Fig 2: Genetic knockout of p35 rescues NMJ defects and motor neuron degeneration in SMA mice. (A–E) Motor neuron axon denervation at NMJs in SMA mice can be rescued by genetic knockout of p35. Colocalization of presynaptic nerve terminals marked by immunostaining with anti-neurofilament (NF) and anti-SV2 antibodies (green in A–D) with postsynaptic AChRs labeled by α-bungarotoxin (αBTX) (red in A–D) shows increased denervation (arrowheads) and partial innervation (arrow) of NMJs on the FDB-2 muscle of SMA mice. The NMJ innervation defect is rescued to the WT level in SMA;p35−/− mice. Quantification in E is from over 120 NMJs on the FDB-2 muscle from each mouse. Five mice for each genotype were used. Denervated, partially innervated, and fully innervated NMJs were analyzed separately. Results are mean ± SEM. ***P < 0.001, one-way ANOVA with Tukey HSD post hoc analysis. (F–J) Motor neuron degeneration in SMA mice is reduced by p35 knockout. Lumbar level spinal cord sections of P9 SMA mice show a near 40% reduction of Islet1-positive (red in F–I) motor neurons compared to WT littermates. Genetic knockout of p35 reduces motor neuron degeneration in SMA;p35−/−mice (J). Data in J are from four to five mice of each genotype in four independent experiments (WT: n = 130; SMA: n = 144; SMA;p35−/−: n = 162; p35−/−: n = 129 sections) and are mean ± SEM. ***P < 0.001, one-way ANOVA with Tukey HSD post hoc analysis.

Fig 3: Genetic knockout of p35 alleviates synaptic defects and hyperexcitability in SMA mouse motor neurons. (A–E) Excitatory glutamatergic synapse loss on SMA spinal cord motor neurons can be rescued by p35 knockout. Colocalization of the glutamatergic synapse marker vGluT1 (green in A–D) with the motor neuron marker ChAT (red in A–D) by immunostaining shows that excitatory synaptic boutons on motor neurons are significantly reduced in SMA mice and rescued to the WT level by genetic knockout of p35 in SMA mice (E). Data in E are quantified from motor neurons of three to four mice from each genotype (WT: n = 42; SMA: n = 51; SMA;p35−/−: n = 54; p35−/−: n = 34 motor neurons) and are mean ± SEM. ***P < 0.001, one-way ANOVA with Tukey HSD post hoc analysis. (F–H) SMA spinal motor neuron hyperexcitability can be reduced by genetic knockout of p35. Example plot of a firing motor neuron recorded during depolarizing current ramps (F). Threshold voltage (green), I-ON (red), and I-OFF (blue) were measured as shown. (G and H). Whole-cell patch-clamp electrophysiological recording of spinal motor neurons from P7-9 SMA mice showed that the amplitude (G) of PIC, which indicates the level of cellular excitability, was significantly increased in SMA motor neurons, and rescued in SMA;p35−/− mice. Threshold voltage (H) of AP firing is significantly more hyperpolarized in SMA motor neurons but not in motor neurons from SMA;p35−/− mice. Quantifications are from four mice of each genotype and are mean ± SEM. *P < 0.05 and **P < 0.01, one-way ANOVA with Tukey HSD post hoc analysis.

Fig 4: p35 degradation in neurons is mediated through the E3 ubiquitin ligase PJA2.(a) GSNO treatment increased p35 polyubiquitination level. Cultured rat cortical neurons were treated with GSNO in the presence of the proteasome inhibitor MG132. The protein lysate was subjected to immunoprecipitation using p35 antibody followed by western blot analysis for polyubiquitin. (b) p35 associated with PJA2 in the mouse hippocampus. Hippocampal lysates of 3-month-old mice were immunoprecipitated (IP) using p35 or PJA2 antibodies (IgG as control) as indicated, and the proteins in the co-immunoprecipitates were examined by western blot analysis. (c,d) p35 degradation is mediated by the E3 ligase PJA2. (c) PJA2 was knocked down in cultured rat cortical neurons, and the endogenous expression of p35 was examined by western blot analysis. (d) Quantification of p35 protein level (relative to the actin level). Data are normalized to the control and represent the mean±s.e.m. of four independent experiments. *P=0.016, **P=0.0018; one-way analysis of variance (ANOVA) with the Student–Newman–Keuls test. (e,f) GSNO promotes p35 degradation in a PJA2-dependent manner. (e) PJA2 was knocked down with PJA2-shRNA (#1) in cultured rat cortical neurons for 4 days, the cells were treated with GSH/GSNO for 1 h and p35 protein level was examined by western blot analysis. (f) Quantification of p35 level (relative to the actin level). Data are normalized to control group and represent the mean±s.e.m. of six independent experiments. ***P=6.81E−7, ###P=6.42E−5; one-way ANOVA with the Student–Newman–Keuls test.

Fig 5: Protein levels of total tau (t-tau), p-tau and its regulating kinases in the hippocampus and cortex after acute hypoxia. Protein levels of p-tau at T181 (C,D), T231 (E,F), S396 (G,H) and cyclin-dependent-like kinase-5 (CDK5) and its activator p35 and p25 (I,J) were increased in the hippocampus and cortex of hypoxic mice. Changes in total-tau (A,B), glycogen synthase kinase-3β (GSK-3β)/phosphorylated GSK-3β (p-GSK-3β) ratio (K,L) were moderate. n = 5 mice in each group. *p < 0.05, **p < 0.01, by two-way ANOVA with Tukey's multiple comparisons test.