Our aging society is confronted with a dramatic increase of patients suffering from tauopathies, which include Alzheimer disease and certain frontotemporal dementias. These disorders are characterized by typical neuropathological lesions including hyperphosphorylation and subsequent aggregation of TAU protein and neuronal cell death. Currently, no mechanism-based cures are available. We generated fluorescently labeled TAU transgenic zebrafish, which rapidly recapitulated key pathological features of tauopathies, including phosphorylation and conformational changes of human TAU protein, tangle formation, neuronal and behavioral disturbances, and cell death. Due to their optical transparency and small size, zebrafish larvae are well suited for both in vivo imaging and drug development. TAU-induced neuronal cell death was imaged by time-lapse microscopy in vivo. Furthermore, we used this zebrafish model to identify compounds targeting the TAU kinase glycogen synthase kinase 3beta (GSK3beta). We identified a newly developed highly active GSK3beta inhibitor, AR-534, by rational drug design. AR-534 reduced TAU phosphorylation in TAU transgenic zebrafish. This transgenic zebrafish model may become a valuable tool for further studies of the neuropathology of dementia.

beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.

Protein N-glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase-like 3 (SPPL3) is an intramembrane-cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N-glycosylation by triggering the proteolytic release of active site-containing ectodomains of glycosidases and glycosyltransferases such as N-acetylglucosaminyltransferase V, β-1,3 N-acetylglucosaminyltransferase 1 and β-1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post-translational process in eukaryotes.

The most frequent genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) is the expansion of a GGGGCC hexanucleotide repeat in a non-coding region of the chromosome 9 open reading frame 72 (C9orf72) locus. The pathological hallmarks observed in C9orf72 repeat expansion carriers are the formation of RNA foci and deposition of dipeptide repeat (DPR) proteins derived from repeat associated non-ATG (RAN) translation. Currently, it is unclear whether formation of RNA foci, DPR translation products, or partial loss of C9orf72 predominantly drive neurotoxicity in vivo. By using a transgenic approach in zebrafish we address if the most frequently found DPR in human ALS/FTLD brain, the poly-Gly-Ala (poly-GA) protein, is toxic in vivo.

We previously investigated the progression of β-amyloid deposition in brain of mice over-expressing amyloid-precursor protein (APP-Swe), a model of Alzheimer's disease (AD), in a longitudinal PET study with the novel β-amyloid tracer [(18)F]-florbetaben. There were certain discrepancies between PET and autoradiographic findings, which seemed to arise from partial volume effects (PVE). Since this phenomenon can lead to bias, most especially in the quantitation of brain microPET studies of mice, we aimed in the present study to investigate the magnitude of PVE on [(18)F]-florbetaben quantitation in murine brain, and to establish and validate a useful correction method (PVEC). Phantom studies with solutions of known radioactivity concentration were performed to measure the full-width-at-half-maximum (FWHM) resolution of the Siemens Inveon DPET and to validate a volume-of-interest (VOI)-based PVEC algorithm. Several VOI-brain-masks were applied to perform in vivo PVEC on [(18)F]-florbetaben data from C57BL/6(N=6) mice, while uncorrected and PVE-corrected data were cross-validated with gamma counting and autoradiography. Next, PVEC was performed on longitudinal PET data set consisting of 43 PET scans in APP-Swe (13-20months) and age-matched wild-type (WT) mice using the previously defined masks. VOI-based cortex-to-cerebellum ratios (SUVR) were compared for uncorrected and PVE-corrected results. Brains from a subset of transgenic mice were ultimately examined by autoradiography ex vivo and histochemistry in vitro as gold standard assessments, and compared to VOI-based PET results. The phantom study indicated a FWHM of 1.72mm. Applying a VOI-brain-mask including extracerebral regions gave robust PVEC, with increased precision of the SUVR results. Cortical SUVR increased with age in APP-Swe mice compared to baseline measurements (16months: +5.5%, p<0.005; 20months: +15.5%, p<0.05) with uncorrected data, and to a substantially greater extent with PVEC (16months: +12.2% p<0.005; 20months: +36.4% p<0.05). WT animals showed no binding changes, irrespective of PVEC. Relative to autoradiographic results, the error [%] for uncorrected cortical SUVR was 18.9% for native PET data, and declined to 4.8% upon PVEC, in high correlation with histochemistry results. We calculate that PVEC increases by 10% statistical power for detecting altered [(18)F]-florbetaben uptake in aging APP-Swe mice in planned studies of disease modifying treatments on amyloidogenesis.

Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.

Fused in sarcoma (FUS) is a nuclear protein that carries a proline-tyrosine nuclear localization signal (PY-NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY-NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN-binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN-mediated nuclear import of ALS-associated FUS mutants. The unmethylated arginine-glycine-glycine domain preceding the PY-NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS-FUS patients contain methylated FUS, while inclusions in FTLD-FUS patients are not methylated. Together with recent findings that FUS co-aggregates with two related proteins of the FET family and TRN in FTLD-FUS but not in ALS-FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.

gamma-Secretase is a protease complex, which catalyzes the final of two subsequent cleavages of the beta-amyloid precursor protein (APP) to release the amyloid-beta peptide (Abeta) implicated in Alzheimer's disease (AD) pathogenesis. In human cells, six gamma-secretase complexes exist, which are composed of either presenilin (PS) 1 or 2, the catalytic subunit, nicastrin, PEN-2, and either APH-1a (as S or L splice variants) or its homolog APH-1b. It is not known whether and how different APH-1 species contribute to the pathogenic activity of gamma-secretase complexes with familial AD (FAD)-associated mutant PS. Here we show that all known gamma-secretase complexes are active in APP processing and that all combinations of APH-1 variants with either FAD mutant PS1 or PS2 support pathogenic Abeta(42) production. Since our data suggest that pathogenic gamma-secretase activity cannot be attributed to a discrete gamma-secretase complex, we propose that all gamma-secretase complexes have to be explored and evaluated for their potential as AD drug target.

Cell-to-cell transmission of protein aggregates is an emerging theme in neurodegenerative disease. Here, we analyze the dipeptide repeat (DPR) proteins that form neuronal inclusions in patients with hexanucleotide repeat expansion C9orf72, the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Sense and antisense transcripts of the (G4C2)n repeat are translated by repeat-associated non-ATG (RAN) translation in all reading frames into five aggregating DPR proteins. We show that the hydrophobic DPR proteins poly-GA, poly-GP, and poly-PA are transmitted between cells using co-culture assays and cell extracts. Moreover, uptake or expression of poly-GA induces nuclear RNA foci in (G4C2)80-expressing cells and patient fibroblasts, suggesting an unexpected positive feedback loop. Exposure to recombinant poly-GA and cerebellar extracts of C9orf72 patients increases repeat RNA levels and seeds aggregation of all DPR proteins in receiver cells expressing (G4C2)80 Treatment with anti-GA antibodies inhibits intracellular poly-GA aggregation and blocks the seeding activity of C9orf72 brain extracts. Poly-GA-directed immunotherapy may thus reduce DPR aggregation and disease progression in C9orf72 ALS/FTD.

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.

Beta secretase (BACE) inhibitors are promising therapeutic compounds currently in clinical phase II/III trials. Preclinical [18F]-florbetaben (FBB) amyloid PET imaging facilitates longitudinal monitoring of amyloidosis in Alzheimer's disease (AD) mouse models. Therefore, we applied this theranostic concept to investigate, by serial FBB PET, the efficacy of a novel BACE1 inhibitor in the PS2APP mouse, which is characterized by early and massive amyloid deposition. Methods: PS2APP and C57BL/6 (WT) mice were assigned to treatment (PS2APP: N=13; WT: N=11) and vehicle control (PS2APP: N=13; WT: N=11) groups at the age of 9.5 months. All animals had a baseline PET scan and follow-up scans at two months and after completion of the four-month treatment period. In addition to this longitudinal analysis of cerebral amyloidosis by PET, we undertook biochemical amyloid peptide quantification and histological amyloid plaque analyses after the final PET session. Results: BACE1 inhibitor-treated transgenic mice revealed a progression of the frontal cortical amyloid signal by 8.4 ± 2.2% during the whole treatment period, which was distinctly lower when compared to vehicle-treated mice (15.3 ± 4.4%, p<0.001). A full inhibition of progression was evident in regions with <3.7% of the increase in controls, whereas regions with >10% of the increase in controls showed only 40% attenuation with BACE1 inhibition. BACE1 inhibition in mice with lower amyloidosis at treatment initiation showed a higher efficacy in attenuating progression to PET. A predominant reduction of small plaques in treated mice indicated a main effect of BACE1 on inhibition of de novo amyloidogenesis. Conclusions: This theranostic study with BACE1 treatment in a transgenic AD model together with amyloid PET monitoring indicated that progression of amyloidosis is more effectively reduced in regions with low initial plaque development and revealed the need of an early treatment initiation during amyloidogenesis.

Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD.

The Apolipoprotein E ε4 allele (i.e. ApoE4) is the strongest genetic risk factor for sporadic Alzheimer's disease (AD). TREM2 (i.e. Triggering receptor expressed on myeloid cells 2) is a microglial transmembrane protein brain that plays a central role in microglia activation in response to AD brain pathologies. Whether higher TREM2-related microglia activity modulates the risk to develop clinical AD is an open question. Thus, the aim of the current study was to assess whether higher sTREM2 attenuates the effects of ApoE4-effects on future cognitive decline and neurodegeneration.

Previous studies have identified a crucial role of the gut microbiome in modifying Alzheimer's disease (AD) progression. However, the mechanisms of microbiome-brain interaction in AD were so far unknown. Here, we identify microbiota-derived short chain fatty acids (SCFA) as microbial metabolites which promote Aβ deposition. Germ-free (GF) AD mice exhibit a substantially reduced Aβ plaque load and markedly reduced SCFA plasma concentrations; conversely, SCFA supplementation to GF AD mice increased the Aβ plaque load to levels of conventionally colonized (specific pathogen-free [SPF]) animals and SCFA supplementation to SPF mice even further exacerbated plaque load. This was accompanied by the pronounced alterations in microglial transcriptomic profile, including upregulation of ApoE. Despite increased microglial recruitment to Aβ plaques upon SCFA supplementation, microglia contained less intracellular Aβ. Taken together, our results demonstrate that microbiota-derived SCFA are critical mediators along the gut-brain axis which promote Aβ deposition likely via modulation of the microglial phenotype.

Therapeutic modulation of TREM2-dependent microglial function might provide an additional strategy to slow the progression of Alzheimer's disease. Although studies in animal models suggest that TREM2 is protective against Alzheimer's pathology, its effect on tau pathology and its potential beneficial role in people with Alzheimer's disease is still unclear. Our aim was to study associations between the dynamics of soluble TREM2, as a biomarker of TREM2 signalling, and amyloid β (Aβ) deposition, tau-related pathology, neuroimaging markers, and cognitive decline, during the progression of autosomal dominant Alzheimer's disease.

Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.

Microglial activation occurs early in Alzheimer's disease (AD) and previous studies reported both detrimental and protective effects of microglia on AD progression. Here, we used CSF sTREM2 to investigate disease stage-dependent drivers of microglial activation and to determine downstream consequences on AD progression. We included 402 patients with measures of earliest beta-amyloid (CSF Aβ1-42 ) and late-stage fibrillary Aβ pathology (amyloid-PET centiloid), as well as sTREM2, p-tau181 , and FDG-PET. To determine disease stage, we stratified participants into early Aβ-accumulators (Aβ CSF+/PET-; n = 70) or late Aβ-accumulators (Aβ CSF+/PET+; n = 201) plus 131 controls. In early Aβ-accumulators, higher centiloid was associated with cross-sectional/longitudinal sTREM2 and p-tau181 increases. Further, higher sTREM2 mediated the association between centiloid and cross-sectional/longitudinal p-tau181 increases and higher sTREM2 was associated with FDG-PET hypermetabolism. In late Aβ-accumulators, we found no association between centiloid and sTREM2 but a cross-sectional association between higher sTREM2, higher p-tau181 and glucose hypometabolism. Our findings suggest that a TREM2-related microglial response follows earliest Aβ fibrillization, manifests in inflammatory glucose hypermetabolism and may facilitate subsequent p-tau181 increases in earliest AD.

With the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states.

Alzheimer's disease (AD) pathology develops many years before the onset of cognitive symptoms. Two pathological processes-aggregation of the amyloid-β (Aβ) peptide into plaques and the microtubule protein tau into neurofibrillary tangles (NFTs)-are hallmarks of the disease. However, other pathological brain processes are thought to be key disease mediators of Aβ plaque and NFT pathology. How these additional pathologies evolve over the course of the disease is currently unknown. Here we show that proteomic measurements in autosomal dominant AD cerebrospinal fluid (CSF) linked to brain protein coexpression can be used to characterize the evolution of AD pathology over a timescale spanning six decades. SMOC1 and SPON1 proteins associated with Aβ plaques were elevated in AD CSF nearly 30 years before the onset of symptoms, followed by changes in synaptic proteins, metabolic proteins, axonal proteins, inflammatory proteins and finally decreases in neurosecretory proteins. The proteome discriminated mutation carriers from noncarriers before symptom onset as well or better than Aβ and tau measures. Our results highlight the multifaceted landscape of AD pathophysiology and its temporal evolution. Such knowledge will be critical for developing precision therapeutic interventions and biomarkers for AD beyond those associated with Aβ and tau.

Cerebrospinal fluid (CSF) YKL40 and sTREM2 are astroglial and microglial activity biomarkers, respectively. We assessed whether CSF YKL40 and sTREM2 baseline levels are associated with longitudinal brain volume and diffusivity changes in cognitively unimpaired adults. Two brain MRI scans of 36 participants (57 to 78-years old, 12 male) were acquired in a 2-year interval. Aβ42, p-tau, YKL40 and sTREM2 concentrations in CSF were determined at baseline. We calculated gray and white matter volume changes per year maps (ΔGM and ΔWM, respectively) by means of longitudinal pairwise registration, and mean diffusivity variation per year (ΔMD) by subtraction. We checked voxel-wise for associations between ΔGM, ΔWM and ΔMD and baseline CSF level of YKL40 and sTREM2 and verified to what extent these associations were modulated by age (YKL40xAGE and sTREM2xAGE interactions). We found a positive association between ΔGM and YKL40 in the left inferior parietal region and no association between sTREM2 and ΔGM. Negative associations were also observed between ΔGM and YKL40xAGE (bilateral frontal areas, left precuneus and left postcentral and supramarginal gyri) and sTREM2xAGE (bilateral temporal and frontal cortex, putamen and left middle cingulate gyrus). We found negative associations between ΔWM and YKL40xAGE (bilateral superior longitudinal fasciculus) and sTREM2xAGE (bilateral superior longitudinal fasciculus, left superior corona radiata, retrolenticular external capsule and forceps minor, among other regions) but none between ΔWM and neither YKL40 nor sTREM2. ΔMD was positively correlated with YKL40 in right orbital region and negatively with sTREM2 in left lingual gyrus and precuneus. In addition, significant associations were found between ΔMD and YKL40xAGE (tail of left hippocampus and surrounding areas and right anterior cingulate gyrus) and sTREM2xAGE (right superior temporal gyrus). Areas showing statistically significant differences were disjoint in analyses involving YKL40 and sTREM2. These results suggest that glial biomarkers exert a relevant and distinct influence in longitudinal brain macro- and microstructural changes in cognitively unimpaired adults, which appears to be modulated by age. In younger subjects increased glial markers (both YKL40 and sTREM2) predict a better outcome, as indicated by a decrease in ΔGM and ΔWM and an increase in ΔMD, whereas in older subjects this association is inverted and higher levels of glial markers are associated with a poorer neuroimaging outcome.