Huntington's disease (HD) is a monogenic autosomal dominant, fatal disorder due to
CAG trinucleotide expansion in exon 1 of the HD gene (HTT) (The Huntington's Disease
Collaborative Research Group, 1993; Figure 1A). Nowadays, there is no cure or effective
treatment for the disease which presents with motor, cognitive and psychiatric dysfunction.
Although typically conceived as a “neurodegenerative disease,” mainly affecting the
GABAergic medium-sized spiny neurons (MSN) of the striatum and deep layers of the
cortex (Rosas et al., 2003), an increasing body of evidence has surfaced indicating
that abnormal neurodevelopment might also have a crucial role in HD (Mehler and Gokhan,
2000, 2001; Humbert, 2010). Neurodegenerative diseases have been classically defined
as chronic and progressive disorders of the nervous system affecting neurologic and
behavioral functions, which start with specific biochemical changes that ultimately
lead to distinct histopathologic and clinical syndromes. On the contrary, neurodevelopmental
disorders result from an anomaly of brain maturation, during fetal or early postnatal
life, which is postulated to alter the structure and/or function of neuronal and synaptic
populations (Harrison, 1995).
Figure 1
CAG expansion in the human
HTT
gene leads to Huntington's Disease (HD): possible contribution of altered non-coding
RNA transcription and aberrant developmental processes are described. (A) Top pie
charts report the proportion of coding and noncoding RNAs in the human genome (Alexander
et al., 2010). Highlighted in the coding portion of the genome, the Huntington's Disease
gene (HTT). Wild type or expanded CAG lengths are indicated by sepia and gray colors,
respectively. Bottom pie charts summarize the relative contribution of single classes
of non-coding RNAs (Baker, 2011) to normal (sepia) or diseased (gray) organism physiology.
The contribution of non-coding RNA transcription to HD process is still not fully
dissected. (B) Schematic representation of human development at different stages [blastocyst
(5 days) and 25 days embryos, 100 days and 5 months fetuses and adult organism] is
shown. Normal (sepia) and mutant huntingtin [expanded HTT gene] (grayscale) expression
is found in the whole organism at any given developmental stage (MacDonald et al.,
2003; Gusella and MacDonald, 2006). Molecular mechanisms (miRNAs, lncRNAs, alterative
splicing, histone modifications and chromatin remodeling) acting since conception
in the organism bearing the mutation are represented by the gray area. (C) A simplified
representation of HD brain development is shown. Developmental stages as in (B). Different
color's shades denote specific brain regions such as telencephalon, diencephalon,
midbrain, and hindbrain which develop differently to originate adult brain structures.
Light gray lines across the head designate coronal sections unveiling inner brain
organization, while puzzle pieces represent various cell types constituting each brain
district. It is evident that the presence of mutant huntingtin (expanded HTT gene)
places any cell of the organism—and of the brain in particular—in a “different biological
state” with subtle cellular and tissue development alterations that culminate in the
overt symptoms appearing in adult life. The changes induced by the expression of CAG-repeat
HTT expansion compromise MSN-generating stem cells specification and prime MSN to
adult neuronal degeneration (Molero et al., 2009; Humbert, 2010).
This opinion piece discusses the existing evidences from HD patients and HD model
systems for a neurodevelopmental component to the neurodegenerative processes of Huntington's
Disease pathophysiology.
The first and most striking observations come from multi-national neuropathological
and neuroimaging studies of prodromal HD, reporting clear brain changes decades prior
to onset of disease: specifically, smaller head circumference (Lee et al., 2012),
caudate and putamen atrophy (Nopoulos et al., 2007; Paulsen et al., 2010), striatal
and cortical white matter abnormalities including increased density of oligodendrocytes
(Gómez-Tortosa et al., 2001) and cortical thinning (Nopoulos et al., 2007) were described.
Moreover, subtle but significant defects in cognitive (Solomon et al., 2007), behavioral
(Duff et al., 2007), and motor function (Hinton et al., 2007) have been identified
in prodromal HD subjects long before a clinical diagnosis. While these observations
might be linked to early neuronal degeneration, the other plausible interpretation
is that they represent manifestations of subtle altered development that prime specific
areas and neuronal subpopulations of the brain to later catastrophic consequences
(Figure 1).
Further indications come from studies of mouse models with inactivation of the HD
orthologue gene Htt. Huntingtin protein, in fact, is expressed in all cell types of
the body, at all developmental stages and it has crucial roles during development
and neurogenesis (Figure 1B). Parallel studies have shown that complete lack of huntingtin
in mice results in embryonic lethality with developmental arrest just post gastrulation
(Duyao et al., 1995; Zeitlin et al., 1995), while severe reduction of huntingtin levels
in heterozygous, Htt hypomorphic mice or Htt conditional knock-out neuronal subpopulations
manifests in abnormal brain development, cognitive and motor abnormalities (Nasir
et al., 1995; White et al., 1997; Auerbach et al., 2001; Godin et al., 2010). Given
this key role of wild-type huntingtin during development, it is not surprising that
studies on genetic engineered mice expressing Htt CAG-expansion pathogenic mutations
show subtle molecular and structural deficits that portend altered developmental processes
and precede the occurrence of neurological symptoms and signs of cell death (Luthi-Carter
et al., 2000; Wheeler et al., 2000; Fossale et al., 2011). Specifically, even in Hdh
knock-in mice, representing a faithful genetic replica of the human HD mutation [expressing
full-length mutant huntingtin at endogenous levels from the native promoter at the
Htt locus], although usually presenting milder molecular and behavioral phenotypes
compared to HD transgenic and fragment lines, one single copy of the mutant Htt allele
is sufficient to cause nuclear accumulation of full-length mutant huntingtin in MSN
as early as 3 weeks of age (Wheeler et al., 2000), molecular changes in pathways related
to energy metabolism and cAMP levels, RNA metabolism (Gines et al., 2003) and measurable
transcription/translation dysfunctions (Fossale et al., 2011) at 3 and 10 weeks of
age, well before the onset of any overt pathological alteration. Strikingly, in the
same Hdh Q111 knock-in HD mouse model, deregulation of the temporal and spatial profiles
of striatal neurogenesis [delayed cell cycle exit and transient aberrant expression
of pluripotency markers in MSN-generating neural stem cells] (Molero et al., 2009)
may expose striatal precursors to inappropriate molecular cues driving them into aberrant
programs of neuronal differentiation, thus reinforcing the notion that altered neurodevelopment
might forecast later MSN susceptibility in HD (Figure 1C).
Additional consistent observations emerged from studies using mouse embryonic stem
cells and neural committed progenitors bearing Htt CAG-expansion mutations (Jacobsen
et al., 2011; Conforti et al., 2013; Biagioli et al., 2015) as well as patient-derived
induced pluripotent stem cells (iPSCs) (The HD iPSC Consortium, 2012), showing measurable
molecular differences and a series of expanded CAG-associated phenotypes that point
toward a central role of wild-type and mutant huntingtin in signal transduction, axonal
guidance, synaptic transmission and neurodevelopment. Specifically, unbiased “- omics”
analyses, probing transcriptomic, chromatin modifications and DNA methylation status
in these cells and their neuronal derivatives, support the hypothesis that wild-type
and mutant huntingtin might affect key chromatin regulators such as DNA and histone
methyl transferases, and demethylases [Polycomb Repressive Complex 2 (PRC2), Mixed
Lineage Leukemia 1–4, JARID1, REST, HYPB-SETD2] (Lee et al., 2013; Ng et al., 2013;
Biagioli et al., 2015). In fact, a growing body of evidence suggests that alterations
of epigenetic modifications constitute a basic molecular mechanism caused by the HD
mutation and responsible for early features of the pathological process. Strikingly,
most of these epigenetic regulators, i.e., Polycomb Complexes, but also other histone
post-transcriptional modifications enzymes, have important roles during transition
from pluripotency to differentiation and neural development, in particular (Jepsen
et al., 2007). While the exact mechanisms of this “huntingtin chromatin function”
are not entirely known, some evidence has accumulated suggesting that wild-type and
CAG-expanded huntingtin may interact, directly or indirectly, with epigenetic co-regulators
and alter their activity (Seong et al., 2010). Interestingly, the DNA methylation
pattern of several promoter regions of genes involved with pluripotency and neural
differentiation (Sox2, Pax6, Nes) was found significantly reduced in presence of mutant
huntingtin (Ng et al., 2013), while the histone methylation status of a class of developmentally-regulated
bivalent promoters associated with “Regulation of Neurogenesis” and “Stress Signaling
and Apoptosis” was altered following the expression of CAG-expanded Htt alleles (Biagioli
et al., 2015). Thus, huntingtin protein might subtly but consistently alter different
aspects of chromatin regulation and transcription during neural development and specification,
explaining the pleiotropic, subtle yet deleterious, effects of mutant huntingtin observed
throughout the life of an HD individual (Figures 1B,C).
Parallel to alterations in the coding portion of the genome, a growing number of studies
is recently showing deregulation of different classes of non-coding RNAs (ncRNAs)—microRNAs
(miRNAs), long non-coding RNAs (lncRNAs), piwi-interacting RNAs (piRNAs), circular
RNAs (circRNAs)—suggesting that they may have a relevant impact on disease onset/progression.
A large percentage of ncRNAs physically interact with various chromatin regulatory
proteins, including PRC2, and other “readers/writers, and erasers” of chromatin modifications,
thus activating or repressing gene expression via a chromatin recruitment mechanism.
The first, intriguing example of such regulatory network is the mammalian X-chromosome
inactivation, where the finely regulated expression of Xist lncRNA is able to recruit
PRC2 to the specific genomic location of the silenced X chromosome to mediate transcriptional
repression. Recently, systematic studies exploring ncRNA function have shown their
involvement in many biological processes also related to embryonic development and
neurogenesis (Qureshi and Mehler, 2012, Sauvageau et al., 2013). Particularly, several
miRNAs, but also lncRNAs have been reported to be enriched in the brain and have key
roles during transition from neural commitment to terminal differentiation (Makeyev
et al., 2007; Sauvageau et al., 2013). Importantly, miRNAs might also target neuronal-specific
transcriptional regulators (i.e., REST and co-REST) as well as brain-enriched alternative-splicing
factors, thus affecting synaptic activity and neuronal function (Makeyev et al., 2007;
Packer et al., 2008). Mutant huntingtin expression has been correlated with alterations
in REST cellular localization and dysregulation of REST-regulated miRNAs (miR-9 and
miR-124) levels (Packer et al., 2008). Specifically, miR-9 and miR-124 levels were
shown to be reduced in the cortical areas of HD post mortem brains where genes related
to neurogenesis were highly overrepresented (Hodges et al., 2006). Moreover, a recent
genome-wide screen of miRNAs in post mortem brains highlighted miRNAs that were differentially
expressed between normal and HD subjects (Hoss et al., 2014). The vast majority of
these miRNAs are highly connected with HOX clusters, a well-studied family of transcription
factors, involved in early brain development, contributing to anterior-posterior positional
establishment. HOX genes and HOX-related miRNAs are tightly regulated by PRC2 proteins
and are found to be altered following mutant huntingtin expression (Seong et al.,
2010). Although the role of development-specific miRNAs and HOX genes in the adult
HD brain is still unclear, one interpretation might suggest aberrant brain development
following mutant huntingtin expression, while, a second plausible explanation might
invoke a reactivation of developmental programs in the later phases of HD degenerative
process where surviving and neurogenic hints try to counteract generalized neuronal
loss.
MiRNAs, however, are not the only class of ncRNAs with developmental potential to
be dysregulated in HD. Several additional studies, in fact, report altered expression
levels of known lncRNAs TUG1, NEAT1, MEG3, and DGCR5 in the brains of HD patients
(Johnson, 2012). MEG3, in particular, is reported to be a REST target with a dynamic
expression during development of the nervous system and associated with PRC2 chromatin
regulator, thus supporting a role for chromatin regulation, non-coding transcription
and neurodevelopment in HD pathogenesis.
Other classes of ncRNA (pi-RNAs and circRNAs), associated with chromatin remodeling
factors and epigenetic modulators, have a clear role during neural development and
differentiation, however their regulation upon expression of mutant huntingtin has
not been analyzed yet.
The analysis of non-coding RNA transcription and its interplay with chromatin regulation
in the context of human neurological disease presents several challenges especially
related to the complexity of ncRNAs in the brain. In fact, the annotation, identification
and functional characterization of ncRNAs continue to be puzzling because of the lack
of a complete understanding of functional domains, their generally low expression
levels, and the poor knowledge of their regulatory regions (Esteller, 2011). International
initiatives such as the Encyclopedia of DNA Elements (ENCODE) or the generation of
knock-out mice for specific ncRNAs (Sauvageau et al., 2013, Goff et al., 2015) represent
the first effort to understand the functional relevance of ncRNAs in the organism
(and brain) physiology and disease. Additional studies in this direction as well as
new and powerful genomic and bioinformatic tools will be crucial to dissect the complexity
of ncRNA brain transcriptome to understand how the interaction between epigenome and
transcriptome will contribute to disease onset and progression and shed light on possible
new paths to therapeutic intervention.
In conclusion, it's time to rethink the simple view of Huntington's Disease as a merely
neurodegenerative disorder to include the idea that expression of the single copy
of the CAG-expanded HTT allele (Figure 1A) is also sufficient to subtly alter development
from conception. The “HD organism” is, at any developmental time point, different
from the “not-HD counterpart” (Figures 1B,C). Like in a puzzle where the HTT-CAG mutation
forces pieces to fit where they do not belong, similarly subtle developmental differences
may give rise to a different panoply of adequately functioning units which may last
for a long time but ultimately not allow the same flexibility with aging, thus aberrant
neurodevelopment might lead to disorganization and dysconnectivity of neurons and
subsequent susceptibility to neurodegenerative processes (Figures 1B,C). Essential
corollaries of this model are not only new approaches to the understanding of the
altered pathologic process, but also new strategies of therapeutic intervention/prevention.
In fact, the recent identification of genetic factors that are able to modify the
age of onset of HD patients, indicate that the HD pathologic process is modifiable
prior to clinical diagnosis (Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium,
2015), thus representing a proof-of-principle of a strategy that is predicated upon
the idea that intervening before onset of clinical signs is likely to be most effective
to finally ameliorate patients quality of life and/or life expectancy.
Author contributions
EK made substantial contributions to conception and design of the work and participated
in drafting the manuscript; MB conceived and supervised the project and wrote the
paper. Authors read and gave final approval of the manuscript version version to be
submitted.
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.