Renewal of oligodendrocyte lineage reverses dysmyelination and CNS neurodegeneration through corrected N-acetylaspartate metabolism

Progress in neurobiology

2023 May 04

Lotun, A;Li, D;Xu, H;Su, Q;Tuncer, S;Sanmiguel, J;Mooney, M;Baer, CE;Ulbrich, R;Eyles, SJ;Strittmatter, L;Hayward, LJ;Gessler, DJ;Gao, G;
PMID: 37149081 | DOI: 10.1016/j.pneurobio.2023.102460

Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.

Generation of a CRF1-Cre transgenic rat and the role of central amygdala CRF1 cells in nociception and anxiety-like behavior

eLife

2022 Apr 07

Weera, MM;Agoglia, AE;Douglass, E;Jiang, Z;Rajamanickam, S;Shackett, RS;Herman, MA;Justice, NJ;Gilpin, NW;
PMID: 35389341 | DOI: 10.7554/eLife.67822

Corticotropin-releasing factor type-1 (CRF1) receptors are critical to stress responses because they allow neurons to respond to CRF released in response to stress. Our understanding of the role of CRF1-expressing neurons in CRF-mediated behaviors has been largely limited to mouse experiments due to the lack of genetic tools available to selectively visualize and manipulate CRF1+ cells in rats. Here, we describe the generation and validation of a transgenic CRF1-Cre-tdTomato rat. We report that Crhr1 and Cre mRNA expression are highly colocalized in both the central amygdala (CeA), composed of mostly GABAergic neurons, and in the basolateral amygdala (BLA), composed of mostly glutamatergic neurons. In the CeA, membrane properties, inhibitory synaptic transmission, and responses to CRF bath application in tdTomato+ neurons are similar to those previously reported in GFP+ cells in CRFR1-GFP mice. We show that stimulatory DREADD receptors can be targeted to CeA CRF1+ cells via virally delivered Cre-dependent transgenes, that transfected Cre/tdTomato+ cells are activated by clozapine-n-oxide in vitro and in vivo, and that activation of these cells in vivo increases anxiety-like and nocifensive behaviors. Outside the amygdala, we show that Cre-tdTomato is expressed in several brain areas across the brain, and that the expression pattern of Cre-tdTomato cells is similar to the known expression pattern of CRF1 cells. Given the accuracy of expression in the CRF1-Cre rat, modern genetic techniques used to investigate the anatomy, physiology, and behavioral function of CRF1+ neurons can now be performed in assays that require the use of rats as the model organism.

A Cre-driver rat model for anatomical and functional analysis of glucagon (Gcg)-expressing cells in the brain and periphery

Molecular metabolism

2022 Nov 08

Zheng, H;López-Ferreras, L;Krieger, JP;Fasul, S;Cea Salazar, V;Valderrama Pena, N;Skibicka, KP;Rinaman, L;
PMID: 36368622 | DOI: 10.1016/j.molmet.2022.101631

The glucagon gene (Gcg) encodes preproglucagon, which is cleaved to form glucagon-like peptide 1 (GLP1) and other mature signaling molecules implicated in metabolic functions. To date there are no transgenic rat models available for precise manipulation of GLP1-expressing cells in the brain and periphery.To visualize and manipulate Gcg-expressing cells in rats, CRISPR/Cas9 was used to express iCre under control of the Gcg promoter. Gcg-Cre rats were bred with tdTomato reporter rats to tag Gcg-expressing cells. Cre-dependent AAVs and RNAscope in situ hybridization were used to evaluate the specificity of iCre expression by GLP1 neurons in the caudal nucleus of the solitary tract (cNTS) and intermediate reticular nucleus (IRt), and by intestinal and pancreatic secretory cells. Food intake was assessed in heterozygous (Het) Gcg-Cre rats after chemogenetic stimulation of cNTS GLP1 neurons expressing an excitatory DREADD.While genotype has minimal effect on body weight or composition in chow-fed Gcg-Cre rats, homozygous (Homo) rats have lower plasma glucose levels. In neonatal and adult Gcg-Cre/tdTom rats, reporter-labeled cells are present in the cNTS and IRt, and in additional brain regions (e.g., basolateral amygdala, piriform cortex) that lack detectable Gcg mRNA in adults but display transient developmental or persistently low Gcg expression. Compared to wildtype (WT) rats, hindbrain Gcg mRNA and GLP1 protein in brain and plasma are markedly reduced in Homo Gcg-Cre rats. Chemogenetic stimulation of cNTS GLP1 neurons reduced overnight chow intake in males but not females, the effect in males was blocked by antagonism of central GLP1 receptors, and hypophagia was enhanced when combined with a subthreshold dose of cholecystokinin-8 to stimulate gastrointestinal vagal afferents.Gcg-Cre rats are a novel and valuable experimental tool for analyzing the development, anatomy, and function of Gcg-expressing cells in the brain and periphery. In addition, Homo Gcg-Cre rats are a unique model for assessing the role of Gcg-encoded proteins in glucose homeostasis and energy metabolism.

Amygdala AVPR1A mediates susceptibility to chronic social isolation in females

bioRxiv : the preprint server for biology

2023 Feb 15

François, M;Delgado, IC;Lafond, A;Lewis, EM;Kuromaru, M;Hassouna, R;Deng, S;Thaker, VV;Dölen, G;Zeltser, LM;
PMID: 36824966 | DOI: 10.1101/2023.02.15.528679

Females are more sensitive to social exclusion, which could contribute to their heightened susceptibility to anxiety disorders. Chronic social isolation stress (CSIS) for at least 7 weeks after puberty induces anxiety-related behavioral adaptations in female mice. Here, we show that Arginine vasopressin receptor 1a ( Avpr1a )-expressing neurons in the central nucleus of the amygdala (CeA) mediate these sex-specific effects, in part, via projections to the caudate putamen. Loss of function studies demonstrate that AVPR1A signaling in the CeA is required for effects of CSIS on anxiety-related behaviors in females but has no effect in males or group housed females. This sex-specificity is mediated by AVP produced by a subpopulation of neurons in the posterodorsal medial nucleus of the amygdala that project to the CeA. Estrogen receptor alpha signaling in these neurons also contributes to preferential sensitivity of females to CSIS. These data support new therapeutic applications for AVPR1A antagonists in women.

Cell-Population Dynamics in Diffuse Gliomas during Gliomagenesis and Its Impact on Patient Survival

Cancers

2022 Dec 26

Nikitin, P;Musina, G;Pekov, S;Kuzin, A;Popov, I;Belyaev, A;Kobyakov, G;Usachev, D;Nikolaev, V;Mikhailov, V;
| DOI: 10.3390/cancers15010145

Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations’ content in diffuse gliomas of different molecular profiles and grades, considering the cell populations’ functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells’ proportion and mesenchymal cell populations’ appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells’ hypoxia response programs’ activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs’ activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas’ pathogenetic tracing up to the practical plane.

Variation in phenotypes from a Bmp-Gata3 genetic pathway is modulated by Shh signaling

PLoS genetics

2021 May 25

Swartz, ME;Lovely, CB;Eberhart, JK;
PMID: 34033651 | DOI: 10.1371/journal.pgen.1009579

We sought to understand how perturbation of signaling pathways and their targets generates variable phenotypes. In humans, GATA3 associates with highly variable defects, such as HDR syndrome, microsomia and choanal atresia. We previously characterized a zebrafish point mutation in gata3 with highly variable craniofacial defects to the posterior palate. This variability could be due to residual Gata3 function, however, we observe the same phenotypic variability in gata3 null mutants. Using hsp:GATA3-GFP transgenics, we demonstrate that Gata3 function is required between 24 and 30 hpf. At this time maxillary neural crest cells fated to generate the palate express gata3. Transplantation experiments show that neural crest cells require Gata3 function for palatal development. Via a candidate approach, we determined if Bmp signaling was upstream of gata3 and if this pathway explained the mutant's phenotypic variation. Using BRE:d2EGFP transgenics, we demonstrate that maxillary neural crest cells are Bmp responsive by 24 hpf. We find that gata3 expression in maxillary neural crest requires Bmp signaling and that blocking Bmp signaling, in hsp:DN-Bmpr1a-GFP embryos, can phenocopy gata3 mutants. Palatal defects are rescued in hsp:DN-Bmpr1a-GFP;hsp:GATA3-GFP double transgenic embryos, collectively demonstrating that gata3 is downstream of Bmp signaling. However, Bmp attenuation does not alter phenotypic variability in gata3 loss-of-function embryos, implicating a different pathway. Due to phenotypes observed in hypomorphic shha mutants, the Sonic Hedgehog (Shh) pathway was a promising candidate for this pathway. Small molecule activators and inhibitors of the Shh pathway lessen and exacerbate, respectively, the phenotypic severity of gata3 mutants. Importantly, inhibition of Shh can cause gata3 haploinsufficiency, as observed in humans. We find that gata3 mutants in a less expressive genetic background have a compensatory upregulation of Shh signaling. These results demonstrate that the level of Shh signaling can modulate the phenotypes observed in gata3 mutants.

Migrating Interneurons Secrete Fractalkine to Promote Oligodendrocyte Formation in the Developing Mammalian Brain

Neuron

2017 May 03

Voronova A, Yuzwa SA, Wang BS, Zahr S, Syal C, Wang J, Kaplan DR, Miller FD.
PMID: 28472653 | DOI: 10.1016/j.neuron.2017.04.018

During development, newborn interneurons migrate throughout the embryonic brain. Here, we provide evidence that these interneurons act in a paracrine fashion to regulate developmental oligodendrocyte formation. Specifically, we show that medial ganglionic eminence (MGE) interneurons secrete factors that promote genesis of oligodendrocytes from glially biased cortical precursors in culture. Moreover, when MGE interneurons are genetically ablated in vivo prior to their migration, this causes a deficit in cortical oligodendrogenesis. Modeling of the interneuron-precursor paracrine interaction using transcriptome data identifies the cytokine fractalkine as responsible for the pro-oligodendrocyte effect in culture. This paracrine interaction is important in vivo, since knockdown of the fractalkine receptor CX3CR1 in embryonic cortical precursors, or constitutive knockout of CX3CR1, causes decreased numbers of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes in the postnatal cortex. Thus, in addition to their role in regulating neuronal excitability, interneurons act in a paracrine fashion to promote the developmental genesis of oligodendrocytes.

Human distal lung maps and lineage hierarchies reveal a bipotent progenitor

Nature

2022 Apr 01

Kadur Lakshminarasimha Murthy, P;Sontake, V;Tata, A;Kobayashi, Y;Macadlo, L;Okuda, K;Conchola, AS;Nakano, S;Gregory, S;Miller, LA;Spence, JR;Engelhardt, JF;Boucher, RC;Rock, JR;Randell, SH;Tata, PR;
PMID: 35355018 | DOI: 10.1038/s41586-022-04541-3

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

A Functional Synonymous Variant in PDGFRA Is Associated with Better Survival in Acral Melanoma

J Cancer

2020 Mar 04

Dai J, Yang L, Xu T, Si L, Cui C, Sheng X, Chi Z, Mao L, Lian B, Tang B, Bai X, Zhou L, Li S, Wang X, Yan X, Kong Y, Guo J
PMID: 32226509 | DOI: 10.7150/jca.43010

Purpose: Polymorphisms of genes in the platelet-derived growth factor (PDGF) signaling pathway have been found to predict cutaneous melanoma (CM) survival, but their clinical effects in acral melanoma (AM) patients have not been explored. The aim of this study was to characterize the functional effect of the tag single-nucleotide polymorphism (SNP) rs2228230:C>T and assess its association with clinical outcomes in AM patients. Methods: The effect of rs2228230:C>T on mRNA structures and codon usage values were evaluated using in silico analyses. PDGF receptor alpha (PDGFRA) expression vectors with the rs2228230:C or rs2228230:T allele were constructed to evaluate the expression and signaling activity of PDGFRA. The expression of PDGFRA in AM samples was measured using in situ RNAscope hybridization and immunohistochemical staining. The association of the rs2228230 genotype with survival was analyzed in two independent AM cohorts. Results: In silico analyses indicated that the rs2228230:T allele increases the minimum free energy and reduces synonymous codon usage. The rs2228230:T allele decreased the expression of PDGFRA by reducing the stability of its mRNA and protein as well as the signaling activity of the MAPK and PI3K/AKT pathways. PDGFRA mRNA and protein expression was significantly reduced in AM tissues with the rs2228230:T allele. The progression-free survival and overall survival of AM patients with the rs2228230:T allele were significantly longer than those of patients with the CC genotype. Conclusion: Our study indicated that rs2228230:T can reduce the expression of PDGFRA and downstream signaling activity and is associated with better survival in AM patients.

Grpr expression defines a population of superficial dorsal horn vertical cells that have a role in both itch and pain

Pain

2022 May 11

Polgár, E;Dickie, AC;Gutierrez-Mecinas, M;Bell, AM;Boyle, KA;Quillet, R;Rashid, EA;Clark, RA;German, MT;Watanabe, M;Riddell, JS;Todd, AJ;
PMID: 35543635 | DOI: 10.1097/j.pain.0000000000002677

Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are thought to transmit nociceptive information. In this study, we have used a GRPRCreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the great majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain- and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.

Pentraxin 3 is a stromally-derived biomarker for detection of pancreatic ductal adenocarcinoma

NPJ precision oncology

2021 Jun 29

Goulart, MR;Watt, J;Siddiqui, I;Lawlor, RT;Imrali, A;Hughes, C;Saad, A;ChinAleong, J;Hurt, C;Cox, C;Salvia, R;Mantovani, A;Crnogorac-Jurcevic, T;Mukherjee, S;Scarpa, A;Allavena, P;Kocher, HM;
PMID: 34188166 | DOI: 10.1038/s41698-021-00192-1

Pancreatic ductal adenocarcinoma (PDAC), characterized by dense desmoplastic stroma laid down by pancreatic stellate cells (PSC), has no reliable diagnostic biomarkers for timely detection. A multi-center cohort of PDAC patients and controls (chronic pancreatitis, intra-ductal papillary neoplasms, gallstones and otherwise healthy) donated serum in an ethically approved manner. Serum PTX3 above 4.34 ng/mL has a higher sensitivity (86%, 95% confidence interval (CI): 65-97%) and specificity (86%, 95% CI: 79-91%), positive predictive value (97%) and likelihood ratio (6.05), and is superior when compared to serum CA19-9 and CEA for detection of PDAC. In vitro and ex vivo analyses of PTX3, in human PDAC samples, PSCs, cell lines and transgenic mouse model for PDAC, suggest that PTX3 originates from stromal cells, mainly PSC. In activated PSC, PTX3 secretion could be downregulated by rendering PSC quiescent using all-trans-retinoic acid (ATRA). PTX3 organizes hyaluronan in conjunction with tumor necrosis factor-stimulated gene 6 (TSG-6) and facilitates stellate and cancer cell invasion. In SCALOP clinical trial (ISRCTN96169987) testing chemo-radiotherapy without stromal targeting, PTX3 had no prognostic or predictive role. However, in STARPAC clinical trial (NCT03307148), stromal modulation by ATRA even at first dose is accompanied with serum PTX3 response in patients who later go on to demonstrate disease control but not those in whom the disease progresses. PTX3 is a putative stromally-derived biomarker for PDAC which warrants further testing in prospective, larger, multi-center cohorts and within clinical trials targeting stroma.

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease

Nature neuroscience

2023 Feb 06

De Schepper, S;Ge, JZ;Crowley, G;Ferreira, LSS;Garceau, D;Toomey, CE;Sokolova, D;Rueda-Carrasco, J;Shin, SH;Kim, JS;Childs, T;Lashley, T;Burden, JJ;Sasner, M;Sala Frigerio, C;Jung, S;Hong, S;
PMID: 36747024 | DOI: 10.1038/s41593-023-01257-z

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-β oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

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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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