Somsankar Dasgupta

Development and characterization of a novel anti-ceramide antibody

Ceramide is emerging as a key sphingolipid that regulates a variety of cellular processes. To facilitate the study of ceramide localization and its interaction with cellular proteins, we have developed a novel antibody against ceramide. Our results indicate that the antibody (rabbit IgG) specifically recognizes ceramide in lipid overlay assays and detects ceramide species with different fatty acid chain lengths that include C2, C8, C16, C18, C20, and C24. The new antibody was compared with the commercially available anti-ceramide antibody (mouse IgM) in immunocytochemistry experiments to study the localization of ceramide. Although both antibodies stain the same regions on the cell membrane, the rabbit IgG reveals the distribution of ceramide in compartments that are not well identified with the commercially available antibody. In addition to staining of ceramide in protrusions of the plasma membrane, the rabbit IgG also detects ceramide in the Golgi apparatus. Pharmacological depletion or increase of ceramide levels results in a corresponding change in staining intensity, confirming the specificity of the antibody. These results indicate that the rabbit IgG is a suitable antibody to determine the localization of ceramide and its interaction with proteins by immunocytochemistry.

Long-chain ceramide is elevated in presenilin 1 (PS1M146V) mouse brain and induces apoptosis in PS1 astrocytes

The pro‐apoptotic sphingolipid ceramide plays an emergent role in the etiology of Alzheimers disease (AD), although its function for neurodegeneration is not known. We determined the concentration and composition of ceramide in hippocampal tissue from newborn presenilin 1 (PS1) knock‐in (PS1M146V) mice, a mouse model for early‐onset familial AD. We found that PS1 tissue contains 3.1 (±0.5)‐fold more total ceramide than wild‐type tissue. In particular, the proportion of C20 and C24 ceramide is increased by 4.0‐ or 8.5‐fold, respectively. The ceramide elevation in PS1 brain is consistent with a 3.7 (±0.5)‐fold increase of the protein level of the neurotrophin receptor p75NTR, which has been suggested to stimulate the hydrolysis of sphingomyelin to generate ceramide. The predominance of C20 and C24 ceramide is concurrent with the elevated gene expression of lass 2 and lass 4, two isoforms of ceramide synthase that generate dihydroceramide with long‐chain fatty acid. Our study indicates that primary cultured astrocytes but not neurons from PS1 mice undergo apoptosis when incubated with C20 ceramide. In contrast, wild‐type astrocytes remain unaffected. The sensitivity of PS1 astrocytes is most likely due to the 9.5 (±0.4)‐fold elevated expression of PAR‐4 (prostate apoptosis response‐4), a protein that inhibits atypical PKCζ/λ in the presence of ceramide. Our results suggest that astroglial death due to ceramide/PAR‐4‐induced apoptosis may critically contribute to the etiology of AD.

Lipids isolated from bone induce the migration of human breast cancer cells

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Highly active antiretroviral therapy and human immunodeficiency virus encephalitis

The ability of highly active antiretroviral therapy (HAART) to prevent the onset of HIV‐associated dementia and to prevent or reduce the neuropathological features of HIV encephalitis (HIVE) remains unclear. Using a severe combined immunodeficient (SCID) mouse model of HIVE, we determined the effects of regular HAART treatment on HIVE. Before studying HAART in infected SCID mice, nonmanipulated SCID mice were treated with a single injection of the HAART cocktail (consisting of zidovudine, lamivudine, and indinavir) to determine optimum dosage and sampling time and to measure antiretroviral levels in the brain. After these preliminary studies, SCID mice that were inoculated with either HIV‐infected or uninfected human monocytes were given intraperitoneal (IP) injections of HAART three times daily over a 1‐ and 2‐week period. All three drugs were detected in the brain using a novel drug extraction technique and a modified high‐performance liquid chromatography method. HAART significantly decreased the amount of astrogliosis and viral load in treated mice compared with mice that received vehicle injections. These studies offer insight into the ability of HAART to treat HIV infection of the brain. Ann Neurol 2005

Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA‐MB‐231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP‐associated exosomes, while siRNA‐mediated knockdown or GW4869‐mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor‐labeled breast cancer stem‐like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

Human embryonic stem and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC, both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes.

Characterization of an apical ceramide-enriched compartment regulating ciliogenesis

The sphingolipid ceramide is essential for the formation, elongation, or maintenance of primary cilia. A novel, apical ceramide-enriched compartment induces formation of a ciliogenic protein complex with Rab11a, which sustains formation and maintenance of primary cilia by preventing deacetylation of microtubules.

Regulation of neural progenitor cell motility by ceramide and potential implications for mouse brain development.

We provide evidence that the sphingolipid ceramide, in addition to its pro‐apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo. Ceramide (N‐palmitoyl d‐erythro sphingosine and N‐oleoyl d‐erythro sphingosine) and the ceramide analog N‐oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti‐ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co‐distributed with its binding partner atypical protein kinase C ζ/λ (aPKC), and Cdc42, α/β‐tubulin, and β‐catenin, three proteins involved in aPKC‐dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide‐mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo, sphingolipid depletion leads to ectopic localization of mitotic or post‐mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo.

Endogenous GD3 ganglioside induces apoptosis in U-1242 MG glioma cells

GD3 ganglioside induces apoptosis in several cell types, but the molecular events through which this occurs are largely unknown. We investigated the apoptotic effects of GD3 expression using U‐1242 MG glioblastoma cells, as these cells synthesize almost exclusively GM3 and GM2 but not GD3. To express GD3 under the control of the TetOn system with minimum leakage, we modified the system by constructing a single tri‐cistronic retrovirus vector containing three genes separated by two internal ribosome entry sites: (a) transcriptional silencer, tTS; (b) mutant of reverse transcriptional activator, rtTA2(S)‐M2 (provided by H. Bujard, Heidelberg, Germany); and (c) enhanced green fluorescent protein (EGFP), as an indicator of the tri‐cistronic gene expression. Using flow cytometry, we selected glioma cells (U1242MG‐GD3 clone) that express high levels of GD3 in response to doxycycline. Expression of GD3 was associated with apoptosis as verified by annexin‐V binding, TdT‐mediated dUTPnick end‐labelling assay (TUNEL), and EGFP degradation. GD3‐induced apoptosis occurred via caspase‐8 activation, as GD3 caused cleavage of caspase‐8 and inhibition of caspase‐8 activation by zlETD‐fmk minimized GD3‐induced apoptosis.

The 5XFAD Mouse Model of Alzheimer's Disease Exhibits an Age-Dependent Increase in Anti-Ceramide IgG and Exogenous Administration of Ceramide Further Increases Anti-Ceramide Titers and Amyloid Plaque Burden.

We present evidence that 5XFAD Alzheimers disease model mice develop an age-dependent increase in antibodies against ceramide, suggesting involvement of autoimmunity against ceramide in Alzheimers disease pathology. To test this, we increased serum anti-ceramide IgG (2-fold) by ceramide administration and analyzed amyloid plaque formation in 5XFAD mice. There were no differences in soluble or total amyloid-β levels. However, females receiving ceramide had increased plaque burden (number, area, and size) compared to controls. Ceramide-treated mice showed an increase of serum exosomes (up to 3-fold using Alix as marker), suggesting that systemic anti-ceramide IgG and exosome levels are correlated with enhanced plaque formation.