ApoE is Secreted as a Lipid Nanoparticle by Mammalian Cells: Implications for Alzheimer’s Disease Pathogenesis

Proteinaceous lipid nanoparticles (LNPs) are the main vehicles for lipid transport between cells in mammals.

“Proteinaceous lipid nanoparticles (LNPs) are the main vehicles for lipid transport between cells in mammals.1–4”

• cites [1] sourcePartly supported

  The “CNS APOE protein structure” and “APOE CNS lipoproteins” sections describe lipid-associated lipoprotein particles and their role in lipid transport. They do not establish that these particles are the main vehicles for lipid transport between cells throughout mammals; that broader, comparative claim is unsupported by this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [2] sourcePartly supported

  The Introduction (page unavailable) says APOE circulates as part of a lipoprotein and plays an essential role in cholesterol and lipid metabolism. The section on APOE and mitochondrial function calls APOE a major modulator of cellular lipoprotein and cholesterol transport. Neither passage establishes that proteinaceous lipid nanoparticles are the main vehicles for lipid transport between cells throughout mammals. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [3] sourceSupported
• cites [4] sourceNot checked — no full text of the source

ApoE is associated with virtually all plasma LNPs, albeit in varying amounts.

“ApoE is associated with virtually all plasma LNPs, albeit in varying amounts.5”

• cites [5] sourcePartly supported

  The Discussion (page unavailable) reports APOE across multiple cerebrospinal-fluid lipoprotein populations of various sizes. That supports a broad association in CSF, but not the claim that APOE is associated with virtually all plasma lipoprotein particles in varying amounts. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

APOE4 lowers the age of onset of Alzheimer's Disease in a dose-dependent fashion.

“APOE4 is the strongest genetic risk factor for AD not only increasing the odds ratio of developing the disease but also lowering the age of onset in a dose-dependent fashion.17, 18”

• cites [17] sourceNot checked — no full text of the source
• cites [18] sourceSupported

Mammalian expression systems retain physiologically relevant secretory, folding and post-translational modification mechanisms.

“Mammalian expression systems retain physiologically relevant secretory, folding and post-translational modification mechanisms, but column-purified recombinant ApoE is invariably prepared as apoprotein.22–25”

• cites [22] sourceNot checked — no full text of the source
• cites [23] sourceCould not verify

  The Results section on apoE3-Jac self-association reports using apoE produced in HEK293 cells, but does not discuss whether mammalian expression preserves physiologically relevant secretion, folding, or post-translational modification mechanisms. Searches for those mechanisms found no passage addressing the claim; page numbers were not available in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [24] sourcePartly supported

  The Methods (Cell Culture; page unavailable) report that HEK293T cells expressed ApoE and that ApoE was detected in their conditioned medium, supporting secretion from a mammalian expression system. The paper does not establish that such systems retain physiologically relevant folding or post-translational modification mechanisms. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [25] sourcePartly supported

  In “Physiologically produced lipidated ApoE reproducibly activates MAP-kinase signaling in human neurons” (page unavailable), the paper reports that HEK293-produced ApoE is secreted and has a glycosylation state, supporting the secretory and post-translational-modification parts of the claim. It also finds that HEK293-produced ApoE has signaling properties similar to physiologically produced ApoE. The copy does not establish that mammalian expression systems retain physiologically relevant folding mechanisms, or make that general claim about expression systems. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The resulting nanoparticles typically lack the characteristic lipid complexity of their plasma- or CSF-derived counterparts.

“the resulting nanoparticles typically lack the characteristic lipid complexity of their plasma- or CSF-derived counterparts.12, 26”

• cites [12] sourcePartly supported

  In “Lipidated, recombinant ApoE lipoproteins resemble astrocyte-secreted ApoE lipoproteins” (page not available), the paper describes nanoparticles assembled from recombinant ApoE and a selected artificial lipid, supporting the claim that these particles have a limited lipid composition. It does not compare their lipid complexity with plasma- or CSF-derived particles or establish that this is typical of resulting nanoparticles generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [26] sourceNot checked — no full text of the source

Column-purified recombinant ApoE is invariably prepared as apoprotein.

“Mammalian expression systems retain physiologically relevant secretory, folding and post-translational modification mechanisms, but column-purified recombinant ApoE is invariably prepared as apoprotein.22–25”

• cites [22] sourceNot checked — no full text of the source
• cites [23] sourceCould not verify

  The Materials and Methods (Cholesterol Efflux Assay; page unavailable) mentions purified human apoE, and the Results (APOE3-Jac variant reduces self-association and increases lipidation; page unavailable) discusses lipid-free apoE. Neither passage says that column purification invariably yields apoprotein; searches for purification and lipid-free recombinant apoE did not establish that point. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [24] sourceCould not verify

  The Methods (Cell Culture) and Results describe ApoE expressed in HEK293T cells and used in conditioned medium, not column-purified ApoE. The Methods (Protein Purification and Labeling) describe column purification of other proteins but do not say whether column-purified recombinant ApoE is invariably an apoprotein. Searches for ApoE apoprotein, lipidation, and column purification found no passage addressing that claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [25] sourceCould not verify

  The 'Production of recombinant proteins' methods describe HEK293- and bacteria-produced ApoE but do not state that column-purified recombinant ApoE is an apoprotein. The 'Lipidated ApoE' methods describe purification of glial lipoproteins, a different preparation. Searches for apoprotein, lipid-free recombinant ApoE, and column purification found no passage establishing the claim. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

APOE4 increases the odds ratio of developing Alzheimer's Disease.

“APOE4 is the strongest genetic risk factor for AD not only increasing the odds ratio of developing the disease but also lowering the age of onset in a dose-dependent fashion.17, 18”

• cites [17] sourceNot checked — no full text of the source
• cites [18] sourceSupported

The apoprotein form of ApoE3 W210* eluted later than Peak 2 of full-length ApoE3 on a preparative SEC column.

“Consistent with earlier reports associating ApoE oligomerization to its C-terminal end domain,43 the apoprotein form of ApoE3 W210* eluted later than Peak 2 of full-length ApoE3 on a preparative SEC column (Figure 6A).”

• cites [43] sourceSupported

The intracellular lipidation mechanism of ApoE is distinct from the mechanism involved in the biosynthesis of ApoB-containing LDL particles.

“indicating that the intracellular lipidation mechanism of ApoE is distinct from its more well-studied counterpart that is involved in the biosynthesis of ApoB-containing LDL particles.41”

• cites [41] sourcePartly supported

  The section ‘Importance of apoB-MTP binding in lipoprotein assembly’ (page unavailable) describes MTP-assisted lipidation of nascent apoB. The paper does not establish how ApoE is lipidated or compare its mechanism with apoB’s, so the claimed distinction is unsupported. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

• AssumptionRests on an assumption: Lomitapide is an FDA-approved drug used to treat hypercholesterolemia based on its ability to inhibit MTTP.

No major isoform differences could be observed in promoting efflux for BODIPY-cholesterol loaded CCF-STTG1.

“Under these conditions, no major isoform differences could be observed (Figure 8D), in agreement with previously reported data.49”

• cites [49] sourceSupported

ApoE is not detected in appreciable quantities in its apo form in physiological fluids.

“Although ApoE is not detected in appreciable quantities in its apo form in physiological fluids, to date all reports describing the biochemical properties of this recombinant protein have deployed lipid-free biosynthetic materials.21–25”

• cites [21] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [23] sourceCould not verify

  The Results section reports cholesterol associated with apoE in human brains, and the Discussion describes lipid transport by apoE-containing particles. Neither passage addresses whether appreciable quantities of lipid-free apoE are detectable in physiological fluids. Searches for lipid-free apoE in plasma or cerebrospinal fluid found no passage establishing that claim; page numbers were unavailable in our copy. (LLM, conf=0.69)

  Experimental — accuracy not yet measured
• cites [24] sourceCould not verify

  The Results and Cell Culture Methods describe ApoE in conditioned medium and its effect on tau uptake, but do not address whether appreciable quantities of lipid-free ApoE occur in physiological fluids. Searches for ApoE’s apo form, lipidation, and physiological fluids found no passage supporting that claim. The copy also does not assess whether prior biochemical reports used lipid-free recombinant ApoE. (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [25] sourceCould not verify

  The Introduction describes ApoE as a major component of circulating lipoproteins, and the section on physiologically produced lipidated ApoE finds it in human-plasma LDL (page numbers unavailable). Neither passage establishes that appreciable quantities of lipid-free ApoE are absent from physiological fluids. Searches for apo-form and lipid-free ApoE did not find that claim in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

To date all reports describing the biochemical properties of this recombinant protein have deployed lipid-free biosynthetic materials.

“Although ApoE is not detected in appreciable quantities in its apo form in physiological fluids, to date all reports describing the biochemical properties of this recombinant protein have deployed lipid-free biosynthetic materials.21–25”

• cites [21] sourceNot checked — no full text of the source
• cites [22] sourceNot checked — no full text of the source
• cites [23] sourcePartly supported

  The Results section (page unavailable) describes biochemical tests of apoE produced in HEK293 cells and bacterially expressed apoE, and discusses self-aggregation of lipid-free apoE. It does not establish that these preparations were all lipid-free or that all reports to date used lipid-free recombinant material. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [24] sourceCould not verify

  The Methods (Cell Culture) describe producing ApoE in HEK293T cells and collecting conditioned medium, while the Results examine its effect on tau uptake. Neither section addresses whether the ApoE was lipid-free or whether all prior biochemical reports used lipid-free recombinant material (searched: lipid-free ApoE, lipidated ApoE). (LLM, conf=0.74)

  Experimental — accuracy not yet measured
• cites [25] sourcePartly supported

  The Methods section (page unavailable) confirms that the paper used recombinant ApoE produced in HEK293 cells and bacteria, but it does not establish that those preparations were lipid-free. The section on physiologically produced lipidated ApoE (page unavailable) also reports testing ApoE-containing glial lipoproteins and LDL. This paper therefore does not support the claim that all such reports used lipid-free materials. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

Several genetic loci with relevance to AD pathogenesis (e.g., PICALM, SORL1, BIN1) encode proteins found in the ER and/or Golgi compartments of cells.

“Second, several genetic loci with relevance to AD pathogenesis (e.g., PICALM, SORL1, BIN1) encode proteins found in the ER and/or Golgi compartments of cells.52, 53”

• cites [52] sourcePartly supported

  The paper reports an association between PICALM and Alzheimer’s disease and identifies BIN1 as another locus of interest (Abstract and Table 2; page unavailable). It discusses their roles in endocytosis, but the passages do not establish that their proteins are found in the ER or Golgi, and they do not address SORL1. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [53] sourceNot checked — no full text of the source

The discovery of disease-relevant alleles that map to two well-studied ATP-dependent lipid pumps, ABCA1 and ABCA7, highlights a potential role for extracellular LNP modification.

“The discovery of disease-relevant alleles that map to two well-studied ATP-dependent lipid pumps, ABCA1 and ABCA7,55, 56 highlights a potential role for extracellular LNP modification.”

• cites [55] sourceSupported
• cites [56] sourceSupported

Compared to ApoE3 or the Alzheimer's Disease-protective ApoE2 variant, the recovered yield of the LNP form of the disease-predisposing ApoE4 variant is higher.

“Compared to ApoE3 or the Alzheimer’s Disease-protective ApoE2 variant, the recovered yield of the LNP form of the disease-predisposing ApoE4 variant is higher.”

The ApoE3 W210* truncation mutant is secreted without lipidation.

“as the ApoE3 W210* truncation mutant is secreted without lipidation”

Secreted ApoE LNPs are markedly better substrates than the apoprotein itself for further growth via the action of ATP-dependent lipid pumps.

“Secreted ApoE LNPs are markedly better substrates than the apoprotein itself for further growth via the action of ATP-dependent lipid pumps.”

The LNP form is the major secreted product from unmodified CCF-STTG1 astrocytoma cells.

“The LNP form is, however, the major secreted product from unmodified CCF-STTG1 astrocytoma cells.”

The LNP yield of the rare disease-protective R251G variant of ApoE4 is comparable to ApoE3 and ApoE2.

“Intriguingly, the LNP yield of the rare disease-protective R251G variant of ApoE4 is comparable to ApoE3 and ApoE2.”

None of the other proteins that co-eluted in Peak 1 were readily identifiable as known constituents of biogenic LNPs.

“None of the other proteins that co-eluted in Peak 1 were readily identifiable as known constituents of biogenic LNPs.”

NSEM allowed direct visualization of stand-alone and antibody-bound LNPs.

“subsequent NSEM allowed direct visualization of stand-alone and antibody-bound LNPs (Figure 1C (right); Supplementary Figure 6).”

ApoE3 was the dominant apolipoprotein in LNPs generated from a recombinant source of this material.

“ApoE3 was in fact the dominant apolipoprotein in LNPs generated from a recombinant source of this material.”

Western blot analysis confirmed the presence of ApoE in concentrated fractions corresponding to both Peaks 1 and 2.

“Western blot analysis confirmed the presence of ApoE in concentrated fractions corresponding to both Peaks 1 and 2”

HepG2 and CCF-STTG1 cells transfected with APOE3 secrete ApoE in both Peak 1 and Peak 2 forms via small-scale SEC.

“When transfected with the APOE3, the supernatants of both cell cultures yielded Peaks 1 and 2 via small-scale SEC (Figure 2).”

The purified SEC fractions of Peaks 1 and 2 are quantitatively stable over the course of the experiment.

“these purified SEC fractions were reinjected onto a SEC column and shown to be quantitatively stable over the course of the experiment (Supplementary Figure 4B).”

ApoE in Peaks 1 and 2 are not interchanging conformers.

“To rule out the possibility that ApoE in Peaks 1 and 2 are interchanging conformers, these purified SEC fractions were reinjected onto a SEC column and shown to be quantitatively stable over the course of the experiment (Supplementary Figure 4B).”

We observed the biogenesis of a well-defined high-molecular weight form of ApoE on size exclusion chromatography in transfected Expi293F cells.

“Over the course of our efforts to produce preparative quantities of ApoE in transfected Expi293F cells, we observed the biogenesis of a well-defined high-molecular weight form of this protein on size exclusion chromatography.”

The yield of ApoE3 in Peak 1 from untransfected cells was two orders of magnitude lower than the overexpressed protein (~0.2 μg per mL culture).

“The yield of ApoE3 in this source of Peak 1 was two orders of magnitude lower than the overexpressed protein (~0.2 μg per mL culture).”

The ApoE protein in Peak 1 binds tightly to antibody HJ15.30.

“the ApoE protein in Peak 1 binds tightly to antibody HJ15.30”

In cultures of untransfected CCF-STTG1 cells, ApoE could only be detected in the fractions corresponding to Peak 1.

“In cultures of untransfected cells, ApoE could only be detected in the fractions corresponding to Peak 1 (Figure 2).”

The LNP size is ~15-20 nm.

“The LNP size (~15–20 nm) is consistent with previous findings with astrocytes from mice, and synthetic, ApoE LNPs.12”

Individual analysis of the ApoE3-containing fractions from the heparin column revealed the presence of Peaks 1 and 2 in all three fractions.

“Individual analysis of the ApoE3-containing fractions from the heparin column revealed the presence of Peaks 1 and 2 in all three fractions (Supplementary Figure 2).”

Two well defined peaks were identified by preparative SEC: one eluting as a high molecular weight species (Peak 1) and the other as a lower molecular weight tetrameric species (~158 kDa, Peak 2).

“Two well defined peaks were identified (Figure 1B), one eluting as a high molecular weight species (hereafter referred to as “Peak 1”) and the other as a lower molecular weight tetrameric species (~158 kDa, hereafter referred to as “Peak 2”) (Figure 1B).”

When Peak 1 of ApoE3 was compared to its Peak 2 form, several phospholipids, sphingomyelins (SM) and ceramides (Cer) were found to be enriched.

“When Peak 1 of ApoE3 was compared to its Peak 2 form, several phospholipids, sphingomyelins (SM) and ceramides (Cer) were found to be enriched (Figure 3B,C).”

Corresponding Peak 1 from negative control untransfected cells did not present detectable fluorescent signal.

“Moreover, corresponding Peak 1 from negative control untransfected cells did not present detectable fluorescent signal”

Lomitapide had no effect on the rate of formation of ApoE3 Peak 1.

“Our studies revealed that lomitapide had no effect on the rate of formation of ApoE3 Peak 1 (Supplementary Figure 9)”

The production of ApoE LNPs by Expi293F cells expressing ApoE3 W210* was markedly impaired.

“The production of ApoE LNPs by Expi293F cells expressing ApoE3 W210* was markedly impaired (Figure 6A,B).”

Enrichment of the BODIPY FL C5-ceramide fluorophore could be detected in ApoE3 Peak 1 but not Peak 2.

“Enrichment of the fluorophore could be detected in ApoE3 Peak 1 but not Peak 2 (Figure 5C).”

• Also stated in: § DISCUSSION · plain

  “only ApoE3 Peak 1 contains the selective perinuclear fluorescent probe”

No cholesterol was detectable in the Peak 2-associated ApoE apoprotein.

“No cholesterol was detectable in the Peak 2-associated ApoE apoprotein (Figure 3A)”

When transfected cells were centrifuged after 24 h and the already secreted ApoE3 was kept in that medium for an additional two days, the apoprotein was the dominant product.

“Similarly, when transfected cells were centrifuged after 24 h and the already secreted ApoE3 was kept in that medium for an additional two days, the apoprotein was the dominant product (Supplementary Figure 8B).”

Overexpression of ApoE3 in Expi293F cells consistently led to a >4-fold enrichment of free cholesterol in Peak 1 fractions.

“Overexpression of ApoE3 in Expi293F cells consistently led to a >4-fold enrichment of free cholesterol in Peak 1 fractions (Figure 3A).”

The Peak 1 fraction of the truncated variant showed no enrichment of cholesterol.

“Furthermore, the Peak 1 fraction of the truncated variant showed no enrichment of cholesterol (Figure 6C).”

After correcting for the amount of cholesterol in the untransfected fraction, the molar ratio of cholesterol:ApoE in LNPs harvested as Peak 1 was 15.9 ± 9.8 (based on four independent transfection experiments each with its own untransfected control).

“After correcting for the amount of cholesterol in the untransfected fraction, the molar ratio of cholesterol:ApoE in LNPs harvested as Peak 1 was 15.9 ± 9.8 (based on four independent transfection experiments each with its own untransfected control).”

Analogous enrichment was observed by comparing ApoE3 Peak 1 to an equivalent sample from an untransfected control.

“Analogous enrichment was observed by comparing ApoE3 Peak 1 to an equivalent sample from an untransfected control (Supplementary Figure 7).”

Expi293F cells express detectable amounts of ABCA1.

“In contrast to prevailing assumptions, we detected the expression of ABCA1 in Expi293F (Figure 5A).”

• Also stated in: § DISCUSSION · plain

  “Our work shows that while Expi293F possesses detectable amounts of ABCA1 expression”

When the growth medium was refreshed every 24 h, the apoprotein remained the dominant product for the next 48 h.

“In contrast, when the growth medium was refreshed every 24 h, the apoprotein remained the dominant product for the next 48 h (Supplementary Figure 8A).”

No cholesteryl ester could be detected in Peak 1 samples.

“nor could any cholesteryl ester be detected in Peak 1 samples.”

The rate of BODIPY-cholesterol efflux in the presence of Peak 1 compared favorably with that promoted by commercially available plasma-derived HDL.

“Indeed, the rate of BODIPY-cholesterol efflux in the presence of Peak 1 compared favorably with that promoted by commercially available plasma-derived HDL (Supplementary Figure 10).”

Peak 1 of each mutant was purified with a comparable ApoE:cholesterol molar ratio (8.3-39 for ApoE2, 9.1-32 for ApoE4, and 9.5-45 for ApoE4 R251G).

“Peak 1 of each mutant was purified with a comparable ApoE:cholesterol molar ratio (8.3–39 for ApoE2, 9.1–32 for ApoE4, and 9.5–45 for ApoE4 R251G).”

Preparative SEC chromatograms for all the isoforms revealed a similar pattern to ApoE3.

“Preparative SEC chromatograms for all the isoforms revealed a similar pattern to ApoE3 (Figure 8A,B).”

Peak 1 undergoes more effective endocytosis than the apoprotein form of ApoE in SH-SY5Y cells.

“As observed by confocal fluorescence microscopy, Peak 1 undergoes more effective endocytosis than the apoprotein form of ApoE (Figure 7C).”

We have identified and biochemically characterized a lipid nanoparticle (LNP) form of ApoE as a primary product of the protein export system in mammalian cells.

“We have identified and biochemically characterized a lipid nanoparticle (LNP) form of ApoE as a primary product of the protein export system in mammalian cells.”

Secreted ApoE LNPs are enriched in ceramide and its derivatives as well as cholesterol and related sterols.

“Our analysis has revealed enrichment of both classes of lipids of growing relevance to AD in secreted ApoE LNPs.”

• AssumptionRests on an assumption: Ceramide and its derivatives as well as cholesterol and related sterols are synthesized in the ER.

A lipid nanoparticle (LNP) form of ApoE is a primary extracellular product of the eukaryotic protein export system.

“We describe a lipid nanoparticle (LNP) form of ApoE as a primary extracellular product of the eukaryotic protein export system.”

The C-terminal domain of ApoE plays a key role in LNP biosynthesis.

“The C-terminal domain of ApoE plays a key role in LNP biosynthesis, as the ApoE3 W210* truncation mutant is secreted without lipidation.”

Bacterial ApoE holoprotein is different from its brain-derived counterpart with respect to key physical and/or chemical properties.

“resulting in holoprotein that is different from its brain-derived counterpart with respect to key physical (e.g., lipid composition) and/or chemical (e.g., post-translational modifications) properties.21”

• cites [21] sourceNot checked — no full text of the source

Expi293F (APOE3/E3) cells do express ApoE3.

“implying that contrary to general understanding Expi293F (APOE3/E3) do express ApoE3 (Supplementary Figure 4A).”

ApoE3 in Peak 1 retains its native conformation.

“Anti-ApoE antibodies were leveraged to show that ApoE3 in Peak 1 retains its native conformation.”

The enrichment of this ceramide analog in Peak 1 is driven by recombinant ApoE3 LNPs expression.

“consistent with the notion that the enrichment of this ceramide analog in Peak 1 is driven by recombinant ApoE3 LNPs expression.”

ApoE3 W210* is a loss-of-function variant as a lipid carrier.

“Our findings support the notion that ApoE3 W210* is a loss-of-function variant as a lipid carrier.”

• AssumptionRests on an assumption: ApoE3 W210* is the most common truncated ApoE variant found in humans and lacks the C-terminal end that plays a major role in lipid-binding.

The distinction between Peak 1 and Peak 2 starts at the earliest stages of biogenesis before the protein reaches the plasma membrane.

“suggesting that the distinction between Peak 1 and Peak 2 starts at the earliest stages of biogenesis before the protein reaches the plasma membrane.”

Our data suggests a likely role for intracellular nucleation.

“At the same time, our data showing that freshly exported ApoE LNPs are considerably better substrates of ATP-dependent lipid pumps than the apoprotein suggests a likely role for intracellular nucleation.”

Secretion of the lipid-free apoprotein may be an artifact of its overexpression.

“our data leads us to propose that secretion of the lipid-free apoprotein may be an artifact of its overexpression”

• AssumptionRests on an assumption: Overexpression of the APOE gene in mammalian cells yields apo-ApoE as the predominant secreted form of the protein.

ApoE export by cells such as astrocytes in the brain and hepatocytes in the liver are regulated in a manner that allows LNP nucleation to occur while the protein is being trafficked through the secretory apparatus of these cells.

“that ApoE export by cells such as astrocytes in the brain and hepatocytes in the liver are regulated in a manner that allows LNP nucleation to occur while the protein is being trafficked through the secretory apparatus of these cells.”

• AssumptionRests on an assumption: The dogma is that the glycosylated tetramer is assembled into an LNP through the action of ATP-dependent lipid pumps (e.g., ABCA1, ABCA7).

This work represents the first study in which a recombinant LNP form of the protein has been purified and analyzed.

“To our knowledge, this work represents the first study in which a recombinant LNP form of the protein has been purified and analyzed.”

• AssumptionRests on an assumption: An analogous mechanism has not been interrogated for ApoE, the dominant protein in HDL-like LNPs found in CSF.