The roles of long non-coding RNAs in CNS diseases with a focus on astroglial-neuronal communication
by Sophie Schröder
Date of Examination:2024-10-01
Date of issue:2025-02-14
Advisor:Prof. Dr. André Fischer
Referee:Prof. Dr. André Fischer
Referee:Prof. Dr. Bernd Wollnik
Persistent Address:
http://dx.doi.org/10.53846/goediss-11072
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Abstract
English
Aging is accompanied by a decline of cognitive functions and represents a major risk factor for the development of neurodegenerative diseases such as Alzheimer’s disease (AD). Key pathological changes linked to AD are the accumulation of amyloid beta (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau, eventually causing neurodegeneration and, ultimately, dementia. All cell types of the central nervous system (CNS), including astrocytes, can be affected by and contribute to disease pathology. Astrocytes, an abundant cell type in the CNS, provide important support functions and can actively shape neuronal and synaptic function under physiological conditions. In response to an insult, astrocytes can acquire a reactive phenotype, which can have both beneficial and detrimental effects. However, the factors determining the outcome of reactive astrocytosis are yet poorly understood. Long non-coding RNAs (lncRNAs) are a heterogenous group of RNA molecules that have a length of more than 200 nucleotides and lack coding potential. Many lncRNAs contain modular functional domains that allow them to interact with DNA loci, RNA molecules, and proteins. By this, they can control virtually all cellular processes, including gene transcription, splicing, messenger RNA (mRNA) stability and translation. Due to their high tissue and cell type specificity, lncRNAs present as promising targets for future drug development strategies. However, a comprehensive understanding of lncRNA function in health and disease is crucial to exploit this potential. Therefore, in this thesis, I characterized the function of astrocytic lncRNAs in the context of AD (project 1) and aging (project 2). In the first project, we employed single nucleus RNA sequencing to identify astrocyte-specific lncRNAs in the human brain. PR domain containing 16 divergent transcript (PRDM16-DT) was the most enriched lncRNA in astrocytes that also has a mouse homolog, which is called Prdm16 opposite strand (Prdm16os). Further analyses revealed its down-regulation in reactive astrocytes and the brains of AD patients, prompting us to perform antisense oligonucleotide (ASO)-mediated knockdown (KD) experiments to investigate its function. The KD of PRDM16-DT/Prdm16os in primary mouse and human induced pluripotent stem cell (iPSC)-derived astrocytes led to a decrease in the expression of genes important for neuronal support and synaptic function, resulting in the impairment of essential astrocytic functions, such as glutamate uptake, lactate secretion and the support of neuronal spine formation. To further elucidate the role of Prdm16os, I then aimed to identify its interaction partners. Suz12, together with Enhancer of zeste homolog 2 (Ezh2) a component of the polycomb repressive complex 2 (Prc2), which mediates histone 3 lysine 27 trimethylation and thereby gene repression, and RE1-Silencing Transcription factor (Rest), a transcription factor that represses the expression of neuronal genes in non-neuronal cells, were found to bind Prdm16os. Using chromatin immunoprecipitation followed by quantitative polymerase chain reaction (qPCR), I showed that Prdm16os acts a decoy for both proteins to prevent them from binding to promoter regions of target genes and thereby allow their expression. Upon Prdm16os KD, Prc2 and Rest can bind more efficiently to the DNA, leading to the down-regulation of genes important for neuronal support. Finally, I aimed to restore Prdm16os expression in reactive astrocytes using Clustered Regularly Interspaced Short Palindromic Repeats-mediated transcriptional activation (CRISPRa). Thereby, I could show that reinstating Prdm16os levels was sufficient to rescue functional deficits induced by cytokine treatment, such as impaired glutamate uptake and decreased lactate secretion. Altogether, these findings indicate that PRDM16-DT, due to its high cell type specificity and its regulation of astrocytic genes important for neuronal support, might serve as a promising drug target in diseases such as AD. In the second project, I analyzed gene expression changes in neuronal and non-neuronal nuclei of the hippocampi of young and old mice, leading to the identification of the lncRNA 3222401L13Rik as a glial lncRNA that showed increased expression levels in astrocytes of aged mice. Similar to the results obtained in the first project, the KD of 3222401L13Rik in primary mouse astrocytes and of its human homolog, ENSG00000272070, in human iPSC-derived astrocytes resulted in the reduced expression of genes mediating neuronal and synaptic support, suggesting, in a broader context, a role of astrocytic lncRNAs in the fine-tuning of the neuronal-astrocytic interaction at the synapse. Moreover, the lncRNA KD resulted in impaired uptake of extracellular glutamate uptake and aberrant Calcium (Ca2+) signaling. In this case, 3222401L13Rik was shown to interact with the transcription factor Neuronal PAS Domain Protein 3 (Npas3), an astrocytic transcription factor that is essential for the expression of genes involved in synapse function. Moreover, I observed that increasing the levels of Npas3 during the KD of 3222401L13Rik could rescue the impaired glutamate uptake and aberrant Ca2+ signaling, indicating that modulating one interaction partner can compensate for reduced levels of the other, an intriguing avenue for future research. Taken together, in the second project, I show that 3222401L13Rik, a lncRNA important for the expression of synapse-associated genes, is up-regulated in astrocytes during aging. This indicates a compensatory mechanism that allows astrocytes to enhance neuronal and synaptic support during aging and to thereby counteract concomitant synaptic loss and neuronal dysfunction.
Keywords: lncRNA; brain; aging; Alzheimer’s disease; astrocytes; non-coding RNA; transcriptomics
