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ObjectiveTo determine the diagnostic and prognostic performance of serum neurofilament light chain (NFL) in amyotrophic lateral sclerosis (ALS).MethodsThis single-centre, prospective, longitudinal study included the following patients: 124 patients with ALS; 50 patients without neurodegenerative diseases; 44 patients with conditions included in the differential diagnosis of ALS (disease controls); 65 patients with other neurodegenerative diseases (20 with frontotemporal dementia, 20 with Alzheimer’s disease, 19 with Parkinson’s disease, 6 with Creutzfeldt-Jakob disease (CJD)). Serum NFL levels were measured using the ultrasensitive single molecule array (Simoa) technology.ResultsSerum NFL levels were higher in ALS in comparison to all other categories except for CJD. A cut-off level of 62 pg/mL discriminated between ALS and all other conditions with 85.5% sensitivity (95% CI 78% to 91.2%) and 81.8% specificity (95% CI 74.9% to 87.4%). Among patients with ALS, serum NFL correlated positively with disease progression rate (rs=0.336, 95% CI 0.14 to 0.506, p=0.0008), and higher levels were associated with shorter survival (p=0.0054). Serum NFL did not differ among patients in different ALS pathological stages as evaluated by diffusion-tensor imaging, and in single patients NFL levels were stable over time.ConclusionsSerum NFL is increased in ALS in comparison to other conditions and can serve as diagnostic and prognostic biomarker. We established a cut-off level for the diagnosis of ALS.

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease. Cytoplasmic fused in sarcoma (FUS) aggregates are pathological hallmarks of FUS-ALS. Proper shuttling between the nucleus and cytoplasm is essential for physiological cell function. However, the initial event in the pathophysiology of FUS-ALS remains enigmatic. Using human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs), we show that impairment of poly(ADP-ribose) polymerase (PARP)-dependent DNA damage response (DDR) signaling due to mutations in the FUS nuclear localization sequence (NLS) induces additional cytoplasmic FUS mislocalization which in turn results in neurodegeneration and FUS aggregate formation. Our work suggests that a key pathophysiologic event in ALS is upstream of aggregate formation. Targeting DDR signaling could lead to novel therapeutic routes for ameliorating ALS. Abnormal cytoplasmic aggregates of FUS are a hallmark of some forms of amyotrophic lateral sclerosis (ALS). Here, using neurons derived from patients with FUS-ALS, the authors demonstrate that impairment of PARP-dependent DNA damage signaling is an event that occurs upstream of neurodegeneration and cytoplasmic aggregate formation in FUS-ALS.

TAR DNA binding protein 43 (TDP-43) is closely related to the pathogenesis of amyotrophic lateral sclerosis (ALS) and translocates to stress granules (SGs). The role of SGs as aggregation-promoting “crucibles” for TDP-43, however, is still under debate. We analyzed TDP-43 mobility and localization under different stress and recovery conditions using live cell single-molecule tracking and super-resolution microscopy. Besides reduced mobility within SGs, a stress induced decrease of TDP-43 mobility in the cytoplasm and the nucleus was observed. Stress removal led to a recovery of TDP-43 mobility, which strongly depended on the stress duration. ‘Stimulated-emission depletion microscopy’ (STED) and ‘tracking and localization microscopy’ (TALM) revealed not only TDP-43 substructures within stress granules but also numerous patches of slow TDP-43 species throughout the cytoplasm. This work provides insights into the aggregation of TDP-43 in living cells and provide evidence suggesting that TDP-43 oligomerization and aggregation takes place in the cytoplasm separate from SGs. Amyotrophic Lateral Sclerosis related TDP-43 protein translocates to stress granules with a concomitant reduction in mobility. Here, the authors use single molecule tracking and find a stress-induced reduction in TDP-43 mobility also in the cytoplasm potentially relevant for TDP-43 aggregation.

Despite extensive effort on studying inflammatory processes in the CNS of Parkinson’s disease (PD) patients, implications of peripheral monocytes are still poorly understood. Here, we set out to obtain a comprehensive picture of circulating myeloid cells in PD patients. We applied a human primary monocyte culture system and flow cytometry-based techniques to determine the state of monocytes from PD patients during disease. We found that the classical monocytes are enriched in the blood of PD patients along with an increase in the monocyte-recruiting chemoattractant protein CCL2. Moreover, we found that monocytes from PD patients display a pathological hyperactivity in response to LPS stimulation that correlates with disease severity. Inflammatory pre-conditioning was also reflected on the transcriptome in PD monocytes using next-generation sequencing. Further, we identified the CD95/CD95L as a key regulator for the PD-associated alteration of circulating monocytes. Pharmacological neutralization of CD95L reverses the dysregulation of monocytic subpopulations in favor of non-classical monocytes. Our results suggest that PD monocytes are in an inflammatory predisposition responding with hyperactivation to a “second hit”. These results provide the first direct evidence that circulating human peripheral blood monocytes are altered in terms of their function and composition in PD patients. This study provides insights into monocyte biology in PD and establishes a basis for future studies on peripheral inflammation.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V)-GFP) cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.

The dysregulation of peripheral immunity in Parkinson’s Disease (PD) includes changes in both the relative numbers and gene expression of T cells. The presence of peripheral T-cell abnormalities in PD is well-documented, but less is known about their association to clinical parameters, such as age, age of onset, progression rate or severity of the disease. We took a detailed look at T-cell numbers, gene expression and activation in cross-sectional cohorts of PD patients and age-matched healthy controls by means of flow cytometry and NanoString gene expression assay. We show that the well-pronounced decrease in relative T-cell numbers in PD blood is mostly driven by a decrease of CD8 + cytotoxic T cells and is primarily associated with the severity of the disease. In addition, we demonstrate that the expression of inflammatory genes in T cells from PD patients is also associated with disease severity. PD T cells presented with increased activation upon stimulation with phytohemagglutinin that also correlated with disease severity. In summary, our data suggest that the consequences of disease severity account for the changes in PD T cells, rather than age, age of onset, duration or the disease progression rate.

ObjectivesRecent advances in amyotrophic lateral sclerosis (ALS) genetics have revealed that mutations in any of more than 25 genes can cause ALS, mostly as an autosomal-dominant Mendelian trait. Detailed knowledge about the genetic architecture of ALS in a specific population will be important for genetic counselling but also for genotype-specific therapeutic interventions.MethodsHere we combined fragment length analysis, repeat-primed PCR, Southern blotting, Sanger sequencing and whole exome sequencing to obtain a comprehensive profile of genetic variants in ALS disease genes in 301 German pedigrees with familial ALS. We report C9orf72 mutations as well as variants in consensus splice sites and non-synonymous variants in protein-coding regions of ALS genes. We furthermore estimate their pathogenicity by taking into account type and frequency of the respective variant as well as segregation within the families.Results49% of our German ALS families carried a likely pathogenic variant in at least one of the earlier identified ALS genes. In 45% of the ALS families, likely pathogenic variants were detected in C9orf72, SOD1, FUS, TARDBP or TBK1, whereas the relative contribution of the other ALS genes in this familial ALS cohort was 4%. We identified several previously unreported rare variants and demonstrated the absence of likely pathogenic variants in some of the recently described ALS disease genes.ConclusionsWe here present a comprehensive genetic characterisation of German familial ALS. The present findings are of importance for genetic counselling in clinical practice, for molecular research and for the design of diagnostic gene panels or genotype-specific therapeutic interventions in Europe.

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease affecting primarily the upper and lower motor neurons. A common feature of all ALS cases is a well-characterized neuroinflammatory reaction within the central nervous system (CNS). However, much less is known about the role of the peripheral immune system and its interplay with CNS resident immune cells in motor neuron degeneration. Here, we characterized peripheral monocytes in both temporal and spatial dimensions of ALS pathogenesis. We found the circulating monocytes to be deregulated in ALS regarding subtype constitution, function and gene expression. Moreover, we show that CNS infiltration of peripheral monocytes correlates with improved motor neuron survival in a genetic ALS mouse model. Furthermore, application of human immunoglobulins or fusion proteins containing only the human Fc, but not the Fab antibody fragment, increased CNS invasion of peripheral monocytes and delayed the disease onset. Our results underline the importance of peripheral monocytes in ALS pathogenesis and are in agreement with a protective role of monocytes in the early phase of the disease. The possibility to boost this beneficial function of peripheral monocytes by application of human immunoglobulins should be evaluated in clinical trials.

TANK-Binding Kinase 1 ( TBK1 ) is involved in autophagy and immune signaling. Dominant loss-of-function mutations in TBK1 have been linked to Amyotrophic Lateral Sclerosis (ALS), Fronto-temporal dementia (FTD), and ALS/FTD. However, pathogenic mechanisms remain unclear, particularly the cell-type specific disease contributions of TBK1 mutations. Here, we show that deleting Tbk1 from mouse motor neurons does not induce transcriptional stress, despite lifelong signs of autophagy deregulations. Conversely, Tbk1 deletion in microglia alters their homeostasis and reactive responses. In both spinal cord and brain, Tbk1 deletion leads to a pro-inflammatory, primed microglial signature with features of ageing and neurodegeneration. While it does not induce or modify ALS-like motor neuron damage, microglial Tbk1 deletion is sufficient to cause early FTD-like social recognition deficits. This phenotype is linked to focal microglial activation and T cell infiltration in the substantia nigra pars reticulata and pallidum. Our results reveal that part of TBK1 -linked FTD disease originates from microglial dysfunction. TBK1 mutations are linked to Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD), but their cell-type specific disease contributions are unclear. Here, the authors show that Tbk1 deletion from mouse microglia shifts them to an aged-like phenotype and causes social recognition deficits.