Biology subjects

Berbari, N. F.

Publications and source records attributed to Berbari, N. F..

5 recordsLinked to original sources

bioRxiv2021

The hedgehog signaling pathway is expressed in the adult mouse hypothalamus and modulated by fasting

The importance of the primary cilium was initially highlighted by the class of human genetic disorders known as ciliopathies. Patients with ciliopathies such as Bardet-Biedl and Alstrom syndrome exhibit hyperphagia-associated obesity as a core clinical phenotype. How primary cilia contribute to energy homeostasis and feeding behavior is complex and unclear, but cilia appear important in both developmental and homeostatic processes. Primary cilia are important signaling centers, required for hedgehog signaling and localization of specific G protein-coupled receptors (GPCRs) with known roles in feeding behavior in mammals. The hedgehog pathway is best known for its role in developmental patterning, but now has recognized roles in adult tissues as well. In the postnatal brain, cilia and hedgehog signaling are important for growth and maintenance of neural progenitors, however, the role of hedgehog signaling in the differentiated adult brain is less clear. Here, we provide a detailed analysis of the expression of core components of the hedgehog signaling pathway in the adult mouse hypothalamus with an emphasis on feeding centers. We show that hedgehog pathway genes continue to be expressed in differentiated neurons important for regulation of feeding behavior. Furthermore, we demonstrate for the first time that pathway activity is regulated at the transcriptional level by fasting. These data suggest that hedgehog signaling is involved in the proper functioning of brain regions which regulate feeding behavior and that hedgehog pathway dysfunction may play a role in the obesity observed in certain ciliopathies. Significance StatementHere we investigate the expression of hedgehog pathway components in the adult mouse hypothalamus. Using dual labeling in situ hybridization we show that core components of the signaling pathway are expressed in multiple neuronal cell types within the hypothalamic feeding centers. Our findings also support previous findings that astrocytes are responsive to hedgehog signaling, as determined by Gli1 and Ptch1 expression. Using qPCR analysis, we show that hypothalamic hedgehog pathway activity is upregulated in response to fasting and this response is nuclei specific. These data not only provide a more detailed understanding of hedgehog pathway expression in the adult mouse hypothalamus but also provide direct evidence of a novel role for hedgehog signaling in the physiological response to fasting.

neuroscience↗

bioRxiv2021

An N-terminal Fusion Allele to Study Melanin Concentrating Hormone Receptor 1

Cilia on neurons play critical roles in both the development and function of the central nervous system (CNS). While it remains challenging to elucidate the precise roles for neuronal cilia, it is clear that a subset of G-protein-coupled receptors (GPCRs) preferentially localize to the cilia membrane. Further, ciliary GPCR signaling has been implicated in regulating a variety of behaviors. Melanin concentrating hormone receptor 1 (MCHR1), is a GPCR expressed centrally in rodents known to be enriched in cilia. Here we have used MCHR1 as a model ciliary GPCR to develop a strategy to fluorescently tag receptors expressed from the endogenous locus in vivo. Using CRISPR/Cas9, we inserted the coding sequence of the fluorescent protein mCherry into the N-terminus of Mchr1. Analysis of the fusion protein (mCherryMCHR1) revealed its localization to neuronal cilia in the CNS, across multiple developmental time points and in various regions of the adult brain. Our approach simultaneously produced fortuitous in/dels altering the Mchr1 start codon resulting in a new MCHR1 knockout line. Functional studies using electrophysiology show a significant alteration of synaptic strength in MCHR1 knockout mice. A reduction in strength is also detected in mice homozygous for the mCherry insertion, suggesting that while the strategy is useful for monitoring the receptor, activity could be altered. However, both lines should aid in studies of MCHR1 function and contribute to our understanding of MCHR1 signaling in the brain. Additionally, this approach could be expanded to aid in the study of other ciliary GPCRs.

neuroscience↗

bioRxiv2021

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and colocalization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis. SUMMARYThe use of Artificial Intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

cell biology↗

bioRxiv2021

Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function.

N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of spinophilin promotes pathological sequelae associated with GluN2B dysfunction. HIGHLIGHTSO_LISpinophilin bidirectionally regulates GluN2B-containing NMDAR function. C_LIO_LILoss of spinophilin in primary hippocampal neurons increases a pro-apoptotic marker. C_LIO_LILoss of spinophilin in vivo decreases measures of spatial cognitive flexibility. C_LI Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of spinophilin, the spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of spinophilin reduces cognitive flexibility in hippocampal dependent tasks. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/424812v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1548b5org.highwire.dtl.DTLVardef@158a0aaorg.highwire.dtl.DTLVardef@19c185dorg.highwire.dtl.DTLVardef@103848b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

A Bbs5 mouse model reveals pituitary cilia contributions to developmental abnormalities.

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl Syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay, and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain which are developmental in origin. A subset of BBS proteins assembles into the BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here we describe two new mouse models for BBS resulting from a congenital null and conditional allele of Bbs5. Bbs5 null mice develop a complex phenotype including craniofacial defects, skeletal shortening, ventriculomegaly, infertility, and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly, and pituitary abnormalities are only found when Bbs5 is mutated prior to P7 indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity independent of the age of Bbs5 loss. Compared to other animal models of BBS, Bbs5 mutant mice exhibit pathologies that suggest a specialized role for Bbs5 in ciliary function.

developmental biology↗