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Activating cannabinoid receptor 2 preserves axonal health through GSK-3β/NRF2 axis in adrenoleukodystrophy

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Janani Parameswaran at Emory University
Janani Parameswaran
Leire Goicoechea
Leire Goicoechea
Leire Goicoechea
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Laura Planas-Serra
Laura Planas-Serra
Laura Planas-Serra
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Antoni Pastor at IMIM Hospital del Mar Medical Research Institute
Antoni Pastor
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Abstract and Figures

Aberrant endocannabinoid signaling accompanies several neurodegenerative disorders, including multiple sclerosis. Here, we report altered endocannabinoid signaling in X-linked adrenoleukodystrophy (X-ALD), a rare neurometabolic demyelinating syndrome caused by malfunction of the peroxisomal ABCD1 transporter, resulting in the accumulation of very long-chain fatty acids (VLCFAs). We found abnormal levels of cannabinoid receptor 2 (CB2r) and related endocannabinoid enzymes in the brain and peripheral blood mononuclear cells (PBMCs) of X-ALD patients and in the spinal cord of a murine model of X-ALD. Preclinical treatment with a selective agonist of CB2r (JWH133) halted axonal degeneration and associated locomotor deficits, along with normalization of microgliosis. Moreover, the drug improved the main metabolic disturbances underlying this model, particularly in redox and lipid homeostatic pathways, including increased lipid droplets in motor neurons, through the modulation of the GSK-3β/NRF2 axis. JWH133 inhibited Reactive Oxygen Species elicited by excess VLCFAs in primary microglial cultures of Abcd1-null mice. Furthermore, we uncovered intertwined redox and CB2r signaling in the murine spinal cords and in patient PBMC samples obtained from a phase II clinical trial with antioxidants (NCT01495260). These findings highlight CB2r signaling as a potential therapeutic target for X-ALD and perhaps other neurodegenerative disorders that present with dysregulated redox and lipid homeostasis.
The ECS is altered in normal-appearing brain white matter (NAWM) of cALD and cAMN patients and the Abcd1⁻ mouse spinal cord. a EC levels (2-AG, 2-LG, 2-OG, AEA, DEA, DGLEA, DHEA, LEA, OEA, and POEA) were measured in NAWM from controls [child (n = 10) and adult controls (n = 13)] and from cALD patients (n = 7) and cAMN patients (n = 13). b Relative gene expression of biosynthetic enzymes (NAPEPLD, DAGLα, and DAGLβ), catabolic enzymes (MAGL and FAAH), and cannabinoid receptors 1 and 2 (CB1r/CNR1 and CB2r/CNR2) was measured by quantitative Reverse transcription-polymerase chain reaction (RT-PCR) in NAWM from controls [child (n = 11) and adult controls (n = 15)], cALD patients (n = 7) and cAMN patients (n = 15). Gene expression was normalized to RPLP0 expression and is represented as the fold change compared to the level in children. c EC (2-AG, 2-LG, 2-OG, AEA, DEA, DHEA, LEA, OEA, PEA, POEA, and SEA) levels were measured in the spinal cords of WT (n = 9) and Abcd1⁻ (n = 8) mice at 12 months of age. d Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptors 1 and 2 (Cb1r/Cnr1, Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 6) and Abcd1⁻ (n = 7) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in WT mice. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). * p < 0.05; ** p < 0.01; *** p < 0.001 after unpaired Student’s t-test or two-way ANOVA followed by Fisher’s post hoc test. #p < 0.05; ##p < 0.01; ###p < 0.001 after the nonparametric Kruskal–Wallis test followed by Wilcoxon’s post hoc test
The ECS is altered in normal-appearing brain white matter (NAWM) of cALD and cAMN patients and the Abcd1⁻ mouse spinal cord. a EC levels (2-AG, 2-LG, 2-OG, AEA, DEA, DGLEA, DHEA, LEA, OEA, and POEA) were measured in NAWM from controls [child (n = 10) and adult controls (n = 13)] and from cALD patients (n = 7) and cAMN patients (n = 13). b Relative gene expression of biosynthetic enzymes (NAPEPLD, DAGLα, and DAGLβ), catabolic enzymes (MAGL and FAAH), and cannabinoid receptors 1 and 2 (CB1r/CNR1 and CB2r/CNR2) was measured by quantitative Reverse transcription-polymerase chain reaction (RT-PCR) in NAWM from controls [child (n = 11) and adult controls (n = 15)], cALD patients (n = 7) and cAMN patients (n = 15). Gene expression was normalized to RPLP0 expression and is represented as the fold change compared to the level in children. c EC (2-AG, 2-LG, 2-OG, AEA, DEA, DHEA, LEA, OEA, PEA, POEA, and SEA) levels were measured in the spinal cords of WT (n = 9) and Abcd1⁻ (n = 8) mice at 12 months of age. d Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptors 1 and 2 (Cb1r/Cnr1, Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 6) and Abcd1⁻ (n = 7) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in WT mice. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). * p < 0.05; ** p < 0.01; *** p < 0.001 after unpaired Student’s t-test or two-way ANOVA followed by Fisher’s post hoc test. #p < 0.05; ##p < 0.01; ###p < 0.001 after the nonparametric Kruskal–Wallis test followed by Wilcoxon’s post hoc test
… 
A CB2r agonist normalizes locomotor deficits and prevents microgliosis and axonal degeneration but not astrogliosis in DKO mice. a, b Bar cross and treadmill tests were performed on a 16-month-old WT (n = 14), Abcd1⁻/Abcd2−/− (DKO) (n = 16) and Abcd1⁻/Abcd2−/−mice treated with a CB2r agonist (DKO + JWH133) (n = 14) and b 18-month-old WT (n = 14), DKO (n = 16) and DKO + JWH133) (n = 14) mice. The number of slips and time (seconds) spent crossing the bar were quantified in the bar cross test. In the treadmill test, the latency to fall off the belt (time until shock) and the number of shocks received were computed after 7 min. c–t Immunohistological analysis of axonal pathology performed in 18-month-old WT, DKO, and DKO + JWH133 mice (n = 4 per genotype and condition). Spinal cord sections were processed for c–e Iba1, f–h GFAP, i–k synaptophysin (SYN), l–n APP, o–q Sudan black, and r–t SMI32 staining. Representative images for WT (c, f, i, l, o, r), DKO (d, g, j, m, p, s) and DKO + JWH133 (e, h, k, n, q, t) are shown. Scale bars = 25 μm (c–t). u Quantification of synaptophysin and APP accumulation and the densities of Iba1⁺ and GFAP⁺ (cells/mm²). Panels r–t are from the anterior horn. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
A CB2r agonist normalizes locomotor deficits and prevents microgliosis and axonal degeneration but not astrogliosis in DKO mice. a, b Bar cross and treadmill tests were performed on a 16-month-old WT (n = 14), Abcd1⁻/Abcd2−/− (DKO) (n = 16) and Abcd1⁻/Abcd2−/−mice treated with a CB2r agonist (DKO + JWH133) (n = 14) and b 18-month-old WT (n = 14), DKO (n = 16) and DKO + JWH133) (n = 14) mice. The number of slips and time (seconds) spent crossing the bar were quantified in the bar cross test. In the treadmill test, the latency to fall off the belt (time until shock) and the number of shocks received were computed after 7 min. c–t Immunohistological analysis of axonal pathology performed in 18-month-old WT, DKO, and DKO + JWH133 mice (n = 4 per genotype and condition). Spinal cord sections were processed for c–e Iba1, f–h GFAP, i–k synaptophysin (SYN), l–n APP, o–q Sudan black, and r–t SMI32 staining. Representative images for WT (c, f, i, l, o, r), DKO (d, g, j, m, p, s) and DKO + JWH133 (e, h, k, n, q, t) are shown. Scale bars = 25 μm (c–t). u Quantification of synaptophysin and APP accumulation and the densities of Iba1⁺ and GFAP⁺ (cells/mm²). Panels r–t are from the anterior horn. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
… 
A CB2r agonist inhibits lipid synthesis, normalizes the excess lipid droplets (LDs) in motor neurons and rescues the GSK-3β/NRF2 axis in Abcd1⁻ mice. a Total H2O2 levels in both primary WT (n = 4 per condition) and Abcd1⁻ (n = 4 per condition) mouse microglia were measured upon C26:0 (50 μM) insult after 24 h in the presence and absence of a CB2r agonist (JWH133 at 100 nM). Quantification is represented as the fold change compared to the WT + vehicle (Veh) group. The complex III inhibitor antimycin was used as a positive control. b Total H2O2 levels were measured upon treatment with C26:0 in the presence and absence of a CB2r agonist (JWH133 at 100 nM) and antagonist (AM-630 at 100 nM) after 24 h in primary Abcd1⁻ (n = 4 per condition) microglia. Quantification is presented as the fold change relative to the Abcd1⁻ + vehicle (Veh) group. c Relative Nrf2, Nqo1, Gsta3, and Gclc gene expression was quantified in the spinal cords of WT (n = 8) and Abcd1⁻ (n = 8) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 5) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in the WT mice. d Representative immunoblot (left) showing the pSer9 GSK-3β and GSK-3β protein levels in the spinal cords of WT (n = 4), Abcd1⁻ (n = 4) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 4) mice at 12 months of age. pSer9 GSK-3β/GSK-3β is presented as the fold change relative to levels in the WT mice (right). e–j Histological analysis performed in 12-month-old e, h WT (n = 4), f, iAbcd1⁻ (n = 5) and g, jAbcd1⁻ plus CB2r agonist (Abcd1⁻ + JWH133) mice (n = 4). Spinal cord histological sections were processed for oil red O (ORO) staining. k Quantification of the surface area of the LDs in spinal cord histological sections from 12-month-old WT, Abcd1⁻ and Abcd1⁻ + JWH133 mice by ImageJ. Scale bar = 25 μm (e–g); scale bar = 5 μm (h–j). l Relative Srebp1c, Fas and Scd1 gene expression were quantified in the spinal cord of WT (n = 8), Abcd1⁻ (n = 8) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 5) mice at 12 months of age. m Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptor 2 (Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + CB2r agonist (JWH133) (n = 5) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in the WT mice. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
A CB2r agonist inhibits lipid synthesis, normalizes the excess lipid droplets (LDs) in motor neurons and rescues the GSK-3β/NRF2 axis in Abcd1⁻ mice. a Total H2O2 levels in both primary WT (n = 4 per condition) and Abcd1⁻ (n = 4 per condition) mouse microglia were measured upon C26:0 (50 μM) insult after 24 h in the presence and absence of a CB2r agonist (JWH133 at 100 nM). Quantification is represented as the fold change compared to the WT + vehicle (Veh) group. The complex III inhibitor antimycin was used as a positive control. b Total H2O2 levels were measured upon treatment with C26:0 in the presence and absence of a CB2r agonist (JWH133 at 100 nM) and antagonist (AM-630 at 100 nM) after 24 h in primary Abcd1⁻ (n = 4 per condition) microglia. Quantification is presented as the fold change relative to the Abcd1⁻ + vehicle (Veh) group. c Relative Nrf2, Nqo1, Gsta3, and Gclc gene expression was quantified in the spinal cords of WT (n = 8) and Abcd1⁻ (n = 8) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 5) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in the WT mice. d Representative immunoblot (left) showing the pSer9 GSK-3β and GSK-3β protein levels in the spinal cords of WT (n = 4), Abcd1⁻ (n = 4) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 4) mice at 12 months of age. pSer9 GSK-3β/GSK-3β is presented as the fold change relative to levels in the WT mice (right). e–j Histological analysis performed in 12-month-old e, h WT (n = 4), f, iAbcd1⁻ (n = 5) and g, jAbcd1⁻ plus CB2r agonist (Abcd1⁻ + JWH133) mice (n = 4). Spinal cord histological sections were processed for oil red O (ORO) staining. k Quantification of the surface area of the LDs in spinal cord histological sections from 12-month-old WT, Abcd1⁻ and Abcd1⁻ + JWH133 mice by ImageJ. Scale bar = 25 μm (e–g); scale bar = 5 μm (h–j). l Relative Srebp1c, Fas and Scd1 gene expression were quantified in the spinal cord of WT (n = 8), Abcd1⁻ (n = 8) and Abcd1⁻  + CB2r agonist (Abcd1⁻  + JWH133) (n = 5) mice at 12 months of age. m Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptor 2 (Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + CB2r agonist (JWH133) (n = 5) mice at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in the WT mice. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
… 
Antioxidants inhibit lipid synthesis, normalize the excess LDs in motor neurons and the ECS pathway and NRF2-target genes in Abcd1⁻ mice. Eight-month-old animals received a combination of antioxidants or chow diet for 4 months. a–f Histological analysis performed in 12-month-old, a, d WT (n = 5), b, eAbcd1⁻ (n = 5) and c, fAbcd1⁻ plus antioxidant (Abcd1⁻  + ANTX) mice (n = 5). Spinal cord histological sections were processed for oil red O (ORO) staining. g Quantification of the surface area of the LDs in spinal cord histological sections from 12-month-old WT, Abcd1⁻ and Abcd1⁻  + ANTX mice by ImageJ. Scale bar = 25 μm (a–c); scale bar = 5 μm (d–f). h Relative Srebp1c, Fas and Scd1 gene expression was quantified in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + ANTX (n = 8) mice at 12 months of age. i Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptors 2 (Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + ANTX mice (n = 8) at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in WT mice. j Relative gene expression of biosynthetic enzymes (NAPEPLD, DAGLα, and DAGLβ), catabolic enzymes (MAGL and FAAH), and CB2R/CNR2 in PBMCs from adult controls (n = 12), AMN patients (n = 11) and AMN patients treated with antioxidants for 12 months (AMN + ANTX) (n = 11). Gene expression was normalized to RPLP0 expression and is presented as the fold change compared to the level in age-matched individuals. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
Antioxidants inhibit lipid synthesis, normalize the excess LDs in motor neurons and the ECS pathway and NRF2-target genes in Abcd1⁻ mice. Eight-month-old animals received a combination of antioxidants or chow diet for 4 months. a–f Histological analysis performed in 12-month-old, a, d WT (n = 5), b, eAbcd1⁻ (n = 5) and c, fAbcd1⁻ plus antioxidant (Abcd1⁻  + ANTX) mice (n = 5). Spinal cord histological sections were processed for oil red O (ORO) staining. g Quantification of the surface area of the LDs in spinal cord histological sections from 12-month-old WT, Abcd1⁻ and Abcd1⁻  + ANTX mice by ImageJ. Scale bar = 25 μm (a–c); scale bar = 5 μm (d–f). h Relative Srebp1c, Fas and Scd1 gene expression was quantified in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + ANTX (n = 8) mice at 12 months of age. i Relative gene expression of biosynthetic enzymes, catabolic enzymes, and cannabinoid receptors 2 (Cb2r/Cnr2) was measured by quantitative RT-PCR in the spinal cords of WT (n = 10), Abcd1⁻ (n = 10), and Abcd1⁻  + ANTX mice (n = 8) at 12 months of age. Gene expression was normalized to Rplp0 expression and is presented as the fold change compared to the level in WT mice. j Relative gene expression of biosynthetic enzymes (NAPEPLD, DAGLα, and DAGLβ), catabolic enzymes (MAGL and FAAH), and CB2R/CNR2 in PBMCs from adult controls (n = 12), AMN patients (n = 11) and AMN patients treated with antioxidants for 12 months (AMN + ANTX) (n = 11). Gene expression was normalized to RPLP0 expression and is presented as the fold change compared to the level in age-matched individuals. Data are represented as box-and-whisker plots (median, interquartile interval, minimum, maximum). *p < 0.05; **p < 0.01; ***p < 0.001 after one-way ANOVA followed by Fisher’s post hoc test
… 
Control of oxidative damage through a CB2 receptor agonist halts axonal degeneration in X-ALD. At baseline (left), ABCD1 malfunction produces an accumulation of VLCFA which generates mitochondrial ROS. Oxidative damage induces GSK-3β phosphorylation, which inhibits NRF2-mediated antioxidant response, exacerbating and chronifying oxidative damage. Redox unbalance induces the SREBP1 transcriptional program and LD synthesis contributing to axonal demise. Treatment with a CB2r agonist (right) inhibits mitochondrial ROS generation in microglia, and reactivates the endogenous antioxidant NRF2 response by keeping GSK-3β unaltered. This improves redox homeostasis and prevents induction of SREBP1c and subsequent LD accumulation, preserving axonal health
Control of oxidative damage through a CB2 receptor agonist halts axonal degeneration in X-ALD. At baseline (left), ABCD1 malfunction produces an accumulation of VLCFA which generates mitochondrial ROS. Oxidative damage induces GSK-3β phosphorylation, which inhibits NRF2-mediated antioxidant response, exacerbating and chronifying oxidative damage. Redox unbalance induces the SREBP1 transcriptional program and LD synthesis contributing to axonal demise. Treatment with a CB2r agonist (right) inhibits mitochondrial ROS generation in microglia, and reactivates the endogenous antioxidant NRF2 response by keeping GSK-3β unaltered. This improves redox homeostasis and prevents induction of SREBP1c and subsequent LD accumulation, preserving axonal health
… 
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Acta Neuropathologica (2022) 144:241–258
https://doi.org/10.1007/s00401-022-02451-2
ORIGINAL PAPER
Activating cannabinoid receptor 2 preserves axonal health
throughGSK‑3β/NRF2 axis inadrenoleukodystrophy
JananiParameswaran1,2,3· LeireGoicoechea1,2,4· LauraPlanas‑Serra1,2· AntoniPastor5· MontserratRuiz1,2·
NoelY.Calingasan6· CristinaGuilera1,2· EsterAso7,8· JordiBoada9· ReinaldPamplona9· ManuelPortero‑Otín9·
RafaeldelaTorre5· IsidreFerrer8,10,11· CarlosCasasnovas1,2,12· AuroraPujol1,2,13 · StéphaneFourcade1,2
Received: 11 January 2022 / Revised: 4 June 2022 / Accepted: 5 June 2022 / Published online: 1 July 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Aberrant endocannabinoid signaling accompanies several neurodegenerative disorders, including multiple sclerosis. Here, we
report altered endocannabinoid signaling in X-linked adrenoleukodystrophy (X-ALD), a rare neurometabolic demyelinating
syndrome caused by malfunction of the peroxisomal ABCD1 transporter, resulting in the accumulation of very long-chain
fatty acids (VLCFAs). We found abnormal levels of cannabinoid receptor 2 (CB2r) and related endocannabinoid enzymes in
the brain and peripheral blood mononuclear cells (PBMCs) of X-ALD patients and in the spinal cord of a murine model of
X-ALD. Preclinical treatment with a selective agonist of CB2r (JWH133) halted axonal degeneration and associated locomo-
tor deficits, along with normalization of microgliosis. Moreover, the drug improved the main metabolic disturbances under-
lying this model, particularly in redox and lipid homeostatic pathways, including increased lipid droplets in motor neurons,
through the modulation of the GSK-3β/NRF2 axis. JWH133 inhibited Reactive Oxygen Species elicited by excess VLCFAs
in primary microglial cultures of Abcd1-null mice. Furthermore, we uncovered intertwined redox and CB2r signaling in the
murine spinal cords and in patient PBMC samples obtained from a phase II clinical trial with antioxidants (NCT01495260).
These findings highlight CB2r signaling as a potential therapeutic target for X-ALD and perhaps other neurodegenerative
disorders that present with dysregulated redox and lipid homeostasis.
Keywords Endocannabinoids· CB2r· GSK-3β/NRF2· X-linked adrenoleukodystrophy· Axonal degeneration· Redox
homeostasis· Lipid droplets
Introduction
The endocannabinoid system (ECS) has attracted increased
attention in the past few decades and has been implicated
in several neurodegenerative disorders and experimental
systems. The ECS consists of cannabinoid receptors 1 and
2 (CB1r and CB2r), endocannabinoid lipid mediators, and
enzymes involved in their metabolism [21].
Cannabinoid receptors are a class of cell membrane
receptors under the G protein-coupled receptor superfam-
ily. Similar to other typical G protein-coupled receptors,
cannabinoid receptors contain seven transmembrane span-
ning domains. Cannabinoid receptors are activated by three
major groups of ligands: endocannabinoids (EC), plant
cannabinoids, and synthetic cannabinoids. Many studies are
currently focused on identifying the neuroprotective role of
synthetic EC-based drugs targeting endocannabinoid com-
ponents within the ECS, comprising both CB1r and CB2r
agonists [21, 42]. CB1r is the predominant CB receptor in
the central nervous system (CNS), and it is mainly expressed
in neurons, astrocytes, and oligodendrocytes. CB2r is mainly
expressed in cells of the immune system (such as B and
T cells, macrophages, and microglia) [18], although recent
evidence has identified CB2r in neurons of individuals with
amyotrophic lateral sclerosis (ALS) [24]. Importantly, the
levels of CB2r were found to be upregulated in various neu-
rodegenerative disease models and patient postmortem tis-
sues, including those of Alzheimer’s disease (AD) [53, 63],
Parkinson’s disease (PD) [63], Huntington’s disease (HD)
[11, 65], ALS [24] and multiple sclerosis (MS) [7, 56]. Such
studies have paved the way for clinical translation.
* Aurora Pujol
apujol@idibell.cat
Extended author information available on the last page of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... In a recent study, abnormal levels of cannabinoid receptor 2 (CB2r) and related endocannabinoid enzymes were found in the brain and peripheral blood mononuclear cells (PBMCs) of X-ALD patients and in the spinal cord of a murine model of X-ALD. They also reported that the use of selective agonist of CB2r improved metabolic disturbances by rescuing GSK-3β-NRF2 axis and improved oxidative stress by induction of NRF2 antioxidant pathway [53]. Trauma or injury to the head triggers or activate symptoms in otherwise asymptomatic or arrested X-ALD patients [54]. ...
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... Our findings are in line with previous reports that have shown CB2R activation to be beneficial in other CNS-related diseases, such as multiple sclerosis and stroke. For instance, in experimental models of demyelinating, neurometabolic disease, CB2R activation effectively inhibited axonal damage through the normalization of altered redox and lipid homeostatic pathways [40]. In stroke models, CB2R activation has exhibited neuroprotective effects by reducing infarct size and inhibiting the inflammatory response [25]. ...
Targeting CB2R in astrocytes for Parkinson's disease therapy: unraveling the Foxg1-mediated neuroprotective mechanism through autophagy-mediated NLRP3 degradation
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  • Dec 2023
  • J NEUROINFLAMM
Background Inflammasomes in astrocytes have been shown to play a crucial role in the pathogenesis of neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Cannabinoid Receptor 2(CB2R), a G protein-coupled receptor (GPCR), is considered a promising therapeutic target in inflammation-related disorders. This study aims to explore the role of CB2R in regulating NOD-like receptor family pyrin domain containing 3 (NLRP3)-mediated neuroinflammation in astrocytes. Methods In an in vivo animal model, specific targeting of astrocytic CB2R was achieved by injecting CB2R-specific adenovirus (or fork head box g1(foxg1) adenovirus) to knock down CB2R or administering CB2R agonists, inhibitors, etc., in the substantia nigra pars compacta (SNc) of mice. A PD mouse model was established using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induction. Animal behavioral tests, western blot, immunofluorescence, and other experiments were performed to assess the loss of midbrain tyrosine hydroxylase (TH) neurons, activation of astrocytes, and activation of the NLRP3 pathway. Primary astrocytes were cultured in vitro, and NLRP3 inflammasomes were activated using 1-methyl-4-phenylpyridinium (MPP ⁺ ) or lipopolysaccharide (LPS) and adenosine triphosphate (ATP). Western blot and ELISA experiments were conducted to assess the release of inflammatory factors. Transcriptomic sequencing and CUT&RUN techniques were employed to study the CB2R regulation of the foxg1 binding site on the autophagy molecule microtubule-associated protein 1 light chain 3 beta (MAP1LC3B). Results Astrocytic CB2R knockdown impaired the motor abilities of MPTP-induced mice, exacerbated the loss of TH neurons, and induced activation of the NLRP3/Caspase-1/interleukin 1 (IL-1β) pathway. Activation of CB2R significantly alleviated motor impairments in mice while reducing NLRP3 deposition on astrocytes. In vitro cell experiments showed that CB2R activation attenuated the activation of the NLRP3/Caspase-1/IL-1β pathway induced by LPS + ATP or MPP ⁺ . Additionally, it inhibited the binding of foxg1 to MAP1LC3B, increased astrocytic autophagy levels, and facilitated NLRP3 degradation through the autophagy–lysosome pathway. Conclusion Activation of CB2R on astrocytes effectively mitigates NLRP3-mediated neuroinflammation and ameliorates the disease characteristics of PD in mice. CB2R represents a potential therapeutic target for treating PD.
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... Moreover, the boosted LD formation in the spinal cord of 1-year-old Abcd1 KO versus WT mice is well linked with the elevated CE-VLCFA levels. Our findings confirm recent observations on LD accumulation in the spinal cord of Abcd1/2 double-KO and Abcd1 KO mice [44,60]. In these studies, LD accumulation was attributed mainly to increased lipogenesis and elevated triglyceride levels due to mTOR/SREBP1c activation, which could be normalised by high-dose biotin treatment [44]. ...
ABCD1 Transporter Deficiency Results in Altered Cholesterol Homeostasis
Article
Full-text available
  • Aug 2023
X-linked adrenoleukodystrophy (X-ALD), the most common peroxisomal disorder, is caused by mutations in the peroxisomal transporter ABCD1, resulting in the accumulation of very long-chain fatty acids (VLCFA). Strongly affected cell types, such as oligodendrocytes, adrenocortical cells and macrophages, exhibit high cholesterol turnover. Here, we investigated how ABCD1 deficiency affects cholesterol metabolism in human X-ALD patient-derived fibroblasts and CNS tissues of Abcd1-deficient mice. Lipidome analyses revealed increased levels of cholesterol esters (CE), containing both saturated VLCFA and mono/polyunsaturated (V)LCFA. The elevated CE(26:0) and CE(26:1) levels remained unchanged in LXR agonist-treated Abcd1 KO mice despite reduced total C26:0. Under high-cholesterol loading, gene expression of SOAT1, converting cholesterol to CE and lipid droplet formation were increased in human X-ALD fibroblasts versus healthy control fibroblasts. However, the expression of NCEH1, catalysing CE hydrolysis and the cholesterol transporter ABCA1 and cholesterol efflux were also upregulated. Elevated Soat1 and Abca1 expression and lipid droplet content were confirmed in the spinal cord of X-ALD mice, where expression of the CNS cholesterol transporter Apoe was also elevated. The extent of peroxisome-lipid droplet co-localisation appeared low and was not impaired by ABCD1-deficiency in cholesterol-loaded primary fibroblasts. Finally, addressing steroidogenesis, progesterone-induced cortisol release was amplified in X-ALD fibroblasts. These results link VLCFA to cholesterol homeostasis and justify further consideration of therapeutic approaches towards reducing VLCFA and cholesterol levels in X-ALD.
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Ataxia with giant axonopathy in Acbd5-deficient mice halted by adeno-associated virus gene therapy
Article
  • Dec 2023
  • BRAIN
Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal β-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.
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A double-blind, placebo-controlled, randomized trial of PXT3003 for the treatment of Charcot–Marie–Tooth type 1A
Article
Full-text available
  • Dec 2021
  • ORPHANET J RARE DIS
Background Charcot–Marie–Tooth disease type 1A (CMT1A) is a rare, orphan, hereditary neuromuscular disorder with no cure and for which only symptomatic treatment is currently available. A previous phase 2 trial has shown preliminary evidence of efficacy for PXT3003 in treating CMT1A. This phase 3, international, randomized, double-blind, placebo-controlled study further investigated the efficacy and safety of high- or low-dose PXT3003 (baclofen/naltrexone/D-sorbitol [mg]: 6/0.70/210 or 3/0.35/105) in treating subjects with mild to moderate CMT1A. Methods In this study, 323 subjects with mild-to-moderate CMT1A were randomly assigned in a 1:1:1 ratio to receive 5 mL of high- or low-dose PXT3003, or placebo, orally twice daily for up to 15 months. Efficacy was assessed using the change in Overall Neuropathy Limitations Scale total score from baseline to months 12 and 15 (primary endpoint). Secondary endpoints included the 10-m walk test and other assessments. The high-dose group was discontinued early due to unexpected crystal formation in the high-dose formulation, which resulted in an unanticipated high discontinuation rate, overall and especially in the high-dose group. The statistical analysis plan was adapted to account for the large amount of missing data before database lock, and a modified full analysis set was used in the main analyses. Two sensitivity analyses were performed to check the interpretation based on the use of the modified full analysis set. Results High-dose PXT3003 demonstrated significant improvement in the Overall Neuropathy Limitations Scale total score vs placebo (mean difference: − 0.37 points; 97.5% CI [− 0.68 to − 0.06]; p = 0.008), and consistent treatment effects were shown in the sensitivity analyses. Both PXT3003 doses were safe and well-tolerated. Conclusion The high-dose group demonstrated a statistically significant improvement in the primary endpoint and a good safety profile. Overall, high-dose PXT3003 is a promising treatment option for patients with Charcot–Marie–Tooth disease type 1A.
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Integrated Management of Multiple Sclerosis Spasticity and Associated Symptoms Using the Spasticity-Plus Syndrome Concept: Results of a Structured Specialists' Discussion Using the Workmat Methodology
Article
Full-text available
  • Sep 2021
Background: Multiple sclerosis (MS) treatment has radically improved over the last years; however, MS symptom management is still challenging. The novel Spasticity-Plus syndrome was conceptualized to frame several spasticity-related symptoms that can be addressed together with broad-spectrum medication, such as certain cannabinoid-based drugs. The aim of this project was to gain insight into Spanish neurologists' clinical experience on MS spasticity and associated symptoms, and to assess the acknowledgment and applicability of the Spasticity-Plus syndrome concept in patients with MS. Methods: Ten online meetings were conducted using the Workmat® methodology to allow structured discussions. Fifty-five Spanish neurologists, experts in MS management, completed and discussed a set of predefined exercises comprising MS symptom assessment and its management in clinical practice, MS symptoms clustering in clinical practice, and their perception of the Spasticity-Plus syndrome concept. This document presents the quantitative and qualitative results of these discussions. Results: The specialists considered that polytherapy is a common concern in MS and that simplifying the management of MS spasticity and associated manifestations could be useful. They generally agreed that MS spasticity should be diagnosed before moderate or severe forms appear. According to the neurologists' clinical experience, symptoms commonly associated with MS spasticity included spasms/cramps (100% of the specialists), pain (85%), bladder dysfunction (62%), bowel dysfunction (42%), sleep disorders (42%), and sexual dysfunction (40%). The multiple correspondence analysis revealed two main symptom clusters: spasticity-spasms/cramps-pain, and ataxia-instability-vertigo. Twelve out of 16 symptoms (75%) were scored >7 in a 0–10 QoL impact scale by the specialists, representing a moderate–high impact. The MS specialists considered that pain, spasticity, spasms/cramps, bladder dysfunction, and depression should be a treatment priority given their frequency and chance of therapeutic success. The neurologists agreed on the usefulness of the new Spasticity-Plus syndrome concept to manage spasticity and associated symptoms together, and their experience with treatments targeting the cannabinoid system was satisfactory. Conclusions: The applicability of the new concept of Spasticity-Plus in MS clinical practice seems possible and may lead to an integrated management of several MS symptoms, thus reducing the treatment burden of disease symptoms.
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Sativex® (nabiximols) cannabinoid oromucosal spray in patients with resistant multiple sclerosis spasticity: the Belgian experience
Article
Full-text available
  • Jun 2021
  • BMC NEUROL
Background This retrospective study evaluates patient-reported outcomes in patients with multiple sclerosis (MS) spasticity who were treated with a cannabinoid oromucosal spray (Sativex®, USAN name: nabiximols) after not sufficiently responding to previous anti-spasticity medications. Methods Of 276 patients from eight centers in Belgium who began treatment prior to 31 December 2017, effectiveness assessment data were available for 238 patients during the test period of 4 to 8/12 weeks, and for smaller patient cohorts with continued treatment for 6/12 months. Results Mean 0–10 spasticity Numerical Rating Scale (NRS) scores improved from 8.1 at baseline to 5.2 (week 4), 4.6 (week 8) and 4.1 (week 12). Mean EuroQoL Visual Analogue Scale (EQ VAS) scores increased from 39 at baseline to 52 (week 4), 57 (week 8) and 59 (week 12). Mean NRS and EQ VAS scores remained in the same 12 weeks’ range in patients with longer-term data. The average dose of cannabinoid oromucosal spray was 6 sprays/day. Most of the 93 out of 276 patients, with initial prescription (33.7%), who discontinued treatment by week 12 did so within the first 8 weeks, mainly due to lack of effectiveness. By week 12, 171 (74%) of the 230 effectiveness evaluable patients reported a clinically meaningful response, corresponding to ≥30% NRS improvement. The tolerability of cannabinoid oromucosal spray was consistent with its known safety profile. Conclusions More than 60% of the patients with MS who started add-on treatment with cannabinoid oromucosal spray reported a clinically relevant symptomatic effect and continued treatment after 12 weeks.
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Lipid Droplets in the Pathogenesis of Hereditary Spastic Paraplegia
Article
Full-text available
  • May 2021
Hereditary spastic paraplegias (HSPs) are genetically heterogeneous conditions caused by the progressive dying back of the longest axons in the central nervous system, the corticospinal axons. A wealth of data in the last decade has unraveled disturbances of lipid droplet (LD) biogenesis, maturation, turnover and contact sites in cellular and animal models with perturbed expression and function of HSP proteins. As ubiquitous organelles that segregate neutral lipid into a phospholipid monolayer, LDs are at the cross-road of several processes including lipid metabolism and trafficking, energy homeostasis, and stress signaling cascades. However, their role in brain cells, especially in neurons remains enigmatic. Here, we review experimental findings linking LD abnormalities to defective function of proteins encoded by HSP genes, and discuss arising questions in the context of the pathogenesis of HSP.
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Adrenoleukodystrophy Newborn Screening in the Netherlands (SCAN Study): The X-Factor
Article
Full-text available
  • Jun 2020
X-linked adrenoleukodystrophy (ALD) is a devastating metabolic disorder affecting the adrenal glands, brain and spinal cord. Males with ALD are at high risk for developing adrenal insufficiency or progressive cerebral white matter lesions (cerebral ALD) at an early age. If untreated, cerebral ALD is often fatal. Women with ALD are not at risk for adrenal insufficiency or cerebral ALD. Newborn screening for ALD in males enables prospective monitoring and timely therapeutic intervention, thereby preventing irreparable damage and saving lives. The Dutch Ministry of Health adopted the advice of the Dutch Health Council to add a boys-only screen for ALD to the newborn screening panel. The recommendation made by the Dutch Health Council to only screen boys, without gathering any unsolicited findings, posed a challenge. We were invited to set up a prospective pilot study that became known as the SCAN study (SCreening for ALD in the Netherlands). The objectives of the SCAN study are: (1) designing a boys-only screening algorithm that identifies males with ALD and without unsolicited findings; (2) integrating this algorithm into the structure of the Dutch newborn screening program without harming the current newborn screening; (3) assessing the practical and ethical implications of screening only boys for ALD; and (4) setting up a comprehensive follow-up that is both patient- and parent-friendly. We successfully developed and validated a screening algorithm that can be integrated into the Dutch newborn screening program. The core of this algorithm is the “X-counter.” The X-counter determines the number of X chromosomes without assessing the presence of a Y chromosome. The X-counter is integrated as second tier in our 4-tier screening algorithm. Furthermore, we ensured that our screening algorithm does not result in unsolicited findings. Finally, we developed a patient- and parent-friendly, multidisciplinary, centralized follow-up protocol. Our boys-only ALD screening algorithm offers a solution for countries that encounter similar ethical considerations, for ALD as well as for other X-linked diseases. For ALD, this alternative boys-only screening algorithm may result in a more rapid inclusion of ALD in newborn screening programs worldwide.
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High‐dose biotin restores redox balance, energy and lipid homeostasis, and axonal health in a model of adrenoleukodystrophy
Article
Full-text available
Biotin is an essential cofactor for carboxylases that regulates energy metabolism. Recently, high‐dose pharmaceutical‐grade biotin (MD1003) was shown to improve clinical parameters in a subset of patients with chronic progressive multiple sclerosis. To gain insight into the mechanisms of action, we investigated the efficacy of high‐dose biotin in a genetic model of chronic axonopathy caused by oxidative damage and bioenergetic failure, the Abcd1‐ mouse model of adrenomyeloneuropathy. High‐dose biotin restored redox homeostasis driven by NRF‐2, mitochondria biogenesis and ATP levels, and reversed axonal demise and locomotor impairment. Moreover, we uncovered a concerted dysregulation of the transcriptional programme for lipid synthesis and degradation in the spinal cord likely driven by aberrant SREBP‐1c/mTORC1 signalling. This resulted in increased triglyceride levels and lipid droplets in motor neurons. High‐dose biotin normalized the hyperactivation of mTORC1, thus restoring lipid homeostasis. These results shed light into the mechanism of action of high‐dose biotin of relevance for neurodegenerative and metabolic disorders..
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Biological basis of cannabinoid medicines
Article
  • Dec 2021
  • SCIENCE
Mechanistic insights into cannabinoid signaling could improve therapeutic applications.
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The brain penetrant PPARγ agonist leriglitazone restores multiple altered pathways in models of X-linked adrenoleukodystrophy
Article
  • Jun 2021
X-linked adrenoleukodystrophy (X-ALD), a potentially fatal neurometabolic disorder with no effective pharmacological treatment, is characterized by clinical manifestations ranging from progressive spinal cord axonopathy [adrenomyeloneuropathy (AMN)] to severe demyelination and neuroinflammation (cerebral ALD-cALD), for which molecular mechanisms are not well known. Leriglitazone is a recently developed brain penetrant full PPARγ agonist that could modulate multiple biological pathways relevant for neuroinflammatory and neurodegenerative diseases, and particularly for X-ALD. We found that leriglitazone decreased oxidative stress, increased adenosine 5′-triphosphate concentration, and exerted neuroprotective effects in primary rodent neurons and astrocytes after very long chain fatty acid–induced toxicity simulating X-ALD. In addition, leriglitazone improved motor function; restored markers of oxidative stress, mitochondrial function, and inflammation in spinal cord tissues from AMN mouse models; and decreased the neurological disability in the EAE neuroinflammatory mouse model. X-ALD monocyte–derived patient macrophages treated with leriglitazone were less skewed toward an inflammatory phenotype, and the adhesion of human X-ALD monocytes to brain endothelial cells decreased after treatment, suggesting the potential of leriglitazone to prevent the progression to pathologically disrupted blood-brain barrier. Leriglitazone increased myelin debris clearance in vitro and increased myelination and oligodendrocyte survival in demyelination-remyelination in vivo models, thus promoting remyelination. Last, leriglitazone was clinically tested in a phase 1 study showing central nervous system target engagement (adiponectin increase) and changes on inflammatory biomarkers in plasma and cerebrospinal fluid. The results of our study support the use of leriglitazone in X-ALD and, more generally, in other neuroinflammatory and neurodegenerative conditions.
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MRI Surveillance of Boys with X‐linked Adrenoleukodystrophy Identified by Newborn Screening: Meta‐analysis and Consensus Guidelines
Article
  • May 2021
Background Among boys with X‐Linked adrenoleukodystrophy, a subset will develop childhood cerebral adrenoleukodystrophy (CCALD). CCALD is typically lethal without hematopoietic stem cell transplant before or soon after symptom onset. We sought to establish evidence‐based guidelines detailing the neuroimaging surveillance of boys with neurologically asymptomatic adrenoleukodystrophy. Methods To establish the most frequent age and diagnostic neuroimaging modality for CCALD, we completed a meta‐analysis of relevant studies published between January 1, 1970 and September 10, 2019. We used the consensus development conference method to incorporate the resulting data into guidelines to inform the timing and techniques for neuroimaging surveillance. Final guideline agreement was defined as >80% consensus. Results One hundred twenty‐three studies met inclusion criteria yielding 1,285 patients. The overall mean age of CCALD diagnosis is 7.91 years old. The median age of CCALD diagnosis calculated from individual patient data is 7.0 years old (IQR: 6.0 – 9.5, n = 349). Ninety percent of patients were diagnosed between 3 and 12. Conventional MRI was most frequently reported, comprised most often of T2‐weighted and contrast‐enhanced T1‐weighted MRI. The expert panel achieved 95.7% consensus on the following surveillance parameters: (1) Obtain an MRI between 12 and 18 months old. (2) Obtain a second MRI 1 year after baseline. (3) Between 3 and 12 years old, obtain a contrast‐enhanced MRI every 6 months. (4) After 12 years, obtain an annual MRI. Conclusion Boys with adrenoleukodystrophy identified early in life should be monitored with serial brain MRIs during the period of highest risk for conversion to CCALD. This article is protected by copyright. All rights reserved.
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Safety and efficacy of lenabasum in a phase 2 randomized, placebo-controlled trial in adults with cystic fibrosis
Article
  • Sep 2020
Background: Few therapies specifically address the chronic airway inflammation in cystic fibrosis (CF) that contributes to progressive destruction of lung tissue and loss of lung function. Lenabasum is a cannabinoid type 2 receptor (CB2) agonist that resolves inflammation in a number of in vitro and in vivo models. Methods: A Phase 2 double-blind, randomized, placebo-controlled study assessed the safety and tolerability of lenabasum in adults with CF. Subjects with FEV1% (ppFEV1) ≥40% predicted were randomized to lenabasum 1 or 5 mg or placebo once daily (QD) (Weeks 1-4), then 20 mg QD, 20 mg twice daily (BID) or placebo (Weeks 5-12), with follow-up at Week 16. Pulmonary exacerbations (PEx) were recorded and biomarkers of blood and lung inflammation were measured. Results: Of 89 subjects randomized, 51 lenabasum and 23 placebo-only subjects completed the study. No deaths or serious or severe adverse events (AE) were considered related to lenabasum. Most AEs were mild/moderate, and the most common were PEx, hemoptysis, dry mouth, and upper respiratory infection. Three lenabasum and one placebo-only subjects discontinued the study for a treatment related AE. New PEx were treated with intravenous antibiotics in 4.0% of lenabasum-treated vs. 11.4% of placebo-treated subjects, during Weeks 1-4 and 5.2% compared to 13.0% during Weeks 5-12 (p<0.2). No significant differences in ppFEV1 were observed between treatment groups. Sputum neutrophils, eosinophils, and neutrophil elastase were numerically reduced, and significant (p<0.05) reductions in IL-8 and immunoglobulin G levels occurred with lenabasum. Conclusions: The safety findings of lenabasum, coupled with biomarker data, support further testing in a larger study with a longer duration.
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