Lingyun Hu

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice.

Background While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits. Research Design and Methods T1-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively. Results STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged. Conclusion Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.

Mitochondrial Superoxide Contributes to Blood Flow and Axonal Transport Deficits in the Tg2576 Mouse Model of Alzheimer's Disease

Background Alzheimers disease (AD) is a neurodegenerative disease characterized by the progressive decline in cognitive functions and the deposition of aggregated amyloid β (Aβ) into senile plaques and the protein tau into tangles. In addition, a general state of oxidation has long been known to be a major hallmark of the disease. What is not known however, are the mechanisms by which oxidative stress contributes to the pathology of AD. Methodology/Principal Findings In the current study, we used a mouse model of AD and genetically boosted its ability to quench free radicals of specific mitochondrial origin. We found that such manipulation conferred to the AD mice protection against vascular as well as neuronal deficits that typically affect them. We also found that the vascular deficits are improved via antioxidant modulation of the endothelial nitric oxide synthase, an enzyme primarily responsible for the production of nitric oxide, while neuronal deficits are improved via modulation of the phosphorylation status of the protein tau, which is a neuronal cytoskeletal stabilizer. Conclusions/Significance These findings directly link free radicals of specific mitochondrial origin to AD-associated vascular and neuronal pathology.

Serine‐derivatized gadonanotubes as magnetic nanoprobes for intracellular labeling

Gadonanotubes (GNTs), which are powerful new T(1)-weighted MRI contrast agents, were derivatized with serine amino acid substituents to produce water-soluble (2 mg ml(-1)) ser-gadonanotubes (ser-GNs) as magnetic nanoprobes for intracellular labeling. The ser-GNTs were used to efficiently label MCF-7 human breast cancer cells (1.5 x 10(9) Gd(3+) ions/cell) with no observable cytotoxicity. Cell pellets derived from the ser-GNT labeled cells give bright T(1)-weighted MR images, confirming that the ser-GNTs are a promising new nanoprobe technology for magnetic cell labeling and possibly for in vivo cellular trafficking.

Convergence of Presenilin- and Tau-Mediated Pathways on Axonal Trafficking and Neuronal Function

Alzheimers disease (AD) is a significant and growing health problem in the aging population. Although definitive mechanisms of pathogenesis remain elusive, genetic and histological clues have implicated the proteins presenilin (PS) and tau as key players in AD development. PS mutations lead to familial AD, and although tau is not mutated in AD, tau pathology is a hallmark of the disease. Axonal transport deficits are a common feature of several neurodegenerative disorders and may represent a point of intersection of PS and tau function. To investigate the contribution of wild-type, as opposed to mutant, tau to axonal transport defects in the context of presenilin loss, we used a mouse model postnatally deficient for PS (PS cDKO) and expressing wild-type human tau (WtTau). The resulting PS cDKO;WtTau mice exhibited early tau pathology and axonal transport deficits that preceded development of these phenotypes in WtTau or PS cDKO mice. These deficits were associated with reduced neurotrophin signaling, defective learning and memory and impaired synaptic plasticity. The combination of these effects accelerated neurodegeneration in PS cDKO;WtTau mice. Our results strongly support a convergent role for PS and tau in axonal transport and neuronal survival and function and implicate their misregulation as a contributor to AD pathogenesis.

Zic3 is required in the extra-cardiac perinodal region of the lateral plate mesoderm for left–right patterning and heart development

Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of Zic3 demonstrates an early role for Zic3 in gastrulation, CNS, cardiac and left-right axial development. The observation of multiple malformations in Zic3(null) mice and the relatively broad expression pattern of Zic3 suggest its important roles in multiple developmental processes. Here, we report that Zic3 is primarily required in epiblast derivatives to affect left-right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in Zic3 deficiency occur not because Zic3 is intrinsically required in the heart but rather because it functions early in the establishment of left-right body axis. In addition, we provide evidence supporting a role for Zic3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of Zic3 during left-right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of Zic3 with signaling pathways involved in the early establishment of laterality.

Genetic Dissection of γ-Secretase-Dependent and-Independent Functions of Presenilin in Regulating Neuronal Cell Cycle and Cell Death

Cell cycle markers have been shown to be upregulated and proposed to lead to apoptosis of postmitotic neurons in Alzheimers disease (AD). Presenilin (PS) plays a critical role in AD pathogenesis, and loss-of-function studies in mice established a potent effect of PS in cell proliferation in peripheral tissues. Whether PS has a similar activity in the neuronal cell cycle has not been investigated. PS exhibits γ-secretase-dependent and -independent functions; the former requires aspartate 257 (D257) as part of the active site, and the latter involves the hydrophilic loop domain encoded by exon 10. We used two novel mouse models, one expressing the PS1 D257A mutation on a postnatal PS conditional knock-out background and the other deleting exon 10 of PS1, to dissect the γ-secretase-dependent and -independent activities of PS in the adult CNS. Whereas γ-secretase plays a dominant role in neuronal survival, our studies reveal potent neuronal cell cycle regulation mediated by the PS1 hydrophilic loop. Although neurons expressing cell cycle markers do not directly succumb to apoptosis, they are more vulnerable under stress conditions. Importantly, our data identify a novel pool of cytoplasmic p53 as a downstream mediator of this cellular vulnerability. These results support a model whereby the PS γ-secretase activity is essential in maintaining neuronal viability, and the PS1 loop domain modulates neuronal homeostasis through cell cycle and cytoplasmic p53 control.

Improvements in a Mouse Model of Alzheimer's Disease Through SOD2 Overexpression are Due to Functional and Not Structural Alterations

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimers disease. We and others have shown that over expression of the mitochondrial antioxidant superoxide dismutase 2 (SOD-2) can improve many of the pathologies in the Tg2576 mouse model of Alzheimers disease that harbors the Swedish mutation in the amyloid precursor protein. However, it is not clear if these improvements are due to functional improvements or structural/anatomical changes. To answer this question, we used diffusion tensor imaging (DTI) to assess the structural integrity of white matter tracts in the control mice, Tg2576 mouse and Tg2576 mice over expressing SOD-2. We observed minimal differences in diffusion parameters with SOD-2 over expression in this model indicating that the improvements we previously reported are due to functional changes and not any alterations to the white matter tractography.

Early changes in the apparent diffusion coefficient (ADC) in a mouse model of Sandhoff's disease occur prior to disease symptoms and behavioral deficits.

Sandhoffs disease is a lysosomal storage disease in which the ganglioside GM2 accumulates in lysosomes. It has been reported that MRI cannot detect abnormalities in spin echo images in clinically presymptomatic Sandhoffs disease patients. Because one of the results of GM2 accumulation is cell swelling and lysosomal distension, our goal was to determine if changes in the diffusion of water is perturbed. We utilized the MRI imaging modality diffusion‐weighted imaging to measure the apparent diffusion coefficient in a mouse models of Sandhoffs disease, the hexb−/− mouse, and determined if diffusion‐weighted imaging could be utilized to detect early changes prior to behavioral or overt disease symptom onset. Here we report for the first time a comprehensive behavioral characterization of the hexb−/− mouse in conjunction with the apparent diffusion coefficient measurement. Our data indicate that the apparent diffusion coefficient decreases in the hexb−/− mouse in many but not all brain regions prior to disease symptoms (<3.5 to 4 months of age) and behavioral deficits (3 months of age). The magnitude of the decrease ranged from 4‐18%. Magn Reson Med, 2009.

P4-196: Presenilin in neuronal survival and neurodegeneration

Background: Presenilins (PS) have been shown to exhibit -secretase dependent and independent activities, the former requires the D257 site and the latter is mediated through the hydrophilic loop domain encoded by exon 10. Mutations in presenilin genes (PS1 and PS2) lead to early-onset of Alzheimer’s disease (AD), and increasing evidence indicates that these mutations lead to a partial loss of function for its physiological activities. Methods: We are using PS conditional double knockout (PScDKO) mice in which both presenilin genes are postnatally deleted in the forebrain to gain insight into the pathophysiology of AD. We are investigating the -secretase dependent and independent functions in adult CNS by genetic rescue using mice containing the PS1D257A mutation or exon 10 loop deletion (PS1 E10), respectively. The in vivo analysis was complemented by primary neuronal culture studies. We examined neuronal cell cycle markers using immunohistochemical methods. Results: The PScDKO mice are viable and exhibit age-related neurodegenerative phenotypes. Cyclin D1 activation and neuronal cell cycle re-entry is one of the earliest hallmarks seen in PScDKO animals and in PS1 null neurons, and this is largely mediated through the PS1 loop domain. However, neurons marked with BrdU do not directly succumb to apoptosis, and -secretase defective mutant PS1D257A leads to neuronal cell death independent of cell cycle re-entry. Nevertheless, cell cycle abnormality enhances the neuronal vulnerability under stress conditions, and our data reveal p53 as a downstream effecter. Conclusions: The results support a model whereby the PS -secretase activity, in a cell cycle independent manner, plays a predominant role in neuronal homeostasis. The PS1 loop domain, through cyclin D1 and p53-dependent mechanisms, facilitates PS1 neuronal function, particularly under stress conditions.