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Streptozotocin-intracerebroventricularly treated rats (STZ-icv) have become an experimental model of sporadic Alzheimer's disease, and demonstrate resembling in cognitive deficits, alterations of Akt/PKB and GSK-3 enzymes of insulin receptor signaling cascade in the brain, as well as alterations of tau protein and accumulation of beta amyloid like aggregates in the brain.

Objectives. The objective of the study was to investigate the expression of enzymes in insulin signalling cascade, protein kinase B (Act/PKB) and downstream glycogen synthase kinase-3 (GSK-3), in the hippocampus of rats with experimental Alzheimer's disease (streptozotocin /STZ/ intracerebroventricularly /icv/ treated rats). Material and Methods. GSK-3 and Act/PKB were measured by Western blot analysis, one month after the icv administration of STZ (1 mg/kg), as well as after the icv administration of glucose transporter 2 (GLUT2) inhibitor 5-thio-D-glucose (TG ; 150 μ g/kg), and their combination (TG+STZ). Cognitive functions were tested by Morris Water Maze Test. Statistical significance was analysed by Kruskal-Wallis ANOVA median test and Mann-Whitney U test, p

The insulin resistance brain state is related to the late-onset sporadic Alzheimer’ s disease, and alterations of insulin receptor (IR) and its downstream phosphatidylinositol-3 kinase signalling pathway have been found in human brain. Confirmation studies have not been performed in an experimental model probably related to the sporadic Alzheimer’ s disease, e.g., rats showing a neuronal IR deficit subsequent to intracerebroventricular (icv) treatment with streptozotocin (STZ). In this study, Western blot analysis performed 1 month after STZ-icv treatment showed an increase (+63%) in the level of phosphorylated glycogen synthase kinase - 3α /β (pGSK-3α /β ) protein in the rat hippocampus, whereas the levels of GSK-3α /β and protein kinase B (Akt/PKB) remained unchanged. Three months after STZ-icv treatment, pGSK-3α /β and Akt/PKB levels tended to decrease (-8% and -9%, respectively). The changes were regionally specific, as a different pattern was found in frontal cortex. Structural alterations were also found, demonstrated as beta amyloid peptide like aggregates in brain capillaries of STZ-icv treated rats. Similar neurochemical changes and cognitive deficits were recorded in rats treated icv with 5-thio-D-glucose, a blocker of glucose transporter 2 (GLUT2) probably involved in brain glucose sensing. These results support the similarity of IR signalling cascade alteration and its consequences in STZ-icv treated rats, to those found in humans with sporadic Alzheimer’ s disease, and suggest a role of GLUT2 in its pathophysiology.

Im Mittelpunkt dieser Arbeit stehen Untersuchungen zur Bildung des Pigments Neuromelanin, das die Ursache für die dunkle Farbgebung der humanen Substantia nigra pars compacta ist. Eine Beteiligung von Neuromelanin an den pathobiochemischen Ereignissen bei Parkinson-Krankheit erklärt das klinische Interesse an Neuromelanin. Die Untersuchungsmöglichkeiten von Neuromelanin sind limitiert: einerseits ist eine chemische Strukturaufklärung aufgrund der Unlöslichkeit dieses amorphen Polymers kaum zu bewerkstelligen, andererseits wird mangels geeigneter biologischer Testsysteme ein Einblick in die Biogenese von Neuromelanin verwehrt. Zurzeit wird die Bildung von Neuromelanin anhand der beiden konkurrierenden Hypothesen als Autoxidation von Dopamin oder durch Beteiligung eines Enzyms („Tyrosinase-Konzept“) erklärt. In dieser Arbeit wurden beide hypothetischen Ansätze bearbeitet, wobei einer enzymatischen Biogenese von Neuromelanin die Präferenz gegeben wird. Zur globalen Untersuchung von Neuromelanin-Granula wurde nun erstmals eine Isolierung der Pigment-haltigen Organelle vorgestellt, die die Basis für eine umfassende Proteomanalyse mittels 1-D-SDS-PAGE und ESI-Tandem-Massenspektrometrie bildete. Mit diesem methodischen Ansatz wurden ingesamt 73 Proteinen identifiziert. Diese waren vor allem lysosomalen Proteinen zuordenbar, z.B. charakteristischen Membranproteinen (LAMP-1), sämtlichen Proteasen, Proteinen des Metabolismus von (Glyco-)Lipiden und Glycoproteinen, aber auch Proteinen des Cytosols und des vesikulären Verkehrs. Entscheidend war die Anwesenheit von Proteinen des Endoplasmatischen Reticulums (ER); Calnexin gilt als ein melanogenes Chaperon, das nicht in Lysosomen vorkommt, dagegen aber in Lysosomen-verwandten Organellen. Im Vergleich mit bereits existierenden Proteinprofilen von Lysosomen und Lysosomen-verwandten Organellen zeigten die in Neuromelanin-Granula identifizierten lysosomalen Proteine und Proteine des ER, dass diese Organellen der humanen Substantia nigra keine konventionellen Lysosomen sind, sondern mit hoher Wahrscheinlichkeit der Gruppe der Lysosomen-verwandten Organellen zuzuordnen sind., This work focusses on the formation of the polyphenolic pigment neuromelanin, which accounts for the dark colour of the human substantia pars compacta. A contribution of neuromelanin to the patho-biochemical events in Parkinson’s disease is suggested, which is the reason for the clinical interest in this pigment. The analytical approaches to investigate neuromelanin are limited: on the one hand, an elucidation of the chemical structure is hardly possible to accomplish due to the insolubility of this amorphous polymer, on the other hand, the lack of appropriate biological test systems prevents insight into the biogenesis of neuromelanin. Currently the formation of neuromelanin is addressed by two competing hypotheses either by autoxidation of dopamine (“autoxidative concept”), or by contribution of an enzyme (“tyrosinase concept”). This work covered both hypothetical approaches, but the concept of an enzymatic biogenesis of neuromelanin is preferred. Aiming at a global analysis of these pigmented organelles, the isolation of neuromelanin granules was introduced for the first time, which formed the basis for an extensive proteomic analysis by 1-D SDS-PAGE and ESI tandem mass spectrometry. Subsequently, the analyses by mass spectrometry facilitated the identification of 73 proteins, most of which were attributable to lysosomal proteins, e.g. characterisitcal membrane proteins (LAMP-1), various proteases, proteins engaged in the metabolism og (glycol)-lipids and glycoproteins, but also cytosolic proteins and proteins involved in vesicular traffic. Intriguingly, there were proteins identified which originate form the endoplasmic reticulum (ER), most notably calnexin, a melanogenic chaperone, which is not found in lysosomes but in lysosome-related organelles. By comparison of yet existing protein profiles of lysosomes and lysosome-related organelles it became clear that neuromelanin granules from the human substatia nigra are no conventional lysosomes, but should rather be attributed to the group of lysosome-related organelles.