AAV9-mediated FIG4 delivery prolongs life span in Charcot Marie Tooth disease type 4J mouse model

The Journal of clinical investigation

2021 Apr 20

Presa, M;Bailey, RM;Davis, C;Murphy, T;Cook, J;Walls, R;Wilpan, H;Bogdanik, L;Lenk, GM;Burgess, RW;Gray, SJ;Lutz, C;
PMID: 33878035 | DOI: 10.1172/JCI137159

Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by recessive, loss-of-function mutations in FIG4, encoding a phosphoinositol(3,5)P2-phosphatase. CMT4J patients have both neuron loss and demyelination in the peripheral nervous system, with vacuolization indicative of endosome/lysosome trafficking defects. Although the disease is highly variable, the onset is often in childhood and FIG4 mutations can dramatically shorten lifespan. There is currently no treatment for CMT4J. Here we present the results of preclinical studies testing a gene therapy approach to restore FIG4 expression. A mouse model of CMT4J, the Fig4-pale tremor (plt) allele, was dosed with a single-stranded AAV9 to deliver a codon-optimized human FIG4 sequence. Untreated, Fig4plt/plt mice have a median survival of approximately 5 weeks. When treated with the AAV9-FIG4 vector at postnatal day 1 or 4, mice survived at least one year, with largely normal gross motor performance and little sign of neuropathy by neurophysiological or histopathological evaluation. When treated at postnatal day 7 or 11, life span was still significantly prolonged and peripheral nerve function was improved, but rescue was less complete. No unanticipated adverse effects were observed. Therefore, AAV9-mediated delivery of FIG4 is a well-tolerated and efficacious strategy in a mouse model of CMT4J.

Hypothalamic Rax+ tanycytes contribute to tissue repair and tumorigenesis upon oncogene activation in mice

Nature communications

2021 Apr 16

Mu, W;Li, S;Xu, J;Guo, X;Wu, H;Chen, Z;Qiao, L;Helfer, G;Lu, F;Liu, C;Wu, QF;
PMID: 33863883 | DOI: 10.1038/s41467-021-22640-z

Hypothalamic tanycytes in median eminence (ME) are emerging as a crucial cell population that regulates endocrine output, energy balance and the diffusion of blood-born molecules. Tanycytes have recently been considered as potential somatic stem cells in the adult mammalian brain, but their regenerative and tumorigenic capacities are largely unknown. Here we found that Rax+ tanycytes in ME of mice are largely quiescent but quickly enter the cell cycle upon neural injury for self-renewal and regeneration. Mechanistically, Igf1r signaling in tanycytes is required for tissue repair under injury conditions. Furthermore, Braf oncogenic activation is sufficient to transform Rax+ tanycytes into actively dividing tumor cells that eventually develop into a papillary craniopharyngioma-like tumor. Together, these findings uncover the regenerative and tumorigenic potential of tanycytes. Our study offers insights into the properties of tanycytes, which may help to manipulate tanycyte biology for regulating hypothalamic function and investigate the pathogenesis of clinically relevant tumors.

Ptchd1 mediates opioid tolerance via cholesterol-dependent effects on μ-opioid receptor trafficking

Nature neuroscience

2022 Aug 18

Maza, N;Wang, D;Kowalski, C;Stoveken, HM;Dao, M;Sial, OK;Giles, AC;Grill, B;Martemyanov, KA;
PMID: 35982154 | DOI: 10.1038/s41593-022-01135-0

Repeated exposure to opioids causes tolerance, which limits their analgesic utility and contributes to overdose and abuse liability. However, the molecular mechanisms underpinning tolerance are not well understood. Here, we used a forward genetic screen in Caenorhabditis elegans for unbiased identification of genes regulating opioid tolerance which revealed a role for PTR-25/Ptchd1. We found that PTR-25/Ptchd1 controls μ-opioid receptor trafficking and that these effects were mediated by the ability of PTR-25/Ptchd1 to control membrane cholesterol content. Electrophysiological studies showed that loss of Ptchd1 in mice reduced opioid-induced desensitization of neurons in several brain regions and the peripheral nervous system. Mice and C. elegans lacking Ptchd1/PTR-25 display similarly augmented responses to opioids. Ptchd1 knockout mice fail to develop analgesic tolerance and have greatly diminished somatic withdrawal. Thus, we propose that Ptchd1 plays an evolutionarily conserved role in protecting the μ-opioid receptor against overstimulation.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1

Cell reports

2022 Jan 11

Han, Y;Villarreal-Ponce, A;Gutierrez, G;Nguyen, Q;Sun, P;Wu, T;Sui, B;Berx, G;Brabletz, T;Kessenbrock, K;Zeng, YA;Watanabe, K;Dai, X;
PMID: 35021086 | DOI: 10.1016/j.celrep.2021.110240

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development

Biochemical and biophysical research communications

2021 Dec 27

Negishi, T;Mihara, N;Chiba, T;D'Armiento, J;Chada, K;Maeda, M;Igarashi, M;Imai, K;
PMID: 34973532 | DOI: 10.1016/j.bbrc.2021.12.093

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2-/- mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2-/- mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens

Nature communications

2021 May 18

Walter, ND;Born, SEM;Robertson, GT;Reichlen, M;Dide-Agossou, C;Ektnitphong, VA;Rossmassler, K;Ramey, ME;Bauman, AA;Ozols, V;Bearrows, SC;Schoolnik, G;Dolganov, G;Garcia, B;Musisi, E;Worodria, W;Huang, L;Davis, JL;Nguyen, NV;Nguyen, HV;Nguyen, ATV;Phan, H;Wilusz, C;Podell, BK;Sanoussi, ND;de Jong, BC;Merle, CS;Affolabi, D;McIlleron, H;Garcia-Cremades, M;Maidji, E;Eshun-Wilson, F;Aguilar-Rodriguez, B;Karthikeyan, D;Mdluli, K;Bansbach, C;Lenaerts, AJ;Savic, RM;Nahid, P;Vásquez, JJ;Voskuil, MI;
PMID: 34006838 | DOI: 10.1038/s41467-021-22833-6

There is urgent need for new drug regimens that more rapidly cure tuberculosis (TB). Existing TB drugs and regimens vary in treatment-shortening activity, but the molecular basis of these differences is unclear, and no existing assay directly quantifies the ability of a drug or regimen to shorten treatment. Here, we show that drugs historically classified as sterilizing and non-sterilizing have distinct impacts on a fundamental aspect of Mycobacterium tuberculosis physiology: ribosomal RNA (rRNA) synthesis. In culture, in mice, and in human studies, measurement of precursor rRNA reveals that sterilizing drugs and highly effective drug regimens profoundly suppress M. tuberculosis rRNA synthesis, whereas non-sterilizing drugs and weaker regimens do not. The rRNA synthesis ratio provides a readout of drug effect that is orthogonal to traditional measures of bacterial burden. We propose that this metric of drug activity may accelerate the development of shorter TB regimens.

DLX1 and the NuRD complex cooperate in enhancer decommissioning and transcriptional repression

Development (Cambridge, England)

2022 Jun 01

Price, JD;Lindtner, S;Ypsilanti, A;Binyameen, F;Johnson, JR;Newton, BW;Krogan, NJ;Rubenstein, JLR;
PMID: 35695185 | DOI: 10.1242/dev.199508

In the developing subpallium, the fate decision between neurons and glia is driven by expression of Dlx1/2 or Olig1/2, respectively, two sets of transcription factors with a mutually repressive relationship. The mechanism by which Dlx1/2 repress progenitor and oligodendrocyte fate, while promoting transcription of genes needed for differentiation, is not fully understood. We identified a motif within DLX1 that binds RBBP4, a NuRD complex subunit. ChIP-seq studies of genomic occupancy of DLX1 and six different members of the NuRD complex show that DLX1 and NuRD colocalize to putative regulatory elements enriched near other transcription factor genes. Loss of Dlx1/2 leads to dysregulation of genome accessibility at putative regulatory elements near genes repressed by Dlx1/2, including Olig2. Consequently, heterozygosity of Dlx1/2 and Rbbp4 leads to an increase in the production of OLIG2+ cells. These findings highlight the importance of the interplay between transcription factors and chromatin remodelers in regulating cell-fate decisions.

Altered cell and RNA isoform diversity in aging Down syndrome brains

Proceedings of the National Academy of Sciences of the United States of America

2021 Nov 23

Palmer, CR;Liu, CS;Romanow, WJ;Lee, MH;Chun, J;
PMID: 34795060 | DOI: 10.1073/pnas.2114326118

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

Investigating immune and non-immune cell interactions in head and neck tumors by single-cell RNA sequencing

Nature communications

2021 Dec 17

Kürten, CHL;Kulkarni, A;Cillo, AR;Santos, PM;Roble, AK;Onkar, S;Reeder, C;Lang, S;Chen, X;Duvvuri, U;Kim, S;Liu, A;Tabib, T;Lafyatis, R;Feng, J;Gao, SJ;Bruno, TC;Vignali, DAA;Lu, X;Bao, R;Vujanovic, L;Ferris, RL;
PMID: 34921143 | DOI: 10.1038/s41467-021-27619-4

Head and neck squamous cell carcinoma (HNSCC) is characterized by complex relations between stromal, epithelial, and immune cells within the tumor microenvironment (TME). To enable the development of more efficacious therapies, we aim to study the heterogeneity, signatures of unique cell populations, and cell-cell interactions of non-immune and immune cell populations in 6 human papillomavirus (HPV)+ and 12 HPV- HNSCC patient tumor and matched peripheral blood specimens using single-cell RNA sequencing. Using this dataset of 134,606 cells, we show cell type-specific signatures associated with inflammation and HPV status, describe the negative prognostic value of fibroblasts with elastic differentiation specifically in the HPV+ TME, predict therapeutically targetable checkpoint receptor-ligand interactions, and show that tumor-associated macrophages are dominant contributors of PD-L1 and other immune checkpoint ligands in the TME. We present a comprehensive single-cell view of cell-intrinsic mechanisms and cell-cell communication shaping the HNSCC microenvironment.

Excitatory and inhibitory modulation of parafacial respiratory neurons in the control of active expiration

Respiratory physiology & neurobiology

2021 Mar 26

Takakura, AC;Malheiros-Lima, MR;Moreira, TS;
PMID: 33781931 | DOI: 10.1016/j.resp.2021.103657

In order to increase ventilation, the respiratory system engages active expiration through recruitment of abdominal muscles. Here, we reviewed the new advances in the modulation of parafacial respiratory (pF) region to trigger active expiration. In addition, we also made a comprehensive discussion of experiments indicating that the lateral aspect of the pF (pFL) is anatomically and functionally distinct from the adjacent and partially overlapping chemosensitive neurons of the ventral aspect of the pF (pFV) also named the retrotrapezoid nucleus. Recent evidence suggest a complex network responsible for the generation of active expiration and neuromodulatory systems that influence its activity. The activity of the pFL is tonically inhibited by inhibitory inputs and also receives excitatory inputs from chemoreceptors (central x peripheral) as well as from catecholaminergic C1 neurons. Therefore, the modulatory inputs and the physiological conditions under which these mechanisms are used to recruit active expiration and increase ventilation need further investigation.

An anti-inflammatory transcriptional cascade conserved from flies to humans

Cell reports

2022 Oct 18

Pavlidaki, A;Panic, R;Monticelli, S;Riet, C;Yuasa, Y;Cattenoz, PB;Nait-Oumesmar, B;Giangrande, A;
PMID: 36261018 | DOI: 10.1016/j.celrep.2022.111506

Innate immunity is an ancestral process that can induce pro- and anti-inflammatory states. A major challenge is to characterize transcriptional cascades that modulate the response to inflammation. Since the Drosophila glial cells missing (Gcm) transcription factor has an anti-inflammatory role, we explored its regulation and evolutionary conservation. Here, we show that the murine Gcm2 (mGcm2) gene is expressed in a subpopulation of aged microglia (chronic inflammation) and upon lysophosphatidylcholine (LPC)-induced central nervous system (CNS) demyelination (acute inflammation). Moreover, mGcm2 conditional knockout mice show an increased inflammatory phenotype upon aging or LPC injection, and hGCM2 is expressed in active demyelinating lesions of patients with multiple sclerosis. Finally, Drosophila Gcm expression is induced upon aging and acute challenge, and its overexpression decreases the inflammatory phenotype. Altogether, these data indicate that the inducible Gcm cascade is conserved from flies to humans and represents a potential therapeutic target in the control of the inflammatory response.

Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function

PLoS biology

2021 Nov 01

Kikel-Coury, NL;Brandt, JP;Correia, IA;O'Dea, MR;DeSantis, DF;Sterling, F;Vaughan, K;Ozcebe, G;Zorlutuna, P;Smith, CJ;
PMID: 34793438 | DOI: 10.1371/journal.pbio.3001444

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest-derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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