Initial findings

(By Dr. Ralf Dressel, Department for Immunogenetics, Georg-August Universität Göttingen and Dr. Fayyazi, the Department of Pathology, Georg-August Universität Göttingen.)

VAMP8 ^-/^- Vti1b^-/^- mice were generated and analyzed with an aim to study the effect of deleting two SNAREs of the same complex on endosomal trafficking. These mice showed a

Chapter 1. Introduction

phenotypic onset in the early post natal life. 66% of the VAMP8 ^-/^- Vti1b^-/^- mice became sick and died before reaching one month of age. Several organs from a VAMP8^-/^- Vti1b^-/ -small and sick 16 day old mouse were excised examined by histochemistry. Liver, kidney, spleen, intestine and brain from this mouse showed no defects. However there was a major defect in the morphology of the thymus (Figure 1.9). The thymus from the age matched control showed two clearly distinguishable areas: the medulla and the cortex (Figure A, B, C). In the thymus from VAMP8^-/^- Vti1b^-/^- mouse however, the cortico medullary boundary was completely disrupted with medullary cells scattered within the cortex (Figure D, E, F).

Figure 1.9. Morphological shows a gross defect in the thymus morphology compared to the wild type control. The thymus from the wild type mouse showed distinct areas:

the lightly stained medulla and a darkly stained cortex (A) with a distinct boundary between the two areas (B).

This demarcation was also seen in the fascein staining that specifically stains the dentritic cells (brown) present in the medulla (C). In the VAMP8^-/ -Vti1b^-/^- small and sick mouse there was no boundary between the cortex and medulla (D) and light pink stained medullary cells appear scattered between the purple stained cortex cells (E). This disruption was also observed in fascein stained sections. The medullary dendritic cells (brown) appear scattered between the cortex (blue) cells (F). (*Histological staining done by Dr. Afshin Fayyazi at the Department of Pathology, Georg-August Universität Göttingen.

VAMP8 Vti1b double knock out high apoptosis compared to the wild type control (Figure 1.10).

Chapter 1. Introduction

Figure 1.10. High number of dead cells in VAMP8-/- Vti1b-/- thymus: TUNNEL Staining to detect dead cells in the thymus sections from VAMP8^-/ -Vti1b^-/^- mouse and compare it to the wild type mouse. The thymus from VAMP8^-/^- Vti1b^-/^- mouse shows a very high number of dead cells that appear as black dots (B). In comparison, the thymus from wild type mouse shows very few dead cells (A). *Histological staining done by Dr. Afshin Fayyazi at the Department of Pathology, Georg-August Universität Göttingen.

A B

Wild type VAMP8 Vti1b double knock out

To study if the disruption in thymus morphology effects the maturation of the T lymphocyte, thymocytes from VAMP8^-/^- Vti1b^-/^- mice were stained with antibodies against CD4 and CD 8 surface antigens. The cells were analyzed by FACS analysis. Out of three independent experiments, two experiments showed a high count for CD4^-CD8^- cells while the third experiment did not show this defect. Additionally it was noticed that the two mice that showed a difference in the CD4^-CD8^- cells also were much lighter than the mouse used in the third experiment.

1.4.4. Initial findings

(Masters Thesis submitted by Namita Kanwar to the Faculty of Biology at Georg August Universität Göttingen)

In the initial work, live births and deaths of VAMP8^-/^- Vti1b^-/^- and VAMP8^-/^- Vti1b⁺/^- mice were recorded. It was observed that VAMP8^-/^- Vti1b^-/^- and VAMP8^-/^- Vti1b⁺/^- pups had high early post-natal mortality. First postnatal month resulted in the loss of nearly 67% of VAMP8^-/^-Vti1b^-/^- and 59% of VAMP8^-/^- Vti1b⁺/^- mice. All the mice were born in normal Mendelian ratios and were normal at birth. However a large population of these mice startedto loose weight between 12-29 days of age and after 2-3 days of constant weight loss, eventually died. At the time of death, these mice were very small (weighed 35-50% of the littermates) and looked very sick. The surviving mice were fertile but lighter than their littermates.

Chapter 1. Introduction

In this study, three ~one month old sick VAMP8^-/^- Vti1b^-/^- mice were analyzedjust before death and compared to VAMP8⁺/^- Vti1b⁺/^- controls. The thymus from these sick mice showed a disruption in morphology as seen before and described in section 1.5.3. There was no cortico-medullary boundary in the thymii of these mice and the medullary region was reduced to remnants. The thymus from these sick VAMP8^-/^- Vti1b^-/^- mice were smaller in size and the cell count was reduced by around 10 folds compared to VAMP8⁺/^- Vti1b⁺/^- controls. FACS analysis showed a disturbance in the developing thymocyte subsets. There was upto 3 folds increase in the percentage of CD4⁺ cells while CD4⁺CD8⁺ cells were reduced by 2 folds the thymus from sick VAMP8^-/^- Vti1b^-/^- mice. However, unlike the findings of the first experiment by Dr. Dressel, there was no increase in the CD4^-CD8 -cells. The analysis of DN1-4 subsets showed a decrease in CD44⁺CD25^- (DN1) and CD44 -CD25⁺ (DN3) cells in VAMP8⁺/^- Vti1b⁺/^- mice. In addition, these thymii also showed increased annexinV positive cells.

The development of thymocytes was also analyzed in four 2 months adult VAMP8^-/^- Vti1b -/^- mice. However, there was no difference in the developing DN1-4 subsets as well as the more mature CD4/CD8 subsets in the thymii from the adult VAMP8^-/^- Vti1b^-/^- mice.

In addition, three experiments were done to analyze the peripheral repertoire of T and B lymphocytes from adult VAMP8^-/^- Vti1b^-/^- mice. There was no defect in the populations of mature CD4⁺ and CD8⁺ T lymphocytes and B lymphocytes in the VAMP8^-/^- Vti1b^-/^- mice compared to VAMP8⁺/^- Vti1b⁺/^- controls.

The activity of several lysosomal enzymes was checked in the liver and kidney of adult VAMP8^-/^- Vti1b^-/^- mice. There was no difference in the activities of hexosaminidase, β-galactosidase, β-mannosidase and β-glucoronidase enzymes in either the liver or the kidney of VAMP8^-/^- Vti1b^-/^- mice compared to the VAMP8⁺/^- Vti1b⁺/^- controls.