Biophysical and structural investigation of the regulation of human GTP cyclohydrolase I by its regulatory protein GFRP.

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

Impaired activation of ALS monocytes by exosomes.

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disease affecting predominantly motor neurons in the spinal cord and motor cortex. Neurodegeneration in ALS is accompanied by a well-characterized neuroinflammatory reaction within the central nervous system and, as described more recently, cells of the peripheral immune system. Particularly monocytes have been implicated in ALS pathogenesis. Exosomes are membrane-enclosed vesicles secreted by various cell types with a diameter of 50-150 nm. Circulating blood exosomes have been shown to be important mediators and regulators of immunity. Therefore, we hypothesize that circulating blood exosomes are putative mediators of monocytic deregulation in ALS. Here we characterize exosomal uptake and the respective immunological reaction of peripheral monocytes from ALS patients and healthy donors using both serum-derived exosomes and TDP-43-loaded exosomes produced in cell culture. We found the pro-inflammatory cytokine secretion by ALS monocytes upon exosomal stimulation to be impaired compared with control monocytes. Moreover, we demonstrate that exosomal TDP-43 induces increased monocytic activation compared with non-aggregation-prone cargo. Therefore, this study underlines the functional deregulation of ALS monocytes and the impact of circulating blood exosomes on monocyte activation.

Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.

Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease.

Age-dependent defects of alpha-synuclein oligomer uptake in microglia and monocytes.

Extracellular alpha-synuclein (αsyn) oligomers, associated to exosomes or free, play an important role in the pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that these extracellular moieties activate microglia leading to enhanced neuronal damage. Despite extensive efforts on studying neuroinflammation in PD, little is known about the impact of age on microglial activation and phagocytosis, especially of extracellular αsyn oligomers. Here, we show that microglia isolated from adult mice, in contrast to microglia from young mice, display phagocytosis deficits of free and exosome-associated αsyn oligomers combined with enhanced TNFα secretion. In addition, we describe a dysregulation of monocyte subpopulations with age in mice and humans. Accordingly, human monocytes from elderly donors also show reduced phagocytic activity of extracellular αsyn. These findings suggest that these age-related alterations may contribute to an increased susceptibility to pathogens or abnormally folded proteins with age in neurodegenerative diseases.

α-synuclein interacts with SOD1 and promotes its oligomerization.

BACKGROUND: Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) are both neurodegenerative diseases leading to impaired execution of movement. α-Synuclein plays a central role in the pathogenesis of PD whereas Cu, Zn superoxide dismutase (SOD1) is a key player in a subset of familial ALS cases. Under pathological conditions both α-synuclein and SOD1 form oligomers and fibrils. In this study we investigated the possible molecular interaction of α-synuclein and SOD1 and its functional and pathological relevance. RESULTS: Using a protein-fragment complementation approach and co-IP, we found that α-synuclein and SOD1 physically interact in living cells, human erythrocytes and mouse brain tissue. Additionally, our data show that disease related mutations in α-synuclein (A30P, A53T) and SOD1 (G85R, G93A) modify the binding of α-synuclein to SOD1. Notably, α-synuclein accelerates SOD1 oligomerization independent of SOD1 activity. CONCLUSION: This study provides evidence for a novel interaction of α-synuclein and SOD1 that might be relevant for neurodegenerative diseases.

TDP-43 is intercellularly transmitted across axon terminals.

Transactive response DNA-binding protein 43 kD (TDP-43) is an aggregation-prone prion-like domain-containing protein and component of pathological intracellular aggregates found in most amyotrophic lateral sclerosis (ALS) patients. TDP-43 oligomers have been postulated to be released and subsequently nucleate TDP-43 oligomerization in recipient cells, which might be the molecular correlate of the systematic symptom spreading observed during ALS progression. We developed a novel protein complementation assay allowing quantification of TDP-43 oligomers in living cells. We demonstrate the exchange of TDP-43 between cell somata and the presence of TDP-43 oligomers in microvesicles/exosomes and show that microvesicular TDP-43 is preferentially taken up by recipient cells where it exerts higher toxicity than free TDP-43. Moreover, studies using microfluidic neuronal cultures suggest both anterograde and retrograde trans-synaptic spreading of TDP-43. Finally, we demonstrate TDP-43 oligomer seeding by TDP-43-containing material derived from both cultured cells and ALS patient brain lysate. Thus, using an innovative detection technique, we provide evidence for preferentially microvesicular uptake as well as both soma-to-soma "horizontal" and bidirectional "vertical" synaptic intercellular transmission and prion-like seeding of TDP-43.

Profilin 1 with the amyotrophic lateral sclerosis associated mutation T109M displays unaltered actin binding and does not affect the actin cytoskeleton.

BACKGROUND: The recent identification of several mutations in PFN1, a protein involved in actin dynamics, strengthens the hypothesis that pathology of amyotrophic lateral sclerosis is linked to cytoskeletal defects. Impaired actin binding is a common denominator of several PFN1 mutations associated with amyotrophic lateral sclerosis, although further mechanisms may also contribute to the death of motor neurons. In this study we examine the actin binding properties of PFN1 carrying the causal T109M mutation and its effects on the actin cytoskeleton. METHODS: Actin binding of PFN1 T109M was examined by co-immunoprecipitation experiments, a split luciferase complementation assay and a pulldown assay with recombinant PFN1. The actin cytoskeleton was investigated by fluorescence microscopy and by ultracentrifuge separation of globular and filamentous actin fractions followed by Western blotting. RESULTS: Using different technical approaches we show that PFN1 T109M displays unaltered actin binding. Furthermore we show that the actin cytoskeleton is not affected by PFN1 carrying the T109M mutation. CONCLUSION: Our data suggest that actin independent mechanisms contribute to the pathogenicity of PFN1 T109M and possibly other PFN1 mutations.

Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia.

Amyotrophic lateral sclerosis (ALS) is a genetically heterogeneous neurodegenerative syndrome hallmarked by adult-onset loss of motor neurons. We performed exome sequencing of 252 familial ALS (fALS) and 827 control individuals. Gene-based rare variant analysis identified an exome-wide significant enrichment of eight loss-of-function (LoF) mutations in TBK1 (encoding TANK-binding kinase 1) in 13 fALS pedigrees. No enrichment of LoF mutations was observed in a targeted mutation screen of 1,010 sporadic ALS and 650 additional control individuals. Linkage analysis in four families gave an aggregate LOD score of 4.6. In vitro experiments confirmed the loss of expression of TBK1 LoF mutant alleles, or loss of interaction of the C-terminal TBK1 coiled-coil domain (CCD2) mutants with the TBK1 adaptor protein optineurin, which has been shown to be involved in ALS pathogenesis. We conclude that haploinsufficiency of TBK1 causes ALS and fronto-temporal dementia.

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

A high-content screen identifies novel compounds that inhibit stress-induced TDP-43 cellular aggregation and associated cytotoxicity.

TDP-43 is an RNA binding protein found to accumulate in the cytoplasm of brain and spinal cord from patients affected with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Nuclear TDP-43 protein regulates transcription through several mechanisms, and under stressed conditions, it forms cytoplasmic aggregates that co-localize with stress granule (SG) proteins in cell culture. These granules are also found in the brain and spinal cord of patients affected with ALS and FTLD. The mechanism through which TDP-43 might contribute to neurodegenerative diseases is poorly understood. To investigate the pathophysiology of TDP-43 aggregation and to isolate potential therapeutic targets, we screened a chemical library of 75,000 compounds using high-content analysis with PC12 cells that inducibly express human TDP-43 tagged with green fluorescent protein (GFP). The screen identified 16 compounds that dose-dependently decreased the TDP-43 inclusions without significant cellular toxicity or changes in total TDP-43 expression levels. To validate the effect, we tested compounds by Western blot analysis and in a Caenorhabditis elegans model that replicates some of the relevant disease phenotypes. The hits from this assay will be useful for elucidating regulation of TDP-43, stress granule response, and possible ALS therapeutics.