Zhuohua Zhang

Transcriptional regulation of APH-1A and increased γ-secretase cleavage of APP and Notch by HIF-1 and hypoxia

The proteolytic cleavage of Alzheimer β‐amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/γ‐secretase complex, which consists of presenilins, nicastrin, APH‐1, and PEN‐2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5′‐flanking region of the human APH‐1A gene and identified a 271‐bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF‐1 to the promoter, which affects the promoter activity. Activation of HIF‐1 by short‐term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH‐1A mRNA and protein expression, accompanied by increased secretion of Aβ and decreased APP CTFs formation, indicative of an increase in γ‐secretase activity. NiCl2 treatments had little effect on APP and the other three components of the γ‐secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH‐1A expression and the γ‐secretase mediated Aβ and Notch NICD generation are regulated by HIF‐1, and the specific control of APH‐1A expression may imply physiological functions uniquely assigned to APH‐1A.— Wang, R., Zhang, Y‐W., Zhang, X., Liu, R., Zhang, X., Hong, S., Xia, K., Xia, J., Zhang, Z., Xu, H. Transcriptional regulation of APH‐1A and increased γ‐secretase cleavage of APP and Notch by HIF‐1 and hypoxia. FASEB J. 20, E614–E622 (2006)

Isorhynchophylline, a natural alkaloid, promotes the degradation of alpha-synuclein in neuronal cells via inducing autophagy

Accumulation of α-synuclein (α-syn) in the brain is a pathogenic feature and also a causative factor of Parkinson disease. Isorhynchophylline (IsoRhy) is a major tetracyclic oxindole alkaloid isolated from the Chinese herbal medicine Uncaria rhynchophylla (Miq.)Jacks (Gouteng in Chinese), which has been used for the treatment of neurological diseases in East Asia for centuries. Here we report a novel function of IsoRhy as a neuronal autophagy inducer. IsoRhy induced autophagy in different neuronal cell lines, including N2a, SH-SY5Y and PC12 cells, and also in primary cortical neurons. Furthermore, IsoRhy induced autophagy in the fat bodies of Drosophila. IsoRhy promoted clearance of wild-type, A53T and A30P α-syn monomers, α-syn oligomers and α-syn/synphilin-1 aggresomes in neuronal cells via the autophagy-lysosome pathway. More importantly, IsoRhy was able to decrease the expression levels of wild-type and A53T α-syn protein in differentiated human dopaminergic neurons. Notably, IsoRhy-induced autophagy was independent of the mTOR pathway but dependent on the function of Beclin 1. Taken together, data from this study raise the possibility that oxindole alkaloid derivatives may serve as a means to stimulate autophagy in neuronal cells, thereby exerting preventive and therapeutic values against neurodegenerative diseases such as Parkinson disease by reducing pathogenic protein aggregates in neurons.

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury: Involvement of PARK2-dependent mitophagy.

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2+/− mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.

Increased Expression of Reticulon 3 in Neurons Leads to Reduced Axonal Transport of β Site Amyloid Precursor Protein-cleaving Enzyme 1

Background: Axonal transport of BACE1 and the regulation of BACE1 synaptic localization remain to be fully characterized. Results: Colocalization of BACE1 with synaptophysin was reduced by overexpression of RTN3. This reduction was due to reduced BACE1 axonal transport. Conclusion: Increased interaction of RTN3 with BACE1 in the soma impacts axonal transport of BACE1. Significance: Changes of BACE1 synaptic localization potentially alter synaptic Aβ generation and amyloid deposition. BACE1 is the sole enzyme responsible for cleaving amyloid precursor protein at the β-secretase site, and this cleavage initiates the generation of β-amyloid peptide (Aβ). Because amyloid precursor protein is predominantly expressed by neurons and deposition of Aβ aggregates in the human brain is highly correlated with the Aβ released at axonal terminals, we focused our investigation of BACE1 localization on the neuritic region. We show that BACE1 was not only enriched in the late Golgi, trans-Golgi network, and early endosomes but also in both axons and dendrites. BACE1 was colocalized with the presynaptic vesicle marker synaptophysin, indicating the presence of BACE1 in synapses. Because the excessive release of Aβ from synapses is attributable to an increase in amyloid deposition, we further explored whether the presence of BACE1 in synapses was regulated by reticulon 3 (RTN3), a protein identified previously as a negative regulator of BACE1. We found that RTN3 is not only localized in the endoplasmic reticulum but also in neuritic regions where no endoplasmic reticulum-shaping proteins are detected, implicating additional functions of RTN3 in neurons. Coexpression of RTN3 with BACE1 in cultured neurons was sufficient to reduce colocalization of BACE1 with synaptophysin. This reduction correlated with decreased anterograde transport of BACE1 in axons in response to overexpressed RTN3. Our results in this study suggest that altered RTN3 levels can impact the axonal transport of BACE1 and demonstrate that reducing axonal transport of BACE1 in axons is a viable strategy for decreasing BACE1 in axonal terminals and, perhaps, reducing amyloid deposition.

Interaction between amyloid precursor protein and Nogo receptors regulates amyloid deposition

Excessive production or accumulation of β‐amyloid (Aβ) peptides in human brains leads to increased amyloid deposition and cognitive dysfunction, which are invariable pathological features in patients with Alzheimers disease (AD). Many cellular factors can regulate the production of Aβ. In this study, we show that a family of proteins named Nogo receptor proteins (NgR1 to NgR3) regulates Aβ production via interaction with amyloid precursor protein (APP). Further mapping of the interacting domain indicates that a small region adjacent to the BACE1 cleavage site of APP mediates interaction of APP with Nogo receptor proteins. Our results also indicate that increased interaction between Nogo receptor and APP reduces surface expression of APP and favors processing of APP by BACE1. When NgR2 was ablated in AD transgenic mice expressing Swedish APP and PS1ΔE9, amyloid deposition was clearly reduced (0.66% of total measured area in APPswe/PS1ΔE9/NgR2–/– mice vs. 0.76% of total measured area in APPswe/PS1ΔE9 mice). Our results demonstrate that down‐regulation of NgR expression is a potential approach for inhibiting amyloid deposition in AD patients.—Zhou, X., Hu, X., He, W., Tang, X., Shi, Q., Zhang, Z., Yan, R. Interaction between amyloid precursor protein and Nogo receptors regulates amyloid deposition. FASEB J. 25, 3146‐3156 (2011). www.fasebj.org

Regulation of Histone Acetylation by Autophagy in Parkinson Disease.

Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP+)-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP+-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP+-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis.

Cystatin C as a potential therapeutic mediator against Parkinson’s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units

Cystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson’s disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T α-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal–vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways.

Impact of RTN3 deficiency on expression of BACE1 and amyloid deposition.

Reticulon 3 (RTN3) has previously been shown to interact with BACE1 and negatively regulate BACE1 activity. To what extent RTN3 deficiency affects BACE1 activity is an intriguing question. In this study, we aimed to address this by generating RTN3-null mice. Mice with complete deficiency of RTN3 grow normally and have no obviously discernible phenotypes. Morphological analyses of RTN3-null mice showed no significant alterations in cellular structure, although RTN3 is recognized as a protein contributing to the shaping of tubular endoplasmic reticulum. Biochemical analysis revealed that RTN3 deficiency increased protein levels of BACE1. This elevation of BACE1 levels correlated with enhanced processing of amyloid precursor protein at the β-secretase site. We also demonstrated that RTN3 deficiency in Alzheimers mouse models facilitates amyloid deposition, further supporting an in vivo role of RTN3 in the regulation of BACE1 activity. Since it has been shown that RTN3 monomer is reduced in brains of Alzheimers patients, our results suggest that long-lasting reduction of RTN3 levels has adverse effects on BACE1 activity and may contribute to Alzheimers pathogenesis.

PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury

ABSTRACT Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.

Neuropeptide Receptors NPR-1 and NPR-2 Regulate Caenorhabditis elegans Avoidance Response to the Plant Stress Hormone Methyl Salicylate

Methyl salicylate (MeSa) is a stress hormone released by plants under attack by pathogens or herbivores . MeSa has been shown to attract predatory insects of herbivores and repel pests. The molecules and neurons underlying animal response to MeSa are not known. Here we found that the nematode Caenorhabditis elegans exhibits a strong avoidance response to MeSa, which requires the activities of two closely related neuropeptide receptors NPR-1 and NPR-2. Molecular analyses suggest that NPR-1 expressed in the RMG inter/motor neurons is required for MeSa avoidance. An NPR-1 ligand FLP-18 is also required. Using a rescuing npr-2 promoter to drive a GFP transgene, we identified that NPR-2 is expressed in multiple sensory and interneurons. Genetic rescue experiments suggest that NPR-2 expressed in the AIZ interneurons is required for MeSa avoidance. We also provide evidence that the AWB sensory neurons might act upstream of RMGs and AIZs to detect MeSa. Our results suggest that NPR-2 has an important role in regulating animal behavior and that NPR-1 and NPR-2 act on distinct interneurons to affect C. elegans avoidance response to MeSa.