Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Lung development and immune status under chronic LPS exposure in rat pups with and without CD26/DPP4 deficiency.

Dipeptidyl-peptidase IV (CD26), a multifactorial integral type II protein, is expressed in the lungs during development and is involved in inflammation processes. We tested whether daily LPS administration influences the CD26-dependent retardation in morphological lung development and induces alterations in the immune status. Newborn Fischer rats with and without CD26 deficiency were nebulized with 1 µg LPS/2 ml NaCl for 10 min from days postpartum (dpp) 3 to 9. We used stereological methods and fluorescence activated cell sorting (FACS) to determine morphological lung maturation and alterations in the pulmonary leukocyte content on dpp 7, 10, and 14. Daily LPS application did not change the lung volume but resulted in a significant retardation of alveolarization in both substrains proved by significantly lower values of septal surface and volume as well as higher mean free distances in airspaces. Looking at the immune status after LPS exposure compared to controls, a significantly higher percentage of B lymphocytes and decrease of CD4+CD25+ T cells were found in both subtypes, on dpp7 a significantly higher percentage of CD4 T+ cells in CD26+ pups, and a significantly higher percentage of monocytes in CD26- pups. The percentage of T cells was significantly higher in the CD26-deficient group on each dpp. Thus, daily postnatal exposition to low doses of LPS for 1 week resulted in a delay in formation of secondary septa, which remained up to dpp 14 in CD26- pups. The retardation was accompanied by moderate parenchymal inflammation and CD26-dependent changes in the pulmonary immune cell composition.

Watching the Vessels Grow: Establishment of Intravital Microscopy in the Arteriovenous Loop Rat Model.

Tissue engineering in reconstructive surgery seeks to generate bioartificial tissue substitutes. The arteriovenous (AV) loop allows the generation of axially vascularized tissue constructs. Cellular mechanisms of this vascularization process are largely unclear. In this study, we developed two different chamber models for intravital microscopy and imaging of the AV loop in the rat. Multiple design variations were implanted and the stability of the chamber and AV loop patency was tested in vivo. Our novel chamber facilitates repetitive observation of the AV loop using fluorescence-enhanced intravital microscopy. This technique can be used for daily evaluation of leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop model on 14 consecutive days. Therefore, our newly developed model for intravital microscopy will provide better understanding of cellular and molecular processes in tissue engineering in the AV loop. Moreover, it supports initiation of the novel approaches for therapeutic applications. Impact statement In the Arteriovenous (AV) loop, axially vascularized tissue can be generated and modified using different tissue engineering approaches. Cellular mechanisms of this vascularization process are largely unclear. We managed to develop an intravital microscopy model for long-term observation of intravascular and perivascular events in the AV loop. Leukocyte-endothelial cell interactions, vascularization, and tissue formation in the AV loop can now be evaluated on a day-to-day basis. This will provide better understanding of cellular and molecular processes happening during tissue engineering within the AV loop.

CD161a-positive natural killer (NK) cells and α-smooth muscle actin-positive myofibroblasts were upregulated by extrarenal DPP4 in a rat model of acute renal rejection.

AIMS: Systemic inhibition of dipeptidyl peptidase 4 (DPP4) showed a protective effect in several transplant models. Here we assessed the specific role of extrarenal DPP4 in renal transplant rejection. METHODS: Kidneys from wildtype (wt) F344 rats were either transplanted in wt Dark Agouti or congenic rats not expressing DPP4. The remaining, not transplanted donor kidney served as healthy controls. To investigate early inflammatory events rats were sacrificed 3 days after transplantation and kidneys were evaluated for inflammatory cells, capillary rarefaction, proliferation, apoptosis and myofibroblasts by immunohistochemistry. RESULTS: Capillary ERG-1-positive endothelial cells were significantly more abundant in renal cortex when transplanted into DPP4 deficient compared to wt recipients. In contrast, TGF-ß and myofibroblasts were reduced by more than 25% in kidneys transplanted into DPP4 deficient compared to wt recipients. Numbers of CD161a-positive NK-cells were significantly lower in allografts in DPP4 deficient compared to wt recipients. Numbers of all other investigated immune cells were not affected by the lack of extrarenal DPP4. CONCLUSION: In early transplant rejection extrarenal DPP4 is involved in the recruitment of NK-cells and early fibrosis. Beneficial effects were less pronounced than reported for systemic DPP4 inhibition, indicating that renal DPP4 is an important player in transplantation-mediated injury.

Thirty Mouse Strain Survey of Voluntary Physical Activity and Energy Expenditure: Influence of Strain, Sex and Day-Night Variation.

We measured indirect calorimetry and activity parameters, VO2 and VCO2 to extract respiratory exchange ratio (RER) and energy expenditure in both sexes of 30 inbred mouse strains of 6 genetic families at 9-13 weeks during one photophase and the subsequent scotophase. We observed a continuous distribution of all traits. While males had higher body weights than females, we observed no sex difference for food and water intake. All strains drank and fed more during the night even if they displayed no day-night difference in activity traits. Several strains showed absent or weak day-night variation in one or more activity traits and these included FVB and 129X1, males of 129S1, SWR, NZW, and SM, and females of SJL. In general females showed higher rearing and ambulatory activity with 6 and 9 strains, respectively, showing a sex difference. Fine motor movements, like grooming, showed less sex differences. RER underlied a strong day-night difference and no sex effect. Only FVB females and males of the RIIIS and SM strain had no day-night variation. Energy expenditure underlies a large day-night variation which was absent in SWR and in FVB females and RIIIS males. In general, female bodies had a tendency to higher energy expenditure values, which became a significant difference in C3H, MAMy, SM, DBA1, and BUB. Our data illustrate the diversity of these traits in male and female inbred mice and provide a resource in the selection of strains for future studies.

Molecular crosstalk between Y5 receptor and neuropeptide Y drives liver cancer.

Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. As a result of globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY receptors represent a highly conserved, stress-activated system involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased expression of NPY5 receptor (Y5R) in HCC, which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peritumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R activation. TGF-ß1 was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R activation and function in liver cancer. The TGF-ß/NPY/Y5R axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.

Dipeptidylpeptidase 4 as a Marker of Activated Fibroblasts and a Potential Target for the Treatment of Fibrosis in Systemic Sclerosis.

OBJECTIVE: Expression of dipeptidylpeptidase 4 (DPP-4) identifies a dermal fibroblast lineage involved in scarring during wound healing. The role of DDP-4 in tissue fibrosis is, however, unknown. The aim of the present study was to evaluate DPP-4 as a potential target for the treatment of fibrosis in patients with systemic sclerosis (SSc). METHODS: Expression of DPP-4 in skin biopsy samples and dermal fibroblasts was analyzed by real-time polymerase chain reaction, immunofluorescence, and Western blot analyses. The activity of DPP-4 was modulated by overexpression, knockdown, and pharmacologic inhibition of DPP4 using sitagliptin and vildagliptin. The effects of DPP4 inhibition were analyzed in human dermal fibroblasts and in different mouse models of SSc (each n = 6). RESULTS: The expression of DPP-4 and the number of DPP-4-positive fibroblasts were increased in the fibrotic skin of SSc patients, in a transforming growth factor ß (TGFß)-dependent manner. DPP-4-positive fibroblasts expressed higher levels of myofibroblast markers and collagen (each P < 0.001 versus healthy controls). Overexpression of DPP4 promoted fibroblast activation, whereas pharmacologic inhibition or genetic inactivation of DPP4 reduced the proliferation, migration, and expression of contractile proteins and release of collagen (each P < 0.001 versus control mice) by interfering with TGFß-induced ERK signaling. DPP4-knockout mice were less sensitive to bleomycin-induced dermal and pulmonary fibrosis (P < 0.0001 versus wild-type controls). Treatment with DPP4 inhibitors promoted regression of fibrosis in mice that had received bleomycin challenge and mice with chronic graft-versus-host disease, and ameliorated fibrosis in TSK1 mice (each P < 0.001 versus untreated controls). These antifibrotic effects were associated with a reduction in inflammation. CONCLUSION: DPP-4 characterizes a population of activated fibroblasts and shows that DPP-4 regulates TGFß-induced fibroblast activation in the fibrotic skin of SSc patients. Inhibition of DPP4 exerts potent antifibrotic effects when administered in well-tolerated doses. As DPP4 inhibitors are already in clinical use for diabetes, these results may have direct translational implications for the treatment of fibrosis in patients with SSc.

Postnatal morphological lung development of wild type and CD26/DPP4 deficient rat pups in dependency of LPS exposure.

BACKGROUND: Rodents are born with morphological immature lungs and an intact surfactant system. CD26/DPP4 is a multifactorial transmembrane integral type II protein, which is involved in physiological and pathophysiological processes and is already expressed during development. CD26/DPP4, called CD26 in the following, is able to enhance or dampen differently triggered inflammation. LPS exposure often used to simulate perinatal infection delays lung development. OBJECTIVE: A perinatal LPS rat model was used to test the hypothesis that CD26 deficiency modulates LPS-induced retardation in morphological lung development. METHODS: New born Fischer CD26 positive (CD26+) and deficient (CD26-) rats were exposed to LPS on postnatal day (day post partum, dpp) 3 and 5. Morphological parameters of lung development were determined stereologically. Lung development was analysed in 7, 10 14 and 21day old rats. RESULTS: Compared to controls LPS application resulted (1) in a mild inflammation independent of the strain, (2) in significantly lower total surface and volume of alveolar septa combined with significantly higher total volume of airspaces and alveolar size on dpp 7 in both substrains. However, compared to controls in LPS treated CD26- rats significant lower values of total septal surface and volume combined with higher values of total parenchymal airspaces and alveolar size were found until the end of classical alveolarization (dpp14). In LPS treated CD26+ rat pups the retardation was abolished already on dpp 10. CONCLUSION: In absence of CD26, LPS enhances the delay of morphological lung development. Morphological recovery was slower after the end of LPS exposure in CD26 deficient lungs.

Differential severity of LPS-induced lung injury in CD26/DPP4 positive and deficient F344 rats.

BACKGROUND: Lipopolysaccharide (LPS) induced inflammation often leads to lung injury, in which pulmonary recruitment of neutrophils plays a pivotal role. Inflammatory processes are influenced by CD26/DPP4, highly expressed in lungs. Asthma induced CD26/DPP4 deficient (CD26/DPP4⁻) Fischer (F) 344 rats suffering from a transport block in the rER caused by a point mutation showed reduced pulmonary inflammation and reduced expression of immunomodulating surfactant proteins (SP). The degree of LPS induced lung injury in CD26/DPP4 deficient rats has not been investigated so far. OBJECTIVE: We hypothesize that LPS induced lung injury leads not only to an attenuated inflammation but also to a reduced SP expression and decreased structural damage in CD26/DPP4⁻ rats. METHODS: Both genotypes were intratracheally instilled with 250 µl LPS or with 250 µl 0.9% NaCl. Nine hours later animals were killed and either bronchoalveolar lavage was carried out to determine inflammatory cells and surface tension or lung blocks were removed and processed for histology, immunohistochemistry, electron microscopy or qRt-PCR analyses and Western Blot analyses. RESULTS: Signs of acute lung injury, such as structural damage of the blood gas barrier occurred only sporadically in both genotypes. LPS-induced CD26/DPP4⁻ rats showed decreased gene expression of SP-A and SP-D and reduced signs of lung inflammation associated with a reduced alveolar influx of macrophages and neutrophils. CONCLUSIONS: Less pulmonary inflammation combined with less structural alterations and minor expression of immunomodulating SP may be an indication of the critical role of CD26/DPP4 in regulating lung inflammation.

Role of hypothalamus-pituitary-adrenal axis modulation in the stress-resilient phenotype of DPP4-deficient rats.

BACKGROUND: Dipeptidyl peptidase 4 (DPP4, CD26) is a moonlighting enzyme responsible for the proteolytic inactivation of neuropeptide Y (NPY), a peptide known for its anxiolytic effect in the central nervous system. Our previous work revealed a stress-resilient phenotype and a potentiation of short-term fear extinction in a congenic rat model deficient for DPP4 activity (DPP4mut). Here, we investigated neuroendocrine mechanisms underlying the phenotype of the DPP4mut animals. We studied the function of the hypothalamus-pituitary-adrenal (HPA) axis including the expression levels of its key genes and explored the possibility of structural NPY system changes. METHODS AND RESULTS: We find decreased expression of Nr3c1 (glucocorticoid receptor - GR) and Fkbp5 (FK506 binding protein 5) in the amygdala and the hypothalamus of the DPP4mut rats, as well as the lower stress-induced peripheral corticosterone (CORT) levels. We detect no significant alterations in basal and DEX-induced CORT levels in the DPP4mut animals. The abundance of NPY-ergic neurons in the basolateral amygdala, dentate gyrus and hippocampus did not differ between the DPP4mut and their wild type littermates. CONCLUSION: DPP4mut rats show blunted CORT response in line with their lower behavioral stress-response profile. These results are consistent with the hypothesis that increased central NPY levels elevate the threshold of stress response. We suggest that changes in the expression levels of key HPA axis genes (Nr3c1 and Fkbp5) are a consequence of the altered stress-perception of DPP4mut animals, thus further contributing to the stress-resilient phenotype.

Novel role of NPY in neuroimmune interaction and lung growth after intrauterine growth restriction.

Individuals with intrauterine growth restriction (IUGR) are at risk for chronic lung disease. Using a rat model, we showed in our previous studies that altered lung structure is related to IL-6/STAT3 signaling. As neuropeptide Y (NPY), a coneurotransmitter of the sympathetic nervous system, regulates proliferation and immune response, we hypothesized that dysregulated NPY after IUGR is linked to IL-6, impaired myofibroblast function, and alveolar growth. IUGR was induced in rats by isocaloric low-protein diet; lungs were analyzed on embryonic day (E) 21, postnatal day (P) 3, P12, and P23. Finally, primary neonatal lung myofibroblasts (pnF) and murine embryonic fibroblasts (MEF) were used to assess proliferation, apoptosis, migration, and IL-6 expression. At E21, NPY and IL-6 expression was decreased, and AKT/PKC and STAT3/AMPKα signaling was reduced. Early reduction of NPY/IL-6 was associated with increased chord length in lungs after IUGR at P3, indicating reduced alveolar formation. At P23, however, IUGR rats exhibited a catch-up of body weight and alveolar growth coupled with more proliferating myofibroblasts. These structural findings after IUGR were linked to activated NPY/PKC, IL-6/AMPKα signaling. Complementary, IUGR-pnF showed increased survival, impaired migration, and reduced IL-6 compared with control-pnF (Co-pnF). In contrast, NPY induced proliferation, migration, and increased IL-6 synthesis in fibroblasts. Additionally, NPY-/- mice showed reduced IL-6 signaling and less proliferation of lung fibroblasts. Our study presents a novel role of NPY during alveolarization: NPY regulates 1) IL-6 and lung STAT3/AMPKα signaling, and 2) proliferation and migration of myofibroblasts. These new insights in pulmonary neuroimmune interaction offer potential strategies to enable lung growth.

Identifying neuropeptide Y (NPY) as the main stress-related substrate of dipeptidyl peptidase 4 (DPP4) in blood circulation.

BACKGROUND: Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease that hydrolyzes dipeptides from the N-terminus of peptides with either proline or alanine at the penultimate position. Substrates of DPP4 include several stress-related neuropeptides implicated in anxiety, depression and schizophrenia. A decline of DPP4-like activity has been reported in sera from depressed patient, but not fully characterized regarding DPP4-like enzymes, therapeutic interventions and protein. METHODS: Sera from 16 melancholic- and 16 non-melancholic-depressed patients were evaluated for DPP4-like activities and the concentration of soluble DPP4 protein before and after treatment by anti-depressive therapies. Post-translational modification of DPP4-isoforms and degradation of NPY, Peptide YY (PYY), Galanin-like peptide (GALP), Orexin B (OrxB), OrxA, pituitary adenylate cyclase-activating polypeptide (PACAP) and substance P (SP) were studied in serum and in ex vivo human blood. N-terminal truncation of biotinylated NPY by endothelial membrane-bound DPP4 versus soluble DPP4 was determined in rat brain perfusates and spiked sera. RESULTS: Lower DPP4 activities in depressed patients were reversed by anti-depressive treatment. In sera, DPP4 contributed to more than 90% of the overall DPP4-like activity and correlated with its protein concentration. NPY displayed equal degradation in serum and blood, and was equally truncated by serum and endothelial DPP4. In addition, GALP and rat OrxB were identified as novel substrates of DPP4. CONCLUSION: NPY is the best DPP4-substrate in blood, being truncated by soluble and membrane DPP4, respectively. The decline of soluble DPP4 in acute depression could be reversed upon anti-depressive treatment. Peptidases from three functional compartments regulate the bioactivity of NPY in blood.

In situ enzymatic activity of transglutaminase isoforms on brain tissue sections of rodents: A new approach to monitor differences in post-translational protein modifications during neurodegeneration.

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the ε-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression.

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.

Reduction in subventricular zone-derived olfactory bulb neurogenesis in a rat model of Huntington's disease is accompanied by striatal invasion of neuroblasts.

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT). The primary neuropathology of HD has been attributed to the preferential degeneration of medium spiny neurons (MSN) in the striatum. Reports on striatal neurogenesis have been a subject of debate; nevertheless, it should be considered as an endogenous attempt to repair the brain. The subventricular zone (SVZ) might offer a close-by region to supply the degenerated striatum with new cells. Previously, we have demonstrated that R6/2 mice, a widely used preclinical model representing an early onset HD, showed reduced olfactory bulb (OB) neurogenesis but induced striatal migration of neuroblasts without affecting the proliferation of neural progenitor cell (NPCs) in the SVZ. The present study revisits these findings, using a clinically more relevant transgenic rat model of late onset HD (tgHD rats) carrying the human HTT gene with 51 CAG repeats and mimicking many of the neuropathological features of HD seen in patients. We demonstrate that cell proliferation is reduced in the SVZ and OB of tgHD rats compared to WT rats. In the OB of tgHD rats, although cell survival was reduced, the frequency of neuronal differentiation was not altered in the granule cell layer (GCL) compared to the WT rats. However, an increased frequency of dopamenergic neuronal differentiation was noticed in the glomerular layer (GLOM) of tgHD rats. Besides this, we observed a selective proliferation of neuroblasts in the adjacent striatum of tgHD rats. There was no evidence for neuronal maturation and survival of these striatal neuroblasts. Therefore, the functional role of these invading neuroblasts still needs to be determined, but they might offer an endogenous alternative for stem or neuronal cell transplantation strategies.

DPP4-deficient congenic rats display blunted stress, improved fear extinction and increased central NPY.

BACKGROUND: Inhibitors of dipeptidyl peptidase 4 (DPP4, CD26) are used for the treatment of type 2 diabetic patients and better glucose tolerance has been confirmed in functionally DPP4-deficient congenic rats (DPP4mut), along with immunological alterations and, interestingly, a stress-resilient phenotype. All these findings are in agreement with the "moonlighting" properties of DPP4, whose proteolytic action is responsible for the inactivation of a number of regulatory peptides including, but not limited to, neuropeptide Y (NPY). Among all candidate substrates, DPP4 displays highest affinity for NPY, an endogenous anxiolytic neurotransmitter that is suggested as a candidate biomarker in post-traumatic stress disorder (PTSD) and depression. METHODS AND RESULTS: Central and peripheral NPY levels were measured by ELISA in DPP4mut and DAwt rats revealing a significantly higher concentration of the peptide in the CSF of DPP4mut animals. This finding positively correlated with the blunted stress phenotype measured on an analgesia-meter. Additionally, when a classical fear-conditioning paradigm was investigated, short-term fear extinction was significantly potentiated in DPP4mut rats as compared to wt controls. CONCLUSIONS: Our findings indicate a positive correlation between reduced stress-responsiveness and increased central NPY, in DPP4mut rats. Most interestingly, the behavioral phenotype extends to facilitation of fear extinction. These observations raise further interest in DPP4-modulating drugs for the potential effect on NPY metabolism, as a therapeutic tool for psychiatric conditions such as anxiety disorders and PTSD.

Differential OVA-induced pulmonary inflammation and unspecific reaction in Dark Agouti (DA) rats contingent on CD26/DPPIV deficiency.

Many disease models have shown that, within the species rat, different strains are differentially susceptible to asthma-induced inflammation depending on the genetic background. Likewise, CD26/DPPIV-deficiency in asthmatic F344 rats has been shown to result in a less pronounced inflammation and in increased Treg cell influx into the lung compared to wild-types. The aim of the present study was to investigate whether the genetic background of the animals interferes with CD26/DPPIV-deficiency in a model of allergic-like inflammation, or whether the deficiency per se is the predominant regulator of the inflammation. Therefore, we hypothesised that CD26/DPPIV-deficient Dark Agouti (DA) rats also exhibit a less pronounced ovalbumin (OVA)-induced inflammation compared to wild-types. After sensitisation with OVA, Al(OH)3 and heat-killed Bordetella pertussis bacilli, animals were challenged three times with 5% aerosolized OVA at intervals of 24h, i.e., on three consecutive days. 24h after the third challenge, animals were sacrificed and examined. In both wild-type and CD26/DPPIV-deficient rat groups, asthma induction led to (1) lung inflammation, (2) significantly increased eosinophil infiltration in the BALF, (3) significantly increased IgE serum levels, (4) a significant increase of inflammatory cytokines, (5) a significant increase of different T cell populations in the lungs and in their draining lymph nodes, as well as to (6) a significantly higher number of all T lymphocyte subtypes in the blood. Thus, the degree of the OVA-induced Th2-driven pulmonary inflammation was similarly pronounced in both wild-type and CD26/DPPIV-deficient DA rats.

Soluble DPP4 originates in part from bone marrow cells and not from the kidney.

Dipeptidyl peptidase 4 (DPP4) is known to inactivate incretins as well as important chemokines and neuropeptides. DPP4 is expressed as a transmembrane protein but also occurs as a soluble enzyme circulating in the blood. However, the origin of the soluble DPP4 (sDPP4) is still unknown. In this study, DPP4 activity was quantified in plasma and extracted from different rat organs. Then, in order to see if the kidney or the bone marrow was the source of sDPP4, kidney or bone marrow transplantation was performed between wildtype (wt) Dark Agouti (DA) and DPP4 deficient congenic rats (n=6-9). Kidney was verified to have the highest DPP4 activity, followed by spleen and lung. In the following three weeks after successful kidney transplantation only transient trace plasma DPP4 activity was detected in DPP4 deficient rats receiving wt kidneys. In addition, DPP4 activity was not diminished in DA wt rats receiving DPP4 deficient kidneys. Both findings indicated that sDPP4 did not originate from the kidney. In contrast, 43±14% (compared to wt) sDPP4 activity was detected in the plasma of DPP4 deficient DA rats that were reconstituted with wt bone marrow cells. Not only leukocyte but also macrophage subpopulations express DPP4 in bone marrow as well as in blood as assessed by flow cytometry. Thus, bone marrow derived cells but not the kidney represent at least one source of sDPP4. And leukocyte or macrophage subpopulations could be potential candidates.

Maternal deprivation decelerates postnatal morphological lung development of F344 rats.

Intensive medical care at premature born infants is often associated with separation of neonates from their mothers. Here, early artificial prolonged separation of rat pups from their dams (Maternal Deprivation, MD) was used to study potential impact on morphological lung maturation. Furthermore, we investigated the influence of an endogenous deficiency of the neuropeptide-cleaving dipeptidyl peptidase IV (DPP4), since the effects of MD are known to be partly mediated via neuropeptidergic effects, hypothesizing that MD will lead to a retardation of postnatal lung development, DPP4-dependendly. We used wild type and CD26/DPP4 deficient rats. For MD, the dam was placed each day into a separate cage for 2 h, while the pups remained in the nest on their own. Morphological lung maturation and cell proliferation at the postnatal days 7, 10, 14, and 21 were determined morphometrically. Maternally deprived wild types showed a retarded postnatal lung development compared with untreated controls in both substrains. During alveolarization, an increased thickness of alveolar septa and a decreased surface of septa about 50% were found. At the end of the morphological lung maturation, the surface of the alveolar septa was decreased at about 25% and the septal thickness remained increased about 20%. The proliferation rate was also decreased about 50% on day 14. However, the MD induced effects were less pronounced in DPP4-deficient rats, due to a significant deceleration already induced by DPP4-deficiency. Thus, MD as a model for postnatal stress experience influences remarkably postnatal development of rats, which is significantly modulated by the DPP4-system.

Metabolic and electrophysiological changes in the basal ganglia of transgenic Huntington's disease rats.

Huntington's disease (HD) is characterized by neuronal loss in the striatum, ultimately leading to an 'imbalance' in the electrical activity of the basal ganglia-thalamocortical circuits. To restore this 'imbalance' in HD patients, which is held responsible for (some) of the motor symptoms, different basal ganglia nuclei have been targeted for surgical therapies, such as ablative surgery and deep brain stimulation. However, evidence to target brain nuclei for surgical therapies in HD is lacking. We reasoned that a neuronal and metabolic mapping of the basal ganglia nuclei could identify a functional substrate for therapeutic interventions. Therefore, the aim of the present study was to investigate the metabolic and neuronal activity of basal ganglia nuclei in a transgenic rat model of HD (tgHD). Subjects were 10-12 month old tgHD rats and wildtype littermates. We examined the striatum, globus pallidus, entopeduncular nucleus, the subthalamic nucleus and substantia nigra at different levels. First, we determined the overall neuronal activity at a supracellular level, by cytochrome oxidase histochemistry. Secondly, we determined the subcellular metabolic activity, by immunohistochemistry for peroxisome proliferator-activated receptor-γ transcription co-activator (PGC-1α), a key player in the mitochondrial machinery. Finally, we performed extracellular single unit recordings in the nuclei to determine the cellular activity. In tgHD rats, optical density analysis showed a significantly increased cytochrome oxidase levels in the globus pallidus and subthalamic nucleus when compared to controls. PGC-1α expression was only enhanced in the subthalamic nucleus and electrophysiological recordings revealed decreased firing frequency of the majority of the neurons in the globus pallidus and increased firing frequency of the majority of the neurons in the subthalamic nucleus. Altogether, our results suggest that the globus pallidus and subthalamic nucleus play a role in the neurobiology of HD and can be potential targets for therapeutic interventions.