Kumi

Monitoring Sustainable Development Goal 14 on the ocean

What was the Ocean University Initiative's contribution to the Ocean Conference?

The participants in the Ocean Conference were invited to make voluntary commitments that aim to contribute to the implementation of SDG 14. Under the #OceanAction21076 engagement, the University of Brest (UBO) committed to propose to the United Nations University the establishment in France of an institute dedicated to the "science and governance of the ocean and coasts" with the support of French oceanographic research organizations, state ministries and local authorities in Brittany. The project will support the voice of the ocean and coasts by joining forces with civil society, with a special focus on capacity building in the Southern ocean. More information is available at: https://oceanconference.un.org/commitments/?id=21076

What is SDG 14 on the ocean? T he SDG 14 -"Conserve and sustainably use the oceans, seas and marine resources for sustainable development" -can be considered as a tool to address sustainable development processes on the ocean in both developed and developing countries and to facilitate action at all levels and with all actors, including civil society, the private sector and the scientific community to strengthen the capacity of the State to achieve the desired outcomes. The SDG 14 targets cover environmental pressures on marine life due to economic activities. Small Island Developing States (SIDS) and coastal communities are especially impacted by these environmental pressures as well as being dependent on marine resources in socio-economic terms.

Why are indicators necessary for the implementation of SDG 14?

A comprehensive framework of indicators is needed for the implementation of SDG 14 to monitor progress, inform policy and ensure the accountability of each stakeholder. The Inter-Agency and Expert Group on SDG indicators (IAEG-SDGs) proposed a global indicator framework that the United Nations General Assembly adopted in March 2016 to track progress at the global level and for collective action towards achieving the 17 SDGs. Global monitoring should be based, as much as possible, on comparable national data that countries should report to the international statistical system. At the global level, the Food and Agriculture Organization (FAO), is the custodian agency for four (14.4.1, 14.6.1, 14.7.1, 14.b) of the 10 SDG 14 indicators 1 . Member States of the United Nations should also develop more detailed indicators at the regional and national levels to track success at these levels.

1 Source: FAO (2017).

14.1 By 2025, prevent and signifi cantly reduce marine pollution of all kinds 14.2 By 2020, sustainably manage and protect marine and coastal ecosystems to avoid signifi cant adverse impacts 14.3 Minimize and address the impacts of ocean acidifi cation, including through enhanced scientifi c cooperation at all levels 14.4 By 2020, effectively regulate harvesting and end overfi shing, illegal, unreported and unregulated fi shing and destructive fi shing practices 14.5 By 2020, conserve at least 10 per cent of coastal and marine areas 14.6 By 2020, prohibit certain forms of fi sheries subsidies which contribute to overcapacity and overfi shing, eliminate subsidies that contribute to illegal, unreported and unregulated fi shing 14.7 By 2030, increase the economic benefi ts to small island developing States and least developed countries from the sustainable use of marine resources T he objectives of South Africa's National Development Plan (NDP) are consistent with those of the SDGs. Both the NDP and the SDGs seek to raise the living standards of the poor. South Africa faces a triple challenge: poverty, inequality and unemployment. In order to address these threats, the NDP seeks to implement a long-term strategy to raise employment and increase opportunities through education, vocational training and health.

2 Source: South Africa (2017). R egarding the ocean, the NDP states that "market and policy failures have resulted in the global economy entering a period of 'ecological deficit', as natural capital (ground water, marine life, terrestrial biodiversity, crop land and grazing) is being degraded, destroyed, or depleted faster than it can be replenished". In South Africa, most fishery resources are already harvested to their limit or over-exploited. They, however, play a key role from a social standpoint. In South Africa, there are about thirty thousand subsistence fishers on the east and south coasts. There are few industrial fisheries, but these employ an equivalent number of people. It is therefore fundamental to manage this resource sustainably (Tables 4 and5).

Indicator 14.5.1D: Percentage of marine and coastal ecosystem types that are wellrepresented in protected areas D efinition: Percentage of marine (benthic and pelagic) and coastal (including estuaries) ecosystem types which are well protected (defined as those ecosystem types for which the full biodiversity target falls within a protected area). Targets are set at 20% for marine and coastal ecosystem types. A pproximately 12% of the overall South African Exclusive Economic Zone are Marine Protected Areas, for the most part (11.7%) concentrated around the Prince Edward Islands. Interdependencies with other SDG targets B efore developing context-specific regional and national indicators, it is important to understand the nature of interdependencies among the SDG 14 targets and between SDG 14 and other SDGs targets, since some targets may reinforce each other, while others may have offsetting effects.

F or instance, in terms of these interdependencies between SDG 14 and other SDG targets, there is potential for trade-offs between healthy oceans (SDG 14), food security (SDG 2) and economic growth and job creation (SDG 8). Economic activities (SDGs 8, 9 and 11) are linked to SDG 14 in the form of pollution, ocean acidification and pressure on marine resources.

O ne way to integrate context-specific trade-offs and synergies between SDG 14 and other SDGs, is to add the relevant ocean-related data to the other SDG indicators. The Baltic Marine Environment Protection Commission, HELCOM, has reported how its indicators can be used to monitor SDGs 2, 6, 9, 11 and 12. These complex links between SDG 14 and other SDGs reveal that there should be stronger collaboration between the scientific and policy communities to properly identify the most important local trade-offs.

Note: Small island states and non-island states that rely heavily on the maritime economy are particularly affected by SDGs 1, 2, 10, 11, 12, 16 and17 The SDG 14 indicators depend on the geographical level of intervention T he spatial levels of intervention for each of the SDG 14 targets determine the most relevant geographical scopes for the development of indicators: subnational, national, transnational, regional or global. Given that few global indicators are currently available, existing regional and national indicators are key for implementing the SDG targets. Moreover, disaggregated indicator data should allow causal inferences, to be made, particularly as regards links within SDG 14 targets, and between SDG 14 targets and other SDG targets. Synergies with multilateral environmental agreements T here are synergies between the SDG 14 targets and key Multilateral Environmental Agreements such as the Convention on Biological Diversity (CBD) and its 20 Aichi targets as well as the Ramsar Convention on wetlands. Exploiting these synergies can be cost effective.

Note:

The transnational intervention level applies when the watershed or the marine protected area are shared between two or more countries. Source: Based on OECD (2017).

Table 6: Synergies between some key multilateral environmental agreements and SDG 14 targets

14. a

 a Increase scientifi c knowledge, develop research capacity and transfer marine technology 14.b Provide access for small-scale artisanal fi shers to marine resources and markets 14.c Enhance the conservation and sustainable use of oceans and their resources by implementing international law The European Institute for Marine Studies (IUEM, UBO) seen from the sea, Plouzané, France (photo by Paul Tréguer, CNRS) Source: UN (2015).

  Indicator 14.5.1A: South African marine protected areas as a percentage of the total Exclusive Economic Zone (EEZ) D efinition: Coverage of Marine Protected Areas (MPAs) in relation to marine areas in the Exclusive Economic Zone.

  ds e a in t e r fa c e In t e g r a t e d C o a s t a l Z o n e s M a n a g e m e n ta in a b le fi s h in g S u s ta in a b le a q u a c u lt u re P o l l u t i o n H e a l t h y s e a c u l t i v a t i o n In it ia l a n d p r o fe s s io n a l t r a in in g E n v ir o n m e n t a l e d u c a t io n Jobs U p s tr e a m m a n a g e m e n t, P o ll u ta n t d is c h a rg e D e s a li n a ti o n , W a te r a c c e of the sea Distribution logistics of sea products S m a ll -s c a le fi s h e r ie s a c c e s s t o m a r k e t B lu e E c o n o m y li n k e d t o lo c a l d e v e lo p e m e t i a l P l a n n i n g S u s t a in a b le fi s h in g a n d a q u a c u lt u re D e c a r b o n iz e d t r a n s p o e s s t o e d u c a t io n a n d t r a in in g G o o d p r a c t ic e Status of Women in maritime activities Marine bio and technical innovations Submarine cabling S u s t a in a b le t o u r is m E x t r a c t io n o f m a r in e r e s s o u r c

  . Elaboration: French Ministry for the Ecological and Inclusive Transition (MTES).

Figure 1 :

 1 Figure 1: Interdependencies between SDG 14 and other SDG targets

Figure 2 :

 2 Figure 2: SDG 14 targets: Main spatial levels of intervention

Table 1 :

 1 SDG 14 targets

		Monitoring Sustainable Development Goal 14 on the ocean
	Global indicators
	relevance, independently of the availability of T he SDG 14 targets were selected for their policy indicators. Consequently, most indicators do not
	yet exist and there is a need to propose and build
	at least one relevant indicator for each target to
	enable monitoring. Eight out of 10 global indicators
	are currently not available for SDG 14 compared
	to 58% for all SDG global indicators. The United
	Nations Statistics Division currently provides
	open access to the two available SDG 14 global
	indicators, namely, 14.4.1 on the proportion of fish
	stocks within biologically sustainable levels and
	14.5.1 on the coverage of protected areas in relation
	Target SDG14 Global Indicator	Source	Availability
	14.1	14.1.1 Index of coastal eutrophication (i) and floating	UNEP in cooperation with IOC-	From 2021
		plastic debris density (ii)	UNESCO
	14.2	14.2.1 Proportion of national exclusive economic zones	UNEP in cooperation with IOC-	From 2021
		managed using ecosystem-based approaches	UNESCO
	14.3	14.3.1 Average marine acidity (pH) measured at agreed	IOC-UNESCO in co-operation with	After 2020
		suite of representative sampling stations	UNEP
	14.4	14.4.1 Proportion of fish stocks within biologically	FAO	1974 -2013
		sustainable levels
	14.5	14.5.1 Coverage of protected areas in relation to	UNEP's World Conservation	2000 -2014
		marine areas	Monitoring Centre, BirdLife Index,
			UICN
	14.6	14.6.1 Progress by countries in the degree of	FAO	After 2017
		implementation of international instruments aiming to combat illegal, unreported and unregulated fishing	OECD
			• Fisheries Support Estimate	2000 -2015
			• Policies against illegal, unreported	From 2018
			and unregulated fishing
	14.7	14.7.1 Sustainable fisheries as a proportion of gross	FAO	No set date
		domestic product in small island developing states,
		least developed countries and all countries
	14.a	14.a.1 Proportion of total research budget allocated to	IOC-UNESCO in cooperation with	From 2018
		research in the field of marine technology	UNEP
	14.b	14.b.1 Progress by countries in the degree of	FAO	From 2016
		application of a legal/regulatory/policy/institutional
		framework which recognizes and protects access rights
		for small-scale fisheries
	14.c	14.

c.1 Number of countries making progress in ratifying, accepting and implementing through legal, policy and institutional frameworks ocean-related instruments that implement international law, as reflected in the United Nations Convention on the Law of the Sea, for the conservation and sustainable use of the oceans and their resources United Nations Division for Ocean Affairs and the Law of the Sea, Office for Legal Affairs No set date Source: OECD (2017).

Table 2 :

 2 SDG 14 global indicators: sources and availability to marine areas. The dates by when the remaining global indicators for SDG 14 should be available are indicated in Table 2. There is, for instance, no set date for the availability of a global indicator for Statistical indicators may be further classified as 'available' (corresponding to global indicators 14.4.1 and 14.5.1), 'proxy' (corresponding to global indicators 14.1.1, 14.2.1 and 14.5.1), 'complementary' (14.1.1, 14.2.1), or 'non-available' (corresponding to global indicators 14.1.1, 14.3.1, 14.7.1 and 14.a.1). The available indicators are identical to the SDG 14 global indicators requested by the IAEG-SDGs. The proxy indicators are close to the SDG 14 global indicators, while being better suited to the French context. The complementary indicators are different from the SDG14 indicators but close to the associated objective. T he French SDG 14 indicators in Table 3 are set to evolve over time. For instance, the SDG 14 national indicator corresponding to global indicator 14.4.1 should include proxy data on the maximum sustainable yield.

	target 14.7 on sustainable fisheries which concerns,
	among other countries, SIDS. Indeed, fisheries in
	SIDS have been threatened by overexploitation,
	land-based pollution and inadequate fisheries'
	monitoring, control and surveillance systems.
	Member States are invited to complement global
	indicators with national indicators that are relevant
	for national policies or for national stakeholders.

Source: French National Council for Statistical Information (2018).

Table 3 :

 3 SDG 14 national indicators in France (June 2018)

	Monitoring Sustainable Development Goal 14 on the ocean
	SDG 14 national indicators in South Africa 2

Table 4 :

 4 South African Protected Areas Database, Department of Environmental Affairs (South Africa, 2017). National Biodiversity Assessment and Prince Edward & Marion Islands 2013, Department of Environmental Affairs (South Africa, 2017). Marine and coastal ecosystems that are well protected (in %)

	Name of Area Prince Edward Islands Mainland South Africa Source: Baseline indicator value (2013): 12.02% Area MPAs (km 2 ) Area EEZ (km 2 ) MPA (%) 180 000 466 880 38.55 4 540 1 068 660 0.42 Combined 184 540 1 535 540 12.02

Source:

Table 5 :

 5 Marine protected areas as a percentage of the total Exclusive Economic Zone

  Some coordination challenges for implementing SDG 14 indicators Monitoring Sustainable Development Goal 14 on the ocean Chemical conventions are: theBasel Convention on the Control of Transboundary Movements of Hazardous Wastes and their Disposal, the Rotterdam Convention on the Prior Informed Consent Procedure for Certain Hazardous Chemicals and Pesticides in International Trade, the Stockholm Convention on Persistent Organic Pollutants and others.

	Multilateral environmental agreements	Synergies with SDG 14 target
	Convention on Biological diversity	14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.a 14.b 14.c
	Convention on Wetlands	14.1 14.2 14.3 14.5 14.7 14.b 14.c
	Convention on the Conservation of Migratory Species of Wild Animals 14.2 14.4 14.5
	United Nations Framework Convention on Climate Change	14.2
	Regional Seas Conventions and Action Plans	14.1 14.2 14.3 14.5 14.7 14.a 14.b 14.c
	Chemicals Conventions	14.1
	Convention on International Trade in Endangered Species of Wild	14.2 14.4
	Flora and Fauna

Note: Source: OECD (2017).

Kitamori

Head of Division Green Growth &

Future research areas

Building on the frontiers of ocean science to develop SDG 14 indicators

A ll of the SDG 14 targets have a strong scientific dimension. The substantial gaps in knowledge and data should not prevent progress in the policy agenda.

Developing innovative approaches for SDG 14 data collection N umerous developing countries' statistical offices were unable to collect, analyze and disseminate data for reporting on the 48 Millennium Goal indicators (2000 -2015). Since the number of SDG indicators is much higher, standing at 244, there is a risk of focusing on less critical or easier to achieve targets.

Valuing marine ecosystem services to implement SDG 14 in synergy with other goals T he concept of ecosystem services can support the implementation of SDG 14 in synergy with other goals.

It can serve as an organizing principle to consider multi-level and cross-sectoral synergies and trade-offs.

The poor appreciation of the benefits provided by marine ecosystems does not encourage investment in their management and conservation by states and coastal communities.

Harmonizing measurement methodologies

A lack of international harmonization of measurement methodologies can have implications both in terms of interpretation and comparability of the data. Marine ecosystem-based indicators in Regional Seas entities are disparate as regards the levels of specificity, the rationales for indicator selection, the degree of sophistication and the use of qualitative information.

Providing incentives for best practice and peer-learning with regards to SDG 14 indicators I t is necessary to define a shared minimum framework involving the responsibility of actors: the United Nations system, governments, the private sector and civil society, but also the different levels of governance within countries and all United Nations agencies.

Identify the specific needs of small island developing states, least developed countries and smallscale fishers H ow can we ensure that the benefits of achieving the targets of ODD 14 reach the most vulnerable countries and social groups?


Angélique Igel-Egalon

Florent Laferrière

Mohammed Moudjou

Mathieu Merzach

Tina Knäpple

Laetitia Herzog

Fabienne Reine

Marie Doumic

Human Rezaei
email: human.rezaei@inra.fr

Vincent Béringue
email: vincent.beringue@inra.fr

Quaternary structural convergence and structural diversification of prion assemblies at the early replication stage

Keywords: prion, neurodegenerative disorders, polymerization, kinetics, diversification

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In terms of pathogenic mechanisms, the prion paradigm unifies a number of age-related, incurable neurodegenerative disorders that are caused by protein misfolding and aggregation [START_REF] Condello | Abeta propagation and strains: Implications for the phenotypic diversity in Alzheimer's disease[END_REF][START_REF] Jucker | Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders[END_REF][START_REF] Jucker | Self-propagation of pathogenic protein aggregates in neurodegenerative diseases[END_REF][START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF]. These disorders include human and animal forms of prion diseases, Alzheimer's disease, Parkinson's disease and Huntington's disease. In principle, host-encoded monomeric proteins or peptides are converted into misfolded and aggregated assemblies, which serve as seeds or templates for further autocatalytic conversion. In prion diseases, the ubiquitously expressed, host-encoded prion protein PrP C is converted into a misfolded, -sheet-rich conformer termed PrP Sc [START_REF] Prusiner | Novel proteinaceous infectious particles cause scrapie[END_REF]. In susceptible host species and in laboratory rodent models, PrP Sc assemblies form stable, structurally distinct PrP Sc conformers [START_REF] Beringue | Prion agent diversity and species barrier[END_REF][START_REF] Bruce | TSE strain variation[END_REF][START_REF] Collinge | A general model of prion strains and their pathogenicity[END_REF][START_REF] Weissmann | Prions on the move[END_REF], known as prion strains, and encode unique stereotypical biological phenotypes [START_REF] Sim | Ultrastructures and strain comparison of under-glycosylated scrapie prion fibrils[END_REF][START_REF] Spassov | Structural differences between TSEs strains investigated by FT-IR spectroscopy[END_REF][START_REF] Telling | Evidence for the conformation of the pathologic isoform of the prion protein enciphering and propagating prion diversity[END_REF][START_REF] Bessen | Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy[END_REF]. The strain-specific structural differences can be observed at the secondary structural level in terms of local structural variation but also at the quaternary level with strain-specific size distributions [START_REF] Silveira | The most infectious prion protein particles[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF][START_REF] Spassov | Structural differences between TSEs strains investigated by FT-IR spectroscopy[END_REF]. A large body of evidence supports the view for further structural diversity within specific prion populations and strains: i) some studies highlight the selection of prion substrains during the transmission of natural isolates [START_REF] Chapuis | Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions[END_REF][START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF][START_REF] Angers | Prion strain mutation determined by prion protein conformational compatibility and primary structure[END_REF] or experimental prion strains [START_REF] Li | Darwinian evolution of prions in cell culture[END_REF] with a species or transmission barrier, ii) size-or density-fractionation studies support the existence of a heterogeneous spectrum of PrP Sc assemblies with distinct tertiary/quaternary structures [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Silveira | The most infectious prion protein particles[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF][START_REF] Kim | Small Protease Sensitive Oligomers of PrP(Sc) in Distinct Human Prions Determine Conversion Rate of PrP(C)[END_REF][START_REF] Tzaban | Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes[END_REF][START_REF] Bett | Biochemical properties of highly neuroinvasive prion strains[END_REF][START_REF] Bett | Enhanced neuroinvasion by smaller, soluble prions[END_REF][START_REF] Sajnani | PK-sensitive PrP is infectious and shares basic structural features with PK-resistant PrP[END_REF] and biological activity (templating activity and infectivity) [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Silveira | The most infectious prion protein particles[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF], and iii) kinetic studies of prion pathogenesis suggest that the formation of neurotoxic PrP Sc species [START_REF] Simoneau | In vitro and in vivo neurotoxicity of prion protein oligomers[END_REF] occurs at the late stage of prion infection but that replicative PrP Sc assemblies are formed at earlier stages [START_REF] Sandberg | Prion propagation and toxicity in vivo occur in two distinct mechanistic phases[END_REF][START_REF] Sandberg | Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked[END_REF]. The prion replication process thus intrinsically allows the structural diversification of PrP Sc assemblies.

While the kinetic aspects of prion replication 'as a whole' have been comprehensively described by measuring infectivity or PrP Sc levels in the brain (see references [START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF][START_REF] Nakaoke | Early appearance but lagged accumulation of detergent-insoluble prion protein in the brains of mice inoculated with a mouse-adapted Creutzfeldt-Jakob disease agent[END_REF] as examples), the processes by which PrP Sc structural diversification and the formation of different subpopulations occur within a given strain remain undescribed and are not mechanistically supported in the actual framework of the prion paradigm. The autocatalytic conversion model proposed by Griffith in 1967 [START_REF] Griffith | Self-replication and scrapie[END_REF], the nucleated-polymerization model described by Lansbury and Caughey in 1995 [START_REF] Lansbury | The chemistry of scrapie infection: implications of the 'ice 9' metaphor[END_REF] and other derived models (e.g., [START_REF] Masel | Quantifying the kinetic parameters of prion replication[END_REF]) merely assume the existence of structurally homogenous assemblies that have absolutely identical propensity to replicate throughout disease progression. These mechanisms intrinsically reduce PrP Sc heterogeneity due to the best replicator selection process [START_REF] Nee | The evolutionary ecology of molecular replicators[END_REF][START_REF] Ohhashi | Molecular basis for diversification of yeast prion strain conformation[END_REF]. A recent high-resolution structural analysis of the N-terminal domain of the yeast prion SuP35 suggests that conformational fluctuations in natively disordered monomeric Sup35 are responsible for the stochastic, structural diversification of Sup35 aggregates [START_REF] Ohhashi | Molecular basis for diversification of yeast prion strain conformation[END_REF]. This idea can be extrapolated to mammalian prion PrP to explain intrastrain structural diversification and strain mutation [START_REF] Collinge | A general model of prion strains and their pathogenicity[END_REF]. However, based on the best replicator selection concept [START_REF] Moulin | Dynamic combinatorial self-replicating systems[END_REF][START_REF] Ojosnegros | Quasispecies as a matter of fact: viruses and beyond[END_REF][START_REF] Nee | The evolutionary ecology of molecular replicators[END_REF], the aforementioned idea does not explain the coevolution of at least two structurally distinct PrP Sc subassemblies within the same environment [START_REF] Chapuis | Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions[END_REF][START_REF] Dur | Divergent prion strain evolution driven by PrPC expression level in transgenic mice[END_REF].

To examine the molecular mechanisms of PrP Sc replication and structural diversification in depth, we explored, with sedimentation velocity (SV)-based methods, the early stage of prion conversion in vivo and in a cell-free system by protein misfolding cyclic amplification (PMCA).

PMCA mimics in vivo prion replication with accelerated kinetics [START_REF] Saborio | Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding[END_REF]. By using several prion strains as templates, we demonstrated that the early stage of prion replication invariably generates two subsets of assemblies, termed Ai and Bi, which differ in proportion, size and structure according to their specific infectivity. The analysis of their kinetics of formation during mb-PMCA combined with kinetic data assimilation revealed the existence of two sequential processes during prion replication. The first process corresponds to a quaternary structural convergence, as it tends to reduce the parental quaternary structure polydispersity to

Results

Small PrP Sc oligomers are formed at the early stage of prion replication

The early phases of prion replication are commonly thought to consist of an elongation/growing process [START_REF] Gaspar | Secondary nucleation of monomers on fibril surface dominates alpha-synuclein aggregation and provides autocatalytic amyloid amplification[END_REF], with the PrP Sc template serving as a base. We studied the size distribution of proteinase K (PK)-resistant PrP Sc (PrP res ) assemblies at the early step of prion replication in the brain by SV in an iodixanol gradient using a previously published methodology [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF][START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF]. The PrP res size distribution at the disease end stage served as the control. Three different host PrP/strain combinations were studied: the 127S cloned scrapie prion strain in ovine PrP tg338 transgenic mice [START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF], the 139A cloned mouse prion strain in mouse PrP tga20 mice [START_REF] Fischer | Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie[END_REF] and the vCJD cloned human prion strain in human PrP tg650 mice [START_REF] Beringue | Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD[END_REF][START_REF] Halliez | Accelerated, spleen-based titration of variant Creutzfeldt-Jakob disease infectivity in transgenic mice expressing human prion protein with sensitivity comparable to that of survival time bioassay[END_REF]. As shown in Figure 1A-C, small oligomers sedimenting between fractions 1 and 4 were preferentially detected at the early stage of pathogenesis, regardless of the strain considered. A second population of oligomers with a larger size distribution and peaking in fractions 8-10 and 18 was observed for 127S. At the disease end stage and for the 3 strains, the small assemblies mostly disappeared at the expense of larger assemblies.

To determine whether the formation of small assemblies in the brain at the early stage of replication can be reproduced by an in vitro bona fide amplification method, we used a highthroughput variant of PMCA (i.e., mb-PMCA), which generates as much infectivity as in the brain at terminal stage of the disease in one unique round of 48 h, with high reproducibility in terms of limiting dilution and the amplification yield [START_REF] Moudjou | Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification[END_REF][START_REF] Moudjou | Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification[END_REF]. When the size distribution of the amplified products was analyzed by SV after one mb-PMCA round, two discrete distributions were observed for the three strains (Figure 1D). The post-PMCA sedimentograms revealed the existence of a major set of small PrP res assemblies sedimenting between fractions 1 and 3 (peak P1) and a minor set of larger assemblies with a well-defined Gaussian distribution centered on fraction 15 (peak P2). The relative proportions of P1 and P2 varied among the three strains; P2 was barely detected in the 139A amplicons. These data indicate that during mb-PMCA amplification, two populations of PrP Sc assemblies are generated that differ according to their quaternary structures, with a predominance of small assemblies.

The bimodal (i.e., generation of two peaks) and discrete behavior of the size distribution as well as the formation of predominantly small assemblies in P1 suggest that the mb-PMCA condition(s) can be a consequence of shearing forces during the sonication step [START_REF] Adachi | Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation[END_REF][START_REF] Okumura | Amyloid fibril disruption by ultrasonic cavitation: nonequilibrium molecular dynamics simulations[END_REF][START_REF] Webster | Detecting the early onset of shearinduced fibril formation of insulin in situ[END_REF] rather than an intrinsic consequence of the replication process. To discriminate between these two possibilities, undiluted 127S seeds (i.e., 20% brain homogenate) were incubated and sonicated in identical mb-PMCA conditions but without the PrP C substrate (i.e., in PrP 0/0 brain lysate).

As shown in Figure 1E, the size distribution analysis of these sonicated 127S seeds in the PrP 0/0 substrate revealed mostly the presence of larger-sized assemblies, as observed upon solubilization at 37°C [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF], thus ruling out the mb-PMCA conditions being at the origin of the formation of small-size assemblies.

Altogether, these observations suggest that in vivo, the early phase of replication for the 127S, 139A and vCJD prion strains generates mainly small-sized assemblies. Similar to in vivo replication, the mb-PMCA amplification condition generates two sets of PrP assemblies that differ in their quaternary structures. The formation of these two groups of assemblies is common to the three strains used here.

P1 and P2 contain two structurally distinct PrP res assemblies

We next asked whether the formation of P2 resulted from a simple condensation of assemblies present in the P1 peak (Oswald ripening process [START_REF] Zhang | Simulations of nucleation and elongation of amyloid fibrils[END_REF]) or from an alternative templating pathway. To address this question, we first examined the influence of the amplification rate on the formation of these two species by varying the concentration of the seed used as the template for the mb-PMCA reaction. We compared the SV-sedimentograms of the mb-PMCA products seeded with 10 -3 to 10 -10 diluted 127S brain homogenate. As shown in Figure 2A, as a function of the seed concentration, the relative amounts of assemblies in P1 decreased as the amounts of those from P2 increased. The variation in the P1 and P2 peak surface area as a function of the logarithm of the dilution factor revealed a quasi-linear decrease in P1 when the P2 peak surface followed a sigmoidal increase (Figure 2B). The sigmoidal increase in P2 to the detriment of the quasi-linear decrease in P1 surface indicates that i) the formation of PrP res assemblies present in P2 follows a seed concentration-dependent cooperative process and that ii) the formation of the P2 peak does not result from the simple condensation of assemblies present in P1 as the variations in P1 and P2 are uncorrelated (Figure 2B). This observation strongly suggests that assemblies forming the P1 and P2 peaks result from distinct polymerization pathways and should therefore be structurally distinct.

To further explore the entanglement between the assemblies forming P1 and P2, we fixed the mb-PMCA regime to favor the formation of the P1 peak by fixing high dilutions of the inoculum seed, followed by quiescent incubations at 37°C for variable periods. As shown with the 127S prions, the SV analysis at defined incubation time points post-PMCA reaction revealed a decrease in the population of P1 in favor of P2 (Figure 2C). At 4 h postincubation, there were equal proportions of assemblies forming P1 and P2. At 24 h, most of the PrP res assemblies were located in the P2 peak. Comparing the distribution in isopycnic gradients [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF] of the PrP res populations at 0h and 24h of quiescent incubation at 37°C revealed a quasi-similar density for PrP res assemblies composing the P1 and the P2 peaks (Figure 2D). This observation leads us to conclude that i) the low sedimentation velocity of the assemblies forming P1 does not result from an interaction with lipids or other low-density molecules and ii) the sedimentation velocity increase of P2 compared to P1 results strictly from a quaternary structure rearrangement through size increase rather than change in compactness reducing the hydrodynamic radius.

As shown in Figure 2E, the formation of assemblies sedimenting in P2 exhibited bimodal behavior (i.e., absence of assemblies of intermediate size) without any significant shift in the P2 peak position, suggesting that the formation of these assemblies resulted from the association with a specific number of assemblies present in P1. Furthermore, the time-dependent surface variation in P1 and P2 showed a sigmoidal shape, indicating that the assemblies present in P2 enhance their own formation according to an autocatalytic process (Figure 2E). Similarly, the 139A and vCJD prions showed a bimodal evolution of P1 to P2 during a 24-h quiescent phase (Figure 2F), arguing in favor of a generic process of transformation.

To determine whether the quaternary structure rearrangement accompanying the transformation of P1 to P2 was in concert with a deeper structural rearrangement in the PrP Sc assemblies, we determined the specific infectivity of the P1 and P2 assemblies. A 127S-PMCA product was fractionated at the end of the reaction or after 48 h of quiescent incubation. Pools of fractions corresponding to the P1 and P2 peaks were inoculated into reporter tg338 mice. The specific infectivity (infectivity per PrP molecule), which is mostly associated to PrP res assemblies [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF], was calculated from the mean survival time using 127S dose-response curves [START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF]. As shown in Figure 2G, the specific infectivity of the P1 peak assemblies was 50-100-fold higher than that of the P2 peak assemblies. This value did not change over a longer period of quiescent incubation (7 days, Figure 2G).

To determine whether the P2 peak assemblies could further evolve, we extended the quiescent phase up to 30 days. For the 127S, 139A and vCJD prion strains, the sedimentogram curves at 7 and 30 days showed a translational shift in the P2 peak to higher fractions, indicative of an isokinetic increase in their mean average sizes (Figure 3, left curves). The difference in the specific infectivity values of the P1 and P2 peak assemblies did not change over a longer period of quiescent incubation (7 days, Figure 2G, 127S strain). This size translation thus contrasts with the bimodal phase and highlights a change in the kinetic regime.

Collectively, these observations indicate that the P1 and P2 peaks contain structurally distinct sets of PrP res assemblies named Ai and Bi. The index i indicates their sizes, with Ai and Bi being the major constituents of the P1 and P2 peaks, respectively. The formation of the Bi assemblies is cooperative and results from a complex kinetic pathway. Upon longer quiescent incubation, a change in the kinetic regime occurs.

The formation of Bi from Ai assemblies requires the presence of PrP C during the quiescent phase Our previous studies revealed that only 30% of the PrP C substrate was converted into PrP Sc after a complete round of mb-PMCA [START_REF] Moudjou | Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification[END_REF][START_REF] Moudjou | Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification[END_REF]. To determine whether the remaining 70% still participated in the transformation of Ai to Bi assemblies during the quiescent phase, PMCA products from the 139A, 127S and vCJD prions were treated with PK to eliminate PrP C before quiescent incubation at 37°C. As shown in Figure 3 (right curves), the amount of Bi assemblies generated during the 48-h quiescent incubation was drastically decreased for the three prion strains in the absence of the PrP C substrate. Further quiescent incubation for 7 and 30 days in the absence of PrP C allowed the formation of low amounts of Bi assemblies for the 127S and 139A prion strains. The fact that the transformation of Ai to Bi assemblies is strongly facilitated by the presence of PrP C suggests that Bi assemblies result from the integration/conversion of PrP C into Ai assemblies. The appearance of a low amount of Bi after a long incubation period without PrP C may result from the leakage of monomers from a conformer cosedimenting with Ai.

Kinetical scheme describing the transformation of Ai to Bi assemblies

To establish a kinetic mechanism and provide a molecular interpretation of the assemblies' dynamics during the quiescent phase, a number of elementary steps were identified based on experimental observations and were used as unavoidable constraints [START_REF] Epstein | An introduction to nonlinear chemical dynamics: oscialltions, waves, patterns and chaos[END_REF]. The first constraint was the existence of two structurally distinct PrP Sc subassemblies, namely, Ai and Bi, with distinct dynamics. Indeed, structurally equivalent assemblies would fail to present a bimodal size distribution, cooperative seed concentration and kinetic evolution or distinct specific infectivity. The second constraint was the existence of a detailed balanced between the PrP Sc assemblies and their elementary subunit (suPrP), as previously shown [START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF] making the size distribution of the PrP Sc assemblies highly dynamic and dependent on the assembly concentration, as shown in Figure 1E. Indeed, SV analysis of the PrP 0/0 brains lysates seeded with 30-fold-diluted 127S-infected brains and submitted to PMCA revealed a quaternary structure rearrangement with a shift in lower molecular weight assemblies according to the detailed balance:

𝑃𝑟𝑃 𝑖 𝑆𝑐 ⇌ 𝑃𝑟𝑃 𝑖-1 𝑆𝑐 + 𝑠𝑢𝑃𝑟𝑃
where 𝑃𝑟𝑃 𝑖 𝑆𝑐 and 𝑃𝑟𝑃 𝑖-1 𝑆𝑐 are the sizes i and i-1 of suPrP Sc , respectively.

Because the existence of suPrP is a generic property of prion strains [START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF], the 3 rd constraint leads us to assume that the Ai and Bi assemblies are in detailed balance with their respective suPrPs (denoted 𝑠𝑢𝑃𝑟𝑃 𝐴 and 𝑠𝑢𝑃𝑟𝑃 𝐵 ) but with distinct equilibrium constants 𝐾 𝑒𝑞 𝐴 and 𝐾 𝑒𝑞 𝐵 .

Thus, at any moment of the process of assembly transformation of Ai to Bi, the following equilibrium should be respected:

𝐴 𝑖 ⇌ 𝐴 𝑖-1 + 𝑠𝑢𝑃𝑟𝑃 𝐴 (1)
𝐵 𝑖 ⇌ 𝐵 𝑖-1 + 𝑠𝑢𝑃𝑟𝑃 𝐵 (2)
The equilibrium constant 𝐾 𝑒𝑞 𝐴𝑖 and 𝐾 𝑒𝑞 𝐵𝑖 governs the respective size distribution of the Ai and Bi assemblies and, thus, the bimodal aspect of the curve. According to our previous SV calibrations with PrP oligomers and globular mass markers [START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF], the size distribution of the Ai and Bi subassemblies were fixed: iA<5 and iB centered around 20 PrP-mers. Due to the limited resolution of SV fractionation for small assemblies, we assumed that Ai and suPrP B cosedimented. The fourth constraint relies on the fact that the transformation of A to B requires PrP C and that the kinetic is cooperative, as shown in Figures 1E and2. This cooperativity implies that B subassemblies facilitate their own formation according to an autocatalytic process. This can be resumed by the following minimalistic autocatalytic process:

𝑠𝑢𝑃𝑟𝑃 𝐴 + 𝑠𝑢𝑃𝑟𝑃 𝐵 ⇌ 𝐶 (3) 𝐶 + 𝑃𝑟𝑃 𝐶 ⟶ 2𝑠𝑢𝑃𝑟𝑃 𝐵 (4)
where C is an active complex reacting with PrP C that generates B assemblies. Considering that suPrP B can condense into B2 [START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF] and according to detailed balance (2), one can establish the reaction model describing the formation of Bi assemblies from the neo-formed suPrP B :

2𝑠𝑢𝑃𝑟𝑃 𝐵 ⇌ 𝐵 2 (5) 𝐵 𝑖 + 𝑠𝑢𝑃𝑟𝑃 𝐵 ⇌ 𝐵 𝑖+1 (6)
Altogether, these six elementary steps constitute the reaction mechanism that describes the transformation of Ai into Bi subassembly species.

To validate the designed mechanism, we translated these elementary reactions into timedependent differential equations (for more details, see Supplementary text) and performed kinetic simulations using the size distribution of the PrP Sc assemblies immediately after cyclic amplification as the initial condition (blue curve in Figure 2A). According to the model, the simulated size distribution variation as a function of time showed bimodal behavior, as was experimentally observed (Figure 4A). Furthermore, the theoretical size distribution centroid presented similar sigmoidal patterns to those of the experimental data (Figure 4B), arguing in favor of an autocatalytic kinetic model describing the overall quaternary structure evolution of PrP Sc assemblies during the quiescent phase. The analysis of the model (for more details, see Supplementary text) revealed that the autocatalytic formation of Bi species occurs at the expense of Ai species and with PrP C consumption (Figure 4 C and D). According to this model, when PrP C is in large excess, Ai constitutes the limiting compound for the formation of Bi assemblies. Therefore, during the quiescent phase, the PrP C to PrP Sc conversion rate is directly proportional to the amount of Ai assemblies (Figure 4D).

Discussion

The mechanisms of prion replication and the dynamics responsible for prion structural diversification in the infected host remain unclear and rarely addressed. In the actual framework of the prion paradigm, the templating process is admitted to occur at the prion assembly interface, leading to an increased size of the complex formed by the template:substrate, out of the fragmentation/depolymerization context. The atypical size distribution observed here at the early stage of the replication process for three distinct prion strains, where accumulation of small-size assemblies dominates, contrasts with this canonical templating model and requires an additional process that considers the dynamics of replication. Furthermore, the existence of a multistep conversion process provides an unexpected approach to reconciling the best replicator [START_REF] Nee | The evolutionary ecology of molecular replicators[END_REF] selection paradigm and diversification process, which is inherent to prion adaptation and evolution.

As shown in vivo for the vCJD, 127S and 139A prion strains, the early stage of the replication process in the brain is dominated by the accumulation of small assemblies, whereas higher-size subsets are mostly detected at the terminal stage of pathogenesis. Such quaternary structural diversity, -and beyond the existence of structurally distinct types of assemblies, as defined by their specific infectivity ( [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF] and Supplemental Figure 1), can be exclusively explained by the existence of a balance between at least two kinetic modes taking place at different stages of the pathogenesis. Both can be governed by evolution or a fluctuation in the replication microenvironment due to the physio-pathological state of the infected animal and/or to the sequential involvement of specific prion-replicating cell types. However, another possibility can lie in the intrinsic and deterministic properties of the PrP replication process to generate structurally distinct types of assemblies. Discriminating between these two nonmutually exclusive hypotheses is technically difficult in vivo. The mb-PMCA as a bona fide amplification method in a more simplified and kinetically controlled context constitutes a relevant method for investigating the intrinsic propensity of the replication process to generate structurally distinct assemblies. Interestingly, and against common belief, the size distribution of the PrP Sc assemblies used as seeds was relatively insensitive to mb-PMCA sonication cycles when a simple dilution displaced the assemblies towards a smaller size (Figure 1E), as previously reported [START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF]. These two observations exclude the contribution of the fragmentation process during the mb-PMCA sonication cycles to the size distribution pattern of PrP Sc assemblies and emphasize the existence of a constitutional dynamic between the PrP Sc subpopulation [START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF],

which should be considered during the replication process. We showed that two sets of PrP Sc assemblies, Ai and Bi, were generated during the mb-PMCA reaction. The Ai and Bi assemblies constitute two structurally distinct PrP Sc subpopulations, as supported by their distinct specific infectivity; the bimodal size distribution instead of a continuum; the effect of initial seed concentration on the respective proportions of Ai and Bi; and the role of PrP C in the transformation of Ai to Bi that indicates that Bi assemblies do not result from simple condensation of Ai assemblies. Therefore, the prion replication process per se intrinsically generates structurally diverse PrP Sc subassemblies.

According to our SV experiments, small-sized PrP Sc assemblies were mainly formed at the early stage of prion replication in the brain and during the mb-PMCA reaction. This was observed with three distinct prion strains (127S, 139A, vCJD) on 3 different PrP genetic backgrounds.

Considering that the PrP Sc assemblies that constitute each strain are structurally distinct, one can ask how distinct PrP Sc assemblies can all generate Ai assemblies that harbor strain structural information while showing the same quaternary structure (at the SV resolution). The first explanation can be the existence of a common narrow subpopulation of PrP Sc (with respect to their quaternary structure) within the three strains that serves as the best replicator and participates in the formation of Ai assemblies. However, the PrP Sc quaternary structure subset that exhibits the highest specific infectivity in vivo (i.e., the best replicator) can be associated with either small-size assemblies (i.e., 127S and 139A [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF] and Supplemental Figure 1A, respectively) or high-molecular-weight assemblies (i.e., vCJD, Supplemental Figure 1B) and is therefore strain-dependent. The existence of a structurally common PrP Sc subpopulation is thus unlikely to be at the origin of the generic formation of a small-size subset in the brain or Ai assemblies in the mb-PMCA condition. Intrinsically, the early steps of the replication process favor the emergence of mainly one subspecies Ai with a highly narrowed size distribution, arguing in favor of a quaternary structural convergence phenomenon during these steps. This structural convergence concerns the PrP domain that governs polymerization (the size of assemblies). However, as the A assemblies harbor the strain structural determinant, one can conclude that Ai assemblies present a certain degree of structural variability, allowing the encoding of strain structural information (Supplemental Figure 1).

All along the quiescent phase and for the three prion strains studied, the Ai assemblies constitute the precursor species in the formation of Bi assemblies. Furthermore, the presence of PrP C is required for the evolution of Ai into Bi assemblies, and according to the kinetic model describing the autocatalytic formation of Bi during the quiescent phase, Ai is the limiting species for conversion when large amounts of PrP C are present (Figure 4C and D and Supplementary text). The bimodal quaternary structure evolution during the quiescent phase is in concert with a specific infectivity decrease that is indicative of a structural rearrangement of species present in P1 and P2 and thus during the transformation of Ai to Bi. Even if the first event conducing to the formation of Bi assemblies remains undetermined, we can assume that Ai can have the intrinsic propensity to spontaneously evolve into Bi assemblies in the presence of PrP C (Figure 5). The cooperative disappearance of P1 in favor of P2 strongly suggests an autocatalytic process for the transformation of Ai to Bi (reactions 3 and 4). This last phenomenon shows the existence of a secondary autocatalytic process, undescribed until now, in the canonical prion replication process [START_REF] Lansbury | The chemistry of scrapie infection: implications of the 'ice 9' metaphor[END_REF]. One can be reasonably envisage that Ai can have the intrinsic propensity to generate Bi assemblies in the presence of PrP C assemblies with a very low efficiency. This parallel pathway to the autocatalytic process can then explain how the first set of Bi assemblies is generated (Figure 5). The existence of a secondary autocatalytic process can be crucially important for maintaining PrP Sc structural diversity throughout the evolution of the pathology.

In the absence of this secondary autocatalytic process, e.g., in the absence of PrP C , the system selects the best replicator and the most thermodynamically stable assemblies. In the presence of PrP C , the system escapes this rule, allowing the specific accumulation of the autocatalytic product (here, the Bi assemblies) rather than the assemblies that are the most thermodynamically stable or have highly specific infectivity. This phenomenon can explain why, for certain prion strains, the most infectious assemblies represent a minor population, while those with the lowest specific infectivity mostly accumulate [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF].

Conclusion

The early step of prion replication for at least three distinct prion strains leads to the formation of small assemblies. The mb-PMCA approach clearly demonstrates the intrinsic properties of the bona fide replication process to generate at least two structurally distinct PrP Sc subassemblies. The deterministic aspect of the replication process to generate a structurally diverse set of assemblies contrasts with the widespread idea that considers the prion diversification process within a given strain (often referred to as the creation of prion quasispecies) as a stochastic event and as a process that is cogoverned by environmental fluctuations [START_REF] Weissmann | Prions on the move[END_REF]. The secondary autocatalytic pathway leading to the formation of Bi subassemblies can participate in prion adaptation during transmission events with species barriers. Considering that the transmitted inoculum initially contains Ai and Bi assemblies, the autocatalytic conversion process of Bi can kinetically drive the adjustment and integration of the new-host PrP C to generate host-adapted Bi assemblies. This hypothesis is supported by our recent observations in which complementation between Ai and Bi subassemblies is required to cross existing species barriers (submitted article).

Methods

Ethics

Animal experiments were conducted in strict accordance with ECC and EU directives 86/009 and 2010/63 and were approved by the local ethics committee of the author's institution (Comethea; permit numbers 12/034 and 15/045).

Transgenic mouse lines and prion strains

The ovine (tg338 line; Val136-Arg154-Gln171 VRQ allele), human (tg650 line; Met129 allele) and mouse (tga20) PrP transgenic lines have been described previously [START_REF] Beringue | Prominent and persistent extraneural infection in human PrP transgenic mice infected with variant CJD[END_REF][START_REF] Fischer | Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie[END_REF][START_REF] Langevin | Marked influence of the route of infection on prion strain apparent phenotype in a scrapie transgenic mouse model[END_REF]. The mouse lines were homozygous and overexpressed approximately 8-, 6-, and 10-fold amounts of heterologous PrP C on a mouse PrP-null background. PrP 0/0 mice were the so-called Zürich-I mice [START_REF] Bueler | Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein[END_REF]. Cloned 127S scrapie, human vCJD and mouse 139A prion strains were serially passaged in tg338, tg650 and tga20 mice, respectively [START_REF] Moudjou | Glycoform-independent prion conversion by highly efficient, cell-based, protein misfolding cyclic amplification[END_REF][START_REF] Moudjou | Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification[END_REF]. These strains were used as pools of mouse-infected brains and prepared as 20% wt/vol homogenates in 5% glucose by use of a tissue homogenizer (Precellys 24 Ribolyzer; Ozyme, France).

Time course analysis of prion accumulation

Eight-week-old female tg338, tg650 and tg20 mice were inoculated intracerebrally in the right cerebral hemisphere with 127S, vCJD or 139A prions (20 μl of a 10% brain homogenate dose).

Infected animals were euthanized by cervical column disruption in triplicate at regular time points and at the terminal stage of disease. Brains were removed and kept for PrP Sc size fractionation.

Miniaturized bead-PMCA assay

The miniaturized bead-PMCA assay [START_REF] Chapuis | Emergence of two prion subtypes in ovine PrP transgenic mice infected with human MM2-cortical Creutzfeldt-Jakob disease prions[END_REF][START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF][START_REF] Moudjou | Highly infectious prions generated by a single round of microplate-based protein misfolding cyclic amplification[END_REF] was used to amplify prions. Briefly, serial tenfold dilutions of 127S, vCJD and 139A prions (mouse brain homogenates diluted in PMCA buffer) were mixed with brain lysates (10% wt/vol) from healthy tg338, tg650 and tga20 mice as respective substrates and subjected to one round of 96 cycles of 30-s sonications (220-240 Watts) followed by 29.5 min of incubation at 37°C. With a >10 4 dilution of the seeds, input PrP Sc is not detected in the mb-PMCA products. PMCA was performed in a 96-well microplate format using a Q700 sonicator (QSonica, USA, Delta Labo, Colombelles, France). For quiescent incubation, the samples were left in the incubator at 37°C for the indicated period of time, without any sonication. To eliminate residual PrP C present in the PMCA products before quiescent incubation, the samples were treated with PK (80 g/ml final concentration). The treatment was stopped by adding 2 mM Pefabloc and 1x EDTA-free protease inhibitor cocktail.

All final products were kept for PrP Sc size fractionation, and aliquots were PK-digested (115 μg/ml final concentration, 0.6% SDS, 1 h, 37°C) prior to immunoblot analyses, as described below.

Sedimentation velocity fractionation

SV experiments were performed as described previously [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF][START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF][START_REF] Igel-Egalon | Reversible unfolding of infectious prion assemblies reveals the existence of an oligomeric elementary brick[END_REF]. Mouse brain homogenates or PMCA products were solubilized by adding an equal volume of solubilization buffer (50 mM HEPES pH 7.4, 300 mM NaCl, 10 mM EDTA, 2 mM DTT, 4% wt/vol dodecyl--Dmaltoside (Sigma)) and incubated for 45 min on ice. Sarkosyl (N-lauryl sarcosine; Fluka) was added to a final concentration of 2% wt/vol, and the incubation continued for an additional 30 min on ice. A total of 150 μl of solubilized samples was loaded atop a 4.8-ml continuous 10-25% iodixanol gradient (Optiprep, Axys-Shield), with a final concentration of 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% Sarkosyl. The gradients were centrifuged at 285,000 g for 45 min in a swinging-bucket SW-55 rotor using an Optima LE-80K ultracentrifuge (Beckman Coulter). Gradients were then manually segregated into 30 equal fractions of 165 μl from the bottom using a peristaltic pump and analyzed by immunoblotting or bioassay for PrP Sc or infectivity, respectively. To avoid any cross-contamination, each piece of equipment was thoroughly decontaminated with 5 M NaOH followed by several rinses in deionized water after each gradient collection [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF].

Isopycnic sedimentation

The entire procedure was performed as described previously [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF]. Mouse brain homogenates or PMCA products were solubilized as described above. For mouse brain homogenates, solubilization was performed at 37°C to mimic PMCA conditions. A total of 220 μl of solubilized material was mixed to reach 40% iodixanol, 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% Sarkosyl final concentration and loaded within a 4.8 ml of 10-60% discontinuous iodixanol gradient with a final concentration of 25 mM HEPES pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.5% Sarkosyl. The gradients were centrifuged at 115 000 g for 17 hours in a swinging-bucket SW-55 rotor using an Optima LE-80K ultracentrifuge (Beckman Coulter). Gradients were then manually segregated into 30 equal fractions of 165 μl from the bottom using a peristaltic pump and analyzed for PrP Sc content by immunoblotting.

Analysis of PrP Sc content by immunoblotting

Aliquots of the SV-fractionated PMCA samples were treated with PK (50 μg/ml final concentration, 1 h, 37°C) before mixing in Laemmli buffer and denaturation at 100°C for 5 min. The samples were run on 12% Bis-Tris Criterion gels (Bio-Rad, Marne la Vallée, France) and electrotransferred onto nitrocellulose membranes. In some instances, denatured samples were spotted onto nitrocellulose membranes using a dot-blot apparatus (Schleicher & Schuell BioScience (Whatman)). Nitrocellulose membranes were probed for PrP with 0.1 μg/ml biotinylated anti-PrP monoclonal antibody Sha31. Immunoreactivity was visualized by chemiluminescence (GE Healthcare). The protein levels were quantified with ImageLab software after acquisition of chemiluminescent signals with a Chemidoc digital imager (Bio-Rad, Marnes-la-Coquette, France). For all SDS-PAGE analyses, a fixed quantity of human recombinant PrP was employed for consistent calibration of the PrP signals in different gels.

Bioassays

The pool of fractions of interest was extemporarily diluted ten-fold in 5% glucose and immediately inoculated via the intracerebral route into reporter tg338 mice (20 μl per pool of fraction, n = 5 mice per pool). Mice showing prion-specific neurological signs were euthanized at the end stage. To confirm prion disease, brains were removed and analyzed for PrP Sc content using the Bio-Rad TsSeE detection kit [START_REF] Dur | A newly identified type of scrapie agent can naturally infect sheep with resistant PrP genotypes[END_REF] prior to immunoblotting, as described above. The survival time was defined as the number of days from inoculation to euthanasia. To estimate what the difference in mean survival times means in terms of infectivity, strain-specific curves correlating the relative infectious dose to survival times were used, as previously described [START_REF] Tixador | The physical relationship between infectivity and prion protein aggregates is strain-dependent[END_REF].

Kinetic simulation

The details of the kinetic simulation are reported in the Supplementary text. Briefly, two distinct sets of assemblies were considered (Ai and Bi). Based on experimental observations, a set of constraints was retained to build biochemical reactions describing the evolution of the quaternary structure of PrP res assemblies. The ordinary differential equations of the biochemical reactions 1 to 6 (in the manuscript) were established and coded in MATLAB for simulations. (D-E) For the sedimentograms from the PMCA products with PrP C substrate (D), the same strains were subjected to a single round of mb-PMCA by using 10 -5 (139A) or 10 -6 (vCJD, 127S) diluted brain homogenates as seed for the reaction. Thirty minutes after the last sonication, the amplified products were solubilized and SV-fractionated. The mean levels of PK-resistant PrP Sc per fraction were obtained from the immunoblot analysis of n=4 independent fractionations of PMCA reactions. The peaks containing PrP Sc assemblies sedimentating in the top and middle fractions were termed P1 and P2, respectively. For the sedimentograms from the PMCA products without PrP C substrate (E), undiluted 127S-infected tg338 brain (20% w/v, red curve) or a 1:32 dilution in PMCA buffer (blue curve) was used as seed, mixed with brain homogenate from PrP 0/0 mice as substrate and subjected to a single round of mb-PMCA before SV fractionation (mean levels from n=3 independent fractionations). (D) PK-resistant PrP Sc isopycnic sedimentograms from PMCA products generated with 127S prions (10 -5 dilution) and immediately fractionated at the end of the PMCA reaction (blue line and symbol) or after a 24h-quiecent incubation at 37°C (red line and symbol). At each time point, the collected samples were frozen. All collected samples were then thawed, fractionated in parallel by sedimentation at the equilibrium [START_REF] Laferriere | Quaternary structure of pathological prion protein as a determining factor of strain-specific prion replication dynamics[END_REF] and analyzed by immunoblot (the mean levels of PK-resistant PrP Sc per fraction were obtained from the immunoblot analysis of n=3 independent fractionations of PMCA reactions). As control, the density profile of PK-resistant PrP Sc assemblies from the brain of terminally sick tg338 mice (solubilization at 37°C to mimic the PMCA conditions) is shown (gray line and symbol).

Legends

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The copyright holder for this preprint (which was this version posted March 20, 2019

Figure 1 .

 1 Figure 1. Size distribution of PrP Sc assemblies from different prion strains at the early

Figure 2 .

 2 Figure 2. Seed concentration-and time-dependent dynamic evolution of the PMCA-

(

  E) Evolution of the percentage of P1 and P2 peaks as a function of the quiescent phase post-(F) PK-resistant PrP Sc sedimentograms from the PMCA products generated with 139A and vCJD prion seeds (10 -5 dilution) and further incubated for a quiescent period of 24 h at 37°C. (G) Specific infectivity of the P1 and P2 peaks post-PMCA reaction and after quiescent incubation. Fractions corresponding to P1 (fractions 1-3) and P2 (fractions 14-16 (days 0 and 2) or 16-18 (day 7)) were pooled and inoculated into groups of reporter tg338 mice at two different dilutions (1:10 and 1:1000) for better accuracy. The specific infectivity of the assemblies was calculated from the mean survival time of the mice using a 127S dose-response curve. *: incomplete attack rate.

Figure 3 .

 3 Figure 3. PrP-dependent generation of Bi assemblies from Ai assemblies

Figure 4 .

 4 Figure 4. Mathematical modeling of the time-dependent dynamic evolution of the PMCA-

(

  B) The time dependency evolution of the simulated centroid (black line) and centroid calculated from experimental sedimentograms of Figure 2D (red circle) show a similar shape, supporting the cooperativity hypothesis of the transformation of Ai into Bi.(C) The simulation of time dependency evolution of the total amount of Ai assemblies (∑ 𝑖𝐴 𝑖 in black), Bi assemblies (∑ 𝑖𝐵 𝑖 in blue) and the monomer (in red) revealed that Ai assemblies constitute the limiting species for the conversion of PrP C during the quiescent phase. In the present simulation framework (for more details, see Supplementary text), only 14% of PrP C is consumed.

Figure 5 .Supplemental Figure 1 .Figure 1 not

 511 Figure 5. Quaternary structural convergence and secondary autocatalytic pathway at the

Acknowledgments

We thank the staff of the Animal facility (INRA-UEAR, Jouy-en-Josas) for animal care. This work was supported by grants from the Fondation pour la Recherche Médicale (Equipe FRM DEQ20150331689), the European Research Council (ERC Starting Grant SKIPPERAD, number 306321), and the Ile de France region (DIM MALINF).