S. Sadiq
Tisch MS Research Center of New YorkAuthor Of 5 Presentations
P0162 - Single cell RNA-seq of MS CSF cells reveals a bias toward expanded CD8 T cells (ID 1407)
Abstract
Background
Multiple Sclerosis (MS) is an immune mediated disease of the central nervous system (CNS). To better understand the role and mechanism of the immune system in MS, we performed single cell RNA-seq (scRNA-seq) on cerebrospinal fluid (CSF) cells of 14 untreated MS patients under the 10x Genomics Chromium Single Cell Immune Profiling system.
Objectives
To immunoprofile MS CSF cells and to identify specific cell types that may be relevant to MS pathogenesis or disease subtype.
Methods
Fourteen untreated MS CSF samples were obtained via lumbar puncture; 7 relapsing remitting MS (RRMS), 5 primary progressive MS (PPMS), and 2 secondary progressive MS (SPMS). Cells were isolated and processed according to 10x Genomics’ protocol. For each sample, 2 libraries were made; a 5’ gene expression library and a VDJ enriched library which targets the αβ chains of the T cell receptor (TCR). The combination of these libraries allows for the identification and analysis of clonally expanded T cells. 10x Genomics’ Cellranger pipeline was used for sequence analysis.
Results
Analyzing the top 15 expanded T cell clones for each sample, 11 of the 14 samples had more expanded CD8 T cell clones than expanded CD4 T cell clones. Of these 11 samples that have more CD8 expanded clones, 9 of the 11 samples actually have more total CD4 T cells than CD8 T cells in the CSF. In examining only highly expanded clones (10 cells or more), 9 of the 14 samples retained this CD8 T cell expansion bias. Our data suggests that expanded T cell clones in MS CSF are more likely to be of the CD8 subtype despite a total T cell ratio that favors CD4 T cells.
Conclusions
Immune clonal expansion suggests a targeted response that may be an integral part of the pathogenesis of MS. A bias of the T cell expansion toward CD8 cells may indicate a more cytotoxic environment in the MS CNS. More samples will be needed to determine if this CD8 bias correlates with a particular subtype of MS or if this is a general MS phenomenon.
P0949 - Cell population analysis in a patient-derived cerebral organoid model of multiple sclerosis. (ID 1704)
Abstract
Background
Multiple sclerosis (MS) is an auto-immune disease characterized by inflammation, demyelination and neural degeneration. While MS etiology is still uncertain, recent studies propose an interplay between genetic and environmental factors but MS genetic determinants are still poorly understood. Recent advances in 3D cerebral organoid cultures, derived from induced pluripotent stem cells (iPSCs), provide new avenues to investigate human disorders. Cerebral organoids contain ventricular structures aligned by neural stem cells, progenitor cells in various stages of differentiation, and neurons in a typical inside-out stratified layout. Furthermore, it has been shown that myelination can be induced in those neurons.
Objectives
We propose here to use human iPSC derived cerebral organoids to study the genetic components of multiple sclerosis. The lack of blood vessels and immune cells in cerebral organoids allows the study of the effect of MS genetic component on neural cells.
Methods
Cerebral organoids were derived from iPS cells of patients with MS. We analyzed stem cell proliferation, migration and differentiation in neuronal and glial lineages in MS organoids compared to healthy control organoids at 42 days in vitro.
Results
MS cerebral organoids seemed to grow faster compared to healthy control organoids, suggesting a higher stem cell proliferation rate. Immunostainings for stem cell marker SOX2 and neuroblast marker DCX revealed that the stem cell pool localized in the Ventricular/Subventricular Zone was larger in MS cerebral organoids compared to control. A lower DCX intensity was detected in MS cerebral organoids, suggesting that MS cerebral organoids might have developed an enlarged stem cell pool at the expense of the neuroblast population. A preliminary quantification of cortical neuron marker CTIP2 did not show a statistically significant difference between MS organoids and healthy controls, suggesting that neuronal maturation might not be affected. An analysis of apoptosis marker CC3 displayed an increase of CC3+ cell numbers in MS organoids, particularly in cortical plate, with little to no cell death in the stem cell pools in both organoid populations. A further analysis of DNA damage and senescence in stem cells as well as oligodendrocyte maturation will be performed.
Conclusions
This study will give new insight on the origin and evolution of the disease and will help to identify potential target for therapeutic strategies designed to promote myelin repair in MS.
P0963 - Filtration of primary progressive MS CSF removes factors causing delayed remyelination of lysolecithin lesions (ID 1799)
Abstract
Background
Primary progressive MS (PPMS) is characterized by disease progression from clinical onset and failure of remyelination. In our prior work, we showed that intrathecal injection of cerebrospinal fluid (CSF) from PPMS patients, but not relapsing-remitting (RRMS) or secondary progressive (SPMS) patients, delayed spontaneous remyelination in lysolecithin-induced lesions. The PPMS CSF factors that impede remyelination have not yet been isolated or characterized. To better understand the size of these undetermined factor(s), we filtered PPMS CSF at 100 kDa and determined the effect of filtered CSF on remyelination in lysolecithin-induced lesions.
Objectives
To investigate whether filtration of PPMS CSF mitigates the delayed remyelination induced by PPMS CSF factors following lysolecithin-induced demyelination.
Methods
CSF obtained from PPMS patients was passed through a tangential flow filtration system for 3 filtration cycles to remove CSF components larger than 100 kDa. Mice underwent a laminectomy at cervical level 5 (C5). 1 μL of 1% lysolecithin was injected into the dorsal column. Five days post injection, 3 μL of PPMS CSF or filtered PPMS CSF were injected into the subarachnoid space at C5. Control mice were injected with saline. Mice were perfused at 12 days post lysolecithin injection and pathology was assessed in the cervical spinal cord.
Results
Intrathecal injection of PPMS CSF at the site of a lysolecithin-induced lesion resulted in significantly larger demyelinated lesions compared to saline controls, as determined by luxol fast blue staining. However, there was no significant difference in lesion volume between filtered PPMS CSF-injected mice and saline controls. This suggests that the 100 kDa filter removed pathological PPMS CSF factor(s) preventing spontaneous remyelination in lysolecithin-induced lesions. Preliminary data suggest that mice injected with filtered PPMS CSF display reduced reactive astrogliosis (GFAP) and microglial activation (Iba1) within the lesion compared to unfiltered PPMS CSF-injected mice. There was no significant difference in numbers of proliferating oligodendrocyte progenitor cells (NG2/Ki67) or mature oligodendrocytes (APC/Olig2).
Conclusions
Our results indicate that PPMS CSF factors responsible for impeding remyelination are larger than 100 kDa and can be removed by filtration. This suggests that CSF pheresis may be a therapeutic option.
P0978 - Metabotropic glutamate receptors are associated with cerebellar dysfunction in multiple sclerosis (ID 1826)
Abstract
Background
Patients with multiple sclerosis (MS) can develop cerebellar dysfunction (CD) with clinical manifestations ranging from mild abnormalities, such as tremor, to severe disabilities involving motor coordination and balance. Progress to understand CD in MS has been hampered by lack of animal models. We developed a rodent cisterna magna injection protocol, using cerebrospinal fluid (CSF) from MS patients with CD, that recapitulate the coordination deficits observed in these patients. Furthermore, since metabotropic glutamate receptors (mGluRs) have been suggested to have a functional relationship with cerebellar discoordination, we examined their impact on motor control disabilities.
Objectives
To develop an animal model that replicates MS-specific cerebellar manifestations, and to investigate the role of mGluRs signaling pathway/s on cerebellar dysfunction in MS.
Methods
We injected into the mice cisterna magna 10 μl of concentrated (20X) CSF. Post-surgery, motor coordination performances of the mice were tested using the rotarod paradigm and their latency to fall from the drum rotating at 24 rpm was recorded. We analyzed 25 CSF samples from MS patients with or without CD. In a pharmacological approach, we examined the mGluRs involvement in CD manifestations in MS by co-injecting CSF samples from MS patients with CD with 3,5-dihydroxyphenylglycine (DHPG) or L(+)-2-amino-4-phosphonobutyric acid (LAP4), two mGluRs agonists.
Results
Our results indicated that the motor coordination of mice injected with CSF from MS patients with CD was significantly impaired in the first four hours post-injection. By contrast, mice injected with CSF from MS patients without CD or mice injected with saline exhibited no abnormalities in their performances on the rotarod. Notably, co-injection experiments with mGluRs agonists DHPG and LAP4, significantly ameliorated the rotarod performances of mice injected with CSF from MS patients with CD.
Conclusions
We developed a novel animal model to study cerebellar dysfunction in MS. We observed that injection of CSF samples from MS patients with CD into the rodent cisterna magna gravely impaired their performance on the rotarod. Our pharmacological approach, using two agonists of mGluRs, suggests that cerebellar dysfunction in MS is the result of impairments in the activation and/or signaling of mGluRs pathways.
P0987 - Pathological effects of primary progressive MS cerebrospinal fluid are antibody-mediated (ID 1021)
Abstract
Background
Primary progressive multiple sclerosis (PPMS) is characterized by unremitting disease progression from disease onset and afflicts 10-15% of MS patients. We previously reported that intrathecal delivery of PPMS cerebrospinal fluid (CSF) from PPMS, but not relapsing-remitting (RRMS) or secondary progressive (SPMS) patients, in mice can induce significant forelimb motor deficits along with demyelination, reactive astrogliosis, and axonal damage in the spinal cord. We also showed that CSF filtration with a 5 kDa molecular weight cutoff (MWCO) filter attenuated the pathology-inducing capacity of PPMS CSF. The PPMS CSF factor(s) responsible for inducing these pathological outcomes have yet to be identified.
Objectives
To identify the factor(s) present in PPMS CSF responsible for inducing motor deficits and spinal cord pathology in mice.
Methods
CSF derived from PPMS patients was pumped through a tangential flow filtration system with 100 kDa MWCO filters for 3 filtration cycles. 8-10 week old female mice underwent laminectomies at cervical levels 4 and 5, then received 3µl injections into the subarachnoid space of either PPMS CSF, filtered PPMS CSF, or filtered PPMS CSF spiked with recombinant IgG antibodies (rAbs) produced from B-cells in PPMS CSF. Control mice were injected with saline. Functional deficits were evaluated by measuring forelimb grip strength, reaching accuracy and tail rigidity at 1 day post injection (DPI), then mice were immediately perfused for histological analyses of the cervical spinal cord.
Results
At 1 DPI, PPMS CSF-injected mice exhibited significantly impaired forelimb function and grip strength compared to saline controls and mice injected with filtered PPMS CSF. Luxol fast blue staining, GFAP and SMI-32 immunostaining showed demyelination, reactive astrogliosis and axonal damage in the dorsal column of PPMS CSF-injected mice, respectively. These pathological changes were not observed in controls or mice injected with filtered PPMS CSF. Mice injected with filtered PPMS CSF spiked with a PPMS rAb developed significant motor deficits at 1 DPI.
Conclusions
Attenuation of the pathology-inducing capacity of PPMS CSF using 100 kDa MWCO filters indicates that the target CSF component is larger than 100 kDa. The addition of PPMS rAbs (150 kDa) into filtered PPMS CSF was able to restore the pathological effects in vivo, suggesting that the induction of motor deficits and pathology by PPMS CSF is antibody-mediated.