Universität zu Köln

Bell-Simons, Michael ORCID: 0000-0003-0681-901X (2025). Domain requirements and mechanisms of axonal Tau sorting. PhD thesis, Universität zu Köln.

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Abstract

Alzheimer’s disease (AD) constitutes a major burden to modern health care systems. Pathological alterations of the microtubule-binding protein Tau are a major hallmark of AD and many other disorders, summarized as tauopathies. Under healthy conditions, Tau regulates the dynamic assembly of axonal microtubule filaments depending on its phosphorylation state, and thus affects essential neuronal functions. Somatodendritic missorting of Tau is an early sign of Tau pathology in AD. Despite the intricate role of intracellular Tau mislocalization under pathological conditions, the mechanisms of axonal Tau sorting and somatodendritic missorting are still poorly understood. Several processes were proposed to affect efficient axonal Tau sorting, including anterograde protein transport or retrograde axonal retention, but their contribution remains elusive. There is also little known about Tau domains, motifs or cellular interaction partners that are required for successful Tau sorting. More knowledge about the underlying processes of axonal Tau sorting would help to develop treatments that can restore physiological sorting in disease conditions and thus attenuate the neurotoxic effects of Tau missorting. Interestingly, the six human Tau isoforms differ in their intracellular sorting behaviour. This distinct localization hint at differential roles of these isoforms in Tau function under health and disease. The exact function of six human Tau isoforms remains still unknown, but previous studies suggested isoform-specific roles in Tau-mediated neuronal toxicity. In addition, tauopathies can be caused already by changes in splicing of the Tau-encoding MAPT gene, resulting in aberrant Tau isoform expression. Thus, we strongly believe that more insights into isoform-specific functions in health and disease could open up new therapeutic avenues. In this study, we aimed i) to determine the relevance of anterograde transport for efficient axonal Tau sorting, ii) to unravel domains, motifs, and binding partners of Tau that are involved in the axonal Tau sorting process, and iii) to identify sorting-specific binding partners between individual Tau isoforms that may hint at distinct roles of Tau isoforms. To address these questions, we established two human neuronal model systems: Human neuroblastoma SH-SY5Y cell line-derived neurons and neurogenin 2 (Ngn2)-transgenic human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons. First, we assessed the role of anterograde transport of somatic Tau in axonal sorting. To this end, we cut the proximal axon of single iPSC-derived and mouse primary neurons with a high-energy UV laser and quantified the levels of somatic Tau accumulation and AT8 phosphorylation upon axon loss. Consistent for both cell models, we found no signs for acute Tau pathology upon axotomy but rather mild reductions of both Tau levels and AT8 phosphorylation at some of the tested time points. These results indicate that impaired anterograde transit due to axon loss does not lead to overt Tau pathology. We argue that these findings either indicate a minor role for anterograde transport of somatically synthesized Tau or underline the capacity of the damaged cell to prevent Tau accumulation. Next, we aimed to unravel domains and motifs of the Tau protein that are required for successful Tau sorting. We generated a library of mutant HA-tagged Tau constructs and aimed to analyse their sorting behaviour. First, we had to establish a setup for efficient axonal sorting of exogenous Tau, an often-faced problem in previous studies. We used a recently engineered MAPT-knock out (MAPT-KO) clone of our iPSC line, and achieved endogenous-like sorting of exogenous 0N3R-Tau with lentiviral gene delivery and a doxycycline-inducible promoter expression. Thus, we were able to analyse the sorting behaviour of our mutant Tau constructs in MAPT-KO iPSC-derived neurons. We observed that large truncations compromise axonal sorting, for instance the Tau N-terminal half accumulated in the soma and formed puncta-like inclusions that were absent for all other constructs. Our results showed that the proline-rich region 2 (PRR2) of Tau is required for efficient Tau sorting. In contrast to previous studies, we showed that efficient axonal sorting is independent of the Tau N-terminal tail and the general microtubule affinity of Tau. We hypothesize that the PRR2 domain is either crucial for mediating sorting-related Tau interactions or We used TurboID-based proximity labelling to compare the interactome of axonally sorted Tau and the non-sorting N-terminal half. While the interactions of the N-terminal half suggested accumulation inside the peroxisomal lumen, we found only several binding partners specific for the sorting Tau isoforms. The chaperone HSP110 activates the major Tau phosphatase PP2A and could promote axonal retention by PP2A-mediated dephosphorylation of Tau, even though our previous sorting analysis showed that Tau mobility had no detectable effect on axonal enrichment. When we analysed the interactomes of the Tau isoforms 0N3R and 0N4R, we identified 0N4R-specific interactors that are involved in the regulation of presynaptic vesicle trafficking and postsynaptic plasticity. Two 0N4R-associated pathways, the CDC42 cascade and RAB11 protein signalling are implicated in AD pathogenesis. Thus, we found evidence that 0N4R or 4R isoforms in general may be involved in synaptic function in health and disease. In conclusion, our study provides evidence for PRR2-dependent axonal Tau sorting independent of the N-terminal tail and the microtubule affinity of Tau. We claim that anterograde Tau transport is more relevant for efficient axonal sorting than axonal retention. Our isoform-specific interactome analysis revealed that 0N4R-Tau is associated with regulators of pre- and postsynaptic activity partially involved in AD pathology. Thus, our work hints at an isoform-specific role of Tau in synaptic function in health and disease. Our findings pave the path for developing tailored treatment approaches that aim to i) prevent or ameliorate Tau missorting and its detrimental effects, and ii) target Tau isoforms responsible for Tau-induced synaptic and neuronal dysfunction.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Bell-Simons, Michael mbell-simons@age.mpg.de https://orcid.org/0000-0003-0681-901X UNSPECIFIED
URN:	urn:nbn:de:hbz:38-784776
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Humangenetik > Institut für Humangenetik
Subjects:	Natural sciences and mathematics
Uncontrolled Keywords:	Keywords Language Alzheimer's Disease UNSPECIFIED Tau sorting UNSPECIFIED Tau pathology UNSPECIFIED
Date of oral exam:	2 September 2024
Referee:	Name Academic Title Zempel, Hans Dr. med. Dr. rer. nat. Kononenko, Natalia Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/78477