Association between a genetic variant of type-1 cannabinoid receptor and inflammatory neurodegeneration in multiple sclerosis

Abstract

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.

Figure 1. CB₁R protein expression in MS lymphocytes.

A. Calibration curve of the ELISA test with different concentrations of CHO-CB₁ cell extracts. Specific binding (SB) of anti-CB₁ antibody to CHO-CB₁ cell extracts followed the equation y = 0.01133×+0.01951 (R² = 0.9775). B. Dispersion graphs of CB₁ protein content in human lymphocytes from short AAT group (white circles and bar) and long AAT group (black circles and bar) of patients. C. Histogram of cumulative data shown in panel B. *p = 0.0335.

Figure 2. CNR1 (AAT)n influences the relationship between inflammation and neuronal atrophy in MS.

A–C. Plot of interaction analysis between CNR1 genotype and previous optic neuritis (ON), analyzing RNFL thickness, MV and LCVA. A significant interaction between genotype and ON condition was found analyzing both RNFL thickness (A) and MV (B), confirming less severe neurodegenerative damage after inflammatory events in subjects with short AAT repeats. A significant interaction between the two conditions (genotype X ON) were also revealed by LCVA analysis (C), supporting the idea that the short AAT repeat genotype is associated with less severe visual impairment secondary to neuroinflammation in MS patients.

Figure 3. Association between lesion load and GM atrophy in MS brain.

There are shown here in red, those brain areas in which local GM volumes are negatively associated with T2-weigthed lesion loads in the whole MS population. This association indicates that MS patients with larger lesion load tend to develop more GM atrophy in the thalamus, the head of the caudate nucleus and the cingulate cortex bilaterally, in the right insular cortex, and in the left postcentral gyrus. Statistical threshold: p values FWE-corrected <0.05. Spatial coordinates (x,y,z) in the figure are in MNI space.

Figure 4. Interaction between the CNR1 genotype and T2-weighted lesion loads on regional GM volumes.

In yellow, there are shown those areas (i.e., left frontal and cingulate cortex, and right temporal cortex) in which an inverse correlation exists between T2-lesion load and local grey matter volumes in individuals with long AAT repeats, but that is absent in those with short AAT repeats. The plot on the top right shows this effect of interaction in the left frontal and cingulate area. This same effect is also confirmed, on a subject by subject basis, by the sctatterplot on the bottom right. Statistical threshold: p values FWE-corrected <0.05. Spatial coordinates (x,y,z) in the figure are in MNI space. Abbreviations: LL = T2-weighted lesion load; GM = grey matter.

Figure 5. CNR1 (AAT)n regulates cognitive abilities in MS patients.

A, B. Plot of interaction analysis between CNR1 genotype and performance at WLG test (A) or ST (B), analyzing the GM volume. These data confirm the relative preservation of neuronal structures after inflammatory events in subjects with short AAT repeats. C. Cognitive impairment was more frequent in MS subjects homozygous for long (AAT)n repeats in CNR1 gene than short AAT repeats group. D. CII was higher among subjects of long AAT group. E–H. Correlation plots between CII (E), WLG (F), ST (G), PASAT (H) and the total number of AAT repeats on the two genes are shown to better demonstrate the association between the number of AAT repeats and cognitive performance. I. Subjects carrying short AAT repeats scored better at D-KEFS, an executive function test.