Howden, J. | bioRxiv Science

bioRxiv2025

Tracking gene expression of single mitochondria in live neurons using nanotweezers

Neurons are highly polarised cells that depend on mitochondria for energy and signalling homeostasis. Importantly, energy and signalling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyse mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction a individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with mitochondrial gene expression tracking and mtDNA profiling. By tracking mitochondrial gene expression in the same neurons over time, we reveal a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to -synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides precise, dynamic measurements of mitochondrial composition and gene expression in vivo at single-organelle resolution, enabling mechanistic studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.

neuroscience↗

bioRxiv2020

KCC2 is required for the survival of mature neurons but not for their development

The K+/Cl- co-transporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A {gamma}-aminobutyric acid receptors GABAARs. In accordance with this, compromised KCC2 activity results in seizures but whether such deficits directly contribute to the subsequent changes in neuronal viability that lead to epileptogenesis, remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression resulted in the rapid activation of the extrinsic apoptotic pathway, in mature hippocampal neurons. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization or dendritic structure. However ablating KCC2 expression in immature neurons was sufficient to prevent the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to loss of KCC2 function. In contrast KCC2 appears to play a minimal role in mediating neuronal development or architecture.

neuroscience↗