Biology subjects

Fak, J. J.

Publications and source records attributed to Fak, J. J..

8 recordsLinked to original sources

bioRxiv2026

Lipid Activation of a Thalamic GPCR Extends Working Memory Time-scales

Working memory capacity is poorly understood. The ability to control and extend working memory time-scales provides the potential to alleviate cognitive decline in disease and aging. Through an unbiased genetic search, we previously identified a thalamic orphan receptor Gpr12 as a potent enhancer of working memory, however its activation mechanism remains poorly understood. Here, we describe the CryoEM structure of Gpr12, revealing a lipidic regulatory site enabling an activated signaling state. By surveying the native lipidic environment in mouse thalamus we identified a class of 20 carbon:4 double bond fatty acid eicosanoids as potential ligands. Cell-based assays confirmed that the endogenous cannabinoid anandamide (AEA), but not other closely related family members or derivatives, robustly activates Gpr12. In vivo imaging during behavior revealed that Gpr12 activation produces a striking molecular state - the persistent suppression of cAMP in thalamus that tracks the duration of memory maintenance. Notably, genetic or pharmacological manipulations that enhance the AEA-Gpr12 signaling axis are sufficient to prolong cAMP suppression and extend the temporal window of memory maintenance. Furthermore, AEA-mediated cAMP suppressions in thalamus support sustained neural activity in PFC, specifically during memory maintenance. Thus, while cannabinoids often impair memory, here we identify an AEA-Gpr12 signaling axis in thalamus that enhances memory, including in primates. These findings identify a lipidic signaling mechanism in thalamus that is sufficient to control and extend working memory duration.

neuroscience↗

bioRxiv2023

A Genetic Locus Mediating Attentional Processing

Attention is required for most higher-order cognitive functions. Prior studies have revealed functional roles for the prefrontal cortex and its extended circuits to enabling attention, but the underlying molecular processes and their impacts on cellular and circuit function remain poorly understood. To develop insights, we here took an unbiased forward genetics approach to identify single genes of large effect on attention. We studied 200 genetically diverse mice on measures of pre-attentive processing and through genetic mapping identified a small locus on chromosome 13 (95%CI: 92.22-94.09 Mb) driving substantial variation (19%) in this trait. Further characterization of the locus revealed a causative gene, Homer1, encoding a synaptic protein, where down-regulation of its short isoforms in prefrontal cortex (PFC) during early postnatal development led to improvements in multiple measures of attention in the adult. Subsequent mechanistic studies revealed that prefrontal Homer1 down-regulation is associated with GABAergic receptor up-regulation in those same cells. This enhanced inhibitory influence, together with dynamic neuromodulatory coupling, led to strikingly low PFC activity at baseline periods of the task but targeted elevations at cue onset, predicting short-latency correct choices. Notably high-Homer1, low-attentional performers, exhibited uniformly elevated PFC activity throughout the task. We thus identify a single gene of large effect on attention - Homer1 - and find that it improves prefrontal inhibitory tone and signal-to-noise (SNR) to enhance attentional performance. A therapeutic strategy focused on reducing prefrontal activity and increasing SNR, rather than uniformly elevating PFC activity, may complement the use of stimulants to improve attention.

neuroscience↗

bioRxiv2021

FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons

Neurons are believed to rely on dendritic localization and translation of mRNAs in order to generate activity-dependent changes in the synaptic plasticity. Here, we develop a strategy combining compartment-specific CLIP and TRAP in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. This revealed transcripts that have differentially localized alternative 3UTR and splicing isoforms. FMRP targets are overrepresented among dendritic mRNAs, and compartment-specific FMRP-CLIP defined 383 dendritic FMRP targets, and also allowed for segregation of whole-cell FMRP targets into functional modules that are locally regulated by FMRP. In the absence of FMRP, dendritic FMRP targets show increased ribosome association, consistent with reported roles for FMRP in translational repression. Together, the data support a model in which distinct patterns of FMRP localization allow it to differentially regulate the expression of nuclear proteins and synaptic proteins within different compartments of a single neuronal cell type.

neuroscience↗

bioRxiv2019

FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

Loss of function of the Fragile X Mental Retardation Protein (FMRP) in human Fragile X Syndrome (FXS) and in model organisms results in phenotypes of abnormal neuronal structure and dynamics, synaptic function and connectivity which may contribute to a state of neuronal, circuit and organism hyperexcitability. Previous in vivo identification of FMRP association with specific mRNA targets in mouse brain revealed that FMRP regulates the translation of a large fraction of the synaptic proteome in both pre- and post-synaptic compartments as well as many transcription factors and chromatin modifying proteins. However, it was not previously possible to determine the ratio of FMRP binding to transcript abundance due to the complexity of different neuronal cell types in whole brain. Moreover, it has been difficult to link the translational regulation of specific targets to model phenotypes or human symptoms. For example, loss-of-function of FMRP in the Purkinje cells of the cerebellum results in three cell autonomous phenotypes related to learning and memory, including enhanced mGluR-LTD at parallel fiber synapses, altered dendritic spines and behavioral deficits in a eyeblink-conditioning learning paradigm shared by human FXS patients. The molecular basis for these and related human Fragile X phenotypes is unknown. To address these critical issues we have developed a new mouse model (the Fmr1 cTAG mouse) in which endogenous FMRP can be conditionally tagged for RNA:protein crosslinking and immunoprecipitation (CLIP) identification of the RNAs with which it interacts in vivo. We used the Fmr1 cTAG mouse to quantitatively evaluate FMRP-mRNA association in Purkinje and cerebellar granule neurons which together comprise the parallel-fiber synapse. We calculated a stoichiometrically ranked list of FMRP RNA binding events by normalizing to ribosome-associated transcript abundance determined by TRAP-seq, and now definitively find that FMRP associates with specific sets of mRNAs which differ between the two cell types. In Purkinje cells, many components of the mGluR signaling pathway are FMRP targets including the top-ranked Purkinje cell mRNA Itpr1, encoding the IP3 receptor, the function of which is critical to proper mGluR-dependent synaptic plasticity. In sum, this novel approach provides the first ranked list of FMRP target mRNAs and further reveals that FMRP regulates a specific set of long neural genes related to relevant cell autonomous phenotypes.\n\nHighlightsO_LIWe have created a mouse model in which endogenous FMRP can be conditionally tagged.\nC_LIO_LIUsing tag-specific CLIP we describe ranked and specific sets of in vivo FMRP mRNA targets in two types of neurons.\nC_LIO_LIThis ranking was used to reveal that FMRP regulates mRNAs with long coding sequences.\nC_LIO_LIFMRP mRNA targets in Purkinje cells, including the top-ranked IP3 receptor, are related to cell-autonomous Fragile X phenotypes.\nC_LIO_LIWe have updated our previous list of whole mouse brain FMRP mRNA targets with more replicates, deeper sequencing and improved analysis\nC_LIO_LIThe use of tagged FMRP in less abundant cell populations allowed identification of novel mRNA targets missed in a whole brain analysis\nC_LI

neuroscience↗

bioRxiv2018

ZFP36 RNA-binding proteins restrain T-cell activation and anti-viral immunity

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in T-cells, which revealed unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through a novel class of AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics in a cell autonomous manner, by attenuating activation marker expression, limiting T-cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T-cell responses to acute viral infection, and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T-cell expansion and effector functions, and suggest ZFP36 inhibition as a novel strategy to enhance immune-based therapies.

molecular biology↗

bioRxiv2018

Differential 3’ Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

neuroscience↗

bioRxiv2018

A master regulatory network restoring brain glutamate homeostasis is coordinately activated in stroke

Post-transcriptional regulation by miRNAs is essential for complex molecular responses to physiological insult and disease. Although many disease-associated miRNAs are known, their global targets and culminating network effects on pathophysiology remain poorly understood. We applied AGO CLIP to systematically elucidate altered miRNA-target interactions in brain following ischemia/reperfusion (I/R) injury. Among 1,190 identified, most prominent was the cumulative loss of target regulation by miR-29 family members. Integration of translational and time-course RNA profiles revealed a dynamic mode of miR-29 target de-regulation, led by acute translational activation and later increase in RNA levels, allowing rapid proteomic changes to take effect. These functional regulatory events rely on canonical and non-canonical miR-29 binding and engage glutamate reuptake signals to control local glutamate levels. These results uncover a miRNA target network that acts acutely to maintain brain homeostasis after ischemic stroke.

neuroscience↗

bioRxiv2018

Neuron-specific cTag-CLIP reveals cell-specific diversity of functional RNA regulation in the brain

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell-types in vivo is unknown. We employed NOVA2 cTag-CLIP to generate functional RBP-RNA maps from single neuronal populations in the mouse brain. Combining cell-type specific data from Nova2-cTag and Nova2 conditional knock-out mice revealed differential NOVA2 regulatory actions (e.g. alternative splicing) on the same transcripts in different neurons, including in cerebellar Purkinje cells, where NOVA2 acts as an essential factor for proper motor coordination and synapse formation. This also led to the discovery of a mechanism by which NOVA2 action leads to different outcomes in different cells on the same transcripts: NOVA2 is able to regulate retained introns, which subsequently serve as scaffolds for another trans-acting splicing factor, PTBP2. Our results describe differential roles and mechanisms by which RBPs mediate RNA diversity in different neurons and consequent functional outcomes within the brain.

neuroscience↗