Biology subjects

Quintino, L.

Publications and source records attributed to Quintino, L..

4 recordsLinked to original sources

bioRxiv2026

CRISPRa-mediated activation of Tfeb in a fibril-induced alpha-synuclein aggregation model

Accumulation of misfolded alpha-synuclein (a-syn) is a key hallmark of Parkinson's disease (PD), making it a key target for novel therapeutic strategies. Here, we use a previously described screening assay to select multiple efficient single guide RNA (sgRNA) combinations targeting transcription factor EB (TFEB) for CRISPR-activation (CRISPRa) using a nuclease-deficient Staphylococcus aureus Cas9 coupled to a truncated VP64-p53-Rta (VPR) activator complex. Selected sgRNA are applied in a rat neuroblastoma in-vitro model of fibril-induced a-syn aggregation with the goal of inducing autophagy and reduce protein aggregation. We successfully induce a-syn aggregation in this model and observe a stagnation in phosphorylated a-syn (p-a-syn) compared to non-phosphorylated a-syn across treatment groups. While we achieve robust expression of Tfeb in the exogenous screening assay and at endogenous mRNA level, we did not observe proportional increase in protein expression. Additionally, we do not observe any additive effects of CRISPRa induction combined with media treatments inducing Tfeb nuclear translocation. Taken together, these findings illustrate difficulties in epigenetic induction of Tfeb using CRISPRa and highlights the need for additional studies exploring this application.

molecular biology↗

bioRxiv2023

A systematic screening assay identifies efficient small guide RNAs for CRISPR activation

CRISPR-mediated gene activation (CRISPRa) encompasses a growing field of biotechnological approaches with exciting implications for gene therapy. However, there is a lack of experimental validation tools for selecting efficient sgRNAs for downstream applications. Here, we present a screening assay capable of identifying efficient single- and double sgRNAs through fluorescence quantification in vitro. In addition, we provide a tailored Golden Gate cloning workflow for streamlined incorporation of selected sgRNA candidates into lentiviral (LVs) or adeno-associated vectors (AAVs). The overall workflow was validated using therapeutically relevant genes for neurodegenerative diseases, such as Tfeb, Adam17, and Sirt1. The most efficient sgRNAs also demonstrated activation of endogenous gene expression at mRNA and protein levels. Further proof- of-principle assays using Tfeb indicated that gene activation was accompanied by increased levels of Lc3b. This data demonstrates the potential of the screening assay to identify functionally efficient sgRNA candidates across multiple genes along with streamlined cloning of viral vectors and may assist in accelerating future developments of CRISPRa-focused applications.

molecular biology↗

bioRxiv2022

Aβ/APP-induced hyperexcitability and dysregulation of homeostatic synaptic plasticity in models of Alzheimer's disease

The proper function of the nervous system is dependent on the appropriate timing of neuronal firing. Synapses continually undergo rapid activity-dependent modifications that require feedback mechanisms to maintain network activity within a window in which communication is energy efficient and meaningful. Homeostatic synaptic plasticity (HSP) and homeostatic intrinsic plasticity (HIP) are such negative feedback mechanisms. Accumulating evidence implicates that Alzheimers disease (AD)-related amyloid precursor protein (APP) and its cleavage product amyloid-beta (A{beta}) play a role in the regulation of neuronal network activity, and in particular HSP. AD features impaired neuronal activity with regional early hyper-activity and A{beta}-dependent hyperexcitability has also been demonstrated in AD transgenic mice. We demonstrate similar hyper-activity in AD transgenic neurons in culture that have elevated levels of both human APP and A{beta}. To examine the individual roles of APP and A{beta} in promoting hyperexcitability we used an APP construct that does not generate A{beta}, or elevated A{beta} levels independently of APP. Increasing either APP or A{beta} in wild type (WT) neurons leads to increased frequency and amplitude of calcium transients. Since HSP/HIP mechanisms normally maintain a setpoint of activity, we examined whether homeostatic synaptic/intrinsic plasticity was altered in AD transgenic neurons. Using methods known to induce HSP/HIP, we demonstrate that APP protein levels are regulated by chronic modulation of activity and show that AD transgenic neurons have an impaired response to global changes in activity. Further, AD transgenic compared to WT neurons failed to adjust the length of their axon initial segments (AIS), an adaptation known to alter excitability. Thus, we present evidence that both APP and A{beta} influence neuronal activity and that mechanisms of HSP/HIP are disrupted in neuronal models of AD.

neuroscience↗

bioRxiv2018

Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

synthetic biology↗