Woojin Scott Kim

Reduced glucocerebrosidase is associated with increased α-synuclein in sporadic Parkinson's disease.

Heterozygous mutations in GBA1, the gene encoding lysosomal glucocerebrosidase, are the most frequent known genetic risk factor for Parkinsons disease. Reduced glucocerebrosidase and α-synuclein accumulation are directly related in cell models of Parkinsons disease. We investigated relationships between Parkinsons disease-specific glucocerebrosidase deficits, glucocerebrosidase-related pathways, and α-synuclein levels in brain tissue from subjects with sporadic Parkinsons disease without GBA1 mutations. Brain regions with and without a Parkinsons disease-related increase in α-synuclein levels were assessed in autopsy samples from subjects with sporadic Parkinsons disease (n = 19) and age- and post-mortem delay-matched controls (n = 10). Levels of glucocerebrosidase, α-synuclein and related lysosomal and autophagic proteins were assessed by western blotting. Glucocerebrosidase enzyme activity was measured using a fluorimetric assay, and glucocerebrosidase and α-synuclein messenger RNA expression determined by quantitative polymerase chain reaction. Related sphingolipids were analysed by mass spectrometry. Multivariate statistical analyses were performed to identify differences between disease groups and regions, with non-parametric correlations used to identify relationships between variables. Glucocerebrosidase protein levels and enzyme activity were selectively reduced in the early stages of Parkinsons disease in regions with increased α-synuclein levels although limited inclusion formation, whereas GBA1 messenger RNA expression was non-selectively reduced in Parkinsons disease. The selective loss of lysosomal glucocerebrosidase was directly related to reduced lysosomal chaperone-mediated autophagy, increased α-synuclein and decreased ceramide. Glucocerebrosidase deficits in sporadic Parkinsons disease are related to the abnormal accumulation of α-synuclein and are associated with substantial alterations in lysosomal chaperone-mediated autophagy pathways and lipid metabolism. Our data suggest that the early selective Parkinsons disease changes are likely a result of the redistribution of cellular membrane proteins leading to a chronic reduction in lysosome function in brain regions vulnerable to Parkinsons disease pathology.

Role of ABCG1 and ABCA1 in regulation of neuronal cholesterol efflux to apolipoprotein-E discs and suppression of amyloid-β peptide generation

Maintenance of an adequate supply of cholesterol is important for neuronal function, whereas excess cholesterol promotes amyloid precursor protein (APP) cleavage generating toxic amyloid-β (Aβ) peptides. To gain insights into the pathways that regulate neuronal cholesterol level, we investigated the potential for reconstituted apolipoprotein E (apoE) discs, resembling nascent lipoprotein complexes in the central nervous system, to stimulate neuronal [3H]cholesterol efflux. ApoE discs potently accelerated cholesterol efflux from primary human neurons and cell lines. The process was saturable (17.5 μg of apoE/ml) and was not influenced by APOE genotype. High performance liquid chromatography analysis of cholesterol and cholesterol metabolites effluxed from neurons indicated that <25% of the released cholesterol was modified to polar products (e.g. 24-hydroxycholesterol) that diffuse from neuronal membranes. Thus, most cholesterol (∼75%) appeared to be effluxed from neurons in a native state via a transporter pathway. ATP-binding cassette transporters ABCA1, ABCA2, and ABCG1 were detected in neurons and neuroblastoma cell lines and expression of these cDNAs revealed that ABCA1 and ABCG1 stimulated cholesterol efflux to apoE discs. In addition, ABCA1 and ABCG1 expression in Chinese hamster ovary cells that stably express human APP significantly reduced Aβ generation, whereas ABCA2 did not modulate either cholesterol efflux or Aβ generation. These data indicate that ABCA1 and ABCG1 play a significant role in the regulation of neuronal cholesterol efflux to apoE discs and in suppression of APP processing to generate Aβ peptides.

Role of ATP-binding cassette transporters in brain lipid transport and neurological disease

The brain is lipid‐rich compared to other organs and although previous studies have highlighted the importance of ATP‐binding cassette (ABC) transporters in the regulation of lipid transport across membranes in peripheral tissues, very little is known regarding ABC transporter function in the CNS. In this study, we bring together recent literature focusing on potential roles for ABC transporters in brain lipid transport and, where appropriate, identify possible links between ABC transporters, lipid transport and neurological disease. Of the 48 transcriptionally active ABC transporters in the human genome, we have focused on 13 transporters (ABCA1, ABCA2, ABCA3, ABCA4, ABCA7 and ABCA8; ABCB1 and ABCB4; ABCD1 and ABCD2; ABCG1, ABCG2, and ABCG4) for which there is evidence suggesting they may contribute in some way to brain lipid transport or homeostasis. The transporters are discussed in terms of their location within brain regions and brain cell types and, where possible, in terms of their known functions and established or proposed association with human neurological diseases. Specific examples of novel treatment strategies for diseases, such as Alzheimer’s disease and X‐linked adrenoleukodystrophy that are based on modulation of ABC transporter function are discussed and we also examine possible functions for specific ABC transporters in human brain development.

Deletion of Abca7 increases cerebral amyloid-β accumulation in the J20 mouse model of Alzheimer's disease

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and has been detected in macrophages, microglia, and neurons. ABCA7 promotes efflux of lipids from cells to apolipoproteins and can also regulate phagocytosis and modulate processing of amyloid precursor protein (APP) to generate the Alzheimers disease (AD) amyloid-β (Aβ) peptide. Genome-wide association studies have indicated that ABCA7 single nucleotide polymorphisms confer increased risk for late-onset AD; however, the role that ABCA7 plays in the brain in the AD context is unknown. In the present study, we crossed ABCA7-deficient (A7−/−) mice with J20 amyloidogenic mice to address this issue. We show that ABCA7 loss doubled insoluble Aβ levels and thioflavine-S–positive plaques in the brain. This was not related to changes in APP processing (assessed by analysis of full-length APP and the APP β C-terminal fragment). Apolipoprotein E regulates cerebral Aβ homeostasis and plaque load; however, the apolipoprotein E concentration was not altered by ABCA7 loss. Spatial reference memory was significantly impaired in both J20 and J20/A7−/− mice compared with wild-type mice; however, there were no cognitive differences between J20 and J20/A7−/− mice. There were also no major differences detected in hippocampal or plaque-associated microglial/macrophage markers between J20 and J20/A7−/− mice, whereas the capacity for bone marrow-derived macrophages derived from A7−/− mice to take up oligomeric Aβ was reduced by 51% compared with wild-type mice. Our results suggest that ABCA7 plays a role in the regulation of Aβ homeostasis in the brain and that this may be related to altered phagocyte function.

The effect of nisin concentration and nutrient depletion on nisin production of Lactococcus lactis.

Abstract The kinetics of nisin production was studied in batch cultures using a construct of Lactococcuslactis subsp. lactis C2SmPrt−, containing a transposon (TnNip) that encodes nisin production. The introduction of TnNip into C2SmPrt−significantly lowered the specific growth rate and the maximum A620 reached was reduced from 15.2 to 11.0. The effect of nisin concentration and nutrient depletion on nisin production of the construct, C2SmPrt−(TnNip), was examined. Nisin production was found to be inhibited by high concentrations of nisin, when grown in excess nutrient, even though growth of the culture continued because nutrient limitation was not operating. However, in low nutrient concentrations nisin production was limited by nutrient depletion. The specific growth rate of C2SmPrt−(TnNip) was altered, by using different nutrient concentrations and different sugars, in order to examine the relationship between nisin production and growth. Nisin production was shown to be growth-associated for most of growth, but near the end of growth, when the specific growth rate was 0.05 h−1 or less, the production ceased.

ATP-binding cassette transporter A7 regulates processing of amyloid precursor protein in vitro

ATP‐binding cassette transporter A7 (ABCA7) is expressed in the brain and, like its closest homolog ABCA1, belongs to the ABCA subfamily of full‐length ABC transporters. ABCA1 promotes cellular cholesterol efflux to lipid‐free apolipoprotein acceptors and also inhibits the production of neurotoxic β‐amyloid (Aβ) peptides in vitro. The potential functions of ABCA7 in the brain are unknown. This study investigated the ability of ABCA7 to regulate cholesterol efflux to extracellular apolipoprotein acceptors and to modulate Aβ production. The transient expression of ABCA7 in human embryonic kidney cells significantly stimulated cholesterol efflux (fourfold) to apolipoprotein E (apoE) discoidal lipid complexes but not to lipid‐free apoE or apoA‐I. ABCA7 also significantly inhibited Aβ secretion from Chinese hamster ovary cells stably expressing human amyloid precursor protein (APP) or APP containing the Swedish K670M671→N670L671 mutations when compared with mock‐transfected cells. Studies with fluorogenic substrates indicated that ABCA7 had no impact on α‐, β‐, or γ‐secretase activities. Live cell imaging of Chinese hamster ovary cells expressing APP‐GFP indicated an apparent retention of APP in a perinuclear location in ABCA7 co‐transfected cells. These studies indicate that ABCA7 has the capacity to stimulate cellular cholesterol efflux to apoE discs and regulate APP processing resulting in an inhibition of Aβ production.

Alpha-synuclein biology in Lewy body diseases

Abstractα-Synuclein is an abundantly expressed neuronal protein that is at the center of focus in understanding a group of neurodegenerative disorders called α-synucleinopathies, which are characterized by the presence of aggregated α-synuclein intracellularly. Primary α-synucleinopathies include Parkinsons disease (PD), dementia with Lewy bodies and multiple system atrophy, with α-synuclein also found secondarily in a number of other diseases, including Alzheimers disease. Understanding how α-synuclein aggregates form in these different disorders is important for the understanding of its pathogenesis in Lewy body diseases. PD is the most prevalent of the α-synucleinopathies and much of the initial research on α-synuclein Lewy body pathology was based on PD but is also relevant to Lewy bodies in other diseases (dementia with Lewy bodies and Alzheimers disease). Polymorphism and mutation studies of SNCA, the gene that encodes α-synuclein, provide much evidence for a causal link between α-synuclein and PD. Among the primary α-synucleinopathies, multiple system atrophy is unique in that α-synuclein deposition occurs in oligodendrocytes rather than neurons. It is unclear whether α-synuclein originates from oligodendrocytes or whether it is transmitted somehow from neurons. α-Synuclein exists as a natively unfolded monomer in the cytosol, but in the presence of lipid membranes it is thought to undergo a conformational change to a folded α-helical secondary structure that is prone to forming dimers and oligomers. Posttranslational modification of α-synuclein, such as phosphorylation, ubiquitination and nitration, has been widely implicated in α-synuclein aggregation process and neurotoxicity. Recent studies using animal and cell models, as well as autopsy studies of patients with neuron transplants, provided compelling evidence for prion-like propagation of α-synuclein. This observation has implications for therapeutic strategies, and much recent effort is focused on developing antibodies that target extracellular α-synuclein.

Assessment of Stress Response of the Probiotic Lactobacillus acidophilus

Different aspects of stress response of Lactobacillus acidophilus were investigated. First, the sublethal and lethal levels of bile, heat, and NaCl stresses were determined. They were 0.05% and 0.5% (bile), 53°C and 60°C (heat), and 2% and 18% (NaCl), respectively. To evaluate the effect of each stress at log phase, log-phase cultures were challenged directly with the lethal level of each stress (control) and were compared to log-phase cultures that were pre-exposed to the sublethal level prior to the exposure at the lethal level (test). Some, if not most, of the cells were killed in the control cultures against each of the three stresses. However, in the test cultures, the number of cells that had survived increased significantly. It appears that L. acidophilus is capable of displaying adaptive response to stress. The adaptive response to one stress was also shown to provide cross-protection against different stresses tested. The effect of each stress on stationary-phase cultures was also investigated. In contrast to log-phase culture, stationary-phase culture was inherently resistant to stress.

Lipid Pathway Alterations in Parkinson's Disease Primary Visual Cortex

Background We present a lipidomics analysis of human Parkinsons disease tissues. We have focused on the primary visual cortex, a region that is devoid of pathological changes and Lewy bodies; and two additional regions, the amygdala and anterior cingulate cortex which contain Lewy bodies at different disease stages but do not have as severe degeneration as the substantia nigra. Methodology/Principal Findings Using liquid chromatography mass spectrometry lipidomics techniques for an initial screen of 200 lipid species, significant changes in 79 sphingolipid, glycerophospholipid and cholesterol species were detected in the visual cortex of Parkinsons disease patients (n = 10) compared to controls (n = 10) as assessed by two-sided unpaired t-test (p-value <0.05). False discovery rate analysis confirmed that 73 of these 79 lipid species were significantly changed in the visual cortex (q-value <0.05). By contrast, changes in 17 and 12 lipid species were identified in the Parkinsons disease amygdala and anterior cingulate cortex, respectively, compared to controls; none of which remained significant after false discovery rate analysis. Using gas chromatography mass spectrometry techniques, 6 out of 7 oxysterols analysed from both non-enzymatic and enzymatic pathways were also selectively increased in the Parkinsons disease visual cortex. Many of these changes in visual cortex lipids were correlated with relevant changes in the expression of genes involved in lipid metabolism and an oxidative stress response as determined by quantitative polymerase chain reaction techniques. Conclusions/Significance The data indicate that changes in lipid metabolism occur in the Parkinsons disease visual cortex in the absence of obvious pathology. This suggests that normalization of lipid metabolism and/or oxidative stress status in the visual cortex may represent a novel route for treatment of non-motor symptoms, such as visual hallucinations, that are experienced by a majority of Parkinsons disease patients.

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

Abstract. When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csps. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested.