Fig 2: Integrated proteomics of human brain reveals NRN1 as a top resilience candidate.A, significant enrichment of modules associated with increased cognitive resilience was identified by PWAS in consensus modules. The dashed red line illustrates the significance cutoff corresponding to a Z score of 1.96 or p = 0.05. Significant, increased resilience modules are highlighted in orange. B, PWAS significant, synaptic modules M5 and M22 positively correlate with cognitive slope, irrespective of the brain region. Bicor and p values (BA6: M22 cor = 0.32, M22 p = 0.00069, M5 cor = 0.4, M5 p = 1.6e-05; BA37: M22 cor = 0.53, M22 p = 3.1e-09, M5 cor = 0.45, M5 p = 9.1e-07). C, differential expression comparing AsymAD and AD groups from M5 and M22 module members. Protein fold-change is the x-coordinate and the -log10p value from one-way ANOVA is the y coordinate for each protein. Proteins above the dashed line (p = 0.05) are considered significantly differentially expressed. Large circles highlight proteins that were significant by PWAS (a = 5e-06). D, NRN1 abundance is significantly reduced in AD. One-way ANOVA (BA6: F = 13.25, p < 0.001; BA37: F = 13.68, p < 0.001) with Tukey test. E, NRN1 abundance correlates positively with global cognitive performance. Bicor and p values (BA6: bicor = 0.53, p = 2.9e-09; BA37: bicor = 0.53, p = 2.2e-09). F, NRN1 abundance correlates positively with cognitive slope. Bicor and p values (BA6: bicor = 0.51, p = 1.8e-08; BA37: bicor = 0.46, p = 6.4e-07). AD, Alzheimer’s disease; AysmAD, asymptomatic AD; BA6, Brodmann area 6; BA37, Brodmann area 37; NRN1, neuritin; PWAS, proteome-wide association study.

Fig 3: Aß42-induced dendritic spine degeneration is blocked by NRN1.A, schematic representation of primary rat hippocampal neuron treatment and dendritic spine morphometric analysis. B, representative maximum-intensity wide-field fluorescent images of hippocampal neurons after deconvolution (left). Corresponding three-dimensional reconstructions of dendrites generated in Neurolucida 360 (right), with dendritic spines color-coded by spine type (blue = thin, orange = stubby, green = mushroom, yellow = filopodia). Scale bar, 5 µm. N = 6 to 8 neurons (one dendrite per neuron) were analyzed per experimental condition. C, dendritic spine density in hippocampal neurons exposed to DMSO, 500 nM Aß42, 150 ng/ml NRN1, or 150 ng/ml NRN1 and 500 nM Aß42. **p < 0.01 (DMSO versus Aß42, actual p = 0.0025) (Aß42versus NRN1 + Aß42, actual p = 0.0026) by one-way ANOVA with Tukey’s test. *p < 0.05 (Aß42versus NRN1, actual p = 0.0177) by one-way ANOVA with Tukey’s test. D, dendritic spine density of thin, stubby, or mushroom spines per 10 µm. *p < 0.05 (DMSO versus Aß42, actual p = 0.0218) by one-way ANOVA with Tukey’s test. (Thin, Aß42versus NRN1+Aß42, actual p = 0.0501) (Mushroom, DMSO versus Aß42, actual p = 0.1514) (Mushroom, Aß42versus NRN1+Aß42, actual p = 0.0598) by one-way ANOVA with Tukey’s test. E, dendritic spine type frequency in hippocampal neurons exposed to DMSO, 500 nM Aß42, 150 ng/ml NRN1, or 150 ng/ml NRN1 and 500 nM Aß42. F, overall dendritic spine length and (G) head diameter. Related data are shown in supplemental Fig. S7. Box plots represent median, 25th, and 75th percentiles. Box hinges represent the interquartile range of the two middle quartiles with a group. Error bars are based on data points 1.5 times the interquartile range from the box hinge. Aß, amyloid-beta; NRN1, neuritin.

Fig 4: NRN1 protects against Aß42-induced neuronal hyperexcitability.A, schematic representation of primary rat hippocampal neuron treatment and single neuron electrophysiology analysis. B, representative raster plots from three units after exposure to DMSO, 500 nM Aß42, 150 ng/ml NRN1, or 150 ng/ml NRN1 and 500 nM Aß42. C, mean firing rate at DIV14 in hippocampal neurons treated with DMSO, compared to baseline (n = 36–54 neurons, unpaired Student’s t test; p = 0.1296). D, mean firing rate at DIV14 in hippocampal neurons treated with 500 nM Aß42, compared to baseline (n = 65–68 neurons, unpaired Student’s t test; p = 0.0022). E, mean firing rate at DIV14 in hippocampal neurons treated with 150 ng/ml NRN1, compared to baseline (n = 32–33 neurons, unpaired Student’s t test; p = 0.0023). F, mean firing rate at DIV14 in hippocampal neurons treated with 150 ng/ml NRN1 and 500 nM Aß42, compared to baseline (n = 100–107 neurons, unpaired Student’s t test; p = 0.0676). Aß, amyloid-beta; NRN1, neuritin.

Fig 5: NRN1 treatment induces changes in the neuronal proteome related to broad synaptic functions.A, schematic representation of rat primary cortical neuronal culture workflow in which neurons were maintained in neurobasal medium for 14 days, treated with 150 ng/ml of NRN1 and analyzed via TMT-MS. B, differential protein expression between NRN1-treated and vehicle-treated neurons (n = 8238 proteins). Proteins above the dashed line (p = 0.05) are considered significantly differentially expressed. Student’s t test was used to calculate p values. Large spots are based on reproducibility-optimized test statistic (ROTS) correction of differentially expressed proteins with 10% or less false discovery rate (FDR). C, gene ontology of significantly differentially expressed proteins in NRN1 treated neurons. A Z-score above 1.96 was considered significant (p < 0.05). NRN1, neuritin; TMT-MS, tandem mass tag mass spectrometry.