## 2 Results
### 3222401L13 Rik Is a Glial-Enriched lncRNA Showing Increased Expression in Astrocytes of Aged Mice

Table 1: Characteristics of lncRNAs deregulated during aging in Neu\(-\)nuclei. the expression pattern of _3222401L13 Rik_ in glial cells by performing Magnetic Activated Cell Sorting (MACS) of astrocytes, oligodendrocyte, and microglia from 3-month-old mice. qPCR analysis showed that _3222401L13 Rik_ expression was comparable across all three glial cell types analyzed (Figure 2D). However, when comparing expression levels in 3-month-old and 16-month-old mice, we observed an increase in _3222401L13 Rik_ levels only in astrocytes but not in oligodendrocytes or microglia (Figure 2E). Consequently, we focused on characterizing the role of 3222401L13 Rik, specifically in astrocytes. Loss of 3222401L13 Rik Leads to Expression Changes in Genes Involved in Neuronal Support and Inflammatory Processes  
IncRNAs can exert a wide range of functions, which primarily depend on their location within the cell, and the identification of their subcellular localization is, therefore, critical for their characterization [30]. Although we identified _3222401L13 Rik_ in Neu\(-\) nuclei, this does not exclude the possibility that it could be prominently expressed in the cytoplasm. Therefore, we investigated the localization of _3222401L13 Rik_ by combining RNAscope for _3222401L13 Rik_ with immunofluorescence staining for the astrocyte marker glial fibrillary  
Figure 2: A _3222401L13 Rik_ is a glial lncRNA that is upregulated in astrocytes during aging. (**A**) Schematic illustration of the genomic localization of _3222401L13 Rik_ in the mouse and _ENSGO0000272070_ in the human genome. (**B**) Expression of the lncRNA _3222401L13 Rik_ in NeuN\(+\) and NeuN\(-\) cells isolated from the hippocampal CA1 region of 3-month-old mice (unpaired \(t\)-test ***\(p<0.0001\)). (**C**) qPCR data showing the expression of _3222401L13 Rik_ in NeuN\(-\) cells from 3- and 16-month-old mice (unpaired \(t\)-test; *\(p<0.05\)). (**D**) Expression of _3222401L13 Rik_ in astrocytes, oligodendrocytes, and microglia isolated from the brains of 3-month-old mice using MACS (One-way ANOVA; ns = not significant). (**E**) Expression of _3222401L13 Rik_ in astrocytes, oligodendrocytes, and microglia isolated from the brains of 3- and 16-month-old mice using MACS technology (unpaired \(t\)-test; *\(p<0.05\), ns = not significant). Data are depicted as mean \(\pm\) standard error. acidic protein (Gfap) and found that 3222401L13 Rik was predominantly located in the nucleus of astrocytes in the adult mouse brain (Figure 3A). Moreover, strikingly, we observed two to three large foci of 3222401L13 Rik per astrocyte (Figure 3A). We confirmed the nuclear localization by performing nuclear and cytoplasmic fractionation of primary mouse astrocytes and measuring the abundance of 3222401L13 Rik in both compartments (Figure 3B). The enrichment of 3222401L13 Rik in the nuclear compartment hints toward the role of this lncRNA in regulating gene transcription. Therefore, we performed a knockdown (KD) of 3222401L13 Rik in primary mouse astrocytes using Gapmers directed against the lncRNA and performed total RNA sequencing to identify transcriptional targets. As a negative control (NC), a Gapmer with no known target in the genome was employed. Gapmers are single-stranded antisense oligonucleotides that mediate RNase H-dependent degradation of RNA, a technique that is especially suitable for targeting nuclear lncRNAs. We confirmed by qPCR that the Gapmers reduced the abundance of 3222401L13 Rik by -75% (Figure 3C). Differential expression analysis after total RNA sequencing revealed that 321 genes were upregulated and 754 genes were downregulated upon 3222401L13 Rik KD (1 log2 FC1 > 0.5, \(p\)-adj. \(<\) 0.05, base mean > 50) (Figure 3D) (Table S3). The upregulated genes were linked with a number of interesting Gene Ontology (GO) terms, including \"Response to interferon-gamma\", \"Positive regulation of nervous system development\", \"Leukocyte migration\", and \"Extracellular matrix organization\" (Figure 3E) (Table S4).