microRNA-34a (miRNA-34a) Mediated Down-Regulation of the Post-synaptic Cytoskeletal Element SHANK3 in Sporadic Alzheimer's Disease (AD)

Abstract

Integrating a combination of bioinformatics, microRNA microfluidic arrays, ELISA analysis, LED Northern, and transfection-luciferase reporter assay data using human neuronal-glial (HNG) cells in primary culture we have discovered a set of up-regulated microRNAs (miRNAs) linked to a small family of down-regulated messenger RNAs (mRNAs) within the superior temporal lobe neocortex (Brodmann A22) of sporadic Alzheimer's disease (AD) brain. At the level of mRNA abundance, the expression of a significant number of human brain genes found to be down-regulated in sporadic AD neocortex appears to be due to the increased abundance of a several brain-abundant inducible miRNAs. These up-regulated miRNAs-including, prominently, miRNA-34a-have complimentary RNA sequences in the 3' untranslated-region (3'-UTR) of their target-mRNAs that results in the pathological down-regulation in the expression of important brain genes. An up-regulated microRNA-34a, already implicated in age-related inflammatory-neurodegeneration-appears to down-regulate key mRNA targets involved in synaptogenesis and synaptic-structure, distinguishing neuronal deficits associated with AD neuropathology. One significantly down-regulated post-synaptic element in AD is the proline-rich SH3 and multiple-ankyrin-repeat domain SHANK3 protein. Bioinformatics, microRNA array analysis and SHANK3-mRNA-3'UTR luciferase-reporter assay confirmed the importance of miRNA-34a in the regulation of SHANK3 expression in HNG cells. This paper reports on recent studies of a miRNA-34a-up-regulation coupled to SHANK3 mRNA down-regulation in sporadic AD superior-temporal lobe compared to age-matched controls. These findings further support our hypothesis of an altered miRNA-mRNA coupled signaling network in AD, much of which is supported, and here reviewed, by recently reported experimental-findings in the scientific literature.

Keywords: Alzheimer's disease (AD); SHANK3 protein; miRNA-34a; neurotransmission; post-synaptic density proteins; superior temporal lobe neocortex (Brodmann A22); synaptic structure in disease; synaptic transmission.

Figure 1

Gene products on human chromosomes 1 and 22 interactively contribute to SHANK3 expression in CNS tissues. (A) Results of miRNA microfluidic array analysis; miRNA-34a is significantly up-regulated in the sporadic AD temporal lobe compared to miRNA-183 and 5S RNA control sncRNA markers; the numbers 1–3 indicates 3 separate control and age-matched AD cases; all female; control mean age 72.1 ± 6.6 years; AD mean age 73.8 ± 8.2 years; N = 3 control and 3 AD: all post mortem intervals <3 h; (B) quantitation of miRNA-34a levels in bar graph format; (C) quantitation of SHANK3 mRNA (using Northern analysis) and SHANK3 protein (using ELISA) in the superior temporal lobe of control and AD as previously described (9, 23); N = 3; *p < 0.001 (ANOVA). (D–G) highly schematicized depiction of the human SHANK3 gene organization at chr 22q13.33 and the human miRNA-34a gene organization at chr 1p36.22; with a P_CT (probability of conserved targeting) or Friedman score of 83 [(24); that has been calculated for all highly conserved miRNA families]; this hsa-miRNA-34a-SHANK3-3′-UTR recognition/interaction is highly favorable and almost certain to occur in the cytoplasm/nucleoplasm of CNS cells; (E) has been modified from (25); the homo sapien (hsa) microRNA-34a (hsa-miRNA-34a) is encoded from the distal end of human chromosome 1p (at chr 1p36.22) and generates a 22 nt mature miRNA-34a species; hence the expression of at least two genes, one cytoskeletal and structural (SHANK3) and one regulatory (miRNA-34a) on human chromosomes 1p and 22q is required for regulating the expression of SHANK3.

Figure 2

Luciferase reporter vector-based studies of miRNA-34a and SHANK3 expression - Functional validation of a miRNA-34a-SHANK3–3′UTR interaction. (A) partial ribonucleotide sequence of the 1986 nt SHANK3-mRNA-3′-UTR is shown in the 5′-3′ direction; the 22 nucleotide (nt) miRNA-34a-SHANK3-3′UTR complementarity-interaction region is indicated by a black underline and the 8 nt SHANK3-mRNA-3′-UTR seed sequence is overlaid in yellow; a single vertical red arrow indicates the 5′ end of a poly A+ tail in the SHANK3 mRNA; the SHANK3 mRNA sequence derived from NM_018965; (B) SHANK3-mRNA-3′UTR expression vector luciferase reporter assay (pLightSwitch-3′UTR; Cat#S801178; Switchgear Genomics, Palo Alto CA); in this vector, the entire 1986 nucleotide SHANK3 3′UTR was ligated into the unique Nhe1-Xho1site; not drawn to scale; (C) HNG cells, 2 weeks in primary culture; neurons (red stain; λ_max = 690 nm), DAPI (blue nuclear stain; λ_max = 470 nm) and glial fibrillary associated protein (GFAP; glial-specific green stain; λ_max = 520 nm); the HNG cell culture is about 60% confluent and at 2 weeks of culture contains 70% neurons and 30% astroglia; human neurons do not culture well in the absence of glia; neurons also show both extensive arborization and display electrical activity (unpublished; Lonza); 40X magnification; HNG cells were transfected with the SHANK3-mRNA-3′UTR expression vector luciferase reporter were treated exogenously with a stabilized miRNA-34a, a scrambled control miRNA-34a (miRNA-34a-sc) or control miRNA-183; see references and text for further details; (D) compared to control, HNG cells transfected with a scrambled (sc) control pLightSwitch-3'UTR vector, the SHANK3-mRNA-3′UTR vector exhibited decreased luciferase signal to a mean of 0.16-fold of controls in the presence of miRNA-34a; this same vector exhibited no change in the presence of the control miRNA-34a-sc or miRNA-183; for each experiment (using different batches of HNG cells) a control luciferase signal was generated and included separate controls with each analysis; in addition a control vector β-actin-3′UTR showed no significant effects on the relative luciferase signal yield after treatment with either miRNA-183 or miRNA-34a (data not shown); a dashed horizontal line set to 1.0 is included for ease of comparison; N = 6; *p < 0.001 (ANOVA). The results suggest a physiologically relevant miRNA-34a-SHANK3-mRNA-3′UTR interaction and a miRNA-34a-mediated down-regulation of SHANK3 expression in HNG cells. This pathogenic interaction may be related to the down-regulation of other immune, inflammatory, and synaptic system genes by up-regulated miRNAs in the CNS resulting in an impairment in trans-synaptic signaling and synaptic cytoarchitecture.