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γ-Secretase is an intramembrane cleaving protease involved in the generation of the Alzheimer's disease (AD)-associated amyloid β peptide (Aβ). γ-Secretase is ubiquitously expressed in different organs, and also in different cell types of the human brain. Besides the involvement in the proteolytic generation of Aβ from the amyloid precursor protein, γ-secretase cleaves many additional protein substrates, suggesting pleiotropic functions under physiological and pathophysiological conditions. Microglia exert important functions during brain development and homeostasis in adulthood, and accumulating evidence indicates that microglia and neuroinflammatory processes contribute to the pathogenesis of neurodegenerative diseases. Recent studies demonstrate functional implications of γ-secretase in microglia, suggesting that alterations in γ-secretase activity could contribute to AD pathogenesis by modulation of microglia and related neuroinflammatory processes during neurodegeneration. In this review, we discuss the involvement of γ-secretase in the regulation of microglial functions, and the potential relevance of these processes under physiological and pathophysiological conditions. This article is part of the series "Beyond Amyloid".

A combination of biochemical, genetic, and imaging approaches is used to show that phosphorylation and lipidation exhibit a complex interplay in sorting of GAP43. Palmitoylation tags GAP43 for global sorting by inducing piggybacking on exocytic vesicles, whereas phosphorylation locally regulates plasma membrane targeting of palmitoylated GAP43., Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43.

Triggering receptor expressed on myeloid cells-2 (TREM2) and its signaling adaptor protein TYROBP/DAP12 play important roles in signal transduction in dendritic cells, osteoclasts, tissue macrophages, and microglia. Recently, TREM2 variants have been shown to be linked to late onset Alzheimer disease. Here, we demonstrate that TREM2 undergoes sequential proteolytic processing by ectodomain shedding and intramembrane proteolysis. The C-terminal fragment (CTF) of TREM2 generated by ectodomain shedding is cleaved by γ-secretase. Importantly, pharmacologic and genetic γ-secretase inhibition resulted in accumulation of TREM2 CTF at the plasma membrane that also interacts with the signaling adaptor protein DAP12. Thus, the accumulated TREM2 CTF thereby might limit the interaction of DAP12 with the functional full-length receptor, resulting in decreased DAP12 phosphorylation and impaired metabolism of phosphatidylinositol 4,5-bisphosphate. Together, these data demonstrate γ-secretase-mediated intramembranous proteolysis of TREM2 and functionally link two Alzheimer disease-associated proteins in one signaling pathway.

Understanding the developmental steps shaping the formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers, is critical. Wnt morphogens are key players in the formation of this specialized peripheral synapse. Yet, the individual and collaborative functions of Wnts as well as their downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain of function studies in culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. In contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR clustering and mRNA downstream activation of the β-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the Planar Cell Polarity (PCP) pathway, which accumulates at embryonic NMJ. Moreover, mice bearing a Vangl2 loss of function mutation (looptail) exhibit a decreased number of AChR clusters and overgrowth of motor axons bypassing AChR clusters. Taken together, our results provide genetic and biochemical evidences that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.

Alexander disease is a fatal neurodegenerative disease caused by mutations in the astrocyte intermediate filament glial fibrillary acidic protein (GFAP). The disease is characterized by elevated levels of GFAP and the formation of protein aggregates, known as Rosenthal fibers, within astrocytes. Lithium has previously been shown to decrease protein aggregates by increasing the autophagy pathway for protein degradation. In addition, lithium has also been reported to decrease activation of the transcription factor STAT3, which is a regulator of GFAP transcription and astrogliogenesis. Here we tested whether lithium treatment would decrease levels of GFAP in a mouse model of Alexander disease. Mice with the Gfap-R236H point mutation were fed lithium food pellets for 4 to 8 weeks. Four weeks of treatment with LiCl at 0.5% in food pellets decreased GFAP protein and transcripts in several brain regions, although with mild side effects and some mortality. Extending the duration of treatment to 8 weeks resulted in higher mortality, and again with a decrease in GFAP in the surviving animals. Indicators of autophagy, such as LC3, were not increased, suggesting that lithium may decrease levels of GFAP through other pathways. Lithium reduced the levels of phosphorylated STAT3, suggesting this as one pathway mediating the effects on GFAP. In conclusion, lithium has the potential to decrease GFAP levels in Alexander disease, but with a narrow therapeutic window separating efficacy and toxicity.

Microglia are resident immune cells in the brain and exert important functions in the regulation of inflammatory processes during infection or cellular damage. Upon activation, microglia undergo complex morphological and functional transitions, including increased motility, phagocytosis and cytokine secretion. Recent findings indicate that exosomes, small vesicles that derive from fusion of multivesicular bodies with the plasma membrane, are involved in secretion of certain cytokines. The presence of specific receptors on the surface of microglia suggests communication with neurons by neurotransmitters. Here, we demonstrate expression of serotonin receptors, including 5-HT2a,b and 5-HT4 in microglial cells and their functional involvement in the modulation of exosome release by serotonin. Our data demonstrate the involvement of cAMP and Ca(2+) dependent signaling pathways in the regulation of exosome secretion. Co-culture of microglia with embryonic stem cell-derived serotonergic neurons further demonstrated functional signaling between neurons and microglia. Together, these data provide evidence for neurotransmitter-dependent signaling pathways in microglial cells that regulate exosome release.

BACKGROUND: Serotonin (5-HT) is well known as widely distributed modulator of developmental processes in both vertebrates and invertebrates. It is also the earliest neurotransmitter to appear during neuronal development. In aquatic invertebrates, which have larvae in their life cycle, 5-HT is involved in regulation of stages transition including larval metamorphosis and settlement. However, molecular and cellular mechanisms underlying developmental transition in aquatic invertebrate species are yet poorly understood. Earlier we demonstrated that in larvae of freshwater molluscs and marine polychaetes, endogenous 5-HT released from the neurons of the apical sensory organ (ASO) in response to external stimuli retarded larval development at premetamorphic stages, and accelerated it at metamorphic stages. Here we used a freshwater snail Helisoma trivolvis to study molecular mechanisms underlying these dual developmental effects of 5-HT. RESULTS: Larval development of H. trivolvis includes transition from premetamorphic to metamorphic stages and shares the main features of metamorphosis with free-swimming aquatic larvae. Three types of 5-HT receptors (5-HT1-, 5-HT4- and 5-HT7-like) are functionally active at premetamorphic (trochophore, veliger) and metamorphic (veliconcha) stages, and expression patterns of these receptors and respective G proteins undergo coordinated changes during development. Stimulation of these receptors modulated cAMP-dependent regulation of cell divisions. Expression of 5-HT4- and 5-HT7-like receptors and their downstream Gs protein was down-regulated during the transition of pre- to metamorphic stage, while expression of 5-HT1 -like receptor and its downstream Gi protein was upregulated. In accordance with relative amount of these receptors, stimulation of 5-HTRs at premetamorphic stages induces developmental retardation, while their stimulation at metamorphic stages induces developmental acceleration. CONCLUSIONS: We present a novel molecular mechanism that underlies stage-specific changes in developmental tempo of H. trivolvis larvae in response to endogenous 5-HT produced by the neurons of the ASO. We suggest that consecutive changes in expression patterns of different receptors and their downstream partners in the course of larval development represent the molecular base of larval transition from premetamorphic (non-competent) to metamorphic (competent) state. peerReviewed

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.

Population-based studies demonstrated that statins might decrease the risk of developing Alzheimer’s disease (AD). Statins inhibit the 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase and thereby de novo synthesis of cholesterol. Cell culture and animal studies indicated that cholesterol affects the proteolytic processing of the amyloid precursor protein and the generation of amyloid-β (Aβ). Recently, we have demonstrated that statins can also stimulate the degradation of Aβ. The statin-induced clearance of Aβ could be attributed to increased release of the insulin-degrading enzyme (IDE) via an exosome-related unconventional secretory pathway. Interestingly, this statin-induced secretion of exosome-associated IDE was independent of cellular cholesterol concentrations, but rather caused by impairment of isoprenoid biosynthesis and protein prenylation. We further identified a new hexapeptide sequence in the C-terminal region of IDE, named the SlyX motif that is critically involved in IDE secretion. Taken these findings together, the increased clearance of Aβ by stimulated secretion of IDE might contribute to the protective effects of statins against AD.

Insulin-degrading enzyme (IDE) is a Zn(2+) metalloprotease with a characteristic inverted catalytic motif. IDE is ubiquitously expressed and degrades peptide substrates including insulin, endorphin, and the amyloid-β peptide. Although IDE is mainly expressed in the cytosol, it can also be found on the cell surface and in secreted form in extracellular fluids. As IDE lacks a characteristic signal sequence that targets the protein to the classical secretory pathway, release of the enzyme involves non-conventional mechanisms. However, functional domains of IDE involved in its secretion remain elusive. By bioinformatical, biochemical, and cell biological methods, we identified a novel amino acid motif ((853)EKPPHY(858)) close to the C terminus of IDE and characterized its function in the non-conventional secretion of the protein. Because of its close homology to an amino acid sequence found in bacterial proteins belonging to the SlyX family, we propose to call it the SlyX motif. Mutagenesis revealed that deletion of this motif strongly decreased the release of IDE, whereas deletion of a potential microbody-targeting signal at the extreme C terminus had little effect on secretion. The combined data indicate that the non-conventional secretion of IDE is regulated by the newly identified SlyX motif.

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β-peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.

The regulation of larval development by starved adults occurs in both freshwater snails, Helisoma trivolvis and marine polychaetes, Platynereis dumerilii. Serotonin (5-HT) links this environmental signal which is detected by larval apical sensory neurons to changes in larval development. A profile of the stage-dependent expression of 5-HT receptors and coupled G proteins is essential in this regulatory mechanism. The final effect on development depends on the modulation of the activity of the larval digestive system.

In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine(1A) receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged alpha(i3) subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G(i)-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Galpha(i) protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca(2+) concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Galpha(i3)-CFP relocation and changes in [Ca(2+)](i) upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.

The mouse 5-hydroxytryptamine4a (5-HT4a) receptor is an unusual member of the G protein-coupled receptor superfamily because it possesses two separate carboxyl-terminal palmitoylation sites, which may allow the receptor to adopt different conformations in an agonist-dependent manner (J Biol Chem 277:2534-2546, 2002). By targeted mutation of the proximal (Cys-328/329) or distal (Cys-386) palmitoylation sites, or a combination of both, we generated 5-HT4a receptor variants with distinct functional characteristics. In this study, we showed that upon 5-HT stimulation, the 5-HT4a receptor undergoes rapid (t(1/2) approximately 2 min) and dose-dependent (EC50 approximately 180 nM) phosphorylation on serine residues by a staurosporine-insensitive receptor kinase. Overexpression of GRK2 significantly reduced the receptor-promoted cAMP formation. The Cys328/329-Ser mutant, which is constitutively active in the absence of ligand, exhibited enhanced receptor phosphorylation under both basal and agonist-stimulated conditions and was more effectively desensitized and internalized via a beta-arrestin-2 mediated pathway compared with the wild-type 5-HT4a. In contrast, G protein activation, phosphorylation, desensitization, and internalization of the other palmitoylation-deficient receptor mutants were affected differently. These findings suggest that palmitoylation plays an important role in modulating 5-HT4a receptor functions and that G protein activation, phosphorylation, desensitization, and internalization depend on the different receptor conformations.

Rare coding variants in the triggering receptor expressed on myeloid cells‐2 (TREM2) gene have been associated with Alzheimer disease (AD) and homozygous TREM2 loss‐of‐function variants have been reported in families with monogenic frontotemporal‐like dementia with/without bone abnormalities. In a whole‐exome sequencing study of a family with probable AD‐type dementia without pathogenic variants in known autosomal dominant dementia disease genes and negative for the apolipoprotein E (APOE) ε4 allele, we identified an extremely rare TREM2 coding variant, that is, a glycine‐to‐tryptophan substitution at amino acid position 145 (NM_018965.3:c.433G>T/p.[Gly145Trp]). This alteration is found in only 1 of 251,150 control alleles in gnomAD. It was present in both severely affected as well as in another putatively affected and one 61 years old as yet unaffected family member suggesting incomplete penetrance and/or a variable age of onset. Gly145 maps to an intrinsically disordered region (IDR) of TREM2 between the immunoglobulin‐like and transmembrane domain. Subsequent cellular studies showed that the variant led to IDR shortening and structural changes of the mutant protein resulting in an impairment of cellular responses upon receptor activation. Our results, suggest that a p.(Gly145Trp)‐induced structural disturbance and functional impairment of TREM2 may contribute to the pathogenesis of an AD‐like form of dementia.

Background Triggering receptor expressed on myeloid cells-2 (TREM2) exerts important functions in the regulation of monocytes, like dendritic cells, osteoclasts, tissue macrophages, and microglia. Mutations in TREM2 are associated with several diseases, including Nasu-Hakola disease, frontotemporal dementia, and Alzheimer’s disease (AD). TREM2 undergoes sequential proteolytic processing by ectodomain shedding and intramembrane proteolysis. Findings We show that inhibition of γ-secretase-dependent cleavage of the TREM2 C-terminal fragment in cellular membranes interferes with TREM2-dependent signaling and cellular function. Inhibition of γ-secretase decreases membrane-proximal signaling and intracellular Ca2+ response. Decreased signaling alters morphological changes and phagocytic activity of cells upon selective stimulation of TREM2. Conclusions The data demonstrate the importance of γ-secretase-dependent intramembrane processing in TREM2-mediated signaling and, thus, a functional relation of two AD-associated proteins. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0479-9) contains supplementary material, which is available to authorized users.