Therapeutic anti-amyloid ß antibodies cause neuronal disturbances.

INTRODUCTION: Recent published clinical trial safety data showed that 41% of Alzheimer patients experienced amyloid-related imaging abnormalities (ARIA), marks of microhemorrhages and edema in the brain, following administration of Biogen's Aduhelm/aducanumab (amino acids 3-7 of the Aß peptide). Similarly, Janssen/Pfizer's Bapineuzumab (amino acids 1-5 of the Aß peptide) and Roche's Gantenerumab (amino acids 2-11/18-27 of the Aß peptide) also displayed ARIA in clinical trials, including microhemorrhage and focal areas of inflammation or vasogenic edema, respectively. The molecular mechanisms underlying ARIA caused by therapeutic anti-Aß antibodies remain largely unknown, however, recent reports demonstrated that therapeutic anti-prion antibodies activate neuronal allergenic proteomes following cross-linking cellular prion protein. METHODS: Here, we report that treatment of human induced pluripotent stem cells- derived neurons (HSCN) from a non-demented donor, co-cultured with human primary microglia with anti-Aß1-6, or anti-Aß17-23 antibodies activate a significant number of allergenic-related proteins as assessed by mass spectrometry. RESULTS: Interestingly, a large proportion of the identified proteins included cytokines such as interleukin (IL)-4, IL-12, and IL-13 suggesting a type-1 hypersensitivity response. Following flow cytometry analysis, several proinflammatory cytokines were significantly elevated following anti-Aß1-6, or anti-Aß17-23 antibody treatment. DISCUSSION: These results justify further and more robust investigation of the molecular mechanisms of ARIA during immunotherapy study trials of AD. HIGHLIGHTS: Allergenic-related proteins are often linked with Alzheimer's disease (AD). We investigated the effects of amyloid beta (Aß) immunotherapy on stem cell derived neurons and primary neuronal cells co-cultured with microglia. Anti-Aß antibody treatment of neurons or neurons co-cultured with microglia led to activation of a substantial number of allergenic-related genes. These allergenic-related genes are associated with endothelial dysfunction possibly responsible for ARIA.

Case Report: Synucleinopathy Associated With Phalaris Neurotoxicity in Sheep.

Chronic intoxication with tryptamine-alkaloid-rich Phalaris species (spp.) pasture plants is known colloquially as Phalaris staggers syndrome, a widely occurring neurological disorder of sheep, cattle, horses, and kangaroos. Of comparative interest, structurally analogous tryptamine-alkaloids cause experimental parkinsonism in primates. This study aimed to investigate the neuropathological changes associated with spontaneous cases of Phalaris staggers in sheep with respect to those encountered in human synucleinopathy. In sheep affected with Phalaris staggers, histological, immunohistochemical, and immunofluorescence analysis revealed significant accumulation of neuromelanin and aggregated α-synuclein in the perikaryon of neurons in the cerebral cortex, thalamus, brainstem, and spinal cord. Neuronal intracytoplasmic Lewy bodies inclusions were not observed in these cases of ovine Phalaris staggers. These important findings established a clear link between synucleinopathy and the neurologic form of Phalaris plant poisoning in sheep, demonstrated in six of six affected sheep. Synucleinopathy is a feature of a number of progressive and fatal neurodegenerative disorders of man and may be a common endpoint of such disorders, which in a variety of ways perturb neuronal function. However, whether primary to the degenerative process or a consequence of it awaits clarification in an appropriate model system.

Cross-Linking Cellular Prion Protein Induces Neuronal Type 2-Like Hypersensitivity.

Background: Previous reports identified proteins associated with 'apoptosis' following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated 'apoptosis', however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response. Methods: Initially, we predicted the allergenicity of the epitope sequences associated with 'neurotoxic' anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of mouse primary neurons (MPN), neuroblastoma cells (N2a) and microglia (N11) cell lines lead to a neuronal allergenic response. Results: In-Silico studies showed that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for previously reported 'neurotoxic' antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, we identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, we identified 8 neuronal allergenic-related proteins following treatment of N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells when compared with untreated cells. Antibody treatment of MPN or MPN co-cultured with antibody-treated N11 led to identifying 10 and 7 allergenic-related proteins when compared with untreated cells. However, comparison with 3F4 antibody treatment revealed 5 and 4 allergenic-related proteins respectively. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Finally, co-culture of N2a or MPN with N11-treated with anti-PrP antibodies resulted in significant accumulation of NO and IL6 but not TNF-α in the cell culture media supernatant. Conclusions: This study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal hypersensitivity response and highlights the important role of microglia in triggering an IgG-mediated neuronal hypersensitivity response. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative disorders to derive safe and targeted biotherapeutics.

Treatment of microglia with Anti-PrP monoclonal antibodies induces neuronal apoptosis in vitro.

Previous reports highlighted the neurotoxic effects caused by some motif-specific anti-PrPC antibodies in vivo and in vitro. In the current study, we investigated the detailed alterations of the proteome with liquid chromatography-mass spectrometry following direct application of anti-PrPC antibodies on mouse neuroblastoma cells (N2a) and mouse primary neuronal (MPN) cells or by cross-linking microglial PrPC with anti-PrPC antibodies prior to co-culture with the N2a/MPN cells. Here, we identified 4 (3 upregulated and 1 downregulated) and 17 (11 upregulated and 6 downregulated) neuronal apoptosis-related proteins following treatment of the N2a and N11 cell lines respectively when compared with untreated cells. In contrast, we identified 1 (upregulated) and 4 (2 upregulated and 2 downregulated) neuronal apoptosis-related proteins following treatment of MPN cells and N11 when compared with untreated cells. Furthermore, we also identified 3 (2 upregulated and 1 downregulated) and 2 (1 upregulated and 1 downregulated) neuronal apoptosis-related related proteins following treatment of MPN cells and N11 when compared to treatment with an anti-PrP antibody that lacks binding specificity for mouse PrP. The apoptotic effect of the anti-PrP antibodies was confirmed with flow cytometry following labelling of Annexin V-FITC. The toxic effects of the anti-PrP antibodies was more intense when antibody-treated N11 were co-cultured with the N2a and the identified apoptosis proteome was shown to be part of the PrPC-interactome. Our observations provide a new insight into the prominent role played by microglia in causing neurotoxic effects following treatment with anti-PrPC antibodies and might be relevant to explain the antibody mediated toxicity observed in other related neurodegenerative diseases such as Alzheimer.

Plant poisoning leads to alpha-synucleinopathy and neuromelanopathy in kangaroos.

The pathogenesis of synucleinopathies, common neuropathological lesions normally associated with some human neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, remains poorly understood. In animals, ingestion of the tryptamine-alkaloid-rich phalaris pastures plants causes a disorder called Phalaris staggers, a neurological syndrome reported in kangaroos. The aim of the study was to characterise the clinical and neuropathological changes associated with spontaneous cases of Phalaris staggers in kangaroos. Gross, histological, ultrastructural and Immunohistochemical studies were performed to demonstrate neuronal accumulation of neuromelanin and aggregated α-synuclein. ELISA and mass spectrometry were used to detect serum-borne α-synuclein and tryptamine alkaloids respectively. We report that neurons in the central and enteric nervous systems of affected kangaroos display extensive accumulation of neuromelanin in the perikaryon without affecting neuronal morphology. Ultrastructural studies confirmed the typical structure of neuromelanin. While we demonstrated strong staining of α-synuclein, restricted to neurons, intracytoplasmic Lewy bodies inclusions were not observed. α-synuclein aggregates levels were shown to be lower in sera of the affected kangaroos compared to unaffected herd mate kangaroos. Finally, mass spectrometry failed to detect the alkaloid toxins in the sera derived from the affected kangaroos. Our preliminary findings warrant further investigation of Phalaris staggers in kangaroos, potentially a valuable large animal model for environmentally-acquired toxic synucleinopathy.

An Aged Canid with Behavioral Deficits Exhibits Blood and Cerebrospinal Fluid Amyloid Beta Oligomers.

Many of the molecular and pathological features associated with human Alzheimer disease (AD) are mirrored in the naturally occurring age-associated neuropathology in the canine species. In aged dogs with declining learned behavior and memory the severity of cognitive dysfunction parallels the progressive build up and location of Aß in the brain. The main aim of this work was to study the biological behavior of soluble oligomers isolated from an aged dog with cognitive dysfunction through investigating their interaction with a human cell line and synthetic Aß peptides. We report that soluble oligomers were specifically detected in the dog's blood and cerebrospinal fluid (CSF) via anti-oligomer- and anti-Aß specific binders. Importantly, our results reveal the potent neurotoxic effects of the dog's CSF on cell viability and the seeding efficiency of the CSF-borne soluble oligomers on the thermodynamic activity and the aggregation kinetics of synthetic human Aß. The value of further characterizing the naturally occurring Alzheimer-like neuropathology in dogs using genetic and molecular tools is discussed.

Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.

Studies of the properties of soluble oligomer species of amyloidogenic proteins, derived from different proteins with little sequence homology, have indicated that they share a common structure and may share similar pathogenic mechanisms. Amyloid ß, tau protein, as well as amyloid precursor protein normally associated with Alzheimer's disease and Parkinson's disease were found in lesions and plaques of multiple sclerosis patients. The objective of the study is to investigate whether brain and cerebrospinal fluid (CSF) samples derived from multiple sclerosis patients demonstrate the presence of soluble oligomers normally associated with protein-misfolding diseases such as Alzheimer's disease. We have used anti-oligomer monoclonal antibodies to immunodetect soluble oligomers in CSF and brain tissues derived from multiple sclerosis patients. In this report, we describe the presence of soluble oligomers in the brain tissue and cerebral spinal fluid of multiple sclerosis patients detected with our monoclonal anti-oligomer antibodies with Western blot and Sandwich enzyme-linked immunosorbent assay (sELISA). These results might suggest that protein aggregation plays a role in multiple sclerosis pathogenesis although further and more refined studies are needed to confirm the role of soluble aggregates in multiple sclerosis.

Potential candidate camelid antibodies for the treatment of protein-misfolding diseases.

Protein-misfolding diseases (PMDs), including Alzheimer's disease would potentially reach epidemic proportion if effective ways to diagnose and treat them were not developed. The quest for effective therapy for PMDs has been ongoing for decades and some of the technologies developed so far show great promise. We report here the development of antibodies by immunization of camelids with prion (PrioV3) and Alzheimer's (PrioAD12, 13 & 120) disease-derived brain material. We show that anti-PrP antibody transmigration across the blood-brain barrier (BBB) was inhibited with phosphatidylinositol-specific phospholipase C (PIPLC). Our camelid anti-prion antibody was also shown to permanently abrogate prion replication in a prion-permissive cell line after crossing the artificial BBB. Furthermore, anti-Aß/tau antibodies were able to bind their specific immunogens with ELISA and immunohistochemistry. Finally, both PrioV3 and PrioAD12 were shown to co-localize with Lamp-1, a marker of late endosomal/lysosomal compartments. These antibodies could prove to be a valuable tool for the neutralization/clearance of PrP(Sc), Aß and tau proteins in cellular compartments of affected neurons and could potentially have wider applicability for the treatment of PMDs.

PrP(Sc)-specific antibodies with the ability to immunodetect prion oligomers.

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0) cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aß and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.

Epitope-specific anti-prion antibodies upregulate apolipoprotein E and disrupt membrane cholesterol homeostasis.

The mechanisms of neuronal degeneration induced by the transformation of normal cellular prion protein (PrP(C)) into disease-associated PrP(Sc) are not fully understood. Previous reports have demonstrated that cross-linking cellular prion protein by anti-PrP(C) antibodies can promote neuronal apoptosis. In this report, we now show that treatment of neuronal cells with anti-prion antibodies leads to sequestration of free cholesterol in cell membranes, significant overexpression of apolipoprotein E, and to cytoplasmic phospholipase A2 activation as well as to production of prostaglandin. These results confirm the in vivo toxic effects and indicate that anti-prion antibody treatment of neurons lead to deleterious effects. Finally, great caution should be exerted when adopting antibody-based therapy for prion diseases.

PrP-specific camel antibodies with the ability to immunodetect intracellular prion protein.

Although there is currently no effective treatment for prion diseases, significant advances have been made in suppressing its progress, using antibodies that block the conversion of PrP(C) into PrP(Sc). In order to be effective in treating individuals that have prion diseases, antibodies must be capable of arresting disease in its late stages. This requires the development of antibodies with higher affinity for PrP(Sc) and systems for effective translocation of antibodies across the blood-brain barrier in order to achieve high concentrations of inhibitor at the site of protein replication. An additional advantage is the ability of these antibodies to access the cytosol of affected cells. To this end, we have generated PrP-specific antibodies (known as PrioV) by immunization of camels with murine scrapie material adsorbed to immunomagnetic beads. The PrioV antibodies display a range of specificities with some recognizing the PrP(27-30) proteinase K-resistant fragment, others specific for PrP(C) and a number with dual binding specificity. Independent of their PrP conformation specificity, one of the PrioV antibodies (PrioV3) was shown to bind PrP(C) in the cytosol of neuroblastoma cells. In marked contrast, conventional anti-PrP antibodies produced in mouse against similar target antigen were unable to cross the neuronal plasma membrane and instead formed a ring around the cells. The PrioV anti-PrP antibodies could prove to be a valuable tool for the neutralization/clearance of PrP(Sc) in intracellular compartments of affected neurons and could potentially have wider applicability for the treatment of so-called protein-misfolding diseases.

A camelid anti-PrP antibody abrogates PrP replication in prion-permissive neuroblastoma cell lines.

The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrP(Sc) would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the "proof-of-principle" for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrP(C). In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrP(Sc) replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.

The Decoy Receptor 3 (DcR3, TNFRSF6B) suppresses Th17 immune responses and is abundant in human cerebrospinal fluid.

The Decoy Receptor 3 (DcR3) is known to compete with the signalling receptors of the Fas ligand (FasL), LIGHT and the TNF-like molecule 1A (TL1A). The primary aim of this study was to provide insights into the role of DcR3 in the modulation of myelin-specific encephalitogenic autoimmune T cell responses. Treatment of PLP-specific lymph node cells with DcR3.Fc protein resulted in a suppression of IFN-g and IL-17, in a reduced proportion of Th17 cells and in a decrease of encephalitogenicity. The Th17 response promoting cytokines IL-6 and IL-23 were suppressed by DcR3.Fc as well. DcR3.Fc-treatment of CD4+ T cells with a defective FasL did not influence the production of IL-17 indicating that DcR3 suppresses IL-17 production by disruption of Fas-FasL interactions. We identified high concentrations of DcR3 in the cerebrospinal fluid (CSF) of patients with various neurological disease states while almost no DcR3 was detected in corresponding serum samples. In conclusion, DcR3 modulates CNS-autoimmunity by interfering with Th17 responses via blockade of Fas-FasL interaction. The anti-inflammatory properties and high DcR3 concentrations in the CSF warrant further investigations in the expression pattern and the function of DcR3 within the CNS.

830 nm laser irradiation induces varicosity formation, reduces mitochondrial membrane potential and blocks fast axonal flow in small and medium diameter rat dorsal root ganglion neurons: implications for the analgesic effects of 830 nm laser.

We report the formation of 830 nm (cw) laser-induced, reversible axonal varicosities, using immunostaining with beta-tubulin, in small and medium diameter, TRPV-1 positive, cultured rat DRG neurons. Laser also induced a progressive and statistically significant decrease (p<0.005) in MMP in mitochondria in and between static axonal varicosities. In cell bodies of the neuron, the decrease in MMP was also statistically significant (p<0.05), but the decrease occurred more slowly. Importantly we also report for the first time that 830 nm (cw) laser blocked fast axonal flow, imaged in real time using confocal laser microscopy and JC-1 as mitotracker. Control neurons in parallel cultures remained unaffected with no varicosity formation and no change in MMP. Mitochondrial movement was continuous and measured along the axons at a rate of 0.8 microm/s (range 0.5-2 microm/s), consistent with fast axonal flow. Photoacceptors in the mitochondrial membrane absorb laser and mediate the transduction of laser energy into electrochemical changes, initiating a secondary cascade of intracellular events. In neurons, this results in a decrease in MMP with a concurrent decrease in available ATP required for nerve function, including maintenance of microtubules and molecular motors, dyneins and kinesins, responsible for fast axonal flow. Laser-induced neural blockade is a consequence of such changes and provide a mechanism for a neural basis of laser-induced pain relief. The repeated application of laser in a clinical setting modulates nociception and reduces pain. The application of laser therapy for chronic pain may provide a non-drug alternative for the management of chronic pain.