Neuron-to-astrocyte proteostatic stress signaling in response to tau pathology

Kevin Llewelyn Batenburg,Wiep Scheper

Maintenance of protein homeostasis or “proteostasis” is essential for the functioning and viability of cells.This is in particular the case for cells like neurons that cannot self-renew and acquire unique functional properties during their lifetime.Cellular proteostatic stress responses are in place to protect cells from damage in case of proteostatic challenges.The integrated stress response (ISR) is one of the key proteostatic stress responses in the cell (Costa-Mattioli and Walter,2020).The ISR is the downstream convergence point for the four stress-induced eIF2α kinases(EIF2AK1–4) that control stress-regulated protein translation via phosphorylation of the translation factor eIF2α.ISR activation results in a transient reduction of global translation while it concomitantly enhances the translation of specific mRNAs,including that encoding the activating transcription factor 4 (ATF4).Together,the translational control mediated by the ISR results in a temporary reduction of the overall protein load and the selectively increased expression of proteins that contribute to restoration of the proteostatic balance.

Neurodegenerative proteinopathies are characterized by the accumulation of aggregated proteins,indicating a major disturbance of intraneuronal proteostasis.Aggregation of tau protein is the key pathological hallmark of neurodegenerative tauopathies,which include Alzheimer’s disease (AD) and a subset of frontotemporal dementias.Because tau is highly enriched in neurons,the pathological tau accumulations are predominantly present in neurons,although in primary tauopathies different extents of tau pathology can be observed in glial cells as well.Tau aggregation results in a direct cell autonomous disruption of neuronal physiology and function.In addition,glial cells are affected by neuronal tau pathology in a cell non-autonomous manner.Astrocytes are the most abundant glial cells in the brain and play crucial roles in the support of neuronal health and function.Astrocytes are increasingly recognized as important contributors to tau-mediated neurodegeneration via a loss of homeostatic function and/or gain of toxic function (Sidoryk-Wegrzynowicz and Strużyńska,2021).

We have recently developed a human induced pluripotent stem cell derived neuron/astrocyte co-culture model for tau pathology (Batenburg et al.,2023a).We used spontaneously aggregating 2N4R tau that forms progressive intraneuronal tau pathology without the addition of exogenous seeds that present a confounding factor for the study of cell non-autonomous responses.We showed the utility of the model for the study of tau-induced neuron-astrocyte signaling and demonstrated the cell non-autonomous accumulation of reactive oxygen species (ROS) and activation of the ISR in astrocytes.In our human co-culture,we showed a direct causal relation between intraneuronal tau aggregation and the astrocytic responses.In addition,we show that this is an early pathogenic response preceding neuronal loss that does not require the presence of microglia or other cell types and suggests the transmission of a factor from neurons with tau pathology.

The cell non-autonomous signaling in our coculture model is induced by tau pathology,but we find no evidence that the secretion of tau itself is involved in the transfer.We hypothesize that the proteostatic disturbance induced by tau aggregation elicits the release of a stress signal from the neurons.This may be one or more protein(s),but can potentially also involve an RNAor lipid-based signal.Proteostatic stress induced by protein aggregation and the activation of stress responses changes the secretome of neurons.For example,neurons under proteostatic stress show increased secretion of the luminal domain of the ER stress-transducing protein CREB3L2-termed TAILS (transmissible activator of increased cell livability under stress;Chung et al.,2022).Extracellular TAILS promote neuronal survival by enhancing the antioxidant resilience in recipient cells,indicating that it mediates a cell nonautonomous anticipatory response.The signaling effects of TAILS in astrocytes have not been investigated,but at least in neurons,TAILS does not result in ISR activation,which may suggest that the neuron-glia signaling we observe employs a different mechanism.Interestingly,TAILS levels are increased in the AD brain,but whether this is derived from tau-induced neuronal secretion is unknown.Our group recently identified Protein Disulfide Isomerases (PDI) as proteins specifically secreted by neurons during proteostatic stress,including tau aggregation (Wolzak et al.,2023).The downstream effects of PDI secretion by neurons with tau pathology on other cells,including astrocytes,are not yet elucidated,but increased PDI levels are detected in cerebrospinal fluid of AD patients and tau-related frontotemporal dementias,indicating that increased PDI secretion occurs in the brain of patients with tau pathology.

It is possible that the neuron-glia signaling in our model is not specific for tau,but a more generic response to disturbed proteostasis.In this respect,it would be interesting to investigate whether ROS accumulation and ISR activation also occur in astrocytes co-cultured with neurons containing other types of protein inclusions associated with neurodegenerative disease,for example,ɑ-synuclein.An interesting neuron-glia interaction with a potentially tau-selective effect is mediated by the apolipoprotein E (ApoE) variant ApoE4,which is the major genetic risk factor for AD.Recently,it was shown that neuronal ApoE4 is involved in tau-induced signaling to different types of glial cells,including astrocytes (Koutsodendris et al.,2023).Although ApoE in the brain is mainly produced by astrocytes,it was shown that neuronspecific deletion of ApoE4 strongly inhibits astrogliosis in a mouse model for intraneuronal tau pathology.However,it is unknown whether this is a direct effect of ApoE4 secreted by neurons,or whether it is indirect,for example via an effect on tau pathology or by affecting lipid homeostasis.Our induced pluripotent stem cell co-culture model allows the incorporation of neurons and astrocytes with different genetic backgrounds.It will therefore be interesting to assess the involvement of the ApoE genotype and related to that the involvement of lipid homeostasis in the tau-induced neuron-astrocyte ROS and ISR signaling.

Our data indicate that the cell non-autonomous stress responses are not readily transferred via conditioned media.This could imply that the tau-induced astrocytic response requires close proximity to and/or direct contact with neurons.Alternatively,this may be a technical artifact due to dilution and/or lability of factor(s) in conditioned media and needs to be further addressed in future studies.An interesting opportunity may be the neuron-astrocyte co-culture of neuronal tau pathology we recently reported,where the cells are co-cultured in a 3D matrix (Batenburg et al.,2023b).This 3D culture more accurately mimics the neuron-astrocyte interactions in the brain,where the astrocytes are in very close contact with neurons.Moreover,diffusion and dilution of factors are limited by the matrix in this culture,mimicking the brain parenchyma.

We did not investigate which eIF2α kinase is responsible for the ISR activation in astrocytes,but the concomitant ROS accumulation in the astrocytes makes it tempting to speculate that this is the stress trigger.HRI (EIF2AK1) is directly activated by ROS and our recent work shows that it plays a key role in the neuron-specific proteostatic stress response (Wolzak et al.,2022).In addition,indirect activation of other eIF2α kinases has also been reported (Costa-Mattioli and Walter,2020).It is also possible that the ROS accumulation and ISR activation are separate events that co-occur but are not functionally connected.Identification of the eIF2α kinase involved will help to identify the triggering stress signals.Recently,neuronal tau pathology was demonstrated to induce the accumulation of double-stranded RNA in astrocytes.DsRNA is the stress trigger for PKR(EIF2AK2) and it would be interesting to investigate whether double-stranded RNA accumulates in astrocytes in our model and whether PKR is the eIF2α kinase responsible for the ATF4 activation that we observe.In addition to transferring a stress signal,an interesting possibility would be the transfer of ATF4 itself.The transcription factor HIF1A,a master regulator of the cellular response to hypoxia,was recently shown to be transferred intercellularly via exosomes (Ferreira et al.,2022).It is unknown whether ATF4 can be transferred between cells,but such a mechanism would allow the transmission of a transcriptional response to recipient cells in the absence of a direct trigger,as an anticipatory response.A different mechanism to induce a transcriptional program in a cell nonautonomous manner is exemplified by TAILS,which stabilizes the transcription factor Nrf2 in recipient cells (Chung et al.,2022).A schematic overview of potential mechanisms for tau-induced neuron-glia signaling leading to ROS accumulation and ISR activation in astrocytes is presented inFigure 1.

Figure 1｜Possible mechanisms for tau-induced neuron-glia signaling.

Modulation of the ISR is extensively investigated as a therapeutic strategy for the treatment of neurodegenerative disease (Costa-Mattioli and Walter,2020),and exciting proof-ofconcept has been obtained in mouse models for neurodegenerative disease,mostly using inhibition of PERK (EIF2AK3).A postulated mechanism for the protective effect of ISR inhibition is the rescue of prolonged attenuation of global protein synthesis.However,this model has recently been challenged by a study demonstrating that astrocyte-selective ISR intervention is protective against neurodegeneration (Smith et al.,2020).This is in line with observations in the human AD entorhinal cortex,where single-cell RNA profiling demonstrated the ISR/ATF4 pathway as one of the key transcriptional programs activated in astrocytes (Grubman et al.,2019).In addition,our lab demonstrated that stress-induced reduction of global protein synthesis is fully intact in PERKdeficient neurons,but not in astrocytes (Wolzak et al.,2022).These data provide support for astrocytes as the primary therapeutic target of ISR interventions for neurodegenerative disease.

The astrocytic response in our tau co-culture model is observed in the absence of neuronal and synaptic loss.Therefore,we model the cell non-autonomous astrocytic contribution to tau pathology prior to neurodegeneration.ISR activation is known to trigger a neuroinflammatory response and may also activate other initially protective pathways.However,prolonged ISR activation in astrocytes may contribute to neurodegeneration,by prolonged pro-inflammatory signaling or loss of homeostatic function of the astrocytes.Along those lines,astrocytes show reduced secretion of the neurotrophic and synaptogenic factor thrombospondin 1 in a mouse model for intraneuronal tau pathology,which has been implicated as a contributor to the neurodegenerative phenotype of these mice(Sidoryk-Wegrzynowicz et al.,2017).

Our study provides a strong incentive to further investigate ISR targeting as a therapeutic strategy for neurodegenerative tauopathies as well as other proteinopathies associated with ISR activation.The cell type-specific stress responses we observed may provide novel,more specific targets that are more amenable to drug intervention.In addition,the identification of molecules involved in neuron-astrocyte stress signaling may provide early disease process-related biomarkers like PDI(Wolzak et al.,2023) that can potentially be used for patient inclusion as well as monitoring of the treatment response in clinical trials.

For stimulating discussions,we thank the members of the Molecular Neurodegeneration group(Department of Functional Genomics,Amsterdam UMC and Vrije Universiteit Amsterdam,Amsterdam,The Netherlands).

The experimental work from our group described in this perspective was supported by ZonMW and Stichting Proefdiervrij (#114022506),PPP Allowance made available by Health～Holland,Top Sector Life Sciences &Health,to stimulate public-private partnerships (#LSHM17014 and LSHM18024),Alzheimer Nederland (grant WE.03-2017-10),European Commission (Joint Programming Initiative Neurodegenerative Diseases/JPco-fuND (ZonMW #733051062),Weston Brain Institute (#NR160014) and ZonMW Memorabel/Alzheimer Nederland (#733050101)(to WS).

Kevin Llewelyn Batenburg,Wiep Scheper*

Amsterdam UMC location Vrije Universiteit Amsterdam,Department of Human Genetics,Amsterdam Neuroscience -Neurodegeneration,Amsterdam,The Netherlands (Batenburg KL,Scheper W)

Department of Functional Genomics,Center for Neurogenomics and Cognitive Research,Vrije Universiteit Amsterdam,Amsterdam Neuroscience -Neurodegeneration,Amsterdam,The Netherlands (Batenburg KL,Scheper W)

*Correspondence to:Wiep Scheper,PhD,w.scheper@amsterdamumc.nl.

https://orcid.org/0000-0002-1431-4559(Wiep Scheper)

Date of submission:March 17,2023

Date of decision:April 19,2023

Date of acceptance:April 24,2023

Date of web publication:May 31,2023

https://doi.org/10.4103/1673-5374.377609

How to cite this article:Batenburg KL,Scheper W(2024) Neuron-to-astrocyte proteostatic stress signaling in response to tau pathology.Neural Regen Res 19(3):505-506.

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