic chains.

    • Protects the membrane from acid hydrolases, including phospholipases.

Main Roles of Lysosomes

  • Degradation of molecules via two main pathways:

    • Endocytic pathway: Phagocytosis (large components) and pinocytosis (small soluble molecules) of extracellular components through vesicles (phagosomes, endosomes).

    • Autophagy pathway: Intracellular degradation of damaged organelles (e.g., mitochondrial degradation) and proteins.

    • Sometimes material goes directly into the lysosome.

  • Fusion with the plasma membrane (calcium-dependent) for membrane repair or secretion (exocytosis).

  • mTORC1 signaling: Located on the lysosomal surface, mediates the balance between anabolism and catabolism, depending on nutrient availability.

    • Nutrient-rich conditions: mTORC1 binds to the membrane and stimulates anabolism by phosphorylating targets and inhibits catabolism by phosphorylating UVRAG, TFEB, and ULK1.

    • Nutrient-low conditions: mTOR becomes inactive, halting phosphorylation, which activates autophagy and lysosome biogenesis, promoting catabolism.

mTORC1 Complex

  • Active (Nutrient-rich):

    • Activates protein translation, nucleotide, and lipid synthesis.

    • Inhibits lysosome-dependent degradation processes.

    • Favors anabolism and inhibits catabolism.

    • Inhibits autophagy initiation and completion.

    • Inhibits lysosomal biogenesis by phosphorylating TFEB.

  • Inactive (Nutrient-low):

    • Inhibits protein translation, nucleotide, and lipid synthesis.

    • Promotes lysosome-dependent degradation processes.

    • Favors catabolism and inhibits anabolism.

    • Non-phosphorylated TFEB is active, inducing the expression of lysosomal and autophagy genes.

Lysosomal Membrane Permeabilization (LMP)

  • Stress-induced loss of lysosomal membrane integrity, leading to the release of molecules and hydrolases into the cytoplasm and cytosolic acidification.

  • Consequences:

    • Acidification of the cytoplasm.

    • Impaired lysosomal degradation efficiency.

    • Degradation of cytosolic molecules.

    • Induction of cell death pathways (apoptosis, necrosis).

  • Can be a component of cell death pathways induced by non-lysosomal stresses.

Types of Stress Inducing LMP

  • Osmotic stress: Lysosome swelling and membrane rupture due to water influx.

  • Pathogens: Pore formation in membranes.

  • Drugs:

    • Weakly basic drugs enter lysosomes, become protonated, and get trapped due to charge, causing osmotic stress.

    • Proton depletion raises lysosomal pH, reducing acid hydrolase activity and causing substrate accumulation.

  • Oxidative stress (ROS): Damage to lysosomal membrane.

  • Abnormal lipid insertion: Defects of metabolism which can create pores.

Consequences of Holes in the Membrane

  • Small elements, like protons, leak out, leading to alkalization.

  • Local acidification of the cytosol due to enzyme leakage, causing damage by degrading molecules.

  • Activation of proteins, like cathepsins, leading to apoptosis, necrosis, ferroptosis, inflammation, and pyroptosis.

    • Cathepsins induce necrosis.

    • ROS + cathepsins induce inflammation and pyroptosis.

    • ROS + iron induce ferroptosis.

  • Type of cell death depends on the released enzymes and the extent of LMP.

Partial vs. Extensive LMP

  • Limited permeabilization: Small pores, no cathepsin release, triggers lysosome repair and clearance (lysophagy) and lysosomal biogenesis; cell survives.

  • Extensive permeabilization: Large pores, massive release of lysosomal content, triggers apoptosis or necrosis; cell dies.

Lysosome Repair and Clearance Mechanisms

  • Defense mechanisms can correct holes and eliminate damages if not too sudden or extensive.

A) Lysosome Repair Pathways

  • 1) ESCRT-mediated lysosomal repair

    • Small pores appear in the lysosomal membranes after stress.

    • Calcium is released into the cytosol, which recruits a calcium sensor called ALG-2 (or PDCD6).

    • ALG-2 recruits the ESCRT machinery, including ESCRT-III, to the vicinity of the damaged area.

    • Subunits of ESCRT-III assemble to form a spiral that brings together membrane parts before and after the pore.

    • The damaged membrane is then pushed inwards, while the limiting membrane seals itself. This internal vesicle-like structure (containing the pore) is then degraded inside the lysosome.

  • 2) The PITT pathway (phosphoinositol-initiated membrane tethering and lipid transport)

    • LMP causes a Ca^{++} leak that triggers the rapid recruitment of phosphatidylinositol 4 kinase type 2α (PI4K2A), which increases PI4P levels in the lysosomal membrane.

    • PI4P recruits several oxysterol-binding protein (OSBP)-related proteins (ORP), which attach the damaged lysosomes to the ER (endoplasmic reticulum) by binding to VAP proteins in the ER membrane.

    • ORP proteins then mediate the exchange of PI4P (in lysosomes) for cholesterol or for phosphatidylserine (PS) coming from the ER.

    • The increase in PS in the lysosomal membrane activates the transfer of additional phospholipids (by ATG2), all these lipid transfers allowing repair of the damaged membrane.

B) Lysophagy: Elimination of Damaged Lysosomes

  • 1) Galectin-dependent lysophagy

    • LAMP1 and LAMP2 (Lysosomal-associated membrane proteins) are heavily glycosylated transmembrane proteins.

    • Galectins are proteins that bind to terminal β-galactose or N-acetyl- β-galactose.

    • Upon LMP, galectins 3, 8, 9 may enter the lysosome and bind to the glycans carried by LAMPs.

    • This triggers the recruitment of ubiquitin ligases and thus the polyubiquitination of LAMPs and other membrane components.

    • These polyubiquitin chains can recruit autophagy adaptors, initiating the packaging of the damaged lysosome in an autophagosome.

    • The damaged lysosome will then be degraded after fusion of this autophagosome with a functional lysosome.

  • 2) Galectin-independent lysophagy pathways

    • Non-canonical pathways have also been identified.

    • The phagophore membrane (which initiates the formation of an autophagosome) can be recruited to the damaged lysosome by several machineries.

C) Induction of Lysosomal Biogenesis

  • Needed to replace eliminated lysosomes after lysophagy. Depends on mTOR activity:

    • Basal conditions (abundant nutrients):

      • mTORC1 is on the lysosome and becomes active.

      • mTOR phosphorylates TFEB (transcription factor), which becomes inactive.

      • Phospho-TFEB binds to a cytosolic chaperone, trapping it in the cytosol.

      • No activation of the CLEAR gene network (lysosomal genes).

    • Stress conditions (including LMP, starvation):

      • mTORC1 detaches from the lysosome; mTOR is inhibited.

      • Calcium is released from damaged lysosomes (via the MCOLN1 channel), activating calcineurin, a cytosolic phosphatase, which dephosphorylates phospho-TFEB.

      • TFEB (dephosphorylated) translocates into the nucleus.

      • TFEB activates the CLEAR gene network, stimulating lysosomal and autophagy biogenesis.

Detecting and Monitoring LMP

A) Monitoring Cathepsin Release in the Cytosol After LMP

  • Use antibodies to identify lysosomal hydrolases like cathepsins.

    • A1) By immunofluorescence detection of cathepsins

      • Antibodies against Cathepsins B, D, and L are used.

      • No damage: Dots in the cell cytoplasm (lysosome).

      • LMP conditions: Hydrolase is diffused in the cytoplasm.

      • Limitations: Requires a lot of LMP to see something, difficult to quantify, low signal due to diffusion.

    • A2) By subcellular fractionation using centrifugation, followed by Western blotting detection of cathepsins in cytosolic and organelle fractions

      • More quantitative.

      • Homogenization to break down the MP, organelles are in suspension.

      • Centrifuge at high speed to pellet organelles, only cytosolic components are in suspension.

      • Identify hydrolases by Western Blotting.

      • Normal: Signal is only in the pellet (organelle fraction).

      • LMP: Signal in the supernatant (cytosolic fraction).

    • A3) LMP may lead to decreased activity of lysosomal enzymes within lysosomes

      • Detection of lysosomal enzyme degradation activity in live cells using fluorogenic substrates.

      • Example: MagicRed probe (cathepsin B and L substrate).

      • Lysosomal hydrolases have highest activity at acidic pH. If the pH increases due to protons release in the cytosol in LMP conditions, their activity decreases => less fluorescent signal.

B) Monitoring of LMP Using Lysomotropic Dyes

  • Red lysotracker probe: Weak base that diffuses into lysosomes where it becomes protonated and fluoresces RED. Detects the pH of lysosome.

    • Control cells: Good signal in lysosome.

    • LMP conditions: Protons leak, less protons, less signal.

    • Limitations: Can have change of pH in other conditions.

C) Endocytosis of Fluorescent Dextrans (Small Molecular Weight Ones)

  • This test can only be used if the endocytic rate is unaltered in stressed cells.

  • Dextran accumulates in the lysosome.

  • With LMP these molecules will leak and the signal will increase in cytosol if the endocytosis speed rate is the same.

D) Galectin Recruitment to Damaged Lysosomes

  • Detection of galectin 3 or 9 by immunofluorescence. The signal becomes punctate if LMP occurs.

    • Normally, these galectins are not present in the cytosol.

    • In early-stage LMP, lysosomal content is exposed to the cytosol, attracting galectins to damaged lysosomes.

    • This recruitment of galectins results in a punctate signal in immunofluorescence.

    • LLOMe (a LMP inducer): Punctuated signal.

    • Triamterene (100 to 500 μM): Increasingly punctuated signal with increasing dose.

E) ESCRT Recruitment

  • Detection of an ESCRT protein involved in lysosome repair by immunofluorescence. The signal becomes punctate if LMP occurs.

LMP: Involvement in Several Diseases

  • Maintaining the integrity and activity of lysosomes is crucial for healthy living.

  • Lysosome-dependent degradation pathways contribute to limit the accumulation of misfolded and aggregated proteins.

  • During normal aging, autophagic rates decline, while misfolded proteins accumulate, contributing to organ dysfunctions.

  • Genetic lysosomal storage diseases are often associated with a decreased lifespan.

  • Lysosomal dysfunction can also contribute to the pathophysiology of several other neurodegenerative diseases.

Genetic Risk Factors for Neurodegenerative Diseases

  • Several mutations in genes can increase lysosomal pH, induce abnormal storage, or alter the repair of damaged lysosomes.

  • Mutated genes involved in autophagy more specifically involved in lysophagy pathways.

  • Lysosomal storage is altered because of accumulation of substrates (accumulation of toxic proteins).

  • You need healthy lysosomes, because protection mechanism are altered.

Environmental Factors

  • Some pollutants also cause LMP: Atmospheric fine nanoparticles, pesticides such as rotenone.

Boosting Lysosome Repair, Clearance and/or Lysosomal Biogenesis as a Treatment Strategy?

A) Boosting autophagy (including lysophagy)?

  • mTOR inhibits mechanisms of autophagy and is the target of rapamycin (= allosteric inhibitor of mTOR).

    • Inhibition of mTOR should also activate TFEB, by other mechanisms, and thus lysosomal biogenesis. Yet, TFEB activation is not induced by rapamycin => Beneficial effects of this drug appear linked to increased clearance rather that increased lysosomal biogenesis.

  • Rapamycin: Drug known to inhibit organ rejection (immunosuppressive). Postulated to be also used to treat neurological diseases.

  • Alzheimer’s disease: Phase II clinical trial ongoing in 2024.

B) Boosting lysosome biogenesis more specifically?

  • Focus on TFEB directly.

  • Preclinical findings: Overexpression of TFEB has been shown to have beneficial effects in animal models.

  • Ongoing search for pharmacological drugs:

    • Trehalose: It is endocytosed and accumulates in lysosomes causing a slight pH increase (limited stress), which promotes TFEB activation.

    • Curcumin: Binds to TFEB and increases its activity.

    • High-throughput search for novel TFEB agonists ongoing

  • Starvation activates TFEB.

C) Caloric Restriction and Exercise

  • Intermittent fasting and caloric restriction?

    • Increase the lifespan of many animal models.

  • Physical exercise induces calcium release from lysosomes and TFEB activation via calcineurin-mediated dephosphorylation.

    • Mouse training: running on a wheel.

D) Can lysosomal membrane repair be stimulated pharmacologically ?

  • An open question.

  • Search for pharmacological agonists or stimulators of PI4K2A.

LMP and Cancer

  • Cancer cells rely on lysosomes for differentiation and proliferation.

  • Lysosomal biogenesis increases during the cancerous transformation.

    • Helps sustain their metabolic needs (rapid metabolism) by providing nutriments.

    • Helps clear toxic molecules and damaged organelles.

    • Promotes lysosomal exocytosis.

      • Helps ECM degradation by lysosomal enzymes, which promotes migration, invasion of cancer cells and angiogenesis.

      • Fibroblasts activation.

      • Promotes Epithelial-to-Mesenchymal transition of other cancer cells:

  • Opposite than with neurological disease, here we want to target lysosomes (boost LMP) and not boost lysosomes.

*Examples of strategies under study (for some cancers):

*   Hypoxanthine and xanthine oxidase injected in the tumor.
*   Alternol administration, which is an activator of xanthine oxidase
*   Increased production of extracellular ROS.
*   Inhibition of acid sphingomyelinase.
*   Uses of nanoparticles
Nanoparticles-based Therapeutic Strategies

  • Variations of nanoparticles-based therapeutic strategies that are under investigation:

    • Nanocarriers that response to Magnetism.

    • Charged nanoparticles that aggregate in the acidic environment of lysosomes.

    • Photoactivable particles that produce ROS once in lysosomes.

    • Endocytosed nanoparticles that act as radioenhancers