Biology subjects

Lisowski, L.

Publications and source records attributed to Lisowski, L..

13 recordsLinked to original sources

bioRxiv2026

Preferential vulnerability of cortical GABAergic interneurons to Nalcn deficiency

Neuronal resting membrane potential (RMP) reflects the balance of leak conductances and varies systematically across cortical cell types. Many GABAergic interneurons exhibit more depolarized RMPs than neighbouring excitatory neurons, but the molecular mechanisms underlying these systematic differences in intrinsic excitability remain incompletely defined. The sodium leak channel non-selective (NALCN) mediates a major fraction of basal sodium conductance and cause neurodevelopmental syndromes characterized by developmental delay and cognitive impairment. Here we define NALCN contribution to cortical circuit development and function with cell-type precision. Using Nalcn-GFP reporter line, we map Nalcn expression across cortical types revealing enrichment in GABAergic hippocampal interneurons relative to hippocampal pyramidal neurons. Using conditional mouse models, we selectively deleted Nalcn in cortical glutamatergic lineages or forebrain GABAergic cells from early embryogenesis. Electrophysiological analysis show that developmental loss of Nalcn preferentially reduces intrinsic excitability across GABAergic interneuron subtypes while sparing pyramidal neurons. To understand the impact of a depolarized GABAergic RMP on brain function, we assessed the behavioral performance of Nalcn-deficient mice that revealed persistent deficits in contextual adaptation and spatial short-term memory. These findings reveal a neuron type-specific function of NALCN in the cerebral cortex and position NALCN as a crucial ion channel regulating basal excitability of GABAergic inhibitory circuits and cortical circuit function.

neuroscience↗

bioRxiv2025

Spatially precise neuron formation via hydrogel mediated modulation of the host astrocyte response

The regenerative capacity of the central nervous system after injury or disease is limited. Advancements in genetic and epigenetic reprogramming of non-neuronal cells into induced neurons presents a promising strategy for neuronal replacement and circuit reconstruction yet is hindered by suboptimal reprogramming efficacy. Here, we have achieved high astrocyte-to-neuron reprogramming efficacy through the ectopic expression of a SOX2 transcription factor using adeno-associated viral vectors. We subsequently designed a hybrid composite biomaterial to act as an implantable reprogramming workshop that perform sequential operations. Once injected, this bespoke system forms a tissue mimetic hydrogel encouraging the ingress of astrocytes to act as our raw material. We demonstrate that the system localized and enhanced the spatially confined delivery of reprogramming factors, entrapping astrocytes to then pass through an intermediate neuroblast state to robustly yield mature neurons. Sustained delivery of valproic acid within the system further promoted neuronal maturation. Overall, using design rules informed by requirements of the reprogramming strategy, we have designed a smart delivery system optimized for both the target tissue and reprogramming factors in a minimally invasive manner to enhance neural repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/695501v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@397e41org.highwire.dtl.DTLVardef@90047forg.highwire.dtl.DTLVardef@1175831org.highwire.dtl.DTLVardef@19f30eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

AAV2 Bypasses Direct Endosomal Escape by Using AAVR to Access the Trans-Golgi Network en Route to the Nucleus.

Vectors based on the adeno-associated virus are widely used as delivery methods in gene therapy applications, yet understanding of the mechanisms governing its intracellular trafficking remains incomplete. Traditional models suggest that AAV escapes from endosomes via membrane disruption, but direct evidence for this process are lacking. Here, we show that AAVR, the essential AAV cell entry receptor, functions as a bona fide retromer cargo. Using in vitro reconstitution assays, we demonstrate that AAVRs cytosolic tail is sufficient to engage the SNX3-retromer complex and drive membrane tubulation, a hallmark of retrograde trafficking. In AAVR-knockout HuH-7 cells, AAV2 particles are internalized but fail to reach the trans-Golgi network (TGN) and support transgene expression. Galectin-8 recruitment assays reveal no evidence of endosomal membrane rupture during productive transduction, distinguishing AAV2 from lytic vectors such as lipid nanoparticles. Moreover, VP1u-deficient and PLA2-mutant AAV2 capsids accumulate at the TGN, indicating that VP1u is dispensable for early trafficking but required for post-TGN progression toward productive transduction. These findings challenge the prevailing model of direct endosomal escape and position AAV transduction as a vesicle-guided, receptor-mediated process.

cell biology↗

bioRxiv2024

State- and circuit-dependent opponent-processing of fear

The presence of valence coding neurons in the basolateral amygdala (BLA) that form distinct projections to other brain regions implies functional opposition between aversion and reward during learning. However, evidence for opponent interactions in fear learning is sparse and may only be apparent under certain conditions. Here we test this possibility by studying the roles of the BLAcentral amygdala (CeA) and BLAnucleus accumbens (Acb) pathways in fear learning in male rats. First, we assessed the organisation of these pathways in the rat brain. BLACeA and BLAAcb pathways were largely segregated in the BLA but shared overlapping molecular profiles. Then we assessed activity of the BLACeA and BLAAcb pathways during two different forms of fear learning - fear learning in a neutral context and fear learning in a reward context. BLA CeA neurons were robustly recruited by footshock regardless of where fear learning occurred whereas recruitment of BLAAcb neurons was state-dependent because footshock only recruited this pathway in a reward context. Finally, we assessed the causal roles of activity in these pathways in fear learning. Photoinhibition of the BLACeA pathway during the footshock US impaired fear learning, regardless of where fear learning occurred. In contrast, photoinhibition of the BLAAcb pathway augmented fear learning, but only in the reward context. Taken together, our findings show circuit- and state-dependent opponent processing of fear. Footshock-driven activity in the BLAAcb pathway can functionally oppose the BLACeA pathway to limit how much fear is learned. Significance StatementHere we identify a fear opponent process in the brain. We show that an aversive event can recruit distinct populations of neurons in the rat basolateral amygdala. One population projects to the central amygdala to promote fear learning. A second population projects to the nucleus accumbens to oppose fear learning. These nucleus accumbens projecting neurons limit how much fear is learned and are candidates for therapeutic targeting to minimize the amount of fear learned after a traumatic experience.

neuroscience↗

bioRxiv2024

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability

Alzheimers disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined induced pluripotent stem cell (iPSC)-derived NGN2 induced neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding the disease pathogenesis.

cell biology↗

bioRxiv2024

Characterisation of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a reversible manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression in various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanised VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 can transduce mouse retinal cells efficiently, reaching the photoreceptors for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity and establish Cas13bt3 as a potential anti-VEGF agent that can achieve long-term control of VEGFA for the treatment of retinal neovascularization.

molecular biology↗

bioRxiv2023

ADEVO: Proof-of-concept of Adenovirus Directed EVOlution by random peptide display on the fiber knob

Directed evolution of viral vectors involves the generation of randomized libraries followed by artificial selection of improved variants. Directed evolution only yielded limited results in adenovirus vector (AdV) development until now, mainly due to insufficient complexities of randomized libraries. Clinical applications of AdVs as gene therapy or oncolytic vectors are still hampered by the predetermined tropism of natural types. To overcome this challenge, we hypothesized that the technology of randomized peptide insertions on the capsid surface can be incorporated into the AdV bioengineering toolbox for vector retargeting. Here we developed Adenovirus Directed EVOlution (ADEVO) protocols based on fiber knob peptide display. As a proof-of-concept, HAdV-C5-derived libraries were constructed following three distinct protocols and selected on A549-DCAR cells that lack the HAdV-C5 primary receptor, with the goal of identifying variants able to infect and lyse these tumor cells more efficiently. All protocols enabled the construction of high complexity libraries with up to 9.6x10^5 unique variants, an approximate 100-fold improvement compared to previously published AdV libraries. After selection, the most enriched variants did not display enhanced infectivity but rather more efficient replication and cell lysis. This warrants investigations into potential unsuspected involvement of the fiber protein in adenovirus replication. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/567388v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@187f7baorg.highwire.dtl.DTLVardef@1990555org.highwire.dtl.DTLVardef@12d3c60org.highwire.dtl.DTLVardef@d703da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Harnessing the power of whole human liver ex situ normothermic perfusion for preclinical AAV vector evaluation

Developing clinically predictive model systems for evaluating gene transfer and gene editing technologies has become increasingly important in the era of personalized medicine. Liver-directed gene therapies present a unique challenge due to the complexity of the human liver. In this work, we describe the application of whole human liver explants in an ex situ normothermic perfusion system to evaluate a set of fourteen natural and bioengineered adeno-associated viral (AAV) vectors directly in human liver, in the presence and absence of neutralizing human sera. Under non-neutralizing conditions, the recently developed AAV variants, AAV-SYD12 and AAV-LK03, emerged as the most functional variants in terms of cellular uptake and transgene expression. However, when assessed in the presence of human plasma containing anti-AAV neutralizing antibodies (NAbs), vectors of human origin, specifically those derived from AAV2/AAV3b, were extensively neutralized, whereas AAV8-derived variants performed efficiently. This study establishes the use of normothermic liver perfusion as an invaluable preclinical model for evaluating liver-targeted gene therapies and providing guidance for making essential decisions that promote the most effective translational programs.

molecular biology↗

bioRxiv2023

A transient protein folding response targets aggregation in the early phase of TDP-43-mediated disease

Understanding the mechanisms that drive TDP-43 pathology is integral to combating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To address this, we sought to determine the timeline of proteomic alterations across disease course in TDP-43 proteinopathy. Using longitudinal quantitative proteomics analysis of cortex samples from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, we identified several distinct protein subsets characterized by temporal alterations in protein abundance across diverse biological pathways, including protein folding, intracellular transport, myelination, and neuronal synaptic function. Remarkably, neurons in the rNLS8 cortex elicited a transitory response primarily comprising protein-folding factors prior to and in the earliest stages of disease progression. This response included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, and proof-of-concept studies showed that DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures. Conversely, knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in the brains and spinal cords of mice. Lastly, the late disease proteomic signatures of rNLS8 mouse cortex strongly correlated with changes in human autopsy-derived TDP-43 proteinopathy tissues, indicating commonality of disease processes. Together, these findings reveal molecular mechanisms that regulate protein levels through distinct stages of ALS and FTLD progression, and suggest that protein folding factors that combat cytoplasmic TDP-43 protein aggregation could be protective in disease. HighlightsO_LIThe first longitudinal map of the cortex proteome throughout TDP-43-driven disease in a mouse model of cytoplasmic TDP-43 proteinopathy (rNLS8 mice). C_LIO_LICytoplasmic TDP-43 accumulation drives many dynamic changes to the cortex proteome, including increases in protein folding factors prior to disease onset. C_LIO_LIThe protein folding factor DNAJB5 decreases TDP-43 aggregation in HEK293 cells and primary cortical neurons and Dnajb5 knockout exacerbates cytoplasmic TDP-43-associated motor impairments in vivo. C_LIO_LIThe proteomic signature of the rNLS8 mouse cortex correlates strongly with postmortem brain tissue from human TDP-43 proteinopathies. C_LIO_LIA new webtool, TDP-map (https://shiny.rcc.uq.edu.au/TDP-map/), allows comparison of transcriptomic and proteomic datasets from mouse and human TDP-43 proteinopathy. C_LI

neuroscience↗

bioRxiv2022

Assessment of pre-clinical liver models based on their ability to predict the liver-tropism of AAV vectors

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors (rAAV). Multiple clinical trials have been undertaken for this target in the past 15 years, however we are still to see market approval of the first liver-targeted AAV-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically- and clinically-predictive preclinical models. To this end, this study reports findings of a functional evaluation of six AAV vectors in twelve preclinical models of the human liver, with the aim to uncover which model is the most relevant for the selection of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

bioengineering↗

bioRxiv2022

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

systems biology↗

bioRxiv2022

A human adenovirus encoding IFN-γ can transduce Tasmanian devil facial tumour cells and upregulate MHC-I

The devil facial tumour disease (DFTD) has led to a massive decline in the wild Tasmanian devil (Sarcophilus harrisii) population. The disease is caused by two independent devil facial tumours (DFT1 and DFT2). These transmissible cancers have a mortality rate of nearly 100%. An adenoviral vector-based vaccine has been proposed as a conservation strategy for the Tasmanian devil. This study aimed to determine if a human adenovirus serotype 5 could express functional transgenes in devil cells. As DFT1 cells do not constitutively express major histocompatibility complex class I (MHC-I), we developed a replication-deficient adenoviral vector that encodes devil interferon gamma (IFN-{gamma}) fused to a fluorescent protein reporter. Our results show that adenoviral-expressed IFN-{gamma} was able to stimulate upregulation of beta-2 microglobulin, a component of MHC-I, on DFT1, DFT2, and devil fibroblast cell lines. This work suggests that human adenoviruses can serve as vaccine platform for devils and potentially other marsupials.

immunology↗

bioRxiv2020

The self-peptide repertoire plays a critical role in transplant tolerance induction

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly-alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically-engineered MHC class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly-recognised pMHC epitopes, and identified 17 strongly immunogenic H-2Kb-associated peptides recognised by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognised by BALB/c (H-2d) mice. As few as five different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

immunology↗