In neurons, mitochondrial quantity and basal cellular respiration are maintained with age, but alterations in other key functions and quantities make these cells susceptible to the pathology of age-related neurodegenerative disease. We observed age-related decreases in cytochrome C, cardiolipin, cytochrome C oxidase (CCO) function, and glutamate response that render cells less capable of responding to stress. Rescue experiments showed that estrogen is a promising treatment in restoring neuron function with age. After finding key differences in CCO, we examined the electron transport chain more closely and found age-related deficits in quantity or function for each individual complex. Our experiments support a lack of endogenous substrates or a failure of upstream complexes to transport electrons to complex IV with age, ultimately leading to age-related neurodegeneration. Reactive oxygen species production may add to the problem by degrading macromolecules such as nucleic acid, cardiolipin, and proteins. Increased ROS may also lead to a redox imbalance in the neuron, reducing the potential for energy production. Also, epigenetic controls such as DNA methylation, histone acetylation ubiquitination and phosphorylation that persist in culture independent of aging hormone levels, vasculature, and immune system may be partly responsible for the observed age-related deficiencies as has been previously observed in aging human muscle (Ronn et al., 2008). This compelling cumulative evidence suggests an age-related deficiency in electron transport via quinones from complexes I to III, and age-related deficiencies in substrates, cofactors, and quantity or function for complex IV. These studies add to the growing body of evidence that dysfunction in the enzyme complexes of the electron transport chain lead to neurodegeneration in senescence-related diseases. In an attempt to integrate our age-related findings with Alzheimer's Disease (AD) pathology, we sequentially isolated the electron transport chain complexes using selective mitochondrial inhibitors in cortical neurons removed from the 3xTg-AD mouse model, which harbors mutations in the PS1, APPSwe and tauP301L genes and follows the proposed temporal development of human AD pathology (Oddo et al., 2003a; 2003b). Overall, we did not detect 3xTg-AD cortical neuron deficits at the four electron transport complexes of mitochondria or in NAD(P)H oxidase (NOX), an extramitochondrial oxygen consumer and regulator of NAD(P)+/NAD(P)H homeostasis (Morre et al., 2000).

Magister Pharmaceuticae - MPharm / Neurodegenerative disorders (NDs) are a range of chronic brain disorders that includes amongst others motor function loss. Parkinson’s disease (PD) is one of the common NDs that has an insidious onset and diagnosed when dopaminergic neurons in the substantia nigra are already lost. The loss creates a deficiency of the dopamine (neurotransmitter) thereby causing neurochemical imbalance resulting in the signs and symptoms of PD. NDs overlap at multiple levels so some of the symptoms overlap as well. NDs currently have no cure yet and current drug therapies only improve the quality of life of the patients by targeting the symptoms mainly. Treatment of PD currently involves different classes of drugs and depending on the stages of the disease, some drugs can be only used as an adjunct therapy. Anti-oxidants and monoamine oxidase inhibitors (MAO-I) are part of the treatment options.

Nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two electronreduction of NO to N2O (2NO + 2H+ + 2e- → N2O + H2O) as part of the process ofdenitrification, the step-wise reduction of nitrate to dinitogen. The NOR-catalyzedreaction is central in the nitrogen cycle, since in this step the N=N double bond isformed. NOR is a deviant heme copper oxidase, located in the cytoplasmic membrane,containing four redox active cofactors. Like cytochrome c oxidase (CcO), NOR canreduce oxygen to water as a side reaction, but in contrast to CcO it does not contributeto the proton motive force that drives the conversion of ADP to ATP by ATP synthase.The active site in the catalytic subunit NorB consists of a non-heme iron FeB and a hemeb3 that are anti-ferromagnetically coupled. Additionally a low-spin heme b in NorB isinvolved in accepting electrons from heme c of NorC, a membrane anchored cytochromec, which is the second subunit of the purified NorBC heterodimer.We have studied the terminal region of the proton entry channel and possible ligands tothe binuclear active site of NOR using the flow-flash technique and could demonstratethat the putative proton channel residues Glu(E)198 and E267 in NorB are essential forproton uptake. We propose that they define the terminal proton channel region close tothe binuclear site. An alanine variant of the fully conserved amino acid residue E202 ofNOR that, according to the model of NOR (47), is located in the vicinity to the active site,is neither essential for catalytic activity nor integrity of the active site.Furthermore, we were able to demonstrate the [NO] dependency of NOR in the reactionbetween fully reduced protein and NO using the flow-flash technique (21, 24). Thebinding of NO to the fully reduced enzyme is clearly concentration dependent,inconsistent with a previously proposed obligatory binding of NO first to FeB before itligates to heme b3, where it, in the first turnover, is reduced by the electrons from theactive site. Further oxidation involves electron transfer from the low-spin hemes, which isaccelerated at lower [NO]. This acceleration at lower substrate concentration is evenlarger at decreased pH. We could demonstrate that substrate inhibition, observed insteady-state measurements, occurs already on oxidizing the fully reduced enzyme,indicating that NO binds to its inhibitory site before electrons can redistribute to theactive site from the low-spin hemes.

Indiana University-Purdue University Indianapolis (IUPUI) / The extracellular matrix (ECM) governs many cellular processes through biochemical and mechanical cues. Particularly, the effect ECM mechanical properties on cells fate has been well established over the years. Many hydrogel systems have been used to mimic the dynamic stiffening processes occurring in ECM. However, changes in ECM stiffness does not fully recapitulate the mechanics of native ECM, as viscoelasticity is also a major factor contributing to ECM dynamic property. This thesis describes the design and characterization of an enzyme-crosslinked hydrogel system that is not only capable of being stiffened on demand, but also can be tuned to obtain viscoelasticity. The first objective of this thesis was to utilize horseradish peroxidase (HRP) to crosslink thiol-norbornene hydrogel and use mushroom tyrosinase (MT) to create secondary DOPA-dimer crosslinks that stiffened the hydrogel. The cytocompatibility of HRP-mediated thiol-norbornene gelation and the effect of stiffening on cell fate was evaluated. The second objective of this thesis represented the first step towards developing a hydrogel system whose viscoelasticity could be dynamically tuned. Thiol-norbornene hydrogel was designed to yield dynamically adaptable boronic ester bonds via partial enzymatic reaction. Thiol-norborne hydrogel was made to contain hydroxyl phenol as well as boronic acid residues within its network. MT, in this case was used to oxidize the hydroxy phenol moieties into DOPA, which then complexed with boronic acid, created dynamic bonds, introducing viscoelasticity to an initial elastic hydrogel.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17895号 / 農博第2018号 / 新制||農||1017(附属図書館) / 学位論文||H25||N4791(農学部図書室) / 30715 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 三芳 秀人, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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