Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy

Abstract

Defective autophagy contributes to Alzheimer disease (AD) pathogenesis although evidence is conflicting on whether multiple stages are impaired. Here, for the first time, we have comprehensively evaluated the entire autophagic process specifically in CA1 pyramidal neurons of hippocampus from early and late-stage AD subjects and nondemented controls. CA1 neurons aspirated by laser capture microdissection were analyzed using a custom-designed microarray comprising 578 neuropathology- and neuroscience-associated genes. Striking upregulation of autophagy-related genes, exceeding that of other gene ontology groups, reflected increases in autophagosome formation and lysosomal biogenesis beginning at early AD stages. Upregulated autophagosome formation was further indicated by elevated gene and protein expression levels for autophagosome components and increased LC3-positive puncta. Increased lysosomal biogenesis was evidenced by activation of MiTF/TFE family transcriptional regulators, particularly TFE3 (transcription factor binding to IGHM enhancer 3) and by elevated expression of their target genes and encoded proteins. Notably, TFEB (transcription factor EB) activation was associated more strongly with glia than neurons. These findings establish that autophagic sequestration is both competent and upregulated in AD. Autophagosome-lysosome fusion is not evidently altered. Despite this early disease response, however, autophagy flux is progressively impeded due to deficient substrate clearance, as reflected by autolysosomal accumulation of LC3-II and SQSTM1/p62 and expansion of autolysosomal size and total area. We propose that sustained induction of autophagy in the face of progressively declining lysosomal clearance of substrates explains the uncommonly robust autophagic pathology and neuritic dystrophy implicated in AD pathogenesis.

Keywords: Alzheimer disease; CA1 pyramidal neurons; MTOR; TFE3; TFEB; autophagosomes; autophagy; dystrophic neurites; hippocampus; lysosomes.

Figure 1.

Changes in expression of autophagy-related genes in hippocampal CA1 pyramidal neurons. (A) Autophagy-related genes showing differential regulation in CA1 neurons via custom-designed microarray analysis of 578 genes. Significantly (p ≤ 0.05) altered in both Br. III/IV and Br. V/VI shown in dark gray; others significantly altered in Br. V/VI only shown in black, no change in gray. (B) Pie chart indicating proportions of significantly upregulated and downregulated autophagy genes on the array platform. (C) Pie chart illustrating a relative paucity of genes upregulated in all 578 transcripts contrasting with the higher proportion that are downregulated. (D) GO analysis of all highly expressed genes in Br. III/IV and Br. V/VI when compared to control group. Autophagy, the most represented term, is highlighted in darker blue. p value cut off 0.05. (E) qPCR validation of selected autophagy/lysosomal genes assayed in the CA1 sector of the hippocampus. Statistical significance is denoted by asterisks with p-values shown determined by one-way ANOVA or for 2 column analysis by Student t test. *p ≤ 0.05; **p ≤ 0.01.

Figure 2.

Evaluation of autophagy induction in AD. (A) Western blot analysis of indices of autophagic induction and autophagosome formation for ULK1, BECN1, PIK3C3/VPS34, ATG7, LC3, and SQSTM1/p62 and degradative products in AD hippocampus. (B) Histogram illustrating quantification of the findings in (A). (C–D) Western blot analysis of FOXO1 and FOXO3 levels in AD and control hippocampal tissue. Samples analyzed from patients as Control (n = 9), from AD Br. III (n = 10) and from AD Br. V (n = 10). Significance determined by one-way ANOVA with post-hoc Dunnett's Comparison to Control test. *p ≤ 0.05; **p ≤ 0.01. Western blots cropped as shown for purposes of clarity.

Figure 3.

Analysis of lysosomal constituents in AD hippocampus. (A) Western blot analysis of structural and enzymatic constituents of lysosomes with quantification shown in (B). Samples analyzed from control patients (n = 9), from AD Br. III (n = 10) and from AD Br. V (n = 10). Significance determined by one-way ANOVA, with post-hoc Dunnett's Comparison to Control test. **p ≤ 0.01. Western blots cropped as shown for purposes of clarity.

Figure 4.

Analysis of TFE3 and TFEB transcription factors in hippocampal CA1 pyramidal neurons and glia. (A) qPCR analysis of CA1 neurons for TFE3 and TFEB. (B) qPCR for both TFEB and TFE3 in hippocampal total RNA. (C) Amplification plots for TFE3 (red) and TFEB (green) showing a 1.2 cycle difference equivalent to a greater than 10-fold higher level of TFE3 mRNA in hippocampal tissue. (D–E) Western blot analysis for TFE3 and TFEB in hippocampal tissue and relative quantification showing higher levels of TFE3. Samples analyzed from control patients (n = 9), from AD Br. III (n = 10) and from AD Br. V (n = 10). (F) Immunocytochemical localization of TFE3 in the hippocampal CA1 region. Cells immunostained for TFE3 and counterstained with Nissl. Black arrowheads indicate translocation of TFE3 in neuronal nuclei; blue arrowheads point to negative TFE3 nuclear staining. Scale bar: 10 μm. (G) Quantification of TFE3 nuclear translocation in glia and neurons. (H) Fluorescent immunolocalization of TFEB (yellow arrowheads for neuronal, blue for glia) with Nissl staining shows neuronal or glial nuclei and with decreasing Nissl staining on nuclear translocation in hippocampal CA1 region. Scale bar: 10 μm. (I) Quantification of TFEB nuclear translocation in glia and neurons. A minimum of 50 fields were quantified per condition. Statistical significance denoted by asterisks with p-values shown determined by one-way ANOVA, with post-hoc Dunnett's Comparison to Control test, or for 2 columns analysis by Student t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 5.

Double-immunofluorescent labeling of the CA1 region with LC3 and CTSD. (A) Immunofluorescent staining for LC3 (green channel) and CTSD (red) in CA1 hippocampal neurons from Control, Br. III and V. Scale bar: 20 μm. (B) Pearson's correlation coefficient of colocalization of CTSD with LC3 in hippocampal CA1 neurons. A minimum of 100 neurons were quantified per condition. (C–D) Characterization of vesicles in Control, Br. III and V CA1 neurons, identified as autophagosome (AP, LC3-positive, CTSD-negative vesicles), autolysosome (AL, LC3- and CTSD-positive vesicles) and lysosome (Ly, LC3-negative, CTSD-positive vesicles). Depicted is the total number and area covered per cell in (C) and (D) respectively. (E) Size distribution of autolysosomes in Control, Br. III and V CA1 neurons. Statistical significance denoted by asterisks with p-values shown determined by one-way ANOVA, with post-hoc Dunnett's Comparison to Control test for grouped analyses, and 2-way ANOVA for size distribution analyses. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 6.

Neuritic dystrophy adjacent to senile plaques in the CA1 region in AD brains. (A) The image of AD Br. V hippocampal CA1 depicts a population of putative dystrophic neurites (white arrowheads in the merged image) surrounding a weakly CTSD-positive senile plaque (P). (B) Z-stack images of consecutive optical slices through the single profile indicated by the box in (A) confirms the typical appearance of a dystrophic neurite, which is shown to contain mainly autophagic vacuoles including LC3-positive AP (green), LC3- and CTSD-positive AL, (yellow-orange vesicles) and less frequent CTSD-positive-only lysosomes (red). Scale bar: 10 μm.

Figure 7.

Expression of cell-type markers in AD hippocampus shows alterations in neuronal and glial content. (A) Representative western blots illustrating neuronal ΤUBB3 and glial GFAP markers in AD and control hippocampus. (B) Relative quantification of markers in hippocampus indicates downregulation of neuronal-specific TUBB3 and upregulation of GFAP, consistent with neuronal cell loss and concomitant astrogliosis. Statistical significance denoted by asterisks with p-values shown determined by one-way ANOVA, Dunnett's test. *p ≤ 0.05; **p ≤ 0.01. Western blots cropped as shown for purposes of clarity.