Amyloid-ß aggregates activate peripheral monocytes in mild cognitive impairment.

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer's Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aß aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer's Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aß aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aß. Hydrodynamic calculations suggest Aß aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Sex-dimorphic neuroprotective effect of CD163 in an α-synuclein mouse model of Parkinson's disease.

Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.

Enhanced production of mesencephalic dopaminergic neurons from lineage-restricted human undifferentiated stem cells.

Current differentiation protocols for generating mesencephalic dopaminergic (mesDA) neurons from human pluripotent stem cells result in grafts containing only a small proportion of mesDA neurons when transplanted in vivo. In this study, we develop lineage-restricted undifferentiated stem cells (LR-USCs) from pluripotent stem cells, which enhances their potential for differentiating into caudal midbrain floor plate progenitors and mesDA neurons. Using a ventral midbrain protocol, 69% of LR-USCs become bona fide caudal midbrain floor plate progenitors, compared to only 25% of human embryonic stem cells (hESCs). Importantly, LR-USCs generate significantly more mesDA neurons under midbrain and hindbrain conditions in vitro and in vivo. We demonstrate that midbrain-patterned LR-USC progenitors transplanted into 6-hydroxydopamine-lesioned rats restore function in a clinically relevant non-pharmacological behavioral test, whereas midbrain-patterned hESC-derived progenitors do not. This strategy demonstrates how lineage restriction can prevent the development of undesirable lineages and enhance the conditions necessary for mesDA neuron generation.

The systemic immune response in Parkinson's disease: focus on the peripheral immune component.

During Parkinson's disease (PD), both the central nervous system (CNS) and peripheral nervous system (PNS) are affected. In parallel, innate immune cells respond early to neuronal changes and alpha-synuclein (α-syn) pathology. Moreover, some of the affected neuronal groups innervate organs with a relevant role in immunity. Consequently, not only microglia, but also peripheral immune cells are altered, resulting in a systemic immune response. Innate and adaptive immune cells may participate in the neurodegenerative process by acting peripherally, infiltrating the brain, or releasing mediators that can protect or harm neurons. However, the sequence of the changes and the significance of each immune compartment in the disease remain to be clarified. In this review, we describe current understanding of the peripheral immune response in PD and discuss the road ahead.

Impact of aging on animal models of Parkinson's disease.

Aging is the biggest risk factor for developing Parkinson's disease (PD), the second most common neurodegenerative disorder. Several animal models have been developed to explore the pathophysiology underlying neurodegeneration and the initiation and spread of alpha-synuclein-related PD pathology, and to investigate biomarkers and therapeutic strategies. However, bench-to-bedside translation of preclinical findings remains suboptimal and successful disease-modifying treatments remain to be discovered. Despite aging being the main risk factor for developing idiopathic PD, most studies employ young animals in their experimental set-up, hereby ignoring age-related cellular and molecular mechanisms at play. Consequently, studies in young animals may not be an accurate reflection of human PD, limiting translational outcomes. Recently, it has been shown that aged animals in PD research demonstrate a higher susceptibility to developing pathology and neurodegeneration, and present with a more disseminated and accelerated disease course, compared to young animals. Here we review recent advances in the investigation of the role of aging in preclinical PD research, including challenges related to aged animal models that are limiting widespread use. Overall, current findings indicate that the use of aged animals may be required to account for age-related interactions in PD pathophysiology. Thus, although the use of older animals has disadvantages, a model that better represents clinical disease within the elderly would be more beneficial in the long run, as it will increase translational value and minimize the risk of therapies failing during clinical studies. Furthermore, we provide recommendations to manage the challenges related to aged animal models.

Increased maternofoetal transfer of antibodies in a murine model of systemic lupus erythematosus, but no immune activation and neuroimmune sequelae in offspring.

Maternally transferred autoantibodies can negatively impact the development and health of offspring, increasing the risk of neurodevelopmental disorders. We used embryo transfers to examine maternofoetal immune imprinting in the autoimmune BXSB/MpJ mouse model. Anti-double-stranded DNA antibodies and total immunoglobulins were measured, using allotypes of the IgG subclass to distinguish maternally transferred antibodies from those produced endogenously. Frequencies of germinal center and plasma cells were analysed by flow cytometry. Microglial morphology in offspring CNS was assessed using immunohistochemistry. In contrast to prior findings, our results indicate that BXSB/MpJ mothers display a mild autoimmune phenotype, which does not significantly impact the offspring.

Neuroinflammation and Immune Changes in Prodromal Parkinson's Disease and Other Synucleinopathies.

Multiple lines of clinical and pre-clinical research support a pathogenic role for neuroinflammation and peripheral immune system dysfunction in Parkinson's disease. In this paper, we have reviewed and summarised the published literature reporting evidence of neuroinflammation and peripheral immune changes in cohorts of patients with isolated REM sleep behaviour disorder and non-manifesting carriers of GBA or LRRK2 gene mutations, who have increased risk for Parkinsonism and synucleinopathies, and could be in the prodromal stage of these conditions. Taken together, the findings of these studies suggest that the early stages of pathology in Parkinsonism involve activation of both the central and peripheral immune systems with significant crosstalk. We consider these findings with respect to those found in patients with clinical Parkinson's disease and discuss their possible pathological roles. Moreover, those factors possibly associated with the immune response, such as the immunomodulatory role of the affected neurotransmitters and the changes in the gut-brain axis, are also considered.

Changes in CD163+, CD11b+, and CCR2+ peripheral monocytes relate to Parkinson's disease and cognition.

Alpha-synuclein pathology is associated with immune activation and neurodegeneration in Parkinson's disease. The immune activation involves not only microglia but also peripheral immune cells, such as mononuclear phagocytes found in blood and infiltrated in the brain. Understanding peripheral immune involvement is essential for developing immunomodulatory treatment. Therefore, we aimed to study circulating mononuclear phagocytes in early- and late-stage Parkinson's disease, defined by disease duration of less or more than five years, respectively, and analyze their association with clinical phenotypes. We performed a cross-sectional multi-color flow cytometry study on 78 sex-balanced individuals with sporadic Parkinson's disease, 28 controls, and longitudinal samples from seven patients and one control. Cell frequencies and surface marker expressions on natural killer cells, monocyte subtypes, and dendritic cells were compared between groups and correlated with standardized clinical scores. We found elevated frequencies and surface levels of migration- (CCR2, CD11b) and phagocytic- (CD163) markers, particularly on classical and intermediate monocytes in early Parkinson's disease. HLA-DR expression was increased in advanced stages of the disease, whereas TLR4 expression was decreased in women with Parkinson's Disease. The disease-associated immune changes of CCR2 and CD11b correlated with worse cognition. Increased TLR2 expression was related to worse motor symptoms. In conclusion, our data highlights the TLR2 relevance in the symptomatic motor presentation of the disease and a role for peripheral CD163+ and migration-competent monocytes in Parkinson's disease cognitive defects. Our study suggests that the peripheral immune system is dynamically altered in Parkinson's disease stages and directly related to both symptoms and the sex bias of the disease.

α-Synuclein Overexpression Increases Dopamine D2/3 Receptor Binding and Immune Activation in a Model of Early Parkinson's Disease.

Progressive degeneration of dopaminergic neurons, immune activation, and α-synuclein pathology characterize Parkinson's disease (PD). We previously reported that unilateral intranigral injection of recombinant adeno-associated viral (rAAV) vectors encoding wild-type human α-synuclein produced a rat model of early PD with dopamine terminal dysfunction. Here we tested the hypothesis that decreases in dopamine result in increased postsynaptic dopamine D2/D3 receptor expression, neuroinflammation, and reduced synaptic vesicle glycoprotein 2A (SV2A) density. Rats were injected with rAAV encoding α-synuclein or green fluorescent protein and subjected to non-pharmacological motor tests, before euthanization at 12 weeks post-injection. We performed: (1) in situ hybridization of nigral tyrosine hydroxylase mRNA, (2) HPLC of striatal dopamine content, and (3) autoradiography with [3H]raclopride, [3H]DTBZ, [3H]GBR12935, [3H]PK11195, and [3H]UCB-J to measure binding at D2/3 receptors, vesicular monoamine transporter 2, dopamine transporters, mitochondrial translocator protein, and SV2A, respectively. rAAV-α-synuclein induced motor asymmetry and reduced tyrosine hydroxylase mRNA and dopamine content in ipsilateral brain regions. This was paralleled by elevated ipsilateral postsynaptic dopamine D2/3 receptor expression and immune activation, with no changes to synaptic SV2A density. In conclusion, α-synuclein overexpression results in dopaminergic degeneration that induced compensatory increases in D2/3 binding and immune activation, recapitulating many of the pathological characteristics of PD.

Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

Monocyte markers correlate with immune and neuronal brain changes in REM sleep behavior disorder.

Synucleinopathies are neurodegenerative diseases with both central and peripheral immune responses. However, whether the peripheral immune changes occur early in disease and their relation to brain events is yet unclear. Isolated rapid-eye-movement (REM) sleep behavior disorder (iRBD) can precede synucleinopathy-related parkinsonism and provides a prodromal phenotype to study early Parkinson's disease events. In this prospective case-control study, we describe monocytic markers in a cohort of iRBD patients that were associated with the brain-imaging markers of inflammation and neuronal dysfunction. Using 11C-PK11195 positron emission tomography (PET), we previously showed increased immune activation in the substantia nigra of iRBD patients, while 18F-DOPA PET detected reduced putaminal dopaminergic function. Here we describe that patients' blood monocytic cells showed increased expression of CD11b, while HLA-DR expression was decreased compared to healthy controls. The iRBD patients had increased classical monocytes and mature natural killer cells. Remarkably, the levels of expression of Toll-like receptor 4 (TLR4) on blood monocytes in iRBD patients were positively correlated with nigral immune activation measured by 11C-PK11195 PET and negatively correlated with putaminal 18F-DOPA uptake; the opposite was seen for the percentage of CD163+ myeloid cells. This suggesting a deleterious role for TLR4 and, conversely, a protective one for the CD163 expression. We show an association between peripheral blood monocytes and brain immune and dopaminergic changes in a synucleinopathy-related disorder, thus suggesting a cross-talk among periphery and brain during the disease.

Periphery and brain, innate and adaptive immunity in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder where alpha-synuclein plays a central role in the death and dysfunction of neurons, both, in central, as well as in the peripheral nervous system. Besides the neuronal events observed in patients, PD also includes a significant immune component. It is suggested that the PD-associated immune response will have consequences on neuronal health, thus opening immunomodulation as a potential therapeutic strategy in PD. The immune changes during the disease occur in the brain, involving microglia, but also in the periphery with changes in cells of the innate immune system, particularly monocytes, as well as those of adaptive immunity, such as T-cells. This realization arises from multiple patient studies, but also from data in animal models of the disease, providing strong evidence for innate and adaptive immune system crosstalk in the central nervous system and periphery in PD. Here we review the data showing that alpha-synuclein plays a crucial role in the activation of the innate and adaptive immune system. We will also describe the studies suggesting that inflammation in PD includes early changes in innate and adaptive immune cells that develop dynamically through time during disease, contributing to neuronal degeneration and symptomatology in patients. This novel finding has contributed to the definition of PD as a multisystem disease that should be approached in a more integratory manner rather than a brain-focused classical approach.

In vivo imaging of synaptic SV2A protein density in healthy and striatal-lesioned rats with [11C]UCB-J PET.

The number of functionally active synapses provides a measure of neural integrity, with reductions observed in neurodegenerative disorders. [11C]UCB-J binds to synaptic vesicle 2A (SV2A) transmembrane protein located in secretory vesicles. We aimed to assess [11C]UCB-J PET as an in vivo biomarker of regional cerebral synaptic SV2A density in rat lesion models of neurodegeneration. Healthy anesthetized rats had [11C]UCB-J PET and arterial blood sampling. We compared different models describing [11C]UCB-J brain uptake kinetics to determine its regional distribution. Blocking studies were performed with levetiracetam (LEV), an antiepileptic SV2A antagonist. Tracer binding was measured in rodent unilateral acute lesion models of Parkinsonism and Huntington's disease, induced with 6-hydroxydopamine (6-OHDA) and quinolinic acid (QA), respectively. [3H]UCB-J autoradiography was performed in postmortem tissue. Rat brain showed high and fast [11C]UCB-J uptake and washout with up to 80% blockade by LEV. [11C]UCB-J PET showed a 6.2% decrease in ipsilateral striatal SV2A binding after 6-OHDA and 39.3% and 55.1% decreases after moderate and high dose QA confirmed by autoradiography. In conclusion, [11C]UCB-J PET provides a good in vivo marker of synaptic SV2A density which can potentially be followed longitudinally along with synaptic responses to putative neuroprotective agents in models of neurodegeneration.

Soluble CD163 Changes Indicate Monocyte Association With Cognitive Deficits in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder with a significant immune component, as demonstrated by changes in immune biomarkers in patients' biofluids. However, which specific cells are responsible for those changes is unclear because most immune biomarkers can be produced by various cell types. OBJECTIVES: The aim of this study was to explore monocyte involvement in PD. METHODS: We investigated the monocyte-specific biomarker sCD163, the soluble form of the receptor CD163, in cerebrospinal fluid (CSF) and serum in two experiments, and compared it with other biomarkers and clinical data. Potential connections between CD163 and alpha-synuclein were studied in vitro. RESULTS: CSF-sCD163 increased in late-stage PD and correlated with the PD biomarkers alpha-synuclein, Tau, and phosphorylated Tau, whereas it inversely correlated with the patients' cognitive scores, supporting monocyte involvement in neurodegeneration and cognition in PD. Serum-sCD163 increased only in female patients, suggesting a sex-distinctive monocyte response. CSF-sCD163 also correlated with molecules associated with adaptive and innate immune system activation and with immune cell recruitment to the brain. Serum-sCD163 correlated with proinflammatory cytokines and acute-phase proteins, suggesting a relation to chronic systemic inflammation. Our in vitro study showed that alpha-synuclein activates macrophages and induces shedding of sCD163, which in turn enhances alpha-synuclein uptake by myeloid cells, potentially participating in its clearance. CONCLUSIONS: Our data present sCD163 as a potential cognition-related biomarker in PD and suggest a role for monocytes in both peripheral and brain immune responses. This may be directly related to alpha-synuclein's proinflammatory capacity but could also have consequences for alpha-synuclein processing. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

PET imaging reveals early and progressive dopaminergic deficits after intra-striatal injection of preformed alpha-synuclein fibrils in rats.

Alpha-synuclein (a-syn) can aggregate and form toxic oligomers and insoluble fibrils which are the main component of Lewy bodies. Intra-neuronal Lewy bodies are a major pathological characteristic of Parkinson's disease (PD). These fibrillar structures can act as seeds and accelerate the aggregation of monomeric a-syn. Indeed, recent studies show that injection of preformed a-syn fibrils (PFF) into the rodent brain can induce aggregation of the endogenous monomeric a-syn resulting in neuronal dysfunction and eventual cell death. We injected 8 µg of murine a-syn PFF, or soluble monomeric a-syn into the right striatum of rats. The animals were monitored behaviourally using the cylinder test, which measures paw asymmetry, and the corridor task that measures lateralized sensorimotor response to sugar treats. In vivo PET imaging was performed after 6, 13 and 22 weeks using [11C]DTBZ, a marker of the vesicular monoamine 2 transporter (VMAT2), and after 15 and 22 weeks using [11C]UCB-J, a marker of synaptic SV2A protein in nerve terminals. Histology was performed at the three time points using antibodies against dopaminergic markers, aggregated a-syn, and MHCII to evaluate the immune response. While the a-syn PFF injection caused only mild behavioural changes, [11C]DTBZ PET showed a significant and progressive decrease of VMAT2 binding in the ipsilateral striatum. This was accompanied by a small progressive decrease in [11C]UCB-J binding in the same area. In addition, our histological analysis revealed a gradual spread of misfolded a-syn pathology in areas anatomically connected to striatum that became bilateral with time. The striatal a-syn PFF injection resulted in a progressive unilateral degeneration of dopamine terminals, and an early and sustained presence of MHCII positive ramified microglia in the ipsilateral striatum and substantia nigra. Our study shows that striatal injections of a-syn fibrils induce progressive pathological synaptic dysfunction prior to cell death that can be detected in vivo with PET. We confirm that intrastriatal injection of a-syn PFFs provides a model of progressive a-syn pathology with loss of dopaminergic and synaptic function accompanied by neuroinflammation, as found in human PD.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Alterations in Blood Monocyte Functions in Parkinson's Disease.

BACKGROUND: PD is a multisystem disease where both central and peripheral nervous systems are affected. This systemic involvement also includes the immune response in PD, which implicates not only microglia in the brain, but also peripheral immune cells, such as monocytes; however, this aspect has been understudied. OBJECTIVES: The purpose of this study was to investigate the PD-related changes in peripheral immune cells, their responsiveness to stimulation, and their ability to release immunomodulatory molecules that might have consequences for the disease progression. METHODS: Using flow cytometry, we investigated the monocytic population in peripheral blood mononuclear cells from PD patients and healthy individuals. We also evaluated the in vitro response to inflammogen lipopolysaccharides and to fibrillar α-synuclein by measuring the expression of CD14, CD163, and HLA-DR and by analysis of soluble immune-related molecules in the supernatant. RESULTS: Peripheral blood immune cells from PD patients had lower survival in culture, but showed a higher monocytic proliferative ability than control cells, which was correlated with shorter disease duration and late disease onset. In addition, PD patients' cells were less responsive to stimulation, as shown by the lack of changes in CD163 and CD14 expression, and by the absence of significant upregulation of anti-inflammatory cytokines in culture. Moreover, PD peripheral immune cells shed lower in vitro levels of soluble CD163, which suggests a less responsive monocytic population and/or an activation status different from control cells. Interestingly, some of the results were sex associated, supporting a differential immune response in females versus males. CONCLUSIONS: Our data suggest that PD involves monocytic changes in blood. These cells show reduced viability and are unresponsive to specific stimuli, which might have a relevant consequence for disease progression. © 2019 International Parkinson and Movement Disorder Society.

Immune system responses in Parkinson's disease: Early and dynamic.

The neuropathological hallmarks of Parkinson's disease (PD) are the degeneration and death of dopamine-producing neurons in the ventral midbrain, the widespread intraneuronal aggregation of alpha-synuclein (α) in Lewy bodies and neurites, neuroinflammation, and gliosis. Signs of microglia activation in the PD brain postmortem as well as during disease development revealed by neuroimaging, implicate immune responses in the pathophysiology of the disease. Intensive research during the last two decades has advanced our understanding of the role of these responses in the disease process, yet many questions remain unanswered. A transformative finding in the field has been the confirmation that in vivo microglia are able to respond directly to pathological a-syn aggregates but also to neuronal dysfunction due to intraneuronal a-syn toxicity well in advance of neuronal death. In addition, clinical research and disease models have revealed the involvement of both the innate and adaptive immune systems. Indeed, the data suggest that PD leads not only to a microglia response, but also to a cellular and humoral peripheral immune response. Together, these findings compel us to consider a more holistic view of the immunological processes associated with the disease. Central and peripheral immune responses aimed at maintaining neuronal health will ultimately have consequences on neuronal survival. We will review here the most significant findings that have contributed to the current understanding of the immune response in PD, which is proposed to occur early, involve peripheral and brain immune cells, evolve as neuronal dysfunction progresses, and is likely to influence disease progression.