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Strom, M.

Publications and source records attributed to Strom, M..

8 recordsLinked to original sources

TRPML1 loss drives lysosomal calcium failure and astrocyte dysfunction across Alzheimer's Disease progression

Astrocytes are among the earliest cells to exhibit dysfunction in Alzheimers disease (AD), developing profound calcium signalling deficits before amyloid plaques have formed, yet the underlying mechanisms remain unknown. Lysosomal dysfunction is a hallmark of AD, but whether it initiates this early functional impairment or arises as a consequence of established pathology remains unresolved. Here, we find that astrocytic cytosolic calcium activity is suppressed prior to amyloid plaque deposition and is accompanied by reduced lysosomal acidification in vivo. Using a lysosome-targeted calcium indicator selectively expressed in astrocytes, we directly visualise lysosomal calcium dynamics in vivo and reveal a profound early loss of lysosomal calcium release, identifying lysosomal failure as an initiating event in astrocyte dysfunction in AD. Reduced expression of the lysosomal calcium channel TRPML1 provides the mechanistic basis for this deficit. Astrocyte-specific restoration of TRPML1 expression rescues lysosomal homeostasis and cytosolic calcium signalling and prevents astrocyte reactivity and morphological hypertrophy. Strikingly, early TRPML1 restoration prevents both the initial calcium hypoactivity observed before plaque formation and the later hyperactivity that characterises post-plaque disease, demonstrating that lysosomal calcium homeostasis stabilises astrocyte function across the disease trajectory. TRPML1 restoration also reduces amyloid plaque burden, indicating that astrocytic lysosomal competence directly shapes disease pathology. These findings identify lysosomal calcium failure as an early organelle-level mechanism linking amyloid stress to astrocyte dysfunction in AD, and position TRPML1-mediated lysosomal calcium signalling as a tractable target for limiting disease progression.

neuroscience↗

A stable subgenomic reporter coronavirus enables transcriptional profiling of bystander cells.

Insertion of fluorescent reporter genes into viral genomes is a powerful tool for monitoring infection. In coronaviruses, this is commonly achieved by replacing accessory open reading frames, thereby deleting endogenous gene functions. An alternative strategy is to manipulate viral RNA synthesis by inserting copies of the viral transcription regulatory sequence (TRS) which drive the transcription of viral subgenomic RNAs. However, coronavirus transcription is tightly regulated, and these modifications frequently disrupt native subgenomic RNA synthesis and attenuate viral growth. Here, we describe a reporter coronavirus that overcomes these limitations. Using human coronavirus (HCoV)-OC43 as a model system, we inserted an mNeonGreen reporter between the Spike and ORF5 coding regions, engineering the TRS and surrounding sequence to minimise off-target effects to transcription. This virus is genetically stable, with wildtype growth kinetics and unaltered subgenomic RNA transcriptional ratios. We developed a flexible reverse genetics system, which allows rapid cloning and virus recovery, supported by optimised HCoV-OC43 culture conditions, for high-titre stock generation, and validated analytical reagents. Our reporter virus enabled sensitive detection and isolation of infected cells, facilitating transcriptomic analyses that distinguish host responses in infected and bystander populations. We found that transcriptional responses to infection of cells in culture were predominantly inflammatory, rather than interferon-mediated, and that bystander cells upregulated pathways associated with cytokine response signalling and cell-cell contact sensing. Together, these tools expand the experimental utility of HCoV-OC43, an important seasonal respiratory pathogen and low containment model for betacoronavirus biology.

microbiology↗

Dlx2 reprograms the transcriptome and laminar position of glia-derivedAscl1-induced interneurons

Direct lineage reprogramming of glial cells into neurons offers a promising strategy to repair diseased brain circuits, but engineering defined neuronal subtypes remains challenging. We found that a phospho-site-deficient Ascl1 variant, Ascl1SA6, but not wildtype Ascl1, induces hallmarks of parvalbumin fast-spiking interneurons, raising the question of how closely these induced neurons resemble canonical cortical interneurons and what transcriptional events underlie this process. Single-cell transcriptomic analysis revealed that Ascl1SA6-induced neurons only partially recapitulated canonical interneuron programs and failed to induce the transcription factor Dlx2 and its downstream targets. Co-expression of Dlx2 with Ascl1SA6 restored a more canonical interneuron-like transcriptome, including genes involved in migration, and resulted in neurons occupying laminar positions more typical of endogenous interneurons. These findings provide molecular insights into how Ascl1 posttranslational modifications regulate its transcriptional activity and demonstrate a strategy to engineer induced cortical interneurons that more closely resemble their native counterparts, offering a framework for layer-specific restoration of inhibitory circuits in neurological diseases.

neuroscience↗

Lactate Blocks Tertiary Lymphoid Structure Formation by Inhibiting B Cell Chemotaxis

Tertiary lymphoid structures (TLS) and B cell infiltration are strong predictors of immunotherapy success across cancers, including triple-negative breast cancer (TNBC). However, immune-cold TNBCs often lack both features. Here, we identify a tumor-intrinsic mechanism that actively suppresses B cell recruitment. Despite evidence of B cell responses in cancer-associated lymph nodes (cLNs), B cells fail to infiltrate TNBC tumors or form TLS. This exclusion is not simply due to chemokine deficiency as exogenous chemokine addition fails to restore B cell migration. Using fractionation and metabolic profiling, we identify lactate as a dominant tumor-secreted metabolite that directly impairs B cell chemotaxis by disrupting mitochondrial metabolism. In vivo, combining lactate inhibition with engineered chemokine secretion promotes cLN-derived B cell infiltration and enables TLS formation, particularly when coupled with CD40 stimulation. Transcriptomics analyses across several human cancer datasets strengthen the association between high glycolytic activity with poor B-cell infiltration in chemokine-rich tumors. Together, our findings reveal lactate as a key metabolic barrier to B cell trafficking and TLS induction, suggesting that metabolic reprogramming may provide an avenue to convert "immune-cold" tumors into TLS-rich, immunologically responsive microenvironments.

cancer biology↗

Barcoded Rabies In Situ Connectomics for high-throughput reconstruction of neural circuits

Sequencing of oligonucleotide barcodes holds promise as a high-throughput approach for reconstructing synaptic connectivity at scale (1). Rabies viruses can act as a vehicle for barcode transmission, thanks to their ability to spread between synaptically connected cells (2, 3). However, applying barcoded rabies viruses to map synaptic connections in vivo has proved challenging (4-7). Here, we develop Barcoded Rabies In Situ Connectomics (BRISC) for high-throughput connectivity mapping in the mouse brain. To ensure that the majority of post-synaptic "starter" neurons are uniquely labeled with distinct barcode sequences, we first generated libraries of rabies viruses with sufficient diversity to label >1000 neurons uniquely. To minimize the probability of barcode transmission between starter neurons, we developed a strategy to tightly control their density. We then applied BRISC to map inputs of single neurons in the primary visual cortex (V1). Using in situ sequencing, we read out the expression of viral barcodes in rabies-infected neurons, while preserving spatial information. We then matched barcode sequences between starter and presynaptic neurons, mapping the inputs of 385 neurons and identifying 7,814 putative synaptic connections. The resulting connectivity matrix revealed layer- and cell-type-specific local connectivity rules and topographic organization of long-range inputs to V1. These results show that BRISC can simultaneously resolve the synaptic connectivity of hundreds of neurons while preserving spatial information, enabling reconstruction of neural circuits at an unprecedented scale.

neuroscience↗

MitoTracker transfers from astrocytes to neurons independently of mitochondria

The neuroprotective transfer of mitochondria from astrocytes to neurons has been primarily investigated by labelling astrocytic mitochondria with the dye MitoTracker. Here we report that MitoTracker transfers to neurons from both astrocytes and astrocyte-conditioned media, independently of mitochondrial transfer. Our observations should prompt an essential re-evaluation of the literature concerning astrocyte-neuron mitochondrial transfer and in other systems in which contact-independent transfer has been observed using mitochondrial dyes.

neuroscience↗

Integration of hunger and hormonal state gates infant-directed aggression

Social behaviour is profoundly shaped by internal physiological states. While significant progress has been made in understanding how individual states such as hunger, stress, or arousal modulate behaviour, animals experience multiple states at any given time. The neural mechanisms that integrate such orthogonal states--and how this integration affects behaviour--remain poorly understood. Here we report how hunger and estrous state converge on neurons in the medial preoptic area (MPOA) to shape infant-directed behaviour. We find that hunger promotes pup-directed aggression in normally non-aggressive virgin female mice. This behavioural switch occurs through inhibition of MPOA neurons, driven by the release of neuropeptide Y (NPY) from Agouti-related peptide-expressing neurons in the arcuate nucleus (ArcAgRP neurons). The propensity for hunger-induced aggression is set by reproductive state, with MPOA neurons detecting changes in progesterone (P4) to estradiol (E2) ratio across the estrous cycle. Hunger and estrous state converge on HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, which sets the baseline activity and excitability of MPOA neurons. Using micro-endoscopic imaging, we confirm these findings in vivo, revealing that MPOA neurons encode a state for pup-directed aggression. This work thus provides a mechanistic understanding of how multiple physiological states are integrated to flexibly control social behaviour.

neuroscience↗

High-throughput screening of human genetic variants by pooled prime editing

Understanding the effects of rare genetic variants remains challenging, both in coding and non-coding regions. While multiplexed assays of variant effect (MAVEs) have enabled scalable functional assessment of variants, established MAVEs are limited by either exogenous expression of variants or constraints of genome editing. Here, we introduce a pooled prime editing (PE) platform in haploid human cells to scalably assay variants in their endogenous context. We first optimized delivery of variants to HAP1 cells, defining optimal pegRNA designs and establishing a co-selection strategy for improved efficiency. We characterize our platform in the context of negative selection by testing over 7,500 pegRNAs targeting SMARCB1 for editing activity and observing depletion of highly active pegRNAs installing loss-of-function variants. We next assess variants in MLH1 via 6-thioguanine selection, assaying 65.3% of all possible SNVs in a 200-bp region spanning exon 10 and distinguishing LoF variants with high accuracy. Lastly, we assay 362 non-coding MLH1 variants across a 60 kb region in a single experiment, identifying pathogenic variants acting via multiple mechanisms with high specificity. Our analyses detail how filtering for highly active pegRNAs can facilitate both positive and negative selection screens. Accordingly, our platform promises to enable highly scalable functional assessment of human variants.

genomics↗