Alzheimer's Disease Pathophysiology in the Retina.

The retina is an emerging CNS target for potential noninvasive diagnosis and tracking of Alzheimer's disease (AD). Studies have identified the pathological hallmarks of AD, including amyloid ß-protein (Aß) deposits and abnormal tau protein isoforms, in the retinas of AD patients and animal models. Moreover, structural and functional vascular abnormalities such as reduced blood flow, vascular Aß deposition, and blood-retinal barrier damage, along with inflammation and neurodegeneration, have been described in retinas of patients with mild cognitive impairment and AD dementia. Histological, biochemical, and clinical studies have demonstrated that the nature and severity of AD pathologies in the retina and brain correspond. Proteomics analysis revealed a similar pattern of dysregulated proteins and biological pathways in the retina and brain of AD patients, with enhanced inflammatory and neurodegenerative processes, impaired oxidative-phosphorylation, and mitochondrial dysfunction. Notably, investigational imaging technologies can now detect AD-specific amyloid deposits, as well as vasculopathy and neurodegeneration in the retina of living AD patients, suggesting alterations at different disease stages and links to brain pathology. Current and exploratory ophthalmic imaging modalities, such as optical coherence tomography (OCT), OCT-angiography, confocal scanning laser ophthalmoscopy, and hyperspectral imaging, may offer promise in the clinical assessment of AD. However, further research is needed to deepen our understanding of AD's impact on the retina and its progression. To advance this field, future studies require replication in larger and diverse cohorts with confirmed AD biomarkers and standardized retinal imaging techniques. This will validate potential retinal biomarkers for AD, aiding in early screening and monitoring.

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.

Neuronal Deposition of Amyloid-ß Oligomers and Hyperphosphorylated Tau Is Closely Connected with Cognitive Dysfunction in Aged Dogs.

BACKGROUND: Canine cognitive dysfunction (CCD) is a progressive syndrome recognized in mature to aged dogs with a variety of neuropathological changes similar to human Alzheimer's disease (AD), for which it is thought to be a good natural model. However, the presence of hyperphosphorylated tau protein (p-Tau) in dogs with CCD has only been demonstrated infrequently. OBJECTIVE: The aim of the present study was to investigate the presence of p-Tau and amyloid-ß oligomer (Aßo) in cerebral cortex and hippocampus of dogs with CCD, with focus on an epitope retrieval protocol to unmask p-Tau. METHODS: Immunohistochemical and immunofluorescence analysis of the cortical and hippocampal regions of five CCD-affected and two nondemented aged dogs using 4G8 anti-Aßp, anti-Aß1 - 42 nanobody (PrioAD13) and AT8 anti-p-Tau (Ser202, Thr205) antibody were used to demonstrate the presence of Aß plaques (Aßp) and Aß1 - 42 oligomers and p-Tau deposits, respectively. RESULTS: The extracellular Aß senile plaques were of the diffuse type which lack the dense core normally seen in human AD. While p-Tau deposits displayed a widespread pattern and closely resembled the typical human neuropathology, they did not co-localize with the Aßp. Of considerable interest, however, widespread intraneuronal deposition of Aß1 - 42 oligomers were exhibited in the frontal cortex and hippocampal region that co-localized with p-Tau. CONCLUSION: Taken together, these findings reveal further shared neuropathologic features of AD and CCD, supporting the case that aged dogs afflicted with CCD offer a relevant model for investigating human AD.

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.

Epitope-specific anti-PrP antibody toxicity: a comparative in-silico study of human and mouse prion proteins.

Despite having therapeutic potential, anti-PrP antibodies caused a major controversy due to their neurotoxic effects. For instance, treating mice with ICSM antibodies delayed prion disease onset, but both were found to be either toxic or innocuous to neurons by researchers following cross-linking PrPC. In order to elucidate and understand the reasons that led to these contradictory outcomes, we conducted a comprehensive in silico study to assess the antibody-specific toxicity. Since most therapeutic anti-PrP antibodies were generated against human truncated recombinant PrP91-231 or full-length mouse PrP23-231, we reasoned that host specificity (human vs murine) of PrPC might influence the nature of the specific epitopes recognized by these antibodies at the structural level possibly explaining the 'toxicity' discrepancies reported previously. Initially, molecular dynamics simulation and pro-motif analysis of full-length human (hu)PrP and mouse (mo)PrP 3D structure displayed conspicuous structural differences between huPrP and moPrP. We identified 10 huPrP and 6 moPrP linear B-cell epitopes from the prion protein 3D structure where 5 out of 10 huPrP and 3 out of 6 moPrP B-cell epitopes were predicted to be potentially toxic in immunoinformatics approaches. Herein, we demonstrate that some of the predicted potentially 'toxic' epitopes identified by the in silico analysis were similar to the epitopes recognized by the toxic antibodies such as ICSM18 (146-159), POM1 (138-147), D18 (133-157), ICSM35 (91-110), D13 (95-103) and POM3 (95-100). This in silico study reveals the role of host specificity of PrPC in epitope-specific anti-PrP antibody toxicity.

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.

Detection of retinal and blood Aß oligomers with nanobodies.

INTRODUCTION: Abnormal retinal changes are increasingly recognized as an early pathological change in Alzheimer's disease (AD). Although amyloid beta oligomers (Aßo) have been shown to accumulate in the blood and retina of AD patients and animals, it is not known whether the early Aßo deposition precedes their accumulation in brain. METHODS AND RESULTS: Using nanobodies targeting Aß1-40 and Aß1-42 oligomers we were able to detect Aß oligomers in the retina and blood but not in the brain of 3-month-old APP/PS1 mice. Furthermore, Aß plaques were detected in the brain but not the retina of 3-month-old APP/PS1 mice. CONCLUSION: These results suggest that retinal accumulation of Aßo originates from peripheral blood and precedes cognitive decline and Aßo deposition in the brain. This provides a very strong basis to develop and implement an "eye test" for early detection of AD using nanobodies targeting retinal Aß.

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.

Age-Specific Retinal and Cerebral Immunodetection of Amyloid-ß Plaques and Oligomers in a Rodent Model of Alzheimer's Disease.

BACKGROUND: Amyloid-ß soluble oligomers (Aßo) are believed to be the cause of the pathophysiology underlying Alzheimer's disease (AD) and are normally detected some two decades before clinical onset of the disease. Retinal pathology associated with AD pathogenesis has previously been reported, including ganglion cell loss, accumulation of Aß deposits in the retina, and reduction of nerve fiber layer thickness as well as abnormalities of the microvasculature. OBJECTIVE: This study's aim is to better understand the relationship between brain and retinal Aßo deposition and in particular to quantify levels of the toxic Aßo as a function of age in the retina of a rodent model of AD. METHODS: Retinas and brain tissue from 5×FAD mice were stained with Congo red, Thioflavin-T (Th-T), and Aß plaque-specific and Aßo-specific antibodies. RESULTS: We show that retinas displayed an age-dependent increase of Th-T-specific amyloid fibrils. Staining with anti-Aß antibody confirmed the presence of the Aß plaques in all 5×FAD retinas tested. In contrast, staining with anti-Aßo antibody showed an age-dependent decrease of retinal Aßo. Of note, Aßo was observed mainly in the retinal nuclear layers. Finally, we confirmed the localization of Aßo to neurons, typically accumulating in late endosomes, indicating possible impairment of the endocytic pathway. CONCLUSION: Our results demonstrate the presence of intraneuronal Aßo in the retina and its accumulation inversely correlated with retinal Aß plaque deposition, indicating an age-related conversion in this animal model. These results support the development of an early AD diagnostic test targeting Aßo in the eye.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma.

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

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.

A Comparative Review of Mixed Mammary Tumors in Mammals.

Mixed tumors are characterized by the histological identification of two or more cell types. Commonly, a mixture of epithelial and myoepithelial cells is included in abundant stroma, which can consist of myxoid, chondroid or bony matrices. Spontaneously arising mixed tumors are rare lesions in the human breast but are common in human salivary glands and canine mammary glands. Subtle histopathological characteristics and overlapping attributes of malignant lesions with other benign lesions can lead to a diagnostic challenge. Mixed tumors can present as benign or malignant. While malignant mixed tumors are quite rare in the human breast they have a poor prognosis. Benign mixed mammary tumors occur more frequently in female dogs than in humans and are usually associated with a good prognosis. This review will provide a comprehensive overview of mixed mammary tumors, across various mammalian species.

Immunoinformatics Approach for Epitope-Based Peptide Vaccine Design and Active Site Prediction against Polyprotein of Emerging Oropouche Virus.

Oropouche virus (OROV) is an emerging pathogen which causes Oropouche fever and meningitis in humans. Several outbreaks of OROV in South America, especially in Brazil, have changed its status as an emerging disease, but no vaccine or specific drug target is available yet. Our approach was to identify the epitope-based vaccine candidates as well as the ligand-binding pockets through the use of immunoinformatics. In this report, we identified both T-cell and B-cell epitopes of the most antigenic OROV polyprotein with the potential to induce both humoral and cell-mediated immunity. Eighteen highly antigenic and immunogenic CD8+ T-cell epitopes were identified, including three 100% conserved epitopes (TSSWGCEEY, CSMCGLIHY, and LAIDTGCLY) as the potential vaccine candidates. The selected epitopes showed 95.77% coverage for the mixed Brazilian population. The docking simulation ensured the binding interaction with high affinity. A total of five highly conserved and nontoxic linear B-cell epitopes "NQKIDLSQL," "HPLSTSQIGDRC," "SHCNLEFTAITADKIMSL," "PEKIPAKEGWLTFSKEHTSSW," and "HHYKPTKNLPHVVPRYH" were selected as potential vaccine candidates. The predicted eight conformational B-cell epitopes represent the accessibility for the entered virus. In the posttherapeutic strategy, ten ligand-binding pockets were identified for effective inhibitor design against emerging OROV infection. Collectively, this research provides novel candidates for epitope-based peptide vaccine design against OROV.

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.

A glycosylphosphatidylinositol analogue reduced prion-derived peptide mediated activation of cytoplasmic phospholipase A2, synapse degeneration and neuronal death.

The pathogenesis of prion diseases includes synapse degeneration and neuronal death. Here we report that pre-treatment with glucosamine-phosphatidylinositol (glucosamine-PI), a synthetic analogue of the glycosylphosphatidylinositol (GPI) anchor that attaches the prion protein (PrP(C)) to plasma membranes, increased the resistance of cultured cortical neurones to the toxic effects of the prion-derived peptide PrP82-146. Pre-treatment with glucosamine-PI reduced the PrP82-146 induced activation of cytoplasmic phospholipase A(2) (cPLA(2)), activation of caspase-3 and synapse degeneration. The addition of glucosamine-PI significantly increased the amount of cholesterol within neuronal membranes consistent with the hypothesis that GPI anchors sequester cholesterol. Whereas in untreated neurones PrP82-146 was found within lipid rafts, in glucosamine-PI treated neurones most PrP82-146 was found in the normal cell membrane and was rerouted into the lysosomes. Complex GPI anchors isolated from PrP(C), Thy-1 or CD55 were also protective against PrP82-146. We conclude that glucosamine-PI, or isolated GPI anchors, can modify local membrane micro-environments that are important in the initiation of signalling events that mediate PrP82-146 induced neurodegeneration.