Systemic deficiency of GM1 ganglioside in Parkinson's disease tissues and its relation to the disease etiology.

Following our initial reports on subnormal levels of GM1 in the substantia nigra and occipital cortex of Parkinson's disease (PD) patients, we have examined additional tissues from such patients and found these are also deficient in the ganglioside. These include innervated tissues intimately involved in PD pathology such as colon, heart and others, somewhat less intimately involved, such as skin and fibroblasts. Finally, we have analyzed GM1 in peripheral blood mononuclear cells, a type of tissue apparently with no direct innervation, and found those too to be deficient in GM1. Those patients were all afflicted with the sporadic form of PD (sPD), and we therefore conclude that systemic deficiency of GM1 is a characteristic of this major type of PD. Age is one factor in GM1 decline but is not sufficient; additional GM1 suppressive factors are involved in producing sPD. We discuss these and why GM1 replacement offers promise as a disease-altering therapy.

Subnormal GM1 in PBMCs: Promise for Early Diagnosis of Parkinson's Disease?

The fact that Parkinson's disease (PD) pathologies are well advanced in most PD patients by the time of clinical elucidation attests to the importance of early diagnosis. Our attempt to achieve this has capitalized on our previous finding that GM1 ganglioside is expressed at subnormal levels in virtually all tissues of sporadic PD (sPD) patients including blood cells. GM1 is present in most vertebrate cells, is especially abundant in neurons where it was shown essential for their effective functioning and long term viability. We have utilized peripheral blood mononuclear cells (PBMCs) which, despite their low GM1, we found to be significantly lower in sPD patients compared to age-matched healthy controls. To quantify GM1 (and GD1a) we used high performance thin-layer chromatography combined with cholera toxin B linked to horseradish peroxidase, followed by densitometric quantification. GM1 was also deficient in PBMCs from PD patients with mutations in the glucocerebrosidase gene (PD-GBA), apparently even lower than in sPD. Reasons are given why we believe these results obtained with patients manifesting fully developed PD will apply as well to PD patients in preclinical stages-a topic for future study. We also suggest that these findings point to a potential disease altering therapy for PD once the early diagnosis is established.

Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition.

Oleaginous fungi are of special interest among microorganisms for the production of lipid feedstocks as they can be cultured on a variety of substrates, particularly waste lingocellulosic materials, and few fungal strains are reported to accumulate inherently higher neutral lipid than bacteria or microalgae. Previously, we have characterized an endophytic filamentous fungus Colletotrichum sp. DM06 that can produce total lipid ranging from 34% to 49% of its dry cell weight (DCW) upon growing with various carbon sources and nutrient-stress conditions. In the present study, we report on the genetic transformation of this fungal strain with the CtDGAT2b gene, which encodes for a catalytically efficient isozyme of type-2 diacylglycerol acyltransferase (DGAT) from oleaginous yeast Candida troplicalis SY005. Besides the increase in size of lipid bodies, total lipid titer by the transformed Colletotrichum (lipid content ∼73% DCW) was found to be ∼1.7-fold more than the wild type (lipid content ∼38% DCW) due to functional activity of the CtDGAT2b transgene when grown under standard condition of growth without imposition of any nutrient-stress. Analysis of lipid fractionation revealed that the neutral lipid titer in transformants increased up to 1.8-, 1.6- and 1.5-fold compared to the wild type when grown under standard, nitrogen stress and phosphorus stress conditions, respectively. Lipid titer of transformed cells was further increased to 1.7-fold following model-based optimization of culture conditions. Taken together, ∼2.9-fold higher lipid titer was achieved in Colletotrichum fungus due to overexpression of a rate-limiting crucial enzyme of lipid biosynthesis coupled with prediction-based bioprocess optimization.

Functional characterization of two structurally novel diacylglycerol acyltransferase2 isozymes responsible for the enhanced production of stearate-rich storage lipid in Candida tropicalis SY005.

Diacylglycerol acyltransferase (DGAT) activity is an essential enzymatic step in the formation of neutral lipid i.e., triacylglycerol in all living cells capable of accumulating storage lipid. Previously, we characterized an oleaginous yeast Candida tropicalis SY005 that yields storage lipid up to 58% under a specific nitrogen-stress condition, when the DGAT-specific transcript is drastically up-regulated. Here we report the identification, differential expression and function of two DGAT2 gene homologues--CtDGAT2a and CtDGAT2b of this C. tropicalis. Two protein isoforms are unique with respect to the presence of five additional stretches of amino acids, besides possessing three highly conserved motifs known in other reported DGAT2 enzymes. Moreover, the CtDGAT2a and CtDGAT2b are characteristically different in amino acid sequences and predicted protein structures. The CtDGAT2b isozyme was found to be catalytically 12.5% more efficient than CtDGAT2a for triacylglycerol production in a heterologous yeast system i.e., Saccharomyces cerevisiae quadruple mutant strain H1246 that is inherently defective in neutral lipid biosynthesis. The CtDGAT2b activity rescued the growth of transformed S. cerevisiae mutant cells, which are usually non-viable in the medium containing free fatty acids by incorporating them into triacylglycerol, and displayed preferential specificity towards saturated acyl species as substrate. Furthermore, we document that the efficiency of triacylglycerol production by CtDGAT2b is differentially affected by deletion, insertion or replacement of amino acids in five regions exclusively present in two CtDGAT2 isozymes. Taken together, our study characterizes two structurally novel DGAT2 isozymes, which are accountable for the enhanced production of storage lipid enriched with saturated fatty acids inherently in C. tropicalis SY005 strain as well as in transformed S. cerevisiae neutral lipid-deficient mutant cells. These two genes certainly will be useful for further investigation on the novel structure-function relationship of DGAT repertoire, and also in metabolic engineering for the enhanced production of lipid feedstock in other organisms.