Seeded aggregation of ANXA11 induces prion-like propagation, TDP-43 co-pathology and nucleocytoplasmic transport defects

This study establishes that ANXA11 is an intrinsically amyloidogenic protein whose prion-like fibrils propagate between cells, induce TDP-43 co-pathology, and disrupt nucleocytoplasmic transport, thereby providing a mechanistic framework for ANXA11-associated ALS and FTD.

The Gist

The Big Picture: A Broken Delivery System in the Brain

Imagine your brain cells (neurons) are like busy, high-tech factories. Inside these factories, there is a critical delivery system that moves important packages (messages and proteins) in and out of the "control room" (the nucleus). If this delivery system breaks, the factory stops working, and the cell eventually dies.

This paper investigates a specific protein called ANXA11. In healthy people, ANXA11 is a helpful worker that helps organize these deliveries. However, in some people with ALS (a disease that kills nerve cells) and FTD (a type of dementia), this protein gets "glitchy."

The researchers discovered that when ANXA11 goes wrong, it doesn't just stop working; it turns into a sticky, infectious mess that spreads from cell to cell, breaks the delivery system, and drags other important proteins down with it.

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The Story of ANXA11: From Liquid to Solid

1. The "Water Droplet" Phase (Liquid-Liquid Phase Separation)

Think of ANXA11 as a drop of water. In a healthy cell, ANXA11 behaves like a liquid droplet. It's fluid, dynamic, and can easily merge with other droplets or split apart to do its job. The researchers showed that if you take pure ANXA11 in a test tube, it naturally forms these liquid droplets.

• Analogy: Imagine a drop of rain on a window. It's round, it moves, and it can merge with other drops. That's healthy ANXA11.

2. The "Hardening" Phase (Liquid-to-Solid Transition)

The problem starts when these liquid droplets sit too long or get stressed. Over time, they stop flowing and turn into something hard and solid, like honey that has crystallized or wet cement drying out.

• The Discovery: The researchers watched these droplets age. They started as fluid (they could move and bounce back after being poked), but after 24 hours, they turned into rigid, solid clumps. These solid clumps are called amyloid fibrils. This is the "bad" form of the protein.

3. The "Zombie" Effect (Prion-Like Propagation)

Here is the scary part: Once ANXA11 turns into these solid, sticky clumps, it becomes contagious.

• The Analogy: Imagine a single zombie in a crowd. If it touches a healthy person, that person turns into a zombie too.
• The Science: The researchers found that these solid ANXA11 clumps can jump from one cell to another. When they enter a healthy cell, they act as a "seed" or a template. They grab the healthy, liquid ANXA11 inside that new cell and force it to turn into a solid clump too. This creates a chain reaction, spreading the damage across the brain.

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The Chain Reaction: Dragging TDP-43 Down

The researchers also found that ANXA11 doesn't just hurt itself; it hurts its neighbors.

• The Neighbor: There is another protein called TDP-43. TDP-43 is already famous for being a villain in ALS and FTD. It usually stays in the control room (nucleus), but when it gets sick, it leaks out and forms toxic clumps.
• The Connection: The study showed that the "zombie" ANXA11 clumps actually force TDP-43 to turn bad too.
• The Analogy: Imagine a group of rowdy kids (ANXA11 clumps) running through a library. They don't just knock over books; they grab the librarian (TDP-43), drag them out of their office, and force them to join the chaos.
• The Result: The ANXA11 clumps cause TDP-43 to become sticky, accumulate in the wrong place, and form toxic aggregates. This explains why patients often have both proteins messed up in their brains.

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The Broken Door: Why the Cell Dies

The final piece of the puzzle is how this kills the cell.

• The Nuclear Pore: The cell has a "gate" called the Nuclear Pore Complex (NPC). It's like a security checkpoint that lets messages (mRNA) leave the control room to be used by the rest of the factory.
• The Blockage: When ANXA11 forms its sticky clumps, it seems to jam the security checkpoint. The researchers found that the proteins that make up the gate (nucleoporins) get pulled into the clumps or move to the wrong place.
• The Consequence: Because the gate is broken, the messages (mRNA) get stuck inside the control room. The rest of the cell doesn't get the instructions it needs to survive.
• The Outcome: The cell starves for instructions, its structure collapses, and it eventually dies.

Summary: The Three-Step Disaster

1. The Transformation: ANXA11 changes from a helpful, fluid worker into a hard, sticky, solid clump.
2. The Spread: These clumps jump between cells, turning healthy proteins into more clumps (like a virus), and they also force TDP-43 to turn bad.
3. The Breakdown: The clumps jam the cell's "delivery gates," trapping important messages inside the nucleus, which leads to the death of the neuron.

Why This Matters

This paper is a breakthrough because it connects the dots. It explains how ANXA11 mutations cause disease (by turning solid and spreading) and why TDP-43 is also found in these patients (because ANXA11 drags it down).

Most importantly, it suggests new ways to treat the disease. Instead of just trying to stop the symptoms, doctors might one day be able to:

• Stop ANXA11 from turning solid (keeping it liquid).
• Stop the clumps from jumping between cells.
• Fix the broken "gates" so messages can get out again.

This research turns a complex mystery into a clear story of a broken delivery system, offering hope for new treatments for ALS and FTD.

Technical Summary

1. Problem Statement

Mutations in the ANXA11 gene are a known cause of familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While recent cryo-electron microscopy (cryo-EM) studies identified heteromeric ANXA11-TDP-43 filaments in patient brains, the fundamental mechanisms linking ANXA11 dysfunction to neurodegeneration remained unclear. Specifically, it was unknown whether:

• ANXA11 possesses intrinsic amyloidogenic properties capable of forming fibrils.
• ANXA11 aggregation follows a prion-like mechanism (self-templating and intercellular propagation).
• ANXA11 aggregates directly induce TDP-43 pathology (co-pathology).
• ANXA11 aggregation disrupts specific cellular pathways, such as nucleocytoplasmic transport, leading to neuronal toxicity.

2. Methodology

The study employed a multi-disciplinary approach combining biophysics, cell biology, proteomics, and stem cell models:

• In Vitro Biophysics: Recombinant full-length ANXA11 was subjected to liquid-liquid phase separation (LLPS) assays under macromolecular crowding (PEG8000). Dynamics were monitored via Fluorescence Recovery After Photobleaching (FRAP), and maturation into solid aggregates was tracked over time. Amyloid formation was confirmed using Thioflavin S (ThS), Congo Red, and Transmission Electron Microscopy (TEM).
• Prion-like Seeding Assays: Real-time Quaking-Induced Conversion (RT-QuIC) was used to test the self-templating capability of ANXA11 preformed fibrils (PFFs).
• Cellular Transmission Models:
  • SH-SY5Y Neuroblastoma Cells: Used to assess intercellular transmission of fluorescently labeled ANXA11 PFFs via co-culture and flow cytometry.
  • Human iPSC-Derived Neurons: Differentiated cortical neurons were used to validate transmission in a disease-relevant human neuronal context.
• Pathological Conversion Assays: HEK293T cells expressing ANXA11-GFP or TDP-43-mCherry were treated with ANXA11 PFFs. Detergent-soluble and -insoluble fractions were analyzed via immunoblotting and dot blotting (using conformation-specific antibodies OC and A11) to assess aggregation and TDP-43 phosphorylation/fragmentation.
• Proximity Labeling Proteomics: A TurboID-based approach was used to map the proteome proximal to ANXA11 aggregates in both soluble and detergent-insoluble fractions to identify aggregation-dependent interactors.
• Functional Assays:
  • Nucleocytoplasmic Transport: Immunofluorescence and Transmission Electron Microscopy (TEM) were used to visualize Nuclear Pore Complex (NPC) integrity and nuclear envelope morphology.
  • mRNA Export: Fluorescence in situ hybridization (FISH) with oligo-dT probes quantified nuclear accumulation of poly(A)+ RNA.
  • Toxicity: Cell viability assays and neurite integrity staining (MAP2/NeuN) were performed on iPSC-derived neurons.

3. Key Contributions

• Intrinsic Amyloidogenicity of ANXA11: The study establishes that ANXA11 undergoes a liquid-to-solid phase transition, maturing from dynamic liquid condensates into stable, amyloid-like fibrils.
• Prion-like Propagation: It demonstrates that ANXA11 fibrils possess self-templating seeding activity and can propagate between cells, including human iPSC-derived neurons.
• Mechanism of TDP-43 Co-pathology: The paper provides evidence that ANXA11 fibrils act as seeds to induce the pathological conversion of TDP-43 (hyperphosphorylation, insolubility, and cytoplasmic aggregation), offering a mechanistic explanation for the heteromeric filaments seen in patients.
• Nucleocytoplasmic Transport Defect: It identifies a direct link between ANXA11 aggregation and the disruption of the Nuclear Pore Complex (NPC), leading to impaired mRNA export and neuronal death.

4. Key Results

• Phase Transition: In vitro, ANXA11 forms liquid droplets that mature into solid, ThS-positive amyloid fibrils over 24 hours. FRAP analysis confirmed the transition from dynamic (liquid) to static (solid) states.
• Seeding and Propagation:
  • RT-QuIC: Sonicated ANXA11 PFFs efficiently seeded the aggregation of monomeric ANXA11, eliminating the lag phase.
  • Transmission: Fluorescently labeled ANXA11 PFFs were internalized by SH-SY5Y cells and iPSC-derived neurons, and subsequently transmitted to neighboring cells in co-culture.
• Induction of TDP-43 Pathology: Treatment with ANXA11 PFFs in TDP-43-expressing cells resulted in:
  • Increased detergent-insoluble TDP-43.
  • Hyperphosphorylation at Ser409/410.
  • Generation of C-terminal fragments (CTFs).
  • Formation of cytoplasmic TDP-43 aggregates.
• Proteomic Rewiring: TurboID proximity labeling revealed that while soluble ANXA11 interacts with membrane trafficking factors, insoluble ANXA11 aggregates specifically recruit Nuclear Pore Complex (NPC) components (e.g., NUP98, NUP107) and nucleocytoplasmic transport factors (e.g., RANGAP1).
• Cellular Dysfunction:
  • NPC Disruption: ANXA11 aggregation caused nuclear envelope invaginations, mislocalization of Lamin B1, and redistribution of nucleoporins (NUP98, NUP107).
  • mRNA Export Failure: FISH analysis showed significant nuclear accumulation of poly(A)+ RNA in aggregate-bearing cells, indicating a block in mRNA export.
  • Neuronal Toxicity: Exposure of iPSC-derived neurons to ANXA11 PFFs led to progressive loss of cell viability and reduced neurite integrity (decreased MAP2 signal).

5. Significance

This study provides a comprehensive mechanistic framework for ANXA11-associated ALS/FTD pathogenesis:

1. Unified Mechanism: It connects the genetic mutation in ANXA11 to neurodegeneration via a specific biophysical pathway: LLPS $\rightarrow$ Liquid-to-Solid Transition $\rightarrow$ Prion-like Propagation.
2. TDP-43 Link: It resolves the mystery of ANXA11-TDP-43 co-pathology by showing that ANXA11 aggregation is an upstream or parallel event that actively seeds and drives TDP-43 pathology.
3. Therapeutic Targets: By identifying nucleocytoplasmic transport defects and NPC remodeling as direct consequences of ANXA11 aggregation, the study highlights these pathways as potential therapeutic entry points. Targeting the phase transition of ANXA11 or preventing its interaction with NPC components could potentially halt disease progression.
4. Disease Relevance: The use of human iPSC-derived neurons confirms that these pathological mechanisms are relevant to human biology, not just cell lines, strengthening the case for ANXA11 as a primary driver in a subset of ALS/FTD cases.