Cortical dysmorphology and reduced cortico-collicular projections in an animal model of autism spectrum disorder.

Autism spectrum disorder is a neurodevelopmental disability that includes sensory disturbances. Hearing is frequently affected and ranges from deafness to hypersensitivity. In utero exposure to the antiepileptic valproic acid is associated with increased risk of autism spectrum disorder in humans and timed valproic acid exposure is a biologically relevant and validated animal model of autism spectrum disorder. Valproic acid-exposed rats have fewer neurons in their auditory brainstem and thalamus, fewer calbindin-positive neurons, reduced ascending projections to the midbrain and thalamus, elevated thresholds, and delayed auditory brainstem responses. Additionally, in the auditory cortex, valproic acid exposure results in abnormal responses, decreased phase-locking, elevated thresholds, and abnormal tonotopic maps. We therefore hypothesized that in utero, valproic acid exposure would result in fewer neurons in auditory cortex, neuronal dysmorphology, fewer calbindin-positive neurons, and reduced connectivity. We approached this hypothesis using morphometric analyses, immunohistochemistry, and retrograde tract tracing. We found thinner cortical layers but no changes in the density of neurons, smaller pyramidal and non-pyramidal neurons in several regions, fewer neurons immunoreactive for calbindin-positive, and fewer cortical neurons projecting to the inferior colliculus. These results support the widespread impact of the auditory system in autism spectrum disorder and valproic acid-exposed animals and emphasize the utility of simple, noninvasive auditory screening for autism spectrum disorder.

[Expression changes of RNA m6A regulators in mouse cerebellum affected by hypobaric hypoxia stimulation].

Objective: To investigate the role of RNA m6A methylation in mediating cerebellar dysplasia through analyzing the phenotypes of the mouse cerebella and the expression of several key m6A regulators upon hypobaric hypoxia treatment. Methods: Five-day old C57/BL6 mice were exposed to hypobaric hypoxia for 9 days. The status of mouse cerebellar development was analyzed by comparing the body weights, brain weights and histological features. Immunostaining of cell-type-specific markers was performed to analyze the cerebellar morphology. Real-time PCR, Western blot and immunohistochemical staining were performed to detect the expression of key m6A regulators in the mouse cerebella. Results: Compared with the control, the body weights, brain weights and cerebellar volumes of hypobaric hypoxic mice were significantly reduced (P<0.01). The expression of specific markers in different cells, including NeuN (mature neuron), Calbindin-D28K (Purkinje cell) and GFAP (astrocyte), was decreased in hypobaric hypoxic mouse cerebella (P<0.01), accompanied with disorganized cellular structure. The expression of methyltransferase METTL3 was significantly down-regulated in the cerebella of hypobaric hypoxic mice (P<0.05). Conclusions: Hypobaric hypoxia stimulation causes mouse cerebellar dysplasia, with structural abnormalities in mature granular neurons, Purkinje cells and astrocytes. Expression of METTL3 is decreased in hypobaric hypoxic mice cerebellum compared with that of normobaric normoxic mice, suggesting that its mediated RNA m6A methylation may play an important role in hypobaric hypoxia-induced mouse cerebellar dysplasia.

Postmortem neuropathology in early Huntington disease.

Two aspects of the neuropathology of early Huntington disease (HD) are examined. Neurons of the neostriatum are counted to determine relative loss in striosomes versus matrix at early stages, including for the first time in preclinical cases. An immunohistochemical procedure is described that tentatively distinguishes early HD from HD mimic disorders in postmortem brains. Counts of striatal projection neurons (SPNs) in striosomes defined by calbindin immunohistochemistry versus counts in the surrounding matrix are reported for 8 Vonsattel grade 0 (including 5 premanifest), 8 grade 1, 2 grade 2 HD, and for 8 control postmortem brains. Mean counts of striosome and matrix SPNs were significantly lower in premanifest grade 0 versus controls, with striosome counts significantly lower than matrix. In 8 grade 1 and 2 grade 2 brains, no striosomes with higher SPN counts than in the surrounding matrix were observed. Comparing dorsal versus ventral neostriatum, SPNs in dorsal striosomes and matrix declined more than ventral, making clear the importance of the dorsoventral site of tissue selection for research studies. A characteristic pattern of expanded polyglutamine-immunopositive inclusions was seen in all HD cases. Inclusions were always present in some SPNs and some pontine nucleus neurons and were absent in Purkinje cells, which showed no obvious cell loss.

Urinary Concentration of Renal Biomarkers in Healthy Term Neonates: Gender Differences in GST-pi Excretion.

BACKGROUND Serum creatinine, the criterion standard in assessment of renal function, is not reliable for the neonatal period because of its dependence on renal immaturity and maternal creatinine levels. Thus, it is important to study other biomarkers of renal function in neonates. The present study aimed to measure the urinary concentration of renal biomarkers: calbindin, clusterin, GST-pi (glutathione-S-transferase-alpha), KIM-1 (kidney injury molecule 1), MCP-1 (monocyte chemoattractant protein-1), and B2M (beta 2-microglobulin) in healthy term neonates. MATERIAL AND METHODS In the study, we included 80 healthy term neonates - 40 females and 40 males. We collected the neonates' urine on their first day of life. Urinary concentrations of calbindin, clusterin, KIM-1, MCP-1, and B2M were assessed using an immunoassay for kidney toxicology research. Because dilution of the urine affects the concentrations of urinary biomarkers, we normalized them to the concentration of urinary creatinine (Cr) and present them as biomarker/Cr ratios. RESULTS We obtained the following values of the assessed biomarker/Cr ratios (median [Q1-Q3]): calbindin/Cr.: 197.04 (56.25-595.17), KIM-1/Cr: 0.09 (0.04-0.18), MCP-1/Cr: 0.05 (0.02-0.14), B2M/Cr: 126.12 (19.03-342.48), GST-pi/Cr in boys: 1.28 (0.46-3.77), GST-pi/Cr in girls: 8.66 (2.51-27.82), clusterin/Cr: 4.55 (1.79-12.97) ng/mg Cr. CONCLUSIONS We showed the urinary levels of calbindin, clusterin, GST-pi, KIM-1, MCP-1, B2M in white, West Slavic, healthy term neonates. We found that in there is an association between female sex and a higher urinary GST-pi excretion, but urinary excretion of calbindin, clusterin, KIM-1, MCP-1, and B2M is sex-independent. The urinary levels of the assessed biomarkers do not depend on the method of delivery.

Vitamin D, Calbindin, and calcium signaling: Unraveling the Alzheimer's connection.

Calcium is a ubiquitous second messenger that is indispensable in regulating neurotransmission and memory formation. A precise intracellular calcium level is achieved through the concerted action of calcium channels, and calcium exerts its effect by binding to an array of calcium-binding proteins, including calmodulin (CAM), calcium-calmodulin complex-dependent protein kinase-II (CAMK-II), calbindin (CAL), and calcineurin (CAN). Calbindin orchestrates a plethora of signaling events that regulate synaptic transmission and depolarizing signals. Vitamin D, an endogenous fat-soluble metabolite, is synthesized in the skin upon exposure to ultraviolet B radiation. It modulates calcium signaling by increasing the expression of the calcium-sensing receptor (CaSR), stimulating phospholipase C activity, and regulating the expression of calcium channels such as TRPV6. Vitamin D also modulates the activity of calcium-binding proteins, including CAM and calbindin, and increases their expression. Calbindin, a high-affinity calcium-binding protein, is involved in calcium buffering and transport in neurons. It has been shown to inhibit apoptosis and caspase-3 activity stimulated by presenilin 1 and 2 in AD. Whereas CAM, another calcium-binding protein, is implicated in regulating neurotransmitter release and memory formation by phosphorylating CAN, CAMK-II, and other calcium-regulated proteins. CAMK-II and CAN regulate actin-induced spine shape changes, which are further modulated by CAM. Low levels of both calbindin and vitamin D are attributed to the pathology of Alzheimer's disease. Further research on vitamin D via calbindin-CAMK-II signaling may provide newer insights, revealing novel therapeutic targets and strategies for treatment.

Isolated loss of the AUTS2 long isoform, brain-wide or targeted to Calbindin-lineage cells, generates a specific suite of brain, behavioral, and molecular pathologies.

Rearrangements within the AUTS2 region are associated with a rare syndromic disorder with intellectual disability, developmental delay, and behavioral abnormalities as core features. In addition, smaller regional variants are linked to wide range of neuropsychiatric disorders, underscoring the gene's essential role in brain development. Like many essential neurodevelopmental genes, AUTS2 is large and complex, generating distinct long (AUTS2-l) and short (AUTS2-s) protein isoforms from alternative promoters. Although evidence suggests unique isoform functions, the contributions of each isoform to specific AUTS2-linked phenotypes have not been clearly resolved. Furthermore, Auts2 is widely expressed across the developing brain, but cell populations most central to disease presentation have not been determined. In this study, we focused on the specific roles of AUTS2-l in brain development, behavior, and postnatal brain gene expression, showing that brain-wide AUTS2-l ablation leads to specific subsets of the recessive pathologies associated with mutations in 3' exons (exons 8-19) that disrupt both major isoforms. We identify downstream genes that could explain expressed phenotypes including hundreds of putative direct AUTS2-l target genes. Furthermore, in contrast to 3' Auts2 mutations which lead to dominant hypoactivity, AUTS2-l loss-of-function is associated with dominant hyperactivity and repetitive behaviors, phenotypes exhibited by many human patients. Finally, we show that AUTS2-l ablation in Calbindin 1-expressing cell lineages is sufficient to yield learning/memory deficits and hyperactivity with abnormal dentate gyrus granule cell maturation, but not other phenotypic effects. These data provide new clues to in vivo AUTS2-l functions and novel information relevant to genotype-phenotype correlations in the human AUTS2 region.

A comparative study on the effect of limestone particle size on performance, ileal digestibility of calcium and phosphorus, and bone characteristics in broilers and pullets.

1. The effects of limestone particle size on growth performance, gastrointestinal tract (GIT) traits, ileal morphology, duodenal gene expression of calbindin, apparent ileal digestibility coefficients (AIDC) of calcium (Ca) and phosphorus (P) and tibia characteristics in broilers and pullets were assessed in broilers and pullets. These birds have different growth rates and likely different responses to parameters, such as particle size.2. A total of 240 chicks aged one day, 120 Ross 308 female broilers, and 120 Hy-Line pullets were allocated randomly into four treatments in a 2 × 2 factorial arrangement with two bird types (broilers vs. pullets) and two limestone particle sizes (<0.5 mm versus 1-2 mm) to give six replicates containing 10 chicks in each from 1 to 21 d of age.3. Feed intake and weight gain were greater (P < 0.001) and feed per gain (FCR) was better (P < 0.001) in broilers compared to pullets from 1 to 21 d of age. Greater villus width (P < 0.01), villus height (P < 0.001) and crypt depth (P < 0.01) were seen for broilers compared to pullets.4. Pullets fed coarse Ca particles had higher calbindin gene expression at 21 d of age (P = 0.05). Both AIDC of Ca and P were higher (P < 0.001) in broilers compared to pullets. The AIDC of Ca from 0.463 to 0.516 was increased (P < 0.05) by feeding coarse limestone particles. A significant interaction was found between bird type and limestone particle size (P < 0.01), where pullets fed coarse Ca particles had higher bone P concentration in tibia than broilers.5. Broilers had better ileum absorptive capacity and growth performance compared to pullets. The AIDC of Ca and P was higher in broilers than in pullets. Increased limestone particle size elevated villus height, AIDC of Ca and concentration of P in the tibia.

Sialyltransferase Mutations Alter the Expression of Calcium-Binding Interneurons in Mice Neocortex, Hippocampus and Striatum.

Gangliosides are major glycans on vertebrate nerve cells, and their metabolic disruption results in congenital disorders with marked cognitive and motor deficits. The sialyltransferase gene St3gal2 is responsible for terminal sialylation of two prominent brain gangliosides in mammals, GD1a and GT1b. In this study, we analyzed the expression of calcium-binding interneurons in primary sensory (somatic, visual, and auditory) and motor areas of the neocortex, hippocampus, and striatum of St3gal2-null mice as well as St3gal3-null and St3gal2/3-double null. Immunohistochemistry with highly specific primary antibodies for GABA, parvalbumin, calretinin, and calbindin were used for interneuron detection. St3gal2-null mice had decreased expression of all three analyzed types of calcium-binding interneurons in all analyzed regions of the neocortex. These results implicate gangliosides GD1a and GT1b in the process of interneuron migration and maturation.

P2Y12 receptor mediates apoptosis and demyelination to affect functional recovery in mice with spinal cord injury.

Among diseases of the central nervous system (CNS), spinal cord injury (SCI) has a high fatality rate. It has been proven that P2Y G protein-coupled purinergic receptors have a neuroprotective role in apoptosis and regeneration inside the damaged spinal cord. The P2Y12 receptor (P2Y12R) has recently been linked to peripheral neuropathy and stroke. However, the role of P2Y12R after SCI remains unclear. Our study randomly divided C57BL/6J female mice into 3 groups: Sham+DMSO, SCI+DMSO, and SCI+MRS2395. MRS2395 as a P2Y12R inhibitor was intraperitoneally injected at a dose of 1.5 mg/kg once daily for 7 days. We showed that the P2Y12R was markedly activated after injury, and it was double labeled with the microglial and neuron. Behavioral tests were employed to assess motor function recovery. By using immunofluorescence staining, the NeuN expression level was detected. The morphology of neurons was observed by hematoxylin-eosin and Nissl staining. P2Y12R, Bax, GFAP, PCNA and calbindin expression levels were detected using Western blot. Meanwhile, mitochondria and myelin sheath were observed by transmission electron microscopy (TEM). Our findings demonstrated that MRS2395 significantly enhanced motor function induced by SCI and that was used to alleviate apoptosis and astrocyte scarring. NeuN positive cells in the SCI group were lower than in the therapy group, although Bax, GFAP, PCNA and calbindin expression levels were considerably higher. Moreover, following MRS2395 therapy, the histological damage was reversed. A notable improvement in myelin sheath and mitochondrial morphology was seen in the therapy group. Together, our findings indicate that activation of P2Y12R in damaged spinal cord may be a critical event and suggest that inhibition of P2Y12R might be a feasible therapeutic strategy for treating SCI.

Pattern of ventral temporal lobe interconnections in rhesus macaques.

The entorhinal cortex (EC, A28) is linked through reciprocal pathways with nearby perirhinal and visual, auditory, and multimodal association cortices in the temporal lobe, in pathways associated with the flow of information for memory processing. The density and laminar organization of these pathways is not well understood in primates. We studied interconnections within the ventral temporal lobe in young adult rhesus monkeys of both sexes with the aid of neural tracers injected in temporal areas (Ts1, Ts2, TE1, area 36, temporal polar area TPro, and area 28) to determine the density and laminar distribution of projection neurons within the temporal lobe. These temporal areas can be categorized into three different cortical types based on their laminar architecture: the sensory association areas Ts1, Ts2, and TE1 have six layers (eulaminate); the perirhinal limbic areas TPro and area 36 have an incipient layer IV (dysgranular); and area 28 lacks layer IV (agranular). We found that (1) temporal areas that are similar in laminar architecture by cortical type are strongly interconnected, and (2) the laminar pattern of connections is dependent on the difference in cortical laminar structure between linked areas. Thus, agranular A28 is more strongly connected with other agranular/dysgranular areas than with eulaminate cortices. Further, A28 predominantly projected via feedback-like pathways that originated in the deep layers, and received feedforward-like projections from areas of greater laminar differentiation, which emanated from the upper layers. Our results are consistent with the Structural Model, which relates the density and laminar distribution of connections to the relationship of the laminar structure between the linked areas. These connections were viewed in the context of the inhibitory microenvironment of A28, which is the key recipient of pathways from the cortex and of the output of hippocampus. Our findings revealed a higher population of calretinin (CR)-expressing neurons in EC, with a significantly higher density in its lateral division. Medial EC had a higher density of CR neurons in the deep layers, particularly in layer Va. In contrast, parvalbumin (PV) neurons were more densely distributed in the deep layers of the lateral subdivisions of rostral EC, especially in layer Va, whereas the densities of calbindin (CB) neurons in the medial and lateral EC were comparable in all layers, except for layer IIIa, in which medial EC had a higher CB population than the lateral. The pattern of connections in the inhibitory microenvironment of EC, which sends and receives input from the hippocampus, may shed light on signal propagation in this network associated with diverse aspects of memory, and disruptions in neurologic and psychiatric diseases that affect this region.

A multifaceted architectural framework of the mouse claustrum complex.

Accurate anatomical characterizations are necessary to investigate neural circuitry on a fine scale, but for the rodent claustrum complex (CLCX), this has yet to be fully accomplished. The CLCX is generally considered to comprise two major subdivisions, the claustrum (CL) and the dorsal endopiriform nucleus (DEn), but regional boundaries to these areas are debated. To address this, we conducted a multifaceted analysis of fiber- and cytoarchitecture, genetic marker expression, and connectivity using mice of both sexes, to create a comprehensive guide for identifying and delineating borders to CLCX, including an online reference atlas. Our data indicated four distinct subregions within CLCX, subdividing both CL and DEn into two. Additionally, we conducted brain-wide tracing of inputs to CLCX using a transgenic mouse line. Immunohistochemical staining against myelin basic protein (MBP), parvalbumin (PV), and calbindin (CB) revealed intricate fiber-architectural patterns enabling precise delineations of CLCX and its subregions. Myelinated fibers were abundant dorsally in CL but absent ventrally, whereas PV expressing fibers occupied the entire CL. CB staining revealed a central gap within CL, also visible anterior to the striatum. The Nr2f2, Npsr1, and Cplx3 genes expressed specifically within different subregions of the CLCX, and Rprm helped delineate the CL-insular border. Furthermore, cells in CL projecting to the retrosplenial cortex were located within the myelin sparse area. By combining own experimental data with digitally available datasets of gene expression and input connectivity, we could demonstrate that the proposed delineation scheme allows anchoring of datasets from different origins to a common reference framework.

Mice are a highly tractable model for studying the claustrum complex (CLCX). However, without a consensus on how to delineate the CLCX in rodents, comparing results between studies is challenging. It is therefore important to expand our anatomical knowledge of the CLCX, to match the level of detail needed to study its functional properties. To improve and expand upon preexisting delineation schemes, we used the combinatorial expression of several markers to create a comprehensive guide to delineate the CLCX and its subregions, including an online reference atlas. This anatomical framework will allow researchers to anchor future experimental data into a common reference space. We demonstrated the power of this new structural framework by combining our own experimental data with digitally available data on gene expression and input connectivity of the CLCX.

The Co-Expression Pattern of Calcium-Binding Proteins with γ-Aminobutyric Acid and Glutamate Transporters in the Amygdala of the Guinea Pig: Evidence for Glutamatergic Subpopulations.

The amygdala has large populations of neurons utilizing specific calcium-binding proteins such as parvalbumin (PV), calbindin (CB), or calretinin (CR). They are considered specialized subsets of γ-aminobutyric acid (GABA) interneurons; however, many of these cells are devoid of GABA or glutamate decarboxylase. The neurotransmitters used by GABA-immunonegative cells are still unknown, but it is suggested that a part may use glutamate. Thus, this study investigates in the amygdala of the guinea pig relationships between PV, CB, or CR-containing cells and GABA transporter (VGAT) or glutamate transporter type 2 (VGLUT2), markers of GABAergic and glutamatergic neurons, respectively. The results show that although most neurons using PV, CB, and CR co-expressed VGAT, each of these populations also had a fraction of VGLUT2 co-expressing cells. For almost all neurons using PV (~90%) co-expressed VGAT, while ~1.5% of them had VGLUT2. The proportion of neurons using CB and VGAT was smaller than that for PV (~80%), while the percentage of cells with VGLUT2 was larger (~4.5%). Finally, only half of the neurons using CR (~53%) co-expressed VGAT, while ~3.5% of them had VGLUT2. In conclusion, the populations of neurons co-expressing PV, CB, and CR are in the amygdala, primarily GABAergic. However, at least a fraction of neurons in each of them co-express VGLUT2, suggesting that these cells may use glutamate. Moreover, the number of PV-, CB-, and CR-containing neurons that may use glutamate is probably larger as they can utilize VGLUT1 or VGLUT3, which are also present in the amygdala.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

Histological and Immunohistochemical Investigation of the Cerebellum in Porcupine and Guinea Pig.

The present study was designed to investigate the cerebellum histology and immunohistochemistry in porcupine (Hystrix cristata) and guinea pig (Cavia porcellus). Two adult porcupines and two adult guinea pigs were used. For general histology, crystal violet and Luxol fast blue stains were applied. For immunohistochemistry, myelin-associated glycoprotein (MAG), neurofilament 200 (NF200), calbindin D-28K, and glial fibrillary-associated protein (GFAP) were investigated. The cerebellar cortex in both species was composed of three cellular layers: molecular, granular, and Purkinje cell (PC) layers. Purkinje cells in the porcupine showed a purple-colored and dark blue-colored cytoplasm in reaction to the crystal violet and Luxol fast blue staining, respectively. In the guinea pig, PC has a uniform reaction to the Luxol fast blue with dark-blue-colored cytoplasm. However, in response to the crystal violet, some PC with dark-purple cytoplasm showed stronger reaction than other PC which showed light-purple cytoplasm. The PC layer in some folia of the porcupine cerebellum was composed of 2-3 layers. The expression rates of calbindin D-28K, MAG, GFAP, and NF200 in the porcupine cerebellum were determined to be 19%, 42.5%, 62%, and 30%, respectively. These values were determined to be 27%, 34%, 43.5%, and 31.5%, respectively, in the guinea pig cerebellum.

Protein Exaptation by Endogenous Retroviral Elements Shapes Tumor Cell Senescence and Downstream Immune Signaling.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immunotherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence-associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbindin reduces CXCL8 chemokine production and downstream neutrophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Characterization of TRPM8-expressing neurons in the adult mouse hypothalamus.

Transient receptor potential melastatin 8 (TRPM8) is a menthol receptor that detects cold temperatures and influences behaviors and autonomic functions under cold stimuli. Despite the well-documented peripheral roles of TRPM8, the evaluation of its central functions is still of great interest. The present study clarifies the nature of a subpopulation of TRPM8-expressing neurons in the adult mice. Combined in situ hybridization and immunohistochemistry revealed that TRPM8-expressing neurons are exclusively positive for glutamate decarboxylase 67 mRNA signals in the lateral septal nucleus (LS) and preoptic area (POA) but produced no positive signal for vesicular glutamate transporter 2. Double labeling immunohistochemistry showed the colocalization of TRPM8 with vesicular GABA transporter at axonal terminals. Immunohistochemistry further revealed that TRPM8-expressing neurons frequently expressed calbindin and calretinin in the LS, but not in the POA. TRPM8-expressing neurons in the POA expressed a prostaglandin E2 receptor, EP3, and neurotensin, whereas expression in the LS was minimal. These results indicate that hypothalamic TRPM8-expressing neurons are inhibitory GABAergic, while the expression profile of calcium-binding proteins, neurotensin, and EP3 differs between the POA and LS.

Shades of gray in human white matter.

Anatomists have long expressed interest in neurons of the white matter, which is by definition supposed to be free of neurons. Hypotheses regarding their biochemical signature and physiological function are mainly derived from animal models. Here, we investigated 15 whole-brain human postmortem specimens, including cognitively normal cases and those with pathologic Alzheimer's disease (AD). Quantitative and qualitative methods were used to investigate differences in neuronal size and density, and the relationship between neuronal processes and vasculature. Double staining was used to evaluate colocalization of neurochemicals. Two topographically distinct populations of neurons emerged: one appearing to arise from developmental subplate neurons and the other embedded within deep, subcortical white matter. Both populations appeared to be neurochemically heterogeneous, showing positive reactivity to acetylcholinesterase (AChE) [but not choline acetyltransferase (ChAT)], neuronal nuclei (NeuN), nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), microtubule-associated protein 2 (MAP-2), somatostatin (SOM), nonphosphorylated neurofilament protein (SMI-32), and calcium-binding proteins calbindin-D28K (CB), calretinin (CRT), and parvalbumin (PV). PV was more richly expressed in superficial as opposed to deep white matter neurons (WMNs); subplate neurons were also significantly larger than their deeper counterparts. NADPH-d, a surrogate for nitric oxide synthase, allowed for the striking morphological visualization of subcortical WMNs. NADPH-d-positive subcortical neurons tended to embrace the outer walls of microvessels, suggesting a functional role in vasodilation. The presence of AChE positivity in these neurons, but not ChAT, suggests that they are cholinoceptive but noncholinergic. WMNs were also significantly smaller in AD compared to control cases. These observations provide a landscape for future systematic investigations.

Comparative features of calretinin, calbindin, and parvalbumin expressing interneurons in mouse and monkey primary visual and frontal cortices.

Fundamental differences in excitatory pyramidal cells across cortical areas and species highlight the implausibility of extrapolation from mouse to primate neurons and cortical networks. Far less is known about comparative regional and species-specific features of neurochemically distinct cortical inhibitory interneurons. Here, we quantified the density, laminar distribution, and somatodendritic morphology of inhibitory interneurons expressing one or more of the calcium-binding proteins (CaBPs) (calretinin [CR], calbindin [CB], and/or parvalbumin [PV]) in mouse (Mus musculus) versus rhesus monkey (Macaca mulatta) in two functionally and cytoarchitectonically distinct regions-the primary visual and frontal cortical areas-using immunofluorescent multilabeling, stereological counting, and 3D reconstructions. There were significantly higher densities of CB+ and PV+ neurons in visual compared to frontal areas in both species. The main species difference was the significantly greater density and proportion of CR+ interneurons and lower extent of CaBP coexpression in monkey compared to mouse cortices. Cluster analyses revealed that the somatodendritic morphology of layer 2-3 inhibitory interneurons is more dependent on CaBP expression than on species and area. Only modest effects of species were observed for CB+ and PV+ interneuron morphologies, while CR+ neurons showed no difference. By contrast to pyramidal cells that show highly distinctive area- and species-specific features, here we found more subtle differences in the distribution and features of interneurons across areas and species. These data yield insight into how nuanced differences in the population organization and properties of neurons may underlie specializations in cortical regions to confer species- and area-specific functional capacities.

Human endogenous retrovirus onco-exaptation counters cancer cell senescence through calbindin.

Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterize most cancer types and are linked with disease outcomes. However, the underlying processes are incompletely understood. Here, we show that elevated transcription of HERVH proviruses predicted survival of lung squamous cell carcinoma (LUSC) and identified an isoform of CALB1, encoding calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression was initiated in preinvasive lesions and associated with their progression. Calbindin loss in LUSC cell lines impaired in vitro and in vivo growth and triggered senescence, consistent with a protumor effect. However, calbindin also directly controlled the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells became the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition were offset by the prevention of SASP and protumor inflammation at later stages.

Aging-associated increased nitric oxide production is a potential cause of inferior eggshell quality produced by aged laying hens.

It is important to prolong the productive life of laying hens without compromising their welfare. Therefore, in this study, we aimed to identify the cause for inferior quality egg production of aged hens by investigating the aging-associated molecular changes related to eggshell formation in the isthmic and uterine mucosae and determining whether nitric oxide plays a role in decreasing the quality of eggs produced by aged hens. Young (35 weeks old) and aged (130 weeks old) White Leghorn laying hens were used in this study to determine the effects of age on the expression of proteins related to eggshell membranes formation in the isthmus and eggshell biomineralization and nitric oxide production in the uterus. Nitric oxide synthesis during the ovulatory cycle was examined in twenty-five laying hens (46-52 weeks old) euthanized at 0, 4, 7, 16, and 24 h after oviposition. S-Nitroso-N-acetylpenicillamine (a nitric oxide donor) was added to the cultured isthmic and uterine mucosal cells to examine the effects of nitric oxide on the expression of genes related to eggshell membranes formation and eggshell biomineralization, respectively. The results showed that the protein abundance of collagen I and V in the isthmic mucosa and collagen V in the eggshell membranes were lower in aged hens than in young hens. The mRNA expression levels of calbindin, osteopontin, and ovocalyxin-36 and the protein abundance of calbindin and carbonic anhydrase-2 were lower in the uterine mucosa of aged hens than in that of young hens. Nitric oxide synthesis was higher in the uterine mucosa of aged hens than in that of young hens. Nitric oxide downregulated the mRNA expression levels of osteopontin and ovocalyxin-36 in cultured uterine mucosal cells. Our results indicated that the eggshell quality decreases with aging due to molecular changes in the uterine mucosa affecting the eggshell membrane formation and eggshell biomineralization. Moreover, nitric oxide overproduction may play a role in this dysfunction.