Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease with an unclear etiology and a high mortality. The unknown IPF pathophysiology reflects in the limited treatment options and the difficulties in modelling the disease. Alveoli dysregulation has been suggested to influence IPF development. Thus, understanding the mechanism of lung repair upon alveolar injury might be the key to reveal IPF pathophysiology. To this end, we aim to develop a mouse model to achieve targeted alveolar cell ablation. Cell ablation was achieved via Adeno Associated Virus serotype 9 (AAV9) expressing the human diphtheria toxin receptor (DTR), which has a specific tropism for alveolar cells. Time course experiment was performed to observe the distinct phases of repair. As a comparison, AAV6.2-DTR that target all lung epithelial cells, was utilized. Weight and SP-D levels were assessed. The cellular dynamics of the epithelial and immune cells were characterized by flow cytometry and single cell RNA sequencing. mRNA sequencing was performed to see the genetic changes during the repair. Both AAV-groups lost weight and had increased SP-D concentration. The highest epithelial cells proliferation occurred on day 4 and the lowest on day 8 post injury. Distinct groups of profibrotic associated genes were serially and time-dependently upregulated. On day 14 post injury, the weight, SP‑D, Ki67+ epithelial cells and gene profiles returned to near initial levels. In conclusion, our AAV-DTR mouse model shows promise in investigating IPF etiology. The longitudinal analysis of selective epithelial injury revealed unique repair characteristics with a near complete lung repair at day 14 post injury.

Thanks to the analysis of an Interspecific Recombinant Congenic Strain (IRCS), we previously defined the Mafq1 quantitative trait locus as an interval on mouse Chromosome 1 associated with male hypofertility and ultrastructural abnormalities. We identified the Spermatogenesis associated protein 3 gene (Spata3 or Tsarg1) as a pertinent candidate within the Mafq1 locus and performed the CRISPR-Cas9 mediated complete deletion of the gene to investigate its function. Male mice deleted for Spata3 were normally fertile in vivo but exhibited a drastic reduction of efficiency in in vitro fertilization assays. Mobility parameters were normal but ultrastructural analyses revealed acrosome defects and an overabundance of lipids droplets in cytoplasmic remnants. The deletion of the Spata3 gene reproduces therefore partially the phenotype of the hypofertile IRCS strain.

CALHM1 (calcium homeostasis modulator 1) forms a plasma membrane ion channel that mediates neuronal excitability in response to changes in extracellular Ca2+ concentration. Six human CALHM homologs exist with no homology to other proteins, although CALHM1 is conserved across >20 species. Here we demonstrate that CALHM1 shares functional and quaternary and secondary structural similarities with connexins and evolutionarily distinct innexins and their vertebrate pannexin homologs. A CALHM1 channel is a hexamer, comprised of six monomers, each of which possesses four transmembrane domains, cytoplasmic amino and carboxyl termini, an amino-terminal helix, and conserved extracellular cysteines. The estimated pore diameter of the CALHM1 channel is ∼14 Å, enabling permeation of large charged molecules. Thus, CALHMs, connexins, and pannexins and innexins are structurally related protein families with shared and distinct functional properties.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fibrosing interstitial lung disease with poor prognosis. The current standard-of-care for IPF is the kinase inhibitor nintedanib, which has a distinct inhibitory profile targeting a range of tyrosine kinases. The mechanism of action of nintedanib has been well characterized for myofibroblast activity, yet remains less clear for the immune populations distributed throughout the lung. Given the proposed role of heterogeneous pulmonary macrophage populations in mediating both protective and pathogenic roles in lung fibrosis, we sought to identify repair-associated macrophage populations in mice influenced by nintedanib after 7 and 14 days post-bleomycin challenge using single cell RNA sequencing. Bleomycin exposure triggered expansion of inflammatory MHCIIhigh macrophage populations, which was partially reversed after nintedanib treatment. Concurrently, nintedanib promoted the expansion of MHCIIlow macrophages, which were linked to attenuation of lung fibrosis. Concomitantly, nintedanib promoted an increased expression of canonical macrophage repair markers in MHCIIlow macrophages. Finally, exposure of inflammatory macrophages to nintedanib in vitro resulted in attenuated expression of MHCII transcripts. In conclusion, a component of nintedanib’s protective mode of action in lung fibrosis relies on expanding distinct MHCIIlow macrophage populations and redirecting them toward a reparative phenotype. This study provides a rationale for further refinement of therapeutic kinase inhibition in fibrotic diseases.

! "Cytochrome P450"(P450と略称)が発見され最初の論文 (1) が発表されたのは 1962年です。50年以上 も昔のことになりますが、現在でも P450に関連した論文が毎年 1,500~1,700も発表されて活発な研究が進 められている "幸運な蛋白質" です。P450の発見の経緯について話題なども含めて解説させていただきます が、詳しい研究経過などは他の総説 (2, 3) を御参照下さい。 ! 私は 1953 年に東京大学理学部化学科を卒業して静岡大学文理学部化学科の助手になりました。静岡大学 文理学部は旧制静岡高等学校が昇格した新制大学の学部で、旧制静岡高等学校の最後の卒業生の一人だった 私には母校への帰還でした。静岡は漆器産業が盛んでしたので私は漆に興味をもって文献を調べたところ、 漆の固化を触媒する酵素 laccase については立ち入った研究がほとんどないことがわかりました。私は漆の laccase の精製から研究を始め、精製した酵素の性質や反応機構の解析などについて 4 報の論文を書き、こ れが私の博士論文になりました。植物の銅酵素 laccaseが私の研究者としての出発点です。

A wide range of evidence indicates that olfactory perception is strongly involved in food intake. However, the polysynaptic circuitry linking the brain areas involved in feeding behavior to the olfactory regions is not well known. The aim of this article was to examine such circuits. Thus, we described, using hodological tools such as transsynaptic viruses (PRV152) transported in a retrograde manner, the long‐distance indirect projections (two to three synapses) onto the main olfactory bulb (MOB). The ß‐subunit of the cholera toxin which is a monosynaptic retrograde tracer was used as a control to be able to differentiate between direct and indirect projections. Our tracing experiments showed that the arcuate nucleus of the hypothalamus, as a major site for regulation of food intake, sends only very indirect projections onto the MOB. Indirect projections to MOB also originate from the solitary nucleus which is involved in energy homeostasis. Other indirect projections have been evidenced in areas of the reward circuit such as VTA and accumbens nucleus. In contrast, direct projections to the MOB arise from melanin‐concentrating hormone and orexin neurons in the lateral hypothalamus. Functional significances of these projections are discussed in relation to the role of food odors in feeding and reward‐related behavior.

The hippocampus is critical for memory formation. The hypothalamic supramammillary nucleus (SuM) sends long-range projections to hippocampal area CA2. While the SuM-CA2 connection is critical for social memory, how this input acts on the local circuit is unknown. Using transgenic mice, we found that SuM axon stimulation elicited mixed excitatory and inhibitory responses in area CA2 pyramidal neurons (PNs). Parvalbumin-expressing basket cells were largely responsible for the feedforward inhibitory drive of SuM over area CA2. Inhibition recruited by the SuM input onto CA2 PNs increased the precision of action potential firing both in conditions of low and high cholinergic tone. Furthermore, SuM stimulation in area CA2 modulated CA1 activity, indicating that synchronized CA2 output drives a pulsed inhibition in area CA1. Hence, the network revealed here lays basis for understanding how SuM activity directly acts on the local hippocampal circuit to allow social memory encoding.

Human post-natal neurodevelopmental delay is often associated with cerebral alterations that can lead, by themselves or associated with peripheral deficits, to premature death. Here, we report the clinical features of 10 patients from six independent families with mutations in the autosomal YIF1B gene encoding a ubiquitous protein involved in anterograde traffic from the endoplasmic reticulum to the cell membrane, and in Golgi apparatus morphology. The patients displayed global developmental delay, motor delay, visual deficits with brain MRI evidence of ventricle enlargement, myelination alterations and cerebellar atrophy. A similar profile was observed in the Yif1b knockout (KO) mouse model developed to identify the cellular alterations involved in the clinical defects. In the CNS, mice lacking Yif1b displayed neuronal reduction, altered myelination of the motor cortex, cerebellar atrophy, enlargement of the ventricles, and subcellular alterations of endoplasmic reticulum and Golgi apparatus compartments. Remarkably, although YIF1B was not detected in primary cilia, biallelic YIF1B mutations caused primary cilia abnormalities in skin fibroblasts from both patients and Yif1b-KO mice, and in ciliary architectural components in the Yif1b-KO brain. Consequently, our findings identify YIF1B as an essential gene in early post-natal development in human, and provide a new genetic target that should be tested in patients developing a neurodevelopmental delay during the first year of life. Thus, our work is the first description of a functional deficit linking Golgipathies and ciliopathies, diseases so far associated exclusively to mutations in genes coding for proteins expressed within the primary cilium or related ultrastructures. We therefore propose that these pathologies should be considered as belonging to a larger class of neurodevelopmental diseases depending on proteins involved in the trafficking of proteins towards specific cell membrane compartments.

Somatostatin (SOM) and somatostatin receptors (SSTR1-4) are present in all olfactory structures, including the olfactory bulb (OB), where SOM modulates physiological gamma rhythms and olfactory discrimination responses. In this work, histological, viral tracing and transgenic approaches were used to characterize SOM cellular targets in the murine OB. We demonstrate that SOM targets all levels of mitral dendritic processes in the OB with somatostatin receptor 2 (SSTR2) detected in the dendrites of previously uncharacterized mitral-like cells. We show that inhibitory interneurons of the glomerular layer (GL) express SSTR4 while SSTR3 is confined to the granule cell layer (GCL). Furthermore, SOM cells in the OB receive synaptic inputs from olfactory cortical afferents. Behavioral studies demonstrate that genetic deletion of SSTR4, SSTR2 or SOM differentially affects olfactory performance. SOM or SSTR4 deletion have no major effect on olfactory behavioral performances while SSTR2 deletion impacts olfactory detection and discrimination behaviors. Altogether, these results describe novel anatomical and behavioral contributions of SOM, SSTR2 and SSTR4 receptors in olfactory processing.

BACKGROUND:Olfactory impairment is reported in mild cognitive impairment (MCI) and Alzheimer's disease (AD) and is associated with hippocampal atrophy. In elderly people, dementia with AD neuropathology and white matter lesions (WML) is common. In this context, olfactory impairment could also depend on the presence of WML.OBJECTIVE:To assess the cross-sectional relationship between olfaction and WML in elderly subjects with MCI.METHODS:Consecutive subjects, >65 years old, diagnosed as MCI after a comprehensive neuropsychological assessment in an expert memory center, with a brain MRI performed within a year and without major depressive state, were included. Olfaction was assessed by the Brief Smell Identification Test (BSIT). Two trained neuroradiologists, blind to cognitive and olfaction status, visually assessed hippocampal atrophy according to Scheltens' scale and WML according to Fazekas criteria.RESULTS:Seventy-five MCI subjects (mean age (SD) = 77.1 (6.2) years, 74.7% of women) were included. After adjustment for age and sex, factors associated with low BSIT scores were older age (p = 0.007), lower BMI (p = 0.08), lower MMSE score (p = 0.05), lower FCRST (p = 0.008), hippocampal atrophy (p = 0.04), periventricular WML (p = 0.007), and deep WML burden (p = 0.005). In multivariate analysis, severe deep WML (OR (95% CI) = 6.29 (1.4-35.13), p = 0.02) remained associated with low BSIT score independently from hippocampal atrophy.CONCLUSION:In elderly MCI subjects, low olfactory performances are associated with WML, whose progression may be slowed by vascular treatments. A longitudinal study to evaluate whether the progression of WML, hippocampal atrophy and low olfactory function, can predict accurately conversion from MCI to dementia is ongoing.

This study investigated whether sst2 gene deletion interacts with age and chronic stress exposure to produce exacerbated emotional and cognitive ageing. Middle-aged (10-12 month) sst2 knockout (sst2KO) and wild-type (WT) mice underwent an unpredictable chronic mild stress (UCMS) procedure for 6 weeks or no stress for control groups. This was followed by a battery of tests to assess emotional and cognitive functions and neuroendocrine status (CORT level). A re-evaluation was performed 6 months later (i.e. with 18-month-old mice). UCMS reproduced neuroendocrine and behavioral features of stress-related disorders such as elevated circulating CORT levels, physical deteriorations, increased anxiety- and depressive-like behaviors and working memory impairments. sst2KO mice displayed behavioral alterations which were similar to stressed WT and exhibited exacerbated changes following UCMS exposure. The evaluations performed in the older mice showed significant long-term effects of UCMS exposure. Old sst2KO mice previously exposed to UCMS exhibited spatial learning and memory accuracy impairments and high levels of anxiety-like behaviors which drastically added to the effects of normal ageing. Spatial abilities and emotionality scores (mean z-scores) measured both at the UCMS outcome and 6 months later were correlated with the initially measured CORT levels in middle-age. The present findings indicate that the deletion of the sst2 receptor gene produces chronic hypercorticosteronemia and exacerbates sensitivity to stressors which over time, have consequences on ageing brain function processes.

The signaling diversity of GABAergic interneurons to post-synaptic neurons is crucial to generate the functional heterogeneity that characterizes brain circuits. Whether this diversity applies to other brain cells, such as the glial cells astrocytes, remains unexplored. Using optogenetics and two-photon functional imaging in the adult mouse neocortex, we here reveal that parvalbumin- and somatostatin-expressing interneurons, two key interneuron classes in the brain, differentially signal to astrocytes inducing weak and robust GABAB receptor-mediated Ca2+ elevations, respectively. Furthermore, the astrocyte response depresses upon parvalbumin interneuron repetitive stimulations and potentiates upon somatostatin interneuron repetitive stimulations, revealing a distinguished astrocyte plasticity. Remarkably, the potentiated response crucially depends on the neuropeptide somatostatin, released by somatostatin interneurons, which activates somatostatin receptors at astrocytic processes. Our study unveils, in the living brain, a hitherto unidentified signaling specificity between interneuron subtypes and astrocytes opening a new perspective into the role of astrocytes as non-neuronal components of inhibitory circuits.

The neuropeptide somatostatin (SOM) is widely expressed in rodent brain and somatostatin-IRES-Cre (SOM-cre) mouse strains are increasingly used to unravel the physiology of SOM-containing neurons. However, while knock-in targeting strategy greatly improves Cre-Lox system accuracy, recent reports have shown that genomic insertion of Cre construct per se can markedly affect physiological function. We show that Cre transgene insertion into the 3' UTR of the somatostatin gene leads to the selective and massive depletion of endogenous SOM in all tested brain regions. It also strongly impacts SOM-related neuroendocrine responses in a similar manner to what has been reported for SST KO mice: increased corticosterone levels after 30-min restraint stress, decreased amplitude and regularity of ultradian growth hormone secretory patterns accompanied by changes in sexually dimorphic liver gene expression (serpina1, Cyp2b9, Cyp2a4, Cyp2d9, and Cyp7b1). In addition to demonstrating the need for examination of the consequences of Cre transgenesis, these results also reveal how this SOM-cre strain may be a useful tool in studying the functional consequences of moderate to low SOM levels as reported in neurological and psychiatric disorders.

Altered brain somatostatin functions recently appeared as key elements for the pathogenesis of stress-related neuropsychiatric disorders. The hippocampus exerts an inhibitory feedback on stress but the mechanisms involved remain unclear. We investigated herein the role of hippocampal somatostatin receptor subtypes in both stress response and behavioral emotionality using C57BL/6, wild type and sst2 or sst4 knockout mice. Inhibitory effects of hippocampal infusions of somatostatin agonists on stress-induced hypothalamo-pituitary-adrenal axis (HPA) activity were tested by monitoring peripheral blood and local hippocampus corticosterone levels, the latter by using microdialysis. Anxiolytic and antidepressant-like effects were determined in the elevated-plus maze, open field, forced swimming, and stress-sensitive beam walking tests. Hippocampal injections of somatostatin analogs and sst2 or sst4, but not sst1 or sst3 receptor agonists produced rapid and sustained inhibition of HPA axis. sst2 agonists selectively produced anxiolytic-like behaviors whereas both sst2 and sst4 agonists had antidepressant-like effects. Consistent with these findings, high corticosterone levels and anxiety were found in sst2KO mice and depressive-like behaviors observed in both sst2KO and sst4KO strains. Both hippocampal sst2 and sst4 receptors selectively inhibit stress-induced HPA axis activation but mediate anxiolytic and antidepressive effects through distinct mechanisms. Such results are to be accounted for in development of pathway-specific somatostatin receptor agents in the treatment of hypercortisolism (Cushing’s disease) and stress-related neuropsychiatric disorders.

Showing partial correlations between the normalized behavioural scores and the normalized volumes of the clusters: normalized scores were obtained taking into account nuisance covariates (i.e. age of the subjects, EL, total GM volume, site of acquisition) for the mean FCSRT (Fig. S1.a-c for TR and S1.d. for DTR) or DMS-48 scores (Fig. S1.e.) and the mean GM volume of the clusters found using FWE correction in the whole group of patients (group ABC). (TIF 278 kb)

Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse Processing of odors in the main olfactory bulb (MOB) is modulated by higher brain afferents depending on the internal state, motivation, memory and emotions. For example satiety or hunger are known to change the perception of food odors. To shed light on this modulation a greater understanding of the underlying circuitry is required. To this aim we conducted tracing experiments in mice. First, stereotaxic injections of monosynaptic retrograde DiI and choleratoxin subunit B (CTb) into the dorsal olfactory bulb (focusing on the granular layer) were performed. In a second approach, we injected the pseudorabies virus 152 (PRV152) (kindly provided by Prof L. Enquist; Princeton University). As a neuronal tracer, this neurotropic virus can spread in synaptically connected neurons, dissecting the entire circuitry. The temporal analysis of the viral distribution allows to determine the number of synapses crossed. Both DiI and CTb confirm all the main centrifugal afferents to the MOB which were previously described in the literature. Thus, except the olfactory tubercle, all regions belonging to the olfactory primary cortex were labeled. Moreover, in the piriform cortex, labeling was mainly located in its dorsal part, confirming the topographical anatomical organization of cortico-bulbar projections. Direct projections arising from orexinergic neurons in the lateral hypothalamus were also observed. Regarding polysynaptic tracing, mice were sacrificed one, two and three days after PRV152 injections. After one day, only some of the direct neuromodulatory afferents were labelled. Thus, the locus coeruleus, which has a very caudal location in the brain close to the forth ventricle, showed already staining. In contrast, other neuromodulatory afferents (arising from raphe nuclei, ventral tegmental area, basal forebrain) were not labeled at this stage. Two days after injection, comparison with DiI/CTb results indicate that all the first-order connections were labeled. At this stage, second-order projections started to appear (for example, the midline thalamic nuclei as reuniens and rhomboid nuclei). Lastly, after three days, an extensive brain labeling occurred although some brain regions still showed a lack of staining. In conclusion, using DiI/CTb tracers that do not cross synapses allow to stain all primary afferents. In contrast, viral tracer migration depends on timing as well as number of connecting synapses. Thus, labeling occurring already one day after injection indicates a strong connection. Accordingly, our results suggest that the stained cells in the locus coeruleus send strong projections to the MOB. Furthermore this method allows to follow up the circuits involved in olfactory modulation. Data analysis of all the other labeled regions, focusing on hypothalamic nuclei and brain areas involved in arousal and food intake, are in progress.

Background: The relationship between the insulin-like growth factor-I (IGF-I) system and Alzheimer's disease (AD) is mostly based on transversal studies. It remains, however, to demonstrate whether IGF-I is associated with cognitive decline over time in AD.Objective: The objective of the study was to analyze the course of cognitive decline of AD subjects over a 24-month period in relation to serum IGF-I and insulin-like growth factor binding protein-3 (IGFBP-3) measured at baseline.Methods: Data are from the SIGAL follow-up study. IGF-I and IGFBP-3 were measured in AD subjects who performed a Mini-Mental State Examination (MMSE) every 6 months for 2 years. MMSE course was analyzed using a mixed model with random intercept and slope function.Results: Among the 200 AD participants, 146 (mean age = 81.1 (standard deviation (SD) = 5.9) years, 62.6% of women) had at least one follow-up visit. Mean IGF-I at baseline was 147.8 (74.2) ng/mL. Hundred forty-six participants (62.6%) had at least one follow-up visit. Mean MMSE was 21.7 (4.7)/30 and dropped on average by 2.28 points per year. MMSE decline was steeper among participants with lower IGF-I. For each decrease of 1 SD of IGF-I, subjects lost an additional 0.63 points per year in MMSE, e.g., participants with IGF-I level of 74 ng/mL lost 2.91 MMSE points per year whereas participants with IGF-I of 222 ng/mL lost 1.65 MMSE points per year. There was no association between IGFBP-3 and cognitive decline.Conclusion: Lower baseline serum IGF-I was associated with faster cognitive decline in AD over a 2-year period.