29 Oct 2024

Far from a static barrier, the brain’s extensive interface with the blood is increasingly being appreciated as an active border, one that serves not only to keep some proteins out, but to let others in (Oct 2024 conference news). Once across, some of these travelers find themselves within the clutches of a specialized type of microglia, according to a preprint posted October 2 on bioRxiv. Through fluorescent labeling of the mouse plasma proteome, scientists led by Tony Wyss-Coray at Stanford University, California, found that certain subsets of microglia readily internalize plasma proteins, and that these cells are most abundant in the hypothalamus, thalamus, and hippocampus.

The microglia, in turn, seem to benefit, revving up metabolism and phagocytosis. The liver protein ApoA-I was particularly good at bolstering the latter. Overall, the findings suggest that the brain’s resident immune cells take cues from proteins coursing through the blood.  

“This study is an exciting new addition to the field of microglial signalling and regulation, particularly in how messengers from the blood can communicate with brain resident immune cells,” commented Róisín McManus of the German Center for Neurodegenerative Diseases in Bonn.

Previously, the Wyss-Coray lab reported that in young, healthy mice, myriad plasma proteins are actively transported into the brain by endothelial cells via receptor-mediated endocytosis (Jul 2020 news). The findings suggested circulating factors could strongly influence brain function and health.

To investigate this, first author Nannan Lu and colleagues looked to the brain’s resident immune cells. They collected and pooled plasma from dozens of mice, then chemically labeled the entire proteome with NHS-Atto647N, a fluorophore dye. They then injected the labeled plasma intravenously into healthy, 3-month-old mice. Twenty hours later, microglia isolated from six different brain regions contained the dye. Uptake was highest in the hypothalamus, where 15 percent of microglia contained dye-labeled proteins. Ten percent of microglia in the thalamus, and 6 percent in the hippocampus, had plasma proteins, whereas less than 3 percent of microglia in the cerebellum, striatum, and cortex did.

Uptake of plasma proteins peaked between 20 and 40 hours after injection. By injecting mice a second time with different fluorophores, the researchers found that almost all the microglia that had internalized the first batch also took in the second, suggesting that they were not taking in these proteins by chance. Co-injecting a pH-sensitive dye along with their labeled plasma proteins revealed that at least some of the internalized proteins were relegated to microglial lysosomes. In the hypothalamus and hippocampus, this uptake plummeted with age, dropping by 60 percent in 2-year-old mice relative to the younger animals.

Microglia loaded with plasma proteins were no closer to capillaries than other microglia, suggesting proximity to blood vessels did not explain the uptake. What, then, distinguished these plasma-positive microglia (PPM) from their plasma-negative (PNM) brethren? To find out, the scientists isolated and sorted microglia, with or without dye-labeled plasma proteins, from the hypothalamus, then ran transcriptomic, proteomic, and lipidomic comparisons.

The PPMs upregulated genes involved in immune responses, chemokine signaling, chemotaxis, endocytosis, and antigen presentation. Proteomics pointed to similar upregulated pathways, as well as to an uptick in proteins involved in metabolism. Lipidomic and metabolomic studies jibed with this, indicating that relative to PNMs, PPMs ramped up mitochondrial metabolic function. Microglia that had gobbled up plasma proteins tended to have larger mitochondria and lysosomes and, in functional assays, phagocytosed more actively.

Do similar microglia exist in the human brain? Perhaps. Searching published single-cell RNA-Seq datasets, the scientists identified a subset of microglia bearing a similar transcriptomic signature as that in the mouse cells that had gorged on plasma proteins. What’s more, when they infused fluorescently labeled plasma proteins into a chimeric mouse model in which microglia had been replaced by human ones, they observed a similar regional pattern of plasma protein uptake, such that human microglia in the hypothalamus took up the most circulating proteins, followed by microglia in the thalamus, hippocampus, cerebellum, striatum, and cortex. This suggested that human microglia can develop an appetite for plasma proteins that is somehow dictated by their local environment within the brain.

Lu and colleagues investigated the repertoire of plasma proteins taken up by microglia, and how those proteins influenced the activity of the cells that consumed them. Many of the internalized proteins belonged to the apolipoprotein family, or were part of blood-clotting pathways. But the microglia were picky. While they ate ApoA-I and ApoA-II, they didn’t touch transferrin or albumin, two highly abundant plasma proteins.

Produced primarily in the liver and intestines, but not in the brain, ApoA proteins form part of high-density lipoprotein (HDL) complexes. These peripheral proteins are known to use transcytosis, mediated by the scavenger receptor BI, to cross into the brain from the blood. Microglia express this receptor on their surface, suggesting they might use it to snag ApoA proteins. In support of this, the researchers found that in cell culture, an antibody to the scavenger receptor prevented ApoA-I uptake into primary brain endothelial cells and microglia.

In cultured, human pluripotent stem-cell-derived microglia, ApoA-I quashed expression of pro-inflammatory genes in response to lipopolysaccharide. ApoA-I also revved microglial phagocytosis, including of fluorescently labeled, fibrillar Aβ, suggesting these microglia might help rein in amyloid plaques.

Finally, in cells isolated from ApoA-I knockout mice, phagocytosis of fluorescently labeled myelin flagged when microglia came from the hypothalamus, but not the cortex. This resonates with the idea that some microglia in the hypothalamus may rely upon plasma proteins, particularly ApoA-I, for optimal phagocytosis. Infusion of these knockout mice with recombinant ApoA-I restored phagocytosis in the microglia that took up the apolipoprotein. Furthermore, microglia that internalized the infused ApoA-I in the knockout mice expressed a bevy of genes associated with antigen presentation, immune response, and phagocytosis, while those that ignored ApoA-I lacked this signature.

Given that microglial phagocytosis of plasma proteins wanes with age, McManus wondered if this explains why microglial phagocytic activity in general slows with age, or with disease (Antignano et al., 2023). “It would be interesting to examine whether this pathway is further modulated in neurodegenerative diseases such as Alzheimer’s, where impaired microglial phagocytosis plays an important role,” she wrote (Podleśny-Drabiniok et al., 2020). She speculated that ApoA-I administration might even have therapeutic benefit (comment below).

Samuel Gandy of Mount Sinai School of Medicine in New York noted that the findings dovetail with previous studies suggesting a protective effect of ApoA-I on AD, reflected in biomarker levels, cholesterol metabolism, and cognitive function (Smach et al., 2011; Lazarus et al., 2015; Montañola et al., 2016). “These studies illustrate the importance of genetic and epigenetic communication linking the peripheral circulation to the function of brain microglia (Endres, 2021),” he wrote.

Lu thinks ApoA-I is responsible for some of the plasma-protein-mediated changes in microglia, but not all. Ongoing experiments in the lab suggest that other plasma proteins may preferentially modulate specific microglial pathways, such as antigen presentation. Future studies will address how the changing plasma proteome skews microglial activity in the context of aging and disease, she said.—Jessica Shugart

Comments

1. • Róisín McManus
     German Center for Neurodegenerative Diseases (DZNE)
   • Posted: 29 Oct 2024

   This new study by Lu and colleagues is an exciting new addition to the field of microglial signalling and regulation, particularly in how messengers from the blood can communicate with brain-resident immune cells.

   This paper builds on previous work from the Wyss-Coray group, who showed that plasma proteins can pass into the parenchyma under healthy conditions (Yang et al., 2020), and they now address how such proteins may be affecting the local environment. To do so, Lu and colleagues fluorescently labelled the plasma from mice, injected it i.v. to recipient mice and 20 hours later, examined which microglial populations had taken up the tagged-plasma proteins. Interestingly, the microglial populations that took up the labelled plasma varied considerably by brain region, with hypothalamic microglia taking up the most. RNA sequencing on the plasma-positive versus plasma-negative microglia from the hypothalamus revealed a significant effect of plasma on the microglial transcriptome, with over 500 genes increased in the plasma-positive microglial cells, many relating to innate immune pathways and antigen presentation.

   Proteomic analysis of the plasma-positive microglia identified over 200 proteins enriched in these cells, with an overlap between the RNA and protein levels of numerous targets. At a functional level, the plasma-positive microglia were more metabolically active, with increased phagocytic activity, demonstrating how functionally distinct this cellular population was. Via a range of confirmation experiments, Lu and colleagues found that the plasma protein Apolipoprotein A-I (ApoA-I) was taken up by microglia in vivo. Importantly, ApoA-I alone could drive many of the changes induced by bulk plasma, including increasing genes that were identified in the original screen and ApoA-I could regulate microglial function including increasing phagocytosis.

   This exciting work highlights the complex signalling occurring between the periphery and the brain, which is an understudied area of research. As the experiments were conducted in young mice, it raises the question of how such processes might change with age and in disease models. Indeed, Lu and colleagues found that the microglia of aged mice took up less labelled-plasma than their young counterparts, and, since these proteins could regulate phagocytosis it may partly explain why microglial phagocytic activity is altered with age (Antignano et al., 2023). It would certainly be interesting to examine whether this pathway is further modulated in neurodegenerative diseases, such as Alzheimer’s, where impaired microglial phagocytosis plays an important role (Podleśny-Drabiniok et al., 2020). As the findings were similar between human and murine microglia, it is interesting to speculate whether ApoA-I administration could have therapeutic benefits to those with neurodegenerative diseases, where successful treatments to stop disease progression are lacking.

   ApoA-I is primarily produced by the intestine and liver, therefore future studies could examine how ApoA-I can act as a messenger to the brain during liver or intestinal diseases such as steatotic liver disease or Crohn’s disease. It is likely that in these diseases the circulating plasma proteins are different to basal healthy conditions, which in turn may modulate the microglia and other brain cells in unknown ways. Altogether this important work brings new light to the complex interplay between the periphery and the brain, with the scope to build on these compelling findings to examine these interactions beyond microglia to other brain cells and to better understand these processes in health and disease.

   References:

   Yang AC, Stevens MY, Chen MB, Lee DP, Stähli D, Gate D, Contrepois K, Chen W, Iram T, Zhang L, Vest RT, Chaney A, Lehallier B, Olsson N, du Bois H, Hsieh R, Cropper HC, Berdnik D, Li L, Wang EY, Traber GM, Bertozzi CR, Luo J, Snyder MP, Elias JE, Quake SR, James ML, Wyss-Coray T. Physiological blood-brain transport is impaired with age by a shift in transcytosis. Nature. 2020 Jul 1; PubMed.

   Antignano I, Liu Y, Offermann N, Capasso M. Aging microglia. Cell Mol Life Sci. 2023 Apr 21;80(5):126. PubMed.

   Podleśny-Drabiniok A, Marcora E, Goate AM. Microglial Phagocytosis: A Disease-Associated Process Emerging from Alzheimer's Disease Genetics. Trends Neurosci. 2020 Dec;43(12):965-979. Epub 2020 Oct 27 PubMed.

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References

News Citations

1. Reconceptualizing the BBB: Is It Time to Swap ‘Barrier’ for ‘Border'? 11 Oct 2024
2. Blood-Brain Barrier Surprise: Proteins Flood into Young Brain 2 Jul 2020

Paper Citations

1. Antignano I, Liu Y, Offermann N, Capasso M. Aging microglia. Cell Mol Life Sci. 2023 Apr 21;80(5):126. PubMed.
2. Podleśny-Drabiniok A, Marcora E, Goate AM. Microglial Phagocytosis: A Disease-Associated Process Emerging from Alzheimer's Disease Genetics. Trends Neurosci. 2020 Dec;43(12):965-979. Epub 2020 Oct 27 PubMed.
3. Smach MA, Edziri H, Charfeddine B, Ben Othman L, Lammouchi T, Ltaief A, Nafati S, Dridi H, Bennamou S, Limem K. Polymorphism in apoA1 Influences High-Density Lipoprotein Cholesterol Levels but Is Not a Major Risk Factor of Alzheimer's Disease. Dement Geriatr Cogn Dis Extra. 2011 Jan;1(1):249-57. PubMed.
4. Lazarus J, Mather KA, Armstrong NJ, Song F, Poljak A, Thalamuthu A, Lee T, Kochan NA, Brodaty H, Wright MJ, Ames D, Sachdev PS, Kwok JB. DNA methylation in the apolipoprotein-A1 gene is associated with episodic memory performance in healthy older individuals. J Alzheimers Dis. 2015;44(1):175-82. PubMed.
5. Montañola A, de Retana SF, López-Rueda A, Merino-Zamorano C, Penalba A, Fernández-Álvarez P, Rodríguez-Luna D, Malagelada A, Pujadas F, Montaner J, Hernández-Guillamon M. ApoA1, ApoJ and ApoE Plasma Levels and Genotype Frequencies in Cerebral Amyloid Angiopathy. Neuromolecular Med. 2016 Mar;18(1):99-108. Epub 2015 Dec 14 PubMed.
6. Endres K. Apolipoprotein A1, the neglected relative of Apolipoprotein E and its potential role in Alzheimer's disease. Neural Regen Res. 2021 Nov;16(11):2141-2148. PubMed.

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