Sébastien S. Hébert

Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression.

Although the role of APP and PSEN genes in genetic Alzheimers disease (AD) cases is well established, fairly little is known about the molecular mechanisms affecting Aβ generation in sporadic AD. Deficiency in Aβ clearance is certainly a possibility, but increased expression of proteins like APP or BACE1/β-secretase may also be associated with the disease. We therefore investigated changes in microRNA (miRNA) expression profiles of sporadic AD patients and found that several miRNAs potentially involved in the regulation of APP and BACE1 expression appeared to be decreased in diseased brain. We show here that miR-29a, -29b-1, and -9 can regulate BACE1 expression in vitro. The miR-29a/b-1 cluster was significantly (and AD-dementia-specific) decreased in AD patients displaying abnormally high BACE1 protein. Similar correlations between expression of this cluster and BACE1 were found during brain development and in primary neuronal cultures. Finally, we provide evidence for a potential causal relationship between miR-29a/b-1 expression and Aβ generation in a cell culture model. We propose that loss of specific miRNAs can contribute to increased BACE1 and Aβ levels in sporadic AD.

Alterations of the microRNA network cause neurodegenerative disease

Brain development crucially depends on the integrity of microRNA (miRNA) pathways, which function at the post-transcriptional level as a rheostat of the transcriptome and proteome of the cell. miRNAs are also involved in many other, more specific, aspects of neuronal function such as neurite outgrowth and synapse formation. Complete loss of miRNA expression in the brain leads to neurodegeneration in several animal models. Evidence from patient material is emerging that miRNA dysregulation could, indeed, contribute to neurodegenerative disorders. The translation of proteins previously implicated in familial forms of disease seems to be under control of miRNAs, and changes in miRNAs might explain how these proteins become affected in sporadic neurodegenerative disease. Thus, miRNAs are moving rapidly center stage as key regulators of neuronal development and function in addition to important contributors to neurodegenerative disorder.

Phosphorylation of the Translation Initiation Factor eIF2α Increases BACE1 Levels and Promotes Amyloidogenesis

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimers disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

MicroRNA regulation of Alzheimer's Amyloid precursor protein expression.

Gene dosage effects of Amyloid precursor protein (APP) can cause familial AD. Recent evidence suggest that microRNA (miRNA) pathways, implicated in gene transcriptional control, could be involved in the development of sporadic Alzheimers disease (AD). We therefore investigated whether miRNAs could participate in the regulation of APP gene expression. We show that miRNAs belonging to the miR-20a family (that is, miR-20a, miR-17-5p and miR-106b) could regulate APP expression in vitro and at the endogenous level in neuronal cell lines. A tight correlation between these miRNAs and APP was found during brain development and in differentiating neurons. We thus identify miRNAs as novel endogenous regulators of APP expression, suggesting that variations in miRNA expression could contribute to changes in APP expression in the brain during development and disease. This possibility is further corroborated by the observation that a statistically significant decrease in miR-106b expression was found in sporadic AD patients.

The amyloid-β precursor protein: integrating structure with biological function

Proteolytic processing of the amyloid‐β precursor protein (APP) generates the Aβ amyloid peptide of Alzheimers disease. The biological function of APP itself remains, however, unclear. In the current review, we study in detail the different subdomains of APP and try to assign functional significance to particular structures identified in the protein.

Genetic ablation of Dicer in adult forebrain neurons results in abnormal tau hyperphosphorylation and neurodegeneration

Type III RNase Dicer is responsible for the maturation and function of microRNA (miRNA) molecules in the cell. It is now well-documented that Dicer and the fine-tuning of the miRNA gene network are important for neuronal integrity. However, the underlying mechanisms involved in neuronal death, particularly in the adult brain, remain poorly defined. Here we show that the absence of Dicer in the adult forebrain is accompanied by a mixed neurodegenerative phenotype. Although neuronal loss is observed in the hippocampus, cellular shrinkage is predominant in the cortex. Interestingly, neuronal degeneration coincides with the hyperphosphorylation of endogenous tau at several epitopes previously associated with neurofibrillary pathology. Transcriptome analysis of enzymes involved in tau phosphorylation identified ERK1 as one of the candidate kinases responsible for this event in vivo. We further demonstrate that miRNAs belonging to the miR-15 family are potent regulators of ERK1 expression in mouse neuronal cells and co-expressed with ERK1/2 in vivo. Finally, we show that miR-15a is specifically downregulated in Alzheimers disease brain. In summary, these results support the hypothesis that changes in the miRNA network may contribute to a neurodegenerative phenotype by affecting tau phosphorylation.

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

γ‐Secretase‐dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase‐3β, Neprilysin and APP, we found no consistent effects of γ‐secretase inhibitors or of genetic deficiencies in the γ‐secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD‐binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Aβ generation in Alzheimers disease will not interfere significantly with the function of these genes.

Amyloid precursor protein promotes post-developmental neurite arborization in the Drosophila brain

The mechanisms regulating the outgrowth of neurites during development, as well as after injury, are key to the understanding of the wiring and functioning of the brain under normal and pathological conditions. The amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimers disease (AD). However, its physiological role in the central nervous system is not known. Many physical interactions between APP and intracellular signalling molecules have been described, but their functional relevance remains unclear. We show here that human APP and Drosophila APP‐Like (APPL) can induce postdevelopmental axonal arborization, which depends critically on a conserved motif in the C‐terminus and requires interaction with the Abelson (Abl) tyrosine kinase. Brain injury induces APPL upregulation in Drosophila neurons, correlating with increased post‐traumatic mortality in appld mutant flies. Finally, we also found interactions between APP and the JNK stress kinase cascade. Our findings suggest a role for APP in axonal outgrowth after traumatic brain injury.

Coordinated and widespread expression of γ-secretase in vivo: evidence for size and molecular heterogeneity

Gamma-secretase is a high molecular weight protein complex composed of four subunits, namely, presenilin (PS; 1 or 2), nicastrin, anterior pharynx defective-1 (Aph-1; A or B), and presenilin enhancer-2 (Pen-2), and is responsible for the cleavage of a number of type-1 transmembrane proteins. A fundamental question is whether different gamma-secretase complexes exist in vivo. We demonstrate here by in situ hybridization and by Northern and Western blotting that the gamma-secretase components are widely distributed in all tissues investigated. The expression of the different subunits seems tightly coregulated. However, some variation in the expression of the Aph-1 proteins is observed, Aph-1A being more general and abundantly distributed than Aph-1B. The previously uncharacterized rodent-specific Aph-1C mRNA is highly expressed in the kidney and testis but not in brain or other tissues, indicating some tissue specificity for the Aph-1 component of the gamma-secretase complex. Blue-native electrophoresis revealed size heterogeneity of the mature gamma-secretase complex in various tissues. Using co-immunoprecipitations and blue-native electrophoresis at endogenous protein levels, we find evidence that several independent gamma-secretase complexes can coexist in the same cell type. In conclusion, our results suggest that gamma-secretase is a heterogeneous family of protein complexes widely expressed in the adult organism.

Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription

A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target genes.