CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis

Background:

Protein aggregation is one of the leading causes of various neurodegenerative disorders¹. In this preprint, the authors focus on a widely studied intrinsically disordered protein called Tau. Tau aggregation gives rise to a family of disorders collectively known as tauopathies. Since most of the tauopathies are acquired in adulthood, factors in the cell milieu govern disease onset and progression. However, early onset drivers of tau aggregation and their mechanistic involvement in disease pathogenesis have not been studied extensively. For example, GWAS studies have uncovered various risk factors but do not provide any information on molecular mechanisms. Additionally, sc-RNA seq can identify differential expression of genes between diseased and normal state but cannot pinpoint the exact role in pathology or early onset.

The focus of this manuscript was to identify the key factors underlying the selective vulnerability of tauopathies among different neuronal cell types. The iPSC-derived neurons used in this study provide an amenable model system for this study since they are physiologically relevant and allow for high-throughput screening experiments. Understanding the specific role of the cellular environment will also help to explain the selective vulnerability of different neuronal cell types to tau aggregation.

Key findings:

Model optimization for high throughput genomic screening

To establish a model system for detecting tau oligomers, the authors examined WT and V377M mutant of tau in iPSC-derived neurons. Neurons were stained for tau oligomers using a T22 antibody, and cells were screened for level of oligomerization using flow cytometry. Neurons expressing both a single and double copy of V377M mutant, showed a significant increase in the accumulation of aggregates compared to WT (see fig.1).

The CRISPRi screens

The authors then knocked down different genes in V322M expressing neurons and sequenced the neurons sorted for different levels of tau oligomerization. This knockdown screen identified various components that were already known to decrease tau oligomer levels (examples: autophagy modulators² and m6A regulatory genes³). One important hit from the screen – showing increased tau oligomer levels – involved the Electron transport chain (ETC). Interestingly, Ubiquitin proteasomal system (UPS)-related factors were also among the top hits, as well as those relating to the GPI-anchor biosynthesis. Additionally, Melvanote – a precursor of cholesterol – decreased tau levels and genes essential for UFMylation, already known to control pathogenic tau levels, also showed up as a hit.

Figure 1 Original(2A) Schematic of the CRISPRi screen performed on iPSC-derived neurons to test the effect on tau oligomerization.

To characterize the top hits more closely, 1,037 genes were pooled into a secondary sgRNA library. The authors then screened both WT and V377M mutant using a total tau antibody and just the WT using T22 antibody. From this, they could conclude that the WT tau screen seemed to be more sensitive to the knockdown of genes involved in mRNA transport, while the V377M screen was enriched for hits regulating the mTOR signaling pathway. Cul5 – an E3 ubiquitin ligase – showed up for both WT and mutant screens. Since Cul5 is highly expressed in excitatory neurons which are relatively resilient to Alzheimer’s disease (AD) ⁴, the authors pointed out that this might contribute to the selective vulnerability of cell types in AD progression.

What is the role of the proteasomal machinery in tau oligomerization?

Since Cul5 and its regulator RNF7 were strong hits in the screen, the authors over-expressed Cul5 and tau in cells to test if they physically interact. Immunoprecipitation revealed that tau co-precipitated with Cul5. RNF7 was also present confirming it to be a functional protein assembly. Now to identify the region in tau recognized by Cul5, supposedly a degron, a number of sub-regions of tau were expressed in the neurons. The authors were able to identify a region (amino acids 80-130) that is recognized by Cul5. They also identified that Cul5 interacted with tau using the adaptor protein SOCS4.

What is the effect of mitochondrial dysfunction on tau oligomerization?

To further their understanding of how the cellular environment promotes tau interaction with the proteasomal machinery, the authors moved their attention to the mitochondrial genes that showed up in the screen. These included FECH, PSAP and FH and interestingly suppression of all these genes increased ROS level in the neurons. The authors then checked how mitochondrial function might be controlling tau oligomer levels by using a pharmacological approach. They could conclude that ROS production is a side-effect of mitochondrial dysfunction which induces tau misprocessing.

To support their findings, the authors induced oxidative stress in the neurons with hydrogen peroxide treatment. Oxidative stress increased tau misprocessing into a 25kDa fragment. Increasing expression of the proteasomal activator PA.28 reduced the levels of the 25kDa tau fragment as expected, suggesting it is the inability of the proteasome to successfully degrade the oxidized proteins during oxidative stress that leads to the accumulation of misprocessed tau fragments.

Figure 2 Original (5C, 5D) Effect of Rotenone on endogenous Tau fragmentation (left). Effect of Rotenone on exogenous GFP-Tau fragmentation in iPSC-derived neurons

Is tau oxidation the cause of its misprocessing?

The authors hypothesized that the N-terminal methiones of tau were oxidized during stress. To test if oxidized tau levels lead to misprocessing of tau every methionine was mutated to leucine. They observed that, with mutant tau expression levels being the same as WT, there was a large decrease in mutant tau fragmentation. Thus, direct tau oxidation during stress forces tau into the proteasome for aberrant degradation.

What is the sequence of the misprocessed tau fragment?

Lastly, to identify the tau sequence created after being misprocessed by the proteasome, the authors purified tau from neurons and digested it with GluC to mimic the proteasome. The sequences were then identified using LC-MS/MS and narrowed down to 9 residues (172-200). This was consistent with the tau biomarker in CSF patients that ends near residue 230⁵. The authors could conclude that N-terminal tau fragments may serve as markers for neuronal oxidative stress that results in changes in proteasomal processivity.

What I liked about the preprint:

I am interested in neuroscience and specifically in understanding how the intracellular environment becomes supportive of disease progression. Understanding the origin and early markers of neurodegeneration can help understand the overall development of diseases into later stages. Proteins like tau have been studied extensively for their involvement in the progression of neurodegenerative diseases. This manuscript highlights how neurons become vulnerable when the cellular machinery is compromised due to internal and external factors leading to oxidative stress. Mitochondrial dysfunction is an important factor that allows the opportunistic misprocessing of tau protein which leads to uncontrolled aggregation in later disease stages. The authors’ CRISPR-based strategy in iPSC-derived neurons allows for high throughput screening in a physiologically relevant system.

 

 

Questions for the authors:

1. As discovered here, oxidative stress can lead to tau fragmentation due to misprocessing. Is it known which cell types in particular are more likely to be affected as they could serve as the sites of onset of tauopathies?

 

2. Regarding the proteasome recognition (degron-like) sequence of tau identified in this manuscript, is it known to be mutated in any tauopathies currently known?

 

3. Are residues of the proteasomal machinery-or even-Cul5 also oxidized (which could be the reason for its aberrant processing of tau)?

 

Future Directions:

Future studies should focus on the effects of early onset tau misprocessing on the propagation of action potentials in the axons. This would provide further insight into cell type vulnerability to protein aggregation eventually causing neurodegeneration. It will also be interesting to see what other protein fragments accumulate due to the misprocessing of the proteasome.

 

References:

1. Ross, C. A. & Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat Med 10, S10–S17 (2004).
2. Caballero, B. et al. Acetylated tau inhibits chaperone-mediated autophagy and promotes tau pathology propagation in mice. Nat Commun 12, 2238 (2021).
3. Jiang, L. et al. Interaction of tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of tauopathy. Mol Cell 81, 4209-4227.e12 (2021).
4. Leng, K. et al. Molecular characterization of selectively vulnerable neurons in Alzheimer’s disease. Nat Neurosci 24, 276–287 (2021).
5. Cicognola, C. et al. Novel tau fragments in cerebrospinal fluid: relation to tangle pathology and cognitive decline in Alzheimer’s disease. Acta Neuropathol 137, 279–296 (2019).

 

doi: https://doi.org/10.1242/prelights.35154

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