Article Figures & Data
Figures
- Figure 1.
Loss of NPRL2 expression in glutamatergic neurons causes early lethality. A, Western blot analysis of NPRL2, NPRL3, DEPDC5, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from brain protein extracts (n = 3 per group). B, Quantification of Western blot signal intensities relative to GAPDH (n = 6 per genotype). Error bars represent the mean ± SD. C, Survival curve of NPRL2 nKO and wild-type littermate controls (n = 12/40, respectively). All NPRL2 nKO animals die 20 d after birth. D, Body mass of NPRL2 nKO and littermate controls (male n = 7/16 and female n = 7/6, respectively). (Extended Data Fig. 1-1). Error bars represent the mean ± SEM a, p < 0.05; c, p < 0.001.
- Figure 2.
NPRL2 regulates dendritic branching of cortical neurons. A, Representative images of Neurolucida tracing of the dendrites of Layer IV–V cortical neurons in NPRL2 nKO and littermate controls (n = 30 neurons/3 mice per genotype). B, Sholl analysis shows a reduced number of intersections, and C reduced cumulative area of dendrites in the NPRL2 nKO compared with littermate controls at P16. D, Soma volume of NPRL2 nKO versus littermate controls. Error bars represent the mean +/- SEM. Scale bar: 50 μm. a, p < 0.05; c, p < 0.001.
- Figure 3.
Electrophysiological properties of NPRL2 nKO neurons. A, Representative tracings of the response samples of control (black) or NPRL2 nKO (red) neurons and protocol of injected depolarization steps. B, The number of APs elicited by different current steps and their (C) frequency. D, Example trace of the first AP from control (black) or NPRL2 nKO (red). First AP (E) amplitude, (F) rise time, and (G) decay slope evoked by different depolarizing steps. RMP (H), rheobase current (I), and capacitance (J) measurements from NPRL2 nKO and controls. NPRL2 nKO = 23 neurons/3 mice; control = 25 neurons/3 mice. Error bars represent the mean +/- SEM. a, p < 0.05; b, p < 0.01.
- Figure 4.
Loss of NPRL2 increases mTORC1 signaling. Representative image of immunohistochemical staining of P-S6 (Ser240/244) and VGLUT2 in the (A) hippocampus and (B) cortex of NPRL2 nKO and control at P16. C, Western blot analysis of pS6, S6, pS6K1, S6K1, pAKT, AKT, and GAPDH from soluble protein extracts (n = 3/3; Extended Data Figs. 4-1, 4-2).
- Figure 5.
Metabolite analysis of P16 brain extracts. Relative abundance of (A) amino acids, and (B) biogenic amines were measured from NPRL2 nKO (white bars) and littermate (black bars) controls using LC-MS/MS (n = 7/10, respectively). Error bars represent the mean ± SEM (Extended Data Figs. 5-1, 5-2) a, p < 0.05; b, p < 0.01; c, p < 0.001.
- Figure 6.
NPRL2 contributes to the regulation of sodium channel expression. A, qRT-PCR analysis of sodium voltage-gated channel α subunits (Scn1A, Scn2A, Scn3A, and Scn5A) and sodium voltage-gated channel β subunits (Scn1B, Scn2B, Scn3B, and Scn4B) in control and NPRL2 nKO brain at P16 (n = 5/4, respectively). B, Western blot analysis of SCN1A and metabotropic glutamate receptor 1 (mGluR1) from membrane fractions of P16 WT and NPRL2 nKO brain (n = 3/3). C, qRT-PCR analysis of Nprl2 and Scn1A from Nprl2loxP/loxP primary neurons infected with control or CRE-expressing lentivirus and treated with 10 nm rapamycin, or DMSO control, for 24 h (n = 6/6; 2 independent experiments; Extended Data Fig. 6-1). Error bars represent the mean +/- SEM. a, p < 0.05; b, p < 0.01; c, p < 0.001.
Tables
Protein target Vendor Catalog # AKT Cell Signaling Technology 9272 DEPDC5 Santa Cruz Biotechnology, Inc. sc-86115 GAPDH Sigma G9295 GFAP Millipore MAB5541 mGluR1 Fisher Scientific 12370 NeuN Millipore AB5541 NPRL2 Santa Cruz Biotechnology, Inc. sc-376986 NPRL3 Abcam ab121346 p70 S6 Kinase Cell Signaling Technology 9202 Phospho-p70 S6 kinase (T389) Cell Signaling Technology 9205 Phospho-AKT (Ser473) Cell Signaling Technology 9271 Phospho-rpS6 (Ser240/244) Cell Signaling Technology 2215 rpS6 Cell Signaling Technology 2217 Scn1A Cell Signaling Technology 18339 VGLUT2 Synaptic Systems 135404 Gene Forward Reverse Bax GTCTCCGGCGAATTGGA TCCACGTCAGCAATCATCCT Dapk2 GCAGGGAGCAGAGCAGAG CCTGGACCATACAATCGGCG Dapk3 CGTAGATGCCGTCTCAGGAT TGGGGACACAGGAGCGTC Kv1.1 CCCGCCAGCGTCCATC CTGCAGCCCTTCTAGGACAC Kv1.3 CTTCTGGTGCGGTTCTTTGC TACCTTGTCGTTCAGCCAGC Kv1.4 GGCTTAACAAGTGATCGCTGC CCAAGGTAGACCCCGGAGAT Kv1.6 GACTGCCAATTTCTGCTTGGG AGTTATTCAGTGGGGAGGCG Kv2.2 AACGTAGGGGGCCTTAACCA TTGTAGTCATCGCACACCTCC Pcna CGAAGGCTTCGACACATACC GGACATGCTGGTGAGGTTCA Scn1A CTGGCTGGACTTCACTGTCAT GATGGTCTTCAGGCCTGGAA Scn1B AACACCAGCGTCGTCAAGAA CCATCTCTGCCACAAGCCAT Scn2A ACCTTTGCGTATGTAACAGAATTT ACAGCTTCTTCACCGACTGG Scn2B CGCCTAACGTCACAGTCTCT GGACACTAAGAGTGGTGGGC Scn3A GGACGTGGGGTCTGAGAATG GTGGTACCGTTACTGTTGCG Scn3B GCTCAGGAAAATGCGTCTGACTAC TGCCTGTACATCACCTCAAGT Scn4B GTGGACAACACGGTGACTCT CACTCCTTCTTCTTCTCTCGGG Scn5A AAGCTAGGCAATTTGTCGGC GTCTTCAGGCCTGGAATAACTG U36B4 CGTCCTCGTTGGAGTGACA CGGTGCGTCAGGGATTG
Extended Data Figure 1-1
Seizure analysis of NPRL2 nKO mice. Quantification of the tonic and tonic-clonic seizures of the NPRL2 nKO mice before their death. Download Figure 1-1, EPS file.
Extended Data Figure 4-1
Quantification of (A) P-S6 signal intensity from cortical immunohistochemistry staining and (B) relative signaling intensity of P-S6:S6 of Western blot analysis. Download Figure 4-1, EPS file.
Extended Data Figure 4-2
Representative image of immunohistochemical staining of P-S6, NeuN, and GFAP in the cortex of (A) control or (B) NPRL2 nKO at P16 (n = 3 per genotype). Scale bar: 25 μm. Download Figure 4-2, EPS file.
Extended Data Figure 5-1
Quantitative gene expression of Bax, Dapk2, Dapk3, and Pcna, in control of NPRL2 nKO brain (n = 5/4 per genotype, respectively). Download Figure 5-1, EPS file.
Extended Data Figure 5-2
Relative abundance of (A) acylcarnitines, (B) sphingolipids, and (C) lysophosphatidylcholines (n = 7/10, respectively). Error bars represent the mean ± SEM. Download Figure 5-2, EPS file.
Extended Data Figure 6-1
Regulation of potassium channel expression. A, qRT-PCR analysis of potassium channels (Kv1.1, Kv1.3, Kv1.4, Kv1.6, Kv2.2) in control and NPRL2 nKO brain at P16 (n = 5/4, respectively). b, p < 0.01. Download Figure 6-1, EPS file.
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