Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition

Characterization and Function of Islet Antigen Presenting Cells during NOD Diabetes

Here we characterized the initial antigen presenting cells (APCs) within the islet of Langerhans to ascertain their identity and functional role as it pertains to autoimmune diabetes. The activation of the adaptive immune system is induced by the innate immune system, and more specifically APCs. Therefore, it is crucial to identify the APCs that are initiating T1D in order to elucidate the break in tolerance and intervene in order to inhibit progression. We have found that there is a resident macrophage that is present in all strains of mice. This islet macrophage has a distinct transcriptional profile that is unique when compared to other non-barrier tissue resident macrophages. The islet resident macrophage’s phenotype is akin to those macrophages found at barrier sites, i.e. the lung and the intestines. The barrier macrophages are constantly in contact with environmental pathogens, but the islet resident macrophage is located in tightly clustered mini-organs that are not in contact with barrier surfaces. We were able to show by RNAseq analysis that the islet resident macrophage is similar to macrophages treated with LPS and, thus, highly inflammatory. Furthermore, transcripts for the inflammatory cytokines TNFα and IL-1β found in islet macrophages were abundant and also were being produced in high amounts as protein. However, we were unable to definitively ascertain any functional role these cytokines have whether that may be inflammatory or homeostatic. Finally, the islet macrophages found in NOD.Rag1-/- mice were homogenous, i.e. single cell qRT-PCR displayed similar gene transcripts being expressed across all cells examined. However, although ~75% of the NOD islet macrophages expressed similar transcripts as the NOD.Rag1-/- macrophages, the remaining macrophages expressed an increase in inflammatory transcripts that are associated with interferon signaling. Finally, when compared to the non-diabetic B6.g7 islet macrophage, the NOD macrophage expressed differing chemokines that could be involved in chemotaxis of autoimmune cells into the islets. In non-diabetic mouse strains, the only leukocyte found within the islets is the resident macrophage. In the NOD mouse, however, at approximately the age of weaning, a CD103+ dendritic cell (DC) is found within the islets. This DC population enters at a similar time that CD3ε+ T cells are entering, however, we find that the initial entry of the CD103+ DC is not dependent on CD3ε+ T cell entry because the CD103+ DCs represent a small fraction of the myeloid compartment even in NOD.Rag1-/- mice. As diabetes progresses, these CD103+ DCs increase and this increase is dependent upon autoreactive CD3ε+ T cell entry. By genetic deletion of the CD103+ DC subset of DCs (cDC1 DCs) by utilizing the Batf3-deficient mouse, diabetes was abolished. The cDC1 DCs were not present in the islets or in any tissue examined, as expected. The protection from diabetes produced by the loss of the cDC1 DCs was absolute. At no timepoint was there infiltration of autoreactive cells to the islet of Langerhans assayed by histology and flow cytometry. The CD3ε+ T cell infiltration never exceeded that of baseline seen in non-diabetic strains. The islet gene expression profile of the NOD.Batf3-/- mouse was essentially identical to the lymphocyte deficient NOD.Rag1-/- islets. The priming of autoreactive CD8+ T cells was extinguished and priming of autoreactive CD4+ T cells was reduced by half. Transfer of naïve autoreactive T cells did not illicit entry into the islets or diabetes. However, diabetic splenocytes were able to confer diabetes when transferred into NOD.Batf3-/- mice. Therefore, the data strongly suggests the block in progression witnessed in the cDC1 DC deficient mouse is a lack of autoreactive T cell priming. In conclusion, we have identified the major APCs residing within the islet of Langerhans in the NOD mouse. During the life of the mouse, there is found a resident macrophage resting in a basal inflammatory state. Upon weaning, a CD103+ DC enters the islet of Langerhans but only in the NOD strain. The NOD islet macrophage expresses aberrant chemokines when compared to non-diabetic strains, which may lead to the initial infiltration of either the CD3ε+ T cells or the CD103+ DCs. When the CD103+ DCs are absent, T1D is halted due to a block in T cell priming. Although the definitive trigger of diabetes is yet to be determined, we believe that within the interplay amongst the islet resident macrophage, the entering T cells, and CD103+ DC is the initiating events. Perturbing one, or all, of the arms of this triad will result in a block in diabetes. How they interact, activate, and propagate the process will serve as the basis for future studies within the lab

Evaluation of focal adhesion kinase as a novel radiosensitising target

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is upregulated in a variety of human cancers. While there is evidence that FAK is implicated in a wide range of crucial cellular processes that are perturbed in malignancy including proliferation, cell cycle, adhesion and invasion, there is limited information regarding the role of FAK in radiation survival. We aimed to evaluate whether FAK is a novel radiosensitising target by studying clonogenicity in wt p53 FAK +/+ versus FAK -/- squamous cell carcinoma (SCC) cell lines generated in this laboratory. Surprisingly, the absence of FAK was associated with increased radioresistance. In this particular context, FAK indirectly inhibits p53 mediated transcriptional regulation of p21 in response to ionising radiation. Why FAK should repress the pro-survival function of p53 is unclear, but this data indicates that inhibition of FAK in combination with radiation may not always be advantageous in the clinical setting and contributes to an increasing body of literature highlighting a close interaction between FAK and p53