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addiction and its radical cure

Pathways from epigenomics and glycobiology towards novel biomarkers of
addiction and its radical cure

human health and has presently reached high community prevalence in
both developing [1] and industrialized nations [2]. Computational
formulation of a biomarker for opioid dependency would be useful for
diagnosis and staging of the severity of the disorder, treatment selection,
treatment comparison and for monitoring progress on treatment.
Central to the question of peripheral biomarker development is the
non-trivial issue of the metric against which it is to be standardized.
From the myriad mechanistic papers on OUD it appears that there are
some central brain mechanisms involved which are then reflected in
other more peripheral phenomena that manifest systemically by both
direct and indirect routes. It seems appropriate in this paper to give
some brief consideration to the central key mechanisms including the
neuronal ensembles and their connectomes to provide a context within
which to place discussion of the peripheral biomarkers which may be
secondarily derived.
Reflecting our major current research interests our group has considered
the potential of emerging epigenetic and glycobiological techniques
for application and further assessment in OUD. This will therefore
form the substance of the present discussion. Not only is there
significant cross-talk between epigenomic and glycobiological regulatory
systems but both areas also touch on other fields such as the
immunostimulation of substance use disorders (SUD’s) [3] and the
endocrinopathy – including sex differences – so that these subjects are
mentioned en passant albeit in such a way so as not to distract from the
main thread of discussion. This broad approach is contributory to the
https://doi.org/10.1016/j.mehy.2018.04.011
Received 16 September 2017; Accepted 11 April 2018
⁎ Corresponding author at: 39 Gladstone Rd., Highgate Hill, Brisbane, Queensland, Australia.
E-mail address: sreece@bigpond.net.au (A.S. Reece).
Medical Hypotheses 116 (2018) 10–21
0306-9877/ © 2018 Published by Elsevier Ltd.
T
main discussion as a useful biomarker should have some predictive
relationship with the diverse and disparate systemic phenomenology of
OUD [4–6].
Importantly many of the central changes are reflected in the blood
[7–9]. Hence the new findings on OUD suggest that some central
changes are reflected in peripheral phenomena. Moreover since both
epigenomics [9,10] and glycobiology [11–14] have been used to derive
highly predictive clinical biomarker indices for complex disorders our
hypothesis is that their combination should provide enhanced power
for discrimination of the presence or absence of OUD-SUD and for severity
ascertainment.
Neuronal ensembles
Classical authors including Donald Hebb had suggested that memories
were likely to be encoded by a sparse and diffusely located network
of neurons which were called cell assemblies [15] and are now
usually known as neuronal ensembles. This is of relevance to addiction
because clinical SUD syndromes are often described as subversions of
normal reward and memory processes [16]. Complementing this work
on memory and motivation in general an elegant series of studies has
been conducted by the National Institute of Drug Abuse Intramural
Research Program in recent years using optogenetic and stereotactic
techniques in transgenic rats demonstrating that drug dependency
syndromes related to nicotine [17], alcohol [18], amphetamines [19]
cocaine [20], food [21] and opioids [22] are related to neuronal ensembles
distributed across the ventral prefrontal cortex, the hippocampus,
the basolateral amygdala, Nucleus Accumbens (NAc) and
ventral tegmental area (VTA). Only about 1% by volume of the number
of neurons in each area is involved in forming the neuronal ensemble.
Neurons are believed to become incorporated into the ensemble based
on receiving the most active input [23] albeit this is an issue of ongoing
discussion and enquiry. Rodent neurons engaged in the neuronal ensemble
(Fig. 1) are marked both by master transcription factors (TF’s)
[24] and by the activation of immediate early genes of which the most
notable is cFos (gene) and its protein product the TF fos and the products
of its various mRNA splice variants [23]. Neurons can be involved
in multiple ensembles in which they partner with different networks of
cells [23].
Importantly interruption of these neuronal ensembles has been
shown to abrogate rodent behavioural states relevant to addictive behaviour
involving both cocaine and opioids [22,25–27]. Interdiction of
addiction-relevant behaviours has been achieved by inactivation of the
– very few – hippocampal cells concerned [22], their VTA [27] or
amygdala [25] counterparts, or re-allocation of hippocampal place cells
to erroneously confound a previously naturally encoded drug-place
preference in rats [26]. Moreover silencing of neurones in the rat orbitofrontal
cortex has been shown to interrupt both context-induced
relapse to heroin [22] and the incubation of heroin craving [28].
Neuronal connectomics
Activity dependent (Hebbian) changes at the synapse have been
described to endorse the finding that “cells that wire together fire together”
in many species both vertebrate and invertebrate [15,29]. Longterm
synaptic potentiation and depression in various forms has been
shown to be a key organic substrate of rodent memory [30,31]. Given
that activity dependent processes in the synapse have been shown to
control plasticity it would follow that there must be a coordination
between the nucleus and the machinery of the synapse to make the
changes long-lasting [29]. This key coordination is thought to be controlled
in the nucleus epigenetically [29] by mechanisms which are still
being explored.
Since neurons have a refractory period after action potential firing,
and frequently receive inhibition, their mutual connections naturally
engender oscillations in neuronal networks which occur at certain
defined frequencies over a wide dynamic range in many species [32].
Gamma (25–100 Hz) and theta (4–10 Hz) waves have been shown to be
particularly important [33]. Moreover significant theta-gamma modulation
occurs such that the phase interaction (or interference) of the
two waves carries information and has been linked to movement initiation
and percept [34] and memory formation [35,36] in many
mammalian species including rodents and primates. These pre-clinical
findings have also been validated in the human: neocortex [37], medial
prefrontal cortex (mPFC) [38], temporal cortex [33], somatosensory
cortex [39], cingulate cortex [40], occipital cortex [41], nucleus accumbens
[42], amygdala [43], insula [39] and hippocampus [44]
many of which are components of the mesolimbic reward circuitry.
These earlier studies were elegantly combined in a recent paper
studying affiliative bonding in monogamous prairie voles [45]. These
workers showed that the theta-gamma modulation of the circuit between
the mPFC and the NAc controlled female affiliative behaviour
with dramatic and sudden slowing of the theta (5–6 Hz)-gamma
(80–84 Hz) coupling during and after mating. Moreover larger increases
in net theta-gamma modulation caused faster displays of affiliative
behaviour. This slowed mPFC-NAc activity persisted after mating and
was predictive of social behaviors. mPFC-generated oscillatory synaptic
plasticity altered NAc-based partner responsiveness which had previously
been shown in this species to be controlled by epigenomic
Fig. 1. Identifying Hippocampal Engram cells and their (B) synaptic connectivity.
Sal – Saline; ChR2 – Channelrhodopsin; Ani – Anisomycin. From:
Ryan TJ, Memory. “Engram Cells Retain Memory under Retrograde Amnesia.”
Science 2015; 348 (6238): 1007–1013. Used by Permission.
A.S. Reece et al. Medical Hypotheses 116 (2018) 10–21
11
mechanisms through oxytocin and prolactin signalling [46]. Although
this work has not been replicated in the SUD field to our knowledge,
this detailed dissection of reward neurophysiology carries obvious
significant implications for understanding, studying, modulating and
potentially one day treating reward related disorders.
Epigenomics
The recent explosion of studies on the epigenetics of SUD’s has
yielded many insights into what were previously poorly understood
pathophysiological mechanisms. This work has been done mainly in
rats with limited validation in human post-mortem brain tissue
[47–49]. Cytosine methylation at CpG dinucleotides usually suppresses
DNA transcription. Histone acetylation negates the positive charge on
the ε-lysine tails rupturing the hydrogen bonds with DNA and moving
them away from the double helix, opening up the double helix and
effectively making genes available to the transcription machinery [8].
Multiple pathways exist coordinating DNA methylation and histone
post-translational modifications [50–53].
It is widely believed that the epigenome is a key locus of gene:
environment interactions [9] in many SUD’s [54,55]. Cell-wide changes
in (non-Hebbian) excitability have been shown to be controlled epigenomically
[29] by several mechanisms. Synaptic insertion of AMPA,
NMDA and metabotropic glutamate receptors in the rat NAc is controlled
epigenomically after chronic morphine correlating closely with
anxiety-like behaviours [56]. Epigenomic mechanisms account for
heightened neuronal excitability in the rat VTA exposed to chronic
opioids mediated via by AMPAR’s, GABAAR’s (GABA A receptors) and
potassium channels [57]. Epigenomically reduced rat VTA μ-opioid
receptors and GABAAR’s cause increased tolerance to both opioids and
benzodiazepines characteristic of clinical OUD [57]. This tolerance was
also related to epigenomically-regulated mechanistic target of rapamycin
(mTOR) -mediated reduction in the size of VTA neuronal somas
[48]. NAc spinogenesis after chronic morphine and cocaine is controlled
epigenomically [47].
These findings have prompted several interventional studies in rats.
Histone deacetylase administration attenuates morphine withdrawal
[49] and modulates behavioural and reward responses to morphine
[58,59]. Addiction-relevant behaviours can be modified by artificial
manipulation of NAc histone post-translational modifications and
chromosomal state (particularly histone 3 lysine 9 dimethylation
(H3K9me2)) using bioengineered transcription factors targeted to the
FosB promoter in a rodent model of chronic cocaine exposure [51].
For example dramatic downregulation of H3K9me2 in the rodent
NAc occurs at splice sites in FosB gene at exon-4 increasing transcription
of ΔFosB which is an unusually long-acting transcription factor
with a half-life of around 12 days as it is phosphorylated and lacks the
101 amino acid C-terminal degron [7,56]. DNA methylation has also
been shown to control rat memory formation [53,60] and inhibition of
the DNA methyltransferases (DNMT) DNMT1 and DNMT3a reduces
neuronal membrane excitability through reduction in potassium channels
in cultured rat neurons [61]. Cortical DNA methylation has been
proposed as a biomarker of rat hippocampal memory [60].
As an example relevant to clinical addiction a useful predictive
whole blood biomarker of current alcohol consumption was derived
from the DNA methylation state of 144 genes amongst Europeans and
165 genes amongst African-ancestry cohorts with many epigenome
wide association study hits occurring in CpG island-shores and enhancers
[9]. Methylcytosine can be oxidized to hydroxymethylcytosine,
formylcytosine [62] and carboxycytosine by the
serial action of the ten-eleven translocase (TET) 1–3 DNA oxidizing
enzymes and others, which has been shown to be important for brain
function and memory formation [63,64], anxiety [65] and cocaine
action [66]. Rat potassium channel upregulation and its accompanying
heightened membrane excitability is TET-3 dependent [61].
RNA forms a further layer of fascinating and complex regulation
within the mechanisms of cellular information control. Cellular RNA’s
occur in both long and short forms. As RNA can bind to itself, it often
folds over and can perform catalytic functions on proteins and other
RNA’s. RNA’s routinely undergo both post-transcriptional splicing in
large spliceosome machines [67] and post-transcriptional modification
using 111 modifications most of which are believed to carry information
[68,69]. Long non-coding RNA’s can be expressed from enhancers
[70] and can form regulatory scaffolds governing the transcriptional
availability of long DNA segments including formation of hybrid DNARNA
triplex scaffolds [70–72], arrange nuclear architecture and coordinate
chromosomal silencing [73].
An epigenomic age biomarker-algorithm has been used to study
numerous neurological and other conditions relevant to SUD in humans
including stress and PTSD [74], neurodegenerative disease [75], HIV
dementia [76], Parkinson’s disease [77], Huntingdon’s disease [78],
Alzheimer’s disease [75,76,79] alcoholism [80,81], insomnia [82] and
menopause [83].
BOX 1
Immune Activation.
Immune activation is a hallmark of addictive disorders. This is
induced by several mechanisms including addictive drugs
themselves [3] and the use of non-sterile injecting equipment
and has been seen in both virally infected and non-infected
patients [91,92,137,138]. Importantly immune activation is
also a major cause of ageing processes [139] and immunosenescence
[140,141] and its related “inflamm-aging”
[142] is also a major peripheral biomarker of it [140,141].
The polyclonal gammopathy of SUD [92,137,143] and its related
changes likely reproduces immunosenescence
[4,5,137,144,145]. This peripheral immune activation is
shown in Fig. 2 redrawn and updated from [92,137,145]
which compares the globulin fraction of human plasma, which
is largely composed of immunoglobulins, in opioid dependent
and control patients over both age and time (see
Supplementary data for statistical analysis). Importantly NF-
κB is the master TF of the immune system in many tissues
[146] and is also involved in sculpting brain networks and
synaptic and dendritic pruning [147,148], and is importantly
involved in cell survival/death and stem cell decisions [146].
Interestingly there are now several reports of the direct involvement
of key epigenomic regulators (ten-eleven translocation
methylcytosine dioxygenase (TET2) and DNMT3a) in
the modulation of immune response [149–151].
The immunostimulatory aspects of drug dependency syndromes
[3] assumes further importance in view of the interaction
of glycosylation processes with the immune system and
particularly circulating immunoglobulins, especially with regard
to their valence determination.
Recent research has revealed intimate interaction at numerous
points between neuronal activity and immune regulation
for not only do neurons have many cytokine and immune
modulator receptors but so too immunocytes carry high
density receptors for neurotransmitters including opioids and
dopamine [152]. This arrangement sets up neuroimmune reflexes
with vagal control of systemic immune resolution including
peritoneal antibacterial activity, and splenic nerve
control of systemic inflammatory tone in rheumatoid arthritis,
metabolic syndrome, essential hypertension, Crohn’s disease
and immunosuppression accompanying spinal cord trauma
[152].
Interestingly it was the recently demonstrated that the
DNA methylation status of GABA receptor genes was associated
with the activity state of the promoters of immune
A.S. Reece et al. Medical Hypotheses 116 (2018) 10–21
12
response genes in circulating monocytes in a manner predictive
of clinical alcohol use [9]. This finding relates the
epigenetic regulation of synaptic activity to a well characterized
and readily accessible major peripheral pathophysiological
process – immunoactivation. These important findings
suggest that peripheral epigenomic biomarkers are reflective
of central neuroinflammatory processes by several pathways.
In conclusion we feel that it is likely that an unbiased
hypothesis free approach to screening of peripheral epigenomic
markers will likely identify genes involved in immune
pathways, and immune-related genes such as the GABA genes
previously implicated in and reflective of alcoholism [9] may
demonstrate useful discriminative value in defining a peripheral
biomarker in OUD/SUD. Similarly we feel that glycomic
changes identified in previous studies in disorders
characterized by immune activation [153–155] may have
some overlap with drug dependency syndromes.
Glycosylation
For over a century OUD has been linked with hyperglycaemia and
there are also many points of interaction between glycobiology and
epigenomics. Hyperglycaemia is both more common [84–87] and more
severe [88] in OUD, a point well recognized by Claude Bernard in 1877
working in dogs [85]. Mechanisms validated in man include physical
inactivity [88], stress hormone signalling [87,89], hypothalamically
mediated preference for fatty and sweet foods [88,90], the proinflammatory
milieu both centrally [3] and systemically [91,92], a likely
pro-senescent state (since atherosclerosis [93,94] and diabetes [85,88]
share common genome wide association study (GWAS) hits at the senescence
locus on chromosome 9q21.3 [95]), acute cerebral hypoxia
[96] and an inhibition of insulin receptor substrate signalling both in
mouse VTA neurons [97] and mouse pancreatic islets [98].
This is relevant pathophysiologically because more than half of all
proteins are glycosylated including cytokines and their receptors and
Fig. 2. Variation in Serum Globulins with (A) Chronological Age and (B) Time in opioid dependent and control patient groups.
A.S. Reece et al. Medical Hypotheses 116 (2018) 10–21
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neurotransmitter receptors [99] which may occur either passively and
non-enzymatically, or mediated by dedicated enzyme control systems
for glycan attachment and removal. O-Glucose-N-Acetyl transferase
(OGT) is one of the major glycosyl transferase enzymes and has been
shown to be essential for embryonic survival in mice [100]. Glycosylation
is often reciprocal with phosphorylation [100]. Glycosylation is
the most complex post-translational modification. There are 120 sugars
in the biological glycan “alphabet” and these often form long and
branched polymers and may bind in multiplex [101,102]. Since sugar
chains are often long and highly charged, they can change all the biophysical
and chemical properties of proteins including their charge,
size, solubility, binding affinities, and polarity (stimulatory to inhibitory),
protein turnover, calcium handling and transcription [99].
Unlike simpler post-translational modifications such as acetylation,
methylation and phosphorylation, the enormous diversity and inherent
complexity of glycosylation reactions makes available to biological
systems nuanced and graded signalling subtlety [99]. Glycans also bind
to DNA and RNA [103,104] and control stem cell proliferation and
migration [105,106] via transmembrane receptors [105] and are implicated
in aging [103]. Extracellular glycans can signal to immune
genes via the epigenome [107]. Gene transcription is downregulated by
interaction of the epigenomic (including sirtuin-histone deacetylase and
Polycomb Repressive Complex, PRC) repressive and glycosylation machinery,
and indeed OGT has been noted to be part of the PRC
[100,108].
Some of the key proteins which are glycosylated and downregulated
include mTOR [109] which coordinates protein synthesis and cell
growth; Mediator kinase complex which transduces signals from promoter
and enhancer TF binding to RNA polymerase II; RNA polymerase
II [100,110] which transcribes DNA; and all the histone proteins [111]
which form the core of the nucleosomes around which DNA is wound.
BOX 2
Feminine Hormonal Factors.
The lateral hypothalamus is involved in morphine reward
mediation [90,156,157] and its neurohormonal output provides
a useful readout on limbic system function albeit contingent
upon multiple peripheral feedback loops [4,158]. The
lateral hypothalamus also coordinates female hormonal cycles
in all mammals including humans [139,158]. Gonadotrophin
releasing hormone has been implicated in lifespan regulation
Fig. 3. Variation in Various Circulating Plasma Glycans by Age and Sex. From: Yu X et al. “Profiling IgG N-glycans as potential biomarker of chronological and
biological ages. A community based study in a Han Chinese population.” Medicine (2016); 95 (28): e4112-e4122. Used by Permission. Key taken from: Lauc G. et al.
(2013), Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers. Plos Genetics
2013 9 (1) e1008225. Used by Permission.
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in the mouse [159]; and FSH has been shown to control mouse
oocyte viability [160] which is also known to determine rodent
lifespan [161,162]. FSH has also been shown to have
metabolic activity and rodent control central visceral fat mass
[163]. During a woman’s reproductive years the LH exceeds
the FSH and this ratio then reverses in the post-menopausal
period implying that the cross-over point is a key measure of
the premenopause. This suggests that the 58% reduction in
the fecund period defined by this ratio in clinical OUD has far
reaching implications [158]. These data are consistent with
the heightened human female sensitivity to OUD noted by
several authors [7,93,158].
There are also significant interactions between gonadotrophic
hormone regulation and glycosylation pathways. LH,
FSH and their receptors are all glycosylated and down regulated
in women [164]. Plasma glycans in women have also
been found to change dramatically with both the menstrual
cycle [165] and with age with significant changes occurring
across the menopausal period in human females [11].
In conclusion we therefore predict that unbiased investigation
of the circulating glycomic profile of OUD/SUD
patients and in particular of their immunoglobulin G subfractions
will reflect in some measure age- and menopausalspecific
changes identified in earlier studies [11,116]. Moreover
it would appear likely that specific investigation of the
glycosylation states of key female hormones such as LH, FSH,
estradiol, progesterone and their respective receptors in circulating
leukocytes, may yield further diagnostic insights and
increase the discriminative power of peripheral biomarkers on
ROC analysis.
Our hypothesis that plasma glycans may be useful biomarkers for
OUD draws support from other literatures. For example as suggested in
Fig. 3 plasma glycans have been shown to vary with human age, they
have been used for development of a biomarker of ageing and important
sex differences have been noted [11]. Plasma glycan profiles
have also been used clinically to develop a biomarker for Parkinson’s
disease where four glycans were selected to derive a biomarker profile
with an 87% sensitivity and 92% specificity for the presence of Parkinsons
disease [13].
Important glycosylation changes on plasma immunoglobulins have
also been shown with inflammatory diseases. Circulating immunoglobulins
of the G class (IgG) are usually glycosylated in various
ways (Fig. 4) forming over 30 glycovariants which may make them
either proinflammatory or immunosuppressive [112]. Principal component
analysis has been used in biomarker development to separate
systemic lupus erythematosus patients from controls where a receiver
operator analysis coefficient of 0.842 was achieved [113].
The majority of our biological processes rely on N-glycosylation of
human proteins. N-glycans affect protein structure and function, and
glycosylation events are known to alter with environmental changes,
age and disease [99]. Variations in N-glycosylation of the IgG complex
have adverse downstream effects on inflammatory pathways known to
be associated with ageing and chronic disease aetiopathogenesis. IgG Nglycan
traits are only partly determined by genetics and so represent
signatures of joint genetic predisposition and environmental influences
across the life-course on overall immune function and wellbeing [99].
The early detection and intervention of disease processes have become
increasingly important to prevent life-long complications associated
with SUD’s, chronic diseases and the subsequent burden on global
health. Studies have shown that selective combinations of IgG N-glycan
structures associate with biological hallmarks of pre-chronic disease
states when biological age exceeds chronological age [11,114]. Studies
have also verified the validity of these IgG N-glycan combinations as
predictive risk profiles and biomarkers of biological ageing in several
human population cohorts of European [114], Chinese [11] and African
descent [113].
The first GWAS of the human N-glycome combined high-throughput
protein glycosylation analysis with GWAS in a mixed European population
cohort study of almost 3000 Croatian and Scottish adults [115].
This performed the most comprehensive association analysis of
common genetic variants with typical glycan profiles and identified
that these genetic variants adversely influence the relative proportions
of different types of N-glycans on human plasma proteins and thereby a
course of events leading to metabolic and inflammatory disorders
[113,115]. In particular, common variants of Hepatocyte Nuclear
Factor 1α (HNF1α) regulate the expression of key fucosyltransferase
and fucose biosynthesis genes, both master regulators of plasma protein
fucosylation influencing N-glycan levels in human plasma. These findings
characterised nine genetic loci associated with specific IgG Nglycan
patterns. These replicated and validated findings further support
the notion that genetic mutations and consequent alterations in the
molecular and enzymatic processes of glycosylation can change glycoprotein
function, triggering inflammatory or autoimmune conditions.
The central role of HNF1α in the regulation of multiple genes involved
in fucosylation may be the molecular mechanism behind the reported
association between common variants of HNF1α and inflammatory
markers, such as C-Reactive Protein, and conditions in which inflammation
plays a key pathogenic role, such as ageing and ageingrelated
chronic diseases including autoimmune diseases (arthritis and
systemic lupus erythematosus), haematological cancers, coronary artery
disease, hypertension and metabolic syndrome [12,14,113–118].
These data are summarized in Fig. 5 depicting the utility of composite
glycan-derived clinical biomarkers for rheumatoid arthritis, systemic
lupus erythematosus and Parkinsons disease as receiver-operator
curves.
For drug abuse syndromes, it is essential that an accurate diagnosis
is obtained and disease progression can be monitored. Immunoglobulin
G (IgG) has the ability to exert both anti-inflammatory and pro-inflammatory
effects, and the N-glycosylation of the fragment crystallizable
portion of IgG is involved in this process. Although there is no
yet a population-based study on profiling of IgG glycan among drug
abuse cases, our study on whether the IgG glycome could be a candidate
biomarker for neurodegenerative disease, i.e., Parkinson’s disease,
could be a good example on such application. Ninety-four communitybased
individuals with Parkinson’s disease and a sex-, age- and ethnically-
matched cohort of 102 individuals with mixed phenotypes, representative
of a “normally” aged Caucasian controls, were investigated
[13]. Plasma IgG glycans were analysed by ultra-performance liquid
chromatography. Overall, seven glycan peaks and 11 derived traits had
statistically significant differences (P < 8.06×10–4) between Parkinson’s
disease cases and healthy controls. Out of the seven significantly
different glycan peaks, four were selected by Akaike’s Information
Criterion to be included in the logistic regression model, with a sensitivity
of 87.2% and a specificity of 92.2% [13]. The study suggested
that there is a reduced capacity for the IgG to inhibit Fcγ-RIIIa binding,
which would allow an increased ability for the IgG to cause antibodydependent
cell cytotoxicity and a possible state of low-grade inflammation
in individuals with Parkinson’s disease. The peripheral IgG
glycome profile changes in PD patients infer an increased pro-inflammatory
capacity. Although it is yet to be determined whether the
immunoglobulins in blood plasma correlate with those in cerebrospinal
fluid, it is evident that the study of systemic inflammation in PD may
provide important information about the neurodegenerative process for
other neurodegenerative disease syndromes such as drug abuse and
provides potential as biomarkers from the perspectives of preventive,
predictive and personalized medicine. In this way it seems likely that
glycomic parameters will comprehend the endocrinopathy, pro-aging
and pro-inflammatory aspects of OUD/SUD’s and significantly enhance
the predictive power of epigenomic peripheral biomarkers.
It is interesting to consider the routes by which central changes of
A.S. Reece et al. Medical Hypotheses 116 (2018) 10–21
15
Parkinson’s disease might be reflected peripherally. Oxidative stress,
immune and epigenetic pathological pathways and immune: stem cell
interactions provide possible clues which may act centrally and also be
detected peripherally.
Therapeutic considerations
These concepts also suggest several very exciting novel therapeutic
pathways into addiction treatment which have not been previously
been explored. These pathophysiological and mechanistic insights into
the fundamental wiring of addictive cells and circuits in turn suggest
several novel potential applications with possible therapeutic implications.
The cells involved are of known cell type – particularly pyramidal
cells in the cortex and hippocampus and medium spiny neurons in the
Nucleus Accumbens. Moreover the ensemble cells have been extensively
characterized by single cell techniques [22,119–123] and
contrasted with surrounding non-activated cells [21]. Each cell type is
controlled by a master transcription factor [24,124]. By definition these
cells are activated by the specific drug of choice. This suggests that the
master transcription factor of these cells could be specifically targeted
by the peripheral injection of the antisense oligonucleotide (ASO’s)
which is complementary to the master TF concerned or targeted by
RNA interference (RNAi) technology and either temporarily or permanently
inactivated. Such a strategy has already been trialled in large
scale phase III clinical trials for the medium and longer term modification
of cholesterol metabolism and the atherogenic process by interfering
with the ANGPTL9 cascade and also targeting coagulation
factors 8 and 9 in haemophilia and for spinal muscular atrophy and
reported in leading medical journals [125–130]. Since the cells are
active there should be a way to make the uptake of the ASO’s dependent
on cell activity as has already been achieved experimentally [121].
Moreover powerful new drugs are being developed by several major
pharmaceutical companies which act on the epigenome [131,132].
They are being developed for disparate applications including weight
reduction the prevention of atherosclerosis, treatment of diabetes and
as powerful new cancer agents which are remarkable for minimally
usually minimally toxic [131,132].
Alternatively new techniques are being developed which allow deep
areas within the substance of the human brain to be specifically targeted
by separate alternating electric or magnetic fields in such a way
that the two fields interfere with each other cancelling out in the more
superficial regions, but reinforcing in the deepest areas of the brain.
Such strategies can be used strategically to map and to target various
brain structures non-invasively within the living awake human brain
Fig. 4. Structural summary of circulating Plasma IgG N-glycans used in (B) Systemic Lupus Erythematosus Biomarker Development. From: Lauc G. et al. (2013), “Loci
associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers.” Plos Genetics 2013 9 (1)
e1008225. Used by Permission.
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[133]. The proof of concept has been established in rats, and such
techniques are now under development for humans [133]. It may be
possible to coordinate targeted information-based therapeutics such as
ASO’s and RNAi and have them uncaged after nanomolecular delivery
in the areas of interest and thus change brain neurophysiology in a
precise and controlled manner for a duration which could be programmed.
It has been demonstrated that cultured neurons can take up
micron-sized particles including nanospheres containing 6000 atoms of
iron in ferritin, and this has been considered as a novel neuroimaging
agent [134]. Clearly such techniques could be re-purposed from diagnostic
application to therapeutic implementation.
The demonstration that addictions – and all memories – depend on
delicately balanced harmonically-tuned cellular networks as their organic
substrate – implies that such finely balanced cellular ensembles
should be susceptible to targeted interference and thus disruption– effectively
re-programming the thinking patterns of the brain! Obviously
the potential to do so – by a technique as simple as a subcutaneous
injection and/or the subsequent application of targeted field potentials
– would require stringent ethical oversight to prevent its inappropriate
application. The coordination of such cutting-edge techniques clearly
opens major new treatment vista’s and may significantly increase the
power of the therapeutic armamentarium of tomorrow.
Hypotheses.
The central hypotheses of this enlarged pathophysiological understanding
are that exciting new pathways to both diagnostic and treatment
modalities may be developed in the near future. We feel that diagnostics
based on epigenetics and glycomics hold unusual promise for
potential bedside application both for the presence of substance dependency
disorders and their severity. Moreover the concepts described
above hold exciting power for therapeutic application to design and
deliver in a fashion targeted over both time and anatomical structure
information-based treatments to disrupt delicate reward-motivational
circuits which form the biological substrate of numerous dependency
syndromes.
In terms of specifying and subsequently testing the hypotheses more
exactly the following guiding remarks may be made:
Diagnostic Biomarkers:
1) Type of future Study: prospective longitudinal – controls and opioid
dependence; also need to include non-smoking and smoking controls
to differentiate out effects of tobacco
2) Direction – both addiction longitudinally and coming off drugs – e.g.
by antagonist supported abstinence such as long acting naltrexone
implant maintenance
3) Aims – to describe in detail epigenomic and glycomic profiles of
opioid addiction
4) Samples – blood – because these are best validated and can readily
be re-sampled
5) Methods to be applied –
i) Epigenomic Next generation sequencing
ii) Glycomics
iii) Genomic – for Mendelian randomization to interrogate causation
6) Outcomes
Machine learning could then be applied to develop:
i) An Epigenomic signature for opioid dependence possibly controlling
the μ-opioid receptor
ii) An Epigenomic signature for tobacco dependence
iii) A Glycomic signature for opioid dependence – possibly involving β-
linked N-acetylglucosamine.
iv) A Glycomic signature for tobacco dependence
v) Causal inferences from both forward and reverse opioid dependence
longitudinal processes
vi) Causal inferences from genomic Mendelian randomization
Biomarkers for ageing, opioid, and tobacco use may be identified by
the application of artificial intelligence machine learning approaches to
epigenomic and glycomic datasets derived from peripheral blood and
the optimization of receiver-operator curve characteristics against doseduration
of substance exposure. β-linked N-acetylglucosamine is a
glycan of particular interest and merits specific attention. Similarly the
μ-opioid receptor gene and its epigenomic and non-coding DNA regulatory
regions merit particular attention and would comprise a useful
focus for Mendelian randomization studies. The use of Mendelian randomization
together with longitudinal samples studying addiction in
both the forward and reverse directions (patients going into addiction
and also coming out of it) would provide a particularly powerful framework
within which to investigate potentially causal relationships.
Treatments
Emerging from (6) (i) above would be epigenomic targets for
therapeutic interdiction in addiction. Since some studies show correlations
between biomarkers circulating in the blood and those in the
reward circuitry of the brain it may be that key insights can be gained
B:
Systemic Lupus
Erythematosus
C:
Parkinsons
Disease
A:
Rheumatoid Arthritis
Fig. 5. Receiver Operating Characteristic Curve for Glycans as Biomarkers of
(A) Rheumatoid Arthritis (coefficient=0.881), (B) Systemic Lupus
Erythematosus (coefficient=0.842) and (C) Parkinsons disease (coefficient=
0.970). From: (A) Sebastian A. et. al. “Glycan Biomarkers for
Rheumatoid Arthritis and its Remission Status in Han Chinese Patients.”
OMICS: A journal of Integrative Biology. 2016, (6): 343–351; (B) Lauc G. et al.,
“Loci associated with N-glycosylation of human immunoglobulin G show
pleiotropy with autoimmune diseases and haematological cancers.” Plos
Genetics 2013, 9: (1) e1008225; (C) Russell A.C. et al. “The N-glycosylation of
immunoglobulin G as a novel biomarker of Parkinson’s disease.” Glycobiology.
2017; 27 (5):501–510. All Used by Permission.
A.S. Reece et al. Medical Hypotheses 116 (2018) 10–21
17
by studies of peripheral biomarkers which reflect key central pathophysiological
processes implicated in the neurocircuitry of opioid dependence.
Adeno- Associated virus No 9 has been shown to allow the delivery
of therapies to the brain and across the blood brain barrier [129,130].
Treatments could be targeted direct to the deep brain structures of the
limbic reward system both by the use of AAV9 vectors and by the use of
electromagnetic uncaging of nanoparticle structures in the circulation
of the limbic reward system [133].
Such targets could then be tested in preclinical animal models prior
to their consideration in humans.
Concluding remarks
These theoretical considerations suggest multidimensional-multidirectional
networked interactions between OUD, epigenomics, glycomics,
immunoactivation and endocrine read-outs of central corticolimbic
status. As the neuronal ensemble networks including their
connectomics are increasingly characterized in the mesocorticolimbic
reward system the application of machine learning algorithms to the
systemic phenomenology of OUD should enable the development of
peripheral-epiphenomenological biomarker sets as was recently demonstrated
for clinical alcoholism [9]. Importantly, central corticolimbic
neuroinflammation [3] has been shown to be predictively relatable
to epigenomic neuroimmune biomarkers within readily
accessible human circulating peripheral monocytes [9]. Since glycanderived
biomarkers have demonstrated utility as predictive clinical
discriminators of several chronic diseases [11–14] their incorporation
along with epigenomic indices should refine and increase the power of
peripherally-sourced algorithms in OUD. Including genomic data for
Mendelian randomization would allow the interrogation of causality
[135] suggesting a comprehensive pan-omic approach to comparative
central-peripheral biomarker algorithm development. As both epigenomics
and glycomics are emerging as powerful multilayered combinatorial
biological control systems reflecting gene-environment interactions
the prospect of their computational combination from
peripherally accessible tissues implies the dawning not only of a new
era of diagnostic insight but also the potential to assess central mechanistically
significant processes. Coordinated and precisely targeted
application of new and emerging therapeutic techniques suggests that
the concepts described herein may find novel application in exciting
new therapies for substance dependency conditions which were previously
considered intransigent and refractory. In time it is conceivable
that other psychiatric disorders such as refractory depression and possibly
post-traumatic stress disorder, may find similar remediation. All of
this suggests a bright future for substance dependence treatment and
the dawning of a new age of diagnostic sophistication and targeted
therapeutic assistance to facilitate change.
Note added in proof
The PINS Prize in Science was awarded by the American Academy
for the Advancement of Science to both the Winner and the Runner up
for execution and demonstration of some of the concepts described in
this paper particularly optogenetic studies in neural engrams of
memory and molecular uncaging of nanoparticles by electromagnetic
fields deep in the brain while this paper was under consideration [136].
Conflict of interest
The authors have no conflict of interest to declare.
Funding statement
No funding was received for the conduct of this study.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.mehy.2018.04.011.
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