Toll-like receptor 4 deficiency facilitates α-synuclein propagation and neurodegeneration in a mouse model of prodromal Parkinson's disease.

The evidence linking innate immunity mechanisms and neurodegenerative diseases is growing, but the specific mechanisms are incompletely understood. Experimental data suggest that microglial TLR4 mediates the uptake and clearance of α-synuclein also termed synucleinophagy. The accumulation of misfolded α-synuclein throughout the brain is central to Parkinson's disease (PD). The distribution and progression of the pathology is often attributed to the propagation of α-synuclein. Here, we apply a classical α-synuclein propagation model of prodromal PD in wild type and TLR4 deficient mice to study the role of TLR4 in the progression of the disease. Our data suggest that TLR4 deficiency facilitates the α-synuclein seed spreading associated with reduced lysosomal activity of microglia. Three months after seed inoculation, more pronounced proteinase K-resistant α-synuclein inclusion pathology is observed in mice with TLR4 deficiency. The facilitated propagation of α-synuclein is associated with early loss of dopamine transporter (DAT) signal in the striatum and loss of dopaminergic neurons in substantia nigra pars compacta of TLR4 deficient mice. These new results support TLR4 signaling as a putative target for disease modification to slow the progression of PD and related disorders.

Targeting α-synuclein by PD03 AFFITOPE® and Anle138b rescues neurodegenerative pathology in a model of multiple system atrophy: clinical relevance.

BACKGROUND: Misfolded oligomeric α-synuclein plays a pivotal role in the pathogenesis of α-synucleinopathies including Parkinson's disease and multiple system atrophy, and its detection parallels activation of microglia and a loss of neurons in the substantia nigra pars compacta. Here we aimed to analyze the therapeutic efficacy of PD03, a new AFFITOPE® immunotherapy approach, either alone or in combination with Anle138b, in a PLP-α-syn mouse model. METHODS: The PLP-α-syn mice were treated with PD03 immunotherapy, Anle138b, or a combination of two. Five months after study initiation, the mice underwent behavioral testing and were sacrificed for neuropathological analysis. The treatment groups were compared to the vehicle group with regard to motor performance, nigral neuronal loss, microglial activation and α-synuclein pathology. RESULTS: The PLP-α-syn mice receiving the PD03 or Anle138b single therapy showed improvement of gait deficits and preservation of nigral dopaminergic neurons associated with the reduced α-synuclein oligomer levels and decreased microglial activation. The combined therapy with Anle138b and PD03 resulted in lower IgG binding in the brain as compared to the single immunotherapy with PD03. CONCLUSIONS: PD03 and Anle138b can selectively target oligomeric α-synuclein, resulting in attenuation of neurodegeneration in the PLP-α-syn mice. Both approaches are potential therapies that should be developed further for disease modification in α-synucleinopathies.

Shock waves promote spinal cord repair via TLR3.

Spinal cord injury (SCI) remains a devastating condition with poor prognosis and very limited treatment options. Affected patients are severely restricted in their daily activities. Shock wave therapy (SWT) has shown potent regenerative properties in bone fractures, wounds, and ischemic myocardium via activation of the innate immune receptor TLR3. Here, we report on the efficacy of SWT for regeneration of SCI. SWT improved motor function and decreased lesion size in WT but not Tlr3-/- mice via inhibition of neuronal degeneration and IL6-dependent recruitment and differentiation of neuronal progenitor cells. Both SWT and TLR3 stimulation enhanced neuronal sprouting and improved neuronal survival, even in human spinal cord cultures. We identified tlr3 as crucial enhancer of spinal cord regeneration in zebrafish. Our findings indicate that TLR3 signaling is involved in neuroprotection and spinal cord repair and suggest that TLR3 stimulation via SWT could become a potent regenerative treatment option.

Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.

Toll-like receptor 4 stimulation with monophosphoryl lipid A ameliorates motor deficits and nigral neurodegeneration triggered by extraneuronal α-synucleinopathy.

BACKGROUND: Alpha-synuclein (α-syn) aggregation represents the pathological hallmark of α-synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Toll-like receptors (TLRs) are a family of highly conserved molecules that recognize pathogen-associated molecular patterns and define the innate immunity response. It was previously shown that TLR4 plays a role in the clearance of α-syn, suggesting that TLR4 up-regulation in microglia may be a natural mechanism to improve the clearance of α-syn. However, administration of TLR4 ligands could also lead to dangerous adverse effects associated with the induction of toxic inflammatory responses. Monophosphoryl lipid A (MPLA) is a TLR4 selective agonist and a potent inducer of phagocytosis which does not trigger strong toxic inflammatory responses as compared to lipopolysaccharide (LPS). We hypothesize that MPLA treatment will lead to increased clearance of α-syn inclusions in the brain of transgenic mice overexpressing α-syn in oligodendrocytes under the proteolipid protein promoter (PLP-α-syn mouse model of MSA), without triggering toxic cytokine release, thus leading to a general amelioration of the pathology. METHODS: Six month old PLP-α-syn mice were randomly allocated to four groups and received weekly intraperitoneal injections of MPLA (50 or 100 µg), LPS or vehicle. After a 12-week treatment period, motor behavior was assessed with the pole test. Brains and plasma samples were collected for neuropathological and immunological analysis. RESULTS: Chronic systemic MPLA treatment of PLP-α-syn mice led to increased uptake of α-syn by microglial cells, a significant motor improvement, rescue of nigral dopaminergic and striatal neurons and region-specific reduction of the density of oligodendroglial α-syn cytoplasmic inclusions in the absence of a marked systemic inflammatory response. CONCLUSION: Our findings demonstrate beneficial effects of chronic MPLA treatment in transgenic PLP-α-syn mice. MPLA appears to be an attractive therapeutic candidate for disease modification trials in MSA and related α-synucleinopathies.

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications.

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.