Accepted Manuscript Human secreted tau increases amyloid-beta production Jessica Bright, Sami Hussain, Vu Dang, Sarah Wright, Bonnie Cooper, Tony Byun, Carla Ramos, Andrew Singh, Graham Parry, Nancy Stagliano, Irene GriswoldPrenner PII:

S0197-4580(14)00602-2

DOI:

10.1016/j.neurobiolaging.2014.09.007

Reference:

NBA 9048

To appear in:

Neurobiology of Aging

Received Date: 4 February 2014 Revised Date:

4 September 2014

Accepted Date: 6 September 2014

Please cite this article as: Bright, J., Hussain, S., Dang, V., Wright, S., Cooper, B., Byun, T., Ramos, C., Singh, A., Parry, G., Stagliano, N., Griswold-Prenner, I., Human secreted tau increases amyloid-beta production, Neurobiology of Aging (2014), doi: 10.1016/j.neurobiolaging.2014.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Human secreted tau increases amyloid-beta production

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Jessica Bright1,2, Sami Hussain1,2, Vu Dang1, Sarah Wright1, Bonnie Cooper1, Tony

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Byun1, Carla Ramos1, Andrew Singh1, Graham Parry1, Nancy Stagliano1 and Irene

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Griswold-Prenner1*

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*Correspondence should be addressed to N.S. ([email protected])

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951 Gateway Blvd, SSF CA 94080

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Telephone: (650) 872-4000

iPierian, Inc.

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11 12 Abstract

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The interaction of amyloid-beta (Aβ) β) and tau in the pathogenesis of Alzheimer’s

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disease (AD) is a subject of intense inquiry, with the bulk of evidence indicating

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that changes in tau are downstream of Aβ β. It has been shown however, that

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human tau overexpression in APP transgenic mice increases Aβ β plaque

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deposition. Here we confirm that human tau increases Aβ β levels. To determine if

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the observed changes in Aβ β levels were due to intracellular or extracellular

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secreted tau (eTau for extracellular tau), we affinity purified secreted tau from AD

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patient derived cortical neuron conditioned media and analyzed it by liquid

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chromatography-mass spectrometry (LC/MS). We found the extracellular species

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to be composed predominantly of a series of N-terminal fragments of tau, with no

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evidence of C-terminal tau fragments. We characterized a subset of high affinity

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tau antibodies, each capable of engaging and neutralizing eTau. We found that

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neutralizing eTau reduces Aβ β levels in vitro in primary human cortical neurons

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where exogenously adding eTau increases Aβ β levels. In vivo, neutralizing human

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tau in two human tau transgenic models also reduced Aβ β levels. We show that the

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human tau insert sequence is sufficient to cause the observed increase in Aβ β

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levels. Our data furthermore suggest that neuronal hyperactivity may be the

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mechanism by which this regulation occurs. We show that neuronal hyperactivity

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regulates both eTau secretion and Aβ β production. Electrophysiological analysis

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shows for the first time that secreted eTau causes neuronal hyperactivity. Its

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induction of hyperactivity may be the mechanism by which eTau regulates Aβ β

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production. Together with previous findings, these data posit a novel connection

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between tau and Aβ β, suggesting a dynamic mechanism of positive feed forward

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regulation. Aβ β drives the disease pathway through tau, with eTau further

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increasing Aβ β levels, perpetuating a destructive cycle.

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Keywords: secreted tau, extracellular tau, eTau, Amyloid-beta (Aβ β), neuronal

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hyperactivity, Alzheimer’s disease, feed forward mechanism, sAPPα α

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42 1. Introduction

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Alzheimer’s disease (AD) is characterized pathologically by amyloid-beta (Aβ) plaques

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and tau dominated neurofibrillary tangles (NFTs). Understandably, the field has focused

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on elucidating the mechanisms regulating these hallmark AD proteins and investigating

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their interplay in disease. Aβ is formed by the sequential cleavage of amyloid precursor

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protein (APP) by β- and γ- secretase. The accumulation of Aβ peptide in extracellular

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plaques is traditionally seen as the primary event in AD, with subsequent NFT formation

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and neuronal and synaptic loss following as a direct result; collectively known as the

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amyloid cascade hypothesis (Hardy and Higgins, 1992; Hardy and Selkoe, 2002).

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Work performed with frontotemporal dementia mutant tau (P301L) transgenic mice

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injected with aggregated Aβ (Gotz et al., 2001) as well as experiments using APP and

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tau bigenic mice (Lewis et al., 2001) indicates an upstream role for Aβ oligomerization in

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initiating tau pathology. In vitro and in vivo models show Aβ induced neurotoxicity and

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Aβ induced axonal transport deficits are ameliorated by dose dependent reductions in

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tau (Rapoport et al., 2002; Vossel et al., 2010). In hippocampal slice preparations Aβ-

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induced impairment of long-term potentiation (LTP) is inhibited in slices generated from

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tau-/- mice (Shipton et al., 2011). In the J20 APP transgenic mouse, reducing

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endogenous mouse tau decreased or eliminated learning and memory deficits,

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exploratory loco motor hyperactivity, interictal spiking, spontaneous seizures, and early

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mortality (Roberson et al., 2007; Roberson et al., 2011). In the APP23 transgenic mice,

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reducing tau dose dependently improved memory in the T-maze and reduced seizures

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and premature mortality (Ittner et al., 2010). The exact downstream mechanism involved

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in tau mediating protection from Aβ toxicity has yet to be shown. Elimination of mouse

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tau in these APP transgenic animals mitigates disease phenotypes but does not alter Aβ

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levels or plaque burden (Roberson et al., 2007; Ittner et al., 2010).

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In contrast to these findings, work done with double transgenic mice expressing both

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human mutant tau and APP showed greater Aβ deposition and NFT-like formation with

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increased neuronal loss in double transgenic animals when compared to single

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transgenic controls (Ribe et al., 2005). Furthermore, Braak and others have shown with

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their analysis of brain slices taken from patient autopsy samples still in the

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presymptomatic stages of AD, Braak-stage I-III (Braak and Braak, 1995), that

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intraneuronal tau alterations precede aggregated Aβ deposition (Braak and Del, 2004;

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Braak et al., 2013; Schonheit et al., 2004). They present evidence that tau tangles

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temporally develop either before or independent of Aβ plaques. These divergent findings

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indicate that the causal link between aberrant APP processing and tau alterations

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remains controversial.

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Recent AD research places tau in the extracellular space under physiological conditions

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(Yamada et al., 2011) and shows endogenous tau to be actively secreted from human

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(Chai et al., 2012) and rat neurons (Pooler et al., 2013). In concert with these

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observations, others have found that tau pathology spreads in AD. Early in disease, tau

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tangles are detectable in the entorhinal cortex and as the disease progresses tau

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pathology spreads into the hippocampus and cortex (Braak and Braak, 1995).

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The spread of tau tangle pathology occurs concomitantly with clinical disease

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progression, moving from memory loss to dementia. In in vivo models in which human

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tau is specifically overexpressed in the entorhinal cortex, tau is secreted and spreads

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along synaptic circuitry, resulting in tau pathology progression from the entorhinal cortex,

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through the hippocampus and into the cortex (Liu et al., 2012; de Calignon A. et al.,

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2012; Dujardin et al., 2014). Furthermore, comparative analysis of AD and healthy

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patient cerebral spinal fluid (CSF) showed a clear increase of amino-terminal (N-

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terminal) tau fragments in AD patient CSF, with no evidence of full-length or carboxyl-

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terminal (C-terminal) tau (Meredith, Jr. et al., 2013). This is in contrast to data

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suggesting that tau in the interstitial fluid (ISF) of P301L mice is full length (Yamada et

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al., 2011).

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Pre-synaptic release of secreted tau from cortical neurons has been shown to be

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mediated by neuronal activity. Yamada et al. used in vivo microdialysis in mouse brain to

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show that increasing neuronal and synaptic activity rapidly increases steady state levels

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of transgenic extracellular tau (Yamada et al., 2014). Additionally, in vitro treatment of rat

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neurons with the sodium channel blocker tetrodotoxin (TTX) prevents AMPA-mediated

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tau release (Pooler et al., 2013).

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In fact both tau and Aβ have been shown to be affected by neuronal hyperactivity. Cirrito

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et al. and others have demonstrated in vitro and in vivo that soluble Aβ levels in brain

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are directly influenced by neuronal, and more specifically, synaptic activity (Cirrito et al.,

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2005; Cirrito et al., 2008; Verges et al., 2011; Kamenetz et al., 2003; DeVos et al.,

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2013). They postulated a negative feedback role for Aβ in healthy neurons, functioning to

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keep neuronal hyperactivity in check by selectively depressing excitatory synaptic

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transmission. Taken together, these findings indicate a direct correlation between

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reductions in tau protein with reductions in neuronal hyperactivity, both of which

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correspond to a decrease in soluble Aβ levels.

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Here we have identified the major forms of human tau secreted from AD patient derived

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cortical neurons to be a pool of secreted N-terminal tau fragments which we call eTau,

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for extracellular tau. We show that eTau, and more specifically the human tau insert

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region, increases soluble Aβ levels while simultaneously lowering the neuroprotective

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product of α-secretase APP cleavage, soluble amyloid precursor protein-α (sAPPα);

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suggesting that eTau modulates Aβ production. We show this eTau/Aβ relationship in

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both primary human in vitro neuronal cultures and in vivo tau transgenic models.

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Additionally, we observed that eTau induces neuronal hyperactivity when applied to

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cortical cultures, and found that reducing or neutralizing eTau prevents aberrant

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neuronal hyperactivity. We suggest that eTau induced hyperactivity is negatively

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impacting neurons and that eTau may elevate Aβ production in AD brain.

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122 2. Materials and Methods

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2.1 Primary human cortical neuron cultures

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Human cortical neuron cultures were prepared as described (Wright et al., 2012) and

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cultured for three weeks in Neurobasal medium with B27 and Glutamax (Invitrogen)

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prior to start of 20 day treatments. Human fetal cerebral cortical tissue was obtained by

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Advanced Bioscience Resources (Alameda, CA) and complied with federal guidelines

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for fetal research and with the Uniformed Anatomical Gift Act.

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2.2 iPSC patient derived cortical neurons (iPSC-CN)

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Induced pluripotent stem cells (iPSC) were generated using the Yamanaka method

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(Takahashi et al., 2007) as previously described (Dimos et al., 2008) from skin biopsies

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taken from a familial presenilin-1 (A260V) AD patient aged 50 and a sporadic AD patient

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aged 60. Briefly, iPSC were differentiated to neurons via dual SMAD (small body size

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mothers against decapentaplegic) inhibition (Chambers et al., 2009) followed by cortical

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neuron differentiation as described (Burkhardt et al., 2013). iPSC derived cortical

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neurons (iPSC-CN) were grown between 70 and 108 days with media changes every

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three days unless otherwise noted. A list of AD patient samples used to generate

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individual iPSC clones, as well as their relevant mutations, is provided in Table 1.

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2.3 Antibodies

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2.3.1 iPierian Tau Antibodies

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Tau sizes are represented based on 2N4R tau sequence length unless otherwise noted.

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IPN001 and IPN002 (iPierian) are mouse monoclonal antibodies generated by standard

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immunization with in vitro aggregated 2N4R tau, followed by clonal selection and

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antibody purification from hybridoma. Both antibodies were selected for their high

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binding affinity to both secreted eTau as well as full length tau (recombinant Tau383,

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0N4R form) and for their ability to neutralize secreted eTau’s electrophysiological

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activity. They both bind to the N-terminus of tau with epitopes spanning amino acids 15-

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24.

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2.3.2 Antibodies

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MC1 (P. Davies) is a conformational monoclonal antibody specific for a conformation

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change in tau found in AD brain and PHF1 (P. Davies) detects the pSer396/404 tau

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epitope present on both normal adult brain tau and paired helical fragment (PHF) tau

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(Jicha et al., 1997). Commercial mouse monoclonal antibodies against tau used are HT7

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(aa 159-163, ThermoScientific), BT2 (aa 194-198, ThermoScientific) and T46 (C-

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terminal tau near amino acids 404-441, Invitrogen). Rabbit polyclonal antibodies used

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were against tau (aa 243-441, Dako) and against APP (C-terminal amino acids, Sigma).

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Mouse monoclonal control antibodies used were to β-Actin (Abcam), α-Tubulin (Cell

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Signaling), mouse IgG (R&D Systems) and mouse IgG whole molecule (Jackson).

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2.4 Secreted Tau Characterization, Purification, and Liquid Chromatography-Mass

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Spectrometry (LC/MS) Analysis

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2.4.1 Secreted Tau Immunoprecipitation

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iPSC derived cortical neurons (iPSC-CN) derived from a familial presenilin-1 (A260V)

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AD patient aged 50 were differentiated for 70 days at which point media conditioned for

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three days was collected for tau immunoprecipitation. Conditioned media was

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centrifuged at 15,000 rpm (4⁰C) and supernatants pre-cleared on a mouse IgG

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(Jackson) Sepharose 4B resin (GEHealthcare) before immunoprecipitation on either a

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N-terminal tau antibody, IPN002, a mid-region tau antibody, HT7, or a C-terminal tau

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antibody, T46, each previously coupled to Sepharose 4B resins. After

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immunoprecipitation, all three bead sets were washed three times with Mammalian

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Protein Extraction Buffer (M-PER, ThermoScientific) and immunoprecipitated protein

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extracted from pelleted beads with 2X Laemmli reducing buffer (Sigma) and separated

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on NuPage Bis-Tris protein gels (Invitrogen). Western blots were probed for tau with

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IPN001 (1 µg/ml), analysis by Odyssey SA software (LiCor). To ensure the tau specificity

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of all three immunoprecipitations, iPSC-CN growth media was incubated with the three

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antibody conjugated resins in parallel with the conditioned media, and the resultant non-

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specific eluate ran as Media control on the IPN001 Western blot.

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2.4.2 Secreted Tau Affinity Isolation

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iPSC derived cortical neurons (iPSC-CN) generated from skin biopsies taken from a

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familial presenilin-1 (A260V) AD patient aged 50 and a sporadic AD patient aged 60

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were cultured for 108 days in total, with media changes every three days. Starting at day

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70 of iPSC-CN culture, conditioned media was collected every three days and pooled for

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secreted tau affinity isolation. Pooled conditioned media was centrifuged at 15,000 rpm

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(4⁰C) and supernatants pre-cleared on a mouse IgG Sepharose 4B resin before tau

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purification on N-terminal IPN002 anti-tau antibody Sepharose 4B coupled resin. Protein

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was eluted with 50 mM sodium citrate, pH 2.3, 150 mM NaCl and neutralized with 1M

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TBS, pH 8.3. The eluate was concentrated and buffer exchanged into phosphate-

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buffered saline (PBS) and here referenced as eTau. A mock protein control was

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generated from a second batch of pooled conditioned media, also pre-cleared over the

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mouse IgG resin before mock affinity purification using mouse IgG resin.

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2.4.3 Liquid Chromatography-Mass Spectrometry Analysis (LC/MS)

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Side-by-side lanes of IPN002 affinity purified eTau were separated on

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reducing/denaturing 10% NuPage gel (Invitrogen), and analyzed by Coomassie stain

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(LiCor) or transferred to nitrocellulose for Western blot analysis with iBlot (Life

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Technologies) and probed for tau with IPN001 (1 µg/ml). Four Coomassie stained/anti-

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tau confirmed bands were excised from the gel and submitted for LC/MS analysis (MS

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Bioworks). Protein was proteolytically digested out of the gel slices with trypsin,

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chymotrypsin or elastase and gel digests were analyzed by nano LC/MS with a Waters

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NanoAcquity HPLC system interfaced to a ThermoFisher Orbitrap Velos Pro. Data were

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searched using Mascot. This analysis was verified using intact protein LC/MS analysis to

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ensure we identified the ends of the tau fragments. Liquid chromatography was

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performed on a C8 column (Agilent) and mass spec analysis performed on a Q-ToF

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Premiere (Waters).

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2.5 eTau Secretion Brefeldin Treatment

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Familial AD PSEN1 (A260V) iPSC-CN were cultured for 70-108 days then washed once

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with growth media prior to addition of fresh growth media with and without 10 µg/mL

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Brefeldin A (Sigma); media was conditioned for four hours at 37⁰C/5%CO2 and collected

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for analysis by IPN001 (1 µg/ml) Western blot. Lysates from treated wells were also

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harvested with 50 µl M-PER and β-actin levels in the lysates were measured by Western

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blot for normalization of eTau data.

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2.6 Primary Human Cortical Neuron Treatments

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Primary human cortical neurons were treated with either 100 nM BACE1 inhibitor

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(BACEi) (Axon Medchem), tetrodotoxin (Tocris), 1 µM Accell siRNA pools against

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human MAPT, BACE1, ADAM9, ADAM10, ADAM17 or Non-Targeting Control (Thermo

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Scientific/Dharmacon, catalog E-012488, E-003747, E-004504, E-004503, E-003453

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respectively ), 50 nM or 500 nM purified secreted tau (eTau), or mock control (iPierian),

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E. coli generated full-length rTau383(0N4R) (rPeptide), synthetic human eTau4 peptide

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(aa 2-68, 0N3R designation ), scrambled human eTau4 peptide control, or the equivalent

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mouse tau sequence (aa 2-57) (Neobiolab), or with 1, 10, or 30 µg/mL protein A purified

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tau antibodies PHF1 and MC1 (Peter Davies), IPN002, or anti-mouse IgG. Treatments

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were diluted in basal media (Neurobasal media with Glutamax and B27, Invitrogen) and

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incubated on neurons for a total of 20 days; conditioned media was harvested every 5

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days and treatments were added again, for a total of 20 days of exposure. Conditioned

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media was stored at -80⁰C until day 20 end of assay for full time course ELISA analysis,

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and cells were lysed in 50 µl M-PER (ThermoScientific) or 50 µl Buffer RLT (Qiagen) for

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protein or mRNA analysis.

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2.7 Lactate Dehydrogenase Assay (LDH)

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LDH activity in in vitro conditioned media from iPSC-CN and human primary cortical

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cultures was determined using the Lactate Dehydrogenase Activity Assay Kit (Sigma).

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25 µl or 50 µl of five day conditioned media was assayed with an equal volume LDH

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reagent and incubated for 30 minutes at room temperature. Plates were read on plate

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reader at 490 nm.

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2.8 Human sAPPα and Aβ40 and Aβ42 ELISAs

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Human sAPPα and Aβ40 and Aβ42 protein levels were measured in primary human

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cortical neuron conditioned media collected every 5 days over a 20 day treatment period

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using specific ELISA assays (Invitrogen #KHB0051), (MilliporeEMD #EZHS40) and

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(MilliporeEMD #EZHS42) respectively.

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2.9 Mouse Aβ40 and Aβ 42 ELISAs

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Mouse Aβ protein levels were measured in mouse cortical brain homogenate or

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interstitial fluid (ISF) using mouse Aβ40 and/or Aβ42 ELISAs (Immuno-Biological

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Laboratories #JP27720 and #JP27721). The mouse Aβ42 ELISA lacked the sensitivity to

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reliably detect Aβ42 in brain tissue.

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2.10 Gene Expression

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Cultured neurons were lysed in Qiagen Buffer RLT and RNA isolated using an RNeasy

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kit (Qiagen). cDNA was reverse-transcribed using the SuperScript VILO cDNA Synthesis

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Kit (Life Technologies). Quantitative RT–PCR was performed using TaqMan Gene

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Expression Assays and Taqman Expression Master Mix (Life Technologies) on an

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Applied Biosystems HT 7900. Total MAPT, BACE1, ADAM9, ADAM10, ADAM17, APP,

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PSEN1 and PSEN2 mRNA expression levels were normalized to RPLPO (housekeeping

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gene) mRNA expression levels and analysed using the ∆∆CT method for relative

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expression. Invitrogen Taqman Gene Expression Assays used Cat#4331182 individual

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ID # MAPT Hs00902194_m1 (for all isoforms MAPT); BACE1 Hs01121195_m1; ADAM9

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Hs00177638_m1; ADAM10 Hs00153853_m1; ADAM17 Hs01041915_m1; APP

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Hs01041915_m1; PSEN1 Hs00997789_m1; PSEN2 Hs01577197_m1; RPLPO

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(Cat#4333761T). A probe recognizing only the 2N4R/1N4R/2N3R/1N3R isoforms of

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MAPT Hs00213484_m1 was used with MAPT Hs00902194_m1 to distinguish fetal

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0N3R MAPT from other MAPT isoforms in both primary human cultures and iPSC-CN

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cultures.

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2.11 Western Blots

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Cultured primary human neurons were lysed in M-PER Buffer. Cell lysates, primary

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cortical neuron conditioned media and iPSC-CN conditioned media, were diluted in 2X

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Laemmli reducing buffer, boiled, separated on NuPage gels and transferred to

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nitrocellulose using iBlot. Western blots were blocked (LiCor) and probed with

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antibodies; IPN001 or IPN002 (1 µg/ml), HT7 (1 µg/ml), PHF1 (1 µg/ml), Dako tau

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(1:3,000), β-actin (1:5,000), α-Tubulin (1:10,000), APP (1:1,000 Sigma) and anti-mouse

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680 or anti-rabbit 800 secondary antibodies (1:10,000, LiCor); analysis by Odyssey SA

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software (LiCor).

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2.12 Animals

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Two transgenic human tau mouse models were used in these studies. Experiments were

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conducted in accordance with the National Institutes of Health Guide for the Care and

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Use of Laboratory Animals guidelines and were approved by the Institutional Animal

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Care and Use Committee. Schematic depicting overall study plans provided in

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Supplementary Figure 3.

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The 8-week study used homozygous JNPL3 mice (Taconic, Tg(Prnp-

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MAPT*P301L)JNPL3Hlmc) as described by (Lewis et al., 2000). The in-life study was

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conducted by Brains On-Line. In brief, the JNPL3 line overexpresses human 0N4R tau

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containing the most common frontotemporal dementia (FTDP-17) mutation P301L on a

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C57BL/6, DBA/2, SW mixed background, driven by the prion promoter. Homozygous

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JNPL3 mice express transgenic tau at levels about two-fold over endogenous tau, and

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demonstrate motor and behavioral deficits as well as age dependent development of

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NFT (Lewis et al., 2000).

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The 26-week study used tau-4R/2N-P301L (van Leuven) mice as described by (Terwel

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et al., 2005). The in-life study was conducted by reMYND. In brief, human 2N4R tau

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containing the FTDP-17 mutation P301L was overexpressed by the mouse thy1

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promoter on a pure FVB genetic background. Mice display an age dependent

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hyperphosphorylation of tau, tau aggregation and tangle formation with concomitant

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development of motor defects.

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2.12.1 Eight week study (JNPL3 P301L mice) - 3.5 month old female JNPL3 mice were

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treated weekly with a single dose of 60 mg/kg irrelevant IgG1 or IPN002 antibody for 8

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weeks by an intraperitoneal (i.p.) injection volume of 10ml/kg. At the end of study mice

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were anesthetized using isoflurane (2%, 800 mL/min O2) and stereotactically-implanted

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with a microdialysis probe in the ventral hippocampus, a polyacrylonitrile membrane with

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3 mm exposed surface and 1 megadalton cutoff (BrainLink, the Netherlands).

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Bupivacain/epinephrine was used for local analgesia and fynadine or carprophen for

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peri-/post-operative analgesia. Push-pull microdialysis sampling was performed 24 hours

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after implantation surgery. Animal probes were connected with FEP tubing to a

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microperfusion pump (Harvard PHD 2000 Syringe pump) and perfused with artificial

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CSF containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2, and

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0.15% BSA at a flow rate of 0.75 µL/min. Microdialysis samples were collected over 60

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minutes; basal samples were collected after stabilization. Dialysis ISF samples were

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centrifuged at 15,000 rpm for 15 minutes at 4⁰C. The animals were sacrificed

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immediately following microdialysis sampling and terminal brain, plasma, and CSF were

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collected (Brains On-Line).

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2.12.2 Twenty-six week study (tau-4R/2N-P301L van Leuven mice)- 3 to 4 month old

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female mice were treated weekly by i.p. injection (10ml/kg) of 20 mg/kg irrelevant IgG1

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or IPN002 antibody for up to 26 weeks (reMYND). At study end mice were sacrificed and

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serum, CSF, and brain collected. CSF was collected via an incision in the neck muscle

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and drawn through a puncture with a 26 gauge needle from the cisterna magna then

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centrifuged at 10,000 x g/10 minutes at 4⁰C. Brain regions were dissected from left

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hemisphere (cerebellum, cortex, hippocampus and hindbrain) for each mouse and

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homogenized in Tris-Buffered Saline (TBS) (Sigma) (1:10 w/v) then centrifuged at

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10,000 x g/10 minutes at 4⁰C. Brain fraction homogenates were diluted to 1 mg/ml in

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TBS and total protein concentration determined by BCA Assay (Pierce); to generate a

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soluble fraction a portion of each homogenate was further spun 1 hour at 100,000 x g

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(4⁰C) and soluble supernatants collected. FVB wild-type cortex was provided by

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reMYND.

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2.13 Tau ELISAs

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2.13.1 Free-tau ELISA – We designed a homogenous alphascreen (Perkin Elmer) assay

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to measure tau not bound by IPN002 in mouse ISF and CSF and in primary human

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cortical culture conditioned media and lysates. The tau antibodies used are HT7 (aa

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159-163) and the N-terminal tau antibody IPN001 (aa 15-24) which competes with

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IPN002 binding and therefore will not interact with tau already bound by IPN002. IPN001

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is biotinylated, and HT7 is conjugated to AlphaLISA acceptor beads (Perkin Elmer).

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Samples are incubated overnight with both antibodies. Streptavidin coated acceptor

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beads (Perkin Elmer) are added for 30 minutes and then the plate is read on Envision

326

plate reader (Perkin Elmer) at excitation 630nm, emission 615 nm. Tau levels were

327

interpolated from an eight point tau protein (rTau383,0N4R) standard curve and

328

analyzed using linear curve fit in Excel.

329

2.13.2 Total tau ELISA – The total tau ELISA uses the tau antibodies HT7 (aa 159-163)

330

to capture and biotinylated BT2 (aa 194-198) for detection. HT7 diluted in PBS is coated

331

on high binding ELISA plates (Costar-Corning) at 1 µg/ml overnight at 4⁰C and plates

332

blocked with 1% Casein (VectorLabs)/PBS for 2 hours at room temperature. Samples

333

are diluted in 0.1%Casein/PBS and incubated overnight at 4⁰C. Tau levels detected with

334

one hour incubation with biotinylated tau antibody BT2, followed by addition of

335

Streptavidin-HRP (Southern Biotech) and then 3,3′,5,5′-tetramethylbenzidine substrate

336

(TMB) (ThermoScientific), assay was stopped with 1M sulfuric acid (Sigma), and the

337

absorbance read on a Beckman plate reader at 450 nm. Tau levels were interpolated

338

from an eight point tau protein (rTau383, 0N4R) standard curve diluted in

339

0.1%Caesin/PBS and analyzed using 4-Parameter Sigmoidal Curve Fit (GraphPad

340

Prism).

341

2.14 Tau Binding Assay

342

To measure the kD of various tau antibodies, high binding ELISA plates were coated

343

overnight at 4⁰C with 100 ng of recombinant Tau383(0N4R) or purified eTau (iPierian)

344

diluted in PBS; wells were then blocked with 1% casein/PBS for 2 hours at room

345

temperature. Tau antibodies (IPN001, IPN002, MC1, PHF1) were diluted to 10 µg/mL in

346

PBS/0.1% casein and 12-point titrations generated, added to the plate, and incubated 2

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hours at room temperature. After washing with 0.05%Tween-20 (Sigma)/PBS, detection

348

antibody goat anti-mouse HRP (Southern Biotech) diluted 1:2,000 in PBS/0.1% casein

349

was added and incubated at room temperature for 1 hour. Wells were washed as above,

350

and TMB substrate (ThermoScientific) added, assay was stopped with 1M sulfuric acid,

351

and absorbance read at 450 nm; kD calculated based on nonlinear regression analysis

352

(GraphPad Prism).

353

2.15 Free IPN002 Levels in CSF and ISF

354

To measure the amount of free IPN002 in mouse CSF or ISF, high binding ELISA plates

355

were coated overnight at 4⁰C with 100 ng of recombinant Tau383(0N4R) diluted in PBS;

356

wells were then blocked with 1% casein/PBS for 2 hours at room temperature. CSF or

357

ISF was diluted 50 or 200 fold in PBS/0.1% casein. Coated plates were washed twice

358

with PBS, then diluted CSF or ISF samples added to the plate in duplicate and incubated

359

2 hours at room temperature. After washing with 0.05%Tween-20 (Sigma)/PBS,

360

detection antibody goat anti-mouse HRP (Southern Biotech) diluted 1:2,000 in

361

PBS/0.1% casein was added and incubated at room temperature for 1 hour. Wells were

362

washed as above, and TMB substrate (ThermoScientific) added, assay was stopped

363

with 1M sulfuric acid, and absorbance read at 450 nm; IPN002 amounts calculated

364

based on dilution factor and linear regression analysis equation of the IPN002 standard

365

titration (GraphPad Prism).

366

2.16 Electrophysiology

367

Whole cell patch clamp recording was performed on primary human cortical cultures and

368

familial AD PSEN1 (A260V) iPSC-CN cultured on a monolayer of normal human

369

astrocytes (Lonza) using micro-pipette (2-5 MOhm) filled with solution containing (mM):

370

K-methyl-sulfate (140), NaCl (10), CaCl2 (1), Mg-ATP (3); Na-GTP (0.4), EGTA (0.2),

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HEPES (10), Phosphocreatine (10) with adjusted pH= 7.3 and mOsm= 305. Neurons

372

were perfused (1-2 ml/min) with artificial cerebral spinal fluid containing (mM): NaCl

373

(140), KCl (2.5), MgCl2 (2) CaCl2 (2), Hepes (10), D-Glucose (10), sucrose (20), adjusted

374

pH= 7.3-4 mOsm = 310. Recordings were made using pClamp-10.3 data acquisition

375

software (Molecular Devices) and MultiClamp 700B amplifier (Axon Instrument; Foster

376

City CA). Puff application of eTau, full-length rTau383 (0N4R), and buffer or mock

377

protein controls was performed using MiniSquirt micro-perfusion system (AutoMate,

378

Berkeley, CA). Bath application of eTau, IPN002 antibody, and buffer controls was

379

performed for other recordings. Off-line data analysis used Clampfit 10.2 analysis

380

software (Molecular Devices). Recordings were conducted at 34-37oC.

381

2.17 Statistical Analysis

382

Statistics were determined using GraphPad Prism software. Data are expressed as

383

mean +/- standard deviation (STD) on a n=3 (unless otherwise noted in figure legends);

384

experiments repeated a minimum of four times. Analysis of tau or Aβ levels, comparing

385

the effects of one treatment versus control, at a single time point, were done by two-

386

tailed unpaired t test with Welchs correction. Analysis of tau, Aβ and sAPPα levels

387

involving repeated measures between multiple treatment groups at different time points

388

was done using one-way ANOVA with Tukeys multiple comparison correction, unless

389

otherwise noted. When significant ( 50% (Figure 1B).

409

We then asked if tau affects α- and/or β- secretase, two proteases opposed in Aβ

410

generation that compete for APP substrate. Colombo et al. show that α- and β-secretase

411

cleavage of their common substrate APP is partially coupled in primary mouse neurons.

412

We confirmed these results in primary human neurons, showing that BACE1 inhibition

413

(BACEi) results in increased production of sAPPα (soluble APP cleaved at the α-

414

secretase site) in the conditioned media, Figure 1C left, as well as increased production

415

of APP CTF-α in the lysate, Supplementary Figure 1C. Furthermore, treatment of

416

primary human neuron cultures with siRNAs directed against BACE1 and ADAM9/10/17

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resulted in opposing effects on cellular Aβ and sAPPα levels (ELISA data

418

Supplementary Figures 1E and 1F).

419

In our primary human neuron cultures we reduced MAPT gene expression and tau

420

protein (Figure 1A) levels over 70% using Accell MAPT siRNA pools. Under these

421

conditions non-targeting siRNA had no effect on sAPPα levels; MAPT knockdown

422

however resulted in a 42% increase in sAPPα (Figure 1C right).

423

Secreted Tau (eTau) identified from AD patient derived iPSC cortical neuron

424

conditioned media is composed of N-terminal tau fragments

425

In light of these findings and recent data showing tau to be secreted from human

426

induced pluripotent stem cell (iPSC) derived cortical neurons (Chai et al., 2012), we

427

characterized tau release from our iPSC cortical neurons (iPSC-CN) derived from AD

428

patients. To do so, we generated iPSC from a skin biopsy taken from a familial

429

presenilin-1 (A260V) AD patient aged 50 then differentiated and cultured iPSC-CN for up

430

to 108 days. To characterize eTau secretion from these iPSC-CN we collected

431

conditioned media beginning at day 70 over time for 96 hours. As shown in the tau

432

Western blot in Figure 2A, tau is present in the conditioned media of iPSC-CN, and

433

quickly accumulates over time.

434

To ensure that the tau found in the conditioned media was not the result of cell lysis we

435

measured the lactate dehydrogenase (LDH) activity in the conditioned media of several

436

of our iPSC-CN lines listed in Table 1, and found all to be free of cell lysis

437

(Supplementary Figure 2A). These data and an absence of other high abundance

438

cytoplasmic proteins such as β-Actin and α-Tubulin (Supplementary Figure 2B) in the

439

conditioned media together confirm the lack of contamination by intracellular proteins in

440

iPSC-CN conditioned media. The tau release we observed from our iPSC-CN is rapid,

441

detectable within 2-5 minutes, and plateaus over 24-96 hours (Figure 2A). iPSC-CN tau

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secretion is not modulated by Brefeldin A treatment, indicating that secretion is occurring

443

independently of the canonical secretory process (Figure 2B).

444

Since iPSC-CNs derived from AD patient samples secrete tau in the absence of

445

neuronal lysis, we characterized the secreted tau species identified in the media. To do

446

so we attempted to immunoprecipitate tau from the media (conditioned 3 days) of 70 day

447

old iPSC-CNs derived from a familial presenilin-1 (A260V) AD patient with either a N-

448

terminal tau antibody (IPN002), a mid-range tau antibody (HT7), or a C-terminal tau

449

antibody (T46). Each of these antibodies efficiently isolates cytoplasmic full length tau

450

(data not shown) but only IPN002 and HT7, not T46, were capable of

451

immunoprecipitating secreted tau (Figure 2C). The C-terminal tau antibody’s failure to

452

isolate secreted tau is shown by IPN001 Western blot analysis of the three

453

immunoprecipitated products; indicating that tau is released from AD iPSC-CN

454

predominately as N-terminal fragments, ranging from 20-28 kDa (Figure 2D) with no

455

evidence of C-terminal reactive fragments. This data is also consistent with reports

456

showing that tau is secreted into AD patient CSF as N-terminal fragments (Meredith, Jr.

457

et al., 2013). We have conducted similar characterizations of eTau secreted from cortical

458

neuron from healthy control subjects and found a similar molecular weight range of eTau

459

fragments as those found for AD patients (data not shown).

460

To verify the 20-28 kDa bands as tau and to identify their sequence, we affinity isolated

461

secreted tau from our iPSC-CN conditioned media with the tau antibody IPN002 and

462

conducted tryptic, chymotrypic and elastase digestion followed by LC/MS analysis on the

463

four major IPN002-affinity isolated, anti-tau reactive bands shown in Figure 2D. We also

464

used intact protein LC/MS to ensure we identified the end of the largest tau fragment.

465

This analysis confirmed that secreted tau is a series of N-terminal tau fragments; all

466

lacking the C-terminal amino acids of tau, including the microtubule binding repeats as

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shown in the sequence alignment in Figure 2E. We named the four major fragments

468

eTau 1, 2, 3 and 4 and refer to the pool of secreted tau as eTau (for extracellular tau).

469

Our iPSC-CNs only express the 0N3R embryonic isoform of tau (Supplementary Figure

470

1B), and LC/MS analysis confirmed the n1 and n2 inserts to be missing in the

471

sequenced fragments, Figure 2E. All forms of eTau start at the second amino acid,

472

alanine, with the N-terminal methionine removed and the alanine acetylated. This is a

473

covalent modification traditionally indicative of a cytosolic protein, providing further

474

evidence that eTau is released independent of the canonical endoplasmic

475

reticulum/golgi secretory process. The four major forms of eTau we identified range in

476

size from 67 amino acids (aa 2-44,103-126, eTau4) to 171 amino acids (aa 2-44, 103-

477

230, eTau1), Figure 2E. Phosphorylation of eTau was rarely detected but when found

478

the residues phosphorylated were consistent with phosphorylation of cytoplasmic tau at

479

residues threonine181 (p181), serine202 (p202), and threonine231 (p231).

480

We further characterized eTau by its reactivity with several tau antibodies, specific

481

affinities are shown in Table 2. Both 0N4R recombinant full length tau-383, and eTau

482

bind with high affinity to the N-terminal tau antibodies MC1 (a conformational specific tau

483

antibody) (Jicha et al., 1997) and IPN002 (generated against aggregated tau, iPierian).

484

Not surprisingly, eTau does not bind to the C-terminal anti-phospho-tau antibody PHF1

485

(Jicha et al., 1997) in either the tau binding assay (Table 2) or by Western blot

486

(Supplementary Figure 2C), indicating that eTau lacks this phosphoepitope, as expected

487

based on LC/MS analysis.

488

eTau regulates amyloid-beta (40 and 42) levels in vitro in primary human neurons

489

We found that this pool of N-terminal eTau fragments is also secreted from human

490

primary cortical neurons at concentrations up to 3-4 nM, when allowed to condition for 5

491

days (data not shown). Total tau ELISA of the conditioned media of human primary

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neurons confirmed this tau secretion, and demonstrated that MAPT knockdown in these

493

primary human neurons significantly reduces secreted eTau levels in the conditioned

494

media over time when compared to non-targeting siRNA control (Figure 3A).

495

To determine if eTau plays a role in regulating Aβ40 and Aβ42, we treated primary human

496

neurons for 20 days (media and treatments replaced every 5 days) with affinity purified

497

eTau from AD patient derived iPSC-CN or mock protein control, and saw a dose and

498

time dependent (as early as 10 days at 500 nM) increase in levels of Aβ40 and Aβ42

499

(Figure 3B). The changes in Aβ levels are small but reproducible over multiple

500

experiments and are statistically significant.

501

Since the conditioned media from primary human neurons, when allowed to condition on

502

cells for five days, already contains secreted eTau at concentration up to 3-4 nM, a large

503

excess of purified secreted eTau (500 nM) appears to be required to generate these

504

additional small changes. Less eTau was needed (75% with siRNA transfection (data not shown). Similar

507

experiments conducted with up to 1 µM of full length rTau383 (0N4R) did not affect Aβ

508

levels (data not shown) suggesting an eTau specific regulation of Aβ levels.

509

eTau regulates sAPPα α levels in vitro in primary human neurons

510

Our data indicate that eTau may be affecting how Aβ is produced. To understand how

511

eTau is affecting Aβ production at the mRNA level we measured the gene expression of

512

APP, BACE1, ADAM9, ADAM10 and ADAM17 in human primary neurons but did not

513

detect any significant gene expression changes after treatment with either MAPT siRNA,

514

purified secreted eTau, or tau binding antibodies (gene expression data not shown).

515

The addition of eTau to primary human neurons for 20 days caused a small but

516

significant, dose dependent decrease in sAPPα levels (Figure 3C). The simultaneous

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increase in Aβ with the decrease in sAPPα indicates that eTau is affecting the

518

production of Aβ in neurons.

519

eTau binding antibodies reduce amyloid-beta (40 and 42), increase sAPPα levels

520

in vitro in primary human neurons

521

We tested each of several antibodies for binding affinity to tau and eTau. As expected all

522

tau antibodies tested bound with good to excellent Kd’s and as expected IPN001,

523

IPN002 bound to eTau, while PHF1 did not bind eTau, Table 2. Unexpectedly, we found

524

that MC1 bound with high affinity to not only tau but also eTau, Table 2. This was

525

unexpected since MC1 is reported to bind to a conformational epitope comprised of both

526

aa 7-9 and aa313-322, but aa313-322 are not present on eTau. This suggests that MC1

527

can also bind to eTau as well as to the conformation epitope described previously. Peter

528

Davies has also found that MC1 binds to the N-termini of tau, in the absence of the C-

529

terminal epitope (personal communication). To confirm therefore that eTau regulates Aβ,

530

we treated our human primary cortical cultures with various tau antibodies. We used the

531

eTau binding antibodies IPN002 and MC1, as well as PHF1, shown not to bind to eTau.

532

We found that when added to cortical neurons, MC1 and IPN002 both time dependently

533

reduced Aβ40 and Aβ42 levels, while the IgG control and PHF1 had no effect (Figure 4A).

534

Additionally, eTau binding antibodies (MC1 and IPN002) increased levels of sAPPα over

535

time, while PHF1 did not (Figure 4B). APP Western blots of the cell lysates of IPN002

536

treated primary human cortical cultures confirm these ELISA results, showing a slight

537

increase in APP CTF-α when compared to IgG control (Supplementary Figure 1C).

538

To demonstrate that our IPN002 antibody is acting extracellularly, specifically binding

539

and neutralizing secreted and not intracellular tau, we used our IPN002-free-tau ELISA

540

assay to analyze the conditioned media and lysate of primary human cortical neurons

541

treated with IPN002. This is an IPN002 competitive tau assay, which only detects tau not

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already bound by IPN002. Analyzing the conditioned media of primary human neurons

543

treated with either IgG control or the N-terminal tau antibody IPN002 every 5 days for 20

544

days, we found that the vast majority of eTau in the conditioned media of these cultures

545

was bound to IPN002, measured as a significant decrease in IPN002-free tau (Figure

546

4C, right). Analyzing the lysates of these cultures we found no change in free tau levels

547

with IPN002 treatment (Figure 4C, left). This indicates that IPN002 is not entering the

548

cell in sufficient quantities to bind/neutralize intracellular tau, and suggests that the

549

antibody’s observed effect on Aβ levels is through its interaction with secreted eTau.

550

These results suggest that eTau plays a role in modulating Aβ levels in vitro. In contrast

551

to the β-secretase inhibitor which rapidly (1 day) and dramatically (75-85%) lowers Aβ

552

levels (Supplementary Figure 1D), IPN002 and MC1 lower Aβ levels more gradually (10

553

days) and modestly (30-40%) (Figure 4A). The gradual effect of these eTau binding

554

antibodies is indicative of a modulatory rather than a direct, inhibitory role.

555

Human synthetic eTau 4 increases Aβ β (40 and 42) levels in vitro in primary human

556

cortical neurons

557

In vivo studies in APP transgenic mice show that reductions of endogenous murine tau

558

do not impact Aβ levels (Roberson et al., 2007; Roberson et al., 2011; Ittner et al.,

559

2010). Therefore, to further understand our findings in the context of these studies

560

involving mouse tau, we asked if human and mouse tau affect Aβ production differently.

561

To do so we generated a synthetic version of human eTau 4, a 67 amino acid peptide

562

corresponding to the shortest secreted N-terminal tau fragment identified by LC/MS from

563

iPSC-CN media. We also synthesized a peptide corresponding to the equivalent amino

564

acid sequence in mouse tau (Figure 5A). Since both our AD patient derived iPSC-CN

565

cultures and our primary human cortical cultures predominately express embryonic

566

0N3R tau (Supplementary Figure 1B) we did not include the n1 and n2 inserts in these

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synthetic sequences. Notably mouse tau is missing the human tau insert, amino acids

568

17-28, which encompasses the majority of the IPN002 epitope (aa 15-24).

569

We treated primary human neurons with either the human or mouse eTau 4 peptide or a

570

scrambled control peptide, and looked for differential effects on Aβ levels. By using

571

synthetic versions of the sequences we eliminated any potential post-translational

572

differences between the two species tau, and treated the same primary human neuronal

573

cultures for direct comparison. Under these conditions human eTau 4 treatment

574

significantly increased Aβ40 and Aβ42 levels (18% and 26% respectively compared to

575

scrambled control), while treatment with mouse eTau 4 did not increase Aβ40 and Aβ42

576

levels (Figure 5B). Again, the increases in Aβ levels with human eTau4 are small but

577

reproducible and highly significant.

578

In fact, by treating the primary human neuronal cultures with an additional peptide

579

encompassing only the human tau insert, we show that the 12 amino acids missing from

580

mouse tau are capable of increasing Aβ40 and Aβ42 to the same level as the entire

581

human eTau 4 peptide (Figure 5B). These data confirm that human eTau increases Aβ

582

production and suggests that the modulation of Aβ production is human eTau specific

583

via an activity conferred by the human tau insert.

584

The eTau antibody, IPN002, reduces Aβ β (40 and 42) levels in vivo

585

To test our hypothesis in vivo, we first used the transgenic P301L tauopathy model

586

(JNPL3, Taconic) that releases mutant human tau into interstitial fluids (ISF) (Figure 6A).

587

We found that tau is present in the ISF of these mice at a truncated size of ~25 kDa,

588

Supplemental Figure 2d, similar to eTau found in the AD patient cortical neuron

589

conditioned media. This is in contrast to the findings by (Yamada et al., 2011) showing

590

full-length tau in the ISF of P301L mice. We treated 3.5 month old JNPL3 mice weekly

591

with either IgG1 control or IPN002 for eight weeks, collected ISF at 5.5 months of age,

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and measured the levels of IPN002 antibody, IPN002-free tau, and Aβ40 and Aβ42. To

593

verify that IPN002 entered the central nervous system (CNS) we measured levels of

594

IPN002 in the plasma and ISF of each animal at study termination and found IPN002

595

present in the ISF in all cases (Figure 6B). Additionally, we found that IPN002 bound

596

almost all the human mutant tau available in the ISF, measured by a 94% reduction in

597

the free tau levels (Figure 6C). Aβ40 and Aβ42 analysis of the animal’s ISF at the end of

598

the eight week treatment showed IPN002 treatment reduced endogenous mouse Aβ 40

599

and Aβ42 levels by ~25% (Figure 6D).

600

We validated these initial findings in a second transgenic P301L tauopathy model, using

601

the tau-4R/2N-P301L (van Leuven) mice, in which secreted human tau levels increase

602

with age, Figure 6E). In this model, 3.5 month old animals have < 10 ng/ml human tau in

603

their cerebrospinal fluid (CSF), by 9.5 months their CSF tau levels increase to an

604

average of 30 ng/ml (Figure 6E). We treated these mice weekly, for up to 26 weeks, with

605

20 mg/kg IgG1 control or IPN002. To verify that IPN002 entered the CNS, we measured

606

levels of IPN002 in the plasma and CSF of each transgenic animal at 9.5 months and

607

found IPN002 present in the CSF in all dosed animals (Figure 6F). CSF analysis by

608

IPN002-free-tau ELISA assay confirmed that human mutant tau was fully engaged by

609

IPN002, measured by a 98% reduction in free tau levels, Figure 6E, IPN002 treated

610

compared to IgG1 control treated mice.

611

We measured Aβ40 levels in both the total homogenate and the soluble fraction of the

612

hindbrain (data not shown) and cortex; the results were consistent between fractions and

613

brain regions. As shown in Figure 6G, Aβ40 levels in the soluble fraction of P301L mouse

614

cortex increase from 14.4 pg/ml at 3.5 months to 22.5 pg/ml at 9.5 months (IgG1 control

615

group). This substantial increase in levels of murine Aβ40 in a tau mouse model may be

616

due either to age, to continued human tau expression, or to a combination of both. To

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distinguish between these possibilities, we measured Aβ 40 levels in the soluble fraction

618

of the cortex of wild type C57/BL6 mice at 4.3 months and 9.5 months of age (Figure

619

6G) and saw no change in Aβ with age. This suggests that the observed increase in

620

mouse Aβ over time in this P301L tau mouse model is due to human tau expression.

621

To determine if elevated levels of Aβ40 could be lowered with the human eTau binding

622

antibody IPN002 we compared the Aβ 40 levels of IgG1 and IPN002 treated mice and

623

found that IPN002 treatment significantly reduced levels of soluble mouse Aβ40 by >

624

25%, Figure 6G. Together these data suggest that human tau released from neurons

625

modulates Aβ production both in vitro and in vivo.

626

TTX treatment lowers Aβ β and eTau levels in primary human cortical neurons in

627

vitro

628

How does eTau modulate Aβ production? In AD, secreted tau is hypothesized to be

629

released pre-synaptically and then taken up post-synaptically, inducing spread, tau

630

pathology, neuronal dysfunction, and neuronal hyperactivity. Kamenetz et al. showed

631

that picrotoxin induced hyperactivity in hippocampal slices dramatically elevated Aβ 40

632

and Aβ42 while TTX treatment was able to lower Aβ levels (Kamenetz et al., 2003);

633

Cirrito et al. found a similar link between heightened neuronal and synaptic activity and

634

increased Aβ production in living mouse ISF (Cirrito et al., 2005; Cirrito et al., 2008).

635

To determine whether our observed increase in Aβ could be the result of eTau induced

636

neuronal hyperactivity, we treated our primary human cortical cultures with the sodium

637

channel blocker TTX. As has been shown previously in vitro and in vivo, TTX is capable

638

of significantly reducing soluble Aβ 40 and Aβ42 in a dose and time dependent manner

639

(Figure 7A), with no change to cell viability (data not shown). In these same cultures,

640

eTau levels analyzed in the conditioned media of the TTX treated neurons were also

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reduced, by 60%, after 10 days of TTX treatment (Figure 7B). eTau in the conditioned

642

media of neurons is significantly reduced by day 10 of TTX treatment; however, Aβ

643

levels are not affected until day 15, suggesting that suppressing neuronal activity

644

reduces eTau levels and subsequently alters Aβ production.

645

eTau induces hyperactivity in human neurons

646

Increased neuronal activity potentiates Aβ levels in vitro and in vivo. This suggests that

647

one mechanism by which eTau can modulate Aβ production is through increasing

648

neuronal activity. To test this hypothesis, we performed whole cell patch clamp

649

recordings to determine the effect eTau has on cortical neuron activity. Application of

650

eTau increased the activity of both iPSCs-derived cortical neurons and primary human

651

cortical neurons (Figure 7C). Interestingly, application of full length tau (rTau383 0N4R)

652

at significantly higher concentrations (up to 3 µM) failed to produce any changes in

653

neuronal activity similar to those caused by eTau treatment. This suggests that the eTau

654

mediated neuronal activity effects may involve a specific interaction of the eTau protein

655

with cortical neurons. This interaction appears to be specific to the N-terminal region of

656

tau as it cannot be replicated with full length tau (rTau383 0N4R). To test this

657

hypothesis, we pre-incubate eTau with the neutralizing antibody IPN002 to block eTau

658

mediated hyperactivity. At a 10:1 molar ratio of IPN002 to eTau, IPN002 inhibited eTau

659

mediated hyperactivity (Figure 7D). Together, these results suggest that eTau can

660

modulate Aβ levels by promoting cortical neuron hyperactivity.

661

4. Discussion

662

In this study we describe the identification of a novel secreted N-terminally truncated tau

663

species (eTau), present both in vitro and in vivo, and show that these secreted tau

664

fragments can regulate Aβ production. We initially purified eTau from the conditioned

665

media of AD patient derived cortical neuron cultures, and found that secreted tau is

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composed predominantly of N-terminal fragments of tau. In vitro we show that treating

667

primary cortical cultures with eTau simultaneously increases Aβ levels while decreasing

668

sAPPα levels. Globally reducing tau with MAPT siRNA or treating neurons with

669

neutralizing eTau antibodies lowers Aβ levels and increases sAPPα levels in parallel.

670

Importantly, we validated these findings in vivo in two tau transgenic (P301L) mouse

671

models. Levels of human tau are elevated in the ISF and CSF of the tau transgenic

672

animals; treating these mice with an eTau-neutralizing antibody (IPN002) reduced

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endogenous murine Aβ in both their ISF and brain. Finally, in vitro we demonstrate that

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eTau treatment increases neuronal hyperactivity. We propose that this eTau driven

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neuronal hyperactivity increase, results not only in increased Aβ secretion, but also in a

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further increase in eTau secretion, and that together eTau and Aβ create a feed forward

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disease mechanism that perpetuates the disease (Supplementary Figure 4).

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We examined the conditioned media of AD patient derived cortical neurons for the

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presence of tau in the absence of neuronal lysis and found tau fragments. Analysis of

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the tau fragments by LC/MS for primary sequence and post-translational modifications

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showed that this secreted tau is composed predominantly of four N-terminal fragments,

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all containing a N-terminal acetylated alanine and a low percentage (

Human secreted tau increases amyloid-beta production.

The interaction of amyloid-beta (Aβ) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence ind...
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