Urinary tract infections trigger synucleinopathy via the innate immune response.

Symptoms in the urogenital organs are common in multiple system atrophy (MSA), also in the years preceding the MSA diagnosis. It is unknown how MSA is triggered and these observations in prodromal MSA led us to hypothesize that synucleinopathy could be triggered by infection of the genitourinary tract causing ɑ-synuclein (ɑSyn) to aggregate in peripheral nerves innervating these organs. As a first proof that peripheral infections could act as a trigger in MSA, this study focused on lower urinary tract infections (UTIs), given the relevance and high frequency of UTIs in prodromal MSA, although other types of infection might also be important triggers of MSA. We performed an epidemiological nested-case control study in the Danish population showing that UTIs are associated with future diagnosis of MSA several years after infection and that it impacts risk in both men and women. Bacterial infection of the urinary bladder triggers synucleinopathy in mice and we propose a novel role of ɑSyn in the innate immune system response to bacteria. Urinary tract infection with uropathogenic E. coli results in the de novo aggregation of ɑSyn during neutrophil infiltration. During the infection, ɑSyn is released extracellularly from neutrophils as part of their extracellular traps. Injection of MSA aggregates into the urinary bladder leads to motor deficits and propagation of ɑSyn pathology to the central nervous system in mice overexpressing oligodendroglial ɑSyn. Repeated UTIs lead to progressive development of synucleinopathy with oligodendroglial involvement in vivo. Our results link bacterial infections with synucleinopathy and show that a host response to environmental triggers can result in ɑSyn pathology that bears semblance to MSA.

Heterozygous GBA D409V and ATP13a2 mutations do not exacerbate pathological α-synuclein spread in the prodromal preformed fibrils model in young mice.

Autophagic dysregulation and lysosomal impairment have been implicated in the pathogenesis of Parkinson's disease, partly due to the identification of mutations in multiple genes involved in these pathways such as GBA, SNCA, ATP13a2 (also known as PARK9), TMEM175 and LRRK2. Mutations resulting in lysosomal dysfunction are proposed to contribute to Parkinson's disease by increasing α-synuclein levels, that in turn may promote aggregation of this protein. Here, we used two different genetic models-one heterozygous for a mutated form of the GBA protein (D409V), and the other heterozygous for an ATP13a2 loss-of-function mutation, to test whether these mutations exacerbate the spread of α-synuclein pathology following injection of α-synuclein preformed fibrils in the olfactory bulb of 12-week-old mice. Contrary to our hypothesis, we found that mice harboring GBA D409V+/- and ATP13a2+/- mutations did not have exacerbated behavioral impairments or histopathology (α-synuclein, LAMP2, and Iba1) when compared to their wildtype littermates. This indicates that in the young mouse brain, neither the GBA D409V mutation or ATP13a2 loss-of-function mutation accelerate the spread of α-synuclein pathology. As a consequence, we postulate that these mutations increase Parkinson's disease risk only by acting in one of the initial, upstream events in the Parkinson's disease pathogenic process. Further, the mutations, and the molecular pathways they impact, appear to play a less important role once the pathogenic process has been triggered and therefore do not specifically influence α-synuclein pathology spread.

Exploring Aß Proteotoxicity and Therapeutic Candidates Using Drosophila melanogaster.

Alzheimer's disease is a widespread and devastating neurological disorder associated with proteotoxic events caused by the misfolding and aggregation of the amyloid-ß peptide. To find therapeutic strategies to combat this disease, Drosophila melanogaster has proved to be an excellent model organism that is able to uncover anti-proteotoxic candidates due to its outstanding genetic toolbox and resemblance to human disease genes. In this review, we highlight the use of Drosophila melanogaster to both study the proteotoxicity of the amyloid-ß peptide and to screen for drug candidates. Expanding the knowledge of how the etiology of Alzheimer's disease is related to proteotoxicity and how drugs can be used to block disease progression will hopefully shed further light on the field in the search for disease-modifying treatments.

Air pollution nanoparticle and alpha-synuclein fibrils synergistically decrease glutamate receptor A1, depending upon nPM batch activity.

Background: Epidemiological studies have variably linked air pollution to increased risk of Parkinson's disease (PD). However, there is little experimental evidence for this association. Alpha-synuclein (α-syn) propagation plays central roles in PD and glutamate receptor A1 (GluA1) is involved in memory and olfaction function. Methods: Each mouse was exposed to one of three different batches of nano-particulate matter (nPM) (300 µg/m3, 5 h/d, 3 d/week), collected at different dates, 2017-2019, in the same urban site. After these experiments, these nPM batches were found to vary in activity. C57BL/6 female mice (3 mo) were injected with pre-formed murine α-synuclein fibrils (PFFs) (0.4 µg), which act as seeds for α-syn aggregation. Two exposure paradigms were used: in Paradigm 1, PFFs were injected into olfactory bulb (OB) prior to 4-week nPM (Batch 5b) exposure and in Paradigm 2, PFFs were injected at 4th week during 10-week nPM exposure (Batches 7 and 9). α-syn pSer129, microglia Iba1, inflammatory cytokines, and Gria1 expression were measured by immunohistochemistry or qPCR assays. Results: As expected, α-syn pSer129 was detected in ipsilateral OB, anterior olfactory nucleus, amygdala and piriform cortex. One of the three batches of nPM caused a trend for elevated α-syn pSer129 in Paradigm 1, but two other batches showed no effect in Paradigm 2. However, the combination of nPM and PFF significantly decreased Gria1 mRNA in both the ipsi- and contra-lateral OB and frontal cortex for the most active two nPM batches. Neither nPM nor PFFs alone induced responses of microglia Iba1 and expression of Gria1 in the OB and cortex. Conclusion: Exposures to ambient nPM had weak effect on α-syn propagation in the brain in current experimental paradigms; however, nPM and α-syn synergistically downregulated the expression of Gria1 in both OB and cortex.

Targeting Chaperone/Co-Chaperone Interactions with Small Molecules: A Novel Approach to Tackle Neurodegenerative Diseases.

The dysfunction of the proteostasis network is a molecular hallmark of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Molecular chaperones are a major component of the proteostasis network and maintain cellular homeostasis by folding client proteins, assisting with intracellular transport, and interfering with protein aggregation or degradation. Heat shock protein 70 kDa (Hsp70) and 90 kDa (Hsp90) are two of the most important chaperones whose functions are dependent on ATP hydrolysis and collaboration with their co-chaperones. Numerous studies implicate Hsp70, Hsp90, and their co-chaperones in neurodegenerative diseases. Targeting the specific protein-protein interactions between chaperones and their particular partner co-chaperones with small molecules provides an opportunity to specifically modulate Hsp70 or Hsp90 function for neurodegenerative diseases. Here, we review the roles of co-chaperones in Hsp70 or Hsp90 chaperone cycles, the impacts of co-chaperones in neurodegenerative diseases, and the development of small molecules modulating chaperone/co-chaperone interactions. We also provide a future perspective of drug development targeting chaperone/co-chaperone interactions for neurodegenerative diseases.

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay.

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular processes. The conserved MEEVD pentapeptide at the C-terminus of Hsp90 is responsible for the interaction with the tetratricopeptide repeat (TPR) motif of co-chaperones. FK506-binding protein (FKBP) 51 and FKBP52 are two similar TPR-motif co-chaperones involved in steroid hormone-dependent diseases with different functions. Therefore, identifying molecules specifically blocking interactions between Hsp90 and FKBP51 or FKBP52 provides a promising therapeutic potential for several human diseases. Here, we describe the protocol for an amplified luminescent proximity homogenous assay to probe interactions between Hsp90 and its partner co-chaperones FKBP51 and FKBP52. First, we have purified the TPR motif-containing proteins FKBP51 and FKBP52 in glutathione S-transferase (GST)-tagged form. Using the glutathione-linked donor beads with GST-fused TPR-motif proteins and the acceptor beads coupled with a 10-mer C-terminal peptide of Hsp90, we have probed protein-protein interactions in a homogeneous environment. We have used this assay to screen small molecules to disrupt Hsp90-FKBP51 or Hsp90-FKBP52 interactions and identified potent and selective Hsp90-FKBP51 interaction inhibitors.

An extended release GLP-1 analogue increases α-synuclein accumulation in a mouse model of prodromal Parkinson's disease.

The repurposing of drugs developed to treat type 2 diabetes for the treatment of Parkinson's disease (PD) was encouraged by the beneficial effect exerted by the glucagon-like peptide 1 (GLP-1) analogue exenatide in a phase 2 clinical trial. The effects of GLP-1 analogues have been investigated extensively using rodent toxin models of PD. However, many of the toxin-based models used lack robust α-synuclein (α-syn) pathology, akin to the Lewy bodies and neurites seen in PD. One prior study has reported a protective effect of a GLP-1 analogue on midbrain dopamine neurons following injection of α-syn preformed fibrils (PFF) into the striatum. Here, we used olfactory bulb injections of PFF as a model of prodromal PD and monitored the effect of a long-acting GLP-1 analogue on the propagation of α-syn pathology in the olfactory system. Thirteen weeks after PFF injection, mice treated with long-acting the GLP-1 analogue had a significant increase in pathological α-syn in brain regions connected to the olfactory bulb, accompanied by signs of microglia activation. Our results suggest that the nature of the neuronal insult and intrinsic properties of the targeted neuronal population markedly influence the effect of GLP-1 analogues.

Deficits in olfactory sensitivity in a mouse model of Parkinson's disease revealed by plethysmography of odor-evoked sniffing.

Hyposmia is evident in over 90% of Parkinson's disease (PD) patients. A characteristic of PD is intraneuronal deposits composed in part of α-synuclein fibrils. Based on the analysis of post-mortem PD patients, Braak and colleagues suggested that early in the disease α-synuclein pathology is present in the dorsal motor nucleus of the vagus, as well as the olfactory bulb and anterior olfactory nucleus, and then later affects other interconnected brain regions. Here, we bilaterally injected α-synuclein preformed fibrils into the olfactory bulbs of wild type male and female mice. Six months after injection, the anterior olfactory nucleus and piriform cortex displayed a high α-synuclein pathology load. We evaluated olfactory perceptual function by monitoring odor-evoked sniffing behavior in a plethysmograph at one-, three- and six-months after injection. No overt impairments in the ability to engage in sniffing were evident in any group, suggesting preservation of the ability to coordinate respiration. At all-time points, females injected with fibrils exhibited reduced odor detection sensitivity, which was observed with the semi-automated plethysmography apparatus, but not a buried pellet test. In future studies, this sensitive methodology for assessing olfactory detection deficits could be used to define how α-synuclein pathology affects other aspects of olfactory perception and to clarify the neuropathological underpinnings of these deficits.

Serum amyloid P component promotes formation of distinct aggregated lysozyme morphologies and reduces toxicity in Drosophila flies expressing F57I lysozyme.

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.

Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aß fly model and the AßPP-BACE1 fly model. In the Aß fly model, the Aß peptide is fused to a secretion sequence and directly overexpressed. In the AßPP-BACE1 model, human AßPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aß peptides and other AßPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aß fly model, this correlates with high Aß1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AßPP-BACE1 fly model, neuronal cell death correlates with low Aß1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AßPP/Aß antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AßPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.

Inhibiting the mitochondrial pyruvate carrier does not ameliorate synucleinopathy in the absence of inflammation or metabolic deficits.

Epidemiological studies suggest a link between type-2 diabetes and Parkinson's disease (PD) risk. Treatment of type-2 diabetes with insulin sensitizing drugs lowers the risk of PD. We previously showed that the insulin sensitizing drug, MSDC-0160, ameliorates pathogenesis in some animal models of PD. MSDC-0160 reversibly binds the mitochondrial pyruvate carrier (MPC) protein complex, which has an anti-inflammatory effect and restores metabolic deficits. Since PD is characterized by the deposition of α-synuclein (αSyn), we hypothesized that inhibiting the MPC might directly inhibit αSyn aggregation in vivo in mammals. To answer if modulation of MPC can reduce the development of αSyn assemblies, and reduce neurodegeneration, we treated two chronic and progressive mouse models; a viral vector-based αSyn overexpressing model and a pre-formed fibril (PFF) αSyn seeding model with MSDC-0160. These two models present distinct types of αSyn pathology but lack inflammatory or autophagy deficits. Contrary to our hypothesis, we found that a modulation of MPC in these models did not reduce the accumulation of αSyn aggregates or mitigate neurotoxicity. Instead, MSDC-0160 changed the post-translational modification and aggregation features of αSyn. These results are consistent with the lack of a direct effect of MPC modulation on synuclein clearance in these models.

Serum Amyloid P Component Ameliorates Neurological Damage Caused by Expressing a Lysozyme Variant in the Central Nervous System of Drosophila melanogaster.

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

AßPP processing results in greater toxicity per amount of Aß1-42 than individually expressed and secreted Aß1-42 in Drosophila melanogaster.

The aggregation of the amyloid-ß (Aß) peptide into fibrillar deposits has long been considered the key neuropathological hallmark of Alzheimer's disease (AD). Aß peptides are generated from proteolytic processing of the transmembrane Aß precursor protein (AßPP) via sequential proteolysis through the ß-secretase activity of ß-site AßPP-cleaving enzyme (BACE1) and by the intramembranous enzyme γ-secretase. For over a decade, Drosophila melanogaster has been used as a model organism to study AD, and two different approaches have been developed to investigate the toxicity caused by AD-associated gene products in vivo In one model, the Aß peptide is directly over-expressed fused to a signal peptide, allowing secretion of the peptide into the extracellular space. In the other model, human AßPP is co-expressed with human BACE1, resulting in production of the Aß peptide through the processing of AßPP by BACE1 and by endogenous fly γ-secretase. Here, we performed a parallel study of flies that expressed the Aß1-42 peptide alone or that co-expressed AßPP and BACE1. Toxic effects (assessed by eye phenotype, longevity and locomotor assays) and levels of the Aß1-42, Aß1-40 and Aß1-38 peptides were examined. Our data reveal that the toxic effect per amount of detected Aß1-42 peptide was higher in the flies co-expressing AßPP and BACE1 than in the Aß1-42-expressing flies, and that the co-existence of Aß1-42 and Aß1-40 in the flies co-expressing AßPP and BACE1 could be of significant importance to the neurotoxic effect detected in these flies. Thus, the toxicity detected in these two fly models seems to have different modes of action and is highly dependent on how and where the peptide is generated rather than on the actual level of the Aß1-42 peptide in the flies. This is important knowledge that needs to be taken into consideration when using Drosophila models to investigate disease mechanisms or therapeutic strategies in AD research.

Beneficial effects of increased lysozyme levels in Alzheimer's disease modelled in Drosophila melanogaster.

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimer's disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome-wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aß1-42 or AßPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aß1-42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aß1-42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aß1-42 and caused a beneficial effect by binding of lysozyme to toxic species of Aß1-42 , which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.