In this review focus is on structural imaging in the Alzheimer’s disease pre-states, particularly cognitively normal (CN) persons at future dementia risk. Findings in mild cognitive impairment (MCI) are described here only for comparison with CN. Cited literature evidence and commentary address issues of structural imaging alterations in CN that precede MCI and AD, regional patterns of such alterations, and the time relationship between structural imaging alterations and the appearance of symptoms of AD, issues relevant to the conduct of future AD prevention trials.

The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine, and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analog (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analog, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development, and provide the first non-lipid inhibitors of human ceramidase activity.

Sphingosine kinases (SK) regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate (S1P); however, the functions of the two isoenzymes (SK1 and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation and migration/invasion. Treatment of A498, Caki-1 or MDA-MB-231 cells with siRNA specific for SK1 or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2, and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein and activity levels of SK1, and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, demonstrating that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the anti-proliferative or anti-migratory effects of the siRNAs. Consistent with these results, differential affects of SK1- and SK2-selective siRNAs on signaling proteins including p53, p21, ERK1, ERK2, FAK and VCAM1 indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy.

Neuroimaging biomarkers that precede cognitive decline have the potential to aid early diagnosis of Alzheimer's disease (AD). A body of diffusion tensor imaging (DTI) work has demonstrated declines in white matter (WM) microstructure in AD and its typical prodromal state, amnestic mild cognitive impairment. The present review summarizes recent evidence suggesting that WM integrity declines are present in individuals at high AD-risk, prior to cognitive decline. The available data suggest that AD-risk is associated with WM integrity declines in a subset of tracts showing decline in symptomatic AD. Specifically, AD-risk has been associated with WM integrity declines in tracts that connect grey matter structures associated with memory function. These tracts include parahippocampal WM, the cinglum, the inferior fronto-occipital fasciculus, and the splenium of the corpus callosum. Preliminary evidence suggests that some AD-risk declines are characterized by increases of radial diffusivity, raising the possibility that a myelin-related pathology may contribute to AD onset. These findings justify future research aimed at a more complete understanding of the neurobiological bases of DTI-based declines in AD. With continued refinement of imaging methods, DTI holds promise as a method to aid identification of presymptomatic AD.

Background/Aims

The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR.

Methods

Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion.

Results

Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25-fold and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54% to 91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/reoxygenation resulted in increases of SK2 mRNA, S1P levels and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes.

Conclusions

SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death and survival after hepatic IR and represents a new target for the treatment of IR injury.

The balance between the pro-apoptotic lipids ceramide and sphingosine and the pro-survival lipid sphingosine 1-phosphate (S1P) is termed the “sphingosine rheostat”. Two isozymes, sphingosine kinase 1 and 2 (SK1 and SK2), are responsible for phosphorylation of pro-apoptotic sphingosine to form pro-survival S1P. We have previously reported the antitumor properties of an SK2 selective inhibitor, ABC294640, alone or in combination with the multikinase inhibitor sorafenib in mouse models of kidney carcinoma and pancreatic adenocarcinoma. Here, we evaluated the combined antitumor effects of the aforementioned drug combination in two mouse models of hepatocellular carcinoma. Although combining the SK2 inhibitor, ABC294640 and sorafenib in vitro only afforded additive drug-drug effects, their combined antitumor properties in the mouse model bearing HepG2 cells mirrored effects previously observed in animals bearing kidney carcinoma and pancreatic adenocarcinoma cells. Combining ABC294640 and sorafenib led to a decrease in the levels of phosphorylated ERK in SK-HEP -1 cells, indicating that the antitumor effect of this drug combination is likely mediated through a suppression of the MAPK pathway in hepatocellular models. We also measured levels of S1P in the plasma of mice treated with two different doses of ABC294640 and sorafenib. We found decreases in the levels of S1P in plasma of mice treated daily with 100 mg/kg of ABC294640 for 5 weeks, and this decrease was not affected by coadministration of sorafenib. Taken together, these data support combining ABC294640 and sorafenib in clinical trials in HCC patients. Furthermore, monitoring levels of S1P may provide a pharmacodynamic marker of ABC294640 activity.

Summary

The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/ 7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.

White matter (WM) microstructural declines have been demonstrated in Alzheimer’s disease and amnestic mild cognitive impairment (aMCI). However, the pattern of WM microstructural changes in aMCI after controlling for WM atrophy is unknown. Here, we address this issue through joint consideration of aMCI alterations in fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity, as well as macrostructural volume in WM and gray matter compartments. Participants were 18 individuals with aMCI and 24 healthy seniors. Voxelwise analyses of diffusion tensor imaging data was carried out using tract-based spatial statistics (TBSS) and voxelwise analyses of high-resolution structural data was conducted using voxel based morphometry. After controlling for WM atrophy, the main pattern of TBSS findings indicated reduced fractional anisotropy with only small alterations in mean diffusivity/radial diffusivity/axial diffusivity. These WM microstructural declines bordered and/or were connected to gray matter structures showing volumetric declines. However, none of the potential relationships between WM integrity and volume in connected gray matter structures was significant, and adding fractional anisotropy information improved the classificatory accuracy of aMCI compared to the use of hippocampal atrophy alone. These results suggest that WM microstructural declines provide unique information not captured by atrophy measures that may aid the magnetic resonance imaging contribution to aMCI detection.

Sphingosine kinases (SKs) are promising new therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate. The functions of the two SK isoenzymes, SK1 and SK2, are not redundant, with genetic ablation of SK2 having more pronounced anticancer effects than removal of SK1. Although several small molecule inhibitors of SKs have been described in the literature, detailed characterization of their molecular and cellular pharmacology, particularly their activities against human SK1 and SK2, have not been completed. Computational modeling of the putative active sites of SK1 and SK2 suggests structural differences that might allow isozyme-selective inhibitors. Therefore, we characterized several SK-inhibitory compounds which revealed differential inhibitory effects on SK1 and SK2 as follows: SKI-II and ABC294735 are SK1/2-dual inhibitors; CB5468139 is a SK1-selective inhibitor; and ABC294640 is a SK2-selective inhibitor. We examined the effects of the SK inhibitors on several biochemical and phenotypic processes in A498 kidney adenocarcinoma cells. The SK2-selective inhibitor ABC294640 demonstrated the most pronounced effects on SK1 and SK2 mRNA expression, decrease of S1P levels, elevation of ceramide levels, cell cycle arrest, and inhibition of proliferation, migration and invasion. ABC294640 also down-regulated the expression or activation of several signaling proteins, including STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to responses to the SK1/2-dual inhibitors. Overall, these results suggest that inhibition of SK2 results in stronger anticancer effects than does inhibition of SK1 or both SK1 and SK2.

The Pim protein kinases play important roles in cancer development and progression, including prostate tumors and hematologic malignancies. To investigate the potential role of these enzymes as anticancer drug targets, we have synthesized novel benzylidene-thiazolidine-2,4-diones that function as potent Pim protein kinase inhibitors. With IC50 values in the nanomolar range, these compounds block the ability of Pim to phosphorylate peptides and proteins in vitro and, when added to DU145 prostate cancer cells overex-pressing Pim, inhibit the ability of this enzyme to phosphorylate a known substrate, the BH3 protein BAD. When added to prostate cancer cell lines, including PC3, DU145, and CWR22Rv1, and human leukemic cells, MV4;11, K562, and U937 cells, these compounds induce G1-S cell cycle arrest and block the antiapoptotic effect of the Pim protein kinase. The cell cycle arrest induced by these compounds is associated with an inhibition of cyclin-dependent kinase 2 and activity and translocation of the Pim-1 substrate p27Kip1, a cyclin-dependent kinase 2 inhibitory protein, to the nucleus. Furthermore, when added to leukemic cells, these compounds synergize with the mammalian target of rapamycin inhibitor rapamycin to decrease the phosphorylation level of the translational repressor 4E-BP1 at sites phosphorylated by mammalian target of rapamycin. Combinations of rapamycin and the benzylidene-thiazolidine-2,4-diones synergistically block the growth of leukemic cells. Thus, these agents represent novel Pim inhibitors and point to an important role for the Pim protein kinases in cell cycle control in multiple types of cancer cells.

Palmitoylation is required for the activities of several cancer-associated proteins, making the palmitoyl acyltransferase (PAT) enzymes that catalyze these reactions potential targets for anticancer therapeutics. In this study, we sought to identify and characterize a human PAT with activity toward N-terminally myristoylated and palmitoylated proteins. NIH/3t3 cells were stably transfected with vectors containing no insert, wild type human DHHC20, or a serine-substituted DHHS20 mutant. Compared with control cells, cells overexpressing wild-type DHHC20 displayed an increase in palmitoylation activity toward a peptide that mimics the N-terminus of myristoylated and palmitoylated proteins, but had no change in activity toward a peptide that mimics the C-terminus of farnesylated and palmitoylated proteins. Cells expressing DHHS20 had no significant change in activity toward either peptide. Overexpression of DHHC20 also caused phenotypic changes consistent with cellular transformation, including colony formation in soft agar, decreased contact inhibition of growth, and increased proliferation under low-serum conditions. Quantitative polymerase chain reaction analyses of human tissues demonstrated that DHHC20 is expressed in a tissue-specific manner, and is overexpressed in several types of human tumors, including ovarian, breast and prostate. Overall, these results demonstrate that DHHC20 is a human N-terminal-myristoyl-directed PAT involved in cellular transformation, that may play a role in cancer.

Reduced task deactivation within regions of the default mode network (DMN) has been frequently reported in Alzheimer’s disease (AD) and amnestic mild cognitive impairment (aMCI). As task deactivations reductions become increasingly used in the study of early AD states, it is important to understand their relationship to atrophy. To address this issue, the present study compared task deactivation reductions during a lexical decision task and atrophy in aMCI, using a series of parallel voxel-wise and region-wise analyses of fMRI and structural data. Our results identified multiple regions within parietal cortex as convergence areas of task deactivation and atrophy in aMCI. Relationships between parietal regions showing overlapping task deactivation reductions and atrophy in aMCI were then explored. Regression analyses demonstrated minimal correlation between task deactivation reductions and either local or global atrophy in aMCI. In addition, a logistic regression model which combined task deactivation reductions and atrophy in parietal DMN regions showed higher classificatory accuracy of aMCI than separate task deactivation or atrophy models. Results suggest that task deactivation reductions and atrophy in parietal regions provide complementary rather than redundant information in aMCI. Future longitudinal studies will be required to assess the utility of combining task deactivation reductions and atrophy in the detection of early AD.

Inflammation mediates/promotes graft injury after liver transplantation (LT). This study investigated the roles of sphingosine kinase-2 (SK2) in inflammation after LT. Liver grafts were stored in UW solution with and without ABC294640 (100 µM), a selective inhibitor of SK2, before implantation. Hepatic sphingosine-1-phosphate (S1P) levels increased ∼4-fold after LT, which was blunted by 40% by ABC294640. Hepatic toll-like receptor-4 (TLR4) expression and nuclear factor-κB (NF-κB) p65 subunit phosphorylation elevated substantially after transplantation. The pro-inflammatory cytokines/chemokines tumor necrosis factor-α, interleukin-1β and C-X-C motif chemokine 10 mRNAs increased 5.9-fold, 6.1-fold and 16-fold, respectively following transplantation, while intrahepatic adhesion molecule-1 increased 5.7-fold and monocytes/macrophage and neutrophil infiltration and expansion of residential macrophage population increased 7.8–13.4 fold, indicating enhanced inflammation. CD4+ T cell infiltration and interferon-γ production also increased. ABC294640 blunted TLR4 expression by 60%, NF-κB activation by 84%, proinflammatory cytokine/chemokine production by 45–72%, adhesion molecule expression by 54% and infiltration of monocytes/macrophages and neutrophils by 62–67%. ABC294640 also largely blocked CD4+ T cell infiltration and interferon-γ production. Focal necrosis and apoptosis occurred after transplantation with serum alanine aminotransferase (ALT) reaching ∼6000 U/L and serum total bilirubin elevating to ∼1.5 mg/dL. Inhibition of SK2 by ABC294640 blunted necrosis by 57%, apoptosis by 74%, ALT release by ∼68%, and hyperbilirubinemia by 74%. Most importantly, ABC294640 also increased survival from ∼25% to ∼85%. In conclusion, SK2 plays an important role in hepatic inflammation responses and graft injury after cold storage/transplantation and represents a new therapeutic target for liver graft failure.

Gangjee et al. recently reported a novel series of 2-amino-4-methyl-5-phenylethyl substituted-7-benzyl-pyrrolo[2,3-d]pyrimidines, some of which exhibited two digit nanomolar antitumor and antimitotic activity and were not subject to P-glycoprotein (Pgp) or Multidrug Resistance Protein 1 (MRP1) mediated tumor resistance (unlike the Vinca alkaloids and taxanes). Some of these compounds, in addition to their antitumor activity, had the ability to reverse the Pgp-mediated resistance to clinically used antimitotic agents. This report consists of an attempt to optimize the various activities of the parent compounds by synthetic variations of the phenyl ring of the 5-phenylethyl side chain. The target compounds were synthesized via a 9-step synthesis involving a Sonogashira reaction. The substituted phenylacetylenes as coupling partners were in turn synthesized from unactivated aryl bromides or iodides. The target compounds exhibited moderate cytotoxicity against MCF-7 tumor cells. However, most of these compounds showed improved cytotoxicity against the resistant NCI/ADR and MCF-7/VP. This study afforded an analog which reversed both Pgp-mediated as well as MRP1-mediated resistance to clinically used antimitotic agents, along with its own antimitotic mediated antitumor activity. In addition, in the NCI-60 cell line panel one of the compounds inhibited the growth of MDA-MD-435 breast cancer cell line at submicromolar concentration.

The National Institute on Aging Preclinical Alzheimer’s disease Workgroup (PADW) has issued a preliminary report with recommendations for classifying preclinical Alzheimer’s disease (pAD) according to 3 early disease stages. Here we examine the PADW recommendations in relation to neuropathological features in a large, consecutive series of cognitively intact elderly persons, autopsied within a year after cognitive testing (n = 126 cognitively intact patients with mean age 83.7 years at death). Subjects were grouped based on a hypothetical construct correlating pathological features with PADW stages. Many cognitively intact individuals were classifiable as pAD (53/126 or 43%), as expected based on epidemiological and biomarker studies. Of these, most (48%) were in “stage 3”, which corresponds to amyloid pathology with early neurodegeneration. As with prior studies, our data indicate that the development of neocortical neurofibrillary tangles is the key pathological event that is not observed in pAD cases: Braak stages III or IV pathology are hence not truly a substrate for “intermediate likelihood” that cognitive impairment is due to Alzheimer’s disease (AD). We also stress the importance of comorbid non-Alzheimer’s disease brain pathologies (hippocampal sclerosis, neocortical alpha-synucleinopathy, cerebrovascular disease, and brains with hippocampal neurofibrillary tangles but no cortical amyloid plaques) that can contribute to the development of cognitive impairment, or which may serve as confounds in the application of the PADW recommendations. While the final recommendations from the PADW working group have not yet been released, this preliminary analysis provides a perspective on those recommendations from a neuropathological point of view.

N-myristoyltransferases (NMT) add myristate to the NH2 termini of certain proteins, thereby regulating their localization and/or biological function. Using RNA interference, this study functionally characterizes the two NMT isozymes in human cells. Unique small interfering RNAs (siRNA) for each isozyme were designed and shown to decrease NMT1 or NMT2 protein levels by at least 90%. Ablation of NMT1 inhibited cell replication associated with a loss of activation of c-Src and its target FAK as well as reduction of signaling through the c-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathway. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assays showed that depletion of either NMT isozyme induced apoptosis, with NMT2 having a 2.5-fold greater effect than NMT1. Western blot analyses revealed that loss of NMT2 shifted the expression of the BCL family of proteins toward apoptosis. Finally, intratumoral injection of siRNA for NMT1 or for both NMT1 and NMT2 inhibited tumor growth in vivo, whereas the same treatment with siRNA for NMT2 or negative control siRNA did not. Overall, the data indicate that NMT1 and NMT2 have only partially overlapping functions and that NMT1 is critical for tumor cell proliferation.

Preclinical Alzheimer’s disease (pAD) reflects neuropathological findings of AD in cognitively normal subjects. The present study represents an effort to determine if differences could be identified in the longitudinal patterns of cognitive performance in persons classified as pAD compared to those who did not meet criteria for AD at autopsy. We included 121 subjects who were cognitively normal from baseline through their last assessment before death and who underwent autopsy. Participants were classified into two groups: pathologically normal (PN; NIA-Reagan low or no-likelihood of AD, n=89) and preclinical AD (pAD; NIA-Reagan criteria of intermediate or high-likelihood of AD in the absence of clinical dementia symptoms, n=32) followed for a mean 7.5 years prior to death. Longitudinal rates and patterns of change in scores on a standard cognitive battery were compared between these two groups. While cognitive results at baseline and last evaluations revealed no clear cross sectional group differences after adjustment for age, APOE status, education, and gender, statistically significant differences between the pAD and PN groups in slope of decline were seen on a composite score of cognitive function. Further analyses showed three components of this score reached significance: constructional praxis, delayed recall of a word list, and category verbal fluency. Despite being clinically viewed as normal at enrollment and at the final exam, there are significant differences in rates of cognitive decline in participants classified as pAD compared to those without this pathology. Longitudinal changes in slope of decline in specific cognitive test measures can serve as non-invasive methods for the detection of pAD.

Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by N-myristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30- and 130-fold lower Km values for Nef than Gag, respectively. Values for Kcat indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower Km for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef–sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef–sgGFP, whereas, depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef–sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence.

Hippocampal sclerosis is a relatively common neuropathological finding (∼10% of individuals over the age of 85 years) characterized by cell loss and gliosis in the hippocampus that is not explained by Alzheimer’s disease. Hippocampal sclerosis pathology can be associated with different underlying causes, and we refer to hippocampal sclerosis in the aged brain as hippocampal sclerosis associated with ageing. Much remains unknown about hippocampal sclerosis associated with ageing. We combined three different large autopsy cohorts: University of Kentucky Alzheimer’s Disease Centre, the Nun Study and the Georgia Centenarian Study to obtain a pool of 1110 patients, all of whom were evaluated neuropathologically at the University of Kentucky. We focused on the subset of cases with neuropathology-confirmed hippocampal sclerosis (n = 106). For individuals aged ≥95 years at death (n = 179 in our sample), each year of life beyond the age of 95 years correlated with increased prevalence of hippocampal sclerosis pathology and decreased prevalence of ‘definite’ Alzheimer’s disease pathology. Aberrant TAR DNA protein 43 immunohistochemistry was seen in 89.9% of hippocampal sclerosis positive patients compared with 9.7% of hippocampal sclerosis negative patients. TAR DNA protein 43 immunohistochemistry can be used to demonstrate that the disease is usually bilateral even when hippocampal sclerosis pathology is not obvious by haematoxylin and eosin stains. TAR DNA protein 43 immunohistochemistry was negative on brain sections from younger individuals (n = 10) after hippocampectomy due to seizures, who had pathologically confirmed hippocampal sclerosis. There was no association between cases with hippocampal sclerosis associated with ageing and apolipoprotein E genotype. Age of death and clinical features of hippocampal sclerosis associated with ageing (with or without aberrant TAR DNA protein 43) were distinct from previously published cases of frontotemporal lobar degeneration TAR DNA protein 43. To help sharpen our ability to discriminate patients with hippocampal sclerosis associated with ageing clinically, the longitudinal cognitive profile of 43 patients with hippocampal sclerosis associated with ageing was compared with the profiles of 75 controls matched for age, gender, education level and apolipoprotein E genotype. These individuals were followed from intake assessment, with 8.2 (average) longitudinal cognitive assessments. A neuropsychological profile with relatively high-verbal fluency but low word list recall distinguished the hippocampal sclerosis associated with ageing group at intake (P < 0.015) and also 5.5–6.5 years before death (P < 0.005). This may provide a first step in clinical differentiation of hippocampal sclerosis associated with ageing versus pure Alzheimer’s disease in their earliest stages. In summary, in the largest series of autopsy-verified patients with hippocampal sclerosis to date, we characterized the clinical and pathological features associated with hippocampal sclerosis associated with ageing.

Resistance to chemotherapy remains a significant obstacle in the treatment of hormone-independent breast cancer. Recent evidence suggests that altered sphingolipid signaling through increased sphingosine kinase activity may be an important mediator of breast cancer drug resistance. Sphingosine kinase-1 (Sphk1) is a proposed key regulator of breast cancer tumorigenesis, proliferation and resistance. There is, however, conflicting data on the role of sphingosine kinase-2 (Sphk2) in cancer biology and resistance, with some suggesting that Sphk2 has an opposing role to that of Sphk1. Here, we studied the effects of the novel selective Sphk2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl) amide), on human breast cancer. ABC294640 blocked both viability and survival at low micromolar IC50 concentrations in the endocrine therapy-resistant MDA-MB-231 and chemoresistant MCF-7TN-R cell systems. Treatment with the inhibitor significantly reduced proliferation, as seen in immunofluorescence staining of Ki-67 in vitro. Interestingly, pharmacological inhibition of Sphk2 induced apoptosis through the intrinsic programmed cell death pathway. Furthermore, ABC294640 also diminished NFκB survival signaling, through decreased activation of the Ser536 phosphorylation site on the p65 subunit. Xenografts of MCF-7TN-R cells growing in immunocompromised mice were utilized to validate the therapeutic efficacy of the sphingosine kinase-2 inhibitor. Treatment with 50 mg of ABC294640/kg completely blocked tumor volume in this model. These results indicate that pharmacological inhibition of Sphk2 with the orally bioavailable selective inhibitor, ABC294640, has therapeutic potential in the treatment of chemoand endocrine therapy-resistant breast cancer.

Mild cognitive impairment (MCI) refers to the clinical state between normal cognition and probable Alzheimer's disease (AD), but persons diagnosed with MCI may progress to non-AD forms of dementia, remain MCI until death, or recover to normal cognition. Risk factors for these various clinical changes, which we term “transitions,” may provide targets for therapeutic interventions. Therefore, it is useful to develop new approaches to assess risk factors for these transitions. Markov models have been used to investigate the transient nature of MCI represented by amnestic single-domain and mixed MCI states, where mixed MCI comprised all other MCI subtypes based on cognitive assessments. The purpose of this study is to expand this risk model by including a clinically determined MCI state as an outcome. Analyses show that several common risk factors play different roles in affecting transitions to MCI and dementia. Notably, APOE-4 increases the risk of transition to clinical MCI but does not affect the risk for a final transition to dementia, and baseline hypertension decreases the risk of transition to dementia from clinical MCI.

MRI diffusion-tensor tracking (DTT) was performed in 17 high-functioning adolescents/adults with autism and 17 pairwise-matched controls. White matter pathways involved in face processing were examined due to the relevance of face perception to the social symptoms of autism, and due to known behavioral and functional imaging findings in autism. The hippocampo-fusiform (HF) and amygdalo-fusiform (AF) pathways had normal size and shape but abnormal microstructure in the autism group. The right HF had reduced across-fiber diffusivity (D-min) compared with controls, opposite to the whole-brain effect of increased D-min. In contrast, left HF, right AF, and left AF had increased D-min and increased along-fiber diffusivity (D-max), more consistent with the whole-brain effect. There was a general loss of lateralization compared with controls. The right HF D-min was markedly low in the autism subgroup with lower Benton face recognition scores, compared with the lower-Benton control subgroup, and compared with the higher-Benton autism subgroup. Similar behavioral relationships were found for performance IQ. Such results suggest an early functionally-significant pathological process in right HF consistent with small-diameter axons (with correspondingly slower neural transmission) and/or higher packing density. In left AF and HF, changes were interpreted as secondary, possibly reflecting axonal loss and/or decreased myelination.

A critical step in the mechanism of action of inflammatory cytokines is the stimulation of sphingolipid metabolism, including activation of sphingosine kinase (SK) which produces the mitogenic and pro-inflammatory lipid sphingosine 1-phosphate (S1P). We have developed orally-bioavailable compounds that effectively inhibit SK activity in vitro, in intact cells and in cancer models in vivo. In the present study, we have assessed the effects of these SK inhibitors on cellular responses to TNFα, and evaluated their efficacies in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice. Using several cell systems, it was shown that the SK inhibitors block the ability of TNFα to: activate NFκB; induce the expression of adhesion proteins; and promote the production of PGE2. In an acute model of DSS-induced ulcerative colitis, the SK inhibitors were equivalent to or more effective than Dipentum in reducing disease progression, colon shortening, and neutrophil infiltration into the colon. The effects of the SK inhibitors were associated with decreased colonic levels of the inflammatory cytokines TNFα, IL-1β, IFN-γ and IL-6, and reduction of S1P levels in the colon. A similar reduction in disease progression was provided by the SK inhibitors in a chronic model of ulcerative colitis in which the mice received three week-long cycles of DSS interspaced with week-long recovery periods. In the chronic model, immunohistochemistry for SK showed increased expression in DSS treated mice (compared to water controls) that was reduced by drug treatment. S1P levels were also elevated in the DSS group and significantly reduced by drug treatment. Together, these data indicate that SK is a critical component in inflammation, and that inhibitors of this enzyme may be useful in the treatment of inflammatory bowel diseases.

Purpose

The increased vascular permeability and pathogenic angiogenesis observed in diabetic retinopathy are induced, at least in part, by local inflammation and vascular endothelial growth factor (VEGF). Therefore, inhibition of signaling from VEGF and tumor necrosis factor α (TNFα) is a promising approach to the treatment of this disease, as well as ocular diseases with similar etiologies, including age-related macular degeneration. A growing body of evidence demonstrates that sphingosine kinase (SK) plays an important role in cellular proliferation and angiogenesis. Therefore, we have examined the effects of SK inhibitors on the responses of retinal endothelial cells (RECs) to VEGF and TNFα, and their therapeutic efficacy in a diabetic retinopathy model.

Methods

The expression and function of SK in bovine and human RECs were examined by immunoblotting. The involvement of SK in mediating responses to VEGF and TNFα was examined using pharmacological inhibitors of SK in cellular and in vivo assays, including a 3-month streptozotocin-induced diabetic retinopathy model in rats.

Results

We demonstrate that SK is present and active in human and bovine RECs, and that SK activity in these cells is stimulated by VEGF. Inhibitors of SK block VEGF-induced production of sphingosine 1-phosphate, and markedly attenuate VEGF-induced proliferation and migration of RECs. Additionally, SK inhibitors are shown to block TNFα-induced expression of adhesion proteins, suppress VEGF-induced vascular leakage in an in vivo mouse model, and reduce retinal vascular leakage in the rat diabetic retinopathy model.

Conclusion

Overall, these studies demonstrate that inhibitors of SK attenuate the effects of proliferative and inflammatory stimuli on RECs both in vitro and in vivo, and so could be significant therapeutics in the treatment of diabetic retinopathy.

Palmitoylated proteins have been implicated in several disease states including Huntington’s, cardiovascular, T-cell mediated immune diseases, and cancer. To proceed with drug discovery efforts in this area, it is necessary to: identify the target enzymes, establish efficient assays for palmitoylation, and conduct high-throughput screening to identify inhibitors. The primary objectives of this review are to examine the types of assays used to study protein palmitoylation and to discuss the known inhibitors of palmitoylation. Six main palmitoylation assays are currently in use. Four assays, radiolabeled palmitate incorporation, fatty acyl exchange chemistry, MALDI-TOF MS and azido-fatty acid labeling are useful in the identification of palmitoylated proteins and palmitoyl acyltransferase (PAT) enzymes. Two other methods, the in vitro palmitoylation (IVP) assay and a cell-based peptide palmitoylation assay, are useful in the identification of PAT enzymes and are more amenable to screening for inhibitors of palmitoylation. To date, two general types of palmitoylation inhibitors have been identified. Lipid-based palmitoylation inhibitors broadly inhibit the palmitoylation of proteins; however, the mechanism of action of these compounds is unknown, and each also has effects on fatty acid biosynthesis. Conversely, several non-lipid palmitoylation inhibitors have been shown to selectively inhibit the palmitoylation of different PAT recognition motifs. The selective nature of these compounds suggests that they may act as protein substrate competitors, and may produce fewer non-specific effects. Therefore, these molecules may serve as lead compounds for the further development of selective inhibitors of palmitoylation, which may lead to new therapeutics for cancer and other diseases.