MicroRNAs (miRNAs), ~22 nt single-stranded non-coding RNAs (ncRNAs) abundant in the
human brain and retina, have emerged as significant post-transcriptional regulators
of messenger RNA (mRNA) abundance and complexity in the human central nervous system
(CNS) in aging, health, and disease. Of the 2050 different miRNAs in the human body
so far identified, only about 25–30 are abundant in either the brain or the retina,
underscoring the high selection pressure carried by RNA sequences located within these
select ncRNAs (1–7). It is noteworthy to point out that: (i) that brain neocortex
and retina share a common neuroectodermal origin; (ii) that brain and retina share
a subfamily of specific miRNA species; and (iii) that the multilayered assemblies
of both neural and retinal cells are targeted by pathogenic processes that drive progressive
pro-inflammatory neurodegeneration (5–9). Indeed, pathologically up-regulated miRNAs
common to both the prototypic age-related inflammatory degeneration of the brain in
Alzheimer’s disease (AD) and of the retina in age-related macular degeneration (AMD)
appear to be associated with deficits in the expression of messenger RNA (mRNA) and
gene families involved in the innate-immune response, inflammation, neurotrophism,
synaptogenesis, and amyloidogenesis (Figure 1). In this “Opinion” paper for the Frontiers
in Neurology Special Research Topic, we will highlight some of the most recent work
in this research area, with emphasis on a family of five up-regulated pro-inflammatory
miRNAs – miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a, and miRNA-155 – that are emerging
as key mechanistic contributors to the AD and AMD process.
Figure 1
(A) Color-coded cluster analysis of significantly up-regulated microRNAs (miRNAs)
in the neocortex of AD (N = 5) versus age-matched controls (N = 5) and (B) in the
whole retina of AMD (N = 5) versus age-matched controls (N = 5); this small family
of “pro-inflammatory” miRNAs consisting of miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a,
and miRNA-155 are often found to be up-regulated approximately twofold or more over
controls (small red arrows); interestingly these miRNAs are inducible and under transcriptional
control by NF-kB [Figure 1 adapted from Ref. (10–13)]; the relative expression levels
for two sequence-related microRNAs, miRNA-146a and miRNA-155 the brain neocortex and
retina are shown in (C) relative to levels for an unchanging brain and retinal control
miRNA-183, which was set to 1.0 and marked by a dashed horizontal line; another abundant
control signal is 5SRNA with a relative signal strength of ~5.0 (shown at ~1/20th
of its actual abundance in the brain and retina; see text) (12, 13). In these samples,
all control and AD neocortical samples were obtained from the superior temporal neocortex
(Brodmann area A22); control and AMD retinal samples were obtained from whole retina;
all control, AD and AMD samples had post-mortem intervals (PMI; death to brain freezing
interval) of 2 h or less (2, 9, 10). Controls were age-matched to moderate-to-late
stages of AD or AMD; increases in specific miRNAs increased as disease stage advanced
[Ref. (11, 12, 14–17); data not shown]; further details on the pathology of these
samples have been recently published (11–20). There were no significant differences
in age, PMI, or RNA yield or quality between either the brain or the retinal tissues.
Of the 12 different homo sapien micro-RNAs (hsa-miRNAs) shown, miRNA-146a and miRNA-155
exhibited the most consistent up-regulation compared with age-matched controls (*p < 0.05;
**p < 0.01, ANOVA); (D) the 3′UTR of the mRNA of complement factor H (CFH); a major
regulator of the innate-immune and inflammatory response, see text; [(21, 22)] is
a prime example of brain and retinal gene expression regulation by multiple and common
miRNAs – miRNA-146a and miRNA-155; (D) shows the complementarity map between miRNA-146a
or miRNA-155 and part of the 232 nt CFH 3′UTR sequence. Overlapping miRNA-146a and
miRNA-155 high-affinity binding sites in the CFH mRNA 3′UTR (each has an energy of
association of less than −22 kcal/mol) that defines an exceptionally stable miRNA–mRNA
interaction and a potentially common CFH mRNA 3′-UTR miRNA regulatory control region
5′-TTTAGTATTAA-3′ (overlaid in green; see text) (12, 13, 23); we cannot exclude the
participation of other human brain- or retina-enriched miRNAs or other small ncRNAs
which may additionally contribute to the neuropathological mechanisms of AD or AMD
pathology; (E) taken together, these recent findings in part define a highly interactive
network of NF-kB-sensitive, up-regulated miRNAs in diseased brain and retina that
can explain much of the observed pathology associated with AD and AMD. The CNS-abundant,
miRNA-125b is a central member of this up-regulated miRNA group that may be in part
responsible for driving deficits in phagocytosis (triggering receptor expressed in
microglial cells; TREM2), innate-immune signaling and chronic inflammation (IkBKG,
CFH), impairments in neurotransmitter packaging and release (synapsin-2; SYN-2), and
neurotrophism (15-lipoxygenase, vitamin D receptor; 15-LOX, VDR). Other NF-kB-sensitive
up-regulated miRNAs (such as miRNA-146a) appear to be responsible for the observed
deficits in NF-kB regulation (IRAK-1, IRAK-2) and/or amyloidogenesis (tertraspanin
12; TSPAN12); these up-regulated miRNAs and down-regulated mRNAs form a highly integrated,
self-perpetuating pathogenic miRNA-mRNA signaling network due to chronic re-activation
of NF-kB stimulation perhaps through the involvement of deficits in IkBKG signaling
(10–13). Inhibition of the NF-kB initiator or individual blocking of the pathogenic
induction of these five miRNAs may provide novel therapeutic benefit for the clinical
management of AD or AMD, however what NF-kB or miRNA inhibition strategies and/or
protocols, or whether they can be utilized either alone or in combination, remain
open to investigation (17, 19, 20, 24, 25). Extensive recent data in human brain cells
in primary culture have indicated that these approaches may neutralize this chronic,
inducible, progressive pathogenic gene expression program to re-establish brain and
retinal cell homeostasis, and ultimately be of novel pharmacological use in the clinical
management of AD and/or AMD (19, 20, 26–30).
Homeostatic levels of specific miRNAs are natural indicators of neurological health
of both the brain and retina (2–10, 31). Recently, multiple independent neurological
research laboratories have provided evidence for the up-regulation of a small group
of five inducible miRNAs in age-related diseases involving a progressive inflammatory
degeneration. That these five miRNAs – miRNA-9, miRNA-34a, miRNA-125b, miRNA-146a,
and miRNA-155 – are up-regulated in both AD and AMD underscores the concept that the
brain and retina share common pathological signaling of a pre-existing subfamily of
miRNAs that individually contribute to various aspects of neurodegenerative disease
(5–12, 31–35). Accumulating evidence, including very recent research findings over
the last 6 months indicate that each of these miRNAs share the following six features:
(i) that they are basally expressed in control brain neocortex and retina (2–9); (ii)
that in vitro they can be induced by a wide range of environmental- and inflammation-linked
physiological stressors, including pro-inflammatory cytokines, amyloid beta (Aβ42)
peptides, neurotoxic metal sulfates, and neurotropic viruses such as herpes simplex
virus-1 (HSV-1) (12, 16, 17, 32–35); (iii) that this group of five pro-inflammatory
miRNAs are over-expressed at least twofold in stressed brain or retinal cells and
in AD or AMD affected tissues (14, 15, 32); (iv) that together, via down-regulation
of multiple mRNA targets (and hence deficits in the expression of genes encoded by
those mRNAs) they regulate various pathophysiological features characteristic of AD
and AMD, including impairments in phagocytosis, synaptogenesis, neurotrophism, NF-kB
signaling and stimulation of progressive inflammation and amyloidogenesis (Figure
1) (7, 12, 13, 23, 26–28, 36); (v) that all five of these pro-inflammatory miRNAs
are under transcriptional control by NF-kB (chiefly the heterotypic p50/p65 dimer)
in human primary neuronal-glial co-cultures, AD and AMD tissues (7, 11–13, 23, 26–28,
36, 37); and (vi) that both NF-kB inhibitors and anti-microRNAs (anti-miRs) effectively
knock down their expression in human brain and retinal cell culture experiments, and
may ultimately be of use therapeutically in the clinical management of AD or AMD (17,
18, 26–29).
Much of the recent research work emphasizing this commonality of the same miRNAs in
basic pathological processes involving brain and retinal degeneration, as exemplified
by miRNA profiling in AD, AMD, and transgenic AD or AMD (TgAD, TgAMD) models, has
been summarized in Figure 1 (5–10, 12, 14, 17, 25, 31–35). First, when compared to
the unchanging 22 nt miRNA-183 and the 120 nt 5S ribosomal RNA (5S rRNA; 5SRNA) control
markers, the five member pro-inflammatory microRNAs miRNA-9, miRNA-34a, miRNA-125b,
miRNA-146a, and miRNA-155 are found to be amongst the most consistently up-regulated
miRNAs in both degenerating human brain neocortex (Figure 1A) and retina (Figure 1B).
Of this group of five pro-inflammatory microRNAs, miRNA-146a and miRNA-155 are typically
found to be increased ~2.5- to 3.3-fold over age-matched controls (Figure 1C). To
add another layer of genetic complexity for post-transcriptional regulation, both
miRNA-146a and miRNA-155 recognize an overlapping 3′ untranslated region (3′UTR) of
the complement factor H (CFH) mRNA (highlighted in green; CFH loss-of-function mutations
or CFH expression deficits are associated with both AD and AMD; see below; Figure
1D). Indeed, the up-regulation of these same five pro-inflammatory miRNAs (yellow
ovals in Figure 1E) appear to form a highly interactive miRNA–mRNA network that can
in part explain the down-regulation of specific brain and retinal genes (black rectangles)
involved in phagocytosis, inflammation, synaptogenesis, neurotrophism, NF-kB signaling,
and amyloidogenesis (Figure 1E; see also the legend to Figure 1 wherein the details
of this highly interactive network are further described).
Alterations in innate-immune signaling are a consistent feature of both AD and AMD
(4, 5, 9, 15). The highly soluble, hydrophilic 155-kDa glycoprotein CFH is one very
illustrative example of an innate-immune repressor and complement control protein
whose abundance and/or activity is significantly down-regulated in both AD and AMD
[(9, 15, 21, 22, 35); see Figure 1D]. CFH (chr 1q32; also known as AC3bINA, adrenomedullin
binding protein-1, AM binding protein-1 factor H, β1H globulin, H factor, and H factor-1)
is an important member of the regulator of complement activation (RCA) group of proteins
encoded within the RCA gene cluster and normally performs a systemic sentinel function
against unscheduled or spontaneous immune system activation (9, 15). CFH mRNA abundance
is down-regulated in AD and/or AMD by a miRNA-146a- and/or miRNA-155–CFH–3′UTR-based
complementarity mechanism and/or by a Y402H loss-of-function mutation (15, 21, 22).
Hence an insufficiency in a homeostatic amount of functioning CFH (as down-regulated
by miRNA-146a and miRNA-155) may have the same end result as the loss-of-function
Y402H mutation in CFH (21, 22). It is important to note that CFH mRNA and hence CFH
gene expression appears to be down-regulated by at least two different miRNAs – miRNA-146a
and/or miRNA-155 – and their differential recognition of overlapping binding sites
in the human CFH mRNA 3′UTR may be dependent on yet-to-be-defined genetic factors
and mechanisms characteristic of individual brain or retinal cells [Figure 1D; (9,
15, 21, 22, 35)].
In summary, it is our opinion that in miRNA research in human degenerative diseases
including AD and AMD, several critical concerns have surfaced: (i) that brain and
retinal miRNAs typically possess limited stabilities, however miRNA half-lives can
be considerably extended via their sequestration into exosomes or the use of other
protective strategies such as adsorption or tertiary folding into RNAse-resistant
structures that may escape initial miRNA detection using traditional methods (17,
18, 23–25); (ii) that accurate quantification of miRNAs is technically feasible although
it still remains challenging due to the small size of mature miRNA isoforms, adsorption
to “inert” surfaces, high sequence homology amongst individual miRNAs, 5′ and 3′ end
polymorphisms, spatial-temporal expression patterns and high dynamic range of miRNA
expression (13, 17, 18, 24); (iii) that miRNA profiling in different AD or AMD studies
suffers from a poor consensus regarding their abundance and complexity; the latter
a very recently acknowledged concern in the field (4–7, 14, 17); and (iv) discrepancies
of miRNA abundances in anatomical areas sampled, variations in patient drug history,
the PMI of the AD and AMD patients and other factors. Together these constitute practical
methodological challenges, especially in the realm of useful biomarkers and diagnostics
for AD or AMD detection (3, 6, 17, 25, 34). Despite these recent concerns data has
begun to filter through on the involvement of distinct miRNA families and miRNA–mRNA
signaling networks linked to innate-immune system alterations, inflammatory, neurotrophic,
and amyloidogenic consequences in AD and AMD. These have steadily yielded a deeper
appreciation into the onset and propagation of complex miRNA–mRNA-modulated biological
networks that directly underlie the pathogenesis of AD and AMD. Lastly, miRNAs are
highly soluble and mobile, and are able to transverse plasma membranes either freely,
adsorbed to carrier molecules or contained within exosomes (17, 19, 23, 25). That
AD and AMD are both progressive “propagating” disease entities suggest a potential
“spreading factor” role for selective miRNAs in the cognitive and visual circuitry,
an evolving research area in which specific combinations of miRNAs may be playing
hitherto unrecognized pathogenic roles.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.