Biology subjects

Hoogenraad, C. C.

Publications and source records attributed to Hoogenraad, C. C..

11 recordsLinked to original sources

bioRxiv2026

Integrated morphological, multi-omics, and functional profiling reveals microglial plasticity driven by AD risk genes

Microglia, the resident macrophages of the central nervous system, are highly dynamic cells essential for brain homeostasis. While genome-wide association studies (GWAS) strongly implicate microglial dysfunction in Alzheimer's disease (AD), the mechanistic links coordinating their diverse transcriptional, morphological, and functional states remain poorly understood. Using human induced pluripotent stem cell-derived microglia (iMicroglia) and single-cell RNA sequencing, we identified six distinct transcriptional profiles and mapped them to specific morphological phenotypes via targeted immunofluorescence, establishing a link between microglial morphology and molecular identity. Transcriptomic and morphological profiling further demonstrated profound microglial plasticity, revealing distinct, stimulus-specific responses to AD-relevant pathologies, including Tau PFF, amyloid-beta, and apoptotic neurons. To assess how AD risk variants perturb these states, we performed high-efficiency CRISPR-Cas9 ribonucleoprotein (RNP) knockouts of specific AD GWAS genes. Bulk RNA-seq profiling revealed extensive transcriptional remodeling following genetic perturbation. Crucially, we show that depletion of these AD risk genes disrupts baseline morpho-transcriptomic coupling and fundamentally alters microglial phagocytic capacity. Together, this study reveals how AD GWAS genes may drive microglia into dysfunctional states characterized by altered morphology, distinct multi-omic signatures, and impaired functions.

neuroscience↗

bioRxiv2026

Soma-seq links intracellular protein states to transcriptional programs in the human brain

Neurodegenerative disease is largely driven by pathological protein states, which cannot be fully inferred from transcript levels. Yet, joint measurement of cytoplasmic proteins and RNA in single cells from archival human brain tissue remains challenging. Here we present Soma-seq, a method integrating transcriptome and antibody-based intracellular protein measurements in single cells from frozen human brain tissue. Applying Soma-seq to Alzheimers disease cortex, we quantified multiple intracellular proteins, including hyperphosphorylated Tau, alongside RNA. Soma-seq resolves a continuous trajectory of pathological progression based on multiplexed protein measurements and reveals associated gene programs. CRISPR perturbation in human iPSC-derived neurons validates Soma-seq-derived candidates and identifies protective factors to Tau aggregation.

neuroscience↗

bioRxiv2024

SARM1 orthosteric base exchange inhibitors cause subinhibitory SARM1 activation

SARM1, an octameric NADase, is a key regulator of axon degeneration and an emerging target in small molecule drug discovery to treat a wide range of neurodegenerative diseases. Recently, a structurally diverse series of adduct-forming, orthosteric SARM1 inhibitors have been discovered. Here, we show that subinhibitory concentrations of these orthosteric inhibitors, under mildly SARM1 activating conditions, cause sustained SARM1 activation. This synergistic adverse effect leads to increased nicotinamide adenine dinucleotide (NAD) consumption, neurodegeneration and release of the biomarker neurofilament-light (NfL) in cultured cortical neurons. In two distinct animal models, we found that low-dose treatment with these orthosteric SARM1 inhibitors results in increased plasma NfL and adverse events when combined with cellular stress or injury conditions. This may present a critical liability for orthosteric SARM1 inhibitors in certain patient populations.

neuroscience↗

bioRxiv2022

Axonal ER tubules regulate local translation via P180/RRBP1-mediated ribosome interactions

Local mRNA translation in axons is critical for the spatial and temporal regulation of the axonal proteome. A wide variety of mRNAs are localized and translated in axons, however how protein synthesis is regulated at specific subcellular sites in axons remains unclear. Here, we establish that the axonal endoplasmic reticulum (ER) supports axonal translation. Axonal ER tubule disruption impairs local translation and ribosome distribution. Using nanoscale resolution imaging, we find that ribosomes make frequent contacts with ER tubules in the axon in a translation-dependent manner and are influenced by specific extrinsic cues. We identify P180/RRBP1 as an axonally distributed ribosome receptor that regulates local translation in an mRNA-dependent manner. Our results establish an important role for the axonal ER in localizing mRNA translation and in dynamically regulating the axonal proteome in response to neuronal stimuli.

neuroscience↗

bioRxiv2020

The mechanism of selective kinesin inhibition by kinesin binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed -solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its -solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.

biochemistry↗

bioRxiv2020

ER - lysosome contacts at a pre-axonal region regulate axonal lysosome availability

Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the ER and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule - lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule - lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function.

cell biology↗

bioRxiv2020

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons

Early neuronal development is a well-coordinated process in which neuronal stem cells differentiate into polarized neurons. This process has been well studied in classical non-human model systems, but to what extent this is recapitulated in human neurons remains unclear. To study neuronal polarization in human neurons, we cultured human iPSC-derived neurons, characterized early developmental stages, measured electrophysiological responses, and systematically profiled transcriptomic and proteomic dynamics during these steps. We found extensive remodeling of the neuron transcriptome and proteome, with altered mRNA expression of ~1,100 genes and different expression profiles of ~1,500 proteins during neuronal differentiation and polarization. We also identified a distinct stage in axon development marked by an increase in microtubule remodeling and apparent relocation of the axon initial segment from the distal to proximal axon. Our comprehensive characterization and quantitative map of transcriptome and proteome dynamics provides a solid framework for studying polarization in human neurons.

neuroscience↗

bioRxiv2019

Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity and functioning

Neurons are among the most highly polarized cell types. They possess structurally and functionally different processes, axon and dendrites, to mediate information flow through the nervous system. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is little understood.\n\nUsing the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cell cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

cell biology↗

bioRxiv2018

MAP7 family proteins are microtubule-tethered allosteric activators of kinesin-1.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1 and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared to MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be co-transported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.\n\nSummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.

cell biology↗

bioRxiv2017

Molecular pathway underlying bouton stabilization by Semaphorin4D during inhibitory synapse formation

Changes in inhibitory connections are essential for experience-dependent circuit adaptations. Defects in inhibitory synapses are linked to neurodevelopmental disorders, but the molecular processes underlying inhibitory synapse formation are not well understood. Here we use high resolution two-photon microscopy in organotypic hippocampal slices to examine the signaling pathways induced by the postsynaptic signaling molecule Semaphorin4D (Sema4D) during inhibitory synapse formation. By monitoring changes in individual GFP-labeled presynaptic boutons we found that the primary action of Sema4D is to induce stabilization of presynaptic boutons within tens of minutes. Stabilizing boutons rapidly recruited synaptic vesicles, which was followed by accumulation of postsynaptic gephyrin. Newly formed inhibitory synapses were complete and functional after 24 hours, as determined by electrophysiology and immunohistochemistry. We further showed that Sema4D signaling is regulated by network activity and can induce a local increase in bouton density, suggesting a possible role in circuit adaptation. We further examined the intracellular signaling cascade triggered by Sema4D and found that bouton stabilization occurred through rapid remodeling of actin, and this could be mimicked by the actin-depolymerizing drug Latrunculin B or by reducing ROCK activity. The intracellular signaling cascade required activation of the receptor tyrosine kinase MET, which is a well-known autism risk factor. Our immunohistochemistry data suggests that MET may be localized to presynaptic inhibitory axons. Together, our data yield important insights in the molecular pathway underlying activity-dependent Sema4D-induced synapse formation and reveal a novel role for MET in inhibitory synapses.\n\nSignificance StatementGABAergic synapses provide the main inhibitory control of neuronal activity in the brain. We make important steps in unraveling the molecular processes that take place when formation of inhibitory synapses is triggered by a specific signaling molecule, Sema4D. We find that this process depends on network activity and involves specific remodeling of the intracellular actin cytoskeleton. We also reveal a previously unknown role for MET in inhibitory synapses. As defects in GABAergic synapses have been implied in many brain disorders, and mutations in MET are strong risk factors for autism, our findings urge for a further investigation of the role of MET at inhibitory synapses.

neuroscience↗

bioRxiv2016

Probing cytoskeletal modulation of passive and active intracellular dynamics using nanobody-functionalized quantum dots

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. Cytoskeletal organization and dynamics are known to modulate cytoplasmic transport processes, but how local transport dynamics depends on the highly heterogeneous intracellular organization of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for transport dynamics in different sub-cellular environments. Rapid imaging of non-functionalized QDs revealed two populations with a 100-fold difference in diffusion constant. Depolymerization of actin increased the fast diffusing fraction, suggesting that slow QDs are trapped inside the actin network. When nanobody-functionalized QDs were targeted to different kinesin motor proteins and moved over microtubules, they did not experience strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 bound QDs displayed subtle directional fluctuations, because the specific subset of stable microtubules used by this motor underwent prominent undulations. Using actin-targeting agents we found that the actin network suppresses most microtubule shape remodeling, rather than promoting them. These results demonstrate how the spatial and structural heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

biophysics↗