Biology subjects

Spitale, R. C.

Publications and source records attributed to Spitale, R. C..

9 recordsLinked to original sources

bioRxiv2026

Upregulation of Calbindin in Adult Inhibitory Neurons Reactivates Critical Period Plasticity in Mouse Visual Cortex

Critical periods are windows of peak learning performance where heightened synaptic plasticity enables fast, robust reorganization in juvenile brain circuits. Unlike adult plasticity which requires ongoing, persistent change in sensory experience, cortical representations can be rapidly changed during critical periods and have enduring effects. Transplantation of GABAergic inhibitory neurons has been shown to restore critical period plasticity to recipient circuits by triggering signalling changes within host inhibitory neurons. Here we transcriptionally profiled host inhibitory neurons in mouse primary visual cortex (V1) to detect gene expression changes during transplant-induced plasticity. Gene ontology enrichment analysis of differentially-expressed (DE) transcripts in host inhibitory neurons revealed synaptic plasticity- and inhibitory neuron development-related profiles. We assessed the protein expression of a top DE candidate, the calcium-binding protein Calbindin (Calb1), across transplant conditions and developmental stages. We found high Calb1 expression during the V1 critical period and during transplant-induced plasticity. To assess the functional significance of transplant-reactivated DE gene activity on visual cortical plasticity, we developed a set of AAVs to manipulate Calb1 expression specifically within inhibitory neurons. Using intrinsic signal imaging to measure ocular dominance plasticity, we found that Calb1 levels in V1 inhibitory neurons determine the extent of visual cortical plasticity. Our study provides evidence that direct induction of critical period-stage gene expression patterns in inhibitory neurons restores juvenile plasticity in targeted adult cortical circuits.

neuroscience↗

bioRxiv2024

Quantification of subcellular RNA localization through direct detection of RNA oxidation

Across cell types and organisms, thousands of RNAs display asymmetric subcellular distributions. The study of this process often requires quantifying abundances of specific RNAs at precise subcellular locations. To analyze subcellular transcriptomes, multiple proximity-based techniques have been developed in which RNAs near a localized bait protein are specifically labeled, facilitating their biotinylation and purification. However, these complex methods are often laborious and require expensive enrichment reagents. To streamline the analysis of localized RNA populations, we developed Oxidation-Induced Nucleotide Conversion sequencing (OINC-seq). In OINC-seq, RNAs near a genetically encoded, localized bait protein are specifically oxidized in a photo-controllable manner. These oxidation events are then directly detected and quantified using high-throughput sequencing and our software package, PIGPEN, without the need for biotin-mediated enrichment. We demonstrate that OINC-seq can induce and quantify RNA oxidation with high specificity in a dose- and light-dependent manner. We further show the spatial specificity of OINC-seq by using it to quantify subcellular transcriptomes associated with the cytoplasm, ER, nucleus, and the inner and outer membranes of mitochondria. Finally, using transgenic zebrafish, we demonstrate that OINC-seq allows proximity-mediated RNA labeling in live animals. In sum, OINC-seq together with PIGPEN provide an accessible workflow for the analysis of localized RNAs across different biological systems.

molecular biology↗

bioRxiv2023

Aberrant splicing in Huntington's disease via disrupted TDP-43 activity accompanied by altered m6A RNA modification.

Huntingtons disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene encoding huntingtin. Prior reports have established a correlation between CAG expanded HTT and altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. Binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Discovery of synapse-specific RNA N6-methyladenosine readers associated with the consolidation of fear extinction memory

The RNA modification N6-methyladenosine (m6A) is critically involved in the regulation of gene activity underlying experience-dependent plasticity, and is necessary for the functional interplay between RNA and RNA binding proteins (RBPs) in the nucleus. However, the complete repertoire of m6A-modified RNA interacting RBPs in the synaptic compartment, and whether they are involved in fear extinction, have yet to be revealed. Using RNA immunoprecipitation followed by mass spectrometry, we discovered 12 novel, synapsespecific, learning-induced m6A readers in the medial prefrontal cortex of male C57/B6 mice. m6A RNA-sequencing also revealed a unique population of learning-related m6A-modified RNAs at the synapse, which includes a variant of the long non-coding RNA (lncRNA) metastasis associated lung adenocarcinoma transcript 1 (Malat1). m6A-modified Malat1 binds to a subset of novel m6A readers, including cytoplasmic FMR1 interacting protein 2 (CYFIP2) and dihydropyrimidase-related protein 2 (DPYSL2) and a cell-type-specific, state-dependent, and synapse-specific reduction in m6A-modified Malat1 disrupts the interaction between Malat1 and DPYSL2 and impairs fear extinction. The consolidation of fear-extinction memory therefore relies on an interaction between m6A-modified Malat1 and select RBPs in the synaptic compartment.

neuroscience↗

bioRxiv2022

Orphan C/D box snoRNAs modulate fear-related memory processes in mice

C/D box small nucleolar RNAs (snoRNAs) comprise a class of small noncoding RNAs with important regulatory effects on cellular RNA function. Although it is well established that snoRNAs coordinate the post-transcriptional modification of pre-ribosomal and small nuclear RNAs by 2-O-methylation, which leads to enhanced RNA stability, whether they are necessary for memory-related processes remains relatively unexplored. Using targeted sequencing, we have identified more than 150 C/D box snoRNAs in the prefrontal cortex of male C57BL/6J mice, 31 of which are differentially expressed in response to fear extinction learning. We have also discovered a subset of snoRNAs, including many orphans, that are enriched in the synaptic compartment, including the orphan snoRNA snord64. An extinction learning-induced increase in synapse-enriched snord64 led to increased 2-O-methylation within the 3-UTR of the mRNA encoding the ubiquitin ligase RNF146. This effect was blocked by snord64 knockdown and was accompanied by attenuated forgetting of conditioned fear and the enhanced retrieval of fear extinction memory. Localized activity of orphan snoRNAs therefore represents a novel mechanism associated with fear-related learning and memory. Significance statementWe have discovered a population of experience-dependent small nucleolar RNAs (snoRNAs), and found that several of these are recruited to the synaptic compartment in response to fear extinction learning. In particular, the orphan snoRNA, snord64, drives the methylation of the mRNA encoding the ubiquitin ligase, RNF146, with a reduction in snord64 attenuating forgetting of conditioned fear and enhancing the retrieval of fear extinction memory. This study reveals a new mechanism of gene regulation associated with fear-related memory that involves the activity of synapse-enriched snoRNAs.

neuroscience↗

bioRxiv2022

Fear extinction is regulated by long noncoding RNA activity at the synapse

Long noncoding RNAs (lncRNAs) represent a multidimensional class of regulatory molecules involved in many aspects of brain function. Emerging evidence indicates that lncRNAs are expressed at the synapse; however, a direct role for their activity in this subcellular compartment in memory formation has yet to be demonstrated. Using lncRNA capture-seq on synaptosomes, we identified a significant number of lncRNAs that accumulate at synapses within the infralimbic prefrontal cortex of adult male C57/Bl6 mice. Among these is a splice variant related to the stress-associated lncRNA, Gas5. RNA immunoprecipitation followed by mass spectrometry and single molecule imaging revealed that this Gas5 isoform, in association with the RNA binding proteins G3bp2 and Caprin1, regulates the activity-dependent trafficking and clustering of RNA granules in dendrites. In addition, we found that cell-type-specific, state-dependent, and synapse-specific knockdown of the Gas5 variant led to impaired fear extinction memory. These findings identify a new mechanism of fear extinction that involves the dynamic interaction between local lncRNA activity and the coordination of RNA condensates in the synaptic compartment.

neuroscience↗

bioRxiv2020

aPKC drives cilia-independent Hedgehog signaling to maintain basal cell carcinoma growth

Primary cilia loss is a common feature of advanced cancers. While primary cilia are necessary to initiate Hedgehog (HH)-driven cancers, how HH pathway activity is maintained in advanced cancers devoid of primary cilia is unclear. Here, we find that HH-driven basal cell carcinoma (BCC) accumulates mutations in Alstrom and Usher syndrome genes. Loss of Alstrom and Usher syndrome gene expression, which are common underlying causes of deafness and blindness, suppresses primary ciliogenesis and HH signaling but enhances expression of atypical protein kinase C iota/lambda (aPKC), a GLI1 kinase necessary for advanced BCC growth. We show that aPKC expression is inversely correlated with primary ciliogenesis and that superficial BCCs display less primary cilia and higher aPKC expression, with the opposite trend in nodular BCC subtypes. Surprisingly, a constitutively active isoform of aPKC but not full-length protein drives HH pathway activity. Overexpression of the constitutively active aPKC variant can maintain HH pathway activity and tumor growth in the absence of primary cilia. Our results suggest tumors enhance isoform-specific expression of aPKC to prevent mutation-induced cessation of tumor growth.

cancer biology↗

bioRxiv2019

Functional conservation of lncRNA JPX despite sequence and structural divergence

Long noncoding RNAs (lncRNAs) have been identified in all eukaryotes and are most abundant in the human genome. However, the functional importance and mechanisms of action for human lncRNAs are largely unknown. Using comparative sequence, structural, and functional analyses, we characterize the evolution and molecular function of human lncRNA JPX. We find that human JPX and its mouse homolog, lncRNA Jpx, have deep divergence in their nucleotide sequences and RNA secondary structures. Despite such differences, both lncRNAs demonstrate robust binding to CTCF, a protein that is central to Jpxs role in X chromosome inactivation. In addition, our functional rescue experiment using Jpx-deletion mutant cells, shows that human JPX can functionally complement the loss of Jpx in mouse embryonic stem cells. Our findings support a model for functional conservation of lncRNAs independent from sequence and structural changes. The study provides mechanistic insight into the evolution of lncRNA function.

molecular biology↗

bioRxiv2019

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Alternative splicing is widely acknowledged to be a crucial regulator of gene expression and is a key contributor to both normal developmental processes and disease states. While cost-effective and accurate for quantification, short-read RNA-seq lacks the ability to resolve full-length transcript isoforms despite increasingly sophisticated computational methods. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) bypass the transcript reconstruction challenges of short reads. Here we introduce TALON, the ENCODE4 pipeline for platform-independent analysis of long-read transcriptomes. We apply TALON to the GM12878 cell line and show that while both PacBio and ONT technologies perform well at full-transcript discovery and quantification, each displayed distinct technical artifacts. We further apply TALON to mouse hippocampus and cortex transcriptomes and find that 422 genes found in these regions have more reads associated with novel isoforms than with annotated ones. We demonstrate that TALON is a capable of tracking both known and novel transcript models as well as their expression levels across datasets for both simple studies and in larger projects. These properties will enable TALON users to move beyond the limitations of short-read data to perform isoform discovery and quantification in a uniform manner on existing and future long-read platforms.

genomics↗