Search

Aim We aimed to investigate the impact of microglial activity and microglial FDG uptake on metabolic connectivity, since microglial activation states determine FDG–PET alterations. Metabolic connectivity refers to a concept of interacting metabolic brain regions and receives growing interest in approaching complex cerebral metabolic networks in neurodegenerative diseases. However, underlying sources of metabolic connectivity remain to be elucidated. Materials and methods We analyzed metabolic networks measured by interregional correlation coefficients (ICCs) of FDG–PET scans in WT mice and in mice with mutations in progranulin (Grn) or triggering receptor expressed on myeloid cells 2 (Trem2) knockouts (−/−) as well as in double mutant Grn−/−/Trem2−/− mice. We selected those rodent models as they represent opposite microglial signatures with disease associated microglia in Grn−/− mice and microglia locked in a homeostatic state in Trem2−/− mice; however, both resulting in lower glucose uptake of the brain. The direct influence of microglia on metabolic networks was further determined by microglia depletion using a CSF1R inhibitor in WT mice at two different ages. Within maps of global mean scaled regional FDG uptake, 24 pre-established volumes of interest were applied and assigned to either cortical or subcortical networks. ICCs of all region pairs were calculated and z-transformed prior to group comparisons. FDG uptake of neurons, microglia, and astrocytes was determined in Grn−/− and WT mice via assessment of single cell tracer uptake (scRadiotracing). Results Microglia depletion by CSF1R inhibition resulted in a strong decrease of metabolic connectivity defined by decrease of mean cortical ICCs in WT mice at both ages studied (6–7 m; p = 0.0148, 9–10 m; p = 0.0191), when compared to vehicle-treated age-matched WT mice. Grn−/−, Trem2−/− and Grn−/−/Trem2−/− mice all displayed reduced FDG–PET signals when compared to WT mice. However, when analyzing metabolic networks, a distinct increase of ICCs was observed in Grn−/− mice when compared to WT mice in cortical (p p Trem2−/− mice did not show significant alterations in metabolic connectivity when compared to WT. Furthermore, the increased metabolic connectivity in Grn−/− mice was completely suppressed in Grn−/−/Trem2−/− mice. Grn−/− mice exhibited a severe loss of neuronal FDG uptake (− 61%, p Grn−/− vs. 22% in WT). Conclusions Presence, absence, and activation of microglia have a strong impact on metabolic connectivity of the mouse brain. Enhanced metabolic connectivity is associated with increased microglial FDG allocation.

19 Pág. Centro de Investigación en Sanidad Animal (CISA), Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune cell surface receptor that regulates microglial function and is involved in the pathophysiology of several neurodegenerative diseases. Its soluble form (sTREM2) results from shedding of the TREM2 ectodomain. The role of TREM2 in prion diseases, a group of rapidly progressive dementias remains to be elucidated. In the present study, we analysed the expression of TREM2 and its main sheddase ADAM10 in the brain of sporadic Creutzfeldt-Jakob disease (sCJD) patients and evaluated the role of CSF and plasma sTREM2 as a potential diagnostic marker of prion disease. Our data indicate that, compared to controls, TREM2 is increased in sCJD patient brains at the mRNA and protein levels in a regional and subtype dependent fashion, and expressed in a subpopulation of microglia. In contrast, ADAM10 is increased at the protein, but not the mRNA level, with a restricted neuronal expression. Elevated CSF sTREM2 is found in sCJD, genetic CJD with mutations E200K and V210I in the prion protein gene (PRNP), and iatrogenic CJD, as compared to healthy controls (HC) (AUC = 0.78-0.90) and neurological controls (AUC = 0.73-0.85), while CSF sTREM2 is unchanged in fatal familial insomnia. sTREM2 in the CSF of cases with Alzheimer's disease, and multiple sclerosis was not significantly altered in our series. CSF sTREM2 concentrations in sCJD are PRNP codon 129 and subtype-related, correlate with CSF 14-3-3 positivity, total-tau and YKL-40, and increase with disease progression. In plasma, sTREM2 is increased in sCJD compared with HC (AUC = 0.80), displaying positive correlations with plasma total-tau, neurofilament light, and YKL-40. We conclude that comparative study of TREM2 in brain and biological fluids of prion diseases reveals TREM2 to be altered in human prion diseases with a potential value in target engagement, patient stratification, and disease monitoring., We thank the HUB-ICO-IDIBELL-Biobank and the CERCA Programme of the Generalitat de Catalunya for institutional support. This study was funded by the Instituto Carlos III (Grants Number CP16/00041 and PI19/00144) to FL. This project was also funded by la Fundació La Marató de TV3 (Grants No. 201821-30, 201821-31and 201821-32 to FL, JCE and EV, respectively) and by the Fondo Europeo de Desarrollo Regional (FEDER) through the Interreg V-A España-Francia-Andorra (POCTEFA 2014–2020) programme (at 65%) to IF. AVP is supported by the Beatriu de Pinós programme (2018-BP-00129) from the Ministry of Business and nowledge of the Government of Catalonia, and cofunded by the EU Horizon 2020 programme under an MSCA grant agreement (801370). HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018-02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862), and the UK Dementia Research Institute at UCL. KB is supported by the Swedish Research Council (#2017-00915), the Swedish State Support for Clinical Research (ADDF), USA (#RDAPB-201809-2016615), the Swedish Alzheimer Foundation (#AF-742881), Hjärnfonden, Sweden (#FO2017-0243), the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986), and the European Union Joint Programme for Neurodegenerative Disorders (JPND2019-466-236). AL is supported by the Ministero della Salute, Italy, for the national surveillance of Creutzfeldt-Jakob disease. This research was also supported in part by the Alberta Synergies in Alzheimer’s and Related Disorders (SynAD) programme which is funded by the Alzheimer Society of Alberta and Northwest Territories through their Hope for Tomorrow programme and the University Hospital Foundation. SynAD operates in partnership with the Neuroscience and Mental Health Institute at the University of Alberta. JADR was supported by grants from the Spanish Ministry of Science, Innovation and Universities (MICINN/FEDER) (RTI2018-099773-B-I00), the CERCA Programme, and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (SGR2017-648) and CIBERNED (CMED2018-2)

Proregenerative responses are required for the restoration of nervous-system functionality in demyelinating diseases such as multiple sclerosis (MS). Yet, the limiting factors responsible for poor CNS repair are only partially understood. Here, we test the impact of a Western diet (WD) on phagocyte function in a mouse model of demyelinating injury that requires microglial innate immune function for a regenerative response to occur. We find that WD feeding triggers an ageing-related, dysfunctional metabolic response that is associated with impaired myelin-debris clearance in microglia, thereby impairing lesion recovery after demyelination. Mechanistically, we detect enhanced transforming growth factor beta (TGFβ) signalling, which suppresses the activation of the liver X receptor (LXR)-regulated genes involved in cholesterol efflux, thereby inhibiting phagocytic clearance of myelin and cholesterol. Blocking TGFβ or promoting triggering receptor expressed on myeloid cells 2 (TREM2) activity restores microglia responsiveness and myelin-debris clearance after demyelinating injury. Thus, we have identified a druggable microglial immune checkpoint mechanism regulating the microglial response to injury that promotes remyelination.

Haploinsufficiency of the progranulin (PGRN)-encoding gene (GRN) causes frontotemporal lobar degeneration (GRN-FTLD) and results in microglial hyperactivation, TREM2 activation, lysosomal dysfunction, and TDP-43 deposition. To understand the contribution of microglial hyperactivation to pathology, we used genetic and pharmacological approaches to suppress TREM2-dependent transition of microglia from a homeostatic to a disease-associated state. Trem2 deficiency in Grn KO mice reduced microglia hyperactivation. To explore antibody-mediated pharmacological modulation of TREM2-dependent microglial states, we identified antagonistic TREM2 antibodies. Treatment of macrophages from GRN-FTLD patients with these antibodies led to reduced TREM2 signaling due to its enhanced shedding. Furthermore, TREM2 antibody-treated PGRN-deficient microglia derived from human-induced pluripotent stem cells showed reduced microglial hyperactivation, TREM2 signaling, and phagocytic activity, but lysosomal dysfunction was not rescued. Similarly, lysosomal dysfunction, lipid dysregulation, and glucose hypometabolism of Grn KO mice were not rescued by TREM2 ablation. Synaptic loss and neurofilament light-chain (NfL) levels, a biomarker for neurodegeneration, were further elevated in the Grn/Trem2 KO cerebrospinal fluid (CSF). These findings suggest that TREM2-dependent microglia hyperactivation in models of GRN deficiency does not promote neurotoxicity, but rather neuroprotection.

Secretases are a group of proteases that are major drug targets considered for the prevention and treatment of Alzheimer's disease (AD). Secretases do not only process the AD-linked neuronal amyloid precursor protein (APP) but also the triggering receptor expressed on myeloid cells 2 (TREM2), thereby controlling microglial functions. This review highlights selected recent discoveries for the α-secretases a disintegrin and metalloprotease 10 (ADAM10) and a disintegrin and metalloprotease 17 (ADAM17), the β-secretase β-site APP cleaving enzyme 1 (BACE1) and γ-secretase and their link to AD. New genetic evidence strengthens the role of α-secretases in AD through cleavage of APP and TREM2. Novel proteins were linked to AD, which control α- and β-secretase activity through transcriptional and post-translational mechanisms. Finally, new opportunities but also challenges are discussed for pharmacologically targeting β- and γ-secretase cleavage of APP and α-secretase cleavage of TREM2 with the aim to prevent or treat AD.

Altres ajuts: Deltaplan Dementie (The Netherlands Organisation for Health Research and Development and Alzheimer Nederland grant numbers 7330550813, 733050103); Dioraphte Foundation (grant number 1402 1300); the European Joint Programme - Neurodegenerative Disease Research (JPND, PreFrontALS); NIH (grant number AG017586); Wyncote Foundation; Arking Family Fund; Invetissements d'avenir (grant number ANR-11-INBS-0011); Schörling Foundation - Swedish FTD Initiative; Swedish Research Council (2015-02926, 2018-02754, 2019-02248); Swedish Alzheimer's Foundation; Brain Foundation; Demensfonden, Stiftelsen för Gamla Tjänarinnor; Stohnes foundation; Region Stockholm (ALF project). Excessive microglial activation might be a central pathological process in GRN-related frontotemporal dementia (FTD-GRN). We measured soluble triggering receptor expressed on myeloid cells 2 (sTREM2), which is shed from disease-associated microglia following cleavage of TREM2, in cerebrospinal fluid of 34 presymptomatic and 35 symptomatic GRN mutation carriers, 6 presymptomatic and 32 symptomatic C9orf72 mutation carriers and 67 healthy noncarriers by ELISA. Although no group differences in sTREM2 levels were observed (GRN: symptomatic (median 5.2 ng/mL, interquartile range [3.9-9.2]) vs. presymptomatic (4.3 ng/mL [2.6-6.1]) vs. noncarriers (4.2 ng/mL [2.6-5.5]): p = 0.059; C9orf72: symptomatic (4.3 [2.9-7.0]) vs. presymptomatic (3.2 [2.2-4.2]) vs. noncarriers: p = 0.294), high levels were seen in a subset of GRN, but not C9orf72, mutation carriers, which might reflect differential TREM2-related microglial activation. Interestingly, 2 presymptomatic carriers with low sTREM2 levels developed symptoms after 1 year, whereas 2 with high levels became symptomatic after >5 years. While sTREM2 is not a promising diagnostic biomarker for FTD-GRN or FTD-C9orf72, further research might elucidate its potential to monitor microglial activity and predict disease progression.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.

Funder: Canadian Institutes of Health Research; Id: http://dx.doi.org/10.13039/501100000024, Funder: Alzheimer's Association (Zenith Award), Funder: UK Alzheimer Society and ARUK, Funder: Wellcome Trust Collaborative Award in Science, Rare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid β peptides (Aβ). Aβ peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post-translational modifications result in the production of several distinct Aβ variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post-translational modifications of Aβ affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aβ, and that phosphorylation of Aβ increases this interaction. Phosphorylation of Aβ also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aβ variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aβ species in preclinical models of Alzheimer's disease.

Progressive accumulation of Amyloid-β (Aβ) deposits in the brain is a characteristic neuropathological hallmark of Alzheimer’s disease (AD). During disease progression, extracellular Aβ plaques undergo specific changes in their composition by the sequential deposition of different modified Aβ species. Microglia are implicated in the restriction of amyloid deposits and play a major role in internalization and degradation of Aβ. Recent studies showed that rare variants of the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) are associated with an increased risk for AD. Post-translational modifications of Aβ could modulate the interaction with TREM2, and the uptake by microglia. Here, we demonstrate that genetic deletion of TREM2 or expression of a disease associated TREM2 variant in mice lead to differential accumulation of modified and non-modified Aβ species in extracellular plaques and intraneuronal deposits. Human brains with rare TREM2 AD risk variants also showed altered deposition of modified Aβ species in the different brain lesions as compared to cases with the common variant of TREM2. These findings indicate that TREM2 plays a critical role in the development and the composition of Aβ deposits, not only in extracellular plaques, but also intraneuronally, that both could contribute to the pathogenesis of AD. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01263-x.

Background: Stem cells‘ (SC) functional heterogeneity and its poorly understood aetiology impedes clinical development of cell-based therapies in regenerative medicine and oncology. Recent studies suggest a strong correlation between the SC migration potential and their therapeutic efficacy in humans. Designating SC migration as a denominator of functional SC heterogeneity, we sought to identify highly migrating subpopulations within different SC classes and evaluate their therapeutic properties in comparison to the parental non-selected cells. Methods: We selected highly migrating subpopulations from mesenchymal and neural SC (sMSC and sNSC), characterized their features including but not limited to migratory potential, trophic factor release and transcriptomic signature. To assess lesion-targeted migration and therapeutic properties of isolated subpopulations in vivo, surgical transplantation and intranasal administration of MSCs in mouse models of glioblastoma and Alzheimer's disease respectively were performed. Findings: Comparison of parental non-selected cells with isolated subpopulations revealed superior motility and migratory potential of sMSC and sNSC in vitro. We identified podoplanin as a major regulator of migratory features of sMSC/sNSC. Podoplanin engineering improved oncovirolytic activity of virus-loaded NSC on distantly located glioblastoma cells. Finally, sMSC displayed more targeted migration to the tumour site in a mouse glioblastoma model and remarkably higher potency to reduce pathological hallmarks and memory deficits in transgenic Alzheimer's disease mice. Interpretation: Functional heterogeneity of SC is associated with their motility and migration potential which can serve as predictors of SC therapeutic efficacy. Funding: This work was supported in part by the Robert Bosch Stiftung (Stuttgart, Germany) and by the IZEPHA grant. CA extern

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer., Pathologic protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells via extracellular vesicles or direct cell-to-cell contact. Here, Liu et al. show that viral glycoproteins can contribute to intercellular proteopathic seed transmission via both routes.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.

Sequence variants of the microglial expressed TREM2 (triggering receptor expressed on myeloid cells 2) are a major risk factor for late onset Alzheimer's disease. TREM2 requires a stable interaction with DAP12 in the membrane to initiate signaling, which is terminated by TREM2 ectodomain shedding and subsequent intramembrane cleavage by γ‐secretase. To understand the structural basis for the specificity of the intramembrane cleavage event, we determined the solution structure of the TREM2 transmembrane helix (TMH). Caused by the presence of a charged amino acid in the membrane region, the TREM2‐TMH adopts a kinked structure with increased flexibility. Charge removal leads to TMH stabilization and reduced dynamics, similar to its structure in complex with DAP12. Strikingly, these dynamical features match with the site of the initial γ‐secretase cleavage event. These data suggest an unprecedented cleavage mechanism by γ‐secretase where flexible TMH regions act as key determinants of substrate cleavage specificity., NMR analyses of the TREM2 transmembrane helix in its native lipid environments reveal the structural basis for site‐specific intramembrane proteolysis of this disease‐associated microglial signaling receptor.

Although human endogenous retroviruses (HERVs) represent a substantial proportion of the human genome and some HERVs, such as HERV-K(HML-2), are reported to be involved in neurological disorders, little is known about their biological function. We report that RNA from an HERV-K(HML-2) envelope gene region binds to and activates human Toll-like receptor (TLR) 8, as well as murine Tlr7, expressed in neurons and microglia, thereby causing neurodegeneration. HERV-K(HML-2) RNA introduced into the cerebrospinal fluid (CSF) of either C57BL/6 wild-type mice or APPPS1 mice, a mouse model for Alzheimer’s disease (AD), resulted in neurodegeneration and microglia accumulation. Tlr7-deficient mice were protected against neurodegenerative effects but were resensitized toward HERV-K(HML-2) RNA when neurons ectopically expressed murine Tlr7 or human TLR8. Transcriptome data sets of human AD brain samples revealed a distinct correlation of upregulated HERV-K(HML-2) and TLR8 RNA expression. HERV-K(HML-2) RNA was detectable more frequently in CSF from individuals with AD compared with controls. Our data establish HERV-K(HML-2) RNA as an endogenous ligand for species-specific TLRs 7/8 and imply a functional contribution of human endogenous retroviral transcripts to neurodegenerative processes, such as AD.

Alzheimer’s disease (AD) is an irreversible, devastating neurodegenerative brain disorder characterized by the loss of neurons and subsequent cognitive decline. Despite considerable progress in the understanding of the pathophysiology of AD, the precise molecular mechanisms that cause the disease remain elusive. By now, there is ample evidence that activated microglia have a critical role in the initiation and progression of AD. The present study describes the identification of Glycoprotein nonmetastatic melanoma protein B (GPNMB) as a novel AD-related factor in both transgenic mice and sporadic AD patients by expression profiling, immunohistochemistry and ELISA measurements. We show that GPNMB levels increase in an age-dependent manner in transgenic AD models showing profound cerebral neuron loss and demonstrate that GPNMB co-localizes with a distinct population of IBA1-positive microglia cells that cluster around amyloid plaques. Our data further indicate that GPNMB is part of a microglia activation state that is only present under neurodegenerative conditions and that is characterized by the up-regulation of a subset of genes including TREM2, APOE and CST7. In agreement, we provide in vitro evidence that soluble Aβ has a direct effect on GPNMB expression in an immortalized microglia cell line. Importantly, we show for the first time that GPNMB is elevated in brain samples and cerebrospinal fluid (CSF) of sporadic AD patients when compared to non-demented controls. The current findings indicate that GPNMB represents a novel disease-associated marker that appears to play a role in the neuroinflammatory response of AD. Electronic supplementary material The online version of this article (10.1186/s40478-018-0612-3) contains supplementary material, which is available to authorized users.

Sequence variations occurring in the gene encoding the triggering receptor expressed on myeloid cells 2 (TREM2) support an essential function of microglia and innate immunity in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. TREM2 matures within the secretory pathway, and its ectodomain is shed on the plasma membrane. Missense mutations in the immunoglobulin (Ig)-like domain such as p.T66M and p.Y38C retain TREM2 within the endoplasmic reticulum and reduce shedding as well as TREM2-dependent phagocytosis. Using mass spectrometry, we have now determined the cleavage site of TREM2. TREM2 is shed by proteases of the ADAM (a disintegrin and metalloproteinase domain containing protein) family C-terminal to histidine 157, a position where an AD-associated coding variant has been discovered (p.H157Y) in the Han Chinese population. Opposite to the characterized mutations within the Ig-like domain, such as p.T66M and p.Y38C, the p.H157Y variant within the stalk region leads to enhanced shedding of TREM2. Elevated ectodomain shedding reduces cell surface full-length TREM2 and lowers TREM2-dependent phagocytosis. Therefore, two seemingly opposite cellular effects of TREM2 variants, namely reduced versus enhanced shedding, result in similar phenotypic outcomes by reducing cell surface TREM2.

A repeat expansion in the non-coding region of C9orf72 gene is the most common mutation causing frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Sense and antisense transcripts are translated into aggregating dipeptide repeat (DPR) proteins in all reading frames (poly-GA,-GP,-GR,-PA and -PR) through an unconventional mechanism. How these changes contribute to cytoplasmic mislocalization and aggregation of TDP-43 and thereby ultimately leading to neuron loss remains unclear. The repeat RNA itself and poly-GR/PR have been linked to impaired nucleocytoplasmic transport. Here, we show that compact cytoplasmic poly-GA aggregates impair nuclear import of a reporter containing the TDP-43 nuclear localization (NLS) signal. However, a reporter containing a non-classical PY-NLS was not affected. Moreover, poly-GA expression prevents TNFα induced nuclear translocation of p65 suggesting that poly-GA predominantly impairs the importin-α/β-dependent pathway. In neurons, prolonged poly-GA expression induces partial mislocalization of TDP-43 into cytoplasmic granules. Rerouting poly-GA to the nucleus prevented TDP-43 mislocalization, suggesting a cytoplasmic mechanism. In rescue experiments, expression of importin-α (KPNA3, KPNA4) or nucleoporins (NUP54, NUP62) restores the nuclear localization of the TDP reporter. Taken together, inhibition of nuclear import of TDP-43 by cytoplasmic poly-GA inclusions causally links the two main aggregating proteins in C9orf72 ALS/FTLD pathogenesis.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin β is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC‐MS/MS analysis of recombinant TREM2 incubated with meprin β revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin β cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b−/− mice compared to WT controls. Isolated BMDMs from Mep1b−/− mice showed significantly increased full‐length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin β deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin β is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2.

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.

Progranulin (PGRN) is predominantly expressed by microglia in the brain, and genetic and experimental evidence suggests a critical role in Alzheimer's disease (AD). We asked whether PGRN expression is changed in a disease severity‐specific manner in AD. We measured PGRN in cerebrospinal fluid (CSF) in two of the best‐characterized AD patient cohorts, namely the Dominant Inherited Alzheimer's Disease Network (DIAN) and the Alzheimer's Disease Neuroimaging Initiative (ADNI). In carriers of AD causing dominant mutations, cross‐sectionally assessed CSF PGRN increased over the course of the disease and significantly differed from non‐carriers 10 years before the expected symptom onset. In late‐onset AD, higher CSF PGRN was associated with more advanced disease stages and cognitive impairment. Higher CSF PGRN was associated with higher CSF soluble TREM2 (triggering receptor expressed on myeloid cells 2) only when there was underlying pathology, but not in controls. In conclusion, we demonstrate that, although CSF PGRN is not a diagnostic biomarker for AD, it may together with sTREM2 reflect microglial activation during the disease.

The sorting of activated receptors into distinct endosomal compartments is essential to activate specific signaling cascades and cellular events including growth and survival. However, the proteins involved in this sorting are not well understood. We discovered a novel role of EndophilinAs in sorting of activated BDNF-TrkB receptors into late endosomal compartments. Mice lacking all three EndophilinAs accumulate Rab7-positive late endosomes. Moreover, EndophilinAs are differentially localized to, co-traffic with, and tubulate, distinct endosomal compartments: In response to BDNF, EndophilinA2 is recruited to both early and late endosomes, EndophilinA3 is recruited to Lamp1-positive late endosomes, and co-trafficks with Rab5 and Rab7 in both the presence and absence of BDNF, while EndophilinA1 colocalizes at lower levels with endosomes. The absence of all three EndophilinAs caused TrkB to accumulate in EEA1 and Rab7-positive endosomes, and impaired BDNF-TrkB-dependent survival signaling cascades. In addition, EndophilinA triple knockout neurons exhibited increased cell death which could not be rescued by exogenous BDNF, in a neurotrophin-dependent survival assay. Thus, EndophilinAs differentially regulate activated receptor sorting via distinct endosomal compartments to promote BDNF-dependent cell survival.

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic β-amyloid (Aβ)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked80% by 1 pM BoNT/A despite cleaving20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.

During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes.

Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprograming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.

A new F-18-labeled phenylthiophenyl derivative specific for imaging of serotonin transporters (SERT) in the brain by positron emission tomography (PET) is described. Fluorinated phenylthiophenyl derivative, ACF, 2-[(2-amino-4-chloro-5-fluorophenyl)thio]-N,N-dimethyl-benzenmethanamine, was prepared by first coupling 2,5-dichloro-4-nitroaniline with 2-mercapto-N,N-dimethylbenzamide. The amino group of the coupled adduct was converted to a fluoro group through a Schiemann reaction. Subsequently, a one pot reduction of both nitro and amide groups by BH(3)-tetrahydrofuran yielded the nonradioactive ACF (yield 25%). In vitro binding assays using cell membrane homogenates of LLC cells expressing SERT, dopamine transporters (DAT), or norepinephrine transporters (NET) showed excellent binding affinity and selectivity for SERT (K(i) = 0.05, 3020, and 650 nM for SERT, DAT, and NET, respectively). For preparation of the [(18)F]ACF, the NH(2) group of the initially coupled adduct was converted to the trimethylammonium salt, which was replaced by [(18)F]fluoride in the presence of Kryptofix 222 and potassium carbonate. The final product, [(18)F]ACF, was obtained after a borane and stannous chloride reduction reaction. The combined two step reaction gave a radiochemical yield of 10-15% (EOB) and a radiochemical purity of >99%. Synthesis of the novel PET tracer, [(18)F]ACF, as a probe for binding to SERT in the brain was successfully achieved. The new tracer [(18)F]ACF showed excellent brain penetration and selective localization after an iv injection in rats (brain uptake at 2, 30, 60, 120, and 240 min was 3.27, 1.28, 0.69, 0.21, and 0.06% dose/organ, respectively). The hypothalamus/cerebellum ratio at 60 min post iv injection was 3.55. This specific localization in the hypothalamus was blocked by pretreatment of (+)McN5652. This novel ligand is a potential PET tracer for in vivo evaluation of SERT in the brain.

Cln3(Delta ex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Delta ex7/8) mice. Homozygous Cln3(Delta ex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Delta ex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post- receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12- 13 week old homozygous Cln3(Delta ex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Delta ex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Delta ex7/8) mice, and male homozygotes had a relative T- cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Delta ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Delta ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Delta ex7/8) mice that merit further study for JNCL biomarker development.

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.