Association between a Genetic Variant of Type-1 Cannabinoid Receptor and Inflammatory Neurodegeneration in Multiple Sclerosis
et al. (2013) Association between a Genetic Variant of Type-1 Cannabinoid Receptor and Inflammatory
Neurodegeneration in Multiple Sclerosis. PLoS ONE 8(12): e82848. doi:10.1371/journal.pone.0082848
Association between a Genetic Variant of Type-1 Cannabinoid Receptor and Inflammatory Neurodegeneration in Multiple Sclerosis
Silvia Rossi 0
Marco Bozzali 0
Monica Bari 0
Francesco Mori 0
Valeria Studer 0
Caterina Motta 0
Fabio Buttari 0
Mara Cercignani 0
Paolo Gravina 0
Nicolina Mastrangelo 0
Maura Castelli 0
Raffaele Mancino 0
Carlo Nucci 0
Fabrizio Sottile 0
Sergio Bernardini 0
Mauro Maccarrone 0
Diego Centonze 0
Celia Oreja-Guevara, University Hospital La Paz, Spain
0 1 Clinica Neurologica, Dipartimento di Medicina dei Sistemi, Universita` Tor Vergata , Rome , Italy , 2 Centro Europeo per la Ricerca sul Cervello (CERC)/Fondazione Santa Lucia , Rome , Italy , 3 Dipartimento di Medicina Sperimentale e Chirurgia, Universita` Tor Vergata , Rome , Italy , 4 Clinical Imaging Sciences Centre, Brighton and Sussex Medical School, University of Sussex , Brighton, East Sussex , United Kingdom , 5 Dipartimento Medicina di Laboratorio, Policlinico Tor Vergata , Rome , Italy , 6 Clinica Oculistica, Dipartimento di Biopatologia, Universita` Tor Vergata , Rome , Italy , 7 IRCCS Centro Neurolesi Bonino Pulejo , Messina , Italy , 8 Dipartimento di Medicina Interna, Universita` Tor Vergata , Rome , Italy , 9 Center of Integrated Research, School of Medicine, Campus Bio-Medico University of Rome , Rome , Italy
Genetic ablation of type-1 cannabinoid receptors (CB1Rs) exacerbates the neurodegenerative damage of experimental autoimmune encephalomyelitis, the rodent model of multiple sclerosis (MS). To address the role on CB1Rs in the pathophysiology of human MS, we first investigated the impact of AAT trinucleotide short tandem repeat polymorphism of CNR1 gene on CB1R cell expression, and secondly on the inflammatory neurodegeneration process responsible for irreversible disability in MS patients. We found that MS patients with long AAT repeats within the CNR1 gene ($12 in both alleles) had more pronounced neuronal degeneration in response to inflammatory white matter damage both in the optic nerve and in the cortex. Optical Coherence Tomography (OCT), in fact, showed more severe alterations of the retinal nerve fiber layer (RNFL) thickness and of the macular volume (MV) after an episode of optic neuritis in MS patients carrying the long AAT genotype of CNR1. MS patients with long AAT repeats also had magnetic resonance imaging (MRI) evidence of increased gray matter damage in response to inflammatory lesions of the white matter, especially in areas with a major role in cognition. In parallel, visual abilities evaluated at the low contrast acuity test, and cognitive performances were negatively influenced by the long AAT CNR1 genotype in our sample of MS patients. Our results demonstrate the biological relevance of the (AAT)n CNR1 repeats in the inflammatory neurodegenerative damage of MS.
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
Type-1 cannabinoid receptors (CB1Rs) are among the most
abundant G protein-coupled receptors in the mammalian brain
, where they play a pivotal role in the control of synaptic
transmission , and in the maintenance of neuronal integrity
[5,6]. Not surprisingly, cannabinoid treatment has been proposed
to contrast the neurodegenerative damage in several
neuroinflammatory diseases . Genetic ablation of CB1Rs exacerbates
the neurodegenerative damage associated with experimental
autoimmune encephalomyelitis (EAE), a reliable mouse model of
multiple sclerosis (MS), by altering synaptic sensitivity to
proinflammatory cytokines released by infiltrating immune cells and
by activated microglia . Inflammation leads to neuronal
damage also in the human brain, and indeed higher frequency and
severity of inflammatory episodes have been associated with
accelerated neurodegeneration and disability accumulation in MS
, but large inter-individual differences among patients
exist. Based on this clinical evidence, we postulated therefore that
genetic differences in CB1R expression and function might
contribute to differential inflammatory neurodegenerative damage
in MS patients, as it occurs in EAE mice.
The gene encoding CB1R (CNR1) is located on chromosome 6,
and shows a microsatellite polymorphism which is an AAT
trinucleotide short tandem repeat (AAT)n, downstream of the
translation site . There is evidence indicating that
microsatellites can affect transcription efficacy in some genes , and if
true also for CNR1, this notion offers the unprecedented
opportunity to address our hypothesis. Of note, although in a
previous study  some clinical measures of disease severity were
unaffected by this microsatellite polymorphism, MS patients with
primary progressive disease course were found to have more
commonly long AAT repeats, in line with the idea that
neurodegenerative damage can be influenced in MS by CB1Rs
Thus, here we first investigated the impact of (AAT)n CNR1
repeats on CB1R expression, and then on the inflammatory
neurodegeneration processes responsible for irreversible disability
in MS patients. Our results provide initial evidence that long ($12
in both alleles) AAT repeats within the CNR1 gene reduce CB1R
expression in MS patients, and exacerbate the impact of
inflammation on neuronal integrity and function in the optic
nerve and in the brain of MS patients.
Materials and Methods
This study complied with the principles of the Declaration of
Helsinki, and was approved by the Ethical Committee of the
Policlinico Universita` Tor Vergata in Rome. All subjects gave
their written informed consent.
A total of 114 central-southern Italian subjects were included in
this study. All had a diagnosis of relapsing-remitting MS . MS
disease onset was defined as the first episode of focal neurological
dysfunction indicative of MS. Relapses were defined as the
development of new or recurrent neurological symptoms not
associated with fever or infection lasting for at least 24 h. Disease
duration was estimated as the number of years from onset to the
last assessment of disability.
At the time of confirmed diagnosis, all MS patients had started
disease-modifying therapy (glatiramer acetate 20 mg s.c. daily,
interferon beta 1a 44 mcg s.c. three times weekly, interferon beta
1a 30 mcg i.m., or interferon beta 1b 250 mcg s.c. every other
day). Mitoxantrone (12 mg/m2 i.v. every 3 months with a life-time
maximum of 140 mg/m2) and natalizumab (300 mg i.v. every
four weeks) were considered as second-line treatments.
Determination of AAT repeats in the CNR1 gene
Peripheral blood samples of MS patients were collected in BD
Vacutainer tubes containing EDTA (Beckton Dickinson, Franklin
Lakes, NJ). Genomic DNA was purified from 200 ml of human
whole blood using MagNA Pure LC DNA Isolation Kit (Roche
Diagnostics GmbH, Mannheim, Germany) in an automated
extractor MagNA Pure LC (Roche Diagnostics) according to the
manufacturers instructions. The CNR1 region containing the
AAT repeats was amplified by polymerase chain reaction (PCR)
from 150 ng of genomic DNA. PCR reaction was performed in a
final volume of 25 ml containing polymerase buffer, 1 mM MgCl2,
0.2 mM of each dNTP, 0.5 pmoles of each primer (sense:
59GTTGCAGTGAGCCAAGATCA-39) and 1.5 U Taq DNA
polymerase (Invitrogen, Madison, USA). Amplification reaction
consisted in an initial denaturation step at 94uC for 5 minutes,
followed by 35 cycles of denaturation at 95uC for 450, annealing at
58uC for 1.5 minutes and elongation at 72uC for 19, and a final
elongation step at 72uC for 79. Sequencing analysis were
performed from 10 ng of PCR products, purified with Agencourt
AMPure PCR Purification kit (Agencourt Bioscience Corporation,
Beverly, MA) in accordance with manufacturers instructions,
using 0.5 pmoles of the sequencing primer
(59-ACCTCCACCCACAAATCAAA-39) and the ABI PRISM BigDye Terminator
v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA). Sequencing reactions consisted in an initial
denaturation step at 96uC for 1 minute, followed by 40 cycles at
96uC for 10 seconds, 50uC for 5 seconds, and 60uC for 4 minutes.
Sequencing products were purified using CleanSEQ dye terminal
removal kit (Agencourt Bioscience Corporation) in accordance
with manufacturers instructions and run on the Applied
Biosystems 3730 DNA Analyzer Instrument (Applied Biosystems).
AAT repeats were counted on the resulting electropherograms.
Determination of CB1R protein expression
In a recent study we have demonstrated that rabbit anti-CB1R
antibodies (cat. 101500; Cayman Chemical Co., Ann Arbor, MI,
USA) recognize a specific band of the expected molecular mass of
CB1R in human peripheral lymphocytes, subjected to 10%
SDSPAGE and electroblotting . Specificity of the anti-CB1R
antibodies was ascertained by preincubating 1 mg of them with
10 mg of the specific blocking peptide (Cayman Chemical Co.),
that was able to fully erase the immunoreactive band . Here,
the same anti-CB1R antibodies were used in quantitative
enzymelinked immunosorbent assays (ELISA), performed on whole cell
lysates (20 mg/well). In addition, in order to draw dose-response
curves and ascertain the linearity range of the ELISA test, different
amounts of human CB1R-transfected Chinese hamster ovarian
cells (CHO-CB1, from Millipore, Bedford. MA, USA) were
analyzed as reported . Briefly, CHO-CB1 extracts (in the
protein range 040 mg/well) were incubated with primary
antiCB1R antibodies (1:500); in negative controls, the same antibodies
were pre-incubated with the specific CB1 blocking peptide (1:10
ratio) for 3 h at room temperature. After incubation with alkaline
phosphatase-conjugated secondary antibody (1:2000 dilution;
BioRad, Hercules, CA, USA), color development of the alkaline
phosphatase reaction was measured at 405 nm, using
p-nitrophenyl phosphate as substrate . A405 values of lymphocyte extracts
were always within the linearity range of the calibration curves
drawn with CHO-CB1R cell homogenates, and were used to
estimate CB1R content in human lymphocytes.
Medical history with respect to visual symptoms was taken from
all MS subjects. Self-report and physician report were confirmed
by record review.
A sample of MS patients with no history of ophthalmological
disease (n = 70) underwent measurement of retinal nerve fiber
layer (RNFL) thickness, Macular Volume (MV) for both eyes using
Stratus Optical Coherence Tomography ([OCTTM] software
version 4.0.2, Carl Zeiss Meditec, Inc.). Briefly, for MV, retinal
thickness was measured automatically as the distance between the
vitreoretinal interface and the anterior boundary of the retinal
pigment epithelium. Stratus OCT images were generated using
the fast map scan protocol consisting of six radial scans spaced 30u
apart, with each scan measuring 6 mm in length. Each image had
a resolution of 10 mm axially and 20 mm transversally. All Stratus
OCT images had a signal strength of 6 mm. RNFL thickness
measurements were read from the automated measurements
generated by the machine using the Fast RNFL analysis. For the
study scanning was performed after pharmacological dilation.
Average RNFL thickness for 360u around the optic disc was
recorded. Values were adjusted for age.
A subset of patients (n = 32) had a clinical history of previous
optic neuritis (ON) in at least one eye, 3 months or more before
examination (ON group). The remaining subjects had never been
affected by ON (n = 38, nON). One randomly chosen eye from
subjects of nON group was included in the study. The
ONaffected eye was chosen for subjects of the ON group.
Visual acuity was measured by a Snellen 20-foot wall chart. All
subjects included had visual acuity values of 1.0 (Snellen
equivalent of 20/20; with or without correction) of both eyes.
Low Contrast Visual Acuity (LCVA) testing was performed using
retroilluminated low-contrast Sloan letter charts (1.25% contrast
at 2 m). Testing was performed by trained technicians experienced
in examination of patients for research studies.
Magnetic resonance imaging (MRI) data acquisition
Thirty-seven out of 114 MS patients had an MRI scan at 3T
(Siemens Magnetom Allegra). The maximum gradient strength is
40 mTm1, with a maximum slew rate of 400mTm-1ms-1. The
MRI session included for every subject: (1) a dual-echo turbo spin
echo (TSE) (TR:6190 ms; TE1: 12 ms; TE2: 109 ms; echo train
length [ETL]: 5; matrix: 2566192; field of view [FOV]:
2306172.5 mm2; 48 contiguous 3 mm thick slices) for lesion
identification and segmentation (scan time: approximately 4 min);
(2) a fluid attenuated inversion recovery (FLAIR) scan (TR:
8170 ms; TE:96; ms; Tl :2100 ms; ETL: 13; same FOV, matrix
and number of slices as TSE) to use as a reference for lesion
identification (scan time: 5 min); (3) morphological 3D
T1weighted magnetization prepared rapid acquisition gradient echo
(MPRAGE) (TE = 2.74 ms, TR = 2500 ms, inversion
time = 900 ms; flip angle = 8u; matrix = 25662086176;
FOV = 25662086176mm3).
MRI lesion segmentation
T2-hyperintense lesions were identified by consensus by two
observers on the short echo images (proton density-weighted) of
the TSE, for every patient. Lesions were outlined on the same scan
using a semi-automated local thresholding contouring software
(Jim 4.0, Xinapse System, Leicester, UK, http://www.xinapse.
com/). FLAIR and T2-weighted scans were always used as a
reference to increase confidence in lesion identification.
Voxel-based morphometry (VBM). The T1-weighted volumes
(MPrage) were processed using the voxel-based morphometry
protocol in SPM8 (http://www.fil.ion.ucl.ac.uk/spm/), an
iterative combination of segmentations and normalizations to produce
a grey matter (GM) probability map in standard space (Montreal
Neurological Institute, or MNI coordinates) . In order to
compensate for compression or expansion which might occur
during warping of images to match the template, GM maps were
modulated by multiplying the intensity of each voxel in the final
images by the Jacobian determinant derived from the deformation
field . GM maps were then smoothed using a 10-mm FWHM
Statistical analysis was performed in SPM8, as detailed below.
For each tissue class (GM, white matter and CSF), every
patients global volume was estimated by integrating the intensity
values over the whole segmented image. The following quantities
were then derived: the total brain volume, computed as the sum of
white and grey matter volume, the intracranial volume, computed
as the sum of the total brain volume and the total CSF volume,
and the brain parenchymal fraction (BPF), equal to the brain
volume to intracranial volume ratio.
Neuropsychological and disability assessment
All 114 patients underwent the neuropsychological and
disability assessment by expert neurologists and
neuropsychologists, who were blinded to laboratory and MRI results. All patients
undergoing MRI examination were clinically and
neuropsychologically examined within 1 week interval.
Expanded Disability Status Scale (EDSS) , a 10-point
disease severity score derived from nine ratings for individual
neurological domains, was administered to all MS patients by a
trained and certified examining neurologist. Progression Index (PI)
was defined as EDSS disease duration.
Cognitive functions were assessed using the Brief Repeatable
Neuropsychological Battery (BRB)  and the Stroop Test (ST)
. The BRB assesses the cognitive domains most frequently
impaired in MS subjects  and incorporates tests of verbal
memory (SRT), visual memory (10/36 SPART), attention,
concentration and speed of information processing (Paced
Auditory Serial Addition Test [PASAT]; Symbol Digit Modality
Test [SDMT]) and verbal fluency (WLG). Moreover, the ST was
administered to evaluate frontal lobe executive functions, which
are not assessed by the BRB . Performance on each test of the
BRB and on the ST was assessed by applying the available Italian
normative values . In particular, failure of a test was defined
when the score was at least two standard deviations (SDs) below
the mean normative values. Consistently with previous works
[28,29], those patients who failed at least three tests were
considered CI (cognitive impaired), and those who failed less than
three tests were considered CP (cognitive preserved). A grading
system was applied to each patients score on each cognitive test,
dependent on the number of SDs below the normative mean (0:
patient scored at or above normative mean; 1: patient scored #1
SD below normative mean; 2: patient scored .1 SD, but #2 SD
below normative mean, etc.). The sum of these grades was
determined across all variables to give the cognitive impairment
index (CII), a single overall measure of cognitive impairment for
each patient . Cognitive tests were repeated after 18
months, using versions B of the BRB. Subjects with cognitive
decline were defined those who had a CII change of $2 points
Higher-order cognitive executive functions were assessed by
Delis-Kaplan Executive Function System (D-KEFS) Sorting Test
 in a subgroup of subjects (n = 62). It measures the examinees
ability of problem-solving behavior. In the free sorting condition,
the examinee is presented with mixed-up cards that display both
stimulus words and various perceptual features. The examinee is
asked to sort the cards into two groups, according to as many
different categorization rules, or concepts as possible and to
describe the concepts used to generate each sort. In the sort
recognition condition, the same sets of cards are each sorted by the
examiner into two groups. We analyzed the total confirmed
correct sorts, which represent the number of correct sorts for
which the verbal description is awarded one or more points and
the combined free description score (FDS), which is based on the
sum of correct description scores in the free sorting and sort
recognition condition [33,34]. Scaled D-KEFS scores were used in
the analysis. Depression was assessed through the Montgomery
and Asberg Depression Rating Scale . Subjects scoring
moderate to severe depression were not included. No subject
was taking psychoactive drugs or substances that might interfere
with neuropsychological performance. Subjects were tested after at
least 3 months from previous relapse and/or detection of active
scans at MRI.
According to our previous report  MS subjects were divided
into two groups according to the AAT repeat polymorphism of
CNR1 gene (short AAT: homozygous or heterozygous for allele
with #11 repeats of AAT triplets; long AAT: homozygous for
allele with $12 repeats of AAT triplets). Differences between two
groups were analyzed using Students t-test, MannWhitney test,
and Fisher exact test, as appropriate. Correlation analysis was
performed by calculating Spearman coefficients. Immunochemical
data were reported as the mean 6 standard error (SE) of
independent determinations, each performed in duplicate. Other
data were presented as mean 6 SD. A p-value (p) of less than 0.05
was considered statistically significant. The average lesion load,
brain volume and BPF were compared between groups using
Students t-tests. VBM statistical analysis was performed using a
full factorial design, where a 2 level factor was used to model the
group defined by the genotype and the lesion load. The interaction
between group and lesion load was also modeled. Age and gender
were entered as nuisance covariates to adjust for potential
confounds. P-values were accepted as significant if lower than
0.05 familywise error rate (FWER), corrected for multiple
comparisons at cluster level. Global GM volumes, derived from
VBM segmentation, were also used to explore potential
interactions between AAT repeat polymorphism of CNR1 gene and
patients performance at the administered cognitive tests.
Multivariate prognostic models were constructed for the cognitive
performance as outcome. The association between genotype and
both overall CI and specific impairment in different cognitive tests
was assessed using multivariate binary logistic regression models.
The association between genotype and cognitive performance as
measured by both the overall CII and D-KEFS variables was
assessed using multivariate linear regression models. Clinical and
demographic variables (disease duration, age, gender, educational
level, EDSS) and genotype were included as predictor variables in
the two models. Age was not entered as potential confound in
analysis of D-KEFS variables, because the values were already
corrected for age. Finally, the assessment of the association
between genotype and CI was replicated in the subgroup of
subjects with shorter disease duration (years of disease ,10) and
less disability (EDSS ,2.5). The association between genotype and
cognitive decline was also assessed by performing a logistic
regression model with disease duration, age, gender, educational
level, EDSS, baseline CII and genotype as predictor variables.
Two-way ANOVAs were performed to analyzed the main effects
of two conditions (genotype versus CI or genotype versus ON) on
the dependent variables (MRI or ophthalmologic variables) and
their interactions. MRI was limited to 37 patients of the whole
studied population, and safety concerns and claustrophobia were
the main reasons to refuse MRI. Similarly, only 70 (21 also had
MRI) out of 114 subjects accepted the ophthalmological
assessment with OCT.
CNR1 (AAT)n controls the expression level of CB1R in MS
To measure the impact of AAT repeats in the CNR1 gene on
CB1R expression, we generated in preliminary experiments a
calibration curve with increasing amounts of CHO-CB1R cell
extracts incubated with anti-CB1R antibodies. Total binding (TB)
of cell extracts was quantified, and was compared to the
nonspecific binding (NSB) of the same samples reacted with
anti-CB1R antibodies that had been pre-incubated with the
blocking peptide. NSB values were then subtracted from TB
values, in order to calculate the specific binding (SB) of the cell
extracts . The dose-dependence curves of TB, NSB, and SB of
different amounts of CHO-CB1R cell extracts are reported in
Fig. 1A, that clearly shows the specificity of the anti-CB1R
antibodies used in this study.
On this background, ELISA tests were used under the same
conditions to calculate SB values, and hence CB1R content, in
lymphocytes from the short (n = 10) and the long AAT
group (n = 12). A significant (p = 0.0335) reduction of CB1R
expression was observed in the long AAT group (CB1R
content = 0.11060.016 mg per mg of protein) with respect to the
short AAT group (CB1R content = 0.15660.011 mg per mg of
total protein) (Fig. 1B,C).
Clinical and genetic characteristics of MS patients
Nine allelic variants of the AAT repeats were found in the
studied population. According to used nomenclature , allele 2
corresponded to (AAT)8, allele 3 to (AAT)9, allele 4 to (AAT)10,
allele 5 to (AAT)11, allele 6 to (AAT)12, allele 7 to (AAT)13, allele 8
to (AAT)14, allele 9 to (AAT)15, allele 10 to (AAT)16. We failed to
detect allele 1 in our sample. The alleles most frequently found
were number 5 (26.7%), number 8 (25.0%) and number 9 (21.1%),
in line with previous observations . We performed the
subsequent analysis classifying CNR1 alleles into a short and a
long group, as previously reported .
The two genotype groups (short and long AAT) did not differ in
terms of the main clinical and demographic characteristics
(Table 1). Of note, PI (EDSS/disease duration) was higher in
the long AAT group (0.5360.45 versus 0.3160.27; p,0.01),
according to the previous reported association between (AAT)n
repeat polymorphism of CNR1 gene and disease progression in
relapsing-remitting MS patients . EDSS and disease duration
were therefore taken into account as confounding factors during
CNR1 (AAT)n influences the relationship between
inflammation and neuronal damage in the optic nerve
Axonal and neuronal loss in MS has been convincingly
associated with reduced RNFL thickness and MV at the OCT.
The availability of these noninvasive measures provides a unique
potential for in vivo investigation of factors associated with axonal
loss secondary to inflammatory demyelination or underlying
primary neurodegeneration, by analyzing optic nerves from ON
eyes or from unaffected eyes, respectively . Thus, we
investigated the possible relationship between genotype and OCT
parameters in MS patients of both short and long AAT repeats
groups. To evaluate the involvement of CNR1 polymorphism on
the structural effects of an inflammatory insult to the optic nerve,
MS patients were classified in those who had previously suffered
from ON in at least one eye (n = 32) and those who had not (nON,
n = 38), on the basis of their clinical history. Two-way ANOVAs
were performed to analyzed the main effects of the two conditions
(genotype versus ON) on the dependent variables (ophthalmologic
variables) and their interactions. A significant main effect of ON
condition was revealed analyzing both RNFL thickness (F = 39.86,
p,0.0001) and MV (F = 25.26, p,0.0001), indicating a damage of
neuronal structures after ON. Interestingly, subjects with short
AAT repeats presented higher values of RNFL thickness and MV
despite ON, suggesting less severe neurodegenerative damage after
inflammatory events. In line with this, a significant interaction
between genotype and ON condition was found (RNFL thickness:
F = 5.57, p = 0.02; MV: F = 11.92, p = 0.001). Conversely,
genotype per se failed to significantly affect OCT parameters (RNFL
thickness: F = 2.13, p = 0.15; MV: F = 2.90, p = 0.09), confirming
the selective involvement of CNR1 polymorphism in limiting
axonal loss secondary to inflammatory demyelination but not
primary neurodegeneration (Fig. 2A,B).
CNR1 (AAT)n influences optic nerve function
Consistent results were obtained at the LCVA test, an emerging
visual functional outcome . In fact, a significant main effect
(F = 13.71, p,0.001) of ON condition and a significant interaction
(F = 12.37, p = 0.001) between the two conditions (genotype X
ns, not significant; M, male; F, female; EDSS, Expanded Disability Status Scale.
ON) were revealed. In line with previous results, genotype failed to
significantly affect LCVA (F = 1.12, p = 0.29) (Fig. 2C). These
findings further support the idea that the short AAT repeat
EDSS median (range) 2.0 (06.0)
EDSS, Expanded Disability Status Scale; BPF, brain parechymal fraction; M, male;
F, female; LL, Lesion Load. No significant between group difference was found
for any of these variables.
genotype is associated with less severe neuronal damage and visual
impairment secondary to neuroinflammation in MS patients.
CNR1 (AAT)n influences the relationship between lesion
load and grey matter atrophy in MS brains
In the population of patients who underwent MRI, no
significant group differences were found between individuals
who carried the short or the long ATT repeat with respect to
mean age, lesion load, total brain volume, BPF, disease duration
and gender distribution (Table 2). Also, no significant differences
were found between the two patient groups in regional GM
volumes (p,0.001, uncorrected). Conversely, a significant
negative association (p,0.05, FWE-corrected) was found between
regional GM volumes and lesion load (Fig. 3) across the whole
population of subjects, indicating that MS patients with larger
lesion load tend to develop more GM atrophy within the
thalamus, the head of the caudate nucleus and the cingulate
cortex bilaterally, in the right insular cortex, and in the left
postcentral gyrus. Interestingly, areas of significant (p,0.05, FWE
corrected) group by lesion load interaction were found in the left
frontal and cingulate cortex and in the right temporal cortex
(Fig. 4, left). In those areas, the inverse correlation between lesion
load and GM matter volume found in patients with the long AAT
genotype was lost in those with short AAT repeats. These results
indicate that, while in the former group patients with higher lesion
load tend to develop more GM atrophy, in the second group this
relationship breaks down, as expected for a relative preservation of
selected GM areas in these subjects. This is also confirmed by the
plot (top, right) and the scatterplot (bottom, right of Fig. 4).
CNR1 (AAT)n regulates cognitive abilities in MS patients
The global GM volume was lower, although not significantly,
among subjects with cognitive impairment (CI: n = 15,
602.8626.6 ml; CP: n = 22, 627.6613.4 ml, p.0.05) and a non
significant inverse correlation was found with CII (n = 37,
r = 20.20, p.0.05). On the other hand, a detrimental role of
the AAT long genotype on global GM volumes was found in
subjects who failed specific tasks of executive functions. In fact a
significant interaction between genotype and the failure of Word
List Generation (WLG) test was revealed analyzing GM volumes,
accounting for approximately 17.17% of the total variance (n = 37,
F = 7.29, p = 0.01; Fig. 4A), suggesting the relative preservation of
neuronal structures in subjects with short AAT repeats. In line
with this, an interaction, despite non statically significant, was
found between genotype and the failure of ST, accounting for
approximately 8.35% of the total variance (n = 37, F = 3.03,
p = 0.09; Fig. 5B). Conversely, no significant interactions were
found on global GM volumes between genotype and failure of
sustained attention tests (PASAT: n = 37, F:1.86, p = 0.18; SDMT:
n = 37, F = 0.05, p = 0.82).
We further explored whether CNR1 (AAT)n polymorphism
affects recruitment of cognitive-related networks by altering
synaptic plasticity. MS patients were therefore classified as CI
(n = 29)] or CP (n = 85) on the basis of their neuropsychological
performance. CNR1 (AAT)n polymorphism was found to predict
cognitive impairment, since the long AAT group had higher
probability of failing more than two cognitive tests at equal values
of age, disease duration, gender, education level and EDSS score.
The response variable (overall CI in our model) was significantly
affected also by disease duration (Table 3). Accordingly, CI
subjects were more frequent in the long AAT group (Fig. 5C;
The same results were obtained when overall cognitive
performance was assessed through the CII independently of the
presence of CI. CII was higher among subjects of the long AAT
group (Fig. 5D) and related to genotype (F = 10.46, p = 0.001).
Also disease duration (F = 14.59, p = 0.0002) and EDSS
(F = 15.24, p = 0.0002) explained a considerable portion of the
CNR1 genotype and cognitive impairment were also associated
to each other when the analysis was restricted to those patients
with less remarkable disability (EDSS,2.5) and shorter disease
duration (years of disease,10). Also in this subgroup of subjects
(n = 62), in fact, genotype (coefficient: 2.70, SE: 1.08, odds ratio
(OR): 14.87, p = 0.01) and disease duration (coefficient: 0.46, SE:
0.22, OR: 1.59, p = 0.03) predicted cognitive impairment. CII was
confirmed to be related to genotype, explaining in this model the
most portion of the total variance (genotype: F = 9.73, p = 0.003;
disease duration: F = 4.77, p = 0.03; EDSS: F:2.81, p = 0.09).
Furthermore, CNR1 polymorphism was associated with higher
risk of impairment on neuropsychological tests exploring executive
functions (WLG and ST) but not on tasks of memory and
sustained attention (Table 4). To better explore the association
between the number of AAT repeats and cognitive performance,
further analyses were performed using the total number of AAT
repeats on the two genes for each subject. Of note, the overall CII,
WLG and ST scores were found to be related to the total number
of AAT repeats for each subject (Fig. 5EG; n = 114; CII: r = 0.26,
p = 0.005; WLG: r = 20.28, p = 0.002; ST: r = 0.23, p = 0.01). No
significant correlations were found analyzing tests of sustained
attention and memory (n = 114; PASAT: r = 0.08, p = 0.40,
Fig. 5H; SDMT: r = 0.02, p = 0.86; Spatial Recall Test [SPART]:
r = 0.05, p = 0.61; Selective Reminding Test [SRT]: r = 20.002,
p = 0.97).
To confirm the impact of CNR1 (AAT)n polymorphism on
executive functions we also administered D-KEFS Sorting test to
the subgroup of patients with shorter disease duration and less
disability (n = 62). D-KEFS Sorting test is a standardized executive
function test designed to assess higher cognitive functions .
Subjects with long AAT repeats had lower FDS and total
confirmed correct sorts, both being related to genotype (FDS:
F = 29.58, p,0.0001; correct sorts: F = 8.88, p = 0.004) (Fig. 5I).
Finally, the long AAT repeat genotype was found to predict
cognitive decline, since this group had higher probability of
worsening in CII at equal values of age, disease duration, gender,
education level, EDSS score and baseline CII. The response
variable was significantly affected also by age and baseline CII
Candidate gene association studies are potentially useful in
determining genetic influences on disease progression in MS and a
neuropsychological outcome measure may more closely relate to
the burden of disease than measures of physical disease
progression. We have already reported that the long alleles of
(AAT)n repeat polymorphism of CNR1 gene (.11 repeats)
represent a genetic risk factor for disease progression in
relapsing-remitting MS .
Here, we have provided initial information on the biological
impact of this polymorphism on CB1R protein expression in a
sample of MS patients, and we have addressed its functional
consequences on inflammation-induced optic nerve and brain
structural damage, as well as on visual and cognitive functioning.
To strengthen our conclusions, however, the impact of CNR1
(AAT)n repeat polymorphism on CB1R mRNA levels should also
be addressed, along with measurements in a larger independent
population of MS patients, by using other CB1R-specific
antibodies, and in healthy individuals. Based on the results of
the present investigation, in fact, we cannot exclude that the
(AAT)n repeat polymorphism affects CB1R function only in the
context of MS disease, and not in control subjects. Brain
inflammation, in fact, can reduce per se CB1R function in EAE
mice , raising the possibility that long (AAT)n repeats within
the CNR1 gene only impact on the effects of the inflammatory
milieu, likely interleukin-1 , on CB1R expression and
function. Also this concept, however, requires further
The hypothesis that CNR1 (AAT)n influences the relationship
between inflammation and neuronal atrophy in MS was first
demonstrated by OCT evaluation. OCT provides noninvasive
means to quantify the structural effects of an inflammatory insult
to the optic nerve, which can then be compared to functional
outcomes, to construct a structural-functional paradigm of central
nervous system (CNS) injury . In fact, OCT can be used to
measure the RNFL thickness and the MV, that are both reduced
following the development of MS and ON [38,45,present work],
and can therefore be used as a correlate of global axonal loss .
Changes in RNFL thickness after ON have been interpreted to
reflect initial axoplasmic flow stasis and subsequent attrition
caused by inflammation in the anterior visual pathway. Recent
studies have shown that the extent of RNFL atrophy correlates
with MRI measures of optic nerve, brain atrophy and disease
activity in MS patients [41,46,47]. Here we showed a significant
association between CNR1 genotype and ON condition, analyzing
the extent of RNFL thickness and MV reduction, thus indicating
the relative preservation of neuronal structures after inflammatory
events in subjects with short AAT repeats. Finally, similar results
were obtained analyzing the LCVA test, an emerging visual
functional outcome . These findings are consistent with the
idea that short AAT repeats in CNR1 gene favor the functional
compensation to the neuronal loss secondary to inflammation in
the CNS of MS patients.
Brain tissue damage presents in MS with macroscopic white
matter lesions, as well as with microscopic damage to the so-called
normal appearing white (NAWM) and grey matter (NAGM)
[48,49]. Damage to the GM is known to be due to either the
accumulation of cortical MS lesions or to the occurrence of
Wallerian degeneration secondary to white matter (WM) damage.
GM damage has been found to play a critical role in determining
motor and cognitive disabilities in MS patients, and in accounting
(at least partially) for the progression from relapsing to secondary
progressive MS course. In the current study, using VBM for
regional GM volumetrics, we found an expected direct association
between lesion load and accumulation of regional GM atrophy,
when considering our MS patients as a whole group. Within the
limitations of the cross-sectional design of this study, and taking
into account the relatively small sample, this confirms that in MS a
relevant proportion of GM damage is explained by accumulation
of macroscopic WM lesions, and the consequent Wallerian
degeneration of GM. However, when considering the interaction
between the genetic pattern of AAT repeats and the relationship
between lesion load and regional GM volumes, we found that the
presence of short AAT repeats had a significant impact on
breaking down this relationship in several cortical locations. This
suggests that the presence of short AAT repeats reduces the WM
damage in MS brains, and its contribution in determining
secondary degeneration of the GM tissue.
Interestingly, the set of GM regions, whose atrophy was found
to be attenuated by the presence of short AAT repeats, includes
areas with a major role in cognition, and in fact we also found that
MS patients with short AAT repeats had less cognitive impairment
than the long AAT patients. Accordingly, we have found that long
AAT MS subjects had a major risk to develop cognitive
impairment and to incur into cognitive decline, at equal values
95% confidence interval
proportion of failure
(short/long AAT group)
95% confidence interval
PASAT, Paced Auditory Serial Addition Test; SDMT, Symbol Digit Modalities Test; SRT-LTS, Selective Reminding Test Long Term Storage; SRT-CLTR, Selective Reminding
Test Consistent Long Term Retrieval; SRT-D, Selective Reminding Test Delayed; SPART, Spatial Recall Test;SPART-D, Spatial Recall Test Delayed; WLG, Word List
Generation; ST, Stroop Test; OR, Odds Ratio.
of age, disease duration, gender, education level and physical
disability. The same results were obtained when overall cognitive
performance was assessed through the CII independently of the
presence of cognitive impairment. Therefore, we confirmed our
results studying the relationship between CNR1 alleles and
cognitive performance in a condition in which possible
confounders, such as disease duration and disability levels, may have a
MS-related cognitive impairment has been consistently
associated with brain atrophy also in the earliest disease stages
[27,50,51], and damage to several GM structures can be
associated with impairment of specific cognitive functions .
Here we have demonstrated that variants of CNR1 gene have a
direct effect on executive functioning measured by WLG test, ST
(inhibition of automatic response), D-KEFS Sorting test (verbal/
nonverbal modality-specific problem-solving skills, ability to
transfer sorting concepts into action and ability to inhibit previous
description responses to engage in flexibility of thinking). Damage
to the frontal and prefrontal cortex leads to impairment in
executive functioning, which normally allows individuals to
effectively engage in complex goal-directed behaviors .
Of note, the contribution of inflammatory WM damage in
determining secondary degeneration of the GM tissue was
attenuated in subjects with short AAT repeats at the level of
cerebral areas involved in these processes. In line with this, these
subjects scored better in executive function tests than the long
AAT group. Of note, the extent of GM atrophy was not
significantly related to global cognitive impairment in our sample,
but a clear interaction between genotype and cognitive
performance was found by analyzing GM volume, suggesting the relative
preservation of neuronal structures in subjects with short AAT
To produce cognitive performance similar to healthy controls,
MS patients require greater recruitment of prefrontal cortical
regions [56,57] and greater deactivation of the anterior cingulate
cortex , the core components of the brains default network,
which consists of brain regions more active during rest or passive
thought than directed cognitive processing. Among healthy
individuals, prefrontal recruitment is positively associated with
age . Therefore, the major extent of GM atrophy secondary to
neuroinflammation observed among subjects homozygous for long
AATn (present work) might disrupt the activation of cerebral
networks essential to limit the negative impact of brain disease on
In conclusion, our study points to CB1R as an interesting
molecular target for preventing neuronal loss and cognitive
impairment in MS as well as in other CNS disorders in which
inflammation-driven neurodegeneration process play a role.
95% confidence interval
CII, Cognitive Impairment Index; EDSS, Expanded Disability Status Scale; SE, Standard Error; OR, Odds Ratio.
Conceived and designed the experiments: SR M. Bozzali M. Bari FM RM
CN SB MM DC. Performed the experiments: SR M. Bari FM VS CM FS
NM M. Cercignani FB PG M. Castelli. Analyzed the data: SR M. Bozzali
M. Cercignani VS CM PG MM. Wrote the paper: DC SR M. Bozzali.
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