Fig 1: Magnitude of brain NAD+ homeostatic disruption correlates with the severity of human and mouse AD(A) NAD+ homeostasis is disrupted in human AD cerebral cortex, relative to control subjects (n = 14–18 per group, ∗∗∗p < 0.001, unpaired t test). Also see Table S1.(B) Greater disruption in human brain NAD+ homeostasis is associated with more p-Tau pathology. Also see Figure S5A and Tables S1 and S2.(C) Greater disruption in human brain NAD+ homeostasis is associated with more oxidative damage, as evidenced by protein carbonylation levels. Also see Figure S5B and Table S2.(D) Greater disruption in human brain NAD+ homeostasis is associated with more neuroinflammation, as evidenced by GFAP levels. Also see Figure S5C and Table S2.(E) Greater disruption in human brain NAD+ homeostasis is associated with more BBB deterioration, as evidenced by ZO-1 levels. Also see Figure S5D and Table S2.(F) Greater disruption in human brain NAD+ homeostasis is associated with more neuronal cell loss, as evidenced by NeuN levels. Also see Figure S5E and Table S2.(G) Greater disruption in human brain NAD+ homeostasis shows a trend of association with more synaptic loss, as evidenced by PSD-95 levels. Also see Figure S5F and Table S2.(H) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more p-tau pathology.(I) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more oxidative damage, as evidenced by protein carbonylation levels.(J) Greater disruption in 6-month-old 5xFAD brain NAD+ homeostasis is associated with more oxidative damage, as evidenced by protein carbonylation levels.(K) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more neuroinflammation, as evidenced by IL-2 levels.(L) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more neuroinflammation, as evidenced by IL-13 levels.(M) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more BBB deterioration, as evidenced by ZO-1 levels.(N) Greater disruption in 6-month-old 5xFAD brain NAD+ homeostasis is associated with more greatly impaired memory, as evidence by discrimination index in the NOR test.(O) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more greatly impaired memory, as evidence by discrimination index in the NOR test.(P) Greater disruption in 6-month-old 5xFAD brain NAD+ homeostasis is associated with more greatly impaired memory, as evidence by the number of platform crossings in the probe memory test of the Morris water maze.(Q) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with more greatly impaired memory, as evidence by the number of platform crossings in the probe memory test of the Morris water maze.(R) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with decreased ability to stay on the accelerating rotating rod, as evidenced by time until falling.(S) Greater disruption in 12-month-old 5xFAD brain NAD+ homeostasis is associated with decreased ability to stay on the accelerating rotating rod, as evidenced by rotation speed of the rod at falling.(T) Levels of the NAD+-synthesizing enzymes glutamine-dependent NAD+ synthetase (NADSYN1) and nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) are reduced in human AD cerebral cortex, relative to control subjects, and present at normal levels in NDAN subjects (n = 73 for control, 80 for AD, and 27 for NDAN; ∗p < 0.05, ∗∗p < 0.01, limma linear regression).(U) Levels of NAD+-consuming enzymes NAD kinase 2 (NADK2), poly (ADP-ribose) polymerase 4 (PARP4), and sirtuin 1 (SIRT1) are increased in human AD cerebral cortex, relative to control subjects, and present at normal levels in NDAN subjects (n = 73 for control, 80 for AD, and 27 for NDAN, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, limma linear regression).See also Figure S5.
Fig 2: Tau pathology leads to neurodegeneration and glial responses in chimeric brains.(A) Representative hMAP2 and Ku80 double staining in cerebral cortex. Control chimeras show intact dendritic architecture, whereas tau-treated chimeras exhibit reduced overall hMAP2+ area and dendritic fragmentation.(B) Quantification of hMAP2+ area in hippocampus and cerebral cortex for control (n = 6), tau 6 mpi (n = 4), and tau 10 mpi (n = 8) chimeras. Data are mean ± SEM; Welch’s ANOVA with Dunnett’s T3 multiple-comparisons test; *p < 0.05, ***p < 0.001.(C) Representative hMAP2 and Homer1 double staining in hippocampus and cerebral cortex in control, tau 6 mpi, and tau 10 mpi chimeras. Homer1 puncta co-localized with hMAP2 are defined as postsynaptic structures associated with human neurons.(D) Quantification of Homer1 puncta co-localized with hMAP2 in hippocampus and cerebral cortex. Control, n = 6; tau 6 mpi, n = 4; tau 10 mpi, n = 4. Data are mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test; *p < 0.05, ****p < 0.0001.(E) Representative GFP, NeuN, and AT8 triple labeling in cortex from control and tau 10 mpi chimeras.(F) Quantification of human neuron density within AT8+ regions (human neurons per AT8+ area), measured in the cortex and hippocampal formation. Control, n = 6; tau 6 mpi, n = 3; tau 10 mpi, n = 4. Data are mean ± SEM; one-way ANOVA with Tukey’s multiple-comparisons test; **p < 0.01, ****p < 0.0001.(G) Representative AT8 and phosphorylated MLKL (pMLKL) double staining in control and tau 6 mpi chimeras. Arrows indicate pMLKL+ neurons.(H) Representative AT8 and p62 double staining in control and tau 10 mpi chimeras. p62 accumulation in tangle-bearing neurons.(I-K) Representative of AT8 and Iba1 double labeling from control chimera and tau 10 mpi chimera. (I) Microglia were found surrounding the punctate AT8-positive phospho-tau deposits. Asterisks point out microglia phagocytosing tau. (J) Arrows highlight microglia-associated AT8+ tau puncta, indicating microglia actively engulfing phospho-tau. (K) Imaris-based 3D reconstruction further indicates microglial engulfment of AT8+ deposits.(L) Representative AT8 and GFAP double staining in control and tau 10 mpi chimeras. Asterisks denote reactive astrocytes associated with punctate AT8+ deposits; the inset indicates astrocytic processes surrounding an AT8+ tangle.Scale bars, as indicated in the panels.
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