Biology subjects

Uenaka, T.

Publications and source records attributed to Uenaka, T..

3 recordsLinked to original sources

bioRxiv2025

Defined human tri-lineage brain microtissues

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic -synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

neuroscience↗

bioRxiv2025

Prevention of Transgene Silencing During Human PluripotentStem Cell Differentiation

While high and stable transgene expression can be achieved in undifferentiated pluripotent stem cells, conventional transgene expression systems are often silenced upon differentiation. Silencing occurs with both randomly integrated transgenes, introduced via transposase or lentiviral methods, and with transgenes targeted to specific genomic sites, including at commonly used safe harbor loci. The challenge to robustly express experimental transgenes in differentiated pluripotent stem cells is a major bottleneck in the field for applications such as CRISPR screening. Here, we conducted a comparative analysis to systematically evaluate the impact of various promoters, transcriptional regulatory elements, insulators, and genomic integration sites on transgene silencing during neuronal differentiation. Our findings reveal that specific combinations of promoters and transcriptional stability elements are able to prevent transgene silencing during differentiation, whereas chromatin insulators had less impact on silencing and three novel safe harbor integration sites performed similarly to the CLYBL locus. Guided by these insights we developed the PiggyBac vector TK4, which showed complete resistance to transgene silencing across various neuronal and microglial differentiation protocols from six different pluripotent stem cell lines, as independently confirmed by seven different laboratories. This construct will be highly useful for assays requiring stable transgene expression during differentiation, and holds the potential for broad applications in various research fields.

neuroscience↗

bioRxiv2024

Restoring hippocampal glucose metabolism rescues cognition across Alzheimer's disease pathologies

Impaired cerebral glucose metabolism is a pathologic feature of Alzheimer Disease (AD), and recent proteomic studies highlight a disruption of glial carbohydrate metabolism with disease progression. Here, we report that inhibition of indoleamine-2,3-dioxygenase 1 (IDO1), which metabolizes tryptophan to kynurenine (KYN) in the first step of the kynurenine pathway, rescues hippocampal memory function and plasticity in preclinical models of amyloid and tau pathology by restoring astrocytic metabolic support of neurons. Activation of IDO1 in astrocytes by amyloid-beta42 and tau oligomers, two major pathological effectors in AD, increases KYN and suppresses glycolysis in an AhR-dependent manner. Conversely, pharmacological IDO1 inhibition restores glycolysis and lactate production. In amyloid-producing APPSwe-PS1{Delta}E9 and 5XFAD mice and in tau-producing P301S mice, IDO1 inhibition restores spatial memory and improves hippocampal glucose metabolism by metabolomic and MALDI-MS analyses. IDO1 blockade also rescues hippocampal long-term potentiation (LTP) in a monocarboxylate transporter (MCT)-dependent manner, suggesting that IDO1 activity disrupts astrocytic metabolic support of neurons. Indeed, in vitro mass-labeling of human astrocytes demonstrates that IDO1 regulates astrocyte generation of lactate that is then taken up by human neurons. In co-cultures of astrocytes and neurons derived from AD subjects, deficient astrocyte lactate transfer to neurons was corrected by IDO1 inhibition, resulting in improved neuronal glucose metabolism. Thus, IDO1 activity disrupts astrocytic metabolic support of neurons across both amyloid and tau pathologies and in a model of AD iPSC-derived neurons. These findings also suggest that IDO1 inhibitors developed for adjunctive therapy in cancer could be repurposed for treatment of amyloid- and tau-mediated neurodegenerative diseases.

neuroscience↗