Systemic inflammation activates coagulation and immune cell infiltration pathways in brains with propagating α-synuclein fibril aggregates.

Synucleinopathies are a group of diseases characterized by brain aggregates of α-synuclein (α-syn). The gradual accumulation of α-syn and the role of inflammation in early-stage pathogenesis remain poorly understood. We explored this interaction by inducing chronic inflammation in a common pre-clinical synucleinopathy mouse model. Three weeks post unilateral intra-striatal injections of human α-syn pre-formed fibrils (PFF), mice underwent repeated intraperitoneal injections of 1 mg/ml lipopolysaccharide (LPS) for 3 weeks. Histological examinations of the ipsilateral site showed phospho-α-syn regional spread and LPS-induced neutrophil recruitment to the brain vasculature. Biochemical assessment of the contralateral site confirmed spreading of α-syn aggregation to frontal cortex and a rise in intracerebral TNF-α, IL-1ß, IL-10 and KC/GRO cytokines levels due to LPS. No LPS-induced exacerbation of α-syn pathology load was observed at this stage. Proteomic analysis was performed contralateral to the PFF injection site using LC-MS/MS. Subsequent downstream Reactome Gene-Set Analysis indicated that α-syn pathology alters mitochondrial metabolism and synaptic signaling. Chronic LPS-induced inflammation further lead to an overrepresentation of pathways related to fibrin clotting as well as integrin and B cell receptor signaling. Western blotting confirmed a PFF-induced increase in fibrinogen brain levels and a PFF + LPS increase in Iba1 levels, indicating activated microglia. Splenocyte profiling revealed changes in T and B cells, monocytes, and neutrophils populations due to LPS treatment in PFF injected animals. In summary, early α-syn pathology impacts energy homeostasis pathways, synaptic signaling and brain fibrinogen levels. Concurrent mild systemic inflammation may prime brain immune pathways in interaction with peripheral immunity.

Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death.

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-ß-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein.

Polo-like kinase 2 modulates α-synuclein protein levels by regulating its mRNA production.

Variations in the α-synuclein-encoding SNCA gene represent the greatest genetic risk factor for Parkinson's disease (PD), and duplications/triplications of SNCA cause autosomal dominant familial PD. These facts closely link brain levels of α-synuclein with the risk of PD, and make lowering α-synuclein levels a therapeutic strategy for the treatment of PD and related synucleinopathies. In this paper, we corroborate previous findings on the ability of overexpressed Polo-like kinase 2 (PLK-2) to decrease cellular α-synuclein, but demonstrate that the process is independent of PLK-2 phosphorylating S129 in α-synuclein because a similar reduction is achieved with the non-phosphorable S129A mutant α-synuclein. Using a specific PLK-2 inhibitor (compound 37), we demonstrate that endogenous PLK-2 phosphorylates S129 only in some cells, but increases α-synuclein protein levels in all tested cell cultures and brain slices. PLK-2 is found to regulate the transcription of α-synuclein mRNA from both the endogenous mouse SNCA gene and transgenic vectors that only contain the open reading frame. Moreover, we are the first to show that regulation of α-synuclein by PLK-2 is of physiological importance since 10days' inhibition of endogenous PLK-2 in wt C57BL/6 mice increases endogenous α-synuclein protein levels. Our findings collectively demonstrate that PLK-2 regulates α-synuclein levels by a previously undescribed transcription-based mechanism. This mechanism is active in cells and brain tissue, opening up for alternative strategies for modulating α-synuclein levels and thereby for the possibility of modifying disease progression in synucleinopaties.

NMDA Receptors Containing the GluN2D Subunit Control Neuronal Function in the Subthalamic Nucleus.

The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)-P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease.

Development of Passive Immunotherapies for Synucleinopathies.

Immunotherapy using antibodies targeting alpha-synuclein has proven to be an effective strategy for ameliorating pathological and behavioral deficits induced by excess pathogenic alpha-synuclein in various animal and/or cellular models. However, the process of selecting the anti-alpha-synuclein antibody with the best potential to treat synucleinopathies in humans is not trivial. Critical to this process is a better understanding of the pathological processes involved in the synucleinopathies and how antibodies are able to influence these. We will give an overview of the first proof-of-concept studies in rodent disease models and discuss challenges associated with developing antibodies against alpha-synuclein resulting from the distribution and structural characteristics of the protein. We will also provide a status on the passive immunization approaches targeting alpha-synuclein that have entered, or are expected to enter, clinical evaluation.

USP30 inhibition induces mitophagy and reduces oxidative stress in parkin-deficient human neurons.

Ubiquitination of mitochondrial proteins plays an important role in the cellular regulation of mitophagy. The E3 ubiquitin ligase parkin (encoded by PARK2) and the ubiquitin-specific protease 30 (USP30) have both been reported to regulate the ubiquitination of outer mitochondrial proteins and thereby mitophagy. Loss of E3 ligase activity is thought to be pathogenic in both sporadic and inherited Parkinson's disease (PD), with loss-of-function mutations in PARK2 being the most frequent cause of autosomal recessive PD. The aim of the present study was to evaluate whether mitophagy induced by USP30 inhibition provides a functional rescue in isogenic human induced pluripotent stem cell-derived dopaminergic neurons with and without PARK2 knockout (KO). Our data show that healthy neurons responded to CCCP-induced mitochondrial damage by clearing the impaired mitochondria and that this process was accelerated by USP30 inhibition. Parkin-deficient neurons showed an impaired mitophagic response to the CCCP challenge, although mitochondrial ubiquitination was enhanced. USP30 inhibition promoted mitophagy in PARK2 KO neurons, independently of whether left in basal conditions or treated with CCCP. In PARK2 KO, as in control neurons, USP30 inhibition balanced oxidative stress levels by reducing excessive production of reactive oxygen species. Interestingly, non-dopaminergic neurons were the main driver of the beneficial effects of USP30 inhibition. Our findings demonstrate that USP30 inhibition is a promising approach to boost mitophagy and improve cellular health, also in parkin-deficient cells, and support the potential relevance of USP30 inhibitors as a novel therapeutic approach in diseases with a need to combat neuronal stress mediated by impaired mitochondria.

Characterization of pSer129-αSyn Pathology and Neurofilament Light-Chain Release across In Vivo, Ex Vivo, and In Vitro Models of Pre-Formed-Fibril-Induced αSyn Aggregation.

Protein aggregation is a predominant feature of many neurodegenerative diseases, including synucleinopathies, which are characterized by cellular inclusions containing α-Synuclein (αSyn) phosphorylated at serine 129 (pSer129). In the present study, we characterized the development of αSyn pre-formed fibril (PFF)-induced pSer129-αSyn pathology in F28tg mice overexpressing human wild-type αSyn, as well as in ex vivo organotypic cultures and in vitro primary cultures from the same mouse model. Concurrently, we collected cerebrospinal fluid (CSF) from mice and conditioned media from ex vivo and in vitro cultures and quantified the levels of neurofilament light chain (NFL), a biomarker of neurodegeneration. We found that the intra-striatal injection of PFFs induces the progressive spread of pSer129-αSyn pathology and microglial activation in vivo, as well as modest increases in NFL levels in the CSF. Similarly, PFF-induced αSyn pathology occurs progressively in ex vivo organotypic slice cultures and is accompanied by significant increases in NFL release into the media. Using in vitro primary hippocampal cultures, we further confirmed that pSer129-αSyn pathology and NFL release occur in a manner that correlates with the fibril dose and the level of the αSyn protein. Overall, we demonstrate that αSyn pathology is associated with NFL release across preclinical models of seeded αSyn aggregation and that the pharmacological inhibition of αSyn aggregation in vitro also significantly reduces NFL release.

Loss of DJ-1 protein stability and cytoprotective function by Parkinson's disease-associated proline-158 deletion.

DJ-1 is a ubiquitous protein regulating cellular viability. Recessive mutations in the PARK7/DJ-1 gene are linked to Parkinson's disease (PD). Although the most dramatic L166P point mutation practically eliminates DJ-1 protein and function, the effects of other PD-linked mutations are subtler. Here, we investigated two recently described PD-associated DJ-1 point mutations, the A179T substitution and the P158Δ in-frame deletion. [A179T]DJ-1 protein was as stable as wild-type [wt]DJ-1, but the P158Δ mutant protein was less stable. In accord with the notion that dimer formation is essential for DJ-1 protein stability, [P158Δ]DJ-1 was impaired in dimer formation. Similar to our previous findings for [M26I]DJ-1, [P158Δ]DJ-1 bound aberrantly to apoptosis signal-regulating kinase 1. Thus, the PD-associated P158Δ mutation destabilizes DJ-1 protein and function. As there is also evidence for an involvement of DJ-1 in multiple system atrophy, a PD-related α-synucleinopathy characterized by oligodendroglial cytoplasmic inclusions, we studied an oligodendroglial cell line stably expressing α-synuclein. α-Synuclein aggregate dependent microtubule retraction upon co-transfection with tubulin polymerization-promoting protein p25α was ameliorated by [wt]DJ-1. In contrast, DJ-1 mutants including P158Δ failed to protect in this system, where we found evidence of apoptosis signal-regulating kinase 1 (ASK1) involvement. In conclusion, the P158Δ point mutation may contribute to neurodegeneration by protein destabilization and hence loss of DJ-1 function.

Establishment of a human induced pluripotent stem cell neuronal model for identification of modulators of A53T α-synuclein levels and aggregation.

Inhibiting formation or promoting degradation of α-synuclein aggregates are among the therapeutical approaches under investigation as disease-modifying treatment strategies for Parkinson's disease. To support these developments, several in vitro models based on seeded α-synuclein aggregation have been established in immortalized cell lines and murine primary neurons. Here, we report on a humanized model with a reproducibility and throughput that enables its use in supporting target identification and validation in pharmacological research. A human induced pluripotent stem cell (iPSC) line was genetically modified to express HA-tagged α-synuclein with the point mutation in position 53 from Alanine to Threonine (A53T) under an inducible system and differentiated into cortical neurons expressing neuronal markers and exhibiting spontaneous activity. Intracellular α-synuclein aggregation was triggered by exposure to exogenous added fibrillated recombinant wild-type human α-synuclein fibrils91 and demonstrated by several endpoints; the formation of Triton-insoluble SDS-soluble α-synuclein, biochemically in a fluorescence resonance energy transfer based aggregation assay and by immunocytochemistry of phosphorylated α-synuclein positive puncta. We demonstrate the feasibility of upscaling the iPSC neuron production for drug discovery and that the model has a suitable dynamic range allowing for both detection of increased and decreased α-synuclein aggregation. Moreover, gene modulation is feasible using siRNAs, making the model suitable for genetic screening for modulators of α-synuclein aggregation. Data on effects of USP8, USP13 and USP9X knockdown on α-synuclein expression and aggregation contradicts published data from immortalized cell lines and murine systems. This highlight the importance of including humanized neuronal models in the confirmation of biological mechanisms in specific variations of Parkinson's disease.

PKR kinase directly regulates tau expression and Alzheimer's disease-related tau phosphorylation.

Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule-stabilizing protein and leaves it increasingly vulnerable to self-assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease-modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR-mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra-exonic regulation of the tau-encoding microtubule-associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full-length- and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.

TNF-α and α-synuclein fibrils differently regulate human astrocyte immune reactivity and impair mitochondrial respiration.

Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.

Measurement of cellular beta-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems.

Amyloid-beta peptide (Abeta), a putatively causative agent of Alzheimer's disease (AD), is proteolytically derived from beta-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease beta-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Abeta production was modified by BACE1 inhibitors similarly to the production of endogenous murine Abeta in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP-APP by BACE1 or alpha-secretases. To render the assay specific for BACE1, the alpha-secretase cleavage site of SEAP-APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP-APP-EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function.

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms. Hence, we analyzed the repertoire of proteins interacting with normal and polyQ expanded ataxin-3 by mass spectrometry. This showed that both normal and polyQ expanded ataxin-3 interacted with components of the protein quality control system and mitochondria. Five proteins showed increased interaction with polyQ expanded ataxin-3 relative to normal and three of these were mitochondrial proteins. The analyses underline the role of ataxin-3 in ubiquitin biology and point towards a role in mitochondrial biology.

Autoimmune antibody decline in Parkinson's disease and Multiple System Atrophy; a step towards immunotherapeutic strategies.

BACKGROUND: Parkinson's' disease (PD) and Multiple System Atrophy (MSA) are progressive brain disorders characterized by intracellular accumulations of α-synuclein and nerve cell loss in specific brain areas. This loss causes problems with movement, balance and/or autonomic functions. Naturally occurring autoantibodies (NAbs) play potentially an important role in clearing or/and blocking circulating pathological proteins. Little is known about the functional properties of anti-α-synuclein NAbs in PD and MSA, and there have been opposing reports regarding their plasma concentrations in these disorders. METHODS: We have investigated the apparent affinity of anti-α-synuclein NAbs in plasma samples from 46 PD patients, 18 MSA patients and 41 controls using competitive enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) set-ups. RESULTS: We found that the occurrence of high affinity anti-α-synuclein NAbs in plasma from PD patients is reduced compared to healthy controls, and nearly absent in plasma from MSA patients. Also, levels of α-synuclein/NAbs immunocomplexes is substantially reduced in plasma from both patient groups. Further, cross binding of anti-α-synuclein NAbs with ß- and γ-synuclein monomers suggest, the high affinity anti-α-synuclein plasma component, seen in healthy individuals, is directed mainly against C-terminal epitopes. Furthermore, we also observed reduced occurrence of high affinity anti-phosphorylated-α-synuclein NAbs in plasma from PD and MSA patients. CONCLUSIONS: One interpretation implies that these patients may have impaired ability to clear and/or block the effects of pathological α-synuclein due to insufficient/absent concentration of NAbs and as such provides a rationale for testing immune-based therapeutic strategies directed against pathological α-synuclein. Following this interpretation, we can hypothesize that high affinity autoantibodies efficiently bind and clear potentially pathological species of α-synuclein in healthy brain, and that this mechanism is impaired or absent in PD and MSA patients.

Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells.

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson's disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50's equivalent to IC50's on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

Antibodies against the C-terminus of α-synuclein modulate its fibrillation.

The 140-residue natively disordered protein α-synuclein (aSN) is a central component in the development of a family of neurodegenerative diseases termed synucleinopathies. This is attributed to its ability to form cytotoxic aggregates such as oligomers and amyloid fibrils. Consequently there have been intense efforts to avoid aggregation or reroute the aggregation pathway using pharmaceutical agents such as small molecules, chaperones and antibodies. aSN's lack of persistent structure in the monomeric state, as well as the multitude of different oligomeric and even different fibrillar states, makes it difficult to raise antibodies that would be efficacious in neutralizing all conformations of aSN. However, the C-terminal 20-40 residues of aSN are a promising epitope for antibody development. It is primarily disordered in both monomeric and aggregated forms, and an anti-C-terminal antibody will therefore be able to bind all forms. Furthermore, it might not interfere with the folding of aSN into membranes, which could be important for its physiological role. Here we report a screen of a series of monoclonal antibodies, which all target the C-terminal of aSN. According to dot blot analyses, different antibodies bound different forms of aSN with different preferences and showed reduced binding to monomeric compared to aggregated (oligomeric and fibrillary) aSN. Consequently they have different effects on aSN's ability to fibrillate and permeabilize membranes. Generally, the antibodies with strongest binding to aggregated aSN in dot blot, also inhibited fibrillation and membrane permeabilization the most, and promoted formation of amorphous aggregates surrounded by small and thin fibers. This suggests that the development of antibodies that targets the C-terminus, exposed in the aggregated forms of aSN, may be beneficial for improved immunotherapy against PD.

Cerebrospinal Fluid Biomarkers of Neurodegeneration Are Decreased or Normal in Narcolepsy.

Objectives: To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. Methods: Twenty-one patients with central hypersomnia (10 type 1 narcolepsy, 5 type 2 narcolepsy, and 6 idiopathic hypersomnia cases), aged 33 years on average and with a disease duration of 2-29 years, and 12 healthy controls underwent CSF analyses of the levels of ß-amyloid, total tau protein (T-tau), phosphorylated tau protein (P-tau181), α-synuclein, neurofilament light chain (NF-L), and chitinase 3-like protein-1 (CHI3L1). Results: Levels of ß-amyloid were lower in patients with type 1 narcolepsy (375.4 ± 143.5 pg/mL) and type 2 narcolepsy (455.9 ± 65.0 pg/mL) compared to controls (697.9 ± 167.3 pg/mL, p < .05). Furthermore, in patients with type 1 narcolepsy, levels of T-tau (79.0 ± 27.5 pg/mL) and P-tau181 (19.1 ± 4.3 pg/mL) were lower than in controls (162.2 ± 49.9 pg/mL and 33.8 ± 9.2 pg/mL, p < .05). Levels of α-synuclein, NF-L, and CHI3L1 in CSF from narcolepsy patients were similar to those of healthy individuals. Conclusion: Six CSF biomarkers of neurodegeneration were decreased or normal in narcolepsy indicating that taupathy, synucleinopathy, and immunopathy are not prevalent in narcolepsy patients with a disease duration of 2-29 years. Lower CSF levels of ß-amyloid, T-tau protein, and P-tau181 in narcolepsy may indicate that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in the central nervous system.

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.A11.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a CAG-repeat expanding mutation in ATXN3. We generated induced pluripotent stem cells (iPSCs) from a SCA3 patient by electroporation of dermal fibroblasts with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of genomically integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. This iPSC line could be useful for the investigation of SCA3 disease mechanisms.

Generation of spinocerebellar ataxia type 3 patient-derived induced pluripotent stem cell line SCA3.B11.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by an expansion of the CAG-repeat in ATXN3. In this study, induced pluripotent stem cells (iPSCs) were generated from SCA3 patient dermal fibroblasts by electroporation with episomal plasmids encoding L-MYC, LIN28, SOX2, KLF4, OCT4 and short hairpin RNA targeting P53. The resulting iPSCs had normal karyotype, were free of integrated episomal plasmids, expressed pluripotency markers, could differentiate into the three germ layers in vitro and retained the disease-causing ATXN3 mutation. Potentially, this iPSC line could be a useful tool for the investigation of SCA3 disease mechanisms.