Presenilin 1 (PS1) located at mitochondrial inner membrane regulates mitochondrial cristae junction proteins arrangement and cristae formation in HEK293 cells

This study demonstrates that Presenilin 1 (PS1) resides in the mitochondrial inner membrane where it regulates cristae junction protein arrangement and cristae formation by preventing ATAD3A oligomerization, thereby maintaining mitochondrial function and preventing dysfunctions associated with Alzheimer's disease.

The Gist

The Big Picture: The "Power Plant" Manager

Imagine your cells are bustling cities, and inside every city, there is a massive Power Plant called the Mitochondrion. This plant generates the electricity (ATP) your body needs to think, move, and survive.

Inside this power plant, there are intricate, folded tunnels called Cristae. Think of these like the winding roads and ramps inside a parking garage. The more organized and efficient these roads are, the better the power plant works.

For a long time, scientists knew about a protein called PS1 (Presenilin 1) because it was a major suspect in causing Alzheimer's disease. They thought PS1 was mostly a "traffic cop" for a different part of the cell (the Endoplasmic Reticulum). But this new study asks: Is PS1 also working inside the Power Plant?

The Discovery: PS1 is the "Foreman" of the Inner Membrane

The researchers found that PS1 isn't just hanging around outside; it is actually stationed on the Inner Wall of the Power Plant. Its job is to act as a Foreman or a Construction Manager.

• The Normal Job: When PS1 is healthy, it keeps the construction crew (other proteins) organized. It makes sure the "roads" (cristae) are built correctly and stay connected.
• The Problem: When PS1 is missing (which happens in certain types of Alzheimer's), the Power Plant goes into chaos.

What Happens When the Foreman is Gone?

The researchers removed PS1 from human cells (HEK293) to see what would happen. Here is the disaster they witnessed, explained through our Power Plant analogy:

1. The Power Goes Out: Without PS1, the plant stops making electricity efficiently. The cells run out of energy (low ATP) and the "voltage" (membrane potential) drops.
2. The Roads Collapse: The beautiful, folded tunnels (cristae) get swollen and messy. Instead of a sleek parking garage, it looks like a collapsed tunnel system.
3. Toxic Smoke: Because the plant is broken, it starts leaking toxic smoke (Reactive Oxygen Species or ROS), which damages the cell.
4. Water Leaks: The system loses control of its water pipes (Calcium), leading to a flood inside the plant.

The Villain: The "Overzealous Security Guard" (ATAD3A)

So, why does the Power Plant collapse when PS1 is gone? The study found a specific protein called ATAD3A.

• The Analogy: Imagine ATAD3A is a Security Guard whose job is to stand at the entrance of the parking ramps (the Cristae Junctions) to keep traffic flowing smoothly.
• The Normal State: PS1 (the Foreman) keeps the Security Guard calm and in the right spot.
• The Chaos: When PS1 is missing, the Security Guard (ATAD3A) gets panicked and starts clumping together with other guards. They form a giant, stubborn mob (oligomerization).
• The Result: This mob blocks the ramps, breaks the connection between the inner and outer walls of the plant, and causes the "blueprint" of the power plant (mitochondrial DNA) to get damaged and lost.

The Fix: Calming the Mob

The researchers tried to fix the problem by introducing a special "peptide" (a tiny piece of protein) that acts like a Peace Officer.

• This Peace Officer told the clumping Security Guards (ATAD3A) to break up and go back to their posts.
• The Result: When the guards stopped clumping, the Power Plant roads were rebuilt, the electricity came back, and the toxic smoke cleared up.

Why This Matters for Alzheimer's

This study is a big deal because it shows a new way Alzheimer's might work.

• Old Theory: Alzheimer's is caused by "plaque" (Aβ) clogging the brain.
• New Theory: Even before the plaque builds up, the "Foreman" (PS1) might be failing to manage the Power Plant's construction crew. This causes the brain cells to lose energy and die from the inside out, long before the plaques appear.

The Takeaway

Think of PS1 as the architect who ensures the power plant's internal roads are built right. Without this architect, the security guards (ATAD3A) panic, block the roads, and shut down the power. By understanding this specific mechanism, scientists might find new ways to treat Alzheimer's by fixing the power plant's internal architecture, rather than just trying to clean up the plaque.

Technical Summary

1. Problem Statement

Alzheimer's Disease (AD) is strongly linked to mutations in the PSEN1 gene, which encodes Presenilin 1 (PS1). While PS1 is well-known as the catalytic core of the $\gamma$-secretase complex involved in Amyloid-$\beta$ (A$\beta$) production, its non-canonical roles in mitochondrial function remain poorly understood.

• The Gap: Although mitochondrial dysfunction (impaired bioenergetics, oxidative stress, calcium dysregulation) is a hallmark of AD, the precise sub-mitochondrial localization of PS1 and the molecular mechanisms by which PS1 deficiency leads to mitochondrial structural collapse are unclear.
• Specific Question: Does PS1 reside in the mitochondrial inner membrane (IMM), and if so, how does its absence affect cristae architecture, protein interactions, and mitochondrial DNA (mtDNA) stability?

2. Methodology

The study utilized a combination of genetic manipulation, sub-cellular fractionation, advanced imaging, and biochemical assays in HEK293 cells and mouse models.

• Cell Models:
  • Generation of PS1 Knockout (PS1 KO) HEK293 cell lines using CRISPR-Cas9.
  • Rescue Experiments: Re-expression of wild-type (WT) PS1 in KO cells to confirm specificity.
  • Intervention: Use of a peptide inhibitor (DA1) targeting ATAD3A oligomerization and siRNA knockdown of ATAD3A.
• Localization & Fractionation:
  • Sub-mitochondrial Fractionation: Density gradient centrifugation to separate Outer Mitochondrial Membrane (OMM), Inner Mitochondrial Membrane (IMM), Intermembrane Space (IMS), and Matrix.
  • Alkali & Protease K Assays: To determine membrane integration and topology.
  • Immunoelectron Microscopy (IEM): Gold-labeled antibodies to visualize PS1 directly on the IMM.
• Functional Assays:
  • Seahorse XF Analyzer: Measured Oxygen Consumption Rate (OCR), ATP synthesis, and proton leakage.
  • Fluorescence Probes: TMRM (membrane potential), Rhod-2 AM (mitochondrial Ca$^{2+}$), and MitoSOX (ROS).
  • MAM Labeling: SPLICS Mt-ER Long P2A plasmid to quantify Mitochondria-Associated Membrane (MAM) area.
• Structural Analysis:
  • Super-Resolution Microscopy (Airyscan): To analyze mitochondrial network branching and vacuolization.
  • Transmission Electron Microscopy (TEM): To visualize cristae ultrastructure.
• Molecular Interactions:
  • Co-Immunoprecipitation (Co-IP) & Mass Spectrometry: To identify PS1 interactors.
  • Non-reducing SDS-PAGE: To detect ATAD3A oligomerization.
  • mtDNA Analysis: qPCR for copy number and long-range PCR for DNA damage/repair efficiency.

3. Key Contributions

1. Definitive Localization: The study provides robust evidence that a fraction of PS1 resides specifically on the Mitochondrial Inner Membrane (IMM), distinct from its ER localization.
2. Novel Mechanism of Cristae Disruption: It identifies that PS1 deficiency triggers the oligomerization of ATAD3A, a key IMM protein, which disrupts the arrangement of cristae junction proteins (MICOS complex, OPA1).
3. $\gamma$-Secretase Independence: The findings establish a $\gamma$-secretase-independent pathway where PS1 regulates mitochondrial homeostasis, offering a new perspective on AD pathogenesis beyond A$\beta$ accumulation.
4. Therapeutic Insight: The study demonstrates that inhibiting ATAD3A oligomerization (via peptide DA1) can rescue mitochondrial defects caused by PS1 loss, suggesting a potential therapeutic target.

4. Key Results

A. PS1 Localization

• PS1 was detected in ER, MAM, and mitochondrial fractions.
• IMM Confirmation: Co-localization with TIM23 (IMM marker), resistance to Proteinase K digestion in intact mitochondria (indicating IMM protection), and direct visualization via IEM confirmed PS1's presence on the mitochondrial inner membrane.

B. Functional Consequences of PS1 Deficiency

• Bioenergetics: PS1 KO cells showed reduced basal OCR, decreased ATP production, and increased proton leakage.
• Homeostasis: Significant loss of mitochondrial membrane potential ($\Delta\Psi$m), elevated mitochondrial Ca$^{2+}$, and increased ROS accumulation.
• Viability: PS1 KO cells exhibited reduced proliferation and increased cell death.

C. Structural Defects

• Morphology: PS1 KO cells displayed fragmented mitochondrial networks, perinuclear clustering, and extensive vacuolization.
• Ultrastructure: TEM revealed swollen cristae and a reduced number of cristae junctions.

D. Molecular Mechanism: The ATAD3A Axis

• Protein Interaction: PS1 interacts with IMM proteins (OPA1, MIC60, ATAD3A, TIM23) but not OMM proteins (MFN2, TOM70).
• ATAD3A Dysregulation:
  • PS1 KO led to increased ATAD3A protein levels and promoted its oligomerization.
  • This oligomerization disrupted the physiological interaction between ATAD3A and cristae junction proteins (MIC60, MIC19, OPA1).
• Downstream Effects:
  • MAM Expansion: PS1 KO cells showed a significantly expanded MAM area.
  • mtDNA Instability: Reduced mtDNA copy number and impaired mtDNA repair capacity (specifically to oxidative stress), while nuclear DNA repair remained intact.
• Rescue Experiments:
  • Overexpressing MFN2 or TIM23 did not rescue the defects.
  • Knocking down ATAD3A or treating with the DA1 peptide (which inhibits ATAD3A oligomerization) successfully restored mtDNA copy numbers, normalized MAM area, rescued membrane potential, and corrected cristae morphology.

5. Significance

This study redefines the role of Presenilin 1 in Alzheimer's Disease by linking it directly to the structural integrity of the mitochondrial inner membrane.

• Pathogenic Mechanism: It proposes a model where PS1 loss leads to ATAD3A oligomerization, which in turn destabilizes the MICOS complex and cristae junctions. This structural failure causes bioenergetic collapse, calcium overload, and mtDNA damage.
• Clinical Relevance: Since ATAD3A oligomerization is observed in AD patients, targeting this specific interaction (rather than just A$\beta$) could offer a novel therapeutic strategy to mitigate mitochondrial failure in neurodegeneration.
• Fundamental Biology: It highlights the critical, non-proteolytic role of PS1 in maintaining organelle architecture and protein network stability within the mitochondria.