A genome-wide short-hairpin RNA (shRNA) screen (Chu et al., Cell 2015) revealed that LDL-derived cholesterol exits lysosomes in part through transient lysosome–peroxisome membrane contacts (LPMCs), bridged by the lysosomal Ca2+-binding protein synaptotagmin VII (Syt7) binding the lipid PI(4,5)P2 on the peroxisomal membrane, and that peroxisomal-disorder cells accumulate lysosomal cholesterol. Later work identified PIP4K2A as the kinase setting peroxisomal PI(4,5)P2 levels, established peroxisome-to-ER cholesterol hand-off at extended-synaptotagmin-mediated contacts, showed peroxisome-to-cilium cholesterol delivery, and found that lysosomal cholesterol accumulation is shared by most peroxisomal disorders and reversible by 2-hydroxypropyl-β-cyclodextrin in mouse models. However, the mechanistic core remains unresolved: what activates Syt7 and regulates LPMC formation (local Ca2+ signals, post-translational modifications, or the peroxisomal proteins from the screen, of which only PIP4K2A and ABCD1 are mechanistically linked to reduced peroxisomal PI(4,5)P2); by what molecular mechanism cholesterol crosses the ~30 nm contact gap (a soluble sterol-transfer protein, a membrane-integral or tether-mediated conduit, or diffusion between apposed membranes); what becomes of LDL-derived cholesterol after it reaches peroxisomes, and how post-lysosomal cholesterol is dynamically distributed among organelles and cell types; and whether defective transport through this route causally contributes to peroxisomal-disorder symptoms and whether promoting cholesterol transport relieves them. This problem asks for the complete mechanistic and pathophysiological dissection of the LPMC cholesterol-transport route.

Background

Cholesterol is an essential membrane lipid of mammalian cells; roughly 60–80% of it resides at the plasma membrane, while cells acquire exogenous cholesterol through receptor-mediated endocytosis of low-density lipoprotein (LDL) particles. In lysosomes — the acidic degradative organelles — LDL cholesteryl esters are hydrolyzed to free cholesterol, which the Niemann-Pick C proteins NPC2 and NPC1 (defective in the neurodegenerative lipid-storage disease Niemann-Pick type C) hand through the lysosomal lumen to the limiting membrane. How cholesterol then leaves the lysosomal membrane and reaches the endoplasmic reticulum (ER), plasma membrane, and other organelles is much less understood: this transport is largely non-vesicular and occurs at membrane contact sites (MCSs), regions where two organelles are apposed within ~10–30 nm and bridged by tether proteins, often together with lipid-transfer proteins that shuttle lipids between the apposed bilayers.

Peroxisomes are single-membrane oxidative organelles best known for very-long-chain fatty acid β-oxidation, ether-lipid (plasmalogen) synthesis, and reactive-oxygen-species metabolism; they physically interact with the ER, mitochondria, lipid droplets, and lysosomes. A genome-wide short-hairpin RNA (shRNA) interference screen designed around amphotericin B — a cholesterol-binding, pore-forming antibiotic whose cytotoxicity reports the arrival of LDL-derived cholesterol at the plasma membrane — identified 341 genes whose knockdown delays post-lysosomal cholesterol transport, with striking enrichment of peroxisome genes (Chu et al., Cell 2015). Nine peroxisomal proteins came out of the screen, and knockdown of each caused lysosomal cholesterol accumulation mimicking NPC1 deficiency. The same study showed that lysosomes form dynamic, LDL-regulated contacts with peroxisomes (lysosome–peroxisome membrane contacts, LPMCs), reconstitutable in vitro from purified organelles, and that these contacts are bridged by synaptotagmin VII (Syt7) — a lysosomal Ca2+-binding protein with cytosolic C2 domains — engaging the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) on the peroxisomal membrane; radioactive-cholesterol transfer assays showed cholesterol moving from lysosome to peroxisome at these contacts. Cells from patients with peroxisomal disorders (Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD), infantile Refsum disease) and corresponding mouse models accumulated lysosomal cholesterol.

Subsequent work from the originating laboratory and from independent groups established several pieces: PIP4K2A, a phosphatidylinositol-5-phosphate 4-kinase, sets peroxisomal PI(4,5)P2 levels, and its knockdown reduces LPMCs and blocks cholesterol egress (Hu et al., 2018); cholesterol continues from peroxisomes to the ER at peroxisome–ER contacts tethered by extended synaptotagmins (E-Syts, ER-resident proteins related to Syt7 whose Ca2+-responsive C2 domains bind peroxisomal PI(4,5)P2), with peroxisomes augmenting lysosome-to-ER cholesterol transfer (Xiao et al., 2019); peroxisomes also deliver cholesterol to the primary cilium — the antenna-like signaling organelle — via a PEX14–Rabin8–Rab10–KIFC3 complex, and Zellweger-syndrome patient cells are ciliary-cholesterol-deficient (Miyamoto et al., 2020); conversion of the peroxisomal cholesterol pool to bile acids was excluded. Functional peroxisomes proved necessary for normal regulation of cholesterol synthesis (Charles et al., 2020). Lysosomal cholesterol accumulation was then found to be a shared biochemical feature of most peroxisomal disorders, reversible by 2-hydroxypropyl-β-cyclodextrin (HPCD, a cholesterol-solubilizing cyclic oligosaccharide), which also ameliorated phenotypes of mice lacking Abcd1, the gene defective in X-linked adrenoleukodystrophy (Dong et al., 2023). Authoritative independent reviews place the peroxisomal route among the established non-vesicular routes out of the late-endosomal/lysosomal system, alongside sterol-transfer-protein routes such as ORP1L (an oxysterol-binding-protein-related sterol-transfer protein) and Aster/GramD1B (Ikonen and Olkkonen, 2023), and Salmonella was shown to subvert the same PI(4,5)P2-dependent contact machinery to siphon lysosomal cholesterol into its vacuole (Raj et al., 2025). Yet, as an independent peroxisome specialist recently summarized, peroxisomes are still only 'proposed' to act as a cholesterol shuttle, and whether altered contacts contribute to disease 'remains to be seen' (Carmichael, 2025).

Problem Statement

The problem, raised by Chu et al. (Cell 2015) and its accompanying preview (Jin et al., 2015) and left unresolved by a decade of follow-up work, is the complete mechanistic and pathophysiological dissection of the lysosome–peroxisome membrane contact (LPMC) route of cholesterol transport. Specifically:

  1. Contact components and regulation. Determine what activates lysosomal Syt7 and brings it into productive engagement with peroxisomal PI(4,5)P2 — for example local Ca2+ signals, post-translational modification, or other inputs — and thereby controls the timing and frequency of LPMC formation; and make a comprehensive assessment of the molecular components of the contact, including whether a three-way lysosome–peroxisome–ER junction is involved, establishing how the peroxisomal proteins whose loss phenocopies NPC1 deficiency (the nine screen hits, together with PIP4K2A and ABCD1) contribute to contact formation and to cholesterol egress from lysosomes. Reduced peroxisomal PI(4,5)P2 is the established mechanism for PIP4K2A and ABCD1 and is one candidate route for the remaining hits, which may instead act on Syt7 engagement, on contact stability or geometry (loss of TMEM135, for example, reduces LPMC number), or indirectly through peroxisome biogenesis or bulk membrane-lipid composition; distinguishing direct enzymatic or lipid-transport activities from these indirect effects is part of the question. (PIP4K2A, identified in 2018, and the cyclodextrin-rescue data of 2023 postdate the 2015 source.)

  2. The transfer step. Establish the molecular mechanism by which cholesterol leaves the lysosomal membrane, crosses the ~30 nm LPMC gap, and inserts into the peroxisomal membrane. Candidate devices include soluble sterol-transfer proteins — the ORP, STARD, GRAMD1 and FABP families, a non-exhaustive list — membrane-integral or tether-mediated conduits spanning the contact, and diffusion between apposed membranes; the in vitro reconstitution in which LPMCs form and lysosome-to-peroxisome transfer is measured operates in the presence of cytosol and metabolic energy (ATP/GTP), which constrains the mechanism that can account for the observed transfer.

  3. Fates and inter-organellar transport. Determine what becomes of LDL-derived cholesterol after it reaches peroxisomes — onward delivery to the ER and to the primary cilium are established, while local esterification, oxidation, delivery to mitochondria, and other routes remain uncharacterized — and how cholesterol is dynamically transported among organelles after leaving the lysosome. Different post-lysosomal pathways, including the parallel sterol-transfer-protein and vesicular routes described in the Background, may function in different cell types or under different conditions, and perhaps only a subset of them is directed to the plasma membrane; their relative contributions are not yet quantified.

  4. Pathophysiology. Determine whether defective cholesterol transport through LPMCs causally contributes — beyond the correlation established so far — to the cholesterol accumulation and clinical manifestations of the different peroxisomal disorders, and whether promoting cholesterol transport (for example with 2-hydroxypropyl-β-cyclodextrin, which so far has preclinical support in Abcd1-knockout mice) relieves those symptoms.

The dissection spans all four strands: strands 1–2 constitute the load-bearing mechanistic core, and strands 3–4 establish the route's place in cellular cholesterol distribution and its medical relevance. Together the strands support one objective — explaining how the LPMC route works and why it matters. The 2018–2023 follow-up work summarized under Background addresses parts of strands 1, 3 and 4.

References

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