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Nutritional influences have been discussed as potential modulators of Parkinson’s disease (PD) pathology through various epidemiological and physiological studies. In animal models, a high-fat diet (HFD) with greater intake of lipid-derived calories leads to accelerated disease onset and progression. The underlying molecular mechanisms of HFD-induced aggravated pathology, however, remain largely unclear. In this study, we aimed to further illuminate the effects of a fat-enriched diet in PD by examining the brainstem and hippocampal transcriptome of alpha-synuclein transgenic mice exposed to a life-long HFD. Investigating individual transcript isoforms, differential gene expression and co-expression clusters, we observed that transcriptional differences between wild-type (WT) and transgenic animals intensified in both regions under HFD. Both brainstem and hippocampus displayed strikingly similar transcriptomic perturbation patterns. Interestingly, expression differences resulted mainly from responses in WT animals to HFD, while these genes remained largely unchanged or were even slightly oppositely regulated by diet in transgenic animals. Genes and co-expressed gene groups exhibiting this dysregulation were linked to metabolic and mitochondrial pathways. Our findings propose the failure of metabolic adaptions as the potential explanation for accelerated disease unfolding under exposure to HFD. From the identified clusters of co-expressed genes, several candidates lend themselves to further functional investigations.

Synucleinopathies are neurodegenerative disorders involving pathological alpha-synuclein (αSyn) protein, including dementia with Lewy bodies, multiple system atrophy and Parkinson's disease (PD). Current in vivo models of synucleinopathy include transgenic mice overexpressing αSyn variants and methods based on administration of aggregated, exogenous αSyn. Combining these techniques offers the ability to study consequences of introducing pathological αSyn into primed neuronal environments likely to develop synucleinopathy. Herein, we characterize the impacts pre-formed fibrils (PFFs) of recombinant, human αSyn have in mice overexpressing human A30P αSyn, a mutation associated with autosomal dominant PD. A30P mouse brain contains detergent insoluble αSyn biochemically similar to PD brain, and these mice develop Lewy-like synucleinopathy with age. Administration of PFFs in A30P mice resulted in regionally-specific accumulations of phosphorylated synuclein, microglial induction and a motor phenotype that differed from PFF-induced effects in wildtype mice. Surprisingly, PFF-induced losses of tyrosine hydroxylase were similar in A30P and wildtype mice. Thus, the PFF-A30P model recapitulates key aspects of synucleinopathy with induction of microglia, creating an appropriate system for evaluating neurodegenerative therapeutics.

Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. While these findings may eventually lead to new therapeutic opportunities, little is known about how these glial responses are reflected by biomarker changes in bodily fluids. Such knowledge, however, appears crucial for patient stratification, as well as monitoring disease progression and treatment responses in clinical trials. Here, we took advantage of well-described mouse models of β-amyloidosis and α-synucleinopathy to explore cerebrospinal fluid (CSF) proteome changes related to their respective proteopathic lesions. Nontargeted liquid chromatography-mass spectrometry revealed that the majority of proteins that undergo age-related changes in CSF of either mouse model were linked to microglia and astrocytes. Specifically, we identified a panel of more than 20 glial-derived proteins that were increased in CSF of aged β-amyloid precursor protein- and α-synuclein-transgenic mice and largely overlap with previously described disease-associated glial genes identified by single-cell transcriptomics. Our results also show that enhanced shedding is responsible for the increase of several of the identified glial CSF proteins as exemplified for TREM2. Notably, the vast majority of these proteins can also be quantified in human CSF and reveal changes in Alzheimer’s disease cohorts. The finding that cellular transcriptome changes translate into corresponding changes of CSF proteins is of clinical relevance, supporting efforts to identify fluid biomarkers that reflect the various functional states of glial responses in cerebral proteopathies, such as Alzheimer’s and Parkinson’s disease.

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.

Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and posttranslational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g., oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases, the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1, which upon activation recruits the ubiquitin ligase parkin. Here, we review mechanisms of mitophagy with an emphasis on posttranslational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on posttranslational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and antiapoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.

While the initial pathology of Parkinson’s disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analyzed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid.

Background: Consecutive adult neurogenesis is a well-known phenomenon in the ventricular–subventricular zone of the lateral wall of the lateral ventricles (V–SVZ) and has been controversially discussed in so-called “non-neurogenic” brain areas such as the periventricular regions (PVRs) of the aqueduct and the fourth ventricle. Dopamine is a known modulator of adult neural stem cell (aNSC) proliferation and dopaminergic neurogenesis in the olfactory bulb, though a possible interplay between local dopaminergic neurodegeneration and induction of aNSC proliferation in mid/hindbrain PVRs is currently enigmatic. Objective/Hypothesis: To analyze the influence of chronic–progressive dopaminergic neurodegeneration on both consecutive adult neurogenesis in the PVRs of the V–SVZ and mid/hindbrain aNSCs in two mechanistically different transgenic animal models of Parkinson´s disease (PD). Methods: We used Thy1-m[A30P]h α synuclein mice and Leu9′Ser hypersensitive α4* nAChR mice to assess the influence of midbrain dopaminergic neuronal loss on neurogenic activity in the PVRs of the V–SVZ, the aqueduct and the fourth ventricle. Results: In both animal models, overall proliferative activity in the V–SVZ was not altered, though the proportion of B2/activated B1 cells on all proliferating cells was reduced in the V–SVZ in Leu9′Ser hypersensitive α4* nAChR mice. Putative aNSCs in the mid/hindbrain PVRs are known to be quiescent in vivo in healthy controls, and dopaminergic deficiency did not induce proliferative activity in these regions in both disease models. Conclusions: Our data do not support an activation of endogenous aNSCs in mid/hindbrain PVRs after local dopaminergic neurodegeneration. Spontaneous endogenous regeneration of dopaminergic cell loss through resident aNSCs is therefore unlikely.

The trans-activation response DNA-binding protein TDP-43 regulates RNA processing and forms neuropathological aggregates in patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Investigating TDP-43 post-translational modifications, we discovered that K84 acetylation reduced nuclear import whereas K136 acetylation impaired RNA binding and splicing capabilities of TDP-43. Such failure of RNA interaction triggered TDP-43 phase separation mediated by the C-terminal low complexity domain, leading to the formation of insoluble aggregates with pathologically phosphorylated and ubiquitinated TDP-43. Confirming the results from site-directed mutagenesis, we succeeded to introduce authentic acetyl-lysine at the identified sites via amber suppression. [AcK84]TDP-43 showed cytoplasmic mislocalization and the aggregation propensity of [acK136]TDP-43 was confirmed. With newly developed antibodies, we found that the nuclear sirtuin SIRT1 can potently deacetylate [acK136]TDP-43. Moreover, SIRT1 reduced the aggregation propensity of [acK136]TDP-43. Thus, distinct lysine acetylations modulate nuclear import, RNA binding and phase separation of TDP-43, suggesting novel regulatory mechanisms for TDP-43 pathogenesis.

Significance Vascular dysfunction, as it develops either during normal aging or vascular disease, remains a major medical problem. The amyloid Medin, which is derived from its precursor protein MFG-E8 (through unknown mechanisms), forms insoluble aggregates in the vasculature of virtually anybody over 50 years of age, and it has been hypothesized that Medin aggregation could contribute to age-associated vascular decline; however, mechanistic analyses have so far been lacking. Our data now demonstrate that reminiscent of humans, mice also develop Medin deposits in an age-dependent manner. Importantly, mice that genetically lack Medin show reduced vascular dysfunction in the aged brain. Therefore, the prevention of Medin accumulation should be investigated as a novel therapeutic approach to preserve vascular health in the aging population.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated alpha-synuclein (alpha S). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of alpha S but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study alpha S conformers among different transgenic (TG) mouse models of alpha-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]alpha S; Thy1-h[A53T]alpha S; Thy1-h[A30P]alpha S; Thy1-m alpha S) that overexpress human or murine alpha S and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal alpha S pathology as evidenced by accumulation of alpha S serine 129 (p-alpha S) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study alpha S conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal alpha S pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of alpha S, but were largely p-alpha S-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded alpha S aggregation. Although nature and significance of microglial inclusions for human alpha-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of alpha-synucleinopathy bears importance for mechanistic and preclinical-translational studies. Funding Agencies|ECEuropean Commission Joint Research CentreEuropean Community (EC); EU/EFPIA/Innovative Medicines Initiative 2 Joint Undertaking (IMPRiND) [116060]; Alexander von Humboldt-FoundationAlexander von Humboldt Foundation; German Academic Exchange Service (DAAD)Deutscher Akademischer Austausch Dienst (DAAD); Swedish Research CouncilSwedish Research Council [2016-00748]

TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193-414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.

The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.

Leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein, is a key causative factor in Parkinson's disease (PD). Identification of novel substrates and the molecular mechanisms underlying the effects of LRRK2 are essential for understanding the pathogenesis of PD. In this study, we showed that LRRK2 played an important role in neuronal cell death by directly phosphorylating and activating apoptosis signal-regulating kinase 1 (ASK1). LRRK2 phosphorylated ASK1 at Thr832 that is adjacent to Thr845, which serves as an autophosphorylation site. Moreover, results of binding and kinase assays showed that LRRK2 acted as a scaffolding protein by interacting with each components of the ASK1-MKK3/6-p38 MAPK pathway through its specific domains and increasing the proximity to downstream targets. Furthermore, LRRK2-induced apoptosis was suppressed by ASK1 inhibition in neuronal stem cells derived from patients with PD. These results clearly indicate that LRRK2 acts as an upstream kinase in the ASK1 pathway and plays an important role in the pathogenesis of PD.

Mitochondrial dysfunction contributes to the pathogenesis of Parkinson’s disease but it is not clear why inherent mitochondrial defects lead specifically to the death of dopaminergic neurons of the mid brain. PINK1 is mitochondrial kinase andPINK1mutations cause early onset Parkinson’s disease.We found that in neuronal progenitors, PINK1 regulates mitochondrial morphology, mitochondrial contact to the endoplasmic reticulum (ER) and the phosphorylation of Miro1. A compensatory metabolic shift towards lipid synthesis provides mitochondria with the components needed for membrane renewal and oxidative phosphorylation, maintaining the mitochondrial network once mature.Cholesterol is increased by loss of PINK1, promoting overall membrane rigidity. This alters the distribution of phosphorylated DAT at synapses and impairs dopamine uptake. PINK1 is required for the phosphorylation of tyrosine hydroxylase at Ser19, dopamine and calcium homeostasis and dopaminergic pacemaking.We suggest a novel mechanism for PINK1 pathogenicity in Parkinson’s disease in addition to but not exclusive of mitophagy. We also provide a basis for potential therapeutics by showing that low doses of the cholesterol depleting drug ß-cyclodextrin reverse PINK1-specific phenotypes.

The abnormal accumulation of α-synuclein aggregates in neurons, nerve fibers, or glial cells is the hallmark of a group of neurodegenerative diseases known collectively as α-synucleinopathies. Clinical, neuropathological, and experimental evidence strongly suggests that α-synuclein plays a role not only as a trigger of pathological processes at disease inception, but also as a mediator of pathological spreading during disease progression. Specific properties of α-synuclein, such as its ability to pass from one neuron to another, its tendency to aggregate, and its potential to generate self-propagating species, have been described and elucidated in animal models and may contribute to the relentless exacerbation of Parkinson's disease pathology in patients. Animal models used for studying α-synuclein accumulation, aggregation, and propagation are mostly based on three approaches: (1) intra-parenchymal inoculations of exogenous α-synuclein (e.g., synthetic α-synuclein fibrils), (2) transgenic mice, and (3) animals (mice or rats) in which α-synuclein overexpression is induced by viral vector injections. Whereas pathological α-synuclein changes are consistently observed in these models, important differences are also found. In particular, pronounced pathology in transgenic mice and viral vector-injected animals does not appear to involve self-propagating α-synuclein species. A critical discussion of these models reveals their strengths and limitations and provides the basis for recommendations concerning their use for future investigations.

α-Synuclein (αSYN) is the neuropathological hallmark protein of Parkinson's disease (PD) and related neurodegenerative disorders. Moreover, the gene encoding αSYN (SNCA) is a major genetic contributor to PD. Interestingly, independent genome-wide association studies also identified SNCA as the most important candidate gene for alcoholism. Furthermore, single-nucleotide-polymorphisms have been associated with alcohol-craving behavior and alcohol-craving patients showed augmented αSYN expression in blood. To investigate the effect of αSYN on the addictive properties of chronic alcohol use, we examined consumption, motivation, and seeking responses induced by environmental stimuli and relapse behavior in transgenic mice expressing the human mutant [A30P]αSYN throughout the brain. The primary reinforcing effects of alcohol under operant self-administration conditions were increased, while consumption and the alcohol deprivation effect were not altered in the transgenic mice. The same mice were subjected to immunohistochemical measurements of immediate-early gene inductions in brain regions involved in addiction-related behaviors. Acute ethanol injection enhanced immunostaining for the phosphorylated form of cAMP response element binding protein in both amygdala and nucleus accumbens of αSYN transgenic mice, while in wild-type mice no effect was visible. However, at the same time, levels of cFos remain unchanged in both genotypes. These results provide experimental confirmation of SNCA as a candidate gene for alcoholism in addition to its known link to PD.

Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] α-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of α-synuclein protofibrils in the CNS. An α-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this α-synuclein transgenic mouse model. As measured by ELISA, 14 month old mice treated for 14 weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic α-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders.

We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.

The gene encoding leucine-rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon-γ (IFN-γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN-γ-mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal-regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN-γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP-1 cells and human peripheral blood monocytes stimulated the ERK5-LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5-dependent IFN-γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages. Leucine-rich repeat kinase 2 (LRRK2) is a major risk factor for the development of Parkinson's disease (PD). However, the role of LRRK2 in the affected neurons remains enigmatic. Recently, LRRK2 has been reported to be strongly expressed in the immune system. Here, we demonstrate that LRRK2 is induced by Interferon gamma via extracellular signal-regulated kinase 5 (ERK5) in macrophages, thus providing new insights in LRRK2 and ERK5 biology.

Parkin is an ubiquitin ligase regulating mitochondrial quality control reactions, including the autophagic removal of depolarized mitochondria (mitophagy). Parkin-mediated protein ubiquitinations may be counteracted by deubiquitinating enzymes (DUBs). We conducted a high-content imaging screen of Parkin translocation to depolarized mitochondria after siRNA mediated silencing of each DUB in Parkin overexpressing HeLa cells. Knockdown of the ubiquitin-specific protease USP36 led to delayed Parkin translocation while only slightly disturbing the ubiquitination of mitochondrial proteins, but final autophagic elimination of mitochondria was severely disrupted. The localization of the nucleolar USP36 was not altered during mitophagy. However, the marker for transcriptional active chromatin, histone 2B Lys120 mono-ubiquitination was found reduced in USP36-silenced cells undergoing mitophagy. We observed a reduction of the mRNA and protein levels of Beclin-1 and its associated autophagy-related key regulator ATG14L in USP36 knockdown cells. Importantly, transfection of active ATG14L into USP36-silenced cells significantly restored Parkin-dependent mitophagy. We propose USP36 as regulator for the Parkin-dependent mitophagy at least in part via the Beclin-1-ATG14L pathway.

Oligomeric forms of the Parkinson9s disease‐causing protein α‐synuclein are suspected to mediate neurodegeneration, but the mechanisms are not understood. The present study of Shrivastava et al (2015) provides a fresh insight into this old mystery. α‐Synuclein oligomers are shown by a combination of top state‐of‐the‐art biochemical and super‐resolution microscopy methods to sequester the neuronal sodium–potassium pump. Such perturbation of ion currents would ultimately lead to Ca 2+ excitotoxicity.

Aggregated α-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Experimental evidence indicates that α-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of α-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when α-synuclein oligomers were added to monomeric α-synuclein. In contrast, exogenously added α-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing α-synuclein that were treated with the oligomers displayed reduced α-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected α-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of α-synuclein oligomers into the neocortex of α-synuclein transgenic mice did not induce formation of proteinase K resistant α-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced α-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.

Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective α-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.

A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

Scientific reports 6(1), 36328 (2016). doi:10.1038/srep36328, Published by Nature Publishing Group, London

TDP-43 is linked to neurodegenerative diseases including frontotemporal dementia and amyotrophic lateral sclerosis. Mostly localized in the nucleus, TDP-43 acts in conjunction with other ribonucleoproteins as a splicing co-factor. Several RNA targets of TDP-43 have been identified so far, but its role(s) in pathogenesis remains unclear. Using Affymetrix exon arrays, we have screened for the first time for splicing events upon TDP-43 knockdown. We found alternative splicing of the ribosomal S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) upon TDP-43 knockdown in non-neuronal and neuronal cell lines. Alternative SKAR splicing depended on the first RNA recognition motif (RRM1) of TDP-43 and on 5 0 -GA-3’ and 5 0 -UG-3 0 repeats within the SKAR pre-mRNA. SKAR is a component of the exon junction complex, which recruits S6K1, thereby facilitating the pioneer round of translation and promoting cell growth. Indeed, we found that expression of the alternatively spliced SKAR enhanced S6K1-dependent signaling pathways and the translational yield of a splicedependent reporter. Consistent with this, TDP-43 knockdown also increased translational yield and significantly increased cell size. This indicates a novel mechanism of deregulated translational control upon TDP-43 deficiency, which might contribute to pathogenesis of the protein aggregation diseases frontotemporal dementia and amyotrophic lateral sclerosis.

Parkinson disease (PD) is a devastating disorder of the nervous system for which no cure exists. Although the exact mechanisms involved in the pathogenesis of PD are unclear, very recently, a novel cellular process has been identified that promises great future potential. Two PD-associated genes have been found to converge on the emerging mitophagy pathway that links the two major cellular dysfunctions implicated in the pathogenesis of PD. Thereby, PINK1 and Parkin physically associate and functionally cooperate to identify and label damaged mitochondria for selective degradation via autophagy. PD-associated mutations in both genes disrupt mitophagy although through different mechanisms, revealing a sequential multistep process. Further key players that tie into this process have been identified and provide the framework for future research aiming at a complete dissection of this neuroprotective pathway. This may not only yield novel targets for therapeutic intervention in PD, but possibly for other neurodegenerative disorders as well.

Mutations inparkin, an E3 ubiquitin ligase, are the most common cause of autosomal-recessive Parkinson's disease (PD). Here, we show that the stress-signaling non-receptor tyrosine kinase c-Abl links parkin to sporadic forms of PD via tyrosine phosphorylation. Under oxidative and dopaminergic stress, c-Abl was activated in cultured neuronal cells and in striatum of adult C57BL/6 mice. Activated c-Abl was found in the striatum of PD patients. Concomitantly, parkin was tyrosine-phosphorylated, causing loss of its ubiquitin ligase and cytoprotective activities, and the accumulation of parkin substrates, AIMP2 (aminoacyl tRNA synthetase complex-interacting multifunctional protein 2) (p38/JTV-1) and FBP-1.STI-571, a selective c-Abl inhibitor, prevented tyrosine phosphorylation of parkin and restored its E3 ligase activity and cytoprotective function bothin vitroandin vivo. Our results suggest that tyrosine phosphorylation of parkin by c-Abl is a major post-translational modification that leads to loss of parkin function and disease progression in sporadic PD. Moreover, inhibition of c-Abl offers new therapeutic opportunities for blocking PD progression.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of autosomal-dominant Parkinson's disease (PD). The second known autosomal-dominant PD gene (SNCA) encodes alpha-synuclein, which is deposited in Lewy bodies, the neuropathological hallmark of PD. LRRK2 contains a kinase domain with homology to mitogen-activated protein kinase kinase kinases (MAPKKKs) and its activity has been suggested to be a key factor in LRRK2-associated PD. Here we investigated the role of LRRK2 in signal transduction pathways to identify putative PD-relevant downstream targets. Over-expression of wild-type [wt]LRRK2 in human embryonic kidney HEK293 cells selectively activated the extracellular signal-regulated kinase (ERK) module. PD-associated mutants G2019S and R1441C, but not kinase-dead LRRK2, induced ERK phosphorylation to the same extent as [wt]LRRK2, indicating that this effect is kinase-dependent. However, ERK activation by mutant R1441C and G2019S was significantly slower than that for [wt]LRRK2, despite similar levels of expression. Furthermore, induction of the ERK module by LRRK2 was associated to a small but significant induction of SNCA, which was suppressed by treatment with the selective MAPK/ERK kinase inhibitor U0126. This pathway linking the two dominant PD genes LRRK2 and SNCA may offer an interesting target for drug therapy in both familial and sporadic disease.

DJ-1 is a neuroprotective gene mutated in recessive Parkinson's disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria, djr-1 (-/-) worms showed stronger phosphorylation of p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.

TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO(LRRK2) self-interaction. Interestingly, ROCO(LRRK2) fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO(LRRK2) reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO(LRRK2) fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.

Parkinson disease (PD)-associated genomic deletions and the destabilizing L166P point mutation lead to loss of the cytoprotective DJ-1 protein. The effects of other PD-associated point mutations are less clear. Here we demonstrate that the M26I mutation reduces DJ-1 expression, particularly in a null background (knockout mouse embryonic fibroblasts). Thus, homozygous M26I mutation causes loss of DJ-1 protein. To determine the cellular consequences, we measured suppression of apoptosis signal-regulating kinase 1 (ASK1) and cytotoxicity for [M26I]DJ-1, and systematically all other DJ-1 methionine and cysteine mutants. C106A mutation of the central redox site specifically abolished binding to ASK1 and the cytoprotective activity of DJ-1. DJ-1 was apparently recruited into the ASK1 signalosome via Cys-106-linked mixed disulfides. The designed higher order oxidation mimicking [C106DD]DJ-1 non-covalently bound to ASK1 even in the absence of hydrogen peroxide and conferred partial cytoprotection. Interestingly, mutations of peripheral redox sites (C46A and C53A) and M26I also led to constitutive ASK1 binding. Cytoprotective [wt]DJ-1 bound to the ASK1 N terminus (which is known to bind another negative regulator, thioredoxin 1), whereas [M26I]DJ-1 bound to aberrant C-terminal site(s). Consequently, the peripheral cysteine mutants retained cytoprotective activity, whereas the PD-associated mutant [M26I]DJ-1 failed to suppress ASK1 activity and nuclear export of the death domain-associated protein Daxx and did not promote cytoprotection. Thus, cytoprotective binding of DJ-1 to ASK1 depends on the central redox-sensitive Cys-106 and may be modulated by peripheral cysteine residues. We suggest that impairments in oxidative conformation changes of DJ-1 might contribute to PD neurodegeneration.

Parkinson's disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood. We investigated the effects of parkin in a human SH-SY5Y neuroblastoma cell culture model of PD, in which transcriptional induction of the enzyme tyrosinase causes a neurotoxic overproduction of cellular DA and its oxidative metabolites. Tyrosinase induction caused formation of reactive oxygen species in the cytosol and mitochondria, and neurotoxicity via activation of apoptotic stress-activated protein kinases and caspase 3. Stable transfection of wild-type parkin suppressed tyrosinase-induced apoptosis, and PD-associated mutations abolished the neuroprotective effect of parkin. Expression of wild-type parkin did not affect reactive oxygen species production, but attenuated the tyrosinase-induced activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as their cognate mitogen-activated protein kinase kinases. PD-associated mutations differentially affected the anti-apoptotic signaling of parkin. Thus, parkin contributes to DAergic neuroprotection by suppression of apoptotic stress-activated protein kinase pathways.

The role of microglial activation in multiple system atrophy (MSA) was investigated in a transgenic mouse model featuring oligodendroglial α-synuclein inclusions and loss of midbrain dopaminergic neurons by means of histopathology and morphometric analysis. Our findings demonstrate early progressive microglial activation in substantia nigra pars compacta (SNc) associated with increased expression of iNOS and correlating with dopaminergic neuronal loss. Suppression of microglial activation by early long-term minocycline treatment protected dopaminergic SNc neurons. The results suggest that oligodendroglial overexpression of α-synuclein may induce neuroinflammation related to nitrosive stress which is likely to contribute to neurodegeneration in MSA. Further, we detected increased toll-like receptor 4 immunoreactivity in both transgenic mice and MSA brains indicating a possible signaling pathway in MSA which needs to be further studied as a candidate target for neuroprotective interventions. © 2007 Movement Disorder Society