Acute Neurotoxicity Models of Prion Disease - American Chemical

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Acute Neurotoxicity Models of Prion Disease ABU MOHAMMED TAUFIQUAL ISLAM, Paul A Adlard, David I. Finkelstein, VICTORIA LEWIS, SILVIA BIGGI, Emiliano Biasini, and Steven J. Collins ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00517 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 3, 2018

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ACS Chemical Neuroscience

Acute Neurotoxicity Models of Prion Disease A M T Islam1, P A Adlard2, D I Finkelstein2, V Lewis1, S Biggi3, *E Biasini3, *S J Collins1,2. 1

Department of Medicine (RMH) and 2Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Australia 3010 and 3CIBIO, University of Trento, Italy. *Corresponding authors: Telephone: +613 8344 1949 Email: [email protected]

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ABSTRACT: Prion diseases are phenotypically diverse, transmissible, neurodegenerative disorders affecting both animals and humans. Misfolding of the normal prion protein (PrPC) into disease-associated conformers (PrPSc) is considered the critical etiological event underpinning prion diseases, with such misfolded isoforms linked to both disease transmission and neurotoxicity. Although important advances in our understanding of prion biology and pathogenesis have occurred over the last 3-4 decades, many fundamental questions remain to be resolved, including consensus regarding the principal pathways subserving neuronal dysfunction, as well as detailed biophysical characterisation of PrPSc species transmitting disease and/or directly associated with neurotoxicity. In vivo and in vitro models have been, and remain, critical to furthering our understanding across many aspects of prion disease pathobiology. Prion animal models are arguably the most authentic in vivo models of neurodegeneration that exist and have provided valuable and multifarious insights into pathogenesis; however, they are expensive and time consuming, and it can be problematic to clearly discern evidence of direct PrPSc neurotoxicity in the overall context of pathogenesis. In vitro models in contrast, generally offer greater tractability and appear more suited to assessments of direct acute neurotoxicity but have until recently been relatively simplistic and overall there remains a relative paucity of validated, biologically-relevant models with heightened reliability as far as translational insights, contributing to difficulties in redressing our knowledge gaps in prion disease pathogenesis. In this review, we overview the spectrum and methodological diversity of in vivo and in vitro models of prion acute toxicity, as well as the pathogenic insights gained from these studies. KEYWORDS: Prion disease, Neurotoxicity, Acute toxicity, PrPC, PrPSc.


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INTRODUCTION:

Prion diseases are phenotypically diverse, transmissible, neurodegenerative disorders affecting humans and a number of animal species (summarised in table1). Creutzfeldt-Jakob disease (CJD) is the most common human form of prion disease, affecting approximately 1-2 persons/million/year and characteristically causing death from dementia and gross motor decline after a mean illness duration of only ~6 months1,2. CJD usually occurs without explanation (sporadic CJD; ~85%), with mutations in the prion protein gene (PRNP; genetic CJD) and horizontal transmission (acquired CJD) explaining the remainder 3. Although generally rare, bovine spongiform encephalopathy (causally linked to the zoonosis variant CJD) and kuru cogently illustrate that under exceptional circumstances the transmissibility of these diseases can lead to dramatic increases in disease incidence4– 6 . It is the combination of a typically devastating clinical course and potential transmissibility that affords prion diseases disproportionate attention across health care settings despite their relative rarity. Similar to other more common neurodegenerative diseases, no proven disease-modifying treatment currently exists, underscoring exigencies in relation to the development of effective therapies. Misfolding of the cellular prion protein (PrPC) into diseaseassociated conformers (PrPSc) is considered the key pathogenic event in prion diseases. Underscoring this fact, transgenic mice in which PrPC expression has been ablated (PrP-knockout mice; PrP0/0) are completely protected against development of prion disease7. PrPC is a cell surface glycoprotein expressed across a broad range of species from fish to mammals, present in almost all tissues but with the highest levels found in the central nervous system (CNS)8,9. The PrPC polypeptide is synthesized directly into the lumen of the endoplasmic reticulum (ER), where the folding machinery of the cell removes signal peptides at the Nterminus (residues 1-22) and the C-terminus (residues 231253) (human sequence numbering), attaches a glycosylphosphatidyl-inositol (GPI) anchor (at serine residue 230) and up to two N-linked oligosaccharide chains (at Asn-181 and/or Asn-197)10,11.The protein also contains a disulphide bond (linking residues 179 and 214)12. PrPC molecules generally travel from the ER to the Golgi apparatus, where the two sugar moieties undergo maturation with considerable elongation and finally the protein is transported to the cell surface and resides in lipid rafts at the plasma membrane13. From the cell surface, PrPC can then enter the endocytic pathway via both clathrin-dependent or -independent mechanisms14,15. Some PrPC molecules (the precise amount varying across different host cells) undergo endogenous proteolytic cleavage by metallo-proteases near residue 111, giving rise to N- and C-terminal fragments called N1 and C1, respectively16,17. Structurally, PrPC is organized into two principal domains: (i) a less ordered N-terminal region (residues 23–125, human sequence) and (ii) a C-terminal globular domain (residues 126–229)12. The former comprises two charged regions (residues 23-27 and 100-110), five histidine-rich octapeptide repeats (residues 51-91) that can bind bivalent metal ions (especially Cu2+) and a highly conserved stretch of hydrophobic amino acids (residues 112131), which has been reported to promote the insertion of the protein into the plasma membrane in particular situations18,19. The C-terminal domain in contrast includes two short βstrands (amino acids 129–133 and 160–163) and three αhelices (amino acids 144–152, 173–194 and 200–227). In contrast to PrPC, PrPSc displays considerable enrichment of βstrand content, perhaps only partly at the expense of α-helical secondary structure20–22 given preservation of α-helices 2 and 3 in some reported models22,23 raising the likelihood that the less structured N-terminus contributes substantially although

studies utilising N-terminally truncated PrPSc (PrP27-30; approximating residues 89-230) still report ~50% β-strand content22. Disease-associated conformers most likely comprise a spectrum of conformations rather than a single structural entity24. Unfortunately, the initiating event(s) and a detailed understanding of the misfolding pathway of PrPC to PrPSc conversion remain to be fully defined. The latter appears to involve a template-directed, auto-catalytic process, with aggregate secondary fragmentation likely to be an important contributor to multimer propagation kinetics25. A number of specific domains, including PrP residues 23-31, 95-125 and 122-140, have been shown to be directly involved in the conversion process26–28. Although prion diseases typically manifest lengthy incubation periods, in vitro and in vivo experimental models suggest that de novo conversion of PrPC commences very soon after inoculation of PrPSc into host cells or tissues 29,30. Despite noteworthy progress in our understanding of prion biology and pathogenesis over the last few decades, many fundamental questions remain unresolved, including consensus regarding the primary function of PrPC in the brain, the principal pathways subserving neuronal dysfunction and detailed biophysical characterisation of PrPSc species transmitting disease and/or directly associated with neurotoxicity. Included among the reported roles for endogenous PrPC in the CNS are neuronal protection 31, transmembrane signalling 32,33, copper and zinc uptake and transport 34,35, as well as synaptic development and temporal maintenance 36–38 and short-term memory and its consolidation 39,40; myelin maintenance also appears an important function of PrPC in the peripheral nervous system 41 . Pathophysiological pathways and the mechanisms implicated in prion diseases are also diverse, with the unfolded protein response 42, heightened oxidative stress, 43,44 proteasomal dysfunction45 and the endoplasmic reticulum stress response46 reported to be involved. It remains to be determined whether various deleterious pathways are usually activated in parallel or a single pathogenic cascade predominates with certain prion strain-host combinations, the latter possibly devolving to the cellular or neuronal level. As with other more common neurodegenerative diseases such as Alzheimer’s disease 47,48 the synapse is likely to be a primary pathophysiological site for evincing the convergence of activated pathogenic cascades49,50 although whether synaptic dysfunction invariably culminates in neuronal loss is unresolved 51. Compelling evidence for PrPSc as the principle, if not exclusive, component of infectious prions exists52–54 although not all findings have clearly aligned with this hypothesis55,56. Analogously, while there is considerable evidence to support a direct causal link for PrPSc to neurotoxicity57,58, possibly through corruption of PrPC function59, important modifying influences have been demonstrated 60 and studies utilising transgenic mice perpetuate controversy as to the level of contingency in relation to ongoing neuronal PrPC expression 61,62 . Further complicating the relationship of PrPSc to neurotoxicity is that while traditionally PrPSc had been considered highly protease-resistant 52, recent evidence supports the existence of protease-sensitive isoforms, which most likely contribute to pathogenesis63,64 and may comprise up to 90% of misfolded PrP in diseased brains63. Details concerning the biophysical characteristics of PrPSc species underpinning transmissibility and neurotoxicity are rudimentary. The insights available through size fractionation and sedimentation velocity fractionation suggest that the most efficient PrPSc species for subserving disease transmission are small oligomers 65,66,which are probably relatively protease-sensitive 63. Even less information is available for putative neurotoxic PrPSc species, but limited



evidence supports that such species are also probably small, protease-sensitive oligomers 67. For the development of effective treatments for prion and other neurodegenerative diseases, the importance of detailed insight into their pathophysiological mechanisms cannot be overstated. Serving this purpose, in vivo and in vitro models have been, and remain, critical to furthering our understanding across many aspects of prion biology and patho-biology. Prion animal models are arguably the most authentic in vivo models of neurodegeneration that exist and have provided valuable and multifarious insights into pathogenesis. However, they are expensive, their characterization generally requires extended periods and it can be problematic to clearly disentangle evidence of direct PrPSc neurotoxicity within the overall context of fully developed pathogenesis. In contrast, in vitro models generally offer greater tractability and convenience, with time-frames of days to a couple of weeks. Consequently, they appear more suited to assessments of direct acute neurotoxicity. Nevertheless, the inherent simplicity of these systems, especially the restricted cell types utilised and the type of preparations employed in many of these studies, leaves concerns regarding their translational relevance. Recently, an in vitro model employing cultured organotypic cerebellar slice explants to assess factors such as those that interfere with PrPSc replication and abrogate cerebellar granule cell loss has been utilised as a model for exploring prion neurotoxicity68,69. Studies utilising this model that relies on de novo PrPSc propagation to generate neurotoxic species over an extended (5-7 week) period, are arguably not ideal for assessing direct acute neurotoxicity. It has been shown, for example, that PrPSc propagation closely correlates with deleterious parallel cellular events such as heightened oxidative stress44, which probably also contribute to pathogenesis but may not represent the direct effects of an acutely neurotoxic prion species. Also, although corruption of normal PrPC function may play a role in prion pathogenesis 70 and studies primarily employing non-PrPSc ligands to PrPC that can induce acute toxicity, such as antiPrP antibodies or antibody fragments71,72 have provided interesting insights, they do not strictly align with investigations dedicated to assessing the neurotoxicity of exogenous prion proteins or cognate fragments in model systems. Overall, there remains a relative paucity of biologically relevant in vitro acute neurotoxicity models of prion disease, contributing to difficulties in redressing extant knowledge gaps in disease mechanisms and pathogenesis. This review offers an illustrative overview of reported models primarily designed to assess the acute direct neurotoxicity of prion proteins or cognate fragments, either ex vivo or not in origin, arbitrarily defined as systems in which the toxicity readout is measured within ≤~3 weeks of exposure.

IN VITRO ACUTE PRION TOXICITY MODELS

A range of neuronal and non-neuronal, primary and immortalised, cell lines as well as neuronal stem cells have been shown to be susceptible to stable infection with prions induced through standard culture conditions 73. Typically, infection is initiated by adding prion containing inoculum (tissue homogenate or cell lysates) to culture medium overlying the cells and allowing the infected culture medium to remain in contact with the cells for a period of many hours to a few days. Although indispensable for sustained infection of cells, PrPC is not essential for initial uptake of PrPSc into cultured cells74,75. As will be described below, in addition to exploiting in vitro cell culture systems to explore the normal function and cell biology of PrPC as well as studying many aspects of conversion of PrPC to PrPSc such as requisite co-

factors and the kinetics and location of propagation, shortterm exposure and chronic prion infection of cells in culture have proven valuable for dissecting the molecular mechanisms underlying neuronal dysfunction and death. Somewhat surprisingly, however, although many cells and cell lines can propagate prion infection, relatively few have been reported to manifest evidence of overt cytopathology in the setting of acute or chronic prion infection46,76,77. In the first report to describe cytopathological changes, this occurred in the setting of chronic Rocky Mountain laboratory (RML) prion strain infection, with a substantial minority of neurons of the GT1 immortalised murine hypothalamic cell line displaying evidence of degeneration, vacuolation and apoptosis, recapitulating some of the histological features observed in human and animal prion disease76; of note, chronic RML infection of the N2a mouse neuroblastoma cell line reported in the same study never developed these morphological changes. Most often, subtle morphological or non-morphological evidence of deleterious cellular consequences has been reported in the setting of acute prion exposure or chronic prion infection, which may only be detectable through heightened assessment vigilance 44,78.

EX VIVO PRION PREPARATIONS FOR ACUTE TOXICITY STUDIES

In an early study of acute neurotoxicity, primary rat cortical neurons (admixed with ~20% GFAP-positive cells) exposed for 12 hours to purified PrPSc preparations generated from the brains of prion-infected hamsters showed significant dosedependent reduction in viability and increased DNA fragmentation (supportive of apoptosis) after prion exposure compared to controls79. Significant and essentially complete protection against toxicity was achieved if cells were pretreated with n-methyl-d-aspartate (NMDA)-receptor blocking compounds, including memantine. Of interest, primary human astrocytes exposed to purified hamster PrPSc under identical conditions did not display reduced viability or apoptotic changes, suggesting the possibility of cell- and/or species-dependent influences on vulnerability to specific prion strains, with memantine treatment of infected N2a cells further clarifying that reduction in PrPSc levels was not the mechanism of protection afforded by the calcium-channel blockers. In contrast to this study, there has been a report of astrocytes being the primary cell type manifesting overt cytopathological evidence of toxicity. Utilising differentiated neurosphere cultures from transgenic (tga/20) mice overexpressing PrPC, Iwamaru and colleagues reported that GFAP-positive astrocytes displayed evidence of reduced viability 12 days following exposure to Chandler strain prions semi-purified from brain homogenates, with shrinkage and cytoplasmic condensation of these cells apparent by day 18 77. Further investigation demonstrated a likely apoptotic basis to the toxicity with enhanced levels of caspase3/7, caspase 8, caspase 9 activities and positive TUNEL staining in these cells coinciding with accumulation of PrPSc. The pancaspase inhibitor z-VAD-fmk was able to partially protect the viability of astrocytes after exposure to prion homogenates albeit with no influence on PrPSc levels, the latter once again demonstrating a potential to rescue cells despite the presence of similar amounts of disease-associated prion protein. In another relatively early study, brain derived, purified, protease-treated hamster scrapie prion protein preparations (PrP27-30) were added on days 2 and 4 to primary mouse mixed cerebellar cell cultures obtained from 6-day-old wildtype and PrP0/0 pups80. Based on the MTT (3- [4, 5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay, a dose-dependent reduction in viability was demonstrated on day 7 following exposure to PrP27-30, which was dependent on PrPC expression. Pre-treatment of the


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primary cultures with 5mM L-leucine methyl ester (selectively lethal to microglia) 2 hours before exposure to 1µg/ml PrP27-30 afforded complete protection in viability suggesting an important role for microglia in the observed neurotoxicity although a recent comprehensive study has demonstrated a contrary outcome with an apparent neuroprotective role for microglia in the setting of prion infection60. Employing N2a cells, Hetz and colleagues46 were able to demonstrate triggering of the endoplasmic reticulum (ER) stress response as a mechanism for inducing apoptosis in acutely prion infected cells. At 48 hours following exposure to purified, proteinase K-treated (PrP27-30) prion preparations derived from the brains of terminally ill 139A strain infected mice, exposed cells manifested shrinkage and aggregation of cell bodies, with the MTS assay confirming a dose-dependent reduction in cell viability across nanomolar concentrations. The development of such acute changes is in contrast to their absence in other reported studies 76 underscoring the likelihood of idiosyncrasy in the vulnerability of certain host cells to particular prion strains. The acute reduction in N2a cell viability was rescued by the iPrP13 peptide known to disrupt the β-sheet secondary structure of PrPSc. Induction of apoptosis was supported by the demonstration of a selective increase in cellular caspase 3 activity peaking at 20 hours following PrP27-30 treatment (without increases in caspase 1 and caspase 8), as well as through detection of externalization of phosphatidylserine to the outer leaflet of the cell membrane in 34% of cells 6 hours following PrP27-30 exposure; the preferential caspase 3 inhibitor Ac-DVED-fmk was able to completely protect prion exposed N2a cells. Additional studies utilizing the fluorophore Fuo-4, demonstrated that the activation of caspase 3 was pre-empted by an increase in intracellular calcium commencing within minutes of exposure to PrP27-30 appearing to be mainly released from the ER. The release of calcium from the ER was associated with activation of the ER-resident caspase 12 and elevated levels of ER chaperones including Grp58 supporting significant ER stress in response to treatment of N2a cells with PrP27-30. N2a cells transfected with a dominant-negative caspase 12 mutant were partially protected against the acute toxic effects of PrP27-30. Importantly, the likely translational relevance of these in vitro findings was demonstrated in both an in vivo model utilizing inoculation with the 139A prion strain and in postmortem brains of patients dying with sporadic and variant CJD. To confidently demonstrate the susceptibility of neurons and astrocytes for propagating prion infection, cerebella from homozygous transgenic 338 (tg338) mice over-expressing ovine PrP ~10-fold on a PrP0/0 background were harvested from 6-day-old pups 81. Bioassay, immunofluorescence and immunoblot techniques confirmed primary cultures highly enriched for granule cell neurons (>95%) or astrocytes (>99%) were equally susceptible and capable of propagating 127S scrapie prions derived from either brain of terminally ill tg338 mice or the MovS2 cell line (an immortalized Schwann cell line originally derived from tg338 mice). Using the combination of microscopic assessment of the percentage of GFAP-negative cells with fragmented nuclei co-localising with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling positivity, it was determined that the rate of apoptosis was approximately doubled in 127S scrapieinfected granule neurons at 28 days post-inoculation, an estimate remarkably similar to the enhanced apoptosis rate observed in chronically M1000 prion-infected rabbit kidney epithelial (RK13) cells 44. Building on previous work by this group demonstrating that intracellular misfolded prion protein impairs the

ubiquitin-proteasome system by stabilising the 20S proteasome in the closed conformation thereby inhibiting substrate entry 45, the role of proteasomal dysfunction in prion pathogenesis was a specific target for further investigations by Kristiansen and co-workers78. Using an LDH release assay to determine that 1µM of the proteasomal inhibitor lactacystin reduced cell viability by ~20% in nonprion infected GT1 and N2aPD88 cells, RML prion infection of these cell lines was found to cause disproportionate and significant cell loss at the same concentration suggesting sensitisation to minor proteasomal dysfunction by prion infection. Previous curing of RML prion infection in the GT1 and N2aPD88 cells using the anti-PrP monoclonal antibody ICSM18 (directed against residues 142-155) restored proteasomal lactacystin vulnerability to the same as mockinfected cells. Of interest, uninfected but transfected N2aPD88 cells over-expressing mouse PrPC and exposed to the same minor 1µM lactacystin proteasomal inhibition developed cytoplasmic aggregates of prion protein but this was not associated with reduced cell viability, which contrasted with the significant toxicity observed in the setting of RML infection and modest proteasomal inhibition of these same cells. Employing a combination of techniques, including fluorescence-activated cell sorting after annexin V and propidium iodide treatment, confirmed that RML infected GT1 cells following 1µM lactacystin exposure were dying from apoptosis mediated by caspase 3 and caspase 8, with PrPSc accumulation in GT1 and N2aPD88 cells within perinuclear vimentin-positive aggresomes co-localising with Hsc70, the 20 S proteasome and ubiquitin demonstrated by immunohistochemistry. Agents that inhibit microtubule dynamics (colchicine and nocodazole) and thereby aggresome formation were found to prevent cell death and caspase 3 and 8 activation without lowering PrPSc levels supporting that the formation of these structures is directly linked to toxicity the caspase-mediated apoptosis in RML infected GT1 and N2aPD88 cells. Exploiting GT1-7 cells chronically infected with various prion strains including Chandler, 22L and Fukuoka prions, Milhavet and colleagues assessed how prion infection altered the host cell‘s ability to cope with oxidative challenges82. GT1-7 cells chronically infected with Chandler prions were found to have increased levels of lipid peroxidation as reflected by malondialdehyde adducts of thiobarbituric acid, while total superoxide dismutase (SOD), manganese SOD (Mn-SOD), glutathione peroxidase (GP) and glutathione reductase (GR) activities were all significantly reduced in comparison to mock-infected GT1-7 cells. To further examine an apparently enhanced vulnerability to oxidative stress in the setting of established prion infection, GT1-7 cells infected with Chandler, 22L and Fukuoka strains were shown at 48 hours after exposure to 1mM buthionine sulfoximine (BSO; an agent known to deplete cells of reduced glutathione; GSH) to have reduced viability assessed by MTT and Trypan blue exclusion assays compared to mock-infected cells. N-acetylcysteine, a known precursor of GSH and a reactive oxygen species scavenger, protected against BSO induced toxicity. On further exploring the explanation for the enhanced susceptibility to oxidative stress using a combination of biochemical techniques, Cu/Zn-SOD and Mn-SOD activities were shown to be reduced in GT1-7 cells infected with 22L and Chandler prions compared to mock-infected Congo red-cured cells, with Cu/Zn-SOD protein levels reduced in the infected cells. Hannaoui and colleagues 83 developed a model to further explore potential varying neuronal tropism displayed by different prion strains. To achieve this, they utilised mouseadapted 139A, 22L and ME7 scrapie prion strains in combination with primary mouse cultures derived from the striatal, cerebellar and cerebral cortical regions of wild-type



C57BL/6 mice, assessing the cultures at time-points up to 28 days post-infection (dpi). Although all three prion strains displayed a broadly similar kinetic pattern of neurotoxicity in cerebellar granule cells as assessed by an MTS assay in combination with immunocytochemistry for MAP2-positive cells and detection of pyknotic nuclei, cerebellar granule cells were shown to be most vulnerable to the neurotoxic effect of 22L prions, which was maximal at 28 dpi. In contrast to the ongoing decline in MAP2-positive neurons from 14 dpi there was a progressive increase in GFAP-positive cells and pyknotic nuclei reflecting astrocytic gliosis and apoptosis, respectively, most marked in cultures exposed to 22L prions. These findings aligned to previous studies supporting that the neuropathological changes evinced with 22L had a stronger tropism for cerebellar neurons. Of additional interest, while PrPSc levels appeared to plateau from 14dpi in 22L prioninfected cerebellar granule cell cultures despite continuing loss of neurons thereafter up to 28 dpi, the less toxic 139A and ME7 prions were associated with progressive accumulation of PrPSc paralleling the ongoing neuronal loss and apoptosis. Further immunocytochemical analysis showed the neurotoxicity in all three prion strains correlated with development of PrPSc aggregates in neurons, with colocalisation studies revealing that this was mostly within lysosomes (as well as in the Golgi in ME7 and 139A infections); there was no correlation with PrPSc aggregates in astrocytes. On turning to primary striatal neuronal cultures and utilising analogous analytical techniques, it was observed that ME7 prions were not propagated in these cells while 139A and 22L both achieved infection but with only 139A prions causing significant loss of neurons from 11dpi and increased apoptosis at 14 dpi albeit without any increase in GFAP-positive cells and with PrPSc levels plateauing from 11 dpi. The authors reported that primary cerebral cortical neuronal cultures only survived for ~18 days limiting assessments of neurotoxicity to a maximum of 11 dpi. Once again employing an analogous approach, ME7 prions were shown not to successfully infect these cells, while 22L and 139A prions propagated in cortical neurons associated with significant cell loss and apoptosis at 11 dpi compared to mock-infected cultures. Similar to striatal neurons there was no significant astrocytic gliosis with 22L and 139A prion infections although PrPSc levels continued to rise in the cultured cells in parallel with the neurotoxic effects. A recent study has reported a new paradigm to assess acute prion neurotoxicity through assessing dendritic spine number and area 84. At 24 hours following exposure to 0.16% brain homogenates derived from wild-type mice dying from RML prion infection, primary murine hippocampal neurons generated from P0 wild-type pups 85 harboured dendritic spines displaying marked retraction, with loss of spines per unit length and reduction in their area. No significant alteration in dendritic spines was observed in primary hippocampal neurons generated from PrP0/0 P0 pups exposed to the same RML-containing brain homogenates or in wildtype primary hippocampal neurons exposed to brain homogenates generated from uninfected age-matched wildtype mice. Although dendritic spine changes were accompanied by evidence of actin collapse, microtubules appeared preserved suggesting preservation of general dendritic morphology. To further support the PrPSc–specific nature of the synaptotoxicity, the authors employed two brain homogenate misfolded prion protein “purification” methods utilising brains from terminally sick RML-infected wild-type mice and uninfected controls; one method employed repeated ultracentrifugation in the presence of Sarkosyl without use of proteases (>50% purity) and the other pronase E in combination with sodium phosphotungstate precipitation (>90% purity). Both PrPSc–enriched preparations caused dendritic spine retraction, with loss of spines per unit length

and reduction in their area in primary hippocampal neuronal cultures from wild-type P0 pups with no significant toxicity observed in primary hippocampal neurons from PrP0/0 P0 pups. Similar results were observed when employing protease-free purified RML-infected brain homogenates treated with proteinase K (PK; 20 µg/ml for 1 hour at 37oC). Of note, use of primary hippocampal neurons from tga/20 P0 pups that over-express PrPC 10-fold did not show greater vulnerability to the toxicity of PK-treated preparations, while primary hippocampal neurons from transgenic mice lacking residues 23-111 (∆PrP23-111PrP) and 23-31 (∆PrP23-31) constructed and maintained on a PrP0/0 background were resistant to the synaptotoxic effects of PrPSc purified without proteases. Based on this result they defined the polybasic Nterminal domain of PrPC as essential for PrPSc induced dendritic spine loss. The heretofore described studies utilising a range of prion strains combined with a diverse spectrum of host cells from immortalised and primary lines on wild-type and transgenic backgrounds have shown that ex vivo preparations can induce acute toxicity in vitro. Employing different detection techniques, such studies have demonstrated that apoptosis appears to be a common pathophysiological mediator with roles also demonstrated for oxidative stress, glutamate receptors, aggresome formation, the ER stress response and impairment of the ubiquitin-proteasome system. Acknowledging the demonstrated tropism or selective neuronal vulnerability to certain prion strains underscores the possibility that some pathogenic pathways or mechanisms may predominate in particular neuronal sub-populations, with the likely modulating influences of other factors such as PrPC expression and microglia awaiting further studies for definitive characterisation.

NON-BIOLOGICALLY OCCURRING SYNTHETIC PRION PEPTIDE FRAGMENT STUDIES

Numerous studies have examined the acute toxic effects of peptides representing domains or segments of full-length PrPC with the prion peptide fragment PrP106-126 by far the most studied based on the belief that this hydrophobic sequence may represent a key toxic component after misfolding of mature PrPC 80,86–89. Unfortunately, justifiable concerns regarding the biological and translational relevance of findings relating to studies employing peptide fragments not naturally occurring in disease, including PrP106-126 have been articulated over an extended period 60,90. Consequently, we will only describe a few illustrative studies. In addition and probably offering greater relevance to pathogenesis, there have been a number of studies utilising truncated prion protein fragments approximating those observed as a consequence of constitutive or endogenous processing of PrPSc 91. In a seminal study by Forloni and co-workers, a range of PrP peptide fragments were synthesised using solid-phase chemistry, including PrP106-126 but also PrP57-64, PrP89106, PrP106-114, PrP127-135 and PrP127-14786. These peptides were then applied to rat primary hippocampal neurons generated from E17 embryos using either a single (“acute”; either day 0 or day 9 after cell plating) or repeated (“chronic”; days 0, 2, 4, 6, and 8 after cell plating) exposure with morphological and biochemical evidence of apoptosis, as well as cell viability using cresyl violet assessed on day 10 following first exposure to the peptide. None of the peptides caused toxicity with acute exposure and only PrP106-126 caused significant, dose-dependent, neuronal loss with chronic exposure. PrP106-126 at concentrations ≥40µM was toxic and associated with hallmark cytopathological features


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of apoptosis such as nuclear chromatin condensation and fragmentation, with the latter confirmed on DNA electrophoresis using agarose gels. Not long after their original report 87, the importance of PrPC expression for PrP106-126 toxicity was re-affirmed by the same authors90 although this finding has not been universal92. In addition, a role for microglia in mediating the toxicity of PrP106-126 was described 90. Employing mixed primary cerebellar cultures generated from neonatal D6 wildtype and PrP0/0 mice with or without co-culture with microglia or astrocytes isolated from cerebral cortices of wild-type and PrP0/0 mice, it was found that 80 µM PrP106126 was not toxic to PrP0/0 primary cerebellar cells and while co-culturing with microglia expressing PrPC proportionally increased toxicity to wild-type cultures as reflected by an MTT viability assay at day 10 after peptide exposure, PrP0/0 microglia and astrocytes expressing PrPC did not enhance PrP106-126 toxicity. Further supporting a role for microglia in delivering the toxicity of PrP106-126 the use of L-leucine methyl ester (LLME) to reduce the population of endogenous microglia in primary cerebellar cultures mitigated the toxicity of PrP106-126 while addition of microglia to LLME-treated primary cerebellar cultures restored the toxic effects of PrP106-126. Additionally, the toxic effects of PrP106-126 were reduced when primary cerebellar cultures were treated with the anti-oxidants vitamin E and N-acetylcysteine supporting that microglia contribute to the toxicity of the PrP106-126 peptide through oxidative damage. Accepting that apoptosis is a common final effector pathway for prion induced neuronal death, Carimalo and colleagues 88 sought to investigate the upstream pathways sub-serving this. These authors utilised primary cortical neurons harvested from wild-type, PrP0/0 and tg338 E14 mice embryos under more stringent culture conditions of reduced serum support to enhance vulnerability to toxic insults likely to trigger apoptosis, as well as sustained anti-mitotic pressure to minimise astrocyte over-growth. As evidenced by the presence of abnormal pyknotic and fragmented nuclei, treatment with 80 µM of aggregated prion peptide fragment PrP106-126 induced 50% apoptotic cell loss by 16 hours and ~70% loss at 24 hours, accompanied by neurite retraction at 16 hours and neurite fragmentation and shrinkage of cell bodies after 24 hours; progressive caspase 3 activation was present from 8 hours after peptide exposure. Additional analyses employing dichlorodihydrofluorescein diacetate, a cell permeable compound that fluoresces upon oxidative cleavage, revealed heightened intracellular oxidative stress by 3 hours following exposure to aggregated PrP106-126, clearly preceding caspase 3 activation and cell morphological changes. The c-Jun N-terminal kinases (JNKs) are members of the family of mitogen-activated protein (MAP) kinases shown to play an important role in regulating apoptosis in neurons93. Notably, aggregated PrP106–126 treatment induced substantial phospho-JNK nuclear translocation and accompanying c-Jun phosphorylation by 8 hours following exposure, preceding evidence of apoptosis of primary cortical neurons. Treatment with 1 µM SP600125 (a specific inhibitor of JNK kinase) and a dominant-negative mutant form of cJun partially protected neurons against PrP106–126-induced cell death further supporting a role for the JNK-c-Jun pathway in acute prion toxicity and apoptosis. Importantly and partially mitigating concerns of biological relevance, phospho-c-Jun was demonstrated in the nuclei of tg338derived primary cortical neurons inoculated with 127S strain prions 7 days previously, which was followed by a significant ~40% loss of neurons at day 14 following exposure.

RECOMBINANT FULL-LENGTH PRION PROTEIN OR RECOMBINANT/SYNTHETIC

BIOLOGICALLY OCCURRING FRAGMENT STUDIES

PEPTIDE

To try to determine whether soluble oligomers of PrP represented the predominant neurotoxic entity, Simoneau and co-workers produced and utilised a range of recombinant proteins including monomers of full-length α-helical mouse and sheep PrP, oligomers of dimeric full-length β-stranded mouse PrP covalently joined head-to-tail through a flexible peptide linker and β-stranded conformers of full-length sheep PrP aggregated into 12-mer and 36-mer oligomers; synthetic mouse PrP105-132 was also utilised as a comparator94. Employing an MTT assay, oligomers of dimeric full-length β-stranded mouse PrP and full-length sheep PrP at 3µM were highly toxic to primary mouse cortical neurons causing ~50% loss after 72 hours, an effect 30-fold more toxic than that observed with synthetic mouse PrP105-132. Appropriate controls demonstrated the linker peptide and His-tag were not responsible for the toxic effects. Of note, the toxic effects of the mouse and sheep PrP oligomers were similar on PrP0/0 primary neuronal cultures suggesting that PrPC expression was not critical for this deleterious effect. Utilising a panel of anti-PrP antibodies directed against various epitopes demonstrated that only antibodies specific for the hydrophobic 106-126 region protected neurons from toxicity with Hoechst 33342 staining revealing condensed and fragmented nuclei supporting death through apoptosis. Importantly, amyloid fibrils of dimeric full-length β-stranded mouse PrP and full-length sheep PrP (generated through ageing) were non-toxic to mouse primary cortical neurons. Recombinant peptide fragments of exact primary sequence recapitulating those occurring as a consequence of α- and βcleavage of full-length PrPC95 were utilised for biophysical and neurotoxicity studies by Johanssen and co-workers91. While fragments analogous to human C1 (PrP112-231; the Cterminal product of α-cleavage) could not be induced into a β-stranded isoform using disulphide bond reduction in a low pH acetate buffer, C2 equivalent fragments (PrP90-231; the C-terminal product of β -cleavage) readily adopted a βstranded conformation. Importantly, despite 4 days of exposure to C1 as monomers or multimeric assemblies there was no evidence of toxicity to primary cortical neuronal cultures (>95% neurons) generated from E14 wild-type mice, while C2 misfolded into soluble β-stranded conformers was toxic at ≥1µM as determined by a cell viability assay; resuspended pellets of insoluble C2 multimers were not toxic. Extending their observations, Johanssen and colleagues additionally employed synthetic peptides PrP111-144, PrP112-144 and PrP 90-144 based on the human mutation Y145STOP 96. Once again, the peptide reminiscent of that occurring with β-cleavage (PrP90-144) was significantly toxic to primary cortical neurons including those from PrP0/0 mice, while synthetic peptides based on α-cleavage (PrP111144 & PrP112-144) displayed no or minimal toxicity. Further biophysical studies, including aggregation assays and SELDI-TOF mass spectrometry suggested that the toxicity of PrP90-144 correlated with persistence of soluble oligomeric species with overall α-cleavage appearing to confer intrinsic protection against potential toxicity of cognate peptides generated through endogenous processing. Employing recombinant, full-length, mouse prion protein refolded into either monomeric α-helical, β-oligomeric or amyloid fibrillar forms, Novitskaya and colleagues were able to demonstrate significant acute neurotoxicity of fibrillar PrP 97 . After 48 hours of exposure, Hoechst-33342 staining revealed fibrillar PrP at 0.5µM was toxic to the four different cell lines studied (SH-SY5Y neuroblastoma, mouse N2a neuroblastoma, mouse fibroblast NIH-3T3 and human NT2 non-differentiated) and in fact caused greater cell loss than that observed with equivalent concentrations of β-oligomeric



PrP, while α-helical PrP showed minimal toxicity compared with controls, which was attributed to minor contamination by β-oligomeric conformers. More dramatic effects were observed when utilising primary rat hippocampal and cerebellar neurons. Primary neurons were incubated for three different durations (24 hours, 2 days, and 4 days) after adding the recombinant PrP preparations at three different concentrations with β-oligomeric and amyloid fibrillar forms evincing comparable toxicity, which was time and dosedependent. Minimal toxicity was again observed for α-helical PrP compared to controls, which was again attributed to minor contamination by β-oligomeric conformers. In addition, the authors employed two different plating protocols for primary neurons, either plating on polylysine for 1 hour or 2 days before treating with the various PrP preparations. The second format allowed cells to grow and produce neurites before incubating with recombinant PrP with both β-oligomeric and amyloid fibrillar preparations causing substantial neurite loss and shortening, which was not observed when exposed to α-helical PrP. In a separate experiment, the authors successfully used 150nM anti-PrP siRNA to reduce PrPC expression in SH-SY5Y cells and found that 24 hours of siRNA treatment eliminated the toxic effect of β-oligomers and amyloid fibrils when assessed with Hoechst-33342 fluorescence staining 48 hours following incubation. This finding suggested that PrPC expression in SH-SY5Y cells was important in mediating the acute toxicity of both β-oligomeric and amyloid fibrillar preparations. Harnessing a range of techniques (eg annexin V, calcein and nuclear fragmentation assays), it was demonstrated that apoptosis appeared to be the primary mechanism of cell death in both the cell lines and primary neurons with little evidence of necrosis as depicted by propidium iodide staining. Further, the authors observed that amyloid fibrillary PrP induced cell aggregation through PrPC-dependent cell surface deposition first evident by 7 hours after treatment followed by failure of neurite outgrowth in such cells and eventually apoptosis; cell aggregation was largely absent following β-oligomeric exposure despite the subsequent occurrence of apoptosis. Elaborating on the finding that cell surface PrPC acts as a receptor to transduce the neurotoxic effects of soluble oligomers of Aβ42 peptides on hippocampal long-term potentiation 98, Resenberger and co-workers undertook a comprehensive study to examine whether PrPC may act in a similar manner for other β-sheet-rich proteins including PrPSc 99 . These authors demonstrated that SH-SY5Y cells expressing mouse PrPC when co-cultured with scrapieinfected mouse neuroblastoma (ScN2a) cells (that release PrPSc into the culture medium) manifested apoptotic cell death with increased active caspase 3 or fragmented nuclei, as well as abnormal mitochondrial morphology as revealed by MitoTracker Red CMXRos. These findings were not observed when SH-SY5Y cells expressing mouse PrPC were co-cultivated with non-prion infected mouse neuroblastoma (N2a) cells. Transiently transfecting SH-SY5Y cells with hamster, human, cervid or bovine PrP to essentially abrogate de novo PrPSc propagation through the “species barrier”, the authors continued to observe significant apoptosis when coculturing with ScN2a cells but not with N2a cells, supporting that toxicity of heterologous PrPSc appeared dependent on cellular PrPC expression but not on PrPSc propagation. Although perhaps not directly pertinent to this review the authors undertook a number of additional experiments assessing the potential role of cell surface PrPC in mediating toxicity for other diverse β-sheet-rich proteins, including: oligomers of Aβ42; the NM region (N-terminal region [N] containing the essential prion-forming determinants and charged middle region [M] enhancing the solubility and stability of the prion form) of the PSI+ prion formed from the essential translation termination factor Sup35 of

Saccharomyces cerevisiae; and artificially designed β- and αpeptides. Illustrating this part of their work, Aβ42 secreted by Chinese hamster ovary (CHO) cells stably transfected with the human amyloid precursor protein carrying the V717F mutation (CHO-7PA2) caused co-cultured SH-SY5Y cells expressing human PrPC to display significantly increased active caspase 3 after 16 hours. Co-culturing using nontransfected CHO cells or SH-SY5Y cells transfected with GPI-anchored GFP was not associated with increased caspase 3 activation and the γ-secretase inhibitor DAPT also prevented the increased apoptosis. Of interest, simultaneous co-culturing of CHO-7PA2 and ScN2a cells with SH-SY5Y cells expressing mouse PrPC did not lead to additive toxicity suggesting that soluble Aβ42 and PrPSc share the same PrPC– related neurotoxicity transduction pathways. Further, through transfecting SH-SY5Y cells with PrP constructs lacking most of the N-terminus (PrP∆27-89) or a heterologous C-terminal transmembrane domain rather than a GPI anchor (PrP-CD4) showed that both regions are important for cell surface PrPC to mediate the toxicity of soluble oligomers of Aβ42. Morevoer, the conformation-dependent antibody A11100 was shown to significantly attenuate the toxicity of both Aβ42 and PrPSc in co-culture experiments, as did pre-incubation of cultures with the NMDA-receptor antagonist memantine. Finally, employing primary cortical neurons from E14.5 and E15.5 wild-type and genetically similar PrP0/0 mice, coculturing with ScN2a cells was shown to impair viability, reduce dendrite length and disturb mitochondrial morphology in wild-type neurons after 4-5 days, which did not occur in PrP0/0 neurons. Adopting an unbiased approach to the size or conformation of the neurotoxic species of prion protein, Zhou and colleagues 101 undertook dilution refolding of recombinant full-length prion protein followed by size-exclusion chromatography and then systematically tested the various derived fractions. Unexpectedly they found the most toxic species was a monomeric, highly α-helical conformer (TPrP), which displayed very modest proteinase-K resistance. Three days of exposure to TPrP at concentrations ≥0.5µg/ml caused reduced cell viability using a luminescence-based assay associated with neuritic retraction and vacuolation in murine PK1 neuroblastoma cells. Further biochemical and morphological studies revealed evidence of apoptosis with increased caspase 8 and caspase 9 activity, as well as evidence of increased autophagolysosome markers (such as the LC3-II/I ratio) recapitulating what was found in the brains of mice dying from prion disease. Although additional murine neuronal cell lines, such as CAD5 and even hippocampal PrP0/0 neurons, were vulnerable to TPrP toxicity at 2.5µg/ml, human and mouse fibroblasts were resistant even at 50µg/ml supporting a relatively selective neuronal susceptibility. The authors also examined the effect of TPrP in vivo (see below) and on organotypic cerebellar slices 69 finding that over 6 days there was significant cell loss and damage. Following up on their studies of the toxic, monomeric, αhelical conformer TPrP, Zhou and colleagues aimed to define the mechanism of neurotoxicity 102. Initial studies involved inhibition of apoptosis (z-VAD pan-caspase inhibitor) and autophagy (eg rapamycin and bafilomycin) with no improvement in the survival of PK1 neuroblastoma cells exposed to TPrP. Similarly, manipulation of lysosomes to increase biogenesis (sucrose) or block their function (chloroquine) and treatments with potent anti-oxidants such as ascorbate had no effect on PK1 cell survival after treatment with TPrP. Comprehensively investigating a somewhat serendipitous observation of improved PK1 cell survival when using cell culture media 2 days following TPrP exposure, the authors observed a dose-dependent protective effect of nicotinamide adenine dinucleotide (NAD+).


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Restoration of neurite regrowth and resorption of vacuoles in PK1 cells was seen starting at 3 hours following NAD+ treatment despite ongoing exposure to TPrP. Further studies employing FK866 (an inhibitor of the salvage synthesis pathway from nicotinamide) confirmed that the rescuing effects of nicotinamide were through its role as a precursor for NAD+, with other studies demonstrating that the protective effects were specific for TPrP with no increased survival after exposure to autophagy modulators or inhibitors of protein translation. Levels of neuronal NAD+ were found to be 10-fold lower after 3 days of TPrP exposure associated with reduced cellular ATP levels. Reinforcing the neuronspecific toxic effects of TPrP, the authors also demonstrated the same toxicity and protective effects of NAD+ in primary cortical neurons while primary astrocytes were unaffected, maintaining normal NAD+ levels. Finally, a number of experiments clarified that the cause of neuronal NAD+ depletion was through mono-ADP-ribosylation of cellular proteins. In vivo studies undertaken are described below. Studies employing recombinant full-length PrP and cognate peptides, as well as those utilising synthetic PrP peptide fragments, especially PrP106-126, have also demonstrated the capacity of misfolded conformers to induce acute cellular toxicity in vitro with a diverse range of, albeit sometimes contradictory, pathogenic insights. Notwithstanding concerns regarding the biological validity of many PrP peptide fragments studied, similar to the previously described studies using ex vivo prion preparations are recurrent mechanistic themes of apoptosis, oxidative stress and a deleterious role for microglia, with additional insights suggesting a protective role for α-cleavage of PrPC and possible involvement of the MAP/JNK family and autophagolysosomes in pathogenesis. Although preponderant data from these studies support the likelihood that misfolded, β-strand-rich, soluble oligomers of PrP represent the main toxic species such findings have been challenged by experiments demonstrating that β-strand-rich fibrillar and highly α-helical forms can be equally toxic, with evidence that the latter operates through NAD+ depletion and not apoptosis or oxidative stress. Studies employing recombinant PrP and synthetic peptide fragments also perpetuate controversy surrounding the role of PrPC expression in acute toxicity with studies reporting independence from PrPSc propagation and contradictory findings in relation to acute toxicity dependence on such expression.

In Vivo Acute Prion Toxicity Models

There are considerably fewer reports of in vivo models examining the acute toxicity of recombinant full-length PrP and cognate peptides, synthetic PrP peptide fragments or ex vivo PrPSc with the majority of such studies primarily utilising morphological techniques to assess neurotoxicity. For their acute in vivo model, Ettaiche and colleagues utilised the retina of adult male Wistar rats 89, an accessible CNS tissue known to be infectious 103 and also serve as a relatively efficient site for successful prion inoculation 104. Notwithstanding previous comments in relation to the uncertain translational relevance of prion peptide fragments without biological correlate, these authors studied the effects of PrP106-126 (human sequence) both freshly prepared and after the peptide had been aged for three days at room temperature employing both vehicle alone (PBS) and soluble PrP89-103 peptide as controls. Three days following intravitreal injection, harvested retinas exposed to 5 nmol aged PrP106-126 demonstrated marked positive terminal deoxynucleotidyl transferase dUTP-end labeling (TUNEL) staining in the ganglion cell and inner and outer nuclear layers in keeping with induction of marked apoptotic cell death by the aged peptide, while freshly prepared PrP106-

126, PrP89-103 and PBS caused only occasional TUNEL positive cells. Further experiments confirmed that the aged PrP106-126 caused dose-dependent toxicity starting at 0.5 nmol. Additionally, supporting apoptotic retinal neuronal death, gel-based DNA electrophoresis revealed characteristic laddering commencing 24 hours after intravitreal injection of 5 nmol aged PrP106-126, which became maximal by 48-72 hours. Electroretinographic studies demonstrated impairment of both a- and b-waves starting one day after treatment with aged PrP106-126 and persisting across days 2, 3 and 7 after exposure while fresh PrP106-126 and PrP89-103 caused no retinal failure. To extend their studies of the neurotoxic effects of recombinant prion proteins into an in vivo model, Simoneau and colleagues utilised the same repertoire of monomers of full-length α-helical mouse and sheep PrP, oligomers and amyloid fibrils of dimeric full-length β-stranded mouse PrP covalently joined head-to-tail through a flexible peptide linker and oligomers (12-mer and 36-mer) and amyloid fibrils of full-length sheep PrP94. The authors stereotactically injected 2 µl of buffer or monomeric α-helical PrP into the left hippocampal CA2 region (controls) and oligomeric or fibrillar PrP conformers into the right hippocampal CA2 region of wild-type C57BL/6 mice or PrP0/0 mice on a C57BL/6 background employing 1 mg/ml of the various PrP preparations. At 24 hours following injection, brains were harvested and screened for evidence of toxicity using gallocyanine staining. As expected, buffer and monomeric αhelical PrP were non-toxic while murine and ovine oligomers were equivalently highly toxic to both wild-type and PrP0/0 mice causing severe loss of pyramidal neurons most pronounced immediately below the injection site; fibrillary forms of PrP were only minimally toxic. Apoptotic neuronal death was demonstrated using ApopTag BrdU that binds to DNA breaks. Although the relevance of monomeric, α-helical TPrP to prion pathogenesis in vivo remains unclear, as part of their assessments of the neurotoxicity of recombinant TPrP, Zhou and co-workers also examined the effects in a mouse model102. In their original report101, 5 days following stereotaxic injection to just above the hippocampus in wildtype C57BL/6 mice, TPrP was found to cause significant dose-dependent loss of pyramidal neurons associated with shrunken, apoptotic cells and occasional vacuolation, with an oligomeric species of PrP 10-fold less neurotoxic while another monomeric α-helical conformer NTPrP was nontoxic similar to the PBS control. The highest amount of TPrP injected (1.4 µg) caused loss of hippocampal pyramidal neurons over a nearly 1 mm segment, which was sometimes complete over short segments of the stratum, with some spillover beyond the hippocampus into the neighbouring dentate gyrus with occasional shrunken apoptotic neurons evident. Employing the same stereotaxic in vivo model, the toxic effect of TPrP on hippocampal pyramidal neurons was reaffirmed in their second report 102 but importantly the authors showed a dose-dependent protective effect of NAD+ when delivered concomitantly. Zhou and colleagues then proceeded to study the effect of NAD+ in the setting of bona fide prion disease. Following routine intracerebral inoculation with 20 µl of a 1% homogenate made from the brains of mice terminally sick from RML prion disease, these mice were then treated with daily intranasal instillation of 30 mg/kg NAD+ starting at 117 or 130 dpi, the latter during the early stage of overt prion disease (hunched posture and hindlimb rigidity evident). No significant increase in survival versus controls was observed for treatment starting at either time-point but NAD+-treated mice treated from 117 dpi displayed slower disease progression while formal assessment of motoric function (Rotarod and Open Field)



starting at 4 days after onset of treatment at 130 dpi showed increased activity in mice receiving NAD+. Pathogenic insights from in vivo acute toxicity studies are very limited due to the paucity of reports. In addition, there are uncertainties regarding the biological validity and translational relevance of the pathogenic insights accompanying the findings based on the choices of prion proteins or peptides tested, illustrated by the use of synthetic PrP106-126 and recombinant highly α-helical monomeric TPrP. Nevertheless, apoptosis was again reported to play a mediating role with soluble β-strand rich oligomers observed to be more toxic than fibrils with the same study finding toxicity was not dependent on PrPC expression. Whether NAD+ depletion is an important pathogenic driver in prion disease remains to be established but exogenous replacement late in animal disease evolution offered minimal benefit.

Conclusions:

Previous drug screening campaigns aimed at defining antiprion compounds primarily utilised simple cell culture systems infected with different prion strains mainly targeting prion replication with limited regard for directly mitigating neurotoxicity. Based on this approach however, when tested in animal models of prion disease, most of these compounds have shown either weak efficacy or prion strain dependence, suggesting that the screening methods employed so far are poor predictors of translatable therapeutic outcome, at least partly due to deficiencies in current models of prion disease utilized, including those used to decipher pathogenesis. Elucidating the pathogenic mechanisms underlying prion diseases remains an indispensably crucial step for designing new and targeted effective compounds or therapeutics capable of halting both prion infectivity and toxicity. In this manuscript, we have critically reviewed different experimental paradigms used to date to study the acute toxicity of prions in vitro, ex vivo or in vivo, highlighting technical aspects, disease relevance and limitations for each method. Clearly, despite the remarkable qualities of some of these assays, there remains a need to develop further robust and biologically relevant approaches to dissecting the neurodegenerative mechanisms underlying prion diseases. Arguably, acute toxicity models more closely recapitulating the complexity of the CNS such as 3-D cultures, tissue explants and in vivo paradigms coupled with the use of ex vivo prion-infected preparations, while technically more challenging, offer a heightened likelihood of translationally relevant insights to facilitate the quest for effective treatments. Thus, we hope that this review serves to further stimulate interest in developing new experimental models aimed at curing prion diseases.

Author Contributions:

A M T Islam – shared primary responsibility for writing the manuscript, sourcing references for inclusion and critically reviewed the final content. P A Adlard - critically reviewed and contributed to the final version of the manuscript. D I Finkelstein - critically reviewed and contributed to the final version of the manuscript. V Lewis - critically reviewed and contributed to the final version of the manuscript. S Biggi - contributed to writing the manuscript and critically reviewed the final version of the manuscript. E Biasini - contributed to writing the manuscript, critically reviewed the final version of the manuscript and shares senior author responsibility for the content of the manuscript. S J Collins – conceived the review, contributed to writing the manuscript, critically reviewed the final version of the

manuscript, sourced references for the review and shares senior author responsibility for the content of the manuscript.

Funding Sources:

The Florey Institute of Neuroscience and Mental Health acknowledge the strong support from the Victorian Government and in particular the funding from the Operational Infrastructure Support Grant. SJC is supported in part by an NHMRC Practitioner Fellowship (#APP1105784). Conflicts of Interest: The authors report no conflicts of interest.

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Animal Prion Diseases

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Host


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Scrapie1 and Atypical Scrapie1

Goat and Sheep.

Bovine Spongiform Encephalopathy (BSE or “Mad Cow Disease”) and Atypical BSE1

Cattle.

Transmissible mink encephalopathy (TME).

Mink.

Feline Spongiform Encephalopathy (FSE)

Domestic cats and Captive wild cats.

Exotic Ungulate Encephalopathy (EUE)

Nyala, Oryx and Kudu.

Chronic Wasting Disease (CWD)1

Cervids.

TSE in non-human Primates (NHP)

Lemurs.

Human Prion Diseases

Host

Sporadic forms Sporadic Creutzfeldt-Jakob Disease (CJD) Sporadic Fatal Insomnia (sFI)

Human.

Genetic forms Familial CJD (fCJD) Gerstmann- Sträussler -Scheinker syndrome (GSS) Fatal Famillial Insomonia (FFI)

Human.

Acquired forms Iatrogenic CJD (iCJD) Variant CJD (vCJD)2 Kuru3

Human.

Table 1: Summary of Animal and Human Prion Diseases. 1. Natural prion diseases, 2. Zoonosis related to bovine spongiform encephalopathy, 3. Largely confined to the Fore linquistic group of the Eastern Highlands of Papua-New Guinea.

Table 2: Summary of acute neurotoxicity models of Prion disease.

In vitro models



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Ex vivo Prion/PrPSc Studies Reference

Prion strain

Host cells

Mueller Eur J Pharmacology 1993

Hamster adapted scrapie (strain not specified).

Iwamaru J Virology 2013

Mainly Chandler but also ME7, 22L BSE and Fukuoka.

Primary rat cortical; primary human astrocytes; N2a line. Neurospheres from PrP0/0 and Tga/20 mice => mainly astrocytes.

Hetz EMBO J 2003

139A => purified by ultracentrifugation and PK treatment.

N2a

48 hours for morphological changes

Giese Brain Pathology 1998

Purified hamster PrP27-30 (strain not specified).

Cronier PNAS 2004

127S scrapie.

Day 7 (exposed days 2 and 4 to PrP27-30) 28 days

Kristiansen J Biol Chem 2005

RML

Primary wildtype mouse cerebellar (not specified whether granule or not). Primary cerebellar granule cells and astrocytes from transgenic tg338 mice (~10X over-express ovine PrP on null background) & PrP knockout mice. GT1, N2aPD88 cell lines, as well as N2aPD88 cells overexpressing mouse PrPC.

Fang PLoS Pathogens 2016

RML; “purified” PrPSc preparations by either pronase E/NaPTA or ultracentrifugation in Sarkosyl. Mock infected & infected with

Milhavet et al PNAS 2000

Primary P0 hippocampal neuronal cultures (for 18-21 days) from PrP0/0, Tga20, Tg(∆23–111), Tg(∆23–31), wildtype C57BL/6. GT1-7 cells chronically prion

Time-frame of toxicity assessment after exposure 12 hours

Within 18 days

Toxicity metrics

Inhibitor treatments

DNA fragmentation; Fluorescein diacetate uptake.

Memantine; MK-801.

Microscopic morphology; LDH release; WST-8 assay; caspase 3/7, caspase 8 and caspase 9 activity assays; TUNEL stain. MTS assay; in situ caspase 3, caspase 8 activity; externalisation of phosphatidlyserine by annexin V; fluorescence intracellular calcium with/without thapsigargin. MTT assay day 7 after exposure.

Pan-caspase zVAD-fmk.

Count % GFAP-neg cells with fragmented nuclei (apoptosis) and TUNEL staining.

Various caspase inhibitors.

L-leucine methyl ester (LLME) => selectively kills microglia. Nil.

24 hours

LDH assay; apoptosis using annexin V binding (flow cytometry), propidium iodide staining, caspase 3 and caspase 8 activity assays; aggresome detection.

24 hours

Number and area of dendritic spines

Caspase inhibitors benzyloxycarbonylDEVD-fmk or benzyloxycarbonylIETD-fmk; proteasomal inhibitors lactacystin & epoxomicin; aggresome microtubule inhibitors colchicine, nocodazole. Recombinant PrP23-110.

48 hours after buthionine

Cell viability with MTT assay; DNA

Congo red treatment of GT1-Chandler


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Hannaoui et al Journal of Virology 2013

Chandler, 22L 87V and Fukuoka prion strains.

infected & mock infected.

sulfoximine and 3morpholinosy dnonimine exposure.

139A, 22L and ME7 prion strains from C57BL/6 mice & mock infection from uninfected C57BL/6 mice.

C57BL/6 primary neuronal striatal (E15), cerebral cortical (E15) and cerebellar granule (P6) cell cultures.

4-28 days post-infection.

fragmentation assay; lipid peroxidation with fluorescence of a malondialdehyde thiobarbituric acid adduct; total, Mn & Cu/Zn SOD activities; glutathione reductase and glutathione peroxidase activities. Cell viability with CellTiter 96 Aqueous One solution (MTS) assay; neuronal and astrocytic numbers by immunoctochemistry using MAP2 and GFAP; apoptotic neurons based on pyknotic/fragmented nuclei.

cells for cure; Nacetylcysteine.

Toxicity metrics


Caspase 3 activity; intracellular ROS using DCFDA assay; morphology – nuclear fragmentation, neurite retraction/fragmentati on; phosphorylated cJun–N-terminal kinase (JNK) translocation; activation of the nuclear c-Jun transcription factor. Microscopically pyknotic nuclei with DNA fragmentation; crystal violet staining for cell viability.

Inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun.

Non-biological prion peptide fragment studies Reference

PrP peptides assessed

Host cells

Carimalo Eur J Neuroscience 2005

PrP106-126 peptide (human); 127S strain prions.

Primary cortical neurons from wildtype, PrP0/0, tg338 E14 embryos.

Forloni Nature 1993

Synthetic PrP106126 (human) but also 57-64, 89106, 106-114, 127-135 & 127147. Synthetic PrP106126 (human); coculturing with microglia purified from wild-type and PrP0/0 mice.

Rat primary hippocampal neurons from E17 embryos.

10 days

Primary cerebellar mixed cultures from neonatal D6 wild-type and PrP0/0 mice.

10 days

Brown Nature 1996

Time frame of toxicity assessment after exposure 4-24 hours

MTT viability assay.

Nil.

LLME (toxic to microglia); antioxidants vitamin E and nacetylcysteine.

Biological prion peptide fragments or full-length protein studies Reference

PrP proteins or endogenous peptides

Host cells

Time-frame of toxicity assessment after


Toxicity metrics



Johanssen Biochemical Journal 2014

Zhou PNAS 2012

Recombinant HuPrP90-231, HuPrP112-231; synthetic 90-144, 111-144 & 112144. Recombinant fulllength mouse PrP => α-helical monomeric; and oligomeric.

Mouse primary cortical neurons wildtype and PrP0/0.

exposure 4 days

Page 22 of 24

Cell count viability assay.

Nil.

Mouse PK1 neuroblastoma cells, CAD5 cells and hippocampal PrPo/o neurons; mouse fibroblastc LD9; human fibroblast 293T cells; also, organotypic cerebellar slices. Mouse PK1 neuroblastoma cells; mouse primary cortical neurons and astrocytes.

2-4 days PK1 cell line; 6 days cerebellar slices.

Cell Titer-Glo (ATP levels) viability assay; phase-contrast microscopy for morphology; fluorescence microscopy for caspases.

Proteinase-K treatment of PrP.

2-4 days PK1 cell line.

Total NAD+/NADH levels; Cell Titer-Glo (ATP levels) & alamar Blue viability assays; phase-contrast microscopy for morphological changes; protein ADP-ribosylation levels.

Morphological assessment of neurites; apoptosis assessed Hoechst 33342 fluorescent nuclear staining & DNA fragmentation, caspase 3 activity, annexin V assay, membrane blebbing (calcein) assay; immunoblot caspase 3 active subunit detection; propidium iodide uptake. 3, [4,5 dimethylthiazol2yl]-2,5 diphenyltetrazolium bromide (MTT) & WST-1 viability assays. Apoptosis using Hoechst 33342 fluorescence.

Numerous including Z-VAD panapoptosis inhibitor; rapamycin (mTOR), 3-MA & bafilomycin autophagy inhibitors; chloroquine lysosomal inhibitor; ascorbic acid antioxidant; nicotinamide; FK866 NAD+ salvage synthesis pathway inhibitor; NAD-ribosyl cyclase inhibitor; 3ABA PARP1 inhibitor. siRNAs directed against human PrP (SH-SY5Y cells).

Zhou Brain 2015

Recombinant fulllength mouse PrP => α-helical monomeric toxic and non-toxic.

Novitskaya JBC 2006

Recombinant fulllength mouse PrP => α-helical monomeric, βoligomeric and amyloid fibrillar forms.

Human SH-SY5Y neuroblastoma cells, mouse N2a neuroblastoma cells, mouse fibroblasts NIH-3T3 cells, and human NT2 (non-differentiated) cells; primary rat hippocampal and cerebellar neurons.

2 days

Simoneau PLoS Pathogens 2007

Recombinant fulllength monomeric mouse and sheep PrP, oligomerised tandem full-length mouse PrP dimer & β-stranded sheep PrP 12-mer and 36-mer oligomers;

Primary mouse cortical neurons.

72 hours


Anti-PrP monoclonal antibodies spanning various epitopes.

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Resenberger EMBO 2011

synthetic mouse PrP105-132. Mouse PrPSc secreted by ScN2a cells; Aβ42 & Aβ42G33A oligomers and high MW aggregates; yeast NM prion protein oligomers (dityrosine crosslinked); designed N-terminally myctagged β-sheet (oligomers) and αhelical peptides.

Chinese hamster ovary (CHO) cells & CHO cell expressing APP751; SH-SY5Y cells & transfected SH-SY5Y cells expressing mouse, hamster, human, cervid & bovine PrP; N2a cells & scrapie infected N2a (ScN2a) cells. Mouse primary cortical neurons wildtype and PrP0/0.

7-16 hours cell lines; 1, 2 and 4 days primary neurons.

Cell lines => apoptosis - count cells positive for active caspase-3 or cells with fragmented nuclei & mitochondrial morphology (using MitoTracker Red CMXRos). Primary neurons mitochondrial morphology (using MitoTracker Red CMXRos) & dendritic lengths in primary neuron cultures.

DAPT; Memantine.

Time-frame of toxicity assessment after exposure 3 days for TUNEL staining and DNA fragmentation; 1,2,3 & 7 days for ERGs.

Toxicity metrics


Terminal deoxynucleotidyl transferase dUTP-end labeling (TUNEL) staining; electrophoresis for DNA fragmentation; electro-retinograms (ERGs). Morphological assessment of neurons (gallocyanine) and apoptosis (ApopTag BrdU kit).

Nil.

In vivo studies Reference

Toxic preparation

Animal model

Ettaiche JBC 2000

PrP106-126 (human) – fresh and aged (3 days at RT); PrP89103.

Adult rat retina using intravitreal injection.

Simoneau PLoS Pathogens 2007

Recombinant fulllength monomeric mouse and sheep PrP, oligomers and fibrils of tandem full-length mouse PrP dimer & oligomers and fibrils of sheep PrP. Recombinant fulllength mouse PrP => α-helical monomeric toxic and non-toxic and oligomeric. Recombinant fulllength mouse PrP => α-helical monomeric toxic and non-toxic.

Stereotaxic injection above CA2 hippocampal region of C57BL/6 wild-type and back-crossed PrP0/0 mice.

24 hours

Stereotaxic injection above hippocampus C57BL/6 wild-type mice.

5 days

Morphological using histology => cell loss, pyknotic nuclei.

Nil.

Stereotaxic injection above hippocampus 12 week-old C57BL/6 mice; routine intracerebral inoculation (ICI) 8 week-old C57BL/6 mice with 1% RML brain homogenate.

5 days

Morphological using histology after Nissl staining => cell loss, pyknotic nuclei; for routine ICI weight, motor activity (Open Field & Rotarod)

Concomitant stereotaxic delivery of NAD+; daily intranasal NAD+ (30mg/kg) starting 117 or 130 dpi.

Zhou PNAS 2012

Zhou Brain 2015


Nil.


“Table of Contents Only”


Page 24 of 24

Acute Neurotoxicity Models of Prion Disease - American Chemical

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