Fig 2: The N-terminal extension of UBE3A dictates its localization. (A) Schematic overview of the chimeric UBE3A constructs in which the N-terminal extensions of mUBE3A-Iso2 and hUBE3a-Iso3 were swapped. (B and C) Localization of chimeric hext3mUBE3A-Iso2 and mext2hUBE3A-Iso3 constructs after transfection in mouse hippocampal AS neurons (B) or into hIPSC-derived AS neurons (C). (D) Schematic overview of the ancestral ‘MATACK’ N-terminal extension (see Fig. 3) and the sequences found in Homo sapiens and Cercopithecidae (Old World Monkeys), Cetartiodactyla (wild boar and whale) and Mus musculus (mouse). (E) Localization of chimeric UBE3A constructs in mouse hippocampal AS neurons. Neurons were transfected at DIV7 with hUBE3A-Iso3 harboring the ancestral ‘MATACK’ extension or the extension of the Cetartiodactyla. All neurons were stained for UBE3A (green) together with MAP2 (pink) and DAPI (blue). Scale bars, 20 µm.

Fig 3: Ascl1 and Ascl1SA6 induce neuronal marker expression when expressed in cortical astrocytes in vivo. (A,B) mCherry co-expression with GFAP (A) or NeuN (B) 14 days post-transduction of AAV8-GFAPshort-mCherry, AAV8-GFAPshort-Ascl1-mCherry, or AAV8-GFAPshort-Ascl1SA6-mCherry. Blue is DAPI counterstain. (C,D) Quantification of the percentage of mCherry+ transduced cells expressing GFAP (C) or NeuN (D) 14 days post-transduction. (E) High magnification images of mCherry+ cells co-stained with MAP2 at 14 days post-transduction of AAV8-GFAPshort-Ascl1-mCherry, or AAV8-GFAPshort-Ascl1SA6-mCherry. Blue is DAPI counterstain. Scale bars = 100 µm. Significance was defined as p-values less than 0.05 and denoted as follows: ns, not significant, <0.05 *, <0.01 **, <0.001 ***.

Fig 4: Visualization of endogenous DBN turnover in neurons using FUNCAT-PLA. a Schematic experimental protocol. Hippocampal neurons were incubated with AHA, and chased with AHA-free medium before performing FUNCAT click chemistry. Immunostaining of DBN and subsequent proximity ligation assay (PLA) performed in situ allow visualization of newly synthesized DBN. b Verification of the FUNCAT-PLA experimental protocol. Fluorescent newly synthesized DBN (FUNCAT-PLA in magenta) in the presence of AHA alone (AHA), AHA and anisomycin (AHA-anisomycin) or methionine alone (Met control). Quantification of the ratio of the FUNCAT-PLA signal over the cell volume marker area (MAP2), relative to AHA-treated neurons is as shown. AHA: n = 53, N = 5; AHA-anisomycin: n = 14, N = 3; Met control: n = 32, N = 5. c Newly synthesized DBN after 2 h pulse with AHA (0 h chase) followed by 24 h chase or 68 h chase. Quantification of the ratio of FUNCAT-PLA signal over cell volume marker area (MAP2), relative to the time-point ‘chase 0 h’ is as shown. 0 h chase: n = 42, N = 3; 24 h chase: n = 40, N = 3; 68 h chase: n = 44, N = 3. All bar graphs are represented as mean ± SEM. Statistics were performed using one-way ANOVA Bonferroni test. ***p < 0.001. N = number of biologically independent experiments, n = total number of neurons. DBN developmentally regulated brain protein, ANOVA analysis of variance

Fig 5: Overexpression of the a2d3 subunit during the first week in vitro promotes axonal outgrowth and branching in young interneurons. A-C, Representative images of GAD67::GFP mouse hippocampal neurons at DIV9 in control conditions (A), as well as after lentiviral infection at DIV2-DIV4 with either pLenti-syn-a2d1::HA (B) or pLenti-syn-a2d3::HA (C). The length and the branching of axons were analyzed exclusively in GAD67-positive interneurons (arrows), which were identified among other neurons (arrowheads) by GFP immunofluorescence. Scale bars, 50 µm. D, Upregulation of the a2d3, but not a2d1, subunit promotes the axonal outgrowth in GAD67-positive interneurons compared with controls. E, Overexpression of a2d3 during the first developmental week leads to twofold increase in axonal branching compared with a2d1-overexpressing or control cultures. GAD67, glutamic acid decarboxylase isoform 67, MAP2, microtubule-associated protein 2. ***p < 0.001. Means and n values are given in Extended Data Figure 8-1.