## 3 Discussion  
In this study, we identify _3222401L13 Rik_ as a glial-enriched lncRNA with elevated expression levels in astrocytes of aged mice. To our knowledge, the function of this lncRNA has not been studied in any cellular system before. However, _3222401L13 Rik_ was found to be upregulated in two distinct RNA-seq datasets related to brain aging. Specifically, upregulation of _3222401L13 Rik_ was observed in ApoD knockout mice, which are characterized by premature brain aging, when bulk cortical tissue was analyzed via RNA-seq [40]. Similarly, upregulation of _3222401L13 Rik_ was detected in the hippocampus of young and old mice, as characterized by both bulk and single-cell RNA-seq [41]. Although neither study specifically highlights _3222401L13 Rik_, these findings support our observation and suggest a role for _3222401L13 Rik_ in brain aging. We found that _3222401L13 Rik_ was highly expressed in glial cells compared to neurons, which aligned with previous research showing that lncRNAs often exhibited tissue-specific expression patterns [19]. The age-associated upregulation of _3222401L13 Rik_ was specific to astrocytes. While we cannot yet explain the underlying molecular mechanisms, it is known that stimuli such as environmental enrichment, aging, and neurodegenerative diseases lead to cell-type-specific changes in gene expression in the brain [42; 43]. Moreover, astrocytes play a crucial role in brain aging, and recent studies suggest that transcriptional changes in astrocytes are a central mechanism for promoting resilience against cognitive decline in Alzheimer's disease [44]. Therefore, we decided to study the function of _3222401L13 Rik_ in astrocytes. It is likely that _3222401L13 Rik_ also regulates important cellular processes in other glial cells, such as microglia and oligodendrocytes, which we plan to address in future research. We found that 3222401L13 Rik is primarily expressed in the nucleus, which aligns with the known role of lncRNAs in gene expression control [13]. Consistent with this, we observed that the knockdown of 3222401L13 Rik in primary mouse astrocytes led to significant changes in gene expression, including upregulation of inflammatory processes and a downregulation of genes associated with neuronal support functions. This finding is consistent with previous studies showing that lncRNAs can have profound effects on astrocytic gene expression control [22; 25]. Strikingly, using RNAscope, we observed that 3222401L13 Rik was localized to two to three nuclear foci per astrocyte. This kind of signal has also been observed in other studies investigating lncRNAs [45; 46]. Moreover, [PERSON] et al. visualized the localization of more than 60 lncRNAs and identified different categories of cellular localization patterns, ranging from bright sub-nuclear foci--similar to the signal we observed for 3222401L13 Rik--to a dispersed nuclear or cytoplasmic distribution [47]. This study further suggests that lncRNAs showing bright nuclear foci are involved in chromatin regulation and are localized to sites of active transcription [47], which aligns with our finding that 3222401L13 Rik regulates the expression of synapse-associated genes. Additionally, lncRNAs are critical for the formation of nuclear condensates with specific functions, such as nuclear speckles [13] which could also explain the localization of lncRNA molecules to nuclear foci. However, further studies will be necessary to elucidate the exact localization of 3222401L13 Rik within the nucleus of astrocytes. In human iPSC-derived astrocytes, knockdown of the homologous lncRNA _ENSG00000272070_ also decreased the expression of genes linked to neuronal support functions. However, no increased expression of genes linked to inflammatory processes was observed. It is important to note that these data are based on qPCR analysis of selected transcripts.