Introduction
The microRNA (miRNA)-guided RNA silencing
pathway is a gene regulatory process present in almost all eukaryotic cells and
based on small non-coding RNAs known as miRNAs [1]. Generated by the
ribonuclease III Dicer, miRNAs are key regulators of gene expression that act
mainly through recognition of specific binding sites generally located in the
3' untranslated region of specific messenger RNAs (mRNAs). Predicted to
regulate up to 90% of the genes in humans [2], miRNAs may control every
cellular processes in all cells and tissues of the human body! Required for the
fine tuning and tight regulation of cellular protein expression, a normal miRNA
function is critical for the maintenance of health and prevention of disease [3]. Deregulation of protein expression
induced by a dysfunctional miRNA-based regulatory system, which may be either
global or miRNA-specific in nature, may thus represent the main etiologic
factor underlying age-related diseases, such as Alzheimer's disease (AD) that
affects the brain (Provost, manuscript submitted).
MicroRNAs and Alzheimer's disease
AD is a slowly progressing, age-related neurodegenerative
disease that currently affects ~2% of the population in industrialized
countries and whose incidence is predicted to increase dramatically over the
next 40 years (http://www.alz.org) [4]. Affecting cholinergic neurons, AD is
characterized by the accumulation of plaques formed of short ß-amyloid (Aß)
peptides in the hippocampal region of the brain [5]. Aß peptides are produced
upon proteolytic cleavage of APP by ß-secretase, also known as ß-site
APP-cleaving enzyme 1 (BACE1), which contributes to the formation of these
plaques [6] (Provost, manuscript submitted).
Post-mortem analyses have revealed upregulation of
BACE1 expression at the protein, but not at the mRNA, level in brains from
patients suffering from AD, as compared to brains from unaffected patients [7],
consistent with an impaired control of BACE1 mRNA translation. In a recent study
from our laboratory, we reported similar observations in an animal model of AD
(APPSwe/PS1 mice) and demonstrated a role for two miRNAs, i.e. miR-298 and
miR-328, in the regulation of BACE1 expression, using mainly transiently
transfected murine neuronal N2a cells in culture [8]. In vivo, we observed
decreased expression levels of miR-298 and miR-328 in the hippocampus of aging
APPSwe/PS1 mice [8], which supports further the possibility that the loss of
miRNA regulation of BACE1 mRNA translation may lead to higher BACE1 protein
expression, an enhanced Aß formation and the development of AD.
Experimental
considerations
Whether these findings can be extrapolated and
transposed to human requires prudence and cautiousness, especially in the
context of multifactorial, age-related diseases like AD, which may result from
an intricate interplay of genetic and environmental factors. Several additional
issues warrant further considerations and need to be taken into account, or
addressed, in order to ascertain our interpretation of miRNA data and, most
importantly, the transposability of our findings to the aging human beings,
such as (i) the nature and source of the biological material under
investigation, (ii) the use and relevance of cellular models, (iii) the use of
primary versus cultured cells, and (iv) the other functions exerted by miRNAs.
Nature and source of the biological material under
investigation
The most obvious limitation here pertains
to the use of mice and the (non-)conservation of miRNA and BACE1 mRNA sequences
and function between species, as discussed previously [8]. In addition,
although very useful for the study of specific aspects of the disease, the
animal models of AD that are currently available, in which the disease is
caused by altering genes involved in Aß metabolism (eg, mutation of the
presenilin 1 gene combined with a chimeric mouse/human APP), only imperfectly
mimic and oversimplify a multifactorial disease as complex as AD. In addition,
whether the observed changes in miRNA levels in the aging AD brain are the
cause or a consequence of the disease (the chicken or the egg dilemma) remains
disputable.
Moreover, since the disease is induced
"artificially", and does not occur or progress "naturally" in these animals,
only the contributory, and not the possible causal, role of miRNAs in the
etiology and/or progression of AD can be investigated. For that purpose, the
targeted deletion or functional alteration of miRNA function, followed by
monitoring of cognitive functions, would be more appropriate.
Animal models may also be more suitable
and provide more insights into the pathogenesis of AD progression as compared
to humans, where brain tissues may only be obtained, and the data collected, at
the time of death, although harvesting brains from subjects of different ages
may partially circumvent this issue. In contrast to studies performed in mice,
results obtained from post-mortem human brain tissues may be markedly
influenced by the time interval between the patients' death and brain tissue
harvesting, due to the relatively short half-lives of some miRNAs (~1 to 3.5
h), as recently reported by Sethi and Lukiw [9].
Use and relevance of cellular models
It is important to underline the utility
of cellular models in complementing molecular, biochemical or animal studies.
As such, cultured neuronal N2a cells, which have been used to obtain most of
the experimental evidences pertaining to the miRNA repression of BACE1
expression [8], are highly relevant and represent the most practical cellular
system to study the molecular mechanisms underlying AD.
Whereas specific cell lines, such as 293 or
293T cells, are immortalized upon transformation with adeno-viruses and/or
simian virus 40 infection, the cell clone Neuro-2a (N2a) was established from a
spontaneous neuroblastoma isolated from the brain of a strain A albino mouse
(please refer to http://www.atcc.org). Cytogenetic analysis of these cells
revealed an unstable karyotype within a stemline range of 94 to 98 chromo-somes
(the cells contain 6 to 10 large chromosomes with median or submedian
centromeres and 2 to 4 minute chromosomes). Although N2a cells are of neuronal
origin, the endogenous biochemical processes in such cells, that may have
undergone tens to hundreds of passages at the time of harvesting, are expected
to differ quite markedly from that of primary neuronal cells, thereby limiting
the scope of the conclusions that can be reached from their use.
Use of primary versus cultured cells
The main issue here pertains to two fundamental
differences distinguishing primary cells or tissues from immortalized, cultured
cells: the genome integrity of the latter and their propensity for cell
division. The implication of these differences in the interpretation of miRNA
data, which was highlighted not too long ago [10], is major: Previously known
as repressors of mRNA translation, miRNAs have also been shown to enhance mRNA
translation upon cell cycle arrest [10]. Whether miRNAs function similarly in
cell cycle-arrested and primary, non-dividing cells remains to be established.
However, the experimental evidences are sufficiently strong to raise a
imperative issue as to whether primary, non-dividing cells, such as cholinergic
neurons, support miRNA repression and/or enhancement of mRNA translation. This
observation also imply that all the miRNA data obtained from cultured cells
should be intrepreted with great caution before transposing them to in vivo
situations, as miRNAs found to repress specific mRNAs in immortalized cultured
cells may exert the exact opposite effects in primary cells or tissues in vivo.
Hence to need to obtain data or additional evidences from primary cells that
either support or challenge our in vitro miRNA data.
Other functions exerted by microRNAs
A recent study by Eiring et al. [11] revealed another
mean by which miRNAs may enhance gene expression. Reported to regulate BACE1
mRNA translation in the context of AD [8], miR-328 has been shown to have a
second function, acting as an RNA decoy by binding to heterogeneous nuclear
ribonucleoprotein E2 and lifting its translational repression of an mRNA
involved in myeloid cell differentiation [11,12]. Therefore, any decrease or
increase in miRNA levels may not yield the expected relief or accentuated
repression of gene expression, respectively. This phenomenon may also explain,
at least in some cases, the lack of any phenotypic changes associated to
specific or global miRNA variations, both in terms of levels and mode of
action, the cumulative effects of which may cancel each other out.
Another level of complexity may be
conferred by the ability of miRNAs to regulate multiple mRNA targets, to exert
indirect effects and to be involved in more complex networks of regulatory
mediators of importance in the pathogenesis of age-related diseases like AD.
Conclusion
and perspectives
Recently, the major research advances
pertaining to the possible role and function of miRNAs in neurodegenerative
diseases, such as AD, has provided novel perspectives to the pathogenesis of
increasingly prevalent, age-related diseases in human. However, further
investigations are required in order to improve our understanding of the
changes that may occur in miRNA biogenesis, metabolism and/or function during
aging, which may be an important contributor to the etiology and progression of
age-related diseases. In this regard, several issues related to the impact of
aging and/or cell division on the integrity and functionality of the miRNA
pathway remain to be explored and might add an additional layer of complexity
to miRNA studies involving mRNA translational regulation: Is the biogenesis
and/or function of miRNAs modified as the cells are dividing, or altered by
age-related processes? Are miRNA genes shut off or turned on, either
specifically or globally, during aging? Do cells acquire or lose functional
miRNAs as they age? As they divide? In that context, prudence and cautiousness
should guide us when interpreting and extrapolating experimental findings
related to miRNAs to a human disease in particular. In AD, for instance,
primary human brain tissues obtained upon death of AD and non-AD patients remain
the most relevant source of biological materials in order to get further
insights into the pathogenesis of AD. However, the utility, versatility and
complementarity of animal and cell culture models, albeit imperfect and coming
with their pros and cons, cannot be ignored, as the insights they provide
simply need to be considered into their proper context with their own
limitations and promises.
P.P. is a Senior Scholar from the Fonds de
la Recherche en Santé du Québec (FRSQ). This work was supported by a Young
Investigator Award from NARSAD, the World's leading charity dedicated to mental
health research, and a Grant from Health Canada/Canadian Institutes of Health
Research (CIHR) (HOP-83069).
The author of this manuscript has no
conflict of interest to declare.