Single‐walled carbon nanotube and graphene nanodelivery of gambogic acid increases its cytotoxicity in breast and pancreatic cancer cells

Graphene and single‐walled carbon nanotubes were used to deliver the natural low‐toxicity drug gambogic acid (GA) to breast and pancreatic cancer cells in vitro, and the effectiveness of this complex in suppressing cellular integrity was assessed. Cytotoxicity was assessed by measuring lactate dehydrogenase release, mitochondria dehydrogenase activity, mitochondrial membrane depolarization, DNA fragmentation, intracellular lipid content, and membrane permeability/caspase activity. The nanomaterials showed no toxicity at the concentrations used, and the antiproliferative effects of GA were significantly enhanced by nanodelivery. The results suggest that these complexes inhibit human breast and pancreatic cancer cells grown in vitro. This analysis represents a first step toward assessing their effectiveness in more complex, targeted, nanodelivery systems. Copyright © 2014 John Wiley & Sons, Ltd.     

Substance use disorders assessed using the Kreek-McHugh-Schluger-Kellogg (KMSK) scale in an urban low-income and predominantly African American sample of primary care patients

The Kreek-McHugh-Schluger-Kellogg (KMSK) scale was developed to quantify self-exposure to opiates, cocaine, alcohol, and tobacco. The original study was limited by a relatively small sample that was not representative of general clinical populations, and did not include marijuana exposure. For the current study, participants were recruited from primary care outpatient clinics in an urban public hospital. The primary measure was the KMSK scale. The Structured Interview for Diagnosis for DSM-IV (SCID) was used as the "gold standard" for substance dependence diagnoses, and the results of KMSK assessments were evaluated using receiver operator characteristic (ROC) analysis. The sample (n = 439) was predominantly African American (90.6%), with mean age (±SD) of 43.1 ± 12.8 years. ROC analyses found that the optimal cutoff scores for alcohol dependence were the same as suggested previously (11), while they were lower for cocaine dependence (10 vs. 11) and opiate dependence (4 vs. 9). The analysis suggested a cutoff score of 8 for marijuana. The KMSK performed well in the current study as a brief tool for evaluating dependence on alcohol, cocaine, marijuana, and opiates in this nonpsychiatric clinic sample of predominantly poor urban African Americans.     

Acid β-glucosidase mutants linked to Gaucher disease, Parkinson disease, and Lewy body dementia alter α-synuclein processing

Objective:

Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α‐synuclein (SNCA). A loss in lysosomal acid–β‐glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk.

Methods:

We analyzed the effects of wild‐type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols.

Results:

We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293‐SNCA cells increased GCase activity, as expected (ie, to 167% in MES‐SNCA, 128% in PC12‐SNCA, and 233% in HEK293‐SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293‐SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8–20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA‐induced SNCA accumulation could be pharmacologically reversed in D409V‐expressing PC12‐SNCA cells by rapamycin, an autophagy‐inducer (≤40%; 10μM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age‐dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]‐null//ps‐transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week‐old cathepsin D‐deficient mice.

Interpretation:

Our results demonstrate that GBA mutants promote SNCA accumulation in a dose‐ and time‐dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders. ANN NEUROL 2011;     

Mutant GBA1 expression and synucleinopathy risk: first insights from cellular and mouse models

Heterozygous mutations in the glucocerebrosidase gene (GBA1) are associated with increased risk for α-synuclein aggregation disorders ('synucleinopathies'), which include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Homozygous GBA1 mutations lead to reduced GBA1 lysosomal activity underlying three variants of Gaucher disease (GD). Despite the wealth of clinical and genetic evidence supporting the association between mutant genotypes and synucleinopathy risk, the precise mechanisms by which GBA1 mutations lead to PD and DLB remain unclear. Here, we summarize recent findings that highlight the complexity of this pathogenetic link. In neural cells, both gain and loss of function mechanisms, as conferred by mutant GBA1 expression and activity loss, respectively, seem to promote aberrant α-synuclein processing. In addition, we draw attention to recent insights gleaned from GD animal models regarding axonal pathology, brain inflammation and memory dysfunction. From a translational perspective, we discuss the concepts of neural enzyme replacement therapy and pharmacological agents as potential treatment strategies for GBA1-associated synucleinopathies. Finally, we touch on the issue whether aberrant α-synuclein species may coregulate GBA1 activity in the vertebrate brain, thereby providing a reverse link, i.e., between an important synucleinopathy risk factor and the enzyme's lysosomal function. In summary, several leads connecting GBA1 mutations with α-synuclein misprocessing have emerged as potential targets for the treatment of GBA1-related synucleinopathies, and possibly, for non-GBA1-associated neurodegenerative diseases.     

Marked differences in neurochemistry and aggregates despite similar behavioural and neuropathological features of Huntington disease in the full-length BACHD and YAC128 mice

The development of animal models of Huntington disease (HD) has enabled studies that help define the molecular aberrations underlying the disease. The BACHD and YAC128 transgenic mouse models of HD harbor a full-length mutant huntingtin (mHTT) and recapitulate many of the behavioural and neuropathological features of the human condition. Here, we demonstrate that while BACHD and YAC128 animals exhibit similar deficits in motor learning and coordination, depressive-like symptoms, striatal volume loss and forebrain weight loss, they show obvious differences in key features characteristic of HD. While YAC128 mice exhibit significant and widespread accumulation of mHTT striatal aggregates, these mHTT aggregates are absent in BACHD mice. Furthermore, the levels of several striatally enriched mRNA for genes, such as DARPP-32, enkephalin, dopamine receptors D1 and D2 and cannabinoid receptor 1, are significantly decreased in YAC128 but not BACHD mice. These findings may reflect sequence differences in the human mHTT transgenes harboured by the BACHD and YAC128 mice, including both single nucleotide polymorphisms as well as differences in the nature of CAA interruptions of the CAG tract. Our findings highlight a similar profile of HD-like behavioural and neuropathological deficits and illuminate differences that inform the use of distinct endpoints in trials of therapeutic agents in the YAC128 and BACHD mice.     

Elevation of serum M-CSF concentrations during pregnancy and ovarian hyperstimulation

Summary. Macrophage colony stimulating factor (CSF-1 or M-CSF) is involved in haemopoiesis and probably in mouse gestation. Sexual steroids induce its production by uterine glandular epithelial cells and its receptor (product of the protooncogene C-FMS) is expressed on placental trophoblastic cells. We measured M-CSF serum levels in 119 pregnant women and in eight women undergoing ovarian hyperstimulation for in vitro fertilization. M-CSF increased early (4–8 weeks) and progressively during gestation. Its rapid elevation during the course of ovarian hyperstimulation suggests that its synthesis is probably induced by sexual steroids. This locally produced M-CSF could play a role in human pregnancy and in the pathogenesis of thrombocytopenias observed during pregnancy.     

Dehiscences of the semicircular canals as discrete third window lesions of the inner ear

The aim of this study was to assess the role of high resolution multi-detector CT in diagnosis of different dehiscences of the semicircular canals (SCC).     

Mobilization of plasma cells in healthy individuals treated with granulocyte colony-stimulating factor for haematopoietic stem cell collection

In mice, the plasma cell (PC) niche in the bone marrow is close to the haematopoietic stem cell (HSC) niche. We investigated whether PCs can be mobilized into the peripheral blood (PB) in healthy donors receiving granulocyte colony-stimulating factor (G-CSF) for the induction of HSC mobilization into the PB. G-CSF increased the count of circulating PCs 6-fold, that of circulating B lymphocytes 4-fold and that of circulating HSCs 44-fold. Mobilized circulating PCs comprised CD138(-) (62·2%) and CD138(+) (37·8%) PCs, the latter being more mature based on increased CD27, CD38 and cytoplasmic immunoglobulin expression. Mobilized PCs had a phenotype close to that of steady-state PB PCs or in vitro generated PCs, but they expressed L-selectin only weakly. Finally, a median value of 0·4 × 10(6) /kg donor PCs - one-thirtieth of the overall PC count in a healthy adult - was grafted into patients, which could contribute to immune memory recovery.