Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.

Epigenetic repression of Wnt receptors in AD: a role for Sirtuin2-induced H4K16ac deacetylation of Frizzled1 and Frizzled7 promoters.

Growing evidence supports a role for deficient Wnt signalling in Alzheimer's disease (AD). First, the Wnt antagonist DKK1 is elevated in AD brains and is required for amyloid-ß-induced synapse loss. Second, LRP6 Wnt co-receptor is required for synapse integrity and three variants of this receptor are linked to late-onset AD. However, the expression/role of other Wnt signalling components remain poorly explored in AD. Wnt receptors Frizzled1 (Fzd1), Fzd5, Fzd7 and Fzd9 are of interest due to their role in synapse formation/plasticity. Our analyses showed reduced FZD1 and FZD7 mRNA levels in the hippocampus of human early AD stages and in the hAPPNLGF/NLGF mouse model. This transcriptional downregulation was accompanied by reduced levels of the pro-transcriptional histone mark H4K16ac and a concomitant increase of its deacetylase Sirt2 at Fzd1 and Fzd7 promoters in AD. In vitro and in vivo inhibition of Sirt2 rescued Fzd1 and Fzd7 mRNA expression and H4K16ac levels at their promoters. In addition, we showed that Sirt2 recruitment to Fzd1 and Fzd7 promoters is dependent on FoxO1 activity in AD, thus acting as a co-repressor. Finally, we found reduced levels of SIRT2 inhibitory phosphorylation in nuclear samples from human early AD stages with a concomitant increase in the SIRT2 phosphatase PP2C. This results in hyperactive nuclear Sirt2 and favours Fzd1 and Fzd7 repression in AD. Collectively, our findings define a novel role for nuclear hyperactivated SIRT2 in repressing Fzd1 and Fzd7 expression via H4K16ac deacetylation in AD. We propose SIRT2 as an attractive target to ameliorate AD pathology.

Nucleus-cytoplasm cross-talk in the aging brain.

Aging is a primary risk factor for fatal neurodegenerative disorders, yet the mechanisms underlying physiological healthy aging and pathological aging, and how these mechanisms can divert one scenario to the other, are not completely understood. In recent years, reports indicate that alterations in nucleocytoplasmic transport may be a hallmark of both healthy and pathological aging. In this review, I summarize recent evidence supporting this information, specifically focusing on the association between the nucleocytoplasmic transport and aging of the brain, indicating both common and case-specific mechanisms and their interplay, and pointing out alterations of these mechanisms as regulatory "switches" for the fate of the aging brain. Importantly, some of these alterations are intervenable druggable targets, paving the way to a future pharmacotherapeutic intervention.

Glycogen synthase kinase-3ß regulates fractalkine production by altering its trafficking from Golgi to plasma membrane: implications for Alzheimer's disease.

Glycogen synthase kinase-3ß (GSK-3ß) is a serine-threonine kinase implicated in multiple processes and signaling pathways. Its dysregulation is associated with different pathological conditions including Alzheimer's disease (AD). Here we demonstrate how changes in GSK-3ß activity and/or levels regulate the production and subsequent secretion of fractalkine, a chemokine involved in the immune response that has been linked to AD and to other different neurological disorders. Treatment of primary cultured neurons with GSK-3ß inhibitors such as lithium and AR-A014418 decreased full-length fractalkine in total cell extracts. Opposite effects were observed after neuron transduction with a lentiviral vector overexpressing the kinase. Biotinylation assays showed that those changes mainly affect the plasma membrane-associated form of the protein, an observation that positively correlates with changes in the levels of its soluble form. These effects were confirmed in lithium-treated wild type (wt) mice and in GSK-3ß transgenic animals, as well as in brain samples from AD patients, evident as age-dependent (animals) or Braak stage dependent changes (humans) in both the membrane-bound and the soluble forms of the protein. Further immunohistochemical analyses demonstrated how GSK-3ß exerts these effects by affecting the trafficking of the chemokine from the Golgi to the plasma membrane, in different and opposite ways when the levels/activity of the kinase are increased or decreased. This work provides for the first time a mechanism linking GSK-3ß and fractalkine both in vitro and in vivo, with important implications for neurological disorders and especially for AD, in which levels of this chemokine might be useful as a diagnostic tool.

Aged PrP null mice show defective processing of neuregulins in the peripheral nervous system.

A prion, a protease-resistant conformer of the cellular prion protein (PrP(C)), is the causative agent of transmissible spongiform encephalopathies or prion diseases. While this property is well established for the aberrantly folded protein, the physiological function of PrP(C) remains elusive. Among different putative functions, the non-pathogenic protein isoform PrP(C) is involved in several cellular processes. Here, we show that PrP(C) regulates the cleavage of neuregulin-1 proteins (NRG1). Neuregulins provide key axonal signals that regulate several processes, including glial cells proliferation, survival and myelination. Interestingly, mice devoid of PrP(C) (Prnp°/°) were recently shown to have a late-onset demyelinating disease in the peripheral nervous system (PNS) but not in the central nervous system (CNS). We found that NRG1 processing is developmentally regulated in the PNS and, by comparing wildtype and Prnp°/° mice, that PrP(C) influences NRG1 processing in old, but not in young, animals. In addition, we found that also the processing of neuregulin-3, another neuregulin family member, is altered in the PNS of Prnp°/° mice. These differences in neuregulin proteins processing are not paralleled in the CNS, thus suggesting a different cellular function for PrP(C) between the CNS and the PNS.

Design of a Potent, Selective, and Brain-Penetrant Inhibitor of Wnt-Deactivating Enzyme Notum by Optimization of a Crystallographic Fragment Hit.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

Developmental influence of the cellular prion protein on the gene expression profile in mouse hippocampus.

The conversion of the cellular prion protein (PrP(C)) to an abnormal and protease-resistant isoform is the key event in prion diseases. Mice lacking PrP(C) are resistant to prion infection, and downregulation of PrP(C) during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrP(C) during prion diseases. However, the silencing of a protein that is widely expressed throughout the central nervous system could be detrimental to brain homeostasis. The physiological role of PrP(C) remains still unclear, but several putative functions (e.g., neuronal development and maintenance) have been proposed. To assess the influence of PrP(C) on gene expression profile in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus at two different developmental stages: newborn (4.5-day-old) and adult (3-mo-old) mice, both from wild-type and Prnp(0/0) animals. Comparing the different datasets allowed us to identify "commonly" co-regulated genes and "uniquely" deregulated genes during postnatal development. The absence of PrP(C) affected several biological pathways, the most representative being cell signaling, cell-cell communication and transduction processes, calcium homeostasis, nervous system development, synaptic transmission, and cell adhesion. However, there was only a moderate alteration of the gene expression profile in our animal models. PrP(C) deficiency did not lead to a dramatic alteration of gene expression profile and produced moderately altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrP(C) levels as therapeutic approach to prion diseases.

Small-molecule inhibitors of carboxylesterase Notum.

Notum has recently been identified as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group from Wnt proteins. There are emerging reports that Notum plays a role in human disease, with published data suggesting that targeting Notum could represent a new therapeutic approach for treating cancer, osteoporosis and neurodegenerative disorders. Complementary hit-finding strategies have been applied with successful approaches that include high-throughput screening, activity-based protein profiling, screening of fragment libraries and virtual screening campaigns. Structural studies are accelerating the discovery of new inhibitors of Notum. Three fit-for-purpose examples are LP-922056, ABC99 and ARUK3001185. The application of these small-molecule inhibitors is helping to further advance an understanding of the role Notum plays in human disease.

E3 ligase mahogunin (MGRN1) influences amyloid precursor protein maturation and secretion.

Altered processing of the Amyloid Precursor Protein (APP) is a well-recognized central pathogenic mechanism in Alzheimer's Disease (AD), and regulation of APP processing is a major focus of research in the AD field. However, how age-associated cellular and molecular changes contribute to changes in the amyloidogenic processing of APP have not been extensively clarified so far. We here provide evidence that the processing of APP is influenced by the e3 ubiquitin ligase Mahogunin (MGRN1), a neuroprotective molecule whose levels decrease with aging. Specifically, the expression of MGRN1 inhibits the maturation of APP by sequestering it in the secretory pathway. This sequestration significantly delayed the proteolytic processing of APP, resulting in a reduced ß-amyloid (Aß) peptide release into the extracellular environment. Accordingly, a reduction of MGRN1 levels in hippocampal neurons, as it occurs during physiological aging, leads to an increased Aß40 and Aß42 release. We therefore propose that age contributes to the amyloidogenic processing of APP by altering its intracellular trafficking along the secretory pathway due in part to the down-regulation of MGRN1.

Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons.

It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons.

Neurodevelopmental expression and localization of the cellular prion protein in the central nervous system of the mouse.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by PrP(Sc), or prion, an abnormally folded form of the cellular prion protein (PrP(C)). The abundant expression of PrP(C) in the central nervous system (CNS) is a requirement for prion replication, yet despite years of intensive research the physiological function of PrP(C) still remains unclear. Several routes of investigation point out a potential role for PrP(C) in axon growth and neuronal development. Thus, we undertook a detailed analysis of the spatial and temporal expression of PrP(C) during mouse CNS development. Our findings show regional differences of the expression of PrP, with some specific white matter structures showing the earliest and highest expression of PrP(C). Indeed, all these regions are part of the thalamolimbic neurocircuitry, suggesting a potential role of PrP(C) in the development and functioning of this specific brain system.

Prion protein paralog doppel protein interacts with alpha-2-macroglobulin: a plausible mechanism for doppel-mediated neurodegeneration.

Doppel protein (Dpl) is a paralog of the cellular form of the prion protein (PrP(C)), together sharing common structural and biochemical properties. Unlike PrP(C), which is abundantly expressed throughout the central nervous system (CNS), Dpl protein expression is not detectable in the CNS. Interestingly, its ectopic expression in the brain elicits neurodegeneration in transgenic mice. Here, by combining native isoelectric focusing plus non-denaturing polyacrylamide gel electrophoresis and mass spectrometry analysis, we identified two Dpl binding partners: rat alpha-1-inhibitor-3 (alpha(1)I(3)) and, by sequence homology, alpha-2-macroglobulin (alpha(2)M), two known plasma metalloproteinase inhibitors. Biochemical investigations excluded the direct interaction of PrP(C) with either alpha(1)I(3) or alpha(2)M. Nevertheless, enzyme-linked immunosorbent assays and surface plasmon resonance experiments revealed a high affinity binding occurring between PrP(C) and Dpl. In light of these findings, we suggest a mechanism for Dpl-induced neurodegeneration in mice expressing Dpl ectopically in the brain, linked to a withdrawal of natural inhibitors of metalloproteinase such as alpha(2)M. Interestingly, alpha(2)M has been proven to be a susceptibility factor in Alzheimer's disease, and as our findings imply, it may also play a relevant role in other neurodegenerative disorders, including prion diseases.