Universität zu Köln

Chintha, Raju (2012). Role of proteolipid protein (PLP/Dm20) and polyunsaturated fatty acids in normal and pathological central nervous system. PhD thesis, Universität zu Köln.

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Abstract

The CNS myelin sheath is an extension of the oligodendrocyte plasma membrane and is composed of 80% lipid and 20% protein. Myelin formation is a very sophisticated and highly conserved process, any disturbance of myelin protein or lipid composition has a strong impact on myelin biogenesis and myelin phenotype, which often results in CNS myelin pathology. The work presented here is divided into two parts: A. An investigation of the role of proteolipid protein (PLP/Dm20) Cys residues in myelin structure and function, and B. The role of PLP/Dm20 and polyunsaturated fatty acids (PUFAs) in CNS myelin morphology and CNS physiology. A. PLP is highly conserved and is the most abundant protein of CNS myelin. Also, point mutations in PLP are known to cause a variety of mild to severe PMD/SPG2 dysmyelinating leukodystrophies both in human and in mice. Some PLP point mutations are directly correlated to the perturbed plasma membrane trafficking of PLP/Dm20 in oligodendroyctes and correlate with the severity of PMD. PLP and its isoform Dm20 have 14 and 12 Cys residues, respectively, and are involved in post-translational S-acylation and the formation of two disulfide bridges. The work presented within this thesis furthers the understanding of the role of the Cys residues in PLP/Dm20. We analysed PLP/Dm20 Cys residue function by replacing Cys residues in PLP/DM20 with Ser residues (i.e., Mut-PLP/Dm20). In silico analyses predicted that the Mut-PLP and Dm20 have impaired transmembrane structure and reduced hydrophobic properties, supported by our in vivo and in vitro analysis which revealed perturbed cell surface targeting of the mutant proteins. Furthermore, in order to understand the role of Mut-PLP in CNS at early embryonic stages, a transgenic mouse mutant is being generated via pronuclear injection of a transgene composed of a Nestin enhancer and MBP promoter regulatory elements along with Mut-PLP cDNA, on a PLP negative genomic background. In addition, to understand the role of di-sulfide bridges in PLP; a knock-in mouse mutant is being generated in which the disulfide bridges of PLP/Dm20 have been deleted. B. PLP is an integral myelin membrane protein, and PUFAs are constituents of phospholipids, which are critical structural components of the CNS myelin membrane. In this thesis, we have expanded the understanding of the significant role of PLP and PUFAs and their impact in the CNS by phenotyping the plp-/-fads2-/- double knock-out mouse model (DM). The DM has been generated and validated on a genomic, RNA and protein level confirming lack of PLP and Fads2 which is a key enzyme for endogenous PUFAs synthesis. The DM exhibited a profound behavioral cataleptic state with mild tremor and seizures starting from 5 months age, and had a significantly reduced life span, compared to the single mutants (plp-/- and fads2-/-) and wild-type counterparts. In the brain and CNS myelin of the DM mice, PUFAs analysis revealed only partially depleted docosahexanoic acid (DHA) levels (40-50%) suggesting that DHA is highly regulated and/or is compensated for depleted PUFAs and was retained tenaciously in membrane structures of the CNS. Intriguingly, an unusual PUFA, eicosatrienoic acid (20:3) was detected, which may substitute for the observed depletion of arachidonic acid. Further analyses of the gene expression profile in the DM brain revealed no significant differences among myelin membrane proteins, desaturases and other myelin specific proteins, thus suggesting no alteration of these proteins in the absence of PLP and PUFAs. The DM mice showed a massive alteration in morphology of the CNS myelin manifesting as hypomyelination and a loss of myelin compaction around axons. In retinal electron micrographs, the retinal pigment epithelium (RPE) region showed no major significant morphological alterations. Retinal function, as assessed by ERG, revealed a decrease in a-wave signals suggesting defects in visual functions. Behavioral physiology studies, as assessed by the rota-rod task and Morris water maze and other similar tasks, showed partial but significant defects in neuromotor coordination/functions. The Morris water maze also revealed significant defects in cognitive ability (i.e., spatial acquisition/learning and in the reference memory). Alterations in behavioral physiology and the reduced lifespan in DM mice may be correlated with the disrupted myelin morphology and hypomyelination of CNS.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Chintha, Raju rajchintha1@yahoo.co.in UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-49478
Date:	27 October 2012
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Biochemie > Institut I für Biochemie
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language Proteolipid protein, Polyunsaturated fatty acids, Myelin, CNS, brain, Lipids, PUFAs English
Date of oral exam:	4 December 2012
Referee:	Name Academic Title Stoffel, Wilhelm Prof. Dr. Dr. Dr. hc mults
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/4947