A delay in vesicle endocytosis by a C-terminal fragment of N-cadherin enhances Aß synaptotoxicity.

Synaptotoxic Aß oligomers are thought to play a major role in the early pathology of Alzheimer´s disease (AD). However, the molecular mechanisms involved in Aß-induced synaptic dysfunction and synapse damage remain largely unclear. Previously, Aß synaptotoxicity has been reported to be enhanced by increased levels of a C-terminal fragment of the synaptic adhesion molecule N-cadherin that is generated by proteolytic shedding of the extracellular domains [1]. To address the molecular mechanisms involved in this process, we have now studied the functional synaptic changes induced by C-terminal fragments (CTF1) of synaptic adhesion proteins. We used synaptophysin-pHluorin (SypHy) fluorescence imaging to monitor synaptic vesicle exo- and endocytosis in cultures of mouse cortical neurons. We increased the levels of C-terminal fragments of synaptic adhesion proteins by pharmacologically inhibiting γ-secretase, which further degrades CTF1 fragments. We found that this intervention caused a delay in synaptic vesicle endocytosis. A similar effect was induced by overexpression of N-cadherin CTF1, but not by overexpression of Neurexin3ß CTF1. Based on these observations, we further studied whether directly modulating synaptic vesicle endocytosis enhances Aß synaptotoxicity. We pharmacologically induced a delayed synaptic vesicle endocytosis by a low concentration of the endocytosis inhibitor dynasore. This treatment enhanced synaptoxicity of Aß oligomers as indicated by a reduced frequency of miniature postsynaptic currents. In conclusion, we propose that delayed endocytosis results in prolonged exposure of synaptic vesicle membranes to the extracellular space, thus enabling enhanced vesicle membrane binding of Aß oligomers. This might in turn promote the endocytic uptake of toxic Aß oligomers and might thus play an important role in intracellular Aß-mediated synaptotoxicity in AD.

Identification of truncated C-terminal fragments of the Alzheimer's disease amyloid protein precursor derived from sequential proteolytic pathways.

Recent evidence supports the emerging hypothesis that the amyloid-ß precursor protein C-terminal fragments (APP-CTFs) and dysregulations in both their qualitative and quantitative productions may actively and directly contribute to the neuronal toxicity in early phases of Alzheimer's disease (AD). These new findings revealed the urgent needs and gaps in better understanding the metabolism and full spectrum of APP-CTFs. In this study, we characterized by mass spectrometry the full patterns of APP-CTFs in different cell types and in the brain of an AD APPPS1 mouse model. In these systems, we first discovered a series of 71-80 amino acids long N-terminally truncated APP-CTFs of unknown functions. We next demonstrated that these N-terminally truncated APP-CTFs are sequentially produced by the proteolytic processing of APP-C80, by an as yet unidentified protease. Finally, these N-terminally truncated APP-CTFs are likely protein substrates recognized and processed by the γ-secretase complex, leading to the production of N-terminally truncated Aß peptides. Together, our findings provide new insights into the metabolism of APP and offer potential new strategies to modulate the production of toxic Aß peptides in AD.

The APMAP interactome reveals new modulators of APP processing and beta-amyloid production that are altered in Alzheimer's disease.

The adipocyte plasma membrane-associated protein APMAP is expressed in the brain where it associates with γ-secretase, a protease responsible for the generation of the amyloid-ß peptides (Aß) implicated in the pathogenesis of Alzheimer's disease (AD). In this study, behavioral investigations revealed spatial learning and memory deficiencies in our newly generated mouse line lacking the protein APMAP. In a mouse model of AD, the constitutive deletion of APMAP worsened the spatial memory phenotype and led to increased Aß production and deposition into senile plaques. To investigate at the molecular level the neurobiological functions of APMAP (memory and Aß formation) and a possible link with the pathological hallmarks of AD (memory impairment and Aß pathology), we next developed a procedure for the high-grade purification of cellular APMAP protein complexes. The biochemical characterization of these complexes revealed a series of new APMAP interactomers. Among these, the heat shock protein HSPA1A and the cation-dependent mannose-6-phosphate receptor (CD-M6PR) negatively regulated APP processing and Aß production, while clusterin, calnexin, arginase-1, PTGFRN and the cation-independent mannose-6-phosphate receptor (CI-M6PR/IGF2R) positively regulated APP and Aß production. Several of the newly identified APMAP interactomers contribute to the autophagy-lysosome system, further supporting an emergent agreement that this pathway can modulate APP metabolism and Aß generation. Importantly, we have also demonstrated increased alternative splicing of APMAP and lowered levels of the Aß controllers HSPA1A and CD-M6PR in human brains from neuropathologically verified AD cases.

The metalloprotease ADAMTS4 generates N-truncated Aß4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.

Induction of Amyloid-ß42 Production by Fipronil and Other Pyrazole Insecticides.

Generation of amyloid-ß peptides (Aßs) by proteolytic cleavage of the amyloid-ß protein precursor (AßPP), especially increased production of Aß42/Aß43 over Aß40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aß production originates from specific mutations of AßPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aßs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aß42/Aß43 over Aß40 and shorter Aßs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aß42/Aß43 production. Nine pyrazole insecticides were found to induce a ß- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aß42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aßs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AßPP by highly purified γ-secretase toward Aß42/Aß43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aß42/Aß43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aß42/Aß43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

The Alzheimer's Disease γ-Secretase Generates Higher 42:40 Ratios for ß-Amyloid Than for p3 Peptides.

Alzheimer's disease is characterized by intracerebral deposition of ß-amyloid (Aß). While Aß40 is the most abundant form, neurotoxicity is mainly mediated by Aß42. Sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases gives rise to full-length Aß (Aß1-x) and N-terminally truncated Aß' (Aß11-x) whereas cleavage by α- and γ-secretases leads to the shorter p3 peptides (Aß17-x). We uncovered significantly higher ratios of 42- versus 40-ending variants for Aß and Aß' than for p3 secreted by mouse neurons and human induced pluripotent stem cell (iPSC)-derived neurons or produced in a cell-free γ-secretase assay with recombinant APP-CTFs. The 42:40 ratio was highest for Aß', followed by Aß and then p3. Mass spectrometry analysis of APP intracellular domains revealed differential processing of APP-C83, APP-C89, and APP-C99 by γ-secretase already at the ε-cleavage stage. This mechanistic insight could aid in developing substrate-targeted modulators of APP-C99 processing to specifically lower the Aß42:Aß40 ratio without compromising γ-secretase function.

Zinc and Copper Differentially Modulate Amyloid Precursor Protein Processing by γ-Secretase and Amyloid-ß Peptide Production.

Recent evidence suggests involvement of biometal homeostasis in the pathological mechanisms in Alzheimer's disease (AD). For example, increased intracellular copper or zinc has been linked to a reduction in secreted levels of the AD-causing amyloid-ß peptide (Aß). However, little is known about whether these biometals modulate the generation of Aß. In the present study we demonstrate in both cell-free and cell-based assays that zinc and copper regulate Aß production by distinct molecular mechanisms affecting the processing by γ-secretase of its Aß precursor protein substrate APP-C99. We found that Zn2+ induces APP-C99 dimerization, which prevents its cleavage by γ-secretase and Aß production, with an IC50 value of 15 µm Importantly, at this concentration, Zn2+ also drastically raised the production of the aggregation-prone Aß43 found in the senile plaques of AD brains and elevated the Aß43:Aß40 ratio, a promising biomarker for neurotoxicity and AD. We further demonstrate that the APP-C99 histidine residues His-6, His-13, and His-14 control the Zn2+-dependent APP-C99 dimerization and inhibition of Aß production, whereas the increased Aß43:Aß40 ratio is substrate dimerization-independent and involves the known Zn2+ binding lysine Lys-28 residue that orientates the APP-C99 transmembrane domain within the lipid bilayer. Unlike zinc, copper inhibited Aß production by directly targeting the subunits presenilin and nicastrin in the γ-secretase complex. Altogether, our data demonstrate that zinc and copper differentially modulate Aß production. They further suggest that dimerization of APP-C99 or the specific targeting of individual residues regulating the production of the long, toxic Aß species, may offer two therapeutic strategies for preventing AD.

Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/γ-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by α- and γ-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. The AXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the γ-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest that AXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing of AXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells.

Shedding of neurexin 3ß ectodomain by ADAM10 releases a soluble fragment that affects the development of newborn neurons.

Neurexins are transmembrane synaptic cell adhesion molecules involved in the development and maturation of neuronal synapses. In the present study, we report that Nrxn3ß is processed by the metalloproteases ADAM10, ADAM17, and by the intramembrane-cleaving protease γ-secretase, producing secreted neurexin3ß (sNrxn3ß) and a single intracellular domain (Nrxn3ß-ICD). We further completed the full characterization of the sites at which Nrxn3ß is processed by these proteases. Supporting the physiological relevance of the Nrxn3ß processing, we demonstrate in vivo a significant effect of the secreted shedding product sNrxn3ß on the morphological development of adult newborn neurons in the mouse hippocampus. We show that sNrxn3ß produced by the cells of the dentate gyrus increases the spine density of newborn neurons whereas sNrxn3ß produced by the newborn neuron itself affects the number of its mossy fiber terminal extensions. These results support a pivotal role of sNrxn3ß in plasticity and network remodeling during neuronal development.

The lipidome associated with the γ-secretase complex is required for its integrity and activity.

γ-Secretase is a multi-subunit membrane protease complex that catalyses the final intramembrane cleavage of the ß-amyloid precursor protein (APP) during the neuronal production of amyloid-ß peptides (Aß), which are implicated as the causative agents of Alzheimer's disease (AD). In the present study, we report the reconstitution of a highly purified, active γ-secretase complex into proteoliposomes without exogenous lipids and provide the first direct evidence for the existence of a microenvironment of 53 molecular species from 11 major lipid classes specifically associated with the γ-secretase complex, including phosphatidylcholine and cholesterol. Importantly, we demonstrate that the pharmacological modulation of certain phospholipids abolishes both the integrity and the enzymatic activity of the intramembrane protease. Together, our findings highlight the importance of a specific lipid microenvironment for the structure and function of γ-secretase.

The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-ß peptides.

Known γ-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer's disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of γ-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (Aß) and that have better pharmacokinetics and an improved safety profile, we completed a screen of ~400 natural products by using cell-based and cell-free γ-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA- (Food and Drug Administration)-approved drug, to be a direct inhibitor of γ-secretase. Micromolar concentrations of DHEC substantially reduced Aß levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 µM, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of γ-secretase but also a candidate for drug repositioning in Alzheimer's disease.

Production of active glycosylation-deficient γ-secretase complex for crystallization studies.

Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD.

Detection of Alzheimer's disease amyloid-beta plaque deposition by deep brain impedance profiling.

OBJECTIVE: Alzheimer disease (AD) is the most common form of neurodegenerative disease in elderly people. Toxic brain amyloid-beta (Aß) aggregates and ensuing cell death are believed to play a central role in the pathogenesis of the disease. In this study, we investigated if we could monitor the presence of these aggregates by performing in situ electrical impedance spectroscopy measurements in AD model mice brains. APPROACH: In this study, electrical impedance spectroscopy measurements were performed post-mortem in APPPS1 transgenic mice brains. This transgenic model is commonly used to study amyloidogenesis, a pathological hallmark of AD. We used flexible probes with embedded micrometric electrodes array to demonstrate the feasibility of detecting senile plaques composed of Aß peptides by localized impedance measurements. MAIN RESULTS: We particularly focused on deep brain structures, such as the hippocampus. Ex vivo experiments using brains from young and old APPPS1 mice lead us to show that impedance measurements clearly correlate with the percentage of Aß plaque load in the brain tissues. We could monitor the effects of aging in the AD APPPS1 mice model. SIGNIFICANCE: We demonstrated that a localized electrical impedance measurement constitutes a valuable technique to monitor the presence of Aß-plaques, which is complementary with existing imaging techniques. This method does not require prior Aß staining, precluding the risk of variations in tissue uptake of dyes or tracers, and consequently ensuring reproducible data collection.

Novel therapeutic strategy for neurodegeneration by blocking Aß seeding mediated aggregation in models of Alzheimer's disease.

Aß accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aß nucleated polymerization is essential for Aß fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aß seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aß seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aß-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aß42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aß seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.

A compressible scaffold for minimally invasive delivery of large intact neuronal networks.

Millimeter to centimeter-sized injectable neural scaffolds based on macroporous cryogels are presented. The polymer-scaffolds are made from alginate and carboxymethyl-cellulose by a novel simple one-pot cryosynthesis. They allow surgical sterility by means of autoclaving, and present native laminin as an attachment motive for neural adhesion and neurite development. They are designed to protect an extended, living neuronal network during compression to a small fraction of the original volume in order to enable minimally invasive delivery. The scaffolds behave as a mechanical meta-material: they are soft at the macroscopic scale, enabling injection through narrow-bore tubing and potentially good cellular scaffold integration in soft target tissues such as the brain. At the same time, the scaffold material has a high local Young modulus, allowing protection of the neuronal network during injection. Based on macroscopic and nanomechanical characterization, the generic geometrical and mechanical design rules are presented, enabling macroporous cellular scaffold injectability.

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aß production in the brain.

The deposition of amyloid-beta (Aß) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aß is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aß production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aß generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aß. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aß production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aß production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.

Inactivation of brain Cofilin-1 by age, Alzheimer's disease and γ-secretase.

Rapid remodeling of the actin cytoskeleton in the pre- and/or post-synaptic compartments is responsible for the regulation of neuronal plasticity,which is an important process for learning and memory. Cofilin1 plays an essential role in these processes and a dysregulation of its activity was associated with the cognitive decline observed during normal aging and Alzheimer's disease (AD). To understand the mechanism(s) regulating Cofilin1 activity we evaluated changes occurring with regard to Cofilin1 and its up-stream regulators Lim kinase-1 (LIMK1) and Slingshot phosphatase-1 (SSH1) in (i) human AD brain, (ii) 1-, 4-, and 10-months old APP/PS1 mice, (iii) wildtype 3-, 8-, 12-, 18- and 26-months old mice, as well as in cellular models including (iv) mouse primary cortical neurons (PCNs, cultured for 5, 10, 15 and 20 days in vitro) and (v) mouse embryonic fibroblasts (MEF). Interestingly,we found an increased Cofilin1 phosphorylation/inactivation with age and AD pathology, both in vivo and in vitro. These changes were associated with a major inactivation of SSH1. Interestingly, inhibition of ã-secretase activity with Compound-E (10 ìM) prevented Cofilin1 phosphorylation/inactivation through an increase of SSH1 activity in PCNs. Similarly, MEF cells double knock-out for ã-secretase catalytic subunits presenilin-1 and -2(MEFDKO) showed a strong decrease of both Cofilin1 and SSH1 phosphorylation,which were rescued by the over expression of human ã-secretase. Together, these results shed new light in understanding the molecular mechanisms promoting Cofilin1 dysregulation, both during aging and AD. They further have the potential to impact the development of therapies to safely treat AD.

Accurate resistivity mouse brain mapping using microelectrode arrays.

Electrical impedance spectroscopy measurements were performed in post-mortem mice brains using a flexible probe with an embedded micrometric electrode array. Combined with a peak resistance frequency method this allowed obtaining intrinsic resistivity values of brain tissues and structures with submillimetric resolution. Reproducible resistivity measurements are reported, which allows the resistivity in the cortex, ventricle, fiber tracts, thalamus and basal ganglia to be differentiated. Measurements of brain slices revealed resistivity profiles correlated with the local density of cell bodies hence allowing to discriminate between the different cortical layers. Finally, impedance measurements were performed on a model of cauterized mouse brain evidencing the possibility to measure the spatial extent and the degree of the tissue denaturation due to the cauterization.

Ferritin H gene deletion in the choroid plexus and forebrain results in hydrocephalus.

Ferritin H, the major iron storage protein, has essential functions in early embryonic development as well as in adult liver and intestine. To address the question whether ferritin H has similarly essential functions in the brain we used the Cre/loxP system to generate mice with a forebrain-specific inactivation of the ferritin H gene. Ferritin H deficiency in most cells of the forebrain including cells of the choroid plexus caused accumulation of cerebrospinal fluid in the lateral ventricles and the subarachnoid space. Brain tissue iron content was unchanged.