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Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain. Several neurodegenerative disorders, including Alzheimer’s disease, arise when protein-cutting enzymes process proteins in the wrong way. The resulting protein fragments can accumulate in nerve cells and cause them to die, leading to symptoms such as memory loss. In the case of Alzheimer’s disease the toxic protein fragment – called amyloid beta – can be produced when one enzyme cuts the amyloid precursor protein. However, the amyloid beta fragment is not made when a different enzyme called ADAM10 cuts the amyloid precursor protein first. There has been a lot of interest in finding drugs that activate ADAM10 to treat Alzheimer’s disease. However, ADAM10 also cuts other proteins on the surface of cells and it is important to know about these proteins if ADAM10 is going to be successfully targeted by a drug. To tackle this issue, Kuhn et al. have now searched for new proteins (or ‘substrates’) that are cut by ADAM10 in mouse nerve cells. The experiments identified proteins that were cut in normal nerve cells, but remained unprocessed in cells where the gene for ADAM10 had been deleted. This search uncovered almost 100 new substrates of ADAM10 that were then validated using biochemical techniques. Among these substrates were many proteins that are normally anchored into the membranes of nerve cells and involved in guiding and positioning these cells in the brain so that they can connect and communicate with each other. Kuhn et al. then deleted the gene for ADAM10 only in the frontmost part of the mouse brain. This led to the nerve cells forming abnormal networks in the regions of the brain that process smells and emotions. Overall the experiments proved that ADAM10 is important not only for the prevention of Alzheimer’s disease, but also for the normal development of the brain. Future studies could now explore how stimulating ADAM10 affects the levels of its substrates. Also, a better understanding of the substrates of ADAM10 may be useful both to predict side effects of drugs that activate ADAM10 and to monitor patients who are responding well to these drugs.

Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of frontotemporal dementia and motor neuron disease. Recently, unconventional non-ATG translation of the expanded hexanucleotide repeat, resulting in the production and aggregation of dipeptide repeat (DPR) proteins (poly-GA, -GR and GP), was identified as a potential pathomechanism of C9ORF72 mutations. Besides accumulation of DPR proteins, the second neuropathological hallmark lesion in C9ORF72 mutation cases is the accumulation of TDP-43. In this study, we characterized novel monoclonal antibodies against poly-GA and performed a detailed analysis of the neuroanatomical distribution of DPR and TDP-43 pathology in a cohort of 35 cases with the C9ORF72 mutation that included a broad spectrum of clinical phenotypes. We found the pattern of DPR pathology to be highly consistent among cases regardless of the phenotype with high DPR load in the cerebellum, all neocortical regions (frontal, motor cortex and occipital) and hippocampus, moderate pathology in subcortical areas and minimal pathology in lower motor neurons. No correlation between DPR pathology and the degree of neurodegeneration was observed, while a good association between TDP-43 pathology with clinical phenotype and degeneration in key anatomical regions was present. Our data confirm that the presence of DPR pathology is intimately related to C9ORF72 mutations. The observed dissociation between DPR inclusion body load and neurodegeneration might suggest inclusion body formation as a potentially protective response to cope with soluble toxic DPR species. Moreover, our data imply that alterations due to the C9ORF72 mutation resulting in TDP-43 accumulation and dysmetabolism as secondary downstream effects likely play a central role in the neurodegenerative process in C9ORF72 pathogenesis.

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis. Significance Cerebral small vessel disease (SVD) is a major cause of stroke and dementia. Hereditary forms, such as cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), may provide insights into key molecular mechanisms and pathways. The serine protease HtrA1, whose activity is impaired in CARASIL, has been proposed to attenuate TGF-β signaling leading to increased pathway activity in diseased arteries. We analyzed HtrA1-deficient mouse brain tissue and mouse and CARASIL patient fibroblasts and found a reduction in signaling activity on various pathway levels suggesting a facilitating role of HtrA1. Moreover, we identified LTBP-1 as a novel HtrA1 substrate and provide evidence for its functional modulation by HtrA1-dependent proteolysis. Our data suggest down-regulation of TGF-β signaling as a key mechanism underlying CARASIL pathogenesis.

In different phases of the transcription cycle, RNA polymerase (Pol) II recruits various factors via its C-terminal domain (CTD), which consists of conserved heptapeptide repeats with the sequence Tyr¹-Ser²-Pro³-Thr⁴ -Ser⁵ -Pro⁶ -Ser⁷. We show that the CTD of transcribing yeast Pol II is phosphorylated at Tyr¹, in addition to Ser², Thr⁴, Ser⁵, and Ser⁷. Tyr¹ phosphorylation stimulates binding of elongation factor Spt6 and impairs recruitment of termination factors Nrd1, Pcf11, and Rtt103. Tyr¹ phosphorylation levels rise downstream of the transcription start site and decrease before the polyadenylation site, largely excluding termination factors from gene bodies. These results show that CTD modifications trigger and block factor recruitment and lead to an extended CTD code that explains transcription cycle coordination on the basis of differential phosphorylation of Tyr¹, Ser², and Ser⁵.

Hexanucleotide repeat expansion in C9orf72 is the most common pathogenic mutation in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite the lack of an ATG start codon, the repeat expansion is translated in all reading frames into dipeptide repeat (DPR) proteins, which form insoluble, ubiquitinated, p62-positive aggregates that are most abundant in the cerebral cortex and cerebellum. To specifically analyze DPR toxicity and aggregation, we expressed DPR proteins from synthetic genes containing a start codon but lacking extensive GGGGCC repeats. Poly-Gly-Ala (GA) formed p62-positive cytoplasmic aggregates, inhibited dendritic arborization and induced apoptosis in primary neurons. Quantitative mass spectrometry analysis to identify poly-GA co-aggregating proteins revealed a significant enrichment of proteins of the ubiquitin–proteasome system. Among the other interacting proteins, we identified the transport factor Unc119, which has been previously linked to neuromuscular and axonal function, as a poly-GA co-aggregating protein. Strikingly, the levels of soluble Unc119 are strongly reduced upon poly-GA expression in neurons, suggesting a loss of function mechanism. Similar to poly-GA expression, Unc119 knockdown inhibits dendritic branching and causes neurotoxicity. Unc119 overexpression partially rescues poly-GA toxicity suggesting that poly-GA expression causes Unc119 loss of function. In C9orf72 patients, Unc119 is detectable in 9.5 % of GA inclusions in the frontal cortex, but only in 1.6 % of GA inclusions in the cerebellum, an area largely spared of neurodegeneration. A fraction of neurons with Unc119 inclusions shows loss of cytosolic staining. Poly-GA-induced Unc119 loss of function may thereby contribute to selective vulnerability of neurons with DPR protein inclusions in the pathogenesis of C9orf72 FTLD/ALS.

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. Defective nuclear import of fused in sarcoma (FUS) is linked to amyotrophic lateral sclerosis (ALS). Arginine methylation of FUS reduces its binding to the nuclear import receptor Transportin leading to cytoplasmic accumulation of methylated FUS in ALS‐FUS patient samples.

Eukaryotic DNA replication initiates during S phase from origins that have been licensed in the preceding G1 phase. Here, we compare ChIP-seq profiles of the licensing factors Orc2, Orc3, Mcm3, and Mcm7 with gene expression, replication timing, and fork directionality profiles obtained by RNA-seq, Repli-seq, and OK-seq. Both, the origin recognition complex (ORC) and the minichromosome maintenance complex (MCM) are significantly and homogeneously depleted from transcribed genes, enriched at gene promoters, and more abundant in early- than in late-replicating domains. Surprisingly, after controlling these variables, no difference in ORC/MCM density is detected between initiation zones, termination zones, unidirectionally replicating regions, and randomly replicating regions. Therefore, ORC/MCM density correlates with replication timing but does not solely regulate the probability of replication initiation. Interestingly, H4K20me3, a histone modification proposed to facilitate late origin licensing, was enriched in late-replicating initiation zones and gene deserts of stochastic replication fork direction. We discuss potential mechanisms specifying when and where replication initiates in human cells.

Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of frontotemporal dementia and motor neuron disease. One consequence of the mutation is the formation of different potentially toxic polypeptides composed of dipeptide repeats (DPR) (poly-GA, -GP, -GR, -PA, -PR) generated by repeat-associated non-ATG (RAN) translation. While previous studies focusing on poly-GA pathology have failed to detect any clinico-pathological correlations in C9ORF72 mutation cases, recent data from animal and cell culture models suggested that it may be only specific DPR species that are toxic and only when accumulated in certain intracellular compartments. Therefore, we performed a systematic clinico-pathological correlative analysis with counting of actual numbers of distinct types of inclusion (neuronal cytoplasmic and intranuclear inclusions, dystrophic neurites) for each DPR protein in relevant brain regions (premotor cortex, lower motor neurons) in a cohort of 35 C9ORF72 mutation cases covering the clinical spectrum from those with pure MND, mixed FTD/MND and pure FTD. While each DPR protein pathology had a similar pattern of anatomical distribution, the total amount of inclusions for each DPR protein varied remarkably (poly-GA > GP > GR > PR/PA), indicating that RAN translation seems to be more effective from sense than from antisense transcripts. Importantly, with the exception of moderate associations for the amount of poly-GA-positive dystrophic neurites with degeneration in the frontal cortex and total burden of poly-GA pathology with disease onset, no relationship was identified for any other DPR protein pathology with degeneration or phenotype. Biochemical analysis revealed a close correlation between insoluble DPR protein species and numbers of visible inclusions, while we did not find any evidence for the presence of soluble DPR protein species. Thus, overall our findings strongly argue against a role of DPR protein aggregation as major and exclusive pathomechanism in C9ORF72 pathogenesis. However, this does not exclude that DPR protein formation might be essential in C9ORF72 pathogenesis in interplay with other consequences associated with the C9ORF72 repeat expansion.

A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly-PR inclusions were rare throughout the brain but significantly more abundant in the CA3/4 region of FTLD cases than in MND cases. Thus, although DPR distribution is not correlated with neurodegeneration spatially, it correlates with neuropathological subtypes.

Innate immune responses are linked to key metabolic pathways, yet the proximal signaling events that connect these systems remain poorly understood. Here we show that phosphofructokinase 1, liver type (PFKL), a rate-limiting enzyme of glycolysis, is phosphorylated at Ser775 in macrophages following several innate stimuli. This phosphorylation increases the catalytic activity of PFKL, as shown by biochemical assays and glycolysis monitoring in cells expressing phosphorylation-defective PFKL variants. Using a genetic mouse model in which PFKL Ser775 phosphorylation cannot take place, we observe that upon activation, glycolysis in macrophages is lower than in the same cell population of wild-type animals. Consistent with their higher glycolytic activity, wild-type cells have higher levels of HIF1α and IL-1β than Pfkl S775A/S775A after LPS treatment. In an in vivo inflammation model, Pfkl S775A/S775A mice show reduced levels of MCP-1 and IL-1β. Our study thus identifies a molecular link between innate immune activation and early induction of glycolysis. Pro-inflammatory signals trigger innate immune pathways with consequential metabolic remodeling. Here authors show that a rate-limiting enzymes of glycolysis, PFKL, is directly regulated by innate stimuli via phosphorylation at Ser775, which modification in turn influences pro-inflammatory cytokine production.