Author Of 3 Presentations
P0311 - Decoding Bruton’s tyrosine kinase signalling in neuroinflammation (ID 1381)
Neuroinflammation in the brain and spinal cord, driven largely by CNS-resident microglia, has been proposed as a significant contributor to disability accumulation in patients living with multiple sclerosis (MS). Bruton’s tyrosine kinase (BTK) is expressed in microglia, as well as in B lymphocytes and monocytes/macrophages found in the periphery. In B cells, this kinase is an essential component of the B-cell receptor signalling pathway regulating proliferation, maturation, antigen presentation, and production of secreted immunoglobulins. We hypothesize that in addition to its role in B cells, BTK regulates microglial deleterious inflammatory signalling; therefore, inhibiting BTK with a brain-penetrant inhibitor may provide therapeutic benefit within the CNS by targeting innate immunity associated with disease progression in MS.
To assess the role of BTK signalling in modulating inflammatory processes in microglial cells both in vitro and in vivo.
Immunohistochemistry, Western blotting, and RNA sequencing monitored BTK or phospho-BTK in primary mouse microglial cells, the rodent model of cuprizone-mediated demyelination, and post-mortem MS brain tissues.
Basal activity of BTK in murine microglial cells in vitro was enhanced by stimulation with immune complexes and silenced with a BTK inhibitor. Transcriptome analysis was used to generate a BTK-dependent transcriptional signature in microglia. In tissue derived from autopsy specimens, immunohistochemistry studies and single-nucleus RNA sequencing demonstrated that BTK was expressed in B cells as well as in microglial cells, with increased levels in MS lesion samples. To further explore the role of BTK in vivo, we identified a BTK-dependent transcriptional profile in brains from cuprizone-treated mice. Oral administration of a brain-penetrant BTK inhibitor downregulated the BTK-dependent gene expression signature in the cuprizone-treated mouse brain. Finally, using post-mortem tissue, we evaluated BTK-dependent activation signatures derived from mouse models in MS samples.
Using the cuprizone-induced toxicity model, we extend our previous findings on the role of BTK in microglia to show that BTK-dependent inflammatory signalling in these cells can be modulated using brain-penetrant BTK inhibitors in vivo, which could abrogate microglia-driven neuroinflammation implicated in disease progression in MS.
STUDY SUPPORT: Sanofi.
P0381 - Reduced sphingosine kinase gene expression in SPMS vs. RRMS astrocytes revealed by single-nucleus RNA-seq (ID 1784)
The sphingosine 1-phosphate (S1P) receptor modulators, siponimod and fingolimod, have overlapping yet also distinct mechanisms underlying their efficacy for treating multiple sclerosis (MS). Siponimod directly binds to S1P receptors (S1P1,5), whereas fingolimod is a prodrug that requires sphingosine kinases (SPHK1/2) to generate active fingolimod-phosphate (engaging S1P1,3,4,5). Both drugs functionally antagonize S1P1 on immune and central nervous system (CNS) cells. Siponimod showed activity in clinical trials of progressive MS (SPMS), whereas fingolimod (trialed in PPMS) did not. However, the mechanistic explanation for the clinical differences between these two S1P receptor modulators is unclear.
Single-cell transcriptome profiles of SPMS and RRMS brains might identify differential gene pathways in relevant cell types that may explain the clinical differences between siponimod vs. fingolimod.
We applied single-nucleus RNA sequencing (snRNA-seq) using 10x Genomics on nuclei derived from region-matched prefrontal cortices of SPMS vs. RRMS-affected brains.
The snRNA-seq yielded 33,197 high quality nuclei that were clustered into major CNS cell types. SPMS brains, as compared to RRMS brains, showed perturbation in sphingolipid pathway genes, and most notably, a downregulation of SPHK1 in astrocytes. Functional proof-of-concept was obtained in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), which resulted in diminished fingolimod efficacy in astrocyte-specific Sphk1/2 double knockout mice.
SPMS brains show reduced SPHK1/2 expression, particularly SPHK1 in astrocytes of SPMS brains. Although it is not clear whether or not this results in reduced local exposure to active fingolimod-P in the MS brain, animal EAE studies are consistent with its functional reduction. These data further support changes in the metabolism of sphingolipids during MS progression that highlight a major metabolic difference for fingolimod vs. siponimod, which could have significant clinical relevance particularly since siponimod does not require SPHK1/2 for receptor activation.
We thank Dr. Shaun R. Coughlin (UCSF) for providing the Sphk1/2flox/flox mice. This work was supported by a grant from Novartis Pharma AG (JC) and the NIH R01NS103940 (YK).
P0584 - Histological analysis of slowly expanding lesions in multiple sclerosis: case report (ID 878)
Slowly expanding lesions (SELs) can be detected on conventional in vivo brain magnetic resonance imaging (MRI). Previous studies suggest that SELs reflect chronic tissue loss in the absence of ongoing acute inflammation. Histopathological characterization of SELs are still not fully investigated.
To characterize SEL regions using in vivo MRIs and postmortem brain tissue, and compare the difference between SEL and non-SEL regions.
We identified an autopsy case with secondary progressive MS (male, age=51 years, disease duration=23 years), who had standardized in vivo MRIs. The interval between the last in vivo MRI and death was 7 weeks. From the last two years of in vivo MRIs, T2 lesions were segmented, and the Jacobian determinants of nonlinear registration between baseline and follow-up scans were calculated. SELs were identified as regions with small local constant and concentric expansion from baseline lesions. We identified 11 regions-of-interest (ROI): 10 T2 lesions (3 SELs and 7 non-SEL) and 1 normal-appearing white matter (NAWM). Using a custom brain cutting box with MRI-visible markers, the in vivo ROIs were localized on the corresponding brain slice. The ROIs were blocked and stained for proteolipid protein, SMI-31/32, and MHC class II. We then evaluated myelin status, axonal diameter, axonal loss, and inflammatory activity in ROIs.
The NAWM region was myelinated, the axonal diameter was 0.74 um, and axonal density was 23.4%. In the SEL regions, the mean axonal diameter was 1.11 um, and mean axonal density was 17.5%. In non-SEL regions, the mean axonal diameter was 0.84 um, and mean axonal density was 15.7%.
Two SEL and 4 non-SEL regions were demyelinated. The demyelinated SEL regions had activated microglia at the lesion edge and were compatible with chronic active lesions. Three demyelinated non-SEL regions also had activated microglia at the edge. One demyelinated non-SEL region was a chronic inactive lesion. No microglia activity was observed in any of the myelinated non-SEL regions. In the myelinated SEL region, the density of activated microglia was higher compared to NAWM.
Not all SEL regions in T2 lesions were demyelinated. SEL also had greater axonal diameters suggesting of axonal swelling. In this case report, all of the demyelinated SEL regions had activated microglia at the lesion edge.