Alzheimer's disease-related amyloid-β induces synaptotoxicity in human ips cell-derived neurons

ABSTRACT Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease

(AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-_β_ (A_β_). However, none of the present

AD iPSC studies could recapitulate the synaptotoxic actions of A_β_, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly

reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of A_β_. We developed a highly efficient immunopurification procedure yielding

immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons

exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible

to the synaptotoxic actions of A_β_. Application of A_β_ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle

clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that A_β_ impaired axonal vesicle clusters. In addition,

electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an A_β_-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA

receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with A_β_ for 8 days did not result in a significant loss of

neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of A_β_-induced

synaptic pathomechanisms and the development of novel therapeutic approaches. SIMILAR CONTENT BEING VIEWED BY OTHERS PROTEIN AGGREGATION AND CALCIUM DYSREGULATION ARE HALLMARKS OF FAMILIAL

PARKINSON’S DISEASE IN MIDBRAIN DOPAMINERGIC NEURONS Article Open access 24 November 2022 HIGH-CONTENT IMAGE-BASED ANALYSIS AND PROTEOMIC PROFILING IDENTIFIES TAU PHOSPHORYLATION INHIBITORS

IN A HUMAN IPSC-DERIVED GLUTAMATERGIC NEURONAL MODEL OF TAUOPATHY Article Open access 23 August 2021 TAU DEPLETION IN HUMAN NEURONS MITIGATES AΒ-DRIVEN TOXICITY Article Open access 15

February 2024 MAIN In Alzheimer's disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.1 Oligomers of the amyloid-_β_ (A_β_) peptide appear to induce

synaptic failure as an early event in the etiology of AD.2, 3, 4 However, despite its well-established synapse-impairing effects in rodent models,5, 6, 7 the synaptotoxic actions of A_β_

most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of A_β_ in cultured rodent neurons and in

transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic A_β_ actions result in the loss of functional

(_α_-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,8, 9, 10 involve long-term depression-like mechanisms,9, 11, 12 and lead to the degradation of the

entire postsynapse (dendritic spines).9, 11, 13 In addition, several distinct presynaptic A_β_ actions on the synaptic vesicle cycle have been described.10, 14 Furthermore, A_β_-induced

impairments of axonal transport regulation and A_β_-induced axon degeneration have been found in rodent neurons.15, 16, 17 This puzzling diversity of A_β_-induced synapse-related defects

raises the question whether all of them are involved in the early pathomechanisms of human AD. In addition to well-established animal systems, the modelling of human neurological disease

pathologies by human induced pluripotent stem cell (hiPSC) technology18 has been proposed as an innovative approach.19, 20, 21 The _in vitro_ differentiation of hiPSCs to excitable neurons

has been reported using a variety of protocols.22, 23, 24 However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.19, 25, 26 In

addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the _in vitro_ differentiation of patient-derived iPSCs has been used to model complex

neurodevelopmental and neurodegenerative diseases.19, 27, 28 Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of A_β_ upon _in vitro_ neuronal

differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.21, 29, 30, 31, 32, 33 Here we describe a hiPSC-based, carefully optimized _in vitro_

differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of A_β_ on human cortical neurons and on human synapses.

RESULTS NEURAL DIFFERENTIATION OF HIPSCS AND IMMUNOPURIFICATION OF HIPSC-DERIVED IMMATURE NEURONS hiPSCs were cultured (Supplementary Figure S1) and _in vitro_ differentiated using an

embryoid body (EB) system similar to published protocols.22 After initial differentiation, EBs were plated on a matrigel substrate leading to the formation of paired box protein 6

(Pax6)-expressing neuroepithelial rosettes (Supplementary Figure S2) that further differentiated to heterogeneous cultures also containing non-neuronal cells (Figures 1a and b). After 6–8

weeks of _in vitro_ differentiation, heterogeneous cultures were dissociated to single cells, which were subjected to immunopurification. Classical immunopanning34 with specific

modifications was performed using the neural cell adhesion molecule (NCAM) antibody VIN-IS-53 to isolate immature neurons expressing NCAM at a high level. To quantify immunopanning

efficiency, dissociated cells without immunopanning (control), dissociated cells isolated by NCAM immunopanning, and dissociated cells non-adherent to the panning plates, respectively

(Figure 1c), were immunocytochemically stained for NCAM and the neuronal marker microtubule-associated protein 2 (MAP2) 1 day after immunopurification (Figures 1d and e). The fraction of

MAP2-positive cells was strongly increased in cells isolated by NCAM immunopanning (91.2±4.3%) as compared with control cells (28.1±20.6%) and to cells non-adherent to the panning plates

(12.2±7.4%) (Figure 1g). The fraction of NCAM-positive cells was also increased by immunopanning (Figure 1f); however, as expected from the low level NCAM expression in neural precursor

cells, the increase was less pronounced as compared with MAP2. We next characterized the immunopurified immature neurons using immunocytochemistry. Staining for cortical marker proteins

revealed that the vast majority of MAP2-positive cells expressed markers of deep layer cortical neurons (Ctip2 (chicken ovalbumin upstream promoter transcription factor-interacting protein

2), Tbr1 (T-box, brain, 1)), while only 5.0±1.4% of the MAP2-positive neurons expressed the upper layer marker special AT-rich sequence-binding protein 2 (Satb2; Figures 1h and i). Similar

to the composition of neuronal cell types in the _in vivo_ cortex, 15.7±1.7% of the MAP2-positive neurons were GABAergic (glutamic acid decarboxylase 67 (GAD67) positive) (Figures 1h and i).

Survival of immature neurons was not affected by the immunopanning procedure (Figure 1c). In summary, NCAM immunopanning of hiPSC-derived heterogeneous cultures resulted in highly purified

MAP2-positive immature deep-layer cortical neurons. MORPHOLOGICAL MATURATION AND ACTION POTENTIAL GENERATION IN HIPSC-DERIVED NEURONS PURIFIED BY IMMUNOPANNING Further cultivation of the

MAP2-positive immature human neurons led to the formation of extended neurites after 1 week and to the formation of a dense neuritic network of mature neurons exhibiting enlarged somata at 8

weeks after immunopurification (Figure 2a). These cultures contained only very few glial cells due to the inhibition of proliferation of non-neuronal cells (see Materials and Methods). To

study whether morphologically maturated iPSC-derived human neurons exhibit essential functional properties typical of cultured cortical neurons, we performed a basic electrophysiological

characterization. Whole-cell patch-clamp recordings (at 8 weeks after immunopurification) of the membrane potential (57.8±0.7 mV resting potential) in current-clamp mode revealed the

generation of action potentials upon injection of depolarizing current in all neurons tested (Figure 2b). To demonstrate the expression of voltage-dependent Na+ currents, we did whole-cell

recordings in voltage-clamp mode at a holding potential of −60 mV. Step depolarizations of the membrane potential elicited typical inward Na+ currents that were blocked by addition of

tetrodotoxin (TTX; 1 _μ_M; Figure 2c). Thus, electrophysiological analysis revealed essential functional properties such as electrical excitability and TTX-sensitive voltage-dependent Na+

currents in hiPSC-derived neurons at 8 weeks after immunopurification. CHARACTERIZATION OF FUNCTIONAL SYNAPSES IN MATURE HIPSC-DERIVED NEURONS PURIFIED BY IMMUNOPANNING At this stage of

maturation, the formation of synaptic structures was indicated by vesicle clusters immunocytochemically stained for the synaptic vesicle-associated proteins VAMP2 (vesicle-associated

membrane protein 2)/synaptobrevin and synapsin I on dendrites (Figures 3a and b). The presence of both glutamate and γ-aminobutyric acid (GABA) containing presynaptic vesicle clusters was

confirmed by a punctate immunostaining on dendrites for the vesicular glutamate transporter 1 (vGlut1, colocalized with postsynaptic postsynaptic density protein 95 (PSD95) puncta) and for

the vesicular GABA transporter (vGAT), respectively (Figures 3c and d). Moreover, the formation of functional synapses was indicated by spontaneous miniature postsynaptic currents (mPSCs)

that were observed by whole-cell patch-clamp recording at −60 mV holding potential (Figures 3e and f). We further characterized spontaneous mPSCs pharmacologically by using specific

antagonists. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) were isolated by addition of gabazine (10 _μ_M) and TTX (1 _μ_M), and were blocked by the AMPA

receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 _μ_M; Figure 3e). GABAA receptor-mediated mPSCs were isolated by addition of DNQX and TTX and were blocked by addition of

gabazine (Figure 3f). This demonstrates the presence of both functional glutamatergic and GABAergic synapses. Taken together, our findings demonstrate that at 8 weeks after

immunopurification our iPSC-derived human neurons exhibit essential functional properties, such as excitability, synaptic activity, and functional glutamatergic and GABAergic synapses. A_Β_

INDUCED LOSS OF CYCLING VESICLE CLUSTERS IN IPSC-DERIVED HUMAN NEURONS To investigate whether functional synapses in iPSC-derived human neurons are susceptible to the deleterious effects of

A_β_, we added A_β_ contained in the supernatant from cultures of 7PA2 Chinese hamster ovary (CHO) cells (expressing human APP751 carrying the familial amyloid precursor protein (APP) V717F

mutation)5, 35, 36 to hiPSC-derived neurons at 8 weeks after immunopurification. Conditioned medium from 7PA2 cells was diluted 1:1 with standard culture medium.37 To test for potential

unspecific effects of the 7PA2 supernatant, we immunodepleted A_β_ from the 7PA2 conditioned medium by using the anti-A_β_ monoclonal antibody IC16.37, 38 A_β_-induced defects were analysed

8 days after A_β_ application. First, we studied the effects of A_β_ on cycling synaptic vesicle clusters in the processes of iPSC-derived human neurons. Cycling vesicle clusters were

stained by the fluorescent dye FM4-64 (_N_-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide).39 Uptake of extracellularly added FM4–64 (10 _μ_M) in

recycling vesicles was stimulated by triggering the exocytosis and endocytosis of synaptic vesicles with extracellular electrical stimulation. After removal of extracellular FM4–64 dye,

FM4–64-stained vesicle clusters were imaged by fluorescence microscopy (Figure 4a). For each FM4–64 staining experiment, the number of fluorescent puncta (i.e., vesicle clusters) was

determined after thresholding the FM4–64 fluorescence image (see Figure 4a) and was multiplied with the mean total fluorescence intensity per punctum, resulting in an overall FM4–64

fluorescence signal per area of neuropil. Strikingly, 8 days after addition of A_β_, we observed a strong, significant reduction in the overall FM4–64 fluorescence signal (Figures 4a and b)

indicating a toxic effect of A_β_ on recycling vesicle clusters. Addition of immunodepleted 7PA2 supernatant did not affect the overall FM4–64 fluorescence signal demonstrating a specific

effect of A_β_ contained in the 7PA2-conditioned medium. The A_β_-induced reduction of the overall FM4–64 fluorescence signal was largely due to a reduction in the number of FM4–64-stained

puncta (vesicle clusters) per area of neuropil as indicated by plotting FM4–64 puncta density _versus_ the mean total fluorescence intensity per punctum (Figure 4b). We also studied the

destaining of FM4–64-stained vesicle clusters by triggering vesicle re-exocytosis with extracellular electrical stimulation (1200 stimuli at 20 Hz (for 60 s)); Figure 4c). The mean

destaining kinetics of FM4–64 puncta were not significantly affected by addition of A_β_, thus further supporting that a reduction in the number of vesicle clusters was the major

A_β_-induced alteration. To further confirm an A_β_-induced impairment of vesicle clusters, we immunocytochemically stained iPSC-derived human neurons for the synaptic vesicle marker vGlut1

after incubation with A_β_. Again, we observed a significant reduction in the density of vGlut1 immunopositive puncta (vesicle clusters) in neurites of A_β_-treated neurons as compared with

vehicle-treated control cultures (Figures 4d and e). Addition of immunodepleted 7PA2 supernatant did not affect the number of vGlut1 puncta, again demonstrating a specific effect of A_β_

contained in the 7PA2-conditioned medium. To check for A_β_-induced changes in the density of processes and in cell density, we fluorescently stained iPSC-derived human neurons by addition

of the membrane permeable dye calcein-AM directly following the FM4–64 staining/destaining experiments (Figure 4f). Eight days after addition of A_β_, the number of neuronal cell bodies and

the density of processes (determined by counting processes crossing a randomly chosen line) were not significantly affected (Figures 4g and h). In summary, our results indicate that addition

of A_β_ results in a strong reduction in the density of vesicle clusters in the neurites of iPSC-derived human neurons. A_Β_ INDUCED FUNCTIONAL IMPAIRMENT OF GLUTAMATERGIC SYNAPSES IN

IPSC-DERIVED HUMAN NEURONS We next studied whether the addition of A_β_ induced deleterious changes at _bona fide_ synaptic sites contacting postsynaptic dendrites. To focus on presynaptic

vesicle clusters, we reanalysed the FM4–64 puncta (from the FM4–64 staining/destaining experiments described above) that contacted the proximal dendrites of iPSC-derived human neurons.

Proximal dendrites arising from neuron somata were identified by calcein-AM staining of whole neurons after FM4–64 staining/destaining experiments, and FM4–64 puncta were analysed after

thresholding the FM4–64 fluorescence image (Figure 5a). Eight days after addition of A_β_, we did not observe a significant change in the dendritic density of FM4–64 puncta or a significant

change in total FM4–64 fluorescence intensity per punctum or a significant change in the overall presynaptic FM4–64 fluorescence signal per dendrite (Figure 5b). Furthermore, the

quantitative analysis of the stimulation-induced destaining of FM4–64 puncta did not reveal any significant changes in exocytosis (Figure 5c). We also reanalysed the presynaptic vGlut1

immunopositive puncta (described above) located on dendrites, which were identified by MAP2 co-immunostaining of dendrites (Figure 5d). Again, 8 days after addition of A_β_, we observed no

significant changes in the density of vGlut1 puncta on dendrites (Figure 5e). Taken together, the analysis of FM4–64 fluorescent and of vGlut1 immunopositive puncta on dendrites thus

revealed no significant presynaptic alterations at a relatively early phase of incubation with A_β_, suggesting that _bona fide_ presynaptic vesicle clusters and functional release

properties were not affected by short-term application of A_β_. We next addressed whether the deleterious effects of A_β_ on functional properties of glutamatergic synapses, which are well

described in rodent neurons, are also observed in iPSC-derived human neurons. We recorded AMPA receptor-mediated mEPSCs (AMPA mEPSCs) using the whole-cell patch-clamp technique (1 _μ_M TTX,

10 _μ_M gabazine added, holding potential: −60 mV). We again applied A_β_ containing supernatant of the 7PA2 CHO cell line at 8 weeks after immunopurification and studied its effects on AMPA

mEPSCs 8 days later. Addition of A_β_ resulted in a strong, significant reduction of the mean amplitude of AMPA mEPSCs (Figures 6a and b), indicating a loss of postsynaptic AMPA receptor

function. This amplitude reduction was not observed upon addition of 7PA2 supernatant immunodepleted of A_β_, demonstrating a specific A_β_ action. Addition of A_β_ did not lead to a

significant reduction of the mean frequency of AMPA mEPSCs (Figures 6a and b). The observed trend to a reduced AMPA mEPSC frequency is likely to be caused by limitations in the detection of

small amplitude minis. To further confirm an A_β_ induced impairment of postsynaptic AMPA receptors, we performed immunocytochemical stainings for GluA1(GluR1) (AMPA receptor subunit 1),

PSD95, and MAP2 (to visualize dendrites) in iPSC-derived human neurons. Eight days addition of A_β_ resulted in a significant reduction of the density of GluA1 immunopositive puncta on

MAP2-immunostained dendrites (Figures 6c and d). Furthermore, the density of synaptic GluA1 immunopositive puncta (colocalized with PSD95 puncta) was also significantly reduced 8 days after

A_β_ application (Figure 6d). The dendritic density of PSD95 immunopositive puncta was not significantly altered (Figures 6c and d). These A_β_-induced changes were not detectable upon

addition of 7PA2 supernatant immunodepleted of A_β_, again demonstrating a specific A_β_ action. Taken together, the observed A_β_-induced reduction of AMPA mEPSC amplitudes and of dendritic

GluA1 expression indicates an early action of A_β_ on postsynaptic AMPA receptors in iPSC-derived human neurons. Interestingly, as indicated by the lack of A_β_ effects on vesicle clusters

on dendrites, an early A_β_-induced presynaptic impairment was not detectable. A_Β_-INDUCED EFFECTS ON CELL SURVIVAL, CELLULAR STRESS AND TAU PROTEIN IN HIPSC-DERIVED NEURONS To study

whether our short-term application of A_β_ compromises cell survival, we stained healthy neurons and nuclei of disrupted cells, respectively by performing a live/dead assay (see Materials

and Methods). Healthy neurons were fluorescently stained by addition of membrane permeable calcein-AM, which is intracellularly converted to fluorescent calcein. Nuclei without an intact

plasma membrane were stained by Ethidium Homodimer 1 (Figure 7a). At 8 days of incubation, we did not observe significant effects of the addition of A_β_ on cell survival (Figure 7b).

Because A_β_-induced cellular stress might be detectable prior to cell death, we studied the expression of marker proteins for endoplasmic reticulum (ER) stress and autophagy by western

blotting analysis. Interestingly, we found a slight A_β_-induced (8 days incubation) increase in the autophagy marker microtubule-associated protein 1A/1B-light chain II (LC3-II) and in the

ER stress markers binding immunoglobulin protein (BiP) and CCAAT-enhancer-binding protein homologous protein (CHOP) (Figures 7c and d). This is in line with the recent description of

increased ER stress in AD patient-derived iPSC lines upon neuronal differentiation.40 We further analysed A_β_-induced changes in tau protein expression and phosphorylation state by western

blotting. Although the expression level of tau protein appeared not to be affected by short-term application of A_β_ (8 days incubation), an increased phosphorylation of tau was clearly

detectable (Figures 7e and f). This finding in human neurons is in line with the A_β_-induced phosphorylation of tau protein, which has been described in mouse models previously.4, 41 In

summary, our results indicate that at an early phase of A_β_ actions defects in synapse function are clearly revealed in iPSC-derived human neurons by changes in postsynaptic AMPA receptors.

In addition, A_β_-induced alterations in neuritic vesicle clusters, in ER stress/autophagy marker proteins, and in tau protein phosphorylation were found. Our work thus establishes that

iPSC-derived human neurons represent an innovative model system to study the molecular mechanisms of A_β_-induced synapse damage in AD. DISCUSSION In this study, we used hiPSCs to establish

an innovative cell culture model of cortical neurons for specific aspects of AD. We analysed the effects of A_β_ on human neurons, human synapses, and synaptic vesicle clusters. We observed

a loss of axonal vesicle clusters, a loss of postsynaptic AMPA receptors, and increased tau protein phosphorylation, thus revealing complex deleterious actions of A_β_. _IN VITRO_

DIFFERENTIATION OF HUMAN IPSCS AND IMMUNOPURIFICATION OF IPSC-DERIVED IMMATURE HUMAN NEURONS _In vitro_ differentiation of human pluripotent stem cells to neurons has been established by

several groups.22, 23, 42 Without addition of specific morphogens/growth factors,43 iPSCs differentiate to dorsal, cortical-like neuronal progenitor cells characterized by Pax6

expression;20, 22, 44 however, other contaminating cell types are also generated. The invention of immunopanning protocols34 enables the highly efficient purification of specific types of

neuronal precursors, neurons, or astrocytes.45, 46, 47 Similarly, the presence of NCAM has been used for selecting neuronally differentiated cells.48, 49 Here we have developed an

immunopanning procedure based on NCAM antibodies, which yielded >90% MAP2-positive immature neurons. Immunocytochemical characterization of the purified neurons revealed typical

cortical-like cultures consisting of mainly glutamaterigc and about 20% GABAergic neurons. FUNCTIONAL MATURATION OF HUMAN IPSC-DERIVED IMMATURE NEURONS, INCLUDING SYNAPSE FORMATION In line

with our results, it has been shown that neurons derived from human iPSCs generate action potentials and exhibit voltage-activated Na+ and K+ currents.19, 24, 44, 50 In addition, our

iPSC-derived human neurons fired repetitive action potentials reminiscent of the firing pattern of regular spiking cortical pyramidal neurons.24 Similar to primary cultured rodent neurons,51

the formation of functional synapses has also been observed in human iPSC-derived neurons; however, reliable synapse formation and function appears to be difficult to achieve.19, 25, 26

Importantly, our iPSC _in vitro_ differentiation protocol resulted reliably in the formation of both functional glutamatergic and GABAergic synapses, thus leading to synaptically active

networks. SYNAPTOTOXIC EFFECTS OF A_Β_ IN HUMAN IPSC-DERIVED NEURONS We used our innovative model system for studying synaptic aspects of AD. Oligomeric A_β_ peptides are thought to be

crucial molecular entities in AD1, 2, 3, 5, 52 and exhibit—in rodent neurons—toxic effects at glutamatergic synapses leading ultimately to synapse loss.8, 11, 37, 52, 53, 54, 55, 56 The

initial deleterious actions of A_β_ on the function of glutamatergic synapses might be specifically localized either presynaptically or postsynaptically. In rodent neurons, an initial

A_β_-induced removal of postsynaptic AMPA receptors by facilitating AMPA receptor endocytosis has been described.9, 10, 12 Furthermore, A_β_ disrupts membrane trafficking and synaptic

recruitment of AMPA receptors by reducing surface expression.57 In line with an initially postsynaptic mechanism, we found a pronounced A_β_-induced reduction in both the amplitudes of

AMPAR-mediated mEPSCs and in postsynaptic AMPA receptor clusters in hiPSC-derived neurons. In addition, also presynaptic mechanisms of action of A_β_ affecting both exocytosis and

endocytosis have been proposed in rodent neurons.10, 14 However, our analysis of both FM-stained and immunocytochemically stained vesicle clusters apposed to dendrites in hiPSC-derived

neurons did not reveal any short-term presynaptic effects of A_β_, thus indicating that A_β_-induced alterations in postsynaptic AMPA receptors might be of crucial importance in the human

disease. In addition to A_β_-induced synaptotoxicity, AD is characterized by the formation of neurofibrillary tangles and tau hyperphosphorylation.58 A_β_-induced tau phosphorylation4, 13,

41 has been proposed to be an early event leading to the formation of neurofibrillary tangles typical of tauopathy.41 In line with familial AD patient-derived iPSC-derived neurons,33 our

data demonstrate that A_β_-induced tau phosphorylation occurs in hiPSC-derived neurons after short-term application of A_β_. Furthermore, we observed a slight A_β_-induced increase in

proteins related to the ER stress-induced unfolded protein response (UPR). UPR includes enhanced expression of chaperones such as BiP, increased degradation of misfolded proteins, and

activates autophagy.59 Failure of this system to restore ER homeostasis initiates apoptotic mechanisms. In line with our results, A_β_ oligomers have been described to induce the UPR.60

EFFECTS OF A_Β_ ON VESICLE CLUSTERS IN HIPSC-DERIVED NEURONS Intriguingly, upon A_β_ application we observed a reduced density of non-synaptic axonal vesicle clusters, which are well known

to undergo exocytosis and endocytosis.61, 62 Because we did not observe any A_β_-induced changes in the FM destaining kinetics (reflecting exocytosis) and in the FM fluorescence intensity

(reflecting endocytosis), an A_β_-induced impairment of the axonal transport of vesicles without axon degeneration appeared to underlie the reduction in the density of axonal vesicle

clusters. In line with this idea, in rodent neurons an impairment of the axonal transport by A_β_ has been observed.15, 16, 17 A_β_-induced defects in the transport of organelles, for

example, mitochondria,63 and in the transport of dense core and synaptic vesicles63, 64 without axon degeneration have been described previously. Thus our results in iPSC-derived human

neurons strongly support an important role of an A_β_-induced impairment in axonal transport of synaptic vesicles at early stages of the human disease. CONCLUSION: HUMAN IPSC DERIVED NEURONS

AS AN INNOVATIVE MODEL SYSTEM TO STUDY TOXIC EFFECTS OF A_Β_ Here we have established hiPSC-derived neurons as an innovative AD model system to study the effects of A_β_. Toxic effects of

A_β_ were complex, including both non-synaptic effects on axonal vesicles and _bona fide_ postsynaptic impairment of AMPA receptors. In addition, an A_β_ induced increase in tau protein

phosphorylation was found. Most importantly, our iPSC-derived human model system will aid the development of novel AD therapeutics based on the investigation of the molecular mechanisms of

deleterious A_β_ actions in human neurons. MATERIALS AND METHODS HIPSC CULTURE AND NEURAL DIFFERENTIATION Experiments were approved by the local ethics committee of the University of

Düsseldorf. Human iPSC line 8/25 (ref. 65) and in some experiments human iPSC line DF6–9–9T.B (WiCell, provided by Dr. J Thomson, University of Wisconsin66) were cultured according to

standard procedures in mTeSr medium (STEMCELL Technologies, Köln, Germany) or in E8 medium67 on hESC qualified matrigel (Corning, Corning, NY, USA) coated cell culture six-well plates

(Sarstedt, Nürnbrecht, Germany). Neural differentiation was performed according to a modified protocol of Li _et al._22 EBs were allowed to form in T25 cell culture flasks (Sarstedt) in

N2B27 medium containing 50% DMEM/F12 (Biochrom, Berlin, Germany), 50% Neurobasal, 2 mM Glutamax, 100 units/ml penicillin and 100 _μ_g/ml streptomycin, 0.1 mM _β_-mercaptoethanol, 1% B27

without vitamin A (all Gibco, Darmstadt, Germany), 3 _μ_g/ml heparin (Sigma Aldrich, St. Louis, MO, USA), and a modified N2 supplement with final concentrations of 7.5 _μ_g/ml human insulin,

50 _μ_g/ml BSA, 8 _μ_g/ml putrescine, 3.1 ng/ml progesterone, 2.6 ng/ml sodium selenite (all Sigma Aldrich), and 25 _μ_g/ml human holo-transferrin (Calbiochem, Darmstadt, Germany). After 14

days of suspension culture, EBs were plated on 100-mm cell culture dishes coated with growth factor reduced matrigel (Corning) in N2B27 medium. Six-to-8 weeks after starting

differentiation, heterogeneous cultures were harvested for immunopanning by scraping in DPBS without calcium and magnesium (PanBiotech, Aidenbach, Germany). IMMUNOPANNING OF HIPSC-DERIVED

IMMATURE NEURONS AND NEURONAL MATURATION Panning plates (100 mm, bacterial petri dish quality; Falcon/Corning, Darmstadt, Germany) were prepared by incubating goat anti mouse IgG+IgM

antibody (Jackson Immunoresearch, Suffolk, UK) at 10 _μ_g/ml in 9 ml of 50 mM Tris HCl/pH 9.5 overnight at 4 °C. After three washing steps with DPBS, the panning plates were incubated

overnight at 4 °C with the primary mouse anti-NCAM antibody (VIN-IS-53, kindly provided by Dr. Peter Andrews, University of Sheffield, UK) (0.55 _μ_g/cm2 in 0.2% BSA Fraction V (Gibco)/DPBS,

7 ml per plate). Plates were washed twice with DPBS and once with 0.02% BSA. Cells harvested from one 100-mm differentiation plate were enzymatically digested in accutase (Sigma Aldrich)

for 30 min. After enzyme removal, cells were dissociated in 0.2% BSA in DPBS supplemented with 1 mg/ml glucose (Sigma Aldrich) and 0.33 mM sodium pyruvate (Gibco), pelleted at 200 g,

resuspended in 0.02% BSA plus glucose and sodium pyruvate, and distributed to four panning plates (7 ml per plate). Cells were incubated on the panning plates for 10 min at RT. Non-adherent

cells were removed by washing 10 times with DPBS. Adherent cells were detached by repeated pipetting (P1000, Eppendorf, Wesseling-Berzdorf, Germany) and rinsing of the plates with 0.02% BSA.

Cells were pelleted at 130 × _g_ for 10 min. Twenty-five thousand cells were plated on PO (1 mg/ml in borate buffer, coating overnight at RT) and laminin (10 mg/ml, coating 3 h at 37 °C)

coated coverslips (12 mm, Assistant, Sondheim/Rhön, Germany) in a 10-_μ_l drop of neuronal culture medium, that is, Neurobasal, 2 mM Glutamax, 1 mM Sodium pyruvate, 100 units/ml penicillin,

and 100 _μ_g/ml streptomycin (all Gibco), 2% NS21 supplement,68 25 _μ_g/ml human insulin, 100 _μ_g/ml BSA, 16 _μ_g/ml putrescine, 62 ng/ml progesterone, 40 ng/ml sodium selenite (all Sigma

Aldrich), 50 _μ_g/ml human holo-transferrin (Calbiochem), and 30 ng/ml BDNF (Peprotech, Hamburg, Germany). Two millilitres of medium was added after neurons had attached (30 min at 37 °C).

To inhibit proliferation of neural stem cells, cultures were treated with 10 _μ_M cytosine _β_-D-arabinofuranoside hydrochloride (Sigma Aldrich) for the first 5 days. After removal of the

mitotic inhibitor, half of the medium was changed once per week over a period of 2–3 months. Evaporation was compensated for by the addition of aqua bidest (Sigma Aldrich). A_Β_ CONTAINING

7PA2-CONDITIONED MEDIUM AND A_Β_ IMMUNODEPLETION All procedures were carried out as described.37 In brief, conditioned neuronal culture medium containing cell-secreted A_β_ was obtained from

a CHO cell line (7PA2) that expresses mutant human APP751 (V717F).35, 36 As determined by ELISA (A_β_40), 7PA2-conditioned medium contained A_β_ at a concentration of 25–50 ng/ml. A_β_

containing 7PA2-conditioned medium was immunodepleted with anti-A_β_ antibody (IC16)38 coupled to NHS-Sepharose (GE Healthcare, Chalfont St Giles, UK) by overnight incubation at 4 °C

followed by centrifugation. Depletion of A_β_ was confirmed by immunoblotting. IMMUNOCYTOCHEMISTRY AND IMMUNOBLOTTING Immunocytochemistry was carried out according to standard protocols, and

quantification of images was carried out as described previously.37 Antibodies used are listed in Supplementary Table S1. DAPI (10 _μ_g/ml) was used to stain nuclei. Immunoblotting was

performed according to standard protocols as described previously.69 Briefly, cells were scraped in 2 × loading buffer (200 mM Tris/HCl pH 6.8, 5% SDS, 20% glycerol, 0.04% bromphenol blue,

200 mM DTT), boiled for 5 min and centrifuged at 16.000 × _g_ for 3 min. Lysates of different samples were adjusted for GAPDH content after analyzing triplicates by SDS-PAGE and

immunoblotting. Adjusted samples were separated by SDS-PAGE (12%, Tris-Glycine) and blotted on nitrocellulose membranes. Immunoblotting and protein detection was performed according to

standard procedures. Membranes were blocked with 10% Roti-Block (Carl Roth, Karlsruhe, Germany) in Tris-buffered saline with 0.1% Tween 20. Proteins of interest were visualized using Western

Bright Quantum ECL (Biozym, Hessisch Oldendorf, Germany) and the Chemi Doc XRS System (Biorad, Munich, Germany). CALCEIN STAINING, FLUORESCENCE IMAGING, AND ANALYSIS OF CELL DEATH For

staining of healthy cells, we incubated cells with the calcein-green component (1/2000) of the LIVE/DEAD Kit (Molecular Probes, Darmstadt, Germany) for 10 min at 37 °C followed by

immunocytochemistry (or direct imaging), which was performed according to standard procedures.37 Fluorescence imaging was carried out on a Zeiss Axiovert 200 M inverted fluorescence

microscope (Zeiss, Oberkochen, Germany) equipped with a 12-bit monochrome CoolSNAP ES2 CCD camera (Photometrics, Tucson, AZ, USA) using the MetaVue and Metamorph (analysis) software

(Visitron Systems, Puchheim, Germany). A_β_-induced cell death was studied by using the LIVE/DEAD Kit (Molecular Probes) according to the manufacturer’s instructions. ELECTROPHYSIOLOGY AND

DATA ANALYSIS Whole-cell patch-clamp recordings were carried out using an EPC7 patch-clamp amplifier (HEKA, Ludwigshafen/Rhein, Germany) and pCLAMP software (Molecular Devices, Sunnyvale,

CA, USA) as described.37, 70 Intracellular solution contained 110 mM KCl, 0.25 mM CaCl2, 10 mM EGTA, and 20 mM HEPES, pH 7.3. The extracellular solution contained 130 mM NaCl, 5 mM KCl, 2.5 

mM CaCl2, 1 mM MgCl2, and 20 mM HEPES, pH 7.3. Action potentials were elicited by depolarizing current injection in current-clamp mode (membrane potential −50 to −60 mV). Voltage-dependent

currents (elicited by step depolarizations) and mPSCs were recorded in voltage-clamp mode (−60 mV holding potential). Quantitative analysis of AMPA mEPSCs was carried out using the Mini

Analysis software (Synaptosoft, Decatur, GA, USA). FLUORESCENCE IMAGING OF CYCLING VESICLE CLUSTERS Cycling vesicle clusters were fluorescently stained by FM4–64 uptake.39 FM4–64 (10 _μ_M)

was added to the extracellular solution (containing in mM: 119 NaCl, 2,5 KCl, 2 CaCl2, 2 MgCl2, 25 HEPES, 30 glucose, pH=7.4), and extracellular stimulation (400 stimuli at 20 Hz) was used

to elicit exocytic/endocytic cycling of vesicles and FM4–64 uptake. Sixty seconds after stimulation, extracellular FM4–64 was removed by washing with extracellular solution (0 mM CaCl2 with

addition of DNQX (10 _μ_M) and D-AP5 (50 _μ_M); 2 min wash, then 2 min wash in the presence of ADVASEP-7 (1 mM), and then 10 min wash), and fluorescent FM4–64 puncta were imaged. For

studying exocytosis of FM4–64 stained vesicles, destaining of FM4–64 puncta was elicited by extracellular electrical stimulation (1200 stimuli at 20 Hz (for 60 s)). After 100 s of

destaining, a second stronger stimulation (2000 stimuli at 20 Hz without imaging (for 100 s)) was carried out to reach background fluorescence levels. Quantitative image analysis was carried

out by defining regions of interest (ROI) around FM4–64 puncta. Total fluorescence intensity was determined for each FM4–64 punctum using the Metamorph software (Molecular Devices). Total

background intensity was determined after the second stimulation within the same ROI and was subtracted from the total fluorescence intensity within the ROI to obtain the total FM4–64

fluorescence signal per punctum. Decay time constants of FM4–64 destaining were determined by monoexponentially fitting (SigmaPlot11 software, Systat Software Inc, San Jose, CA, USA) the

mean fluorescence decay after averaging the normalized (intensity at the start of stimulation was set to 100%) destaining curves of FM4–64 puncta obtained from a field of view (Figure 4) or

a proximal dendrite (Figure 5). STATISTICS All data are given as means±S.E.M. Results were statistically analysed using one-way ANOVA with Holm–Sidak _posthoc_ test (SigmaPlot11 software).

ABBREVIATIONS * A_β_: amyloid-_β_ * AD: Alzheimer's disease * AMPA: _α_-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid * APP: amyloid precursor protein * BiP: binding immunoglobulin

protein * CHO: Chinese hamster ovary * CHOP: CCAAT-enhancer-binding protein homologous protein * Ctip2: chicken ovalbumin upstream promoter transcription factor-interacting protein 2 *

DNQX: 6,7-dinitroquinoxaline-2,3-dione * EB: embryoid body * EPSC: excitatory postsynaptic current * ER: endoplasmic reticulum * FM4–64: _N_-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)

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density protein 95 * Satb2: special AT-rich sequence-binding protein 2 * Tbr1: T-box, brain, 1 * TTX: tetrodotoxin * UPR: unfolded protein response * VAMP2: vesicle-associated membrane

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Scholar  Download references ACKNOWLEDGEMENTS We thank Dr. PW Andrews for providing hybridoma supernatant containing NCAM antibody, and Dr. H Zaehres, Dr. H Schöler, Dr. P Wernet, and Dr. G

Kögler for providing human iPSCs (USSC-iPSCs line 08/25). We also thank Dr. DJ Selkoe for providing 7PA2 CHO cells, and Dr. C Korth and Dr. A Müller-Schiffmann for providing IC16 antibody

for A_β_ immunodepletion. We further thank M Bohndorf for excellent technical assistance. This work was supported by the Forschungskommission (No. 52/2011), Medical Faculty,

Heinrich-Heine-University Düsseldorf and the Strategischer Forschungsfonds, Heinrich-Heine-University Düsseldorf. AUTHOR INFORMATION Author notes * A Andreyeva and B van Stegen: These

authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Institute of Neuro- and Sensory Physiology, Medical Faculty, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany K

Nieweg, A Andreyeva, B van Stegen & K Gottmann * Institute of Pharmacology and Clinical Pharmacy, Phillips University, Marburg, Germany K Nieweg & G Tanriöver Authors * K Nieweg View

author publications You can also search for this author inPubMed Google Scholar * A Andreyeva View author publications You can also search for this author inPubMed Google Scholar * B van

Stegen View author publications You can also search for this author inPubMed Google Scholar * G Tanriöver View author publications You can also search for this author inPubMed Google Scholar

* K Gottmann View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHORS Correspondence to K Nieweg or K Gottmann. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by A Verkhratsky Supplementary Information accompanies this paper on Cell Death and Disease

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amyloid-_β_ induces synaptotoxicity in human iPS cell-derived neurons. _Cell Death Dis_ 6, e1709 (2015). https://doi.org/10.1038/cddis.2015.72 Download citation * Received: 02 August 2014 *

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