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Glucokinase neurons of the paraventricular nucleus of the thalamus sense glucose and decrease food consumption

iScience

2021 Oct 01

Kessler, S;Labouèbe, G;Croizier, S;Gaspari, S;Tarussio, D;Thorens, B;
| DOI: 10.1016/j.isci.2021.103122

The paraventricular nucleus of the thalamus (PVT) controls goal-oriented behavior through its connections to the nucleus accumbens (NAc). We previously characterized Glut2aPVT neurons that are activated by hypoglycemia, and which increase sucrose seeking behavior through their glutamatergic projections to the NAc. Here, we identified glucokinase (Gck)-expressing neurons of the PVT (GckaPVT) and generated a mouse line expressing the Cre recombinase from the glucokinase locus (GckCre/+ mice). Ex vivo calcium imaging and whole-cell patch clamp recordings revealed that GckaPVT neurons that project to the NAc were mostly activated by hyperglycemia. Their chemogenetic inhibition or optogenetic stimulation, respectively, enhanced food intake or decreased sucrose-seeking behavior. Collectively, our results describe a neuronal population of Gck-expressing neurons in the PVT, which has opposite glucose sensing properties and control over feeding behavior than the previously characterized Glut2aPVT neurons. This study allows a better understanding of the complex regulation of feeding behavior by the PVT.

NK-B cell cross talk induces CXCR5 expression on natural killer cells

iScience

2021 Oct 01

Rascle, P;Jacquelin, B;Petitdemange, C;Contreras, V;Planchais, C;Lazzerini, M;Dereuddre-Bosquet, N;Le Grand, R;Mouquet, H;Huot, N;Müller-Trutwin, M;
| DOI: 10.1016/j.isci.2021.103109

B cell follicles (BCFs) in lymph nodes (LNs) are generally exempt of CD8+ T and NK cells. African green monkeys (AGMs), a natural host of simian immunodeficiency virus (SIV), display NK cell-mediated viral control in BCF. NK cell migration into BCF in chronically SIVagm-infected AGM is associated with CXCR5+ NK cells. We aimed to identify the mechanism leading to CXCR5 expression on NK cells. We show that CXCR5+ NK cells in LN were induced following SIVagm infection. CXCR5+ NK cells accumulated preferentially in BCF with proliferating B cells. Autologous NK-B cell co-cultures in transwell chambers induced CXCR5+ NK cells. Transcriptome analysis of CXCR5+ NK cells revealed expression of bcl6 and IL6R. IL-6 induced CXCR5 on AGM and human NK cells. IL6 mRNA was detected in LN at higher levels during SIVagm than SIVmac infection and often produced by plasma cells. Our study reveals a mechanism of B cell-dependent NK cell regulation.

Daily coordination of orexinergic gating in the rat superior colliculus-Implications for intrinsic clock activities in the visual system

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Oct 01

Chrobok, L;Jeczmien-Lazur, JS;Bubka, M;Pradel, K;Klekocinska, A;Klich, JD;Ridla Rahim, A;Myung, J;Kepczynski, M;Lewandowski, MH;
PMID: 34533886 | DOI: 10.1096/fj.202100779RR

The orexinergic system delivers excitation for multiple brain centers to facilitate behavioral arousal, with its malfunction resulting in narcolepsy, somnolence, and notably, visual hallucinations. Since the circadian clock underlies the daily arousal, a timed coordination is expected between the orexin system and its target subcortical visual system, including the superior colliculus (SC). Here, we use a combination of electrophysiological, immunohistochemical, and molecular approaches across 24 h, together with the neuronal tract-tracing methods to investigate the daily coordination between the orexin system and the rodent SC. Higher orexinergic input was found to occur nocturnally in the superficial layers of the SC, in time for nocturnal silencing of spontaneous firing in this visual brain area. We identify autonomous daily and circadian expression of clock genes in the SC, which may underlie these day-night changes. Additionally, we establish the lateral hypothalamic origin of the orexin innervation to the SC and that the SC neurons robustly respond to orexin A via OX2 receptor in both excitatory and GABAA receptor-dependent inhibitory manners. Together, our evidence elucidates the combination of intrinsic and extrinsic clock mechanisms that shape the daily function of the visual layers of the SC.

Longitudinal peripheral tissue RNA-Seq transcriptomic profiling, hyperalgesia, and wound healing in the rat plantar surgical incision model

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Oct 01

Goto, T;Sapio, MR;Maric, D;Robinson, JM;Domenichiello, AF;Saligan, LN;Mannes, AJ;Iadarola, MJ;
PMID: 34499774 | DOI: 10.1096/fj.202100347R

Postoperative pain and delayed healing in surgical wounds, which require complex management strategies have understudied complicated mechanisms. Here we investigated temporal changes in behavior, tissue structure, and transcriptomic profiles in a rat model of a surgical incision, using hyperalgesic behavioral tests, histological analyses, and next-generation RNA sequencing, respectively. The most rapidly (1 hour) expressed genes were the chemokines, Cxcl1 and Cxcl2. Consequently, infiltrating leukocytes were abundantly observed starting at 6 and peaking at 24 hours after incising which was supported by histological analysis and appearance of the neutrophil markers, S100a8 and S100a9. At this time, hyperalgesia was at a peak and overall transcriptional activity was most highly activated. At the 1-day timepoint, Nppb, coding for natriuretic peptide precursor B, was the most strongly upregulated gene and was localized by in situ hybridization to the epidermal keratinocytes at the margins of the incision. Nppb was basically unaffected in a peripheral inflammation model transcriptomic dataset. At the late phase of wound healing, five secreted, incision-specific peptidases, Mmp2, Aebp1, Mmp23, Adamts7, and Adamtsl1, showed increased expression, supporting the idea of a sustained tissue remodeling process. Transcripts that are specifically upregulated at each timepoint in the incision model may be potential candidates for either biomarkers or therapeutic targets for wound pain and wound healing. This study incorporates the examination of longitudinal temporal molecular responses, corresponding anatomical localization, and hyperalgesic behavioral alterations in the surgical incision model that together provide important and novel foundational knowledge to understand mechanisms of wound pain and wound healing.

DMD- TREATMENT

Neuromuscular Disorders

2021 Oct 01

Gushchina, L;Bradley, A;Vetter, T;Frair, E;Bellinger, C;Simmons, T;Rohan, N;Wein, N;Flanigan, K;
| DOI: 10.1016/j.nmd.2021.07.171

Exon 2 duplications of the DMD gene, encoding the dystrophin protein, account for around 6-11% of all duplication mutations associated with X-linked Duchenne muscular dystrophy (DMD). As part of the preclinical development of a U7snRNA vector currently in a clinical trial (ClinicalTrials.gov NCT04240314), we have previously evaluated the therapeutic efficacy, absence of off-target splicing effects in AAV9.U7snRNA-mediated skipping of exon 2 in a murine Dmd model, and lack of toxicity in non-human primates. Here we report that 3-month-old Dup2 mice systemically injected with scAAV9.U7.ACCA vector, containing four copies of U7snRNA targeted to the exon 2 splice acceptor and splice donor sites, showed efficient exon 2 skipping, long-term dystrophin expression, and skeletal muscle function correction 18-months post vector administration. The RT-PCR data showed that a single vector injection (3E13 vg/kg) resulted in significant exon 2 skipping in tibialis anterior (TA), diaphragm (Dia) and heart tissues, showing an average of 46%, 32% and 73% total therapeutic transcripts, respectively. To determine the degree of functional rescue, in situ and in vitro physiology studies on TA and Dia muscles were performed. Both Dia and TA from 21-month-old control Dup2 mice exhibited a functional deficit with a significant reduction in specific force output (45-61%) compared with Bl6 mice. The significant force drop was also observed in those mice compared with Bl6 following a rigorous fatigue protocol. The single vector infusion resulted in a dramatic improvement in specific force output up to 64-76% in Dia and TA, and better protection of the TA muscle (up to 73%) from repeated fatigue. Overall, our results confirm that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in straight muscles from Dup2 mice and functional recovery of TA and Dia muscles 18-month post vector administration.

DMD-BRAIN: EP. 133 Expression and localization of dystrophin isoforms transcripts in human adult control brain areas.

Neuromuscular

2021 Oct 01

Falzarano, M;Rachele, R;Mietto, M;
| DOI: 10.1016/j.nmd.2021.07.158

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease due to pathogenic variants in the DMD gene. DMD often is associated with cognitive and neuro-behavioural co-morbidities, which pathogenesis and genotype-phenotype relationship are only partially understood. Multiple DMD isoforms, differentially affected based on the mutation site, play a role in these co-morbidities, based on their prominent (Dp71) or exclusive (Dp140) brain expression. All known dystrophin isoforms were analysed in 24 normal adult human brain areas using TaqMan assays on TissueScan cDNA array (OriGene). Ct were used to build a heatmap of dystrophin isoforms expression with values ranging from 40 (lowest) to 27 (highest) Ct. A specific BaseScope™ ZZ probe was also used to detect and localize the Dp71 in a formalin-fixed paraffin-embedded cerebellum. Three main dystrophin isoform clusters were identified. Cluster 1 includes Dp116, Dp427p1, Dp260.1 that show low expression in all brain areas. Cluster 2 includes Dp260.2, Dp427m, Dp140 with intermediate expression levels. Cluster 3 consists of Dp427b, Dp427p2, Dp71 that have the overall high expression in most brain areas. Hierarchical clustering also highlighted brain areas with overall low dystrophin expression, such as telencephalon (frontal, temporal and occipital lobe, amygdala, caudate, and choroid plexus), diencephalon (thalamus and hypothalamus), and myelencephalon (medulla). By contrast, the cerebellum showed the highest expression of all DMD isoforms. BaseScope on cerebellum showed Dp71 expression in molecular, granular, and Purkinje layers, white matter and blood vessels. We showed that adult human brain areas have differential enrichments for expression of specific dystrophin isoforms, and that BaseScope has high sensitivity in detecting Dp71 in fixed bran tissues. This information on the regional and cellular pattern of expression of the multiple dystrophin isoforms, may contribute to the understanding of the DMD brain co-morbidities.

DMD/BMD-OUTCOME MEASURES: EP.132 Double the trouble: familial hyperlipidemia and Becker muscular dystrophy with a hemizygous nonsense mutation in the dystrophin (DMD) gene

Neuromuscular

2021 Oct 01

Sayar, Y;Yildirim, M;Bektas, O;Sut, NY;
| DOI: 10.1016/j.nmd.2021.07.157

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by the absence of dystrophin. It is characterized by progressive skeletal and cardiac muscle weakness that usually becomes apparent between the ages of 5 and 15. Familial hypercholesterolemia (FH) is an autosomal dominant disorder of lipid metabolism present from birth. It is characterized by remarkably high low-density lipoprotein cholesterol (LDL-C) levels causing premature coronary heart disease. We report a 9-year-old boy with familial hyperlipidemia who presented with creatinine kinase elevation. He was born after uneventful pregnancy and delivery with consanguineous marriage of his parents. The motor developmental milestones were normal. On physical and neurological examination at the age of 9, he had only mild pseudohypertrophy in the gastrocnemius muscles. Gowers’ sign was negative. He had no myotonia or muscle weakness. The cardiological evaluation was unremarkable. Serum creatine kinase (CK) level was elevated (2889 UI/L). Moreover, serum triglyceride (183 mg/dl), VLDL cholesterol (37 mg/dl), LDL cholesterol (212 mg/dl), and total cholesterol (289 mg/dl) were elevated. We identified a heterozygous mutation of c.1246 C>T (p.R416W) (paternal) in the LDLR gene and a hemizygous nonsense mutation of c.71G>A (p.W24) in the DMD gene. Based on the clinical manifestations, examination, and laboratory findings, we diagnosed BMD and familial hyperlipidemia. We described a case with familial hyperlipidemia presented with serum creatinine kinase (CK) elevation and identified hemizygous nonsense mutation of c.71G>A p.W24 in the dystrophin gene.

DMD/BMD- OUTCOME MEASURES

Neuromuscular Disorders

2021 Oct 01

Schrama, E;Koeks, Z;van de Velde, N;Kan, H;Krom, Y;van Duyvenvoorde, H;Niks, E;
| DOI: 10.1016/j.nmd.2021.07.156

To study the epidemiology of Becker muscular dystrophy (BMD) in the Netherlands, we searched the database of the laboratory for diagnostic genome analysis (LDGA) of the Leiden university medical centre (LUMC), where genetic testing has been concentrated since the 1980’s, and the Dutch dystrophinopathy database (DDD) for patients diagnosed with BMD. We expect that together these databases contain the vast majority of BMD patients and can thus be used to approximate the incidence of BMD. We collected 380 BMD patients of whom 80 were found in both the LDGA database and the DDD, 208 were only known in the LDGA and 92 only registered in the DDD. An estimate of the incidence per decade was calculated using the date of birth and seemed stable from 1970 until 2009 at around 3 per 100,000 live births (range 2.81-3.09). Interestingly the incidence between 2000-2009 was calculated at 3.06 per 100,000 live births, while part of the BMD patients in this age category is at the time of writing most likely not yet diagnosed, therefore we expect that this number is an underestimate. This might be due to advances in genetic testing due to the availability of gene panels, resulting in a younger age at diagnosis. Preliminary analysis on the type of mutation showed that the vast majority of 66% is a deletion, followed by 14% of duplications, 10% point mutations. Of these deletions and duplications, 87% are predicted not to shift the reading frame of the DMD gene (in-frame). In the majority of out-of-frame mutations it concerns an exon 3-7 deletion. Ongoing analyses will focus on the proportion of de novo mutations age at diagnosis, and survival. In conclusion, this preliminary data suggests that advances in and availability of genetic testing may have resulted in a lower age at diagnosis of BMD patients. It also emphasizes that a combination of genetic diagnostic registries and clinical registries is required to obtain a complete overview of the epidemiology of BMD.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2

Stem Cell Research

2021 Oct 01

Zhu, S;Stanslowsky, N;Fernández-Trillo, J;Mamo, T;Yu, P;Kalmbach, N;Ritter, B;Eggenschwiler, R;Ouwendijk, W;Mzinza, D;Tan, L;Leffler, A;Spohn, M;Brown, R;Kropp, K;Kaever, V;Ha, T;Narayanan, P;Grundhoff, A;Förster, R;Schambach, A;Verjans, G;Schmidt, M;Kispert, A;Cantz, T;Gomis, A;Wegner, F;Viejo-Borbolla, A;
| DOI: 10.1016/j.scr.2021.102535

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

ALK Gene Rearrangements in Lung Adenocarcinomas: Concordance of Immunohistochemistry, Fluorescence In Situ Hybridization, RNA In Situ Hybridization, and RNA Next-Generation Sequencing Testing

JTO Clinical and Research Reports

2021 Oct 01

Canterbury, C;Fernandes, H;Crapanzano, J;Murty, V;Mansukhani, M;Shu, C;Szabolcs, M;Saqi, A;
| DOI: 10.1016/j.jtocrr.2021.100223

Introduction The 2018 updated molecular testing guidelines for patients with advanced lung cancer incorporated ALK immunohistochemistry (IHC) analysis as an equivalent to fluorescence in situ hybridization (FISH) method recommended in 2013. Nevertheless, no specific recommendation for alternative methods was proposed owing to insufficient data. The aim of this study was to compare the results of ALK IHC, FISH, RNA next-generation sequencing (NGS), and RNA in situ hybridization (ISH) with available clinical data. Methods A search for lung carcinomas with ALK testing by greater than or equal to one modality (i.e., ALK IHC, FISH, NGS) was performed; a subset underwent RNA ISH. When available, clinical data were recorded. Results The results were concordant among all performed testing modalities in 86 of 90 cases (95.6%). Of the four discordant cases, two were ALK positive by FISH but negative by IHC, RNA NGS, and RNA ISH. The remaining two cases failed RNA NGS testing, one was IHC negative, FISH positive, RNA ISH negative and the second was IHC positive, FISH positive, RNA ISH equivocal. RNA NGS identified one rare and one novel ALK fusion. Sufficient therapy data were available in 10 cases treated with tyrosine kinase inhibitors; three had disease progression, including one with discordant results (FISH positive, RNA NGS negative, IHC negative, RNA ISH negative) and two with concordant ALK positivity among all modalities. Conclusions Our results reveal high concordance among IHC, RNA NGS, and RNA ISH. In cases of discordance with available RNA NGS, FISH result was positive whereas IHC and ISH results were negative. On the basis of our data, multimodality testing is recommended to identify discrepant results and patients (un)likely to respond to tyrosine kinase inhibitors.

Hemodynamic phenotyping of transgenic rats with ubiquitous expression of an angiotensin-(1-7)-producing fusion protein

Clinical science (London, England : 1979)

2021 Sep 30

Alves, DT;Mendes, LF;Sampaio, WO;Coimbra-Campos, LMC;Vieira, MAR;Ferreira, AJ;Martins, AS;Popova, E;Todiras, M;Qadri, F;Alenina, N;Bader, M;Santos, RAS;Campagnole-Santos, MJ;
PMID: 34494083 | DOI: 10.1042/CS20210599

Activation of the angiotensin (Ang)-converting enzyme (ACE) 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system (RAS) induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay (RIA) were similar to control Sprague-Dawley (SD) rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure (MAP), assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma atrial natriuretic peptide (ANP) and higher urinary sodium concentration. Moreover, evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decrease in total peripheral resistance (TPR). TG7371 rats, on the other hand, also presented increased cardiac and global sympathetic tone, increased plasma vasopressin (AVP) levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower blood pressure (BP). Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.

Somatostatin Interneurons of the Insula Mediate QR2-Dependent Novel Taste Memory Enhancement

eNeuro

2021 Sep 29

Gould, NL;Kolatt Chandran, S;Kayyal, H;Edry, E;Rosenblum, K;
PMID: 34518366 | DOI: 10.1523/ENEURO.0152-21.2021

Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information.

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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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