Zhihou Liang

Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome

Adults with Down syndrome (DS) develop Alzheimer neurofibrillary degeneration in the brain, but the underlying molecular mechanism is unknown. Here, we report that the presence of an extra copy of the dual‐specificity tyrosine‐phosphorylated and regulated kinase 1A (Dyrk1A) gene due to trisomy 21 resulted in overexpression of Dyrk1A and elevated kinase activity in DS brain. Dyrk1A phosphorylated tau at several sites, and these sites were hyperphosphory‐lated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase‐3β (GSK‐3β). Dyrk1A‐induced tau phosphorylation inhibited taus biological activity and promoted its self‐aggregation. In Ts65Dn mouse brain, an extra copy of the Dyrk1A gene caused increased expression and activity of Dyrk1A and resulted in increased tau phosphorylation. These findings strongly suggest a novel mechanism by which the overexpression of Dyrk1A in DS brain causes neurofibrillary degeneration via hyperphosphorylating tau. Liu, F., Liang, Z., Wegiel, J., Hwang, Y.‐W., Iqbal, K., Grundke‐Iqbal, I., Ramakrishna, N., Gong, C.‐X. Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome. FASEB J. 22, 3224–3233 (2008)

Down-regulation of cAMP-dependent protein kinase by over-activated calpain in Alzheimer disease brain

Impaired cognition and memory may be associated with down‐regulation of cAMP‐response element‐binding protein (CREB) in the brain in patients with Alzheimer disease, but the molecular mechanism leading to the down‐regulation is not understood. In this study, we found a selective reduction in the levels of the regulatory subunits (RIIα and RIIβ) and the catalytic subunit (Cβ) as well as the enzymatic activity of cAMP‐dependent protein kinase (PKA), which is the major positive regulator of CREB. We also observed that PKA subunits were proteolyzed by calpain and the levels of PKA subunits correlated negatively with calpain activation in the human brain. These findings led us to propose that in the brain in patients with Alzheimer disease, over‐activation of calpain because of calcium dysregulation causes increased degradation and thus decreased activity of PKA, which, in turn, contributes to down‐regulation of CREB and impaired cognition and memory.

A non-transgenic mouse model (icv-STZ mouse) of Alzheimer's disease: similarities to and differences from the transgenic model (3xTg-AD mouse).

Alzheimer’s disease (AD) can be divided into sporadic AD (SAD) and familial AD (FAD). Most AD cases are sporadic and result from multiple etiologic factors, including environmental, genetic, and metabolic factors, whereas FAD is caused by mutations in the presenilins or amyloid-β (Aβ) precursor protein (APP) genes. A commonly used animal model for AD is the 3xTg-AD transgenic mouse model, which harbors mutated presenilin 1, APP, and tau genes and thus represents a model of FAD. There is an unmet need in the field to characterize animal models representing different AD mechanisms, so that potential drugs for SAD can be evaluated preclinically in these animal models. A mouse model generated by intracerebroventricular (icv) administration of streptozocin (STZ), the icv-STZ mouse, shows many aspects of SAD. In this study, we compared the non-cognitive and cognitive behaviors as well as biochemical and immunohistochemical alterations between the icv-STZ mouse and the 3xTg-AD mouse. We found that both mouse models showed increased exploratory activity as well as impaired learning and spatial memory. Both models also demonstrated neuroinflammation, altered synaptic proteins and insulin/IGF-1 (insulin-like growth factor-1) signaling, and increased hyperphosphorylated tau in the brain. The most prominent brain abnormality in the icv-STZ mouse was neuroinflammation, and in the 3xTg-AD mouse it was elevation of hyperphosphorylated tau. These observations demonstrate the behavioral and neuropathological similarities and differences between the icv-STZ mouse and the 3xTg-AD mouse models and will help guide future studies using these two mouse models for the development of AD drugs.

VEGF-expressing human umbilical cord mesenchymal stem cells, an improved therapy strategy for Parkinson’s disease

The umbilical cord provides a rich source of primitive mesenchymal stem cells (human umbilical cord mesenchymal stem cells (HUMSCs)), which have the potential for transplantation-based treatments of Parkinsons Disease (PD). Our pervious study indicated that adenovirus-associated virus-mediated intrastriatal delivery of human vascular endothelial growth factor 165 (VEGF 165) conferred molecular protection to the dopaminergic system. As both VEGF and HUMSCs displayed limited neuroprotection, in this study we investigated whether HUMSCs combined with VEGF expression could offer enhanced neuroprotection. HUMSCs were modified by adenovirus-mediated VEGF gene transfer, and subsequently transplanted into rotenone-lesioned striatum of hemiparkinsonian rats. As a result, HUMSCs differentiated into dopaminergic neuron-like cells on the basis of neuron-specific enolase (NSE) (neuronal marker), glial fibrillary acidic protein (GFAP) (astrocyte marker), nestin (neural stem cell marker) and tyrosine hydroxylase (TH) (dopaminergic marker) expression. Further, VEGF expression significantly enhanced the dopaminergic differentiation of HUMSCs in vivo. HUMSC transplantation ameliorated apomorphine-evoked rotations and reduced the loss of dopaminergic neurons in the lesioned substantia nigra (SNc), which was enhanced significantly by VEGF expression in HUMSCs. These findings present the suitability of HUMSC as a vector for gene therapy and suggest that stem cell engineering with VEGF may improve the transplantation strategy for the treatment of PD.

Anesthesia Induces Phosphorylation of Tau

Abnormal hyperphosphorylation and aggregation of microtubule-associated protein tau play a crucial role in neurodegeneration of Alzheimers disease (AD). Anesthesia has been associated with cognitive impairment and the risk for AD. Here we investigated the effects of anesthesia on site-specific tau phosphorylation and the possible mechanisms. We found that anesthesia for short periods (30 sec to 5 min) induced tau phosphorylation at Thr181, Ser199, Thr205, Thr212, Ser262, and Ser404 to small, but significant, extents, which appeared to result from anesthesia-induced activation of stress-activated protein kinases. Anesthesia for a longer time (1~h) induced much more dramatic phosphorylation of tau at the above sites, and the further phosphorylation may be associated with hypothermia induced by anesthesia. Anesthesia-induced tau phosphorylation appears to be specific because the increased phosphorylation was only seen at half of the tau phosphorylation sites studied and was not observed in global brain proteins. These studies clarified the dynamic changes of tau phosphorylation at various sites and, thus, served as a fundamental guide for future studies on tau phosphorylation by using brains of anesthetized experimental animals. Our findings also provide a possible mechanism by which anesthesia may cause postoperative cognitive impairment and increase the risk for AD.

Potential autophagy enhancers attenuate rotenone-induced toxicity in SH-SY5Y.

Recent studies have shown that autophagy upregulation may be a tractable therapeutic intervention for clearing the disease-causing proteins, including α-synuclein, ubiquitin, and other misfolded or aggregated proteins in Parkinsons disease (PD). In this study, we explored a novel pharmacotherapeutic approach to treating PD by utilizing potential autophagy enhancers valproic acid (VPA) and carbamazepine (CBZ). Pretreatment with VPA (3 mM) and CBZ (50 μM) along with positive control rapamycin (Rap, 0.2 μM) or lithium (LiCl, 10 mM) significantly enhanced cell viability, decreased rotenone-induced nuclear fragmentation and apoptosis, ameliorated the decrease in mitochondrial membrane potential, reduced reactive oxygen species generation in the human neuroblastoma SH-SY5Y cells. Specifically, the numbers of lysosomes and autophagic vacuolar organelles were increased and the microtubule-associated protein 1 light chain 3-II (LC3-II) expression was up-regulated by VPA, CBZ, Rap, and LiCl (53%, 31%, 72%, and 63%), suggesting that these agents activated autophagic pathways. Moreover, pretreatment with the autophagy inhibitor chloroquine (Chl, 10 μM) remarkably strengthened rotenone toxicity in these cells. Our results suggest that VPA and CBZ, the most commonly used anti-epilepsy and mood-stabilizing medications with low-risk and easy administration might be potential therapeutics for PD.

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

A clinically-related animal model of Parkinsons disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research.

PKA modulates GSK-3β- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

Phosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3β (GSK‐3β), cyclin‐dependent protein kinase 5 (cdk5) and cAMP‐dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK‐3β, but the site‐specific modulation of GSK‐3β‐catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK‐3β‐catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter‐regulation of tau phosphorylation by the three major tau kinases.

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation.

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimers disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.

Intranasal insulin restores insulin signaling, increases synaptic proteins, and reduces Aβ level and microglia activation in the brains of 3xTg-AD mice.

Decreased brain insulin signaling has been found recently in Alzheimers disease (AD). Intranasal administration of insulin, which delivers the drug directly into the brain, improves memory and cognition in both animal studies and small clinical trials. However, the underlying mechanisms are unknown. Here, we treated 9-month-old 3xTg-AD mice, a commonly used mouse model of AD, with daily intranasal administration of insulin for seven days and then studied brain abnormalities of the mice biochemically and immunohistochemically. We found that intranasal insulin restored insulin signaling, increased the levels of synaptic proteins, and reduced Aβ40 level and microglia activation in the brains of 3xTg-AD mice. However, this treatment did not affect the levels of glucose transporters and O-GlcNAcylation or tau phosphorylation. Our findings provide a mechanistic insight into the beneficial effects of intranasal insulin treatment and support continuous clinical trials of intranasal insulin for the treatment of AD.