The UNC5C T835M mutation associated with Alzheimer's disease leads to neurodegeneration involving oxidative stress and hippocampal atrophy in aged mice

Abstract

Alzheimer's disease (AD) is characterized by amyloid plaques, neurofibrillary tangles, and synaptic and neuronal loss. Recently, a rare autosomal dominant coding mutation, T835M, in the Un-coordinated 5c (UNC5C) netrin receptor gene was segregated with late-onset AD (LOAD). Overexpression of T835M in primary hippocampal neurons increased cell death in response to neurotoxic stimuli including beta-amyloid (Aβ) suggesting a mechanism by which T835M may confer increased risk of LOAD. However, the molecular mechanism of T835M-mediated cell death remained under explored. Toward this end, we generated a mouse T835M knock-in (Unc5c^KI/KI) model and employed biochemical and histological analyses to understand the molecular mechanism of T835M-mediated pathogenesis in late onset Alzheimer's disease. We show that homozygous KI mice have significantly reduced hippocampal volume, increased ventricular volume, dendritic disorganization (CA1 region) and reduced UNC5C protein level by 12-18 months of age. Further, we show that the neuronal cell death is observed in the Unc5c^KI/KI mice by 12 months of age by TUNEL analysis and activated Caspase 3/7 assay. Proteomic analysis of hippocampal samples showed upregulation of oxidative stress and downregulation of chaperone proteins at 18 months corroborating the biochemical and histological results showing increased c-Jun N-terminal Kinase (JNK) phosphorylation, NADPH oxidase, and decreased Netrin1 levels. Moreover, Unc5c^KI/KI mice also show morphological changes in the astrocytes with increased number of branched processes, reduced GFAP levels, and significantly increased activation of microglia. Overall, these results suggest that T835M mutation causes neurodegeneration by creating an oxidative stress environment leading to synaptic degeneration and weakened astrocytes, thereby leading to neuronal cell death via apoptosis. Furthermore, to assess the effects of amyloid pathology on the mutation, we crossed Unc5c^KI/KI mice with App^{NL-G-F/NL-G-F} mice and observed an exacerbation of mutation-associated changes along with increased levels of Aβ₄₂, suggesting that the T835M mutation increases the susceptibility of neurons to cell death and elevated Aβ₄₂ levels, thus promoting AD pathogenesis. Understanding the molecular mechanism of cell death in regions susceptible to neurodegeneration such as the hippocampus could shed light on the players and pathways involved in cell death in AD pathogenesis and therefore could inform therapeutic approaches for AD.

Fig. 1

Unc5c^KI/KI mice show significant hippocampal atrophy with age. A. Coronal sections of hemibrains immunostained for NeuN (green) in Unc5c^+/+ and Unc5c^KI/KI mice at 6, 12, and 18 months. Scale bar, 1.16 mm. Three different bregma positions (−1.34 mm (anterior), −1.70 mm (center), −2.06 mm (posterior)) were chosen for each animal to do the area analysis. Hippocampus is highlighted by dashed yellow region. B. Quantification of hippocampal area by ImageJ from 6–18 months. Blue circles—males; pink triangle—females. n = 4 females, 2 males (6 months), n = 5 females, 4 males (12 months), n = 6 females, 2 males (18 months). C. Schematic representation of the longitudinal MRI study. n = 8/genotype (4 males, 4 females) D. (Left) MRI slices with superimposed segmented regions of interest (Hippocampus (Blue), ventricle (red)) visualizing the changes in ventricle and hippocampus size over the course of ~ 5–6 months (13 to 18 month) for Unc5c^+/+ (top) and Unc5c^KI/KI (bottom) mice. (Right) Representative 3D rendered MR images are shown for both Unc5c^+/+ and Unc5c^KI/KI mice at 18 months. E–F. Quantification of the change over time (13 months to 18 months) in ventricular volume (E) and hippocampal volume (F). G. Representative MRI image slice showing the cortical thickness measurement. Red lines indicate various positions where the thickness was measured. Measurements (in white) in three different regions are similar in both Unc5c^+/+ and Unc5c^KI/KI mice at 18 months. H. Quantification of cortical thickness in Unc5c^+/+ and Unc5c^KI/KI mice at 13 and 18 months. I. Representative 2D MRI slices depicting the brains of Unc5c^+/+ and Unc5c.^KI/KI mice with superimposed fractional anisotropy (FA) patterns thresholded at ~ 0.2 (red) across the whole brain. J. Quantification of the change over time (∆) in FA/FA_WT (13 months to 18 months). Statistics calculated using two-tailed unpaired student’s t-tests and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. ns = non-significant. *p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and ****p-value of ≤ 0.0001

Fig. 2

Unc5c^KI/KI mice have axonal and synaptic degeneration and dendritic disorganization A. Immunoblots of presynaptic/axonal and postsynaptic proteins in the hippocampal samples of 18-month-old Unc5c^+/+ and Unc5c^KI/KI mice. B-F. Quantification of the immunoblots in (A) normalized to GAPDH. Blue circles—males; pink triangle – females. n = 4–5 females, 3–4 males/genotype. G. CA1 region of hippocampus stained for post-synaptic proteins such as PSD95 at 3-months and 18-months and MAP2 at 18 months in Unc5c^+/+ and Unc5c^KI/KI mice. H, I. Quantification of mean fluorescence intensity of PSD95 (H) and MAP2 (I). J, K Graph showing the distribution of orientation of dendrites with respect to the CA1 nuclear layer obtained at 3 months (J) and 18 months (K) in Unc5c^+/+ (black) and Unc5c^KI/KI (red) mice. n = 6 (Unc5c^+/+), n = 7 (Unc5c.^KI/KI). Statistics calculated using two-tailed unpaired student’s t-tests and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. ns = non-significant. p-value ≤ 0.05, *p-value ≤ 0.01, **p-value ≤ 0.001, and ****p-value of ≤ 0.0001

Fig. 3

Proteomics reveal upregulation of oxidative stress and down-regulation of chaperone proteins in Unc5c^KI/KI mice hippocampi. A. Volcano plot of up-and down-regulated proteins obtained by TMT-MS of hippocampal homogenates of Unc5c^+/+ and Unc5c^KI/KI mice at 18 months (n = 5 females/genotype). Number of up-regulated (red) and down-regulated proteins (blue) are shown along with total number of proteins obtained. B, C. DAVID analysis of up-regulated proteins (B) and down-regulated proteins (C) showing the most significant GO terms. D, E. List of proteins under each significantly up-regulated (D) and down-regulated (E) biological process/GO term in DAVID analysis. F. Immunoblot analysis of hippocampal homogenates from 18-month Unc5c^+/+ and Unc5c^KI/KI mice for proteins (underlined in red in D) under each of the GO categories listed in D. G-I. Quantification of the immunoblots for UQCRB (G), CALM1 (H) and CAPZB (I) normalized to β-tubulin (n = 5 females/genotype). J. Immunoblot analysis of hippocampal homogenates from 18-month Unc5c^+/+ and Unc5c.^KI/KI mice for proteins (underlined in red in E) under each of the GO categories listed in E. K-M. Quantification of the immunoblots for GFAP (K), HSPD1 (L) and CACNB4 (M) normalized to β-tubulin (n = 5 females/genotype). Statistics calculated using two-tailed unpaired student’s t-tests. Data are presented as mean ± SEM. ns = non-significant. p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, and ***p-value of ≤ 0.0001

Fig. 4

Increased neuronal apoptosis in Unc5c^KI/KI mice A. Immunoblot analysis using an UNC5C-specific antibody on 12-month-old hippocampal samples from Unc5c^+/+ and Unc5c^KI/KI mice. Unc5c^KO/KO (Unc5c^−/−) is used as a negative control. The Full-length (FL) UNC5 C band was observed around 115 kDa. Note two additional lower bands that are specific to the UNC5C antibody labeled Cleaved 1 (CL1) and Cleaved 2 (CL2) above and below the 50 k D marker, respectively. N = 10 (5 females, 5 males) B. Quantification of FL, CL1, CL2 and combined (CL1 + CL2) bands of UNC5C normalized to GAPDH and presented as arbitrary units (a.u). C. Quantification of the ratios of CL1, CL2, and combined CL1 + CL2 to FL bands. Blue circles – males; pink triangles – pink. D. Confocal images of CA1 region from 18m Unc5c^+/+ and Unc5c^KI/KI mice stained for TUNEL-positive neurons (NeuN, magenta; TUNEL⁺, red). Scale bar, 100 μm. Sections around −1.70 mm Bregma position were chosen for analysis. White boxed region in upper panels is enlarged in lower panels. Yellow arrowheads show TUNEL⁺ cells, of which some are NeuN⁺ (white arrowheads). Scale bar, 20 μm. E, F. Quantification of number of TUNEL⁺ neurons (TUNEL⁺ NeuN⁺) (E) and non-neuronal TUNEL⁺ cells (TUNEL⁺ NeuN⁻) (F) in hippocampal sections of Unc5c^+/+ (black) and Unc5c^KI/KI (red) mice. Blue circles—males; pink triangle—females. N = 5–7 males, n = 5–8 females/genotype/age. G. Quantification of the %NeuN covered area in the hippocampus at 18 months. n = 6 mice/genotype (2 sections/animal). H. Quantification of caspase-3 activity assay expressed as relative fluorescent units. n = 9–10 mice/genotype I. Immunoblot analysis of hippocampal homogenates from Unc5c^+/+ and Unc5c.^KI/KI mice for proteins involved in UNC5 C T835M-mediated apoptosis pathway at 12 months. J-N. Quantification of immunoblot signals for Protein kinase-D (PKD) (J), phospho-JNK/JNK (K), cycline-dependent kinase (CDK5) (L), NADPH oxidase (NOX1) (M), Netrin1 (NTN1) (N) normalized to GAPDH. Blue circles—males; pink triangle—females. n = 3–5 females, n = 2–5 males/genotype. Statistics calculated using two-tailed unpaired student’s t-tests, multiple t-test, two-way ANOVA using Sidak’s multiple comparisons test (for panels B and C) and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. ns = non-significant. *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, and ****p-value of ≤ 0.0001

Fig. 5

Astrocyte morphology is significantly altered in the Unc5c^KI/KI mice. A-C. Confocal images of CA1 region of Unc5c^+/+ and Unc5c^KI/KI mice at 3 months (A), 12 months (B) and 18 months of age (C) immunostained for GFAP (astrocytes). Scale bar, 33 μm. Sections around −1.70 mm Bregma position were chosen for analysis. White-dashed boxed region is enlarged in A’ (3 months), B’ (12 months), and C’ (18 months). Scale bar, 50 μm. D. Quantification of mean fluorescent intensity of GFAP in the CA1 region of hippocampus at 3–6 months, 12 months and 18 months in Unc5c^+/+ and Unc5c.^KI/KI mice. E. Quantification of the % GFAP covered area in the hippocampus at 12 months and 18 months. Blue circles—males; pink triangle—females. n = 5–7 females, 5–7 males/genotype/age (2 sections/animal were used in the analysis). F. Quantification of number of astrocytes in the CA1 region of hippocampus at 18 months. n = 4 females, 3 males. Statistics calculated using two-tailed unpaired student’s t-tests and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. Only comparisons with significant p-value are indicated. * p-value ≤ 0.05, ** p-value ≤ 0.01, ** p-value ≤ 0.001, and **** p-value of ≤ 0.0001

Fig. 6

Microglia show increased activation in Unc5c^KI/KI mice. A. Confocal microscope images of CA1 of Unc5c^+/+ and Unc5c^KI/KI mice at 18 months of age immunostained for Iba1 (red), CD68 (magenta) and C1q (green). Scale bar, 33 μm. B. Higher magnification of images in A showing increased activation of microglia (C1q⁺, CD68⁺) in the Unc5c^KI/KI mice. Scale bar, 7 μm. C, D, F. Quantification of mean fluorescence intensity of Iba1 (C), CD68 (D), and C1q (F) in Unc5c^+/+ and Unc5c^KI/KI mice. E, G. Quantification of number of microglia (Iba1⁺) that are CD68⁺ (E) and both CD68⁺ and C1q.⁺ (G). Blue circles—males; pink triangle—females. n = 4–6 females, 3–5 males/genotype/age (2 sections/animal were used in the analysis). Statistics calculated using two-tailed unpaired student’s t-tests. Data are presented as mean ± SEM. Only comparisons with significant p-value are indicated. *p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value of ≤ 0.0001

Fig. 7

Neurodegeneration and UNC5C-mediated apoptosis are exacerbated in dKI mice. A. Confocal microscope images of CA1 of NLGF and dKI mice at 6 and12 months of age immunostained for NeuN (red). B. Quantification of the %NeuN covered area in the hippocampus at 6 and 12 months. n = 5 mice/genotype/age (2 sections/animal). C. Quantification of cleaved Caspase-3 activity assay expressed as relative fluorescent units in NLGF and dKI mice at 6 and 12 months. n = 9–10 mice/genotype/age. D. Coronal sections of hemibrains immunostained with LAMP1 (green), 3D6 (red), and Aβ₄₂ (white) from NLGF and dKI mice at 12 months. Scale bar, 500 μm. Inset in each panel is a high-magnification image of the CA1 region outlined by a dashed box in the respective low-magnification image. D’. Outline of a plaque with the ‘halo’ marked by LAMP1 (green) and Aβ₄₂ (white) to indicate how the diameter was measured. The diameter of the plaque core (Aβ₄₂) is indicated by solid line while the dashed line is the diameter of LAMP1. E–G. diameter of the dystrophic neurites (LAMP1 – Aβ₄₂ diameter) (E), Quantification of LAMP1/Aβ₄₂ fill area (F), and 3D6 fill area (G) of NLGF and dKI mice at 12 months. n = 5 mice/genotype/age (3–5 sections/animal). H–K. MSD ELISA results of Aβ species – Aβ₃₈ (H), Aβ₄₀ (I), Aβ₄₂ (J) and Aβ₄₂/Aβ₄₀ (K) of hippocampal homogenates from 6-month and 12-month-old mice. L. Immunoblot of hippocampal homogenates from NLGF and dKI mice for proteins involved in the UNC5C T835M pathway at 6 and 12 months. M-P. For immunoblots in L, quantification of NADPH oxidase (NOX1) (M), phospho-JNK/total JNK (N), cyclin-dependent kinase (CDK5) (O), Netrin1 (P) normalized to GAPDH. Blue circles—males; pink triangle—females. n = 5 mice/genotype/age. Statistics calculated using two-tailed unpaired student’s t-test and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. Only data with significant p values are indicated. * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value of ≤ 0.0001

Fig. 8

Synaptic abnormalities and dendritic disorganization are exacerbated in dKI mice. A. Immunoblot blot analysis of synaptic/axonal proteins in NLGF and dKI mice – PSD95, Synaptophysin (SYP), BACE1, GFAP and SMI312, at 6 and 12 months. n = 5/sex/genotype/age. B-F. Quantification of immunoblot signals for proteins in A normalized to GAPDH. G. Immunofluorescence microscopy for neuronal/synaptic proteins – BACE1, GFAP, SMI312 (phosphorylated neurofilament), PSD95, synaptophysin, and APP in NLGF and dKI mice at 12 months. Yellow star in panels with SMI312 and GFAP indicate the position of amyloid plaque. H. Graph showing the distribution of orientation of dendrites emerging from CA1 neuronal layer in NLGF and dKI mice at 12 months. There was a significant difference in the regions closer to 0° from (−8.5° to 3.5°) I. Overlay of the graph in H and graph in Fig. 2L showing dendritic orientation in NLGF and dKI mice at 12 months as compared to that of Unc5c^+/+ and Unc5c.^KI/KI at 18 months as shown in Fig. 2L. Blue circles—males; pink triangle—females. n = 5/sex/genotype/age. Statistics calculated using two-tailed unpaired student’s t-test and ordinary one-way ANOVA using Tukey’s multiple comparison tests with Bartlett’s test correction. Data are presented as mean ± SEM. Only data with significant p values are indicated. *p-value ≤ 0.05, *p-value ≤ 0.01, *** p-value ≤ 0.001, and **** p-value of ≤ 0.0001

Fig. 9

Mechanism of UNC5C T835M-mediated cell death and neurodegeneration. (Left) In WT mice (Unc5c^+/+), UNC5C performs its function as dependence receptor in axon guidance pathway in the presence of its ligand Netrin1. (Right) UNC5C T835M (Unc5c^KI/KI) mutation leads to neurodegeneration via synaptic degeneration, disorganization and apoptotic cell death in neurons, reduced GFAP levels and changed morphology with more processes in astrocytes and activated microglia. The mutation results in decreased and increased PKD and CDK5 levels, respectively, leading to activation of the JNK pathway, with further increase in NADPH oxidase (NOX1) creating an oxidative stress environment. With reduced chaperone proteins, NOX1 could trigger the activation of cleaved caspase-3 leading to apoptosis. The UNC5C T835M mutation also results in decreased Netrin1, which could elevate Aβ₄₂ production, in addition to increased apoptosis. Overall, this pathway leads to increased neuronal loss, reduced hippocampal volume and increased ventricular volume in Unc5c^KI/KI mice. Thus, Unc5c^KI/KI mice provide an ideal environment for the study of various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, or Huntington’s disease. UNC5C-mediated neurodegeneration, oxidative stress, and apoptosis are exacerbated in the presence of β-amyloid. Images created with BioRender.com