New mechanism of Mol Cell︱ Alzheimer's disease: Tau protein oligomerization induces nuclear cell transport of RNA binding protein HNRNPA2B1 and mediates enhancement of m6A-RNA modification

Written by Jiang Lulu, edited by Wang Sizhen, the misfolding and aggregation of the microtubule-associated protein Tau (MAPT) is the core feature of Alzheimer's disease (AD) [1]
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When neurons undergo degeneration, they exhibit characteristic pathological changes, such as the accumulation of neurofibrillary tangles formed by misfolded Tau protein
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The accumulation of Tau protein is closely related to the cognitive decline of AD patients
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These research advances have led to the development of powerful diagnostic methods and potential drugs
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However, neurofibrillary tangles are only part of the pathology of AD.
Scientists have been working hard to understand how neurofibrillary tangles cause nerve cell damage, which is important for developing the best drugs and diagnostic methods for AD
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 In healthy neurons, Tau protein dynamically binds to tubulin to stabilize microtubules
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In the disease state, Tau protein misfolds, which contains many forms, including Tau oligomerization, phosphorylation, acetylation, formation of fiber aggregates, etc.
[2]
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A large number of documents describe the pathological changes of Tau during disease, but there are few studies describing the biological functions of Tau in stress and disease states
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More and more evidences show that Tau oligomers are a key type of neuronal damage that can induce RNA binding proteins to aggregate and combine to form stress bodies [3]
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The reversible binding of stress bodies can help clear misfolded protein complexes, but when they accumulate chronically and continuously, they will lead to extensive inhibition of protein synthesis in neurons [4]
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At present, the molecular mechanism of oligomer Tau mediated this reaction is still unclear
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 On August 27, 2021, in the latest paper published online in Molecular Cell with the title "Interaction of Tau with HNRNPA2B1 and N6-methyladenosine RNA mediates the progression of Tauopathy", Lulu Jiang of Boston University, USA ( Article 1), Benjamin Wolozin (corresponding author) and others have studied these scientific issues
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They reported that in the process of Tau oligomerization, it will capture the RNA binding protein HNRNPA2B1 and methylated RNA transcripts, that is, N6-methyladenosine (m6A) modified RNA to form Tau oligomers-HNRNPA2B1-m6A RNA complex regulates stress response and inhibits protein synthesis
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In AD models and patient brains, this complex persists and undergoes pathological changes, leading to Tau fiber formation, nuclear membrane destruction, and progressive neurodegeneration
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Previous studies have also shown that Tau protein accumulation can cause protein transfer from the nucleus to the cytoplasm [5]
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Tau captures RNA-binding proteins and their transcripts and drags them into the cytoplasm [3, 4]
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Should this behavior be blamed on Tau oligomers? To find out, first, the first author Lulu Jiang and his colleagues created a human wild-type Tau, a synthetic lentiviral expression vector of 4R1N Tau: 4R1N Tau and mCherry (a fluorescent protein that is easy to detect) and a A cryptochrome peptide Cry2Olig oligomerized under light was fused, and the lentiviral vector was transfected into primary cultured mouse neurons for the expression of human Tau protein composition (Figure 1A)
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 Then, the scientists used optogenetic methods to irradiate blue light onto the cells to promote Tau protein oligomerization (Figure 1A)
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Under a 20µW blue light with a wavelength of 488λ for 20 minutes, it is sufficient to induce neurons to produce stable Tau oligomers.
The control group showed that such low-dose light alone did not cause cell changes
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After 60 minutes, Tau begins to form neurofibrillary tangles that can be detected by ThioS
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Will the accumulation of these Tau proteins stimulate neuronal regression? In fact, after 60 minutes of light, the nerve dendrites shrank and deformed; the caspase-3 enzyme activity in neurons increased, which is a sign of programmed cell death; lactate dehydrogenase LDH was also detected in the neuronal culture medium.
This is a harbinger of neuronal degeneration
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 In the process of Tau oligomerization, what molecular biological processes take place in neurons? The authors found that Tau is phosphorylated at sites T181 and S262 (detected by antibodies AT270 and 12E8, respectively)
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Immunoprecipitation experiments showed that Tau fibrils contained nuclear membrane protein lamin B2 and its receptors, suggesting that the integrity of cell membranes was disturbed, and the puromycin method detected that protein synthesis in neurons was inhibited
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In order to find out the cause of these molecular events, Jiang and his colleagues used mass spectrometry to analyze the protein-protein interaction during Tau oligomerization (Figure 1B)
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They exposed neuronal cultures to light for 0, 20, or 60 minutes, then immunoprecipitated mCherry, pulling down Tau neurofibrillary tangles and any other proteins that bind to them
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The results showed that after 20 minutes of light, Tau protein captured nuclear envelope proteins and proteins involved in RNA metabolism and RNA splicing; after 1 hour of light, proteins involved in cell death also appeared in the immunoprecipitation complex
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Figure 1: Experimental procedure (A); mass spectrometry analysis of Tau oligomer capture protein in cultured neurons exposed to light for 20 (B left) or 60 minutes (B right), HNRNPA2B1 accumulated in large amounts
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(Picture quoted from: Jiang L, et al.
, Mol Cell.
2021) Generally, HNRNPA2B1 is located in the nucleus, where it binds and stabilizes RNA, such as N6-methyladenosine (m6A) modified RNA [6]
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As the most abundant mRNA modification, this methylation controls mRNA metabolism
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This attracted the attention of Jiang et al
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Previous studies have also shown that m6A RNA is up-regulated in the brain of AD mouse model APP/PS1 mice [7]
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Could HNRNPA2B1 and m6A-RNA be trapped together by Tau fibers? Scientists added fluorescent anti-HNRNPA2B1 and anti-m6A antibodies to neurons and exposed them to light for 60 minutes.
They found that proteins and RNA co-localized with Tau fibrils in the cytoplasm, suggesting that HNRNPA2B1 might drag m6A RNA together
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To verify this, the scientists knocked out about half of HNRNPA2B1 by adding small interfering RNA to the cultured neurons.
The results showed that the m6A RNA of Tau oligomers was 40% less
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Knockdown of HNRNPA2B1 also reduced the enzymatic activity of caspase-3 and the amount of damaged DNA, suggesting that this protein may play a role in the toxicity and neurodegeneration of Tau
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 The above molecular events are triggered by artificially induced Tau oligomers.
Do they also occur in animal models? In fact, when HNRNPA2B1 and lamin B2 were immunoprecipitated with tangles from the brains of six-month-old mice, Jiang and colleagues found similar changes in the PS19/P301S model
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The amount of m6A RNA in the tangles in three-, six- and nine-month-old mice is also increasing
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So, can knocking down HNRNPA2B1 protect mice? The scientists injected shRNA targeting the HNRNPA2B1 transcript into the hippocampus of three-month-old mice
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Two weeks later, they injected Tau oligomers extracted from other P301S mice to promote the spread of tangles.
Three weeks later, they examined brain slices
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Compared with the P301S control, there are fewer m6A and active caspase-3 in the hippocampus after knockdown, and fewer Tau oligomers
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From this the authors concluded: HNRNPA2B1 contributes to Tau toxicity, and the triplet may contribute to the formation of oligomers
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 So what about in human cases of AD? Are there HNRNPA2B1 and m6A RNA in human tangles? The researchers obtained postmortem temporal cortex tissues from four controls and 14 people with AD from the AD Centers of Boston University and Emory University
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For each stage of disease progression (Braak), they have samples from three people
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The scientists labeled the tissues with antibodies against Tau, HNRNPA2B1, and m6A
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In the control, HNRNPA2B1 and m6A are localized in the nucleus, but in the later stage of AD, HNRNPA2B1 and m6A RNA are in the cytoplasm, and they are co-localized with Tau (Figure 2)
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The strongest co-localization of the three occurred in Braak stage II-IV
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m6A is widely distributed throughout the cytoplasm in advanced diseases, suggesting that it may be free from HNRNPA2B1 and Tau in the later stages of the disease
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Figure 2 Tau oligomers (red), RNA-binding protein HNRNPA2B1 (purple), and m6A-modified RNA (green) in the brains of AD patients accumulate and increase as the disease progresses (Braak stages) and show co-localization characteristics
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(Picture quoted from: Jiang L, et al.
, Mol Cell.
2021) In the late stage of Braak, the amount of methylated RNA seems to be higher
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Comparing BraakVI stage with I stage, Jiang found that the total m6A RNA isolated from brain tissue increased by 4.
5 times
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In particular, Tau co-precipitation, the scientists found that VI stage m6A six times I stage
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Similarly, they found that although the case and control group had similar total amounts of HNRNPA2B1, the frontal cortex tissue of Braak stage IV cases had twice as many HNRNPA2B1 binding Tau oligomers
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The authors attribute the difference to AD tissues having more Tau oligomers and more HNRNPA2B1 in the cytoplasm than in the nucleus
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Figure 3 The conclusion of the article: Tau oligomerization causes nucleocytoplasmic transfer of RNA-binding protein and its read-modified RNA, which induces inhibition of protein synthesis and impaired cell membrane integrity (Picture quoted from: Jiang L, et al.
, Mol Cell .
2021) Conclusion and discussion of the article, inspiration and prospects.
In summary, this study proved to provide the following three important information (Figure 3): 1.
In neurons, the process of oligomerization combines methylated RNA and RNA The protein pulls into the cytoplasm and forms a stress complex
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2.
Knockdown of the RNA binding protein HNRNPA2B1 can inhibit protein misfolding in neurons
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3.
In the AD brain, as the disease worsens, m6A methylated RNA will increase and accumulate
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 This work opens up a new way for researchers to use it to explore the process of AD, and in the process may develop new methods to treat the disease
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At the same time, there are still many issues waiting for more exploration and research
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For example, given that methylated mRNA seems to be important for tangling and toxicity, what are these transcripts and what do they do? It is still unclear, and m6A RNA will need to be sequenced in the future to find out
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 In addition, besides AD, what about other protein-related diseases? Is there the same pathological change? Can HNRNPA2B1 or m6A be used for the differential diagnosis of diseases? These are all problems that need to be solved urgently
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 More importantly, can it be possible to develop new disease treatments by regulating the m6A modification of RNA? Looking forward to more studies to explain in the future
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In addition, on September 3, 2021, this article was cited as a Research Highlight by Nature Reviews Molecular Cell Biology, with the title "Tau oligomers are linked to m6A-RNA" [8]
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038Lulu Jiang (left, first author), Benjamin Wolozin (right, corresponding author) (photo provided from: Benjamin Wolozin Lab) Selected previous articles [1] Cereb Cortex | Li Tao's research group reported The abnormality of the cortical myelin covariation network with the deep characteristics of the cerebral cortex in schizophrenia [2] Cell︱ hold hands, advance and retreat together! The formation of a cellular network between microglia to work together to degrade pathological α-syn [3] lipids and Alzheimer's disease! The lack of myelin in the central nervous system in adulthood can lead to Alzheimer’s disease-like neuroinflammation and cognitive impairment [4] eLife︱ sorting protein SNX27 regulates AMPA receptor transport through synaptic adhesion protein LRFN2 Mechanism [5] Brain︱ new method! Plasma soluble TREM2 can be used as a potential detection marker for white matter damage in cerebral small vessel disease [6] EMBO J︱neuron Miro1 protein deletion destroys mitochondrial autophagy and overactivates the integrated stress response [7] Science frontier review interpretation︱nicotinic acetylcholine The regulatory mechanism of receptor-assisted molecules and the application prospects of disease treatment and transformation [8] Cereb Cortex︱ oxytocin can regulate the individualized processing of facial identities and the classification of facial races in the early facial regions of the brain [9] Nat Commun | Qi Xin Project The group revealed the molecular mechanism of the compound CHIR99021 to treat Huntington’s disease by regulating mitochondrial function [10] Neurosci Bull︱ synapse-associated protein Dlg1 improves depression-like behavior in mice by inhibiting microglia activation [11] Brain | For the first time! PAX6 may be a key factor in the pathogenesis of Alzheimer's disease and a new therapeutic target [12] Sci Adv︱ blockbuster! DNA methylation protein DNMT1 mutation can induce neurodegenerative diseases [13] Cereb Cortex︱MET tyrosine kinase signal transduction timing abnormality is a key mechanism affecting the development and behavior of normal cortical neural circuits in mice [14] Nat Biomed Eng︱ The team of academician Ye Yuru develops a new strategy for whole-brain gene editing-mediated treatment of Alzheimer's disease [15] Luo Liqun Science's heavy review System Interpretation ︱ Neural circuit structure-a high-quality scientific research training course recommendation for a system that makes the brain "computer" run at high speed 【1】Data graph help guide! How good is it to learn these software? 【2】Single-cell sequencing data analysis and project design network practical class (October 16-17)【3】JAMA Neurol︱Attention! Young people are more likely to suffer from "Alzheimer's disease"? [4] Patch clamp and optogenetics and calcium imaging technology seminar (October 30-31) References (slide up and down to view) [1] Scheltens, P.
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