When studying neurodegenerative disease in human tissue, scientists often have trouble pinning down what went wrong first, since they must usually look at postmortem brain samples. Newer research techniques, such as the generation of cerebral organoids from human stem cells, are helping parse out the initial steps on the pathway to disease. Case in point: In the September 13 Stem Cell Reports, researchers led by Kenneth Kosik at the University of California, Santa Barbara, in collaboration with Sally Temple at the Neural Stem Cell Institute of Rensselaer, New York, describe the use of organoids to study the effect of pathogenic tau mutations. Over the first few months in culture, the main effect of these mutations was to boost cholesterol production by astrocytes. Though the researchers do not know the mechanisms involved, the data fit with a growing body of research showing disruption of lipid metabolism in neurodegenerative disease.
- Human cerebral organoids allow the study of neuron-astrocyte interactions.
- Mutant tau, which is mainly neuronal, increases cholesterol production in astrocytes.
- Is this boost compensatory or harmful?
The findings may point toward new research directions, Kosik believes. “This shows it is possible to use human brain organoids to study a human neurodegenerative disease,” he told Alzforum. This, even though organoids contain many more progenitor and newborn cells than do older human brains.
Julia TCW at Boston University noted that most studies of tauopathy have focused on neurons. She believes the new findings of dysregulated cholesterol in astrocytes are significant and could suggest new therapeutic targets for early intervention.
Cholesterol Boom. Astrocytes (red) carrying mutant tau (bottom) make much more HMGCS1 (green), an enzyme in the cholesterol biosynthesis pathway, compared to cells with wild-type tau (top). [Courtesy of Glasauer et al., Stem Cell Reports.]
Protocols for generating cerebral organoids have been around for a decade, with recent technical advances making them more reproducible and reliable (Aug 2013 news; Jun 2019 news). Kosik and colleagues employed a protocol developed at Stanford University that produces long-lived spherical organoids enriched for pyramidal cortical neurons and astrocytes (Yoon et al., 2019).
First author Stella Glasauer used induced pluripotent stem cells made from three people with heterozygous V337M tau mutations, two with heterozygous R406W, and one with homozygous R406W to generate dozens of cerebral organoids. In people, these mutations cause frontotemporal dementia. By three months in culture, the organoids consisted of mostly excitatory pyramidal cells, with smaller populations of astrocytes and inhibitory interneurons. Glasauer and colleagues then isolated cells from pooled samples of three to four genetically identical organoids for single-cell RNA-Seq. They compared tau mutant organoids with genetically corrected isogenic controls at several different ages. They analyzed around 1,200 cells per sample, for a total of 76,111 cells.
In excitatory neurons with mutant tau, 60 genes went down and 81 genes up compared with isogenic controls. Suppressed genes were mostly associated with glycolysis, the biochemical process that produces energy in the absence of oxygen, while several of the elevated genes were mitochondrial. In inhibitory neurons, GABA receptors and glycolytic genes were suppressed.
Lipids Gone Wild. Most gene expression changes in human cerebral organoids carrying mutant tau were in astrocytes, where 142 genes were down (blue dots) and 112 up (red dots) compared to controls organoids; in particular, multiple genes responsible for cholesterol metabolism and fatty acid synthesis were highly elevated (boxes). [Courtesy of Glasauer et al., Stem Cell Reports.]
However, most changes were in astrocytes, where 142 genes were down and 112 up (see image above). In particular, numerous genes involved in cholesterol synthesis, metabolism, and transport were highly expressed in astrocytes with mutant tau. These changes were more pronounced in cells with homozygous than heterozygous R406W. They also became more dramatic with age, with cholesterol synthesis genes more elevated in 8-month-old organoids than in 4-month-olds. In addition, genes responsible for fatty acid synthesis went up.
Curiously, APOE expression was low in astrocytes with mutant tau. This was true regardless of whether they carried APOE2, 3, or 4. Some previous studies have linked APOE4 to disrupted lipid metabolism in astrocytes, but this was in the context of Alzheimer’s disease, in the presence of wild-type tau (Apr 2019 conference news; Aug 2019 news; Nov 2021 conference news).
The authors verified some of these RNA-Seq findings at the lipid level, using liquid chromatography mass spectrometry to identify several cholesterol pathway components in organoids and isogenic controls made from three of the cell lines. They detected high levels of cholesterol, three of its precursors, and one metabolite, in 7-month-old mutant tau organoids, but not in 4-month-old. This suggested that expression changes do affect lipid synthesis, but with a time delay, since the transcripts were already elevated at four months.
How does mutant tau juice cholesterol metabolism? While this is unknown, Kosik noted that neurons need high amounts of cholesterol in their plasma membrane, and this is produced by astrocytes. He speculated that mutant tau oligomers might damage neuronal membranes, provoking astrocytes to ramp up cholesterol production to compensate and repair the damage. A previous study found that cholesterol in neuronal membranes helps keep out tau aggregates, protecting cells (May 2022 news). Conversely, it is equally possible that elevated cholesterol is harmful, since its immediate metabolites, cholesterol esters, have been shown to be neurotoxic and promote tau phosphorylation (Feb 2019 news).
In future work, Kosik will further characterize the phenotype of mutant tau organoids, particularly their lipid composition, to figure out whether these changes are harmful or helpful. He will also look for early signs of tau pathology, such as hyperphosphorylation or aggregation. A previous study of V337M cerebral organoids reported hyperphosphorylated tau and loss of excitatory neurons by six months in culture, suggesting these structures can reproduce some of the features of FTD (Bowles et al., 2021).—Madolyn Bowman Rogers
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- Bowles KR, Silva MC, Whitney K, Bertucci T, Berlind JE, Lai JD, Garza JC, Boles NC, Mahali S, Strang KH, Marsh JA, Chen C, Pugh DA, Liu Y, Gordon RE, Goderie SK, Chowdhury R, Lotz S, Lane K, Crary JF, Haggarty SJ, Karch CM, Ichida JK, Goate AM, Temple S. ELAVL4, splicing, and glutamatergic dysfunction precede neuron loss in MAPT mutation cerebral organoids. Cell. 2021 Aug 19;184(17):4547-4563.e17. Epub 2021 Jul 26 PubMed.
- Glasauer SM, Goderie SK, Rauch JN, Guzman E, Audouard M, Bertucci T, Joy S, Rommelfanger E, Luna G, Keane-Rivera E, Lotz S, Borden S, Armando AM, Quehenberger O, Temple S, Kosik KS. Human tau mutations in cerebral organoids induce a progressive dyshomeostasis of cholesterol. Stem Cell Reports. 2022 Sep 13;17(9):2127-2140. Epub 2022 Aug 18 PubMed.