Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression - PubMed (original) (raw)

Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression

Stephen D Ginsberg et al. Biol Psychiatry. 2010.

Abstract

Background: Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimer's disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis.

Methods: Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection was performed using postmortem brain tissue. Validation was achieved using real-time quantitative polymerase chain reaction and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via small interference RNA.

Results: Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively upregulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal quantitative polymerase chain reaction and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the downregulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB downregulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD.

Conclusions: These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long-term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression.

Copyright © 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

TC RNA amplification procedure and LCM of CA1 hippocampal neurons. A. Schematic overview of the TC RNA amplification procedure. mRNA extracted from LCM captured cells (green line) and the TC primer serve as templates for the first strand synthesis with poly d(T) acting as a primer. First strand cDNA consists of three portions, the 5′ end comprised of the poly d(T), the mRNA complementary portion in the middle (purple line), and the 3′ end is comprised of the TC primer complementary to the cDNA (denoted as TC′). The TC′ portion hybridizes with the TC primer present in the reaction and forms a double stranded region which provides a functional RNA synthesis promoter for in vitro transcription and robust RNA amplification. B. Neurofilament-immunoreactive CA1 neurons in the hippocampal pyramidal cell layer from a subject with AD prior to LCM. Tissue sections were dehydrated but not coverslipped to enable proper execution of the LCM process. Scale bar: 50 mm. C. Slightly larger image in B following LCM of an individual CA1 neuron. Scale bar: 50 mm.

Figure 2

Figure 2

Differential regulation of select rab GTPases and neurotrophin receptors during the progression of AD. Boxplots (NCI, green; MCI, blue; AD, red) indicating relative expression levels of select rab GTPases and neurotrophin receptors. Significant up regulation of rab4, rab5, and rab7 is depicted along with significant down regulation of rab3, TrkB, and TrkC.

Figure 3

Figure 3

Association between select rab GTPase and neurotrophin receptor gene expression levels within CA1 neurons and antemortem cognitive measures in the same subjects. Scatterplots illustrate the relationship of gene expression levels and MMSE (left panel) and GCS scores (right panel) from individuals classified as NCI (green squares), MCI (blue triangles), and AD (red circles). A. A highly significant negative association exists with rab4 and MMSE (p < 0.0001) and GCS performance (p < 0.0002). B. A highly significant negative association exists with rab5 and MMSE (p < 0.001) and GCS performance (p < 0.001). C. A significant negative association exists with rab7 and MMSE (p < 0.01) and GCS performance (p < 0.001). D. rab24 expression levels are inversely correlated with MMSE (p < 0.0004) and GCS (p < 0.0002) scores. E. A significant association between rab3 gene expression down regulation and poor MMSE (p < 0.002) and GCS (p < 0.005) performance was observed. F. Down regulation of TrkB expression correlated with MMSE (p < 0.02) and GCS (p < 0.0001) performance. G. Down regulation of TrkC expression correlated with MMSE (p < 0.003) and GCS (p < 0.0004) performance.

Figure 4

Figure 4

Histograms showing expression of TrkB and EGFR levels in fibroblasts infected with rab5 viral vector constructs and expression of select rab GTPases following TrkB siRNA in cultured human fibroblasts. A. Upper left panel; down regulation of TrkB-FL is observed in fibroblasts infected with rab5wt (single asterisk denotes p < 0.005) and the constitutively active construct rab5Q79L (asterisk) compared to mock transfected (sucrose) control (LacZ) transfected cells. TrkB-FL down regulation was also found in the rab5wt and rabQ79L constructs compared to the dominant negative construct rab5S34N (double asterisk denotes p < 0.01). Upper right panel; down regulation of TrkB-T1 is seen in fibroblasts infected with rab5wt (single asterisk denotes p < 0.007) and the constitutively active construct rab5Q79L (asterisk) compared to mock transfected (sucrose) control (LacZ) transfected cells. Similar to TrkB-FL, TrkB-T1 down regulation was observed in the rab5wt and rabQ79L constructs compared to rab5S34N (double asterisk denotes p < 0.02). Lower left panel; down regulation of TrkB is observed via qPCR in fibroblasts infected with rab5wt and rab5Q79L (single asterisk denotes p < 0.001) compared to control (LacZ) infected cells and to the dominant negative rab5S34N construct (double asterisk denotes p < 0.04), suggesting that rab5 overexpression regulates TrkB at the transcriptional level. Lower right panel; in contrast to differential TrkB expression levels, no significant differences in related EGFR expression were observed with any of the fibroblasts infected with rab5 constructs or controls. TrkB and EGFR levels were normalized to TUBB ± SD for quantitative immunoblot analysis. B. qPCR analysis following TrkB siRNA (Silencer 753) indicated significant knockdown of TrkB expression {34.4% ± 0.9 (SEM); p < 0.001; asterisk} compared to vector treated cells. In contrast, no significant regulation of Gapdh (118.8% ± 7.8), rab4 (86.4% ± 7.8), rab5 (98.5% ± 11.5), or rab7 (114.9% ± 9.3) levels was observed following TrkB siRNA. C. Upper panel; immunoblot analysis demonstrated significant knockdown of TrkB expression (56.4% ± 4.1 (SEM); p < 0.005; asterisk), but not rab5 expression (95.0% ± 3.0), following TrkB siRNA. Lower panel; representative immunoblots demonstrating knockdown of TrkB expression with no significant alterations in rab5 or GAPDH expression.

Figure 5

Figure 5

Schematic diagram illustrating gene expression level changes within components of the endosomal-lysosomal system and neurotrophins at the cell surface and within signaling endosomes of vulnerable CA1 pyramidal neurons in MCI and AD. Genes demonstrating up regulation are denoted with a plus sign (one plus sign for ‘late’ changes observed only in AD and two plus signs indicating ‘early’ up regulation seen in MCI and AD). Genes demonstrating down regulation are denoted with a minus sign (one minus sign for ‘late’ changes observed only in AD and two minus signs indicating ‘early’ up regulation seen in MCI and AD).

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