Hong-an Li

Peroxynitrite induces Alzheimer-like tau modifications and accumulation in rat brain and its underlying mechanisms

To investigate the upstream effector that led to tau hyperphosphorylation, nitration, and accumulation as seen in Alzheimers disease brain, and the underlying mechanisms, we bilaterally injected SIN‐1, a recognized peroxynitrite donor, into the hippocampus of rat brain. We observed that the level of nitrated and hyperphosphorylated tau was markedly increased in rat hippocampus 24 h after drug administration, and these alterations were prevented by preinjection of uric acid, a natural scavenger of peroxynitrite. Concomitantly, we detected a significant activation in glycogen synthase kinase‐3β (GSK‐3β) and p38 MAPKs, including p38α, p38β, and p38δ, but no obvious change was measured in the activity of p38γ, ERK, and c‐Jun amino‐terminal kinase (JNK). Both nitrated tau and hyperphosphorylated tau were aggregated in the hippocampus, in which the activity of 20S proteasome was significantly arrested in SIN‐1‐injected rats. Further studies demonstrated that the hyperphosphorylated tau was degraded as efficiently as normal tau by 20S proteasome, but the nitrated tau with an unorderly secondary structure became more resistant to the proteolysis. These results provide the first in vivo evidence showing that peroxynitrite simultaneously induces tau hyperphosphorylation, nitration, and accumulation, and that activation of GSK‐3β, p38α, p38β, p38δ isoforms and the inhibition of proteasome activity are respectively responsible for the peroxynitrite‐induced tau hyperphosphorylation and accumulation. Our findings reveal a common upstream stimulator and a potential therapeutic target for Alzheimer‐like neurodegeneration.—Zhang, Y‐J., Xu, Y‐F., Liu, Y‐H., Yin, J., Li, H‐L., Wang, Q., Wang, J‐Z. Peroxynitrite induces Alzheimer‐like tau modifications and accumulation in rat brain and its underlying mechanisms. FASEB J. 20, 1431–1442 (2006)

Effects of endogenous beta-amyloid overproduction on tau phosphorylation in cell culture.

Alzheimers disease is characterized by β‐amyloid (Aβ) overproduction and tau hyperphosphorylation. Recent studies have shown that synthetic Aβ promotes tau phosphorylation in vitro. However, whether endogenously overproduced Aβ promotes tau phosphorylation and the underlying mechanisms remain unknown. Here, we used mouse neuroblastoma N2a stably expressing wild‐type amyloid precursor protein (APPwt) or the Swedish mutant APP (APPswe) to determine the alterations of phosphorylated tau and the related protein kinases. We found that phosphorylation of tau at paired helical filament (PHF)‐1, pSer396 and pThr231 epitopes was significantly increased in cells transfected with APPwt and APPswe, which produced higher levels of Aβ than cells transfected with vector or amyloid precursor‐like protein 1. The activity of glycogen synthase kinase‐3 (GSK‐3) was up‐regulated with a concomitant reduction in the inhibitory phosphorylation of GSK‐3 at its N‐terminal Ser9 residue. In contrast, the activity of cyclin‐dependent kinase‐5 (CDK‐5) and protein kinase C (PKC) was down‐regulated. Inhibition of GSK‐3 by LiCl, but not inhibition of CDK‐5 by roscovitine, arrested Aβ secretion and tau phosphorylation. Inhibition of PKC by GF‐109203X activated GSK‐3, whereas activation of PKC by phorbol‐12,13‐dibutyrate inhibited GSK‐3. These results suggest that endogenously overproduced Aβ induces increased tau phosphorylation through activation of GSK‐3, and that inactivation of PKC is at least one of the mechanisms involved in GSK‐3 activation.

Protein Phosphatase 2A Facilitates Axonogenesis by Dephosphorylating CRMP2

Protein phosphatase 2A (PP2A) is indispensable in development, and deficits of PP2A and deterioration of neuronal axons have been observed in several neurodegenerative disorders, but the direct link between PP2A and the neuronal axon development is still missing. Here, we show that PP2A is essential for axon development in transfected rat brain and the dissociated hippocampal neurons. Upregulation of PP2A catalytic subunit (PP2Ac) not only promotes formation and elongation of the functional axons but also rescues axon retardation induced by PP2A inhibition. PP2A can dephosphorylate collapsin response mediator protein-2 (CRMP2) that implements the axon polarization, whereas constitutive expression of phosphomimic-CRMP2 abrogates the effect of PP2A upregulation. We also demonstrate that PP2Ac is enriched in the distal axon of the hippocampal neurons. Our results reveal a mechanistic link between PP2A and axonogenesis/axonopathy, suggesting that upregulation of PP2A may be a promising therapeutic for some neurodegenerative disorders.

I2PP2A regulates p53 and Akt correlatively and leads the neurons to abort apoptosis

A chronic neuron loss is the cardinal pathology in Alzheimer disease (AD), but it is still not understood why most neurons in AD brain do not accomplish apoptosis even though they are actually exposed to an environment with enriched proapoptotic factors. Protein phosphatase-2A inhibitor-2 (I(2)(PP2A)), an endogenous PP2A inhibitor, is significantly increased in AD brain, but the role of I(2)(PP2A) in AD-like neuron loss is elusive. Here, we show that I(2)(PP2A) regulates p53 and Akt correlatively. The mechanisms involve activated transcription and p38 MAPK activities. More importantly, we demonstrate that the simultaneous activation of Akt induced by I(2)(PP2A) counteracts the hyperactivated p53-induced cell apoptosis. Furthermore, I(2)(PP2A), p53 and Akt are all elevated in the brain of mouse model and AD patients. Our results suggest that the increased I(2)(PP2A) may trigger apoptosis by p53 upregulation, but due to simultaneous activation of Akt, the neurons are aborted from the apoptotic pathway. This finding contributes to the understanding of why most neurons in AD brain do not undergo apoptosis.

Inhibition of protein phosphatases induces transport deficits and axonopathy.

The activity of protein phosphatase (PP)‐2A and PP‐1 decreased in the brains of Alzheimer’s disease and inhibition of the phosphatases led to spatial memory deficit in rats. However, the molecular basis underlying memory impairment of the phosphatase inhibition is elusive. In the present study, we observed a selective inhibition of PP‐2A and PP‐1 with Calyculin A (CA) not only caused hyperphosphorylation of cytoskeletal proteins, but also impaired the transport of pEGFP‐labeled neurofilament‐M subunit in the axon‐like processes of neuroblastoma N2a cells and resulted in accumulation of neurofilament in the cell bodies. To analyze the morphological alteration of the cells during inhibition of the phosphatases, we established a cell model showing steady outgrowth of axon‐like cell processes and employed a stereological system to analyze the retraction of the processes. We found CA treatment inhibited outgrowth of the cell processes and prolonged treatment with CA caused retraction of the processes and meanwhile, the early neurodegenerative varicosities were also obvious in the CA‐treated cells. We conclude suppression of PP‐2A and PP‐1 by CA not only damages intracellular transport but also leads to cell degeneration, which may serve as the functional and structural elements for the memory deficits induced by suppression of the phosphatases.

Overexpression of Tau Proteins Antagonizes Amyloid-β-Potentiated Apoptosis Through Mitochondria-Caspase-3 Pathway in N2a Cells

It has been a puzzle why the tangle-bearing neurons in Alzheimers disease (AD) brain do not die preferentially of apoptosis even though they are actually challenged by multiple proapoptotic factors. Recently, we have reported that phosphorylation of tau can antagonize apoptosis induced by exogenous apoptotic inducers. Amyloid-beta (Abeta), a recognized endogenous proapoptotic factor, is significantly increased in the AD brains, however, it is not known whether tau could abate the Abeta-potentiated apoptosis. Here, we observed that the cells bearing high level of Abeta were more vulnerable than the controls to H2O2-induced apoptosis, and this effect of Abeta was associated with decrease of Bcl-2, elevation of Bax and cytosolic cytochrome-c, as well as activation of caspase-3, suggesting that Abeta could potentiate the oxidant-induced cell apoptosis with involvement of mitochondria-caspase-3 pathway. More importantly, we also found that expression of tau that became hyperphosphorylated could reduce the Abeta-potentiated apoptosis with simultaneous preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity, implying that overexpression of tau that became hyperphosphorylated can attenuate the Abeta-potentiated cell apoptosis through mitochondria-caspase-3 pathway. These findings provide an explanation of the chronic nature of neurodegeneration of neurons with neurofibrillary pathology of abnormal hyperphosphorylated tau in AD and related tauopathies.

Tau overexpression inhibits cell apoptosis with the mechanisms involving multiple viability-related factors.

The formation of neurofibrillary tangles, mainly composed of hyperphosphorylated tau protein, is a hallmark in the brain of human tauopathies, including Alzheimers disease (AD). Although neurons bearing neurofibrillary tangles are constantly exposed to various apoptotic stimuli, they do not appear to preferentially die by apoptosis. The underlying mechanism for such resistance to apoptosis remains elusive. Previously, we studied the role of tau phosphorylation in apoptosis and found that tau hyperphosphorylation by glycogen synthase kinase-3 (GSK-3) rendered cells more resistant to apoptosis. In this study, we show that the overexpression of tau without any exogenous activation of kinases also confers increased resistance to apoptosis in both N2a cells and in a tau transgenic mouse model. Mechanistically, the overexpression of tau was associated with a reduced p53 level, decreased release of cytochrome C from mitochondria, and inhibition of caspases-9/-3. Additionally, a decreased phosphorylation and increased nuclear translocation of beta-catenin were also detected in N2a/tau cells, and knockdown of beta-catenin eliminated the anti-apoptotic effect of tau. Furthermore, tau was spontaneously hyperphosphorylated upon overexpression and by staurosporine treatment. The phosphorylation level of p53 decreased upon tau overexpression, and a more profound reduction of the phosphorylated p53 was detected when the cells were treated with lithium and roscovitine, inhibitors of GSK-3 and cyclin-dependent kinase-5 (Cdk-5). These results suggest that the overexpression of tau, which may be hyperphosphorylated by endogenous GSK-3 and Cdk-5, is anti-apoptotic by mechanisms involving modulation of multiple anti-apoptotic factors, including beta-catenin and p53-mitochondria-caspase-mediated apoptotic pathways.

Tau Dephosphorylation Potentiates Apoptosis by Mechanisms Involving a Failed Dephosphorylation/Activation of Bcl-2

Phosphorylation of tau, a major microtubule-associated protein, has been recently discovered to affect cell apoptosis. While the phosphorylation of tau is dynamically regulated, the role of tau dephosphorylation in cell viability is elusive. Here, we observed that the cells bearing high level of the dephosphorylated tau at Tau-1 epitope were more vulnerable to the apoptosis induced by staurosporine, camptothecin, and hydrogen peroxide, though the general outcome of tau expression was still anti-apoptotic. Further studies demonstrate that co-expression of tau and protein phosphatase 2A catalytic subunit (PP2Ac), the most active tau phosphatase, potentiates cell apoptosis with a correlatively increased dephosphorylation of tau and phosphorylation of Bcl-2 at Ser87 (pS87-Bcl2, the inactive form of the anti-apoptotic factor), whereas expression of PP2Ac alone in the absence of tau decreases the levels of pS87-Bcl2 and cleaved PARP, markers of early apoptosis. Finally, both tau and Bcl-2 were co-immunoprecipitated with PP2Ac, but the binding level of Bcl-2 with PP2Ac decreased prominently when tau was co-expressed. These data suggest that tau dephosphorylation by PP2Ac facilitates cell apoptosis with the mechanisms involving a failed dephosphorylation/activation of Bcl-2.

Humanin attenuates Alzheimer-like cognitive deficits and pathological changes induced by amyloid β-peptide in rats

Amyloid β-peptide (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer’s disease (AD). Humanin (HN) is a secretory peptide that inhibits the neurotoxicity of Aβ. However, the mechanism(s) by which HN exerts its neuroprotection against Aβ-induced ADlike pathological changes and memory deficits are yet to be completely defined. In the present study, we provided evidence that treatment of rats with HN increases the number of dendritic branches and the density of dendritic spines, and upregulates pre- and post-synaptic protein levels; these effects lead to enhanced long-term potentiation and amelioration of the memory deficits induced by Aβ1–42. HN also attenuated Aβ1–42-induced tau hyperphosphorylation, apparently by inhibiting the phosphorylation of Tyr307 on the inhibitory protein phosphatase-2A (PP2A) catalytic subunit and thereby activating PP2A. HN also inhibited apoptosis and reduced the oxidative stress induced by Aβ1–42. These findings provide novel mechanisms of action for the ability of HN to protect against Aβ1–42-induced AD-like pathological changes and memory deficits.

Opposite monosynaptic scaling of BLP–vCA1 inputs governs hopefulness- and helplessness-modulated spatial learning and memory

Different emotional states lead to distinct behavioural consequences even when faced with the same challenging events. Emotions affect learning and memory capacities, but the underlying neurobiological mechanisms remain elusive. Here we establish models of learned helplessness (LHL) and learned hopefulness (LHF) by exposing animals to inescapable foot shocks or with anticipated avoidance trainings. The LHF animals show spatial memory potentiation with excitatory monosynaptic upscaling between posterior basolateral amygdale (BLP) and ventral hippocampal CA1 (vCA1), whereas the LHL show memory deficits with an attenuated BLP–vCA1 connection. Optogenetic disruption of BLP–vCA1 inputs abolishes the effects of LHF and impairs synaptic plasticity. By contrast, targeted BLP–vCA1 stimulation rescues the LHL-induced memory deficits and mimics the effects of LHF. BLP–vCA1 stimulation increases synaptic transmission and dendritic plasticity with the upregulation of CREB and intrasynaptic AMPA receptors in CA1. These findings indicate that opposite excitatory monosynaptic scaling of BLP–vCA1 controls LHF- and LHL-modulated spatial memory, revealing circuit-specific mechanisms linking emotions to memory.