Control of Prion Uptake by Bone Morphogenetic Protein Signaling

This study identifies the Bone Morphogenetic Protein (BMP) signaling pathway as a critical regulator of prion uptake, demonstrating that both genetic activation and pharmacological inhibition of this pathway respectively enhance or suppress prion internalization and propagation in human cell models.

The Gist

The Big Picture: How Prions "Break In"

Imagine a prion as a mischievous, shape-shifting burglar. Unlike a normal virus that carries its own blueprints, a prion is just a piece of protein that has folded into the wrong shape. Once it gets inside a healthy cell, it acts like a "bad influence," forcing all the healthy proteins in that cell to fold into the same wrong shape. This causes a chain reaction that destroys brain cells, leading to fatal diseases like Mad Cow Disease or Creutzfeldt-Jakob Disease.

For a long time, scientists knew how these burglars replicated once they were inside. But the big mystery was: How do they get in the door in the first place?

This paper is like a massive security audit. The researchers wanted to find out which "locks" or "doormats" on the surface of a cell allow these prion burglars to enter.

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The Experiment: The "Do-It-Yourself" Security Test

To solve this mystery without risking a real outbreak, the scientists created a clever, safe laboratory setup:

1. The Safe House: They used human brain cells (neuroblastoma cells) but surgically removed the gene that makes the "victim" protein (PrP). Without this protein, the prions can't replicate or cause disease inside these cells. This made the experiment safe to handle.
2. The Burglar: They created synthetic prions in a lab using sheep proteins. They painted these prions with fluorescent green paint (like glow-in-the-dark stickers) so they could see exactly when and where they entered the cells.
3. The Search: They used a powerful genetic tool called CRISPR-activation. Imagine the cell's DNA as a giant library of instruction manuals. The researchers used a robot librarian to randomly "turn up the volume" on every single manual in the library, one by one.
   • They asked: "If we make the cell produce more of Protein X, does the green prion burglar get in easier?"
   • They also asked: "If we make the cell produce more of Protein Y, does the burglar get blocked?"

The Big Discovery: The "BMP" Key

After testing thousands of genes, they found a surprising pattern. The most important factor wasn't a specific "prion receptor" (a door handle made just for prions). Instead, it was a whole signaling system called Bone Morphogenetic Protein (BMP) signaling.

Here is the analogy:

• The Cell is a house.
• The Prion is a burglar trying to sneak in.
• BMP Signaling is the house's security system and front door mechanism.

The researchers found that:

• Turning UP the BMP system (making the door mechanism more active) made the house much easier to break into. The prions flooded in.
• Turning DOWN the BMP system (using drugs to lock the mechanism) made the house very secure. The prions couldn't get in.

Specifically, they found that when the cell's "BMP receptors" (the door handles) were activated, the cell became a "super-uptaker," swallowing up the prions. When they blocked these receptors with drugs (like Noggin and Dorsomorphin), the prions were stuck outside.

Why This Matters

1. It's Not Just One Key: Scientists thought there might be one specific "prion receptor." Instead, they found that the cell's general "state of mind" (controlled by BMP signaling) determines if it's open to invasion. It's like a house that is more vulnerable to burglary when the lights are off and the alarm is disabled, rather than because of a specific broken lock.
2. A New Way to Fight Disease: Since BMP signaling is a pathway we can control with drugs, this opens a new door for treatment. If we can give patients a drug that temporarily "locks the door" (inhibits BMP signaling), we might be able to stop prions from entering cells in the first place. This could stop the disease before it starts spreading through the brain.
3. Safety First: The fact that they could do this with sheep prions in a safe lab setting proves we can study these deadly diseases without needing to handle the most dangerous human samples.

The Takeaway

Think of prion disease as a house invasion. This paper discovered that the house doesn't have a specific "prion door." Instead, the house has a general security system (BMP signaling). If the security system is active, the burglars (prions) can't get in. If the system is turned off, the burglars walk right in.

The scientists have now found the switch to that security system. By learning how to flip that switch, we might finally have a way to lock the doors against these deadly protein burglars.

Technical Summary

1. Problem Statement

Prion diseases (Transmissible Spongiform Encephalopathies) are fatal neurodegenerative disorders caused by the misfolded prion protein ($PrP^{Sc}$). A critical, yet poorly understood, step in the prion life cycle is the cellular uptake of infectious prions by host cells. While the conversion of normal prion protein ($PrP^C$) to the pathogenic form is well-studied, the specific molecular mechanisms and host factors governing the initial internalization of prions remain largely unidentified.

• Gap: Unlike other neurodegenerative aggregates (e.g., Tau, $\alpha$-synuclein), no specific receptors or signaling pathways have been definitively validated as therapeutic targets for prion entry.
• Challenge: Studying prion uptake is difficult due to biohazards associated with human prions and the lack of homogeneous, infectious, and fluorescently labeled prion preparations suitable for high-throughput screening.

2. Methodology

The authors developed a multi-faceted approach combining synthetic biology, CRISPR screening, and pharmacological validation.

A. Generation of Infectious Fluorescent Prions

• Recombinant Prion Production: They produced full-length recombinant ovine PrP (VRQ polymorphism) in E. coli.
• PMSA: Using Protein Misfolding Shaking Amplification (PMSA) with dextran as a cofactor, they generated infectious aggregates in vitro.
• Validation: These aggregates were confirmed to be infectious via intracerebral inoculation in TgVole and Tg338 transgenic mice, showing characteristic incubation periods, PK-resistant $PrP^{Sc}$, and spongiform pathology.
• Labeling: Aggregates were conjugated with AlexaFluor488 (prion488) and pHrodo Red to allow single-cell resolution tracking of internalization via flow cytometry.

B. Cell Model System

• SH-KOvp64 Cells: A human neuroblastoma (SH-SY5Y) line with CRISPR-mediated knockout of the PRNP gene (preventing $PrP^C$ expression and subsequent replication) was engineered to express a constitutively active dCas9-VP64 transactivator.
• Rationale: This system isolates the uptake step from downstream replication, allowing the study of entry mechanisms without confounding amplification.

C. Genome-Wide CRISPR Activation Screen

• Library: Used the T.gonfio library, a quadruple-guide RNA (qgRNA) human CRISPR activation library targeting all transcription start sites of the protein-coding genome.
• Workflow:
  1. Transduced SH-KOvp64 cells with the library.
  2. Exposed cells to prion488 for 24 hours.
  3. Sorted cells into Prion488-positive (high uptake) and Prion488-negative (low uptake) populations via FACS.
  4. Sequenced genomic DNA to identify enriched or depleted guides.
• Specificity Controls: Validated hits against other aggregates (Tau, $\alpha$-synuclein, A$\beta$) and non-aggregated endocytic probes (Transferrin, Dextran, BioParticles) to distinguish prion-specific mechanisms from general endocytosis.

D. Pharmacological and Genetic Validation

• Genetic: Arrayed CRISPRa screens confirmed top hits.
• Pharmacological: Used Noggin (extracellular BMP antagonist) and Dorsomorphin (BMP receptor kinase inhibitor) to suppress BMP signaling.
• Propagation Assays: Tested BMP inhibition in chronically infected cell lines (HovH and HovL) expressing ovine PrP to assess effects on $PrP^{Sc}$ accumulation.

3. Key Results

A. Identification of BMP Signaling as a Modulator

• Screen Output: The primary screen identified 43 modifiers of prion uptake.
• Top Hit: Bone Morphogenetic Protein (BMP) signaling emerged as the most significant pathway.
  • Enhancers: Transcriptional activation of BMP receptors (BMPR1B, BMPR2, ACVRL1), co-receptors (RGMA), and the effector SMAD1 increased prion uptake.
  • Inhibitors: Activation of the inhibitory effector SMAD6 reduced uptake.
• Validation:
  • Genetic: Overexpression of BMP receptors/SMAD1 increased uptake; SMAD6 decreased it.
  • Pharmacological: Treatment with Noggin and Dorsomorphin significantly reduced prion internalization in multiple cell lines (SH-SY5Y, LN229 glioblastoma, HEK-293A).
  • Mechanism: Upregulation of BMP genes increased phosphorylation of SMAD1/5 (pSMAD1/5) and expression of ID genes, confirming canonical pathway engagement.

B. Specificity of Uptake Mechanisms

• Cargo Selectivity: While some hits (e.g., KRAS, MET) affected general endocytosis, others like the transcription factor FOXI3 selectively enhanced prion and fibril uptake without affecting fluid-phase markers (Transferrin/Dextran).
• Pathway Independence: Prion uptake was not dependent on clathrin-mediated endocytosis (Transferrin uptake was unaffected by hits), suggesting prions utilize specific or alternative endocytic routes (e.g., macropinocytosis, phagocytosis-like pathways).

C. Impact on Prion Propagation

• In chronically infected cell lines (HovH/HovL), pharmacological inhibition of BMP signaling (Noggin + Dorsomorphin) led to:
  • A reduction in the number of $PrP^{Sc}$-positive cells.
  • A decrease in total and PK-resistant $PrP^{Sc}$ levels (confirmed by Western Blot).
• This indicates that BMP signaling is not only required for initial entry but also sustains the steady-state burden of prions in infected cells.

D. Relevance to Other Diseases

• The authors analyzed genetic datasets for Alzheimer's (AD), ALS, and Parkinson's (PD) and found no significant enrichment of BMP pathway genes in these conditions, suggesting the BMP-prion link may be specific to prion biology or context-dependent.
• However, several hit genes (BMPR2, MECOM, GSX2, LRIG1) mapped to genomic regions associated with sporadic Creutzfeldt-Jakob disease (sCJD) risk.

4. Significance and Contributions

1. Novel Mechanism Discovery: This is the first study to identify the BMP signaling pathway as a critical regulator of prion cellular uptake. It shifts the paradigm from searching for a single "prion receptor" to understanding how host signaling states (cell state) modulate susceptibility to infection.
2. Methodological Advance: The development of fluorescent, infectious recombinant ovine prions allows for high-throughput, biohazard-reduced screening of prion entry mechanisms, overcoming the limitations of brain-derived prions.
3. Therapeutic Potential: The findings suggest that pharmacological inhibition of BMP signaling (using existing inhibitors like Noggin or Dorsomorphin) could be a viable strategy to block the early steps of prion infection and reduce prion propagation in infected tissues.
4. Cell State Dependence: The study highlights that prion susceptibility is not static but is dynamically regulated by transcriptional programs and signaling pathways, offering new angles for understanding strain-specific tropism and disease progression.

Conclusion

De Cecco et al. successfully utilized a CRISPR-activation screen to uncover that BMP signaling positively regulates prion uptake. By demonstrating that inhibiting this pathway reduces both prion internalization and subsequent accumulation of infectious prions, the study provides a robust mechanistic framework and a potential therapeutic avenue for interfering with prion disease propagation.