## 2 Results
### 3222401L13 Rik Is Important for Glutamate Uptake, Ca\({}^{2+}\) Signaling, and Neuronal Support  
Although it would be interesting to study further the upregulated genes linked to inflammatory processes, the majority of deregulated genes were decreased after the KD of 3222401L13 Rik, suggesting that this lncRNA could act as an activator of gene transcription under physiological conditions. The pathways linked to these downregulated genes indicate a potential function of 3222401L13 Rik in mediating neuronal support. Therefore, we decided to study in the context of this work the role of 3222401L13 Rik in gene activation and the regulation of neuronal support. In this regard, an important role of astrocytes is the removal of glutamate from the extracellular space, which is mainly mediated by glutamate transporter 1 (Glt-1) and glutamate aspartate transporter (Glast). We found that the levels of both transporters were reduced on the RNA (Figure 4A) and the protein level (Figure 4B). The results of the experiments are presented in Figure 4B. The results of the experiments are presented in Figure 4C. The results of the experiments are presented in Figure 4D. The results of the experiments are presented in Figure 4D. following the KD of _3222401L13 Rik_. In line with this, the uptake of glutamate from the extracellular space was also impaired in primary astrocytes in the absence of _3222401L13 Rik_ (Figure 4C). Another important feature of astrocytes is the ability to generate transient increases in intracellular calcium (Ca\({}^{2+}\)) levels in response to neuronal activity, which can induce the release of various gliotransmitters, e.g., glutamate, ATP, and GABA [32]. These gliotransmitters can, in turn, have a wide range of effects on neurons, such as the synchronization of action potential firing [33] or the regulation of synaptic vesicle release [34]. Pathological alterations of astrocytic Ca\({}^{2+}\) dynamics have been described in different contexts, e.g., in aging and Alzheimer's disease [35;36]. Therefore, we measured Ca\({}^{2+}\) levels in response to adenosine triphosphate (ATP) stimulation and found that the increase in intracellular Ca\({}^{2+}\) was reduced after the KD of _3222401L13 Rik_ compared to astrocytes treated with NC Ga  
Figure 4: The KD of 3222401L13 Rik affects glutamate uptake, Ca\({}^{2+}\) signaling, and the support of neuronal function. (**A**) qPCR showing the expression levels of the glutamate transporters _Glt-1_ and _Glast_ after the KD of _3222401L13 Rik_ in primary astrocytes (unpaired \(t\)-test; ***\(p<0.001\)). (**B**) Left panel: Representative immunoblot of Glt-1 and Glast following the KD of 3222401L13 Rik. in primary astrocytes. Right panel: Quantification of the left panel (unpaired \(t\)-test; *\(p<0.05\), ***\(p<0.001\)). (**C**) Glutamate uptake of primary astrocytes after the KD of 3222401L13 Rik (unpaired \(t\)-test; *\(p<0.01\)). (**D**) Increase in intracellular Ca\({}^{2+}\) levels in response to ATP treatment after the KD of 3222401L13 Rik (unpaired \(t\)-test; ***\(p<0.0001\)). (**E**) Survival of neurons after treatment with 100 \(\mu\)M glutamate cultured alone or co-cultured with NC or KD astrocytes (One-way ANOVA; *\(p<0.05\), ***\(p<0.001\), ns = not significant). (**F**) Left panel: Representative images of dendrite and spine labeling of neurons cultured alone or co-cultured with NC or KD astrocytes. Right panel: Quantification of spines shown in the left panel (One-way ANOVA; ***\(p<0.0001\); ns = not significant). Data are depicted as mean \(\pm\) standard error. NC: negative control. KD: knockdown. Since our previous results suggested that the lncRNA _3222401L13 Rik_ was important for maintaining neuronal support functions of astrocytes, we next asked whether the loss of _3222401L13 Rik_ in astrocytes affected neurons. Therefore, we cultured primary neurons alone or together with astrocytes treated with either control (+ NC astrocytes) or _3222401L13 Rik_ KD Gapmers (+ KD astrocytes).