Biology subjects

Lundberg, C.

Publications and source records attributed to Lundberg, C..

3 recordsLinked to original sources

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↗

bioRxiv2023

Hallmark molecular and pathological features of POLG disease are recapitulated in cerebral organoids

In our research, we developed a 3D brain organoid model to study POLG-related encephalopathy, a mitochondrial disease stemming from POLG gene mutations. We utilized induced pluripotent stem cells (iPSCs) derived from patients with these mutations to generate cortical organoids, which exhibited typical POLG disease features, such as altered morphology, neuronal loss, and mtDNA depletion. We also identified significant dysregulation in pathways crucial for neuronal development and function, alongside upregulated NOTCH and JAK-STAT signaling pathways. Metformin treatment ameliorated many of these abnormalities, except for the persistent affliction of inhibitory DA GLU neurons. This novel model effectively mirrors both the molecular and pathological attributes of POLG disease, providing a valuable tool for mechanistic understanding and therapeutic screening for POLG-related disorders and other conditions characterized by compromised neuronal mtDNA maintenance and complex I deficiency. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/558087v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1759de3org.highwire.dtl.DTLVardef@d0a3dborg.highwire.dtl.DTLVardef@1b58eaborg.highwire.dtl.DTLVardef@a986a8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe have successfully developed cortical organoid model that represents POLG-related disease. C_LIO_LIThis model effectively replicates both histological and molecular signatures seen in the brains of patients. C_LIO_LIThe cortical organoid model displays a range of features common in POLG-related disease, including neurodegeneration, mtDNA depletion, and neuronal complex I deficiency. C_LIO_LIThe use of metformin supplementation in this model improved mitochondria protein and reduced cell death. C_LI

cell 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↗