Biology subjects

Arsic, A.

Publications and source records attributed to Arsic, A..

5 recordsLinked to original sources

bioRxiv2022

The tail domain of neurofilament light chain accumulates in neuronal nuclei during oxidative injury

Neurofilament light chain (NFL), one of the major subunits of neuron-specific intermediate filaments, is an important component of the neuronal cytoskeleton. Mutations in the NEFL gene and disturbances of NFL turnover or transport are associated with the pathophysiology of various diseases of the nervous system. Also, NFL is released from neurons as a result of neuroaxonal damage and NFL levels in cerebrospinal fluid and blood have emerged as a biomarker of the progression of numerous neurological diseases. One common underlying factor in these diseases is oxidative stress. Although both NFL and oxidative injury play a role in the development of neurological diseases, the effect of oxidative stress on NFL at the cellular level has not been extensively studied. Here, by investigating the response of neurofilaments to in vitro oxidative injury, we discovered that NFL localizes in the nuclei of primary neurons. We show that this depends on calpain activity and determine that a proteolytic fragment of the tail domain, not the full-length NFL, is that which accumulates in the nuclei. Furthermore, we demonstrate that the recombinant NFL tail domain accumulates in the nuclei of neurons and neuroblastoma cells, even in the absence of oxidative injury. Finally, we show that the tail domain of NFL interacts with the DNA in the nuclei of living cells, suggesting that NFL plays a role in the regulation of gene expression in response to oxidative injury.

neuroscience↗

bioRxiv2022

Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry

The axon initial segment (AIS) is a highly specialized neuronal compartment that regulates the generation of action potentials and maintenance of neuronal polarity. Despite its importance, live imaging of the AIS is challenging due to the limited number of suitable labeling methods. To overcome this limitation, we established a novel approach for live labeling of the AIS using unnatural amino acids (UAAs) and bioorthogonal click chemistry. The small size of the UAAs and the possibility of introducing them virtually anywhere into the target proteins make this method particularly suitable for live labeling and imaging of complex and spatially restricted proteins. With this approach, we labeled two large AIS components, the 186 kDa isoform of neurofascin (NF186) and the 260 kDa voltage-gated sodium channel (NaV1.6), and performed widefield and confocal microscopy in fixed and living neurons. Moreover, we demonstrated the applicability of this method by studying the localization of two epilepsy-causing NaV1.6 variants with a loss-of-function effect. Finally, to further improve the efficiency of the UAA incorporation, we developed adeno-associated viral (AAV) vectors for click labeling in primary neurons. The use of AAV vectors will facilitate the transfer of UAA-based click labeling technology to more complex biological systems, such as organotypic slice cultures, organoids, and animal models.

neuroscience↗

bioRxiv2021

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

Modern light microscopy, including super-resolution techniques, brought about a demand for small labeling tags that bring the fluorophore closer to the target. This challenge can be addressed by labeling unnatural amino acids (UAAs) with click chemistry. UAAs are site-specifically incorporated into a protein of interest by genetic code expansion. If the UAA carries a strained alkene or alkyne moiety it can be conjugated to a tetrazine-bearing fluorophore via a strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC), a variant of bioorthogonal click chemistry. The minimal size of the incorporated tag and the possibility to couple the fluorophores directly to the protein of interest with single-residue precision make SPIEDAC live-cell labeling unique. However, until now, this type of labeling has not been used in complex, non-dividing cells, such as neurons. Using neurofilament light chain as a target protein, we established SPIEDAC labeling in living primary neurons and applied it for fixed-cell, live-cell, dual-color pulse--chase and super-resolution microscopy. We also show that SPIEDAC labeling can be combined with CRISPR/Cas9 genome engineering for tagging endogenous NFL. Due to its versatile nature and compatibility with advanced microscopy techniques, we anticipate that SPIEDAC labeling will contribute to novel discoveries in neurobiology.

neuroscience↗

bioRxiv2020

Interferon-γ signaling synergizes with LRRK2 in human neurons and microglia

Increasing evidence suggests a role for interferons (IFNs) in neurodegeneration. Parkinsons disease (PD) associated kinase LRRK2 has been implicated in IFN type II (IFN) response in infections and nigral neuronal loss. However, whether and how LRRK2 synergizes with IFN-{gamma} still remains unclear. Here, we employed dopaminergic (DA) neurons and microglia differentiated from patient induced pluripotent stem cells to unravel the role of IFN-{gamma} in LRRK2-PD. We show that IFN-{gamma} induces LRRK2 expression in both DA neurons and microglial cells. LRRK2-G2019S, the most common PD-associated mutation, sensitizes DA neurons to IFN-{gamma} by decreasing AKT phosphorylation. IFN-{gamma} suppresses NFAT activity in both neurons and microglia and synergistically enhances LRRK2-induced defects of NFAT activation. Furthermore, LRRK2-G2019S negatively regulates NFAT via calcium and microtubule dynamics. Importantly, we uncover functional consequences of the reduction of NFAT activity in both cell types, namely defects of neurite elongation and alteration of microglial activation profile and motility. We propose that synergistic IFN-{gamma}/LRRK2 activation serves as a direct link between inflammation and neurodegeneration in PD.

neuroscience↗

bioRxiv2018

Seeing in the dark: Vectashield quenches Alexa Fluor 647 fluorescence, but does not hinder dSTORM imaging

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent ON state. This helps identifying structures of interests. Subsequently, labelled samples are brought to blinking, which is necessary for localization of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor (AF) 647, blinks in Vectashield. However, after adding Vectashield to our samples, we noticed that the fluorescence intensity of AF647 and its improved variant, AF647+, is quenched. Since structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) practical advice on how to use Vectashield for dSTORM imaging of AF647 and AF647+.

biophysics↗