Zhiping Shao

A presenilin-1/γ-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions

E‐cadherin controls a wide array of cellular behaviors including cell–cell adhesion, differentiation and tissue development. Here we show that presenilin‐1 (PS1), a protein involved in Alzheimers disease, controls a γ‐secretase‐like cleavage of E‐cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E‐cadherin residues Leu731 and Arg732 at the membrane–cytoplasm interface. The PS1/γ‐secretase system cleaves both the full‐length E‐cadherin and a transmembrane C‐terminal fragment, derived from a metalloproteinase cleavage after the E‐cadherin ectodomain residue Pro700. The PS1/γ‐secretase cleavage dissociates E‐cadherins, β‐catenin and α‐catenin from the cytoskeleton, thus promoting disassembly of the E‐cadherin–catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E‐cadherin to the cytosol and increases the levels of soluble β‐ and α‐catenins. Thus, the PS1/γ‐secretase system stimulates disassembly of the E‐cadherin– catenin complex and increases the cytosolic pool of β‐catenin, a key regulator of the Wnt signaling pathway.

PS1 activates PI3K thus inhibiting GSK‐3 activity and tau overphosphorylation: effects of FAD mutations

Phosphatidylinositol 3‐kinase (PI3K) promotes cell survival and communication by activating its downstream effector Akt kinase. Here we show that PS1, a protein involved in familial Alzheimers disease (FAD), promotes cell survival by activating the PI3K/Akt cell survival signaling. This function of PS1 is unaffected by γ‐secretase inhibitors. Pharmacological and genetic evidence indicates that PS1 acts upstream of Akt, at or before PI3K kinase. PS1 forms complexes with the p85 subunit of PI3K and promotes cadherin/PI3K association. Furthermore, conditions that inhibit this association prevent the PS1‐induced PI3K/Akt activation, indicating that PS1 stimulates PI3K/Akt signaling by promoting cadherin/PI3K association. By activating PI3K/Akt signaling, PS1 promotes phosphorylation/inactivation of glycogen synthase kinase‐3 (GSK‐3), suppresses GSK‐3‐dependent phosphorylation of tau at residues overphosphorylated in AD and prevents apoptosis of confluent cells. PS1 FAD mutations inhibit the PS1‐dependent PI3K/Akt activation, thus promoting GSK‐3 activity and tau overphosphorylation at AD‐related residues. Our data raise the possibility that PS1 may prevent development of AD pathology by activating the PI3K/Akt signaling pathway. In contrast, FAD mutations may promote AD pathology by inhibiting this pathway.

Extracellular progranulin protects cortical neurons from toxic insults by activating survival signaling.

To reduce damage from toxic insults such as glutamate excitotoxicity and oxidative stresses, neurons may deploy an array of neuroprotective mechanisms. Recent reports show that progranulin (PGRN) gene null or missense mutations leading to inactive protein, are linked to frontotemporal lobar degeneration (FTLD), suggesting that survival of certain neuronal populations needs full expression of functional PGRN. Here we show that extracellular PGRN stimulates phosphorylation/activation of the neuronal MEK/extracellular regulated kinase (ERK)/p90 ribosomal S6 kinase (p90RSK) and phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathways and rescues cortical neurons from cell death induced by glutamate or oxidative stress. Pharmacological inhibition of MEK/ERK/p90RSK signaling blocks the PGRN-induced phosphorylation and neuroprotection against glutamate toxicity while inhibition of either MEK/ERK/p90RSK or PI3K/Akt blocks PGRN protection against neurotoxin MPP(+). Inhibition of both pathways had synergistic effects on PGRN-dependent neuroprotection against MPP(+) toxicity suggesting both pathways contribute to the neuroprotective activities of PGRN. Extracellular PGRN is remarkably stable in neuronal cultures indicating neuroprotective activities are associated with full-length protein. Together, our data show that extracellular PGRN acts as a neuroprotective factor and support the hypothesis that in FTLD reduction of functional brain PGRN results in reduced survival signaling and decreased neuronal protection against excitotoxicity and oxidative stress leading to accelerated neuronal cell death. That extracellular PGRN has neuroprotective functions against toxic insults suggests that in vitro preparations of this protein may be used therapeutically.

Wild-Type But Not FAD Mutant Presenilin-1 Prevents Neuronal Degeneration by Promoting Phosphatidylinositol 3-Kinase Neuroprotective Signaling

The role of presenilin-1 (PS1) in neuronal phosphatidylinositol 3-kinase (PI3K)/Akt signaling was investigated in primary neuronal cultures from wild-type (WT) and PS1 null (PS1−/−) embryonic mouse brains. Here we show that in PS1−/− cultures, the onset of neuronal maturation coincides with a decrease in the PI3K-dependent phosphorylation-activation of Akt and phosphorylation-inactivation of glycogen synthase kinase-3 (GSK-3). Mature PS1−/− neurons show increased activation of apoptotic caspase-3 and progressive degeneration preceded by dendritic retraction. Expression of exogenous WT PS1 or constitutively active Akt in PS1−/− neurons stimulates PI3K signaling and suppresses both caspase-3 activity and dendrite retraction. The survival effects of PS1 are sensitive to inhibitors of PI3K kinase but insensitive to γ-secretase inhibitors. Familial Alzheimer disease (FAD) mutations suppress the ability of PS1 to promote PI3K/AKT signaling, prevent phosphorylation/inactivation of GSK-3 and promote activation of caspase-3. These mutation effects are reversed upon coexpression of constitutively active Akt. Together, our data indicate that the neuroprotective role of PS1 depends on its ability to activate the PI3K/Akt signaling pathway and that PS1 FAD mutations increase GSK-3 activity and promote neuronal apoptosis by inhibiting the function of PS1 in this pathway. These observations suggest that stimulation of PI3K/Akt signaling may be beneficial to FAD patients.

Presenilin mediates neuroprotective functions of ephrinB and brain-derived neurotrophic factor and regulates ligand-induced internalization and metabolism of EphB2 and TrkB receptors.

Activation of EphB receptors by ephrinB (efnB) ligands on neuronal cell surface regulates important functions, including neurite outgrowth, axonal guidance, and synaptic plasticity. Here, we show that efnB rescues primary cortical neuronal cultures from necrotic cell death induced by glutamate excitotoxicity and that this function depends on EphB receptors. Importantly, the neuroprotective function of the efnB/EphB system depends on presenilin 1 (PS1), a protein that plays crucial roles in Alzheimers disease (AD) neurodegeneration. Furthermore, absence of one PS1 allele results in significantly decreased neuroprotection, indicating that both PS1 alleles are necessary for full expression of the neuroprotective activity of the efnB/EphB system. We also show that the ability of brain-derived neurotrophic factor (BDNF) to protect neuronal cultures from glutamate-induced cell death depends on PS1. Neuroprotective functions of both efnB and BDNF, however, were independent of γ-secretase activity. Absence of PS1 decreases cell surface expression of neuronal TrkB and EphB2 without affecting total cellular levels of the receptors. Furthermore, PS1-knockout neurons show defective ligand-dependent internalization and decreased ligand-induced degradation of TrkB and Eph receptors. Our data show that PS1 mediates the neuroprotective activities of efnB and BDNF against excitotoxicity and regulates surface expression and ligand-induced metabolism of their cognate receptors. Together, our observations indicate that PS1 promotes neuronal survival by regulating neuroprotective functions of ligand-receptor systems.

Inhibitors of γ-secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates

γ‐Secretase inhibitors (GSIs) are drugs used in research to inhibit production of Aβ and in clinical trials to treat Alzheimers disease (AD). They inhibit proteolytic activities of γ‐secretase noncompetitively by unknown mechanisms. Here, we used cortical neuronal cultures expressing endogenous levels of enzymes and substrates to study the effects of GSIs on the structure and function of γ‐secretase. We show that GSIs stabilize the interactions between the C‐terminal fragment of presenilin (PS‐CTF), the central component of the γ‐secretase complex, and its partners the APH‐1/nicastrin and PS1‐NTF/PEN‐2 subcomplexes. This stabilization dose‐dependently correlates with inhibition of N‐cadherin cleavage, a process limited by enzyme availability. In contrast, production of amyloid precursor protein (APP) intracellular domain (AICD) is insensitive to low concentrations of GSIs and is limited by substrate availability. Interestingly, APP is processed by both PS1‐and PS2‐containing γ‐secretase complexes, while N‐cadherin and ephrinB1 are processed only by PS1‐containing complexes. Paradoxically, low concentrations of GSIs specifically increased the levels of Aβ without affecting its catabolism, indicating increased Aβ production. Our data reveal a mechanism of γ‐secretase inhibition by GSIs and provide evidence that distinct γ‐secretase complexes process specific substrates. Furthermore, our observations have implications for GSIs as therapeutics because processing of functionally important substrates may be inhibited at lower concentrations than Aβ.—Barthet, G., Shioi, J., Shao, Z., Ren, Y., Georgakopoulos, A., Robakis, N. K. Inhibitors of γ‐secretase stabilize the complex and differentially affect processing of amyloid precursor protein and other substrates. FASEB J. 25, 2937–2946 (2011). www.fasebj.org

Open chromatin profiling of human postmortem brain infers functional roles for non-coding schizophrenia loci

&NA; Open chromatin provides access to DNA‐binding proteins for the correct spatiotemporal regulation of gene expression. Mapping chromatin accessibility has been widely used to identify the location of cis regulatory elements (CREs) including promoters and enhancers. CREs show tissue‐ and cell‐type specificity and disease‐associated variants are often enriched for CREs in the tissues and cells that pertain to a given disease. To better understand the role of CREs in neuropsychiatric disorders we applied the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC‐seq) to neuronal and non‐neuronal nuclei isolated from frozen postmortem human brain by fluorescence‐activated nuclear sorting (FANS). Most of the identified open chromatin regions (OCRs) are differentially accessible between neurons and non‐neurons, and show enrichment with known cell type markers, promoters and enhancers. Relative to those of non‐neurons, neuronal OCRs are more evolutionarily conserved and are enriched in distal regulatory elements. Transcription factor (TF) footprinting analysis identifies differences in the regulome between neuronal and non‐neuronal cells and ascribes putative functional roles to a number of non‐coding schizophrenia (SCZ) risk variants. Among the identified variants is a Single Nucleotide Polymorphism (SNP) proximal to the gene encoding SNX19. In vitro experiments reveal that this SNP leads to an increase in transcriptional activity. As elevated expression of SNX19 has been associated with SCZ, our data provide evidence that the identified SNP contributes to disease. These results represent the first analysis of OCRs and TF‐binding sites in distinct populations of postmortem human brain cells and further our understanding of the regulome and the impact of neuropsychiatric disease‐associated genetic risk variants.

Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via γ-secretase-independent transcriptional mechanisms

Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1‐null (PS1‐/‐) brains have reduced amounts of this receptor. PS1‐/‐ cortical neurons contain little EGFR and show no epidermal growth factor‐induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1‐/‐neurons, and PS1‐/‐ brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down‐regulation of PS1 decreases this mRNA. These effects are neuron specific, as PS1 affects the EGFR of neither glial nor fibroblast cells. In addition, PS1 controls EGFR through novel mechanisms shared with neither γ‐secretase nor PS2. Our data reveal that PS1 functions as a positive transcriptional regulator of neuronal EGFR controlling its expression in a cell‐specific manner. Severe downregulation of EGFR may contribute to developmental abnormalities and lethal phenotype found in PS1, but not PS2, null mice. Furthermore, PS1 may affect neuroprotection and Alzheimer disease by controlling survival signaling of neuronal EGFR.—Bruban, J., Voloudakis, G., Huang, Q., Kajiwara, Y., Al Rahim, M., Yoon, Y., Shioi,J., Gama Sosa, M. A., Shao, Z., Georgakopoulos, A., Robakis, N. K. Presenilin 1 is necessary for neuronal, but not glial, EGFR expression and neuroprotection via 7‐secretase‐independent transcriptional mechanisms. FASEB J. 29, 3702‐3712 (2015). www.fasebj.org

P4-195: Presenilin1 prevents neurodegeneration by stimulating PI3K/Akt neuroprotective signaling: Effects of PS1 familial Alzheimer's disease mutations

and G384A were subjected to treatment with different GSMs, and A 42 and A 38 levels were analyzed by ELISA, mass spectrometry or high-resolution SDS-urea electrophoresis. Results: After sulindac sulfide treatment, all mutant cell lines displayed a strongly reduced A 42response as compared to WT-PS1 expressing cells. Conversely, A 38 levels were increased to similar levels in the mutant cell lines. These results were confirmed with the structurally divergent GSM ibuprofen. Furthermore, mass spectrometry and SDS-urea electrophoresis did not reveal compensatory changes in levels of other secreted or intracellular A peptides. Conclusions: These data provide evidence that A 42 and A 38 can be generated independently by -secretase, and argue against a precursor-product relationship between these peptides.