Selective inhibitors of the PSEN1-gamma-secretase complex.

Clinical development of γ-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimer's disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific γ-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1-APH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different γ-secretase complexes might provide safer drugs in the future.

PSEN1-selective gamma-secretase inhibition in combination with kinase or XPO-1 inhibitors effectively targets T cell acute lymphoblastic leukemia.

BACKGROUND: T cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype that comprises 10-15% of childhood and 20-25% of adult ALL cases. Over 70% of T-ALL patients harbor activating mutations in the NOTCH1 signaling pathway and are predicted to be sensitive to gamma-secretase inhibitors. We have recently demonstrated that selective inhibition of PSEN1-containing gamma-secretase complexes can overcome the dose-limiting toxicity associated with broad gamma-secretase inhibitors. In this study, we developed combination treatment strategies with the PSEN1-selective gamma-secretase inhibitor MRK-560 and other targeted agents (kinase inhibitors ruxolitinib and imatinib; XPO-1 inhibitor KPT-8602/eltanexor) for the treatment of T-ALL. METHODS: We treated T-ALL cell lines in vitro and T-ALL patient-derived xenograft (PDX) models in vivo with MRK-560 alone or in combination with other targeted inhibitors (ruxolitinib, imatinib or KPT-8602/eltanexor). We determined effects on proliferation of the cell lines and leukemia development and survival in the PDX models. RESULTS: All NOTCH1-signaling-dependent T-ALL cell lines were sensitive to MRK-560 and its combination with ruxolitinib or imatinib in JAK1- or ABL1-dependent cell lines synergistically inhibited leukemia proliferation. We also observed strong synergy between MRK-560 and KPT-8602 (eltanexor) in all NOTCH1-dependent T-ALL cell lines. Such synergy was also observed in vivo in a variety of T-ALL PDX models with NOTCH1 or FBXW7 mutations. Combination treatment significantly reduced leukemic infiltration in vivo and resulted in a survival benefit when compared to single treatment groups. We did not observe weight loss or goblet cell hyperplasia in single drug or combination treated mice when compared to control. CONCLUSIONS: These data demonstrate that the antileukemic effect of PSEN1-selective gamma-secretase inhibition can be synergistically enhanced by the addition of other targeted inhibitors. The combination of MRK-560 with KPT-8602 is a highly effective treatment combination, which circumvents the need for the identification of additional mutations and provides a clear survival benefit in vivo. These promising preclinical data warrant further development of combination treatment strategies for T-ALL based on PSEN1-selective gamma-secretase inhibition.

Chronic Treatment with 50 mg/kg Cannabidiol Improves Cognition and Moderately Reduces Aß40 Levels in 12-Month-Old Male AßPPswe/PS1ΔE9 Transgenic Mice.

Alzheimer's disease (AD) is characterized by progressive cognitive decline and pathologically by the accumulation of amyloid-ß (Aß) and tau hyperphosphorylation causing neurodegeneration and neuroinflammation. Current AD treatments do not stop or reverse the disease progression, highlighting the need for more effective therapeutics. The phytocannabinoid cannabidiol (CBD) has demonstrated antioxidant, anti-inflammatory, and neuroprotective properties. Furthermore, chronic CBD treatment (20 mg/kg) reverses social and object recognition memory deficits in the AßPPxPS1 transgenic mouse model with only limited effects on AD-relevant brain pathology. Importantly, studies have indicated that CBD works in a dose-dependent manner. Thus, this study determined the chronic effects of 50 mg/kg CBD in male AßPPxPS1 mice. 12-month-old mice were treated with 50 mg/kg CBD or vehicle via daily intraperitoneal injections for 3 weeks prior to behavioral testing. A variety of cognitive domains including object and social recognition, spatial and fear-associated memory were evaluated. Pathological brain analyses for AD-relevant markers were conducted using ELISA and western blot. Vehicle-treated male AßPPxPS1 mice demonstrated impaired social recognition memory and reversal spatial learning. These deficits were restored after CBD treatment. Chronic CBD tended to reduce insoluble Aß40 levels in the hippocampus of AßPPxPS1 mice but had no effect on neuroinflammation, neurodegeneration, or PPARγ markers in the cortex. This study demonstrates that therapeutic-like effects of 50 mg/kg CBD on social recognition memory and spatial learning deficits in AßPPxPS1 mice are accompanied by moderate brain region-specific reductions in insoluble Aß40 levels. The findings emphasize the clinical relevance of CBD treatment in AD; however, the underlying mechanisms involved require further investigation.

Deficits in Enrichment-Dependent Neurogenesis and Enhanced Anxiety Behaviors Mediated by Expression of Alzheimer's Disease-Linked Ps1 Variants Are Rescued by Microglial Depletion.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that presently affects an estimated 5.7 million Americans. Understanding the basis for this disease is key for the development of a future successful treatment. In this effort, we previously reported that mouse prion protein-promoter-driven, ubiquitous expression of familial AD (FAD)-linked human PSEN1 variants in transgenic mice impairs environmental enrichment (EE)-induced proliferation and neurogenesis of adult hippocampal neural progenitor cells (AHNPCs) and in a non-cell autonomous manner. These findings were confirmed in PS1M146V/+ mice that harbor an FAD-linked mutation in the endogenous PSEN1 gene. We now demonstrate that CSF1R antagonist-mediated microglial depletion in transgenic male mice expressing mutant presenilin 1 (PS1) or PS1M146V/+ "knock-in" mice leads to a complete rescue of deficits in proliferation, differentiation and survival of AHNPCs. Moreover, microglia depletion suppressed the heightened baseline anxiety behavior observed in transgenic mice expressing mutant PS1 and PS1M146V/+ mice to levels observed in mice expressing wild-type human PS1 or nontransgenic mice, respectively. These findings demonstrate that in mice expressing FAD-linked PS1, microglia play a critical role in the regulation of EE-dependent AHNPC proliferation and neurogenesis and the modulation of affective behaviors.SIGNIFICANCE STATEMENT Inheritance of mutations in genes encoding presenilin 1 (PS1) causes familial Alzheimer's disease (FAD). Mutant PS1 expression enhances the levels and assembly of toxic Aß42 peptides and impairs the self-renewal and neuronal differentiation of adult hippocampal neural progenitor cells (AHNPCs) following environmental enrichment (EE) that is associated with heightened baseline anxiety. We now show that microglial depletion fully restores the EE-mediated impairments in AHNPC phenotypes and suppresses the heightened baseline anxiety observed in mice expressing FAD-linked PS1. Thus, we conclude that the memory deficits and anxiety-related behaviors in patients with PS1 mutations is a reflection not just of an increase in the levels of Aß42 peptides, but to impairments in the self-renewal and neuronal differentiation of AHNPCs that modulate affective behaviors.

Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Reduced cerebral glucose utilization is found in aged individuals and often is an early sign of neurodegeneration. Here, we show that under glucose deprivation (GD) conditions, decreased expression of presenilin 1 (PS1) results in decreased neuronal survival, whereas increased PS1 increases neuronal survival. Inhibition of γ-secretase also decreases neuronal survival under GD conditions, which suggests the PS1/γ-secretase system protects neurons from GD-induced death. We also show that neuronal levels of the survival protein, phosphoprotein enriched in astrocytes at ∼15 kDa (PEA15), and its mRNA are regulated by PS1/γ-secretase. Furthermore, down-regulation of PEA15 decreases neuronal survival under reduced glucose conditions, whereas exogenous PEA15 increases neuronal survival even in the absence of PS1, which indicates that PEA15 promotes neuronal survival under GD conditions. The absence or reduction of PS1, as well as γ-secretase inhibitors, increases neuronal miR-212, which targets PEA15 mRNA. PS1/γ-secretase activates the transcription factor, cAMP response element-binding protein, regulating miR-212, which targets PEA15 mRNA. Taken together, our data show that under conditions of reduced glucose, the PS1/γ-secretase system decreases neuronal losses by suppressing miR-212 and increasing its target survival factor, PEA15. These observations have implications for mechanisms of neuronal death under conditions of reduced glucose and may provide targets for intervention in neurodegenerative disorders.-Huang, Q., Voloudakis, G., Ren, Y., Yoon, Y., Zhang, E., Kajiwara, Y., Shao, Z., Xuan, Z., Lebedev, D., Georgakopoulos, A., Robakis, N. K. Presenilin1/γ-secretase protects neurons from glucose deprivation-induced death by regulating miR-212 and PEA15.

Mitochondrion to endoplasmic reticulum apposition length in zebrafish embryo spinal progenitors is unchanged in response to perturbations associated with Alzheimer's disease.

Mutations in the human genes PRESENILIN1 (PSEN1), PRESENILIN2 (PSEN2) and AMYLOID BETA A4 PRECURSOR PROTEIN (APP) have been identified in familial Alzheimer's disease (AD). The length of mitochondrion-endoplasmic reticulum (M-ER) appositions is increased in Psen1-/-/Psen2-/- double knockout murine embryonic fibroblasts and in fibroblasts from AD-affected individuals. Development of an easily accessible, genetically manipulable, in vivo system for studying M-ER appositions would be valuable so we attempted to manipulate M-ER apposition length in zebrafish (Danio rerio) embryos. We injected fertilized zebrafish eggs with antisense morpholino oligonucleotides (MOs) that inhibit expression of zebrafish familial AD gene orthologues psen1 and psen2. Furthermore, we treated zebrafish embryos with DAPT (a highly specific γ-secretase inhibitor) or with sodium azide (to mimic partially hypoxic conditions). We then analyzed M-ER apposition in an identified, presumably proliferative neural cell type using electron microscopy. Our analysis showed no significant differences in M-ER apposition lengths at 48 hours post fertilization (hpf) between psen1 & psen2 MO co-injected embryos, embryos treated with DAPT, or sodium azide, and control embryos. Instead, the distribution of M-ER apposition lengths into different length classes was close to identical. However, this indicates that it is feasible to reproducibly measure M-ER size distributions in zebrafish embryos. While our observations differ from those of murine and human studies, this may be due to differences in cellular differentiation and metabolic state, cell age, or species-specific responses. In particular, by focusing on a presumably proliferative embryonic cell type, we may have selected a cell heavily already reliant on anaerobic glycolysis and less responsive to factors affecting M-ER apposition. Future examination of more differentiated, more secretory cell types may reveal measurable responses of M-ER apposition to environmental and genetic manipulation.

Protective Effect of Genistein against Neuronal Degeneration in ApoE-/- Mice Fed a High-Fat Diet.

Altered cholesterol metabolism is believed to play a causal role in major pathophysiological changes in neurodegeneration. Several studies have demonstrated that the absence of apolipoprotein E (ApoE), a predominant apolipoprotein in the brain, leads to an increased susceptibility to neurodegeneration. Previously, we observed that genistein, a soy isoflavone, significantly alleviated apoptosis and tau hyperphosphorylation in SH-SY5Y cells. Therefore, we investigated the neuroprotective effects of dietary genistein supplementation (0.5 g/kg diet) in the cortex and hippocampus of wild-type C57BL/6 (WT) and ApoE knockout (ApoE-/-) mice fed a high-fat diet (HFD) for 24 weeks. Genistein supplementation alleviated neuroinflammation and peripheral and brain insulin resistance. Reductions in oxidative and endoplasmic reticulum stress were also observed in ApoE-/- mice fed a genistein-supplemented diet. Beta-secretase 1 and presenilin 1 mRNA levels and beta-amyloid peptide (Aß) protein levels were reduced in response to genistein supplementation in ApoE-/- mice but not in WT mice. Although the absence of ApoE did not increase tau hyperphosphorylation, genistein supplementation reduced tau hyperphosphorylation in both WT and ApoE-/- mice. Consistent with this result, we also observed that genistein alleviated activity of c-Jun N-terminal kinase and glycogen synthase kinase 3ß, which are involved in tau hyperphosphorylation. Taken together, these results demonstrate that genistein alleviated neuroinflammation, Aß deposition, and hyperphosphorylation in ApoE-/- mice fed an HFD.

The Zebrafish Equivalent of Alzheimer's Disease-Associated PRESENILIN Isoform PS2V Regulates Inflammatory and Other Responses to Hypoxic Stress.

Dominant mutations in the PRESENILIN genes PSEN1 and PSEN2 cause familial Alzheimer's disease (fAD) that usually shows onset before 65 years of age. In contrast, genetic variation at the PSEN1 and PSEN2 loci does not appear to contribute to risk for the sporadic, late onset form of the disease (sAD), leading to doubts that these genes play a role in the majority of AD cases. However, a truncated isoform of PSEN2, PS2V, is upregulated in sAD brains and is induced by hypoxia and high cholesterol intake. PS2V can increase γ-secretase activity and suppress the unfolded protein response (UPR), but detailed analysis of its function has been hindered by lack of a suitable, genetically manipulable animal model since mice and rats lack this PRESENILIN isoform. We recently showed that zebrafish possess an isoform, PS1IV, that is cognate to human PS2V. Using an antisense morpholino oligonucleotide, we can block specifically the induction of PS1IV that normally occurs under hypoxia. Here, we exploit this ability to identify gene regulatory networks that are modulated by PS1IV. When PS1IV is absent under hypoxia-like conditions, we observe changes in expression of genes controlling inflammation (particularly sAD-associated IL1B and CCR5), vascular development, the UPR, protein synthesis, calcium homeostasis, catecholamine biosynthesis, TOR signaling, and cell proliferation. Our results imply an important role for PS2V in sAD as a component of a pathological mechanism that includes hypoxia/oxidative stress and support investigation of the role of PS2V in other diseases, including schizophrenia, when these are implicated in the pathology.

The expression of presenilin 1 enhances carcinogenesis and metastasis in gastric cancer.

Presenilin 1 (PS-1, encoded by PSEN1) is a part of the gamma- (γ-) secretase complex. Mutations in PSEN1 cause the majority of cases of familial Alzheimer's disease (FAD). Although in recent years PS-1 has been implicated as a tumor enhancer in various cancers, nothing is known regarding its role in gastric cancer (GC). In the present study, we investigate the role and clinical significance of PS-1 in GC. We observed that PS-1 was significantly upregulated and amplified in GC tissues and cell lines, and its aberrant expression was positively correlated with lymph node metastasis and with poor overall survival. Furthermore, PS-1 promoted tumor invasion and metastasis of GC both in vitro and vivo without affecting the proliferation of GC cells (MGC-803 and MKN-45). The results of treatment with the γ-secretase inhibitor DAPT were consistent with the outcomes of PS-1 silencing. PS-1/γ-secretase cleaves E-cadherin and releases its bound protein partner, ß-catenin, from the actin cytoskeleton, thereby allowing it to translocate into the nucleus and to activate the TCF/LEF-1 transcriptional activator, which may promote GC invasion and metastasis.In conclusion, PS-1 promotes invasion and metastasis in GC and may represent a novel prognostic biomarker and potential therapeutic target for GC treatment.

Inhibition of amyloid precursor protein secretases reduces recovery after spinal cord injury.

Amyloid-ß (Aß) is produced through the enzymatic cleavage of amyloid precursor protein (APP) by ß (Bace1) and γ-secretases. The accumulation and aggregation of Aß as amyloid plaques is the hallmark pathology of Alzheimer׳s disease and has been found in other neurological disorders, such as traumatic brain injury and multiple sclerosis. Although the role of Aß after injury is not well understood, several studies have reported a negative correlation between Aß formation and functional outcome. In this study we show that levels of APP, the enzymes cleaving APP (Bace1 and γ-secretase), and Aß are significantly increased from 1 to 3 days after impact spinal cord injury (SCI) in mice. To determine the role of Aß after SCI, we reduced or inhibited Aß in vivo through pharmacological (using DAPT) or genetic (Bace1 knockout mice) approaches. We found that these interventions significantly impaired functional recovery as evaluated by white matter sparing and behavioral testing. These data are consistent with a beneficial role for Aß after SCI.

Dissociated presenilin-1 and TACE processing of ErbB4 in lung alveolar type II cell differentiation.

Neuregulin (NRG) stimulation of ErbB4 signaling is important for type II cell surfactant synthesis. ErbB4 may mediate gene expression via a non-canonical pathway involving enzymatic cleavage releasing its intracellular domain (4ICD) for nuclear trafficking and gene regulation. The accepted model for release of 4ICD is consecutive cleavage by Tumor necrosis factor alpha Converting Enzyme (TACE) and γ-secretase enzymes. Here, we show that 4ICD mediates surfactant synthesis and its release by γ-secretase is not dependent on previous TACE cleavage. We used siRNA to silence Presenilin-1 (PSEN-1) expression in a mouse lung type II epithelial cell line (MLE12 cells), and both siRNA knockdown and chemical inhibition of TACE. Knockdown of PSEN-1 significantly decreased baseline and NRG-stimulated surfactant phospholipid synthesis, expression of the surfactant proteins SP-B and SP-C, as well as 4ICD levels, with no change in ErbB4 ectodomain shedding. Neither siRNA knockdown nor chemical inhibition of TACE inhibited 4ICD release or surfactant synthesis. PSEN-1 cleavage of ErbB4 for non-canonical signaling through 4ICD release does not require prior cleavage by TACE.

Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes.

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.

Trans-dominant negative effects of pathogenic PSEN1 mutations on γ-secretase activity and Aß production.

Mutations in the PSEN1 gene encoding Presenilin-1 (PS1) are the predominant cause of familial Alzheimer's disease (FAD), but the underlying mechanisms remain unresolved. To reconcile the dominant action of pathogenic PSEN1 mutations with evidence that they confer a loss of mutant protein function, we tested the hypothesis that PSEN1 mutations interfere with γ-secretase activity in a dominant-negative manner. Here, we show that pathogenic PSEN1 mutations act in cis to impair mutant PS1 function and act in trans to inhibit wild-type PS1 function. Coexpression of mutant and wild-type PS1 at equal gene dosage in presenilin-deficient mouse embryo fibroblasts resulted in trans-dominant-negative inhibition of wild-type PS1 activity, suppressing γ-secretase-dependent cleavage of APP and Notch. Surprisingly, mutant PS1 could stimulate production of Aß42 by wild-type PS1 while decreasing its production of Aß40. Mutant and wild-type PS1 efficiently coimmunoprecipitated, suggesting that mutant PS1 interferes with wild-type PS1 activity via physical interaction. These results support the conclusion that mutant PS1 causes wild-type PS1 to adopt an altered conformation with impaired catalytic activity and substrate specificity. Our findings reveal a novel mechanism of action for pathogenic PSEN1 mutations and suggest that dominant-negative inhibition of presenilin activity plays an important role in FAD pathogenesis.

Presenilin 1/γ-secretase modulates P-cadherin processing and influences cell adhesion in oral squamous cell carcinoma cell lines.

P-cadherin belongs to a family of Ca(2+)-dependent homophilic cell-cell adhesion proteins that are important for correct cellular localization and tissue integrity in the oral epithelium. P-cadherin is only expressed in the basal and suprabasal cell layers of the oral epithelium, but in advanced oral squamous cell carcinoma (OSCC), a reduced membranous and an enhanced cytoplasmic truncated P-cadherin level is observed. In this study, we investigated the impact of presenilin (PS) 1/γ-secretase on P-cadherin processing in OSCC. Western blot analyses showed an enhanced PS1 expression in OSCC cell lines and in primary oral keratinocytes (POK) isolated from primary OSCC tissue (OSCC POK) compared with POKs isolated from normal oral mucosa. Immunocytochemical stainings and co-immunoprecipitation experiments revealed a cytoplasmic colocalization and a direct interaction of P-cadherin and PS1 in OSCC POKs. Blocking of PS1/γ-secretase activity by the PS1/γ-secretase inhibitors and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, another specific γ-secretase inhibitor yielded a 100 kDa P-cadherin band on western blots of OSCC cell line extracts. Small interfering RNA knockdown of PS1 equally generated a 100 kDa P-cadherin form in OSCC POKs. Mass spectrometry analyses and experiments with the glycosylation inhibitor tunicamycin characterized the appearing 100 kDa P-cadherin band as the unglycosylated full-length form of P-cadherin. On the functional level, cell attachment assays demonstrated an enhanced cell adhesion after PS1/γ-secretase inhibition only in the transiently P-cadherin expressing OSCC cell line PCI52 but not in the PCI52 control cells. In summary, our results show that PS1/γ-secretase contributes to P-cadherin processing and to reduced cell adhesion in OSCC.

Intracranial injection of Gammagard, a human IVIg, modulates the inflammatory response of the brain and lowers Aß in APP/PS1 mice along a different time course than anti-Aß antibodies.

Gammagard IVIg is a therapeutic approach to treat Alzheimer's disease currently in phase 3 clinical trials. Despite the reported efficacy of the approach the mechanism of action is poorly understood. We have previously shown that intracranial injection of anti-Aß antibodies into the frontal cortex and hippocampus reveals important information regarding the time course of events once the agent is in the brain. In the current study we compared IVIg, mouse-pooled IgG, and the anti-Aß antibody 6E10 injected intracranially into the frontal cortex and hippocampus of 7-month-old APP/PS1 mice. We established a time course of events ranging from 1 to 21 d postinjection. IVIg and pooled mouse IgG both significantly reduced Aß deposition to the same degree as the 6E10 anti-Aß antibody; however, the clearance was much slower to occur, happening between the 3 and 7 d time points. In contrast, as we have previously shown, Aß reductions were apparent with the 6E10 anti-Aß group at the 1 d time point. Also, neuroinflammatory profiles were significantly altered by the antibody treatments. APP/PS1 transgenic mice at 7 months of age typically exhibit an M2a inflammatory phenotype. All antibody treatments stimulated an M2b response, yet anti-Aß antibody was a more rapid change. Because the neuroinflammatory switch occurs before the detectable reductions in amyloid deposition, we hypothesize that the IVIg and pooled mouse IgG act as immune modulators and this immune modulation is responsible for the reductions in amyloid pathology.

Antisense directed against PS-1 gene decreases brain oxidative markers in aged senescence accelerated mice (SAMP8) and reverses learning and memory impairment: a proteomics study.

Amyloid ß-peptide (Aß) plays a central role in the pathophysiology of Alzheimer's disease (AD) through the induction of oxidative stress. This peptide is produced by proteolytic cleavage of amyloid precursor protein (APP) by the action of ß- and γ-secretases. Previous studies demonstrated that reduction of Aß, using an antisense oligonucleotide (AO) directed against the Aß region of APP, reduced oxidative stress-mediated damage and prevented or reverted cognitive deficits in senescence-accelerated prone mice (SAMP8), a useful animal model for investigating the events related to Aß pathology and possibly to the early phase of AD. In the current study, aged SAMP8 were treated by AO directed against PS-1, a component of the γ-secretase complex, and tested for learning and memory in T-maze foot shock avoidance and novel object recognition. Brain tissue was collected to identify the decrease of oxidative stress and to evaluate the proteins that are differently expressed and oxidized after the reduction in free radical levels induced by Aß. We used both expression proteomics and redox proteomics approaches. In brain of AO-treated mice a decrease of oxidative stress markers was found, and the proteins identified by proteomics as expressed differently or nitrated are involved in processes known to be impaired in AD. Our results suggest that the treatment with AO directed against PS-1 in old SAMP8 mice reverses learning and memory deficits and reduces Aß-mediated oxidative stress with restoration to the normal condition and identifies possible pharmacological targets to combat this devastating dementing disease.

Upregulated function of mitochondria-associated ER membranes in Alzheimer disease.

Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by γ-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of γ-secretase, and γ-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and γ-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.

Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.

In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.

Amyloid precursor protein (APP) mediated regulation of ganglioside homeostasis linking Alzheimer's disease pathology with ganglioside metabolism.

Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aß production, are enriched in amyloid plaques and bind amyloid beta (Aß). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aß and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aß to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aß release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aß and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aß production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aß generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aß-GM3 complex and AICD-mediated repression of GD3S transcription.

Encapsulated VEGF-secreting cells enhance proliferation of neuronal progenitors in the hippocampus of AßPP/Ps1 mice.

Vascular endothelial growth factor (VEGF) promotes neurogenesis in the adult hippocampus, but the way in which this process occurs in the Alzheimer's disease (AD) brain is still unknown. We examined the proliferation of neuronal precursors with an ex vivo approach, using encapsulated VEGF secreting cells, in AßPP/PS1 mice, a mouse model of AD. Overexpression of VEGF and VEGF receptor flk-1 was observed in the cerebral cortex from VEGF microcapsules-treated AßPP/PS1 mice at 1, 3 and 6 months after VEGF-microcapsule implantation. Stereological counting of 5-bromodeoxyuridine positive cells revealed that encapsulated VEGF secreting cells significantly enhanced cellular proliferation in the hippocampal dentate gyrus (DG). The number of neuronal precursors in VEGF microcapsules-treated AßPP/PS1 mice was also greater, and this effect remains after 6 months. We also confirmed that encapsulated VEGF secreting cells also stimulated angiogenesis in the cerebral cortex and hippocampal dentate gyrus. In addition, we found that VEGF-microcapsule treatment was associated with a depressed expression and activity of acetylcholinesterase in the hippocampus of AßPP/PS1 mice, a similar pattern as first-line medications for the treatment of AD. We conclude that stereologically-implanted VEGF-microcapsules exert an acute and long-standing neurotrophic effects, and could be utilized to improve potential therapies to control the progression of AD.