Manipulation of Body Fat by Passive and Active Immunisation Against the Adipocyte

The aim of the work described in this thesis was to devise a technique for the reduction of body fat by passive or active immunisation against the adipocyte. Passive immunisation of rats with an antiserum, raised in sheep, against rat adipocyte plasma membranes (A/S 83) caused a 50% reduction in parametrial adipocyte number which persisted, at least until 24 weeks after treatment. This was accompanied by a reduction in parametrial adipocyte size which persisted for 8 weeks but had recovered by 24 weeks, resulting in a 50% reduction in parametrial adipose mass at 24 weeks. Subcutaneous and peri-renal adipose depots were, initially, less affected and had recovered their total mass by 24 weeks after treatment. Differential effects on different adipose depots were found to be due largely to the site of injection of the antiserum. Body composition analysis showed that total body fat was reduced by 30% 8 weeks after treatment and lost fat was replaced by protein and water. Side effects of A/S 83 were limited to a reduction in food intake on the first day of treatment, which gradually recovered to reach normal levels by 4 days and transient proteinurea with fluctuations in body weight about 2 weeks after treatment in some rats. The initial effects of A/S 83 on adipose tissue and food intake were dependent on the presence of circulating complement. A doubling of serum free fatty acids and triglycerides occurred 6-24 h after treatment, then returned to normal levels by 48 h after treatment and was probably evidence of adipocyte destruction. Treatment with A/S 83 did not affect the ability of rats to undergo a normal pregnancy and lactation. More recent bleeds of the same sheep did not reproduce the in vivo effects of earlier bleeds of A/S 83 and so 2 additional antisera were raised against rat adipocyte plasma membranes. These new antisera, while reproducing the effects of A/S 83 on adipose tissue, had the additional side effects of anaesthetic-like effects immediately after administration and the production of gross liver abnormalities. The presence or absence of side effects could not be correlated with strength of binding to nervous tissue or hepatocyte plasma membranes, as determined by ELISA and Western blotting. An attempt was made to prepare rat adipocyte specific antigens by affinity purification of anti-(adipocyte plasma membrane) antibodies, adsorption of those antibodies with non-adipose tissues and the use of the adipocyte specific antibodies to affinity purify antigens from solubilized adipocyte plasma membranes. Six polypeptides, with molecular weights of 53-96 kD, were considerably enriched in the adipocyte specific antigen preparation. Antisera raised against the adipocyte specific antigens showed some binding to non-adipose tissues and antisera raised against hepatocyte and erythrocyte plasma membranes showed some binding to adipocyte specific antigens, which cast doubt on the true adipocyte specificity of some of the components of the adipocyte specific antigen preparation. However, the anti-(adipocyte specific antigen) antiserum showed considerably more adipocyte specificity than antisera raised against whole adipocyte plasma membranes, had fat-reducing properties in vivo and did not cause liver abnormalities. Adsorption of anti-(adipocyte plasma membrane) antisera with liver homogenate provided an antiserum that retained its effects on adipose tissue but no longer caused liver abnormalities. It is, therefore, likely that adipocyte specific antisera can have fat-reducing properties, but both the anti-(adipocyte specific antigen) antiserum and the adsorbed antiserum reproduced the anaesthetic-like effects of anti-(adipocyte plasma membrane) antisera and caused a reduction in food intake on the first day of treatment. Antisera raised against hepatocyte and erythrocyte plasma membranes had no effects on adipose tissue. Attempts were made to reproduce the effects of passive immunisation by means of active immunisation against the adipocyte, as this might prove a more practical approach for the treatment of large species. Rats were immunised with rat adipocyte plasma membranes or adipocyte specific antigens conjugated to BSA, or with BSA alone in complete Freunds adjuvant. Despite the absence of a convincing demonstration of circulating anti-(adipocyte plasma membrane) antibodies, adipocyte plasma membrane- and adipocyte specific antigen-treated rats showed a 40-50% reduction in adipocyte numbers and a compensatory increase in adipocyte size. In only 1 out of 3 groups of rats did active immunisation induce a reduction in adipose mass and, in this group, body weight was also reduced. (Abstract shortened by ProQuest.)

Gold Particle Analyser: Detection and quantitative assessment of electron microscopy gold probes

Gold particle probes are an essential electron microscopy tool to examine protein localisation, as well as protein trafficking. They can be introduced into living cells when conjugated to a protein that is endocytosed or to an antibody against a cell surface protein. Alternatively, gold particles can be introduced into fixed cells or tissue when conjugated to antibodies, immunoglobulin binding molecules or chemical probes applied to permeabilised samples or electron microscopy sections. Colloidal gold particles that have not been enlarged through chemical (gold or silver) enhancement are typically spherical and can be prepared in a range of specific sizes, allowing multiple proteins to be localised within a single sample. The typically homogeneous shape and size of the colloidal gold makes them ideal for computer assisted detection and analysis. Here we demonstrate a program developed to automatically identify two sizes of gold particle and perform a range of analyses that includes (i) distribution and cluster analysis; (ii) selection and analysis of gold particles allocated close to or either side of a membrane; (iii) measurement of organelle size; (iv) estimation of the number of gold particles within an aggregate and (v) the detection of chemically enhanced irregular sized and shaped gold particles. We show this easy-to-use program can greatly assist electron microscopists, to reliably and efficiently analyse gold particles within their images

Annexin 11 is required for midbody formation and completion of the terminal phase of cytokinesis

Annexins are Ca2+-binding, membrane-fusogenic proteins with diverse but poorly understood functions. Here, we show that during cell cycle progression annexin 11 translocates from the nucleus to the spindle poles in metaphase and to the spindle midzone in anaphase. Annexin 11 is recruited to the midbody in late telophase, where it forms part of the detergent-resistant matrix that also contains CHO1. To investigate the significance of these observations, we used RNA interference to deplete cells of annexin 11. A combination of confocal and video time-lapse microscopy revealed that cells lacking annexin 11 fail to establish a functional midbody. Instead, daughter cells remain connected by intercellular bridges that contain bundled microtubules and cytoplasmic organelles but exclude normal midbody components such as MKLP1 and Aurora B. Annexin 11–depleted cells failed to complete cytokinesis and died by apoptosis. These findings demonstrate an essential role for annexin 11 in the terminal phase of cytokinesis

EGF receptor trafficking: consequences for signaling and cancer

The ligand-stimulated epidermal growth factor receptor (EGFR) has been extensively studied in the analysis of molecular mechanisms regulating endocytic traffic and the role of that traffic in signal transduction. Although such studies have largely focused on mitogenic signaling and dysregulated traffic in tumorigenesis, there is growing interest in the potential role of EGFR traffic in cell survival and the consequent response to cancer therapy. Here we review recent advances in our understanding of molecular mechanisms regulating ligand-stimulated EGFR activation, internalization, and post-endocytic sorting. The role of EGFR overexpression/mutation and new modulators of EGFR traffic in cancer and the response to cancer therapeutics are also discussed. Finally, we speculate on the relationship between EGFR traffic and cell survival

Hrs- and CD63-dependent competing mechanisms make different sized endosomal intraluminal vesicles.

Multivesicular endosomes/bodies (MVBs) contain intraluminal vesicles (ILVs) that bud away from the cytoplasm. Multiple mechanisms of ILV formation have been identified, but the relationship between different populations of ILVs and MVBs remains unclear. Here, we show in HeLa cells that different ILV subpopulations can be distinguished by size. EGF stimulation promotes the formation of large ESCRT-dependent ILVs, whereas depletion of the ESCRT-0 component, Hrs, promotes the formation of a uniformly sized population of small ILVs, the formation of which requires CD63. CD63 has previously been implicated in ESCRT-independent sorting of PMEL in MVBs and transfected PMEL is present on the small ILVs that form on Hrs depletion. Upregulation of CD63-dependent ILV formation by Hrs depletion indicates that Hrs and CD63 regulate competing machineries required for the generation of distinct ILV subpopulations. Taken together our results indicate that ILV size is influenced by their cargo and mechanism of formation and suggest a competitive relationship between ESCRT-dependent and -independent mechanisms of ILV formation within single MVBs

ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation.

Intracellular amyloid-β (Aβ) accumulation is a key feature of early Alzheimer's disease and precedes the appearance of Aβ in extracellular plaques. Aβ is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aβ production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aβ production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aβ accumulation. This was accompanied by dramatically decreased Aβ secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aβ accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aβ secretion

Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.publishersversionpublishe

Cholesterol overload: contact sites to the rescue!

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER

Changes in Mitochondrial Size and Morphology in the RPE and Photoreceptors of the Developing and Ageing Zebrafish

Mitochondria are essential adenosine triphosphate (ATP)-generating cellular organelles. In the retina, they are highly numerous in the photoreceptors and retinal pigment epithelium (RPE) due to their high energetic requirements. Fission and fusion of the mitochondria within these cells allow them to adapt to changing demands over the lifespan of the organism. Using transmission electron microscopy, we examined the mitochondrial ultrastructure of zebrafish photoreceptors and RPE from 5 days post fertilisation (dpf) through to late adulthood (3 years). Notably, mitochondria in the youngest animals were large and irregular shaped with a loose cristae architecture, but by 8 dpf they had reduced in size and expanded in number with more defined cristae. Investigation of temporal gene expression of several mitochondrial-related markers indicated fission as the dominant mechanism contributing to the changes observed over time. This is likely to be due to continued mitochondrial stress resulting from the oxidative environment of the retina and prolonged light exposure. We have characterised retinal mitochondrial ageing in a key vertebrate model organism, that provides a basis for future studies of retinal diseases that are linked to mitochondrial dysfunction

Aβ accumulation causes MVB enlargement and is modelled by dominant negative VPS4A.

BACKGROUND: Alzheimer's disease (AD)-linked β-amyloid (Aβ) accumulates in multivesicular bodies (MVBs) with the onset of AD pathogenesis. Alterations in endosomes are among the earliest changes associated with AD but the mechanism(s) that cause endosome enlargement and the effects of MVB dysfunction on Aβ accumulation and tau pathology are incompletely understood. METHODS: MVB size and Aβ fibrils in primary neurons were visualized by electron microscopy and confocal fluorescent microscopy. MVB-dysfunction, modelled by expression of dominant negative VPS4A (dnVPS4A), was analysed by biochemical methods and exosome isolation. RESULTS: Here we show that AD transgenic neurons have enlarged MVBs compared to wild type neurons. Uptake of exogenous Aβ also leads to enlarged MVBs in wild type neurons and generates fibril-like structures in endocytic vesicles. With time fibrillar oligomers/fibrils can extend out of the endocytic vesicles and are eventually detectable extracellularly. Further, endosomal sorting complexes required for transport (ESCRT) components were found associated with amyloid plaques in AD transgenic mice. The phenotypes previously reported in AD transgenic neurons, with net increased intracellular levels and reduced secretion of Aβ, were mimicked by blocking recycling of ESCRT-III by dnVPS4A. DnVPS4A further resembled AD pathology by increasing tau phosphorylation at serine 396 and increasing markers of autophagy. CONCLUSIONS: We demonstrate that Aβ leads to MVB enlargement and that amyloid fibres can form within the endocytic pathway of neurons. These results are consistent with the scenario of the endosome-lysosome system representing the site of initiation of Aβ aggregation. In turn, a dominant negative form of the CHMP2B-interacting protein VPS4A, which alters MVBs, leads to accumulation and aggregation of Aβ as well as tau phosphorylation, mimicking the cellular changes in AD