Biology subjects

Zempel, H.

Publications and source records attributed to Zempel, H..

6 recordsLinked to original sources

bioRxiv2024

Expression of recombinant human glutamylating TTLLs in human cells leads to differential tubulin glutamylation patterns, with only TTLL6 disrupting microtubule dynamics

Polyglutamylation, a post-translational modification (PTM) catalyzed by a subset of Tubulin Tyrosine Ligase-Like (TTLL) family enzymes, regulates microtubule dynamics through its influence on interactions with microtubule-associated, motor, and severing proteins, and has recently also been implicated in genetic and neurodegenerative diseases. In this study, we characterized the glutamylation activity of various glutamylating TTLLs in human embryonic kidney 293T (HEK293T) cells, revealing distinct patterns of mono- and polyglutamylation among TTLL family members, with TTLL4 and TTLL11 exhibiting the strongest chain initiation and elongation activities, respectively. We found that TTLL6 expression uniquely decreased microtubule stability, with live-cell imaging of end-binding protein (EB3) showing a TTLL6-induced decrease in microtubule stability. To explore therapeutic modulation of TTLL activity, we tested LDC10, a novel TTLL inhibitor, which successfully blocked glutamylation across all TTLLs investigated in this study, while also reversing the microtubule-destabilizing effects of TTLL6. These findings identify a potential pathogenic role of TTLL6 in microtubule dynamics and highlight LDC10 as a promising pharmacological tool to counteract TTLL-induced microtubule destabilization.

cell biology↗

bioRxiv2024

Knockdown of TTLL1 reduces Aβ-induced TAU pathology in human iPSC-derived cortical neurons

Microtubules play a crucial role in neuronal structure and function, with their stability and dynamics regulated by posttranslational modifications (PTMs) such as polyglutamylation. In Alzheimer disease (AD), the microtubule-associated protein TAU becomes mislocalized into the somatodendritic compartment ( TAU missorting), dissociates from microtubules, aggregates into neurofibrillary tangles, and contributes to microtubule destabilization and neuronal death. Here, we investigated the role of Tubulin-Tyrosine-Ligase-Like proteins (TTLLs) in TAU missorting and microtubule dysregulation using human induced pluripotent stem cell (hiPSC)-derived cortical neurons treated with oligomeric amyloid-beta (oA{beta}) to replicate AD-like conditions. TTLL1, TTLL4, TTLL6 were selectively knocked down (KD) to assess their impact on TAU missorting and microtubule stability. Fluorescence resonance energy transfer (FRET) microscopy was used to examine interactions between TAU and TTLL proteins. We observed TAU missorting, increased tubulin polyglutamylation, decreased microtubule stability, and synaptic declustering in oA{beta}-treated neurons. TTLL1 KD significantly reduced TAU missorting, tubulin polyglutamylation, and synaptic disintegration, while TTLL4 KD showed moderate effects, and TTLL6 KD restored microtubule acetylation. Importantly, TTLL KD did not impair neuritic networks, dendritic complexity, or neuronal activity. FRET microscopy revealed a potential interaction between TAU and TTLL1, but not other TTLLs, suggesting a direct role of TTLL1 in TAU-mediated toxicity. Our findings indicate that targeting TTLL1, either alone or in combination with other TTLLs, may be a promising therapeutic strategy to counteract microtubule and synaptic dysfunction in AD and related neurodegenerative disorders.

neuroscience↗

bioRxiv2024

Axonal Tau sorting depends on the PRR2 domain and 0N4R-specific interactions hint at distinct roles of Tau isoforms in synaptic plasticity

Tau pathology is a major hallmark of Alzheimers disease (AD) and related diseases, called tauopathies. While Tau is normally enriched in axons, somatodendritic missorting of the microtubule-associated protein is a key event in early disease development. Tau missorting promotes synaptic loss and neuronal dysfunction but the mechanisms underlying both normal axonal sorting and pathological missorting remain unclear. Interestingly, the disease-associated Tau brain isoforms show different axodendritic distribution, but the distinct role of these isoforms in health and disease largely unknown. Here, we aimed to identify domains or motifs of Tau and cellular binding partners that are required for efficient axonal Tau sorting, and we studied the differences of the isoform-specific Tau interactome. By using human MAPT-KO induced pluripotent stem cell (iPSC)-derived glutamatergic neurons, we analyzed the sorting behavior of more than 20 truncated or phosphorylation-mutant Tau constructs, and we used TurboID-based proximity labelling and proteomics to identify sorting- and isoform-specific Tau interactors. We found that efficient axonal Tau sorting was independent of the N-terminal tail, the C-terminal repeat domains, and the general microtubule affinity of Tau. In contrast, the presence of the proline-rich region 2 (PRR2) was necessary for successful sorting. Our interactome data revealed peroxisomal accumulation of the Tau N-terminal half, while axonal Tau interacted with the PP2A activator HSP110. When we compared the interactome of 0N3R- and 0N4R-Tau, we observed specific interactions of 0N4R-Tau with regulators of presynaptic exocytosis and postsynaptic plasticity, which are partially associated with AD pathogenesis, such as members of the CDC42 pathway and the RAB11 proteins, while 0N3R-Tau bound to MAP4 and other cytoskeletal elements. In sum, our study postulates that axonal Tau sorting relies on the PRR2 domain but not on microtubule affinity, and unravels a potential isoform-specific role in synaptic function and AD-related dysfunction.

neuroscience↗

bioRxiv2020

Endo-lysosomal Aβ concentration and pH enable formation of Aβ oligomers that potently induce Tau missorting

Amyloid-β peptide (Aβ) forms metastable oligomers >50 kD, termed AβOs or protofibrils, that are more effective than Aβ amyloid fibrils at triggering Alzheimer’s disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aβ accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AβO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AβO formation and the high endo-lysosomal Aβ concentration together enable extensive AβO formation of Aβ42 under physiological conditions. Exploiting the enhanced AβO formation of the dimeric Aβ variant dimAβ we furthermore demonstrate targeting of AβOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AβOs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Axonal TAU sorting in SH-SY5Y-derived neurons requires the C-terminus of TAU but is independent of Ankyrin G

Somatodendritic missorting of the axonal protein TAU is a hallmark of Alzheimers disease and related tauopathies. Cultured rodent primary neurons and iPSC-derived neurons are used for studying mechanisms of neuronal polarity, including TAU trafficking. However, these models are expensive, time-consuming and/or require the sacrification of animals. In this study, we evaluated four differentiation procedures to generate mature neuron cultures from human SH-SY5Y neuroblastoma cells, in comparison to mouse primary neurons, and tested their TAU sorting capacity. We show that SH-SY5Y-derived neurons, differentiated with sequential RA/BDNF treatment, are suitable for investigating axonal TAU sorting. These human neurons show pronounced neuronal polarity, axodendritic outgrowth, expression of the neuronal maturation markers TAU and MAP2, and, importantly, efficient axonal sorting of endogenous and transfected human wild type TAU, similar to primary neurons. We demonstrate that axonal TAU enrichment requires the presence of the C-terminal half, as a C-terminus-lacking construct (N-term-TAUHA) is not axonally enriched in both neuronal cell models. Moreover, SH-SY5Y-derived neurons do not show formation of a classical axon initial segment (AIS), indicated by the lack of Ankyrin G (ANKG) and tripartite motif-containing protein 46 (TRIM46) at the proximal axon, which suggests that successful axonal TAU sorting is independent of classical AIS formation. Taken together, our results suggest i) that SH-SY5Y-derived neurons are a valuable human neuronal cell model for studying TAU sorting, which is readily accessible at low cost and without animal need, and that ii) the mechanisms of axonal TAU targeting require the TAU C-terminal half but are independent of ANKG or TRIM46 enrichment at the proximal axon.

neuroscience↗

bioRxiv2020

Subcellular localization of TAU isoforms and their influence on microtubule dynamics

In the adult human brain, six isoforms of the microtubule-associated protein TAU are expressed, which result from alternative splicing of exons 2, 3 and 10 of the MAPT gene. These isoforms differ in the number of N-terminal inserts (0N, 1N, 2N) and C-terminal repeat domains (3R or 4R) and are differentially expressed depending on the brain region and developmental stage. Although all TAU isoforms can aggregate and form neurofibrillary tangles, some tauopathies, such as Picks Disease and Progressive Supranuclear Palsy, are characterized by the accumulation of specific TAU isoforms. The influence of the individual TAU isoforms in a cellular context, however, is understudied. In this report, we investigated the subcellular localization of the human-specific TAU isoforms in primary neurons, and analyzed TAU isoform-specific effects on cell area and microtubule dynamics in SH-SY5Y neuroblastoma cells. Our results show that 2N-TAU isoforms are particularly retained from axonal sorting and that axonal enrichment is independent from the number of repeat domains, but that the additional repeat domain of 4R-TAU isoform results in a general reduction of cell size and an increase of microtubule counts in cells expressing 4R-TAU isoforms. Our study points out that individual TAU isoforms may influence microtubule dynamics differentially both by different sorting patterns as well as by direct effects on microtubule dynamics.

neuroscience↗