Lisa Zondler

Inflammatory dysregulation of blood monocytes in Parkinson’s disease patients

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

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.

Serum microRNAs in patients with genetic amyotrophic lateral sclerosis and pre-manifest mutation carriers

Knowledge about the nature of pathomolecular alterations preceding onset of symptoms in amyotrophic lateral sclerosis is largely lacking. It could not only pave the way for the discovery of valuable therapeutic targets but might also govern future concepts of pre-manifest disease modifying treatments. MicroRNAs are central regulators of transcriptome plasticity and participate in pathogenic cascades and/or mirror cellular adaptation to insults. We obtained comprehensive expression profiles of microRNAs in the serum of patients with familial amyotrophic lateral sclerosis, asymptomatic mutation carriers and healthy control subjects. We observed a strikingly homogenous microRNA profile in patients with familial amyotrophic lateral sclerosis that was largely independent from the underlying disease gene. Moreover, we identified 24 significantly downregulated microRNAs in pre-manifest amyotrophic lateral sclerosis mutation carriers up to two decades or more before the estimated time window of disease onset; 91.7% of the downregulated microRNAs in mutation carriers overlapped with the patients with familial amyotrophic lateral sclerosis. Bioinformatic analysis revealed a consensus sequence motif present in the vast majority of downregulated microRNAs identified in this study. Our data thus suggest specific common denominators regarding molecular pathogenesis of different amyotrophic lateral sclerosis genes. We describe the earliest pathomolecular alterations in amyotrophic lateral sclerosis mutation carriers known to date, which provide a basis for the discovery of novel therapeutic targets and strongly argue for studies evaluating presymptomatic disease-modifying treatment in amyotrophic lateral sclerosis.

Peripheral monocytes are functionally altered and invade the CNS in ALS patients.

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

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.

α-synuclein interacts with SOD1 and promotes its oligomerization

BackgroundParkinson’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.ResultsUsing 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.ConclusionThis study provides evidence for a novel interaction of α-synuclein and SOD1 that might be relevant for neurodegenerative diseases.

Proteasome impairment by α-synuclein

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

Age Increases Monocyte Adhesion on Collagen

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxβ2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin β2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.

Monocyte subtypes in ALS

Toll-like receptors (TLRs) are a category of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) derived from infected microorganisms. The recognition is followed by the activation of intracellular signaling cascades and leads to the production of type I interferons (IFNs), which are key cytokines in antiviral immunity. Subsequently, IFNs exert antiviral effects by inducing hundreds of IFN-stimulated genes (ISGs). TLR3, a member of TLRs, which recognizes double-stranded RNA (dsRNA), is expressed in astrocytes. The activation of astroglial TLR3 induces the expression of IFN-β, a major type I IFN, and various proinflammatory cytokines and chemokines including CXCL10. ISG56 and ISG54 are members of ISGs. Little is known about the role of ISG56 and ISG54 in TLR3 signaling in astrocytes. In addition, there have been no reports on the expression and function of ISG54 in astrocytes. Therefore, the aim of this study is to elucidate TLR3dependent ISG54 and ISG56 expressions in astrocytes. We initially confirmed that polyinosinic–polycytidylic acid (poly IC), an authentic dsRNA, up-regulated the expression of ISG54 and ISG56 in U373MG human astrocytoma cells. This reaction was mediated by the TLR3/ IFN-β pathway. We also found that ISG56 positively regulates the expression of ISG54, retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Silencing of ISG54, ISG56, RIG-I and MDA5 markedly inhibited the expression of a chemokine CXCL10 in response to poly IC. These results suggest that ISG54 and ISG56 are involved in the induction of CXCL10 in TLR3/ IFN-β signaling by cooperating with RIG-I and MDA5. ISG54 and ISG56 may contribute to both immune and inflammatory reactions elicited by the TLR3/IFN-β signaling pathway in astrocytes, and may play an important role in neuroimmunology.

Inflammatory dysregulation of blood monocytes in Parkinson's disease patients

Background: Plovamer acetate (PA) is a copolymer mixture of 4 amino acids of defined ratio rationally designed to have improved efficacy over glatiramer acetate (GA). Aside from binding to major histocompatibility complex (MHC) II molecules, both copolymers (PA and GA) have been proposed to act directly on monocytes through an MHC II-independent mechanism. Objective: To compare the effect of PA versus GA in the in vitro polarization of human monocytes to M1 and M2 macrophage subsets. Methods: Monocytes from human blood were polarized for 6 days towards M1 and M2 phenotypes in a medium containing granulocyte macrophage colony-stimulating factor and macrophage colonystimulating factor, respectively. M1 cultures were activated using interferon gamma plus lipopolysaccharide, and M2a or M2c using interleukin (IL)-4 or IL-10, respectively. RNA was isolated and multiple transcripts were analyzed to demonstrate M1/M2 polarization. The effect of exposure to PA or GA during the period of polarization was then assessed. Results: Human monocytes were successfully polarized to M1/M2 subsets as indicated by the pattern of transcripts induced after activation. CD206 and CD163 expression was detected by fluorescence-activated cell sorting on M2 cells, reliably discriminating them from M1 cells. Released cytokines tumor necrosis factor alpha (TNFalpha), IL12p70, and IL-1beta served as markers of activated M1 cells, and these, together with COX2 and CD80, were also detected by qPCR in M1 cells. PA inhibited the expression of TNFalpha, COX2, and IL12p40 by M1-activated cells, while GA either increased the expression or had no effect. Conversely, PA significantly potentiated the expression of CD163 and CD209 by M2activated cells, while GA had no effect. Conclusion: M1/M2 polarizing cultures were successfully established with the use of a variety of markers to distinguish the different lineages. When PA or GA was included during the polarization period, PA had a negative effect on the expression of M1-associated markers and a positive effect on the expression of M2-associated markers. GA, in contrast, enhanced M1 markers and had no effect on M2 markers. Together, these findings suggest that unlike GA, PA suppresses the establishment of a proinflammatory M1 phenotype, while enhancing an anti-inflammatory M2 phenotype, supporting the development of PA as a disease-modifying drug.