Purkinje Cell Protein Research Articles

Cerebellum Purkinje cell vulnerability in aged rats with memory impairment.

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.

Immunohistochemical field parcellation of the human hippocampus along its antero-posterior axis

The primate hippocampus includes the dentate gyrus, cornu ammonis (CA), and subiculum. CA is subdivided into four fields (CA1-CA3, plus CA3h/hilus of the dentate gyrus) with specific pyramidal cell morphology and connections. Work in non-human mammals has shown that hippocampal connectivity is precisely patterned both in the laminar and longitudinal axes. One of the main handicaps in the study of neuropathological semiology in the human hippocampus is the lack of clear laminar and longitudinal borders. The aim of this study was to explore a histochemical segmentation of the adult human hippocampus, integrating field (medio-lateral), laminar, and anteroposterior longitudinal patterning. We provide criteria for head-body-tail field and subfield parcellation of the human hippocampus based on immunodetection of Rabphilin3a (Rph3a), Purkinje-cell protein 4 (PCP4), Chromogranin A and Regulation of G protein signaling-14 (RGS-14). Notably, Rph3a and PCP4 allow to identify the border between CA3 and CA2, while Chromogranin A and RGS-14 give specific staining of CA2. We also provide novel histological data about the composition of human-specific regions of the anterior and posterior hippocampus. The data are given with stereotaxic coordinates along the longitudinal axis. This study provides novel insights for a detailed region-specific parcellation of the human hippocampus useful for human brain imaging and neuropathology.

Astrocyte-like subpopulation of NG2 glia in the adult mouse cortex exhibits characteristics of neural progenitor cells.

Glial cells expressing neuron-glial antigen 2 (NG2), also known as oligodendrocyte progenitor cells (OPCs), play a critical role in maintaining brain health. However, their ability to differentiate after ischemic injury is poorly understood. The aim of this study was to investigate the properties and functions of NG2 glia in the ischemic brain. Using transgenic mice, we selectively labeled NG2-expressing cells and their progeny in both healthy brain and after focal cerebral ischemia (FCI). Using single-cell RNA sequencing, we classified the labeled glial cells into five distinct subpopulations based on their gene expression patterns. Additionally, we examined the membrane properties of these cells using the patch-clamp technique. Of the identified subpopulations, three were identified as OPCs, whereas the fourth subpopulation had characteristics indicative of cells likely to develop into oligodendrocytes. The fifth subpopulation of NG2 glia showed astrocytic markers and had similarities to neural progenitor cells. Interestingly, this subpopulation was present in both healthy and post-ischemic tissue; however, its gene expression profile changed after ischemia, with increased numbers of genes related to neurogenesis. Immunohistochemical analysis confirmed the temporal expression of neurogenic genes and showed an increased presence of NG2 cells positive for Purkinje cell protein-4 at the periphery of the ischemic lesion 12 days after FCI, as well as NeuN-positive NG2 cells 28 and 60 days after injury. These results suggest the potential development of neuron-like cells arising from NG2 glia in the ischemic tissue. Our study provides insights into the plasticity of NG2 glia and their capacity for neurogenesis after stroke.

Screening and validation of plasma cell-derived, purinergic, and calcium signalling-related genetic signature to predict prognosis and PD-L1/PD-1 blockade responses in lung adenocarcinoma.

This study aims at screening and validation of prospective genetic signature for lung adenocarcinoma (LUAD) prognosis and treatment. The immune-related genes (IRGs) were obtained from The Cancer Genome Atlas (TCGA) dataset where a total of 535 LUAD and 59 control samples were included. A risk model was then developed for the risk stratification of LUAD patients. The immune cell infiltration, clinical outcomes, and the therapeuticefficacy of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) blockade were compared between high and low-risk groups.Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used to explore the biological processes and signalling pathways associated with the IRGs. Finally, IRGs mRNA levels were assayed by reverse transcription quantitative real-time PCR (RT-qPCR)in LUAD and relevant cell lines. Two IRGs, P2RX1 (purinergic receptor P2X 1) and PCP4 (Purkinje cell protein 4), were screened from a module that possesses the highest correlation with plasma cells. RT-qPCR verified the expression of the two IRGs in plasmacytoma cell RPMI 8226 but not in LUAD cells. A higher risk score is associated with a lower infiltration of immune cells. Kaplan-Meier and nomogram analysis showed that the high-risk group has a lower survival rate than the low-risk cohort. Furthermore, the high-risk group had a worse response rate to PD-L1/PD-1 blockade. GSVA and GSEA-GO results indicated that a lower risk score is linked to signalling pathways and biological functions promoting immune response and inflammation. In contrast, a higher risk score is associated with signalling cascades promoting tumour growth. The immune-related prognostic model based on P2RX1 and PCP4 is conducive to predicting the therapeutic response of PD-L1/PD-1 blockade and clinical outcomes of LUAD.

PCP4 Promotes Alzheimer's Disease Pathogenesis by Affecting Amyloid-β Protein Precursor Processing.

Down syndrome (DS) is caused by an extra copy of all or part of chromosome 21. The patients with DS develop typical Alzheimer's disease (AD) neuropathology, indicating the role of genes on human chromosome 21 (HSA21) in the pathogenesis of AD. Purkinje cell protein 4 (PCP4), also known as brain-specific protein 19, is a critical gene located on HSA21. However, the role of PCP4 in DS and AD pathogenesis is not clear. To explore the role of PCP4 in amyloid-β protein precursor (AβPP) processing in AD. In this study, we investigated the role of PCP4 in AD progression in vitro and in vivo. In vitro experiments, we overexpressed PCP4 in human Swedish mutant AβPP stable expression or neural cell lines. In vitro experiments, APP23/PS45 double transgenic mice were selected and treated with AAV-PCP4. Multiple topics were detected by western blot, RT-PCR, immunohistochemical and behavioral test. We found that PCP4 expression was altered in AD. PCP4 was overexpressed in APP23/PS45 transgenic mice and PCP4 affected the processing of AβPP. The production of amyloid-β protein (Aβ) was also promoted by PCP4. The upregulation of endogenous AβPP expression and the downregulation of ADAM10 were due to the transcriptional regulation of PCP4. In addition, PCP4 increased Aβ deposition and neural plaque formation in the brain, and exuberated learning and memory impairment in transgenic AD model mice. Our finding reveals that PCP4 contributes to the pathogenesis of AD by affecting AβPP processing and suggests PCP4 as a novel therapeutic target for AD by targeting Aβ pathology.

Defining hippocampal area CA2 in the fox (Vulpes vulpes) brain.

Since 1959, the Russian Farm-Fox study has bred foxes to be either tame or, more recently, aggressive, and scientists have used them to gain insight into the brain structures associated with these behavioral features. In mice, hippocampal area CA2 has emerged as one of the essential regulators of social aggression, and so to eventually determine whether we could identify differences in CA2 between tame and aggressive foxes, we first sought to identify CA2 in foxes (Vulpes vulpes). As no clearly defined area of CA2 has been described in species such as cats, dogs, or pigs, it was not at all clear whether CA2 could be identified in foxes. In this study, we cut sections of temporal lobes from male and female red foxes, perpendicular to the long axis of the hippocampus, and stained them with markers of CA2 pyramidal cells commonly used in tissue from rats and mice. We observed that antibodies against Purkinje cell protein 4 best stained the pyramidal cells in the area spanning the end of the mossy fibers and the beginning of the pyramidal cells lacking mossy fibers, resembling the pattern seen in rats and mice. Our findings indicate that foxes do have a "molecularly defined" CA2, and further, they suggest that other carnivores like dogs and cats might as well. With this being the case, these foxes could be useful in future studies looking at CA2 as it relates to aggression.

Morphological and molecular markers of mouse area CA2 along the proximodistal and dorsoventral hippocampal axes.

Hippocampal area CA2 is a molecularly and functionally distinct region of the hippocampus that has classically been defined as the area with large pyramidal neurons lacking input from the dentate gyrus and the thorny excrescences (TEs) characteristic of CA3 neurons. A modern definition of CA2, however, makes use of the expression of several molecular markers that distinguish it from neighboring CA3 and CA1. Using immunohistochemistry, we sought to characterize the staining patterns of commonly used CA2 markers along the dorsal-ventral hippocampal axis and determine how these markers align along the proximodistal axis. We used a region of CA2 that stained for both Regulator of G-protein Signaling 14 (RGS14) and Purkinje Cell Protein 4 (PCP4; "double-labeled zone" [DLZ]) as a reference. Here, we report that certain commonly used CA2 molecular markers may be better suited for drawing distinct boundaries between CA2/3 and CA2/1. For example, RGS14+ and STEP+ neurons showed minimal to no extension into area CA1 while areas stained with VGluT2 and Wisteria Floribunda agglutinin were consistently smaller than the DLZ/CA2 borders by ~100μ on the CA1 or CA3 sides respectively. In addition, these patterns are dependent on position along the dorsal-ventral hippocampal axis such that PCP4 labeling often extended beyond the distal border of the DLZ into CA1. Finally, we found that, consistent with previous findings, mossy fibers innervate a subset of RGS14 positive neurons (~65%-70%) and that mossy fiber bouton number and relative size in CA2 are less than that of boutons in CA3. Unexpectedly, we did find evidence of some complex spines on apical dendrites in CA2, though much fewer in number than in CA3. Our results indicate that certain molecular markers may be better suited than others when defining the proximal and distal borders of area CA2 and that the presence or absence of complex spines alone may not be suitable as a distinguishing feature differentiating CA3 from CA2 neurons.

Molecular Mechanism of Long Noncoding RNA SNHG14 in Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells through the NEDD4L/FOXA2/PCP4 Axis.

Bone marrow-derived mesenchymal stem cells (BMSCs) have a superior potential of osteogenic differentiation (OD) and a promising stem cell type to treat bone defects. This study sought to investigate the molecular mechanism of long noncoding RNA small nucleolar RNA host gene 14 (SNHG14) in OD of BMSCs. Western blot analysis or RT-qPCR showed that SNHG14, neural precursor cell expressed developmentally downregulated 4-like (NEDD4L), and Purkinje cell protein 4 (PCP4) were upregulated whereas forkhead box A2 (FOXA2) was declined in OD of BMSCs. RT-qPCR and cell staining showed that SNHG14 downregulation repressed OD of BMSCs, as manifested by reductions in osteopontin and osteocalcin levels, the mineralization degree, and alkaline phosphatase activity. RNA/Co/chromatin immunoprecipitation and dual-luciferase assays and determination of mRNA stability and ubiquitination level showed that SNHG14 bound to human antigen R improves NEDD4L mRNA stability and expression, further promoted FOXA2 ubiquitination to inhibit FOXA2 expression, and then reduced FOXA2 enrichment on the PCP4 promoter to upregulate PCP4 transcription. Functional rescue experiments showed that the overexpression of NEDD4L or PCP4 and knockdown of FOXA2 both attenuated the inhibition of SNHG14 downregulation on OD of BMSCs. Overall, our findings suggested that SNHG14 promoted OD of BMSCs through the NEDD4L/FOXA2/PCP4 axis.

Exercise mitigates calpain induced Purkinje cell loss in diabetes

Calpain-1 is a ubiquitous calcium dependent cysteine protease and found in cytoplasm as well as mitochondria. We have earlier reported that active calpain-1 is translocated from cytosol to mitochondria and activates MMP9. Calpain-1 activation is detrimental to the heart in several different ways, but there is little evidence that it can degrade Purkinje cell protein (PCP-4) and impair contractility in diabetes. Our hypothesis is that in diabetes, PCP-4 is degraded by calpain-1, causing contractile dysfunction that can be mitigated by exercise. To test this hypothesis, we recruited four groups of mice, 1) db/+ control, 2) db/+ with exercise, 3) db/db, 4) db/db with exercise. The mice were exercised on treadmill for 8 weeks as per American Veterinary Research Guidelines. Adding calcium to isolated cardiomyocytes caused them to lose shape and die. Compared with live myocytes, we observed high calpain-1 levels as well as significantly lower levels of PCP-4 and increased levels of calmodulin and calmodulin kinase II (CaMKII) in dead myocytes. We used the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) plasmid to knock down calpain-1 in HL-1 myocytes which restored the levels of PCP-4 along with calmodulin and CaMKII. In vivo, we found upregulated levels of calpain-1 in db/db mice (diabetic) as compared to db/+ which were mitigated in the exercised mice. Conclusively our data strongly suggests that in diabetes there is high induction of calpain-1 with degrades PCP-4, a protein important for contractility and exercise can mitigate this.

Circ_0019693 promotes osteogenic differentiation of bone marrow mesenchymal stem cell and enhances osteogenesis-coupled angiogenesis via regulating microRNA-942-5p-targeted purkinje cell protein 4 in the development of osteoporosis

ABSTRACT Circular RNA (circRNA) is a crucial regulator in multiple human diseases, including osteoporosis (OP). However, the function of numerous circRNAs remains unclear. This study aimed to explore the role and mechanism of circ_0019693 in bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteogenesis-coupled angiogenesis. The expression of circ_0019693, miR-942-5p and purkinje cell protein 4 (PCP4) was measured using quantitative real-time PCR (qPCR) or Western blot. Osteogenic differentiation was monitored according to the protein levels of RUNX family transcription factor 2 (RUNX2), osteopontin (OPN) and osteocalcin (OCN) by Western blot analysis, and the activity of alkaline phosphatase (ALP). Angiogenesis was evaluated by tube formation assay. The targeting relationship between miR-942-5p and circ_0019693 or PCP4 was identified using pull-down, dual-luciferase reporter, and RNA immunoprecipitation assays. Circ_0019693 was downregulated in serum samples and bone tissues from OP patients relative to normal subjects. Circ_0019693 expression was enhanced in the stages of BMSC osteogenic differentiation. Circ_0019693 overexpression enhanced the activity of ALP and the expression of RUNX2, OPN and OCN, and its overexpression also promoted angiogenesis. However, circ_0019693 knockdown played the opposite effects. MiR-942-5p was ensured to be a target of circ_0019693, and miR-942-5p enrichment reversed the effects of circ_0019693. In addition, PCP4 was a target of miR-942-5p, and miR-942-5p inhibitor-promoted BMSC osteogenic differentiation and angiogenesis were partly repressed by PCP4 knockdown. In conclusion, circ_0019693 promotes BMSC osteogenic differentiation osteogenesis-coupled angiogenesis via regulating miR-942-5p-targeted PCP4, thus blocking the development of OP.

PCP4/PEP19 and HER2 Are Novel Prognostic Markers in Mucoepidermoid Carcinoma of the Salivary Gland.

Simple SummaryMucoepidermoid carcinoma (MEC) is the most common malignancy of all salivary neoplasms, and no effective treatment strategy for MEC has been established other than resection. In this study, we showed that Purkinje cell protein (PCP) 4/peptide (PEP) 19 and human epidermal growth factor receptor 2 (HER2) are predicted to play important roles in the pathogenesis and progression of MEC. The detection of PCP4/PEP19 and HER2 may be useful for providing more effective treatments against MEC.Mucoepidermoid carcinoma (MEC) is one of the most common malignant salivary gland carcinomas, but no effective treatment strategy has been established other than surgical resection. Purkinje cell protein (PCP) 4/peptide (PEP) 19 is a calmodulin-binding antiapoptotic peptide that is expressed and inhibits apoptosis in human breast cancer cells. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor that has been implicated in the pathogenesis of many carcinomas, particularly breast and gastric carcinomas. In the present study, we performed immunohistochemical analyses of samples from 73 patients who underwent surgical resection for MEC of the salivary gland using antibodies against PCP4/PEP19 and HER2. PCP4/PEP19 expression was related to better prognosis, while HER2 expression was associated with worse prognosis. Patients that were PCP4/PEP19-positive and HER2-negative showed similar outcomes to PCP4/PEP19 and HER2 alone. Therefore, PCP4/PEP19 and HER2 are predicted to play important roles in the pathogenesis and progression of MEC.

Laminar Organization of the Entorhinal Cortex in Macaque Monkeys Based on Cell-Type-Specific Markers and Connectivity.

The entorhinal cortex (EC) is a major gateway between the hippocampus and telencephalic structures, and plays a critical role in memory and navigation. Through the use of various molecular markers and genetic tools, neuron types constituting EC are well studied in rodents, and their layer-dependent distributions, connections, and functions have also been characterized. In primates, however, such cell-type-specific understandings are lagging. To bridge the gap between rodents and primates, here we provide the first cell-type-based global map of EC in macaque monkeys. The laminar organization of the monkey EC was systematically examined and compared with that of the rodent EC by using immunohistochemistry for molecular markers which have been well characterized in the rodent EC: reelin, calbindin, and Purkinje cell protein 4 (PCP4). We further employed retrograde neuron labeling from the nucleus accumbens and amygdala to identify the EC output layer. This cell-type-based approach enabled us to apply the latest laminar definition of rodent EC to monkeys. Based on the similarity of the laminar organization, the monkey EC can be divided into two subdivisions: rostral and caudal EC. These subdivisions likely correspond to the lateral and medial EC in rodents, respectively. In addition, we found an overall absence of a clear laminar arrangement of layer V neurons in the rostral EC, unlike rodents. The cell-type-based architectural map provided in this study will accelerate the application of genetic tools in monkeys for better understanding of the role of EC in memory and navigation.

Autoantibodies Against the Purkinje Cell Protein RGS8 in Paraneoplastic Cerebellar Syndrome

ObjectiveTo describe the identification of regulator of G-protein signaling 8 (RGS8) as an autoantibody target in patients with cerebellar syndrome associated with lymphoma.MethodsSera of 4 patients with a very similar unclassified reactivity against cerebellar Purkinje cells were used in antigen identification experiments. Immunoprecipitations with cerebellar lysates followed by mass spectrometry identified the autoantigen, which was verified by recombinant immunofluorescence assay, immunoblot, and ELISA with the recombinant protein.ResultsThe sera and CSF of 4 patients stained the Purkinje cells and molecular layer of the cerebellum. RGS8 was identified as the target antigen in all 4 sera. In a neutralization experiment, recombinant human RGS8 was able to neutralize the autoantibodies' tissue reaction. Patient sera and CSF showed a specific reactivity against recombinant RGS8 in ELISA and immunoblot, whereas no such reactivity was detectable in the controls. Clinical data were available for 2 of the 4 patients, remarkably both presented with cerebellar syndrome accompanied by B-cell lymphoma of the stomach (patient 1, 53 years) or Hodgkin lymphoma (patient 2, 74 years).ConclusionOur results indicate that autoantibodies against the intracellular Purkinje cell protein RGS8 represent new markers for paraneoplastic cerebellar syndrome associated with lymphoma.Classification of EvidenceThis study provided Class IV evidence that autoantibodies against the intracellular Purkinje cell protein RGS8 are associated with paraneoplastic cerebellar syndrome in lymphoma.

GNG13 Is a Potential Marker of the State of Health of Alzheimer's Disease Patients' Cerebellum.

Brain regions such as the cerebellum (CB) have been neglected for a long time in the study of Alzheimer's disease (AD) pathogenesis. In reference to a new emerging hypothesis according to which there is an altered cerebellar synaptic processing in AD, we verified the possible role played by new biomarkers in the CB of AD patients compared with not-demented healthy control subjects (NDHS). Using a bioinformatics approach, we have collected several microarray datasets and obtained 626 cerebella sample biopsies belonging to subjects who did not die from causes related to neurological diseases and 199 cerebella belonging to AD. The analysis of logical relations between the transcriptome dataset highlighted guanine nucleotide-binding protein (G protein) gamma 13 (GNG13) as a potential new biomarker for Purkinje cells (PCs). We havecorrelated GNG13 expression levels with already widely existing bibliography of PC marker genes, such as Purkinje cell protein 2 (PCP2), Purkinje cell protein 4 (PCP4), and cerebellin 3 (CBLN3). We showed that expression levels of GNG13 and PCP2, PCP4, and CBLN3 were significantly correlated with each other in NDHS and in AD and significantly reduced in AD patients compared with NDHS subjects. In addition, we highlighted a negative correlation between the expression levels of PC biomarkers and age. From the outcome of our investigation, it is possible to conclude that the identification of GNG13 as a potentially biomarker in PCs represents also a state of health of CB, in association with the expression of PCP2, PCP4, and CBLN3.

Loss of the ER membrane protein complex subunit Emc3 leads to retinal bipolar cell degeneration in aged mice.

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved protein complex involved in inserting the transmembrane domain of membrane proteins into membranes in the ER. EMC3 is an essential component of EMC and is important for rhodopsin synthesis in photoreceptor cells. However, the in vivo function of Emc3 in bipolar cells (BCs) has not been determined. To explore the role of Emc3 in BCs, we generated a BC-specific Emc3 knockout mouse model (named Emc3 cKO) using the Purkinje cell protein 2 (Pcp2) Cre line. Although normal electroretinography (ERG) b-waves were observed in Emc3 cKO mice at 6 months of age, Emc3 cKO mice exhibited reduced b-wave amplitudes at 12 months of age, as determined by scotopic and photopic ERG, and progressive death of BCs, whereas the ERG a-wave amplitudes were preserved. PKCa staining of retinal cryosections from Emc3 cKO mice revealed death of rod BCs. Loss of Emc3 led to the presence of the synaptic protein mGLuR6 in the outer nuclear layer (ONL). Immunostaining analysis of presynaptic protein postsynaptic density protein 95 (PSD95) revealed rod terminals retracted to the ONL in Emc3 cKO mice at 12 months of age. In addition, deletion of Emc3 resulted in elevated glial fibrillary acidic protein, indicating reactive gliosis in the retina. Our data demonstrate that loss of Emc3 in BCs leads to decreased ERG response, increased astrogliosis and disruption of the retinal inner nuclear layer in mice of 12 months of age. Taken together, our studies indicate that Emc3 is not required for the development of BCs but is important for long-term survival of BCs.

Ancestral regulatory mechanisms specify conserved midbrain circuitry in arthropods and vertebrates

Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. Here, we identify a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. We show that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.

Cerebellar Morphology and Behavioral Profiles in Mice Lacking Heparan Sulfate Ndst Gene Function.

Disruption of the Heparan sulfate (HS)-biosynthetic gene N-acetylglucosamine N-Deacetylase/N-sulfotransferase 1 (Ndst1) during nervous system development causes malformations that are composites of those caused by mutations of multiple HS binding growth factors and morphogens. However, the role of Ndst function in adult brain physiology is less explored. Therefore, we generated mice bearing a Purkinje-cell-specific deletion in Ndst1 gene function by using Cre/loxP technology under the control of the Purkinje cell protein 2 (Pcp2/L7) promotor, which results in HS undersulfation. We observed that mutant mice did not show overt changes in the density or organization of Purkinje cells in the adult cerebellum, and behavioral tests also demonstrated normal cerebellar function. This suggested that postnatal Purkinje cell development and homeostasis are independent of Ndst1 function, or that impaired HS sulfation upon deletion of Ndst1 function may be compensated for by other Purkinje cell-expressed Ndst isoforms. To test the latter possibility, we additionally deleted the second Purkinje-cell expressed Ndst family member, Ndst2. This selectively abolished reproductive capacity of compound mutant female, but not male, mice, suggesting that ovulation, gestation, or female reproductive behavior specifically depends on Ndst-dependent HS sulfation in cells types that express Cre under Pcp2/L7 promotor control.

A qualitative solution with quantitative potential for the mouse hippocampal cortex flatmap problem

The hippocampal formation (HPF) is a focus of intense experimental investigation, particularly because of its roles in conscious memory consolidation, spatial navigation, emotion, and motivated behaviors. However, the HPF has a complex three-dimensional geometry resulting from extreme curvature of its layers, and this presents a challenge for investigators seeking to decipher hippocampal structure and function at cellular and molecular scales (neuronal circuitry, gene expression, and other properties). Previously, this problem was solved qualitatively for the rat by constructing a physical surface model of the HPF based on histological sections, and then deriving from the model a flatmap. Its usefulness is exemplified by previous studies that used it to display topological relationships between different components of intrahippocampal circuitry derived from experimental pathway-tracing experiments. Here the rat HPF flatmap was used as a starting point to construct an analogous flatmap for the mouse, where the great majority of experimental hippocampal research is currently performed. A detailed account of underlying knowledge and principles is provided, including for hippocampal terminology, and development from an embryonic nonfolded sheet into differentiated multiple adjacent cortical areas, giving rise to the adult shape. To demonstrate its utility, the mouse flatmap was used to display the results of pathway-tracing experiments showing the dentate gyrus mossy fiber projection, and its relationship to the intrahippocampal Purkinje cell protein 4 gene-expression pattern. Finally, requirements for constructing a computer graphics quantitative intrahippocampal flatmap, with accompanying intrahippocampal coordinate system, are presented; they should be applicable to all mammals, including human.

MiR-122 Exerts Inhibitory Effects on Osteoblast Proliferation/Differentiation in Osteoporosis by Activating the PCP4-Mediated JNK Pathway

Osteoporosis is characterized by the reduction of bone mineral density and deterioration of bone quality which leads to high risk of fractures. Some microRNAs (miRNAs) have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass maintenance. We aimed to clarify whether miR-122 could regulate osteoblast differentiation in ovariectomized rats with osteoporosis. miR-122 was upregulated and Purkinje cell protein 4 (PCP4) was downregulated in ovariectomized rats. PCP4 was identified as a target of miR-122 by dual-luciferase reporter gene assay. We transfected isolated osteoblasts from ovariectomized rats with miR-122 mimic or inhibitor or PCP4 overexpression vectors. Proliferation and differentiation of osteoblasts were repressed by the overexpression of miR-122 but enhanced by overexpression of PCP4. miR-122 could induce the activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, while PCP4 blocked this pathway. Rescue experiments further demonstrated that the inhibiting effects of miR-122 on osteoblast differentiation could be compensated by activation of the PCP4 or inhibition of JNK signaling pathway. Collectively, our data imply that miR-122 inhibits osteoblast proliferation and differentiation in rats with osteoporosis, highlighting a novel therapeutic target for osteoporotic patients.

Overexpression of human wtTDP-43 causes impairment in hippocampal plasticity and behavioral deficits in CAMKII-tTa transgenic mouse model

AimsThe current study utilizes the adeno-associated viral gene transfer system in the CAMKIIα-tTA mouse model to overexpress human wild type TDP-43 (wtTDP-43) and α-synuclein (α-Syn) proteins. The co-existence of these proteins is evident in the pathology of neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Parkinson disease (PD), and dementia with Lewy bodies (DLB). MethodsThe novel bicistronic recombinant adeno-associated virus (rAAV) serotype 9 drives wtTDP-43 and α-Syn expression in the hippocampus via “TetO” CMV promoter. Behavior, electrophysiology, and biochemical and histological assays were used to validate neuropathology. ResultsWe report that overexpression of wtTDP-43 but not α-Syn contributes to hippocampal CA2–specific pyramidal neuronal loss and overall hippocampal atrophy. Further, we report a reduction of hippocampal long-term potentiation and decline in learning and memory performance of wtTDP-43 expressing mice. Elevated wtTDP-43 levels induced selective degeneration of Purkinje cell protein 4 (PCP-4) positive neurons while both wtTDP-43 and α-Syn expression reduced subsets of the glutamate receptor expression in the hippocampus. ConclusionsOverall, our findings suggest the significant vulnerability of hippocampal neurons toward elevated wtTDP-43 levels possibly via PCP-4 and GluR-dependent calcium signaling pathways. Further, we report that wtTDP-43 expression induced selective CA2 subfield degeneration, contributing to the deterioration of the hippocampal-dependent cognitive phenotype.