Biology subjects

Coughlin, G. M.

Publications and source records attributed to Coughlin, G. M..

5 recordsLinked to original sources

bioRxiv2025

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

bioRxiv2025

Structural basis of liver de-targeting and neuronal tropism of CNS-targeted AAV capsids

Crossing the blood-brain barrier while minimizing liver transduction is a key challenge in developing safe adeno-associated virus (AAV) vectors for treating brain disorders. In mice, the engineered capsid PHP.eB shows enhanced brain transduction, while the further engineered CAP-B10 is also de-targeted from astrocytes and liver. Here, we solve cryo-EM structures of CAP-B10 and its complex with AAV receptor (AAVR) domain PKD2, at 2.22 and 2.20 [A] resolutions, respectively. These structures reveal a structural motif that hinders AAVR binding, which we confirm by measuring affinities. We show that this motif is transferable to other capsids by solving cryo-EM structures of AAV9-X1 and AAV9-X1.1, without and with PKD2, at 3.09, 2.51, and 2.18 [A], respectively. Using this structural information, we designed and validated novel AAV variants with reduced liver and altered brain cell tropism in vivo. Overall, our findings demonstrate that rationally modulating AAVR affinity can alter liver targeting and cellular tropism.

molecular biology↗

bioRxiv2025

Molecularly-guided spatial proteomics captures single-cell identity and heterogeneity of the nervous system

Single-cell spatial proteomics (scSP) holds substantial potential for profiling healthy and diseased tissues. The emerging method of molecularly-guided unbiased scSP has mostly been applied to peripheral somatic tissues. Here, we optimize and apply scSP to the healthy and diseased mammalian brain, using molecularly-guided laser capture microdissection and unbiased mass spectrometry. We systematically evaluate the effects of tissue fixation, marker staining, and sample input size on proteome coverage and quantitative accuracy. We benchmark this workflow by profiling region-specific neuronal proteomes and describing the response of non-neuronal cells to acute brain injury. Across these applications, we integrate complementary transcriptomic resources to evaluate cross-modality trends and refine neuronal proteomic results by filtering out protein signals likely arising from non-neuronal cells, an essential consideration in heterogeneous tissues such as the brain. Finally, we leverage this approach to resolve proteomic differences between dopaminergic neuron subpopulations with differential vulnerability to Parkinsons disease and to uncover disease-specific disruptions in -synuclein-aggregate-bearing single dopaminergic neurons. Together, these data demonstrate the utility of scSP in neuroscience research for understanding fundamental biology and the molecular drivers of neurological conditions.

neuroscience↗

bioRxiv2023

Spatial genomics of AAVs reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo

Integrating cell type-specific regulatory elements (e.g. enhancers) with recombinant adeno-associated viruses (AAVs) can provide broad and efficient genetic access to specific cell types. However, the packaging capacity of AAVs restricts the size of both the enhancers and the cargo that can be delivered. Transcriptional crosstalk offers a novel paradigm for cell type-specific expression of large cargo, by separating distally-acting regulatory elements into a second AAV genome. Here, we identify and profile transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain. To understand transcriptional crosstalk, we develop spatial genomics methods to identify and localize AAV genomes and their concatemeric forms in cultured cells and in tissue. Using these methods, we construct detailed views of the dynamics of AAV transduction and demonstrate that transcriptional crosstalk is dependent upon concatemer formation. Finally, we leverage transcriptional crosstalk to drive expression of a large Cas9 cargo in a cell type-specific manner with systemically-administered engineered AAVs and demonstrate AAV-delivered, minimally-invasive, cell type-specific gene editing in wildtype animals that recapitulates known disease phenotypes. HighlightsO_LITranscriptional crosstalk between enhancers and promoters delivered in trans by AAVs is a generalized phenomenon. C_LIO_LISpatial genomics techniques, AAV-Zombie and SpECTr, reveal that AAV genome concatemerization facilitates transcriptional crosstalk. C_LIO_LITranscriptional crosstalk can be leveraged for minimally-invasive, targeted AAV delivery of large cargo, including machinery for CRISPR-based gene editing and manipulation. C_LIO_LITranscriptional crosstalk enables cell-type specific gene disruption in wildtype animals, recapitulating behavioural phenotypes of genetic knockouts. C_LI

bioengineering↗

bioRxiv2023

Dysregulated mammalian estrus cycle rescued by timed activation of VIP neurons in the circadian pacemaker and late afternoon light exposure

Jet lag and shift work disrupt the menstrual cycle and decrease fertility. The circadian pacemaker, the suprachiasmatic nucleus (SCN), is known to modulate ovulation, but the mechanism is unclear. Here we explore this connection by tracking the dynamics of vasoactive intestinal peptide (VIP)-expressing neurons in the SCN in freely-behaving mice. We show that SCNVIP activity is time-of-day- and sex-dependent, and estrous-state-dependent in late afternoon, gating downstream activation of GnRH neurons. Afternoon light, as well as specific activation of SCNVIP neurons, rescues estrous cycle regularity and egg release in animals in altered light conditions, emphasizing the role of SCNVIP neurons as a time-dependent light-responsive switch. Our results reveal the dynamic mechanism by which SCNVIP neurons mediate light responses to regulate estrous states and demonstrate light-induced fertility rescue. One Sentence SummaryModulating and recording the activity of suprachiasmatic VIP neurons in freely behaving mice reveals their regulation of fertility by mediating the response to late afternoon light.

neuroscience↗