Neuronal cell type specific roles for Nprl2 in neurodevelopmental disorder-relevant behaviors

Abstract

Loss of function in the subunits of the GTPase-activating protein (GAP) activity toward Rags-1 (GATOR1) complex, an amino-acid sensitive negative regulator of the mechanistic target of rapamycin complex 1 (mTORC1), is implicated in both genetic familial epilepsies and Neurodevelopmental Disorders (NDDs) (Baldassari et al., 2018). Previous studies have found seizure phenotypes and increased activity resulting from conditional deletion of GATOR1 function from forebrain excitatory neurons (Yuskaitis et al., 2018; Dentel et al., 2022); however, studies focused on understanding mechanisms contributing to NDD-relevant behaviors are lacking, especially studies understanding the contributions of GATOR1's critical GAP catalytic subunit, nitrogen permease regulator like-2 (Nprl2). Given the clinical phenotypes observed in patients with Nprl2 mutations, in this study, we sought to investigate the neuronal cell type contributions of Nprl2 to NDD behaviors. We conditionally deleted Nprl2 broadly in most neurons (Synapsin1^cre), in inhibitory neurons only (Vgat^cre), and in Purkinje cells within the cerebellum (L7^cre). Broad neuronal deletion of Nprl2 resulted in seizures, social and learning deficits, and hyperactivity. In contrast, deleting Nprl2 from inhibitory neurons led to increased motor learning, hyperactive behavior, in addition to social and learning deficits. Lastly, Purkinje cell (PC) loss of Nprl2 also led to learning and social deficits but did not affect locomotor activity. These phenotypes enhance understanding of the spectrum of disease found in human populations with GATOR1 loss of function and highlight the significance of distinct cellular populations to NDD-related behaviors. SIGNIFICANCE STATEMENT: We aim to elucidate the neuronal-specific contributions of nitrogen permease regulator like-2 (Nprl2) to its neurodevelopmental disorder (NDD)-relevant phenotypes. We conditionally deleted Nprl2 broadly in neurons (Syn1^cre), in inhibitory neurons (Vgat^cre), and in cerebellar Purkinje cells (L7^cre). We identify seizures only in the Syn1^cre conditional mutant (cKO); hyperactivity, learning difficulties, social deficits, and impulsivity in the Syn1^cre and Vgat^cre cKOs; and social deficits, and fear learning deficits in L7^cre cKOs. To our knowledge, we are the first to describe the behavioral contributions of Nprl2's function across multiple cell types. Our findings highlight both critical roles for Nprl2 in learning and behavior and also distinct contributions of select neuronal populations to these NDD-relevant behaviors.

Keywords: Behavior; Nprl2; mTORC1.

Figure 1.. Broad-neuronal loss of Nprl2 results in early mortality and seizures

(A) Western blot analysis of NPRL2, pS6, and S6 in whole-cell lysates of cortical tissue, 3–5-month-old mice. 15 ug of protein per well. Quantification includes n= 3 controls, 3 mutant, , Unpaired student t-test. NPRL2 and pS6/S6 were normalized to β-actin. (B) Representative immunohistochemistry for NeuN (purple), pS6 (green), and DAPI (blue) staining in the neocortex. Scale bar 500 um. (C-E) Conditional knockout (cKO) mutants body weights, brain weights and brain-to-body ratios compared with control littermates. Unpaired student t-test, n=8 controls, 4 mutants. (F) Kaplan-Meier survival curve of control and mutant mice. Log rank Mantel Cox test, n=42 controls, 34 mutants. (G) Audiogenic seizures scores age P17-P20. Unpaired student t-test n=15 controls, 10 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 2.. Broad-neuronal loss of Nprl2 results in hyperactivity, social, and learning deficits.

NO= novel object, NA= novel animal, FA= familiar animal. (A) Time to fall from rotarod in seconds, Unpaired student t-test n=15 controls, 9 mutants. (B) Hindlimb clasping score, Unpaired student t-test. n= 5 controls and 7 mutants. (C) Open field distance in 1 minute time bins, 2-way ANOVA with Sidaks multiple comparisons, n=15 controls, 9 mutants. (D) Open field time in center, Unpaired student t-test n=15 controls, 9 mutants. (E) Elevated Plus Maze (EPM), Unpaired student t-test n=15 controls, 9 mutants (F) Light/dark box, 2-way ANOVA with Sidaks multiple comparisons, n=15 controls, 9 mutants. (G-H) Three-Chambered Social test, (G) social approach (H) social novelty test, 2-way ANOVA with Sidaks multiple comparisons, n=13 controls, 9 mutants. (I) Social olfaction tests, 2-way ANOVA with Sidaks multiple comparisons, n=9 controls, 6 mutants. (J) Water-y-maze, number correct responses out of 15 attempts. T1-T3 are days with the platform on one side, and R1-R3 are the three days of when the platform position has been reversed and is placed on the opposite side, 2-way ANOVA with Sidaks multiple comparisons, n=15 controls, 9 mutants. (K) Context and (L) cued fear conditioning, Unpaired student t-test n=15 controls, 9 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 3.. Inhibitory-neuronal loss of Nprl2 leads to increased mTORC1 activity in inhibitory cells and greater brain weight

(A) Representative immunohistochemistry for Vgat (Red), pS6 (green), and DAPI (blue) staining in the neocortex. Scale bar 500 um. (B-D) Conditional knockout (cKO) mutants body weights, brain weights and brain-to-body ratios compared with control littermates. Unpaired student t-test, n=6 controls and 6 mutants. (E) Kaplan-Meier survival curve of control and mutant mice. Log rank Mantel Cox test, n=17 controls and 13 mutants. (F) Audiogenic seizures scores age P17-P20, Unpaired student t-test, n=5 controls and 4 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 4.. Inhibitory-neuronal loss of Nprl2 leads to hyperactivity, decreased social interests and difficult learning acquisition

NO= novel object, NA= novel animal, FA= familiar animal. (A) Time to fall from rotarod in seconds, Unpaired student t-test, n=19 controls and 16 mutants. (B) Open field distance in 1 minute time bins, 2-way ANOVA with Sidaks multiple comparisons, n=15 control and 13 mutants. (C) Open field time in center, Unpaired student t-test, n=15 control and 13 mutants. (D) Elevated plus maze (EPM), time spent in open arms, Unpaired student t-test, n=19 controls, 16 mutant. (E) Light/dark box, 2-way ANOVA with Sidaks multiple comparisons, n= 16 controls and 14 mutants. (F-G) Three-Chambered Social test, (F) social approach, (G) social novelty test, 2-way ANOVA with Sidaks multiple comparisons, n=11 controls, 12 mutants. (H) Social olfaction test, 2-way ANOVA with Sidaks multiple comparisons, n=9 in each group. (I) Water-y-maze, number correct responses out of 15. T1-T3 are days with the platform on one side, and R1-R3 are the three days of when the platform position has been reversed and is placed on the opposite side. 2-way ANOVA with Sidaks multiple comparisons, n=17 controls and 13 mutants, (J) Context and (K) cued fear conditioning, Unpaired student t-test, n=19 controls and 16 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 5.. Cerebellar-PC loss of Nprl2 leads to increased mTORC1 activity in PCs and greater CB/body weight ratios

(A) Representative images of pS6 (green), and calbindin (purple) staining in right crus 1 of the cerebellum. Scale bar 500 um. (B-D) Conditional knockout (cKO) mutants body weights, brain weights and brain-to-body ratios compared with control littermates. Unpaired student t-test, n= 9 controls, 9 mutants. (E) Kaplan-Meier survival curve of control and mutant mice. Log rank Mantel Cox test, n= 13 controls and 13 mutants. (F) Audiogenic seizures scores age P17-P20, Unpaired student t-test, n= 11 controls, 19 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 6.. Cerebellar-PC loss of Nprl2 leads to inflexible behavior and social deficits

NO= novel object, NA= novel animal, FA= familiar animal. (A) Time to fall from rotarod in seconds, Unpaired student t-test, n=27 controls and 20 mutants. (B) Open field distance in 1 minute time bins, 2-way ANOVA with Sidaks multiple comparisons, n=27 control and 20 mutants. (C) Open field time in center, Unpaired student t-test, n=27 control and 20 mutants (D) Elevated plus maze (EPM), time spent in open arms, Unpaired student t-test, n=14 controls, 13 mutants. (E) Light/dark box, 2-way ANOVA with Sidaks multiple comparisons box n= 27 controls and 20 mutants. (F-G) Three-Chambered Social test, (F) social approach, (G) social novelty test, 2-way ANOVA with Sidaks multiple comparisons, n= 13 controls, 15 mutants. (H) Social olfaction test, 2-way ANOVA with Sidaks multiple comparisons, n=4 controls and 7 mutants. (I) Water-y-maze, number correct responses out of 15, T1-T3 are days with the platform on one side, and R1-R3 are the three days of when the platform position has been reversed and is placed on the opposite side. 2-way ANOVA with Sidaks multiple comparisons, n=27 controls and 20 mutants, (J) Context and (K) cued fear conditioning, Unpaired student t-test, 6 controls, and 5 mutants. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; *p<0.0001

Figure 7.. L7^cre; Nprl2^fl/fl PCs are less excitable

(A) Input resistance, n= 25 cells each group. (B) Mutant PCs have decreased spontaneous firing. Mann Whitney test. (C) and decreased excitability. Whiskers represent minimum to maximum; box represents 25–75 percentile; line represents median. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001

Figure 8.. Summary of behaviors in Nprl2 cKOs

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