Loss of lrrk2 impairs dopamine catabolism, cell proliferation, and neuronal regeneration in the zebrafish brain

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age)

Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration

The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour ?-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer’s disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases

Microbiological and chemical monitoring of Marsala base wine obtained by spontaneous fermentation during large-scale production

The present work was undertaken to evaluate the effect of the natural winemaking on the microbial and chemical composition of Marsala base wine. To this purpose, a large-scale vinification process of Grillo grape cultivar was monitored from harvesting to the final product. Total yeasts (TY) showed a rapid increase after must pressing and reached values almost superimposable to those registered during the conventional winemakings. Lactic acid bacteria (LAB) were registered at the highest levels simultaneously to yeast growth at the beginning of the process. Saccharomyces cerevisiae was the species found at the highest concentrations in all samples analysed. Several strains (n= 16) was registered at high levels during the alcoholic fermentation and/or aging of wine; only two of them were detected on the grape surface. Lactobacillus plantarum was the LAB species most frequently isolated during the entire vinification process. Ethanol content was approximately 14% (v/v) at the end of vinification. The value of pH did not greatly vary during the process and the volatile acidity (VA) was detected at low concentrations during the entire transformation. The concentration of malic acid rapidly decreased during the AF; on the other hand, lactic acid showed an irregular trend during the entire process. trans-caffeil tartaric acid was the most abundant hydroxycinnamoyl tartaric acid and volatile organic compounds (VOC) were mainly represented by isoamylic alcohol and isobutanol

Clinical patterns of hepatocellular carcinoma in nonalcoholic fatty liver disease: A multicenter prospective study

107noNonalcoholic fatty liver disease (NAFLD) represents the hepatic manifestation of metabolic syndrome and may evolve into hepatocellular carcinoma (HCC). Only scanty clinical information is available on HCC in NAFLD. The aim of this multicenter observational prospective study was to assess the clinical features of patients with NAFLD-related HCC (NAFLD-HCC) and to compare them to those of hepatitis C virus (HCV)-related HCC. A total of 756 patients with either NAFLD (145) or HCV-related chronic liver disease (611) were enrolled in secondary care Italian centers. Survival was modeled according to clinical parameters, lead-time bias, and propensity analysis. Compared to HCV, HCC in NAFLD patients had a larger volume, showed more often an infiltrative pattern, and was detected outside specific surveillance. Cirrhosis was present in only about 50% of NAFLD-HCC patients, in contrast to the near totality of HCV-HCC. Regardless of tumor stage, survival was significantly shorter (P = 0.017) in patients with NAFLD-HCC, 25.5 months (95% confidence interval 21.9-29.1), than in those with HCV-HCC, 33.7 months (95% confidence interval 31.9-35.4). To eliminate possible confounders, a propensity score analysis was performed, which showed no more significant difference between the two groups. Additionally, analysis of patients within Milan criteria submitted to curative treatments did not show any difference in survival between NAFLD-HCC and HCV-HCC (respectively, 38.6 versus 41.0 months, P = nonsignificant) Conclusions: NAFLD-HCC is more often detected at a later tumor stage and could arise also in the absence of cirrhosis, but after patient matching, it has a similar survival rate compared to HCV infection; a future challenge will be to identify patients with NAFLD who require more stringent surveillance in order to offer the most timely and effective treatment. (Hepatology 2016;63:827-838)openopenPiscaglia F.; Svegliati-Baroni G.; Barchetti A.; Pecorelli A.; Marinelli S.; Tiribelli C.; Bellentani S.; Bernardi M.; Biselli M.; Caraceni P.; Domenicali M.; Garuti F.; Gramenzi A.; Lenzi B.; Magalotti D.; Cescon M.; Ravaioli M.; Del Poggio P.; Olmi S.; Rapaccini G.L.; Balsamo C.; Di Nolfo M.A.; Vavassori E.; Alberti A.; Benvegnau L.; Gatta A.; Giacomin A.; Vanin V.; Pozzan C.; Maddalo G.; Giampalma E.; Cappelli A.; Golfieri R.; Mosconi C.; Renzulli M.; Roselli P.; Dell'isola S.; Ialungo A.M.; Risso D.; Marenco S.; Sammito G.; Bruzzone L.; Bosco G.; Grieco A.; Pompili M.; Rinninella E.; Siciliano M.; Chiaramonte M.; Guarino M.; Camma C.; Maida M.; Costantino A.; Barcellona M.R.; Schiada L.; Gemini S.; Lanzi A.; Stefanini G.F.; Dall'aglio A.C.; Cappa F.M.; Suzzi A.; Mussetto A.; Treossi O.; Missale G.; Porro E.; Mismas V.; Vivaldi C.; Bolondi L.; Zoli M.; Granito A.; Malagotti D.; Tovoli F.; Trevisani F.; Venerandi L.; Brandi G.; Cucchetti A.; Bugianesi E.; Vanni E.; Mezzabotta L.; Cabibbo G.; Petta S.; Fracanzani A.; Fargion S.; Marra F.; Fani B.; Biasini E.; Sacco R.; Morisco F.; Caporaso N.; Colombo M.; D'ambrosio R.; Croce L.S.; Patti R.; Giannini E.G.; Loria P.; Lonardo A.; Baldelli E.; Miele L.; Farinati F.; Borzio M.; Dionigi E.; Soardo G.; Caturelli E.; Ciccarese F.; Virdone R.; Affronti A.; Foschi F.G.; Borzio F.Piscaglia, F.; Svegliati-Baroni, G.; Barchetti, A.; Pecorelli, A.; Marinelli, S.; Tiribelli, C.; Bellentani, S.; Bernardi, M.; Biselli, M.; Caraceni, P.; Domenicali, M.; Garuti, F.; Gramenzi, A.; Lenzi, B.; Magalotti, D.; Cescon, M.; Ravaioli, M.; Del Poggio, P.; Olmi, S.; Rapaccini, G. L.; Balsamo, C.; Di Nolfo, M. A.; Vavassori, E.; Alberti, A.; Benvegnau, L.; Gatta, A.; Giacomin, A.; Vanin, V.; Pozzan, C.; Maddalo, G.; Giampalma, E.; Cappelli, A.; Golfieri, R.; Mosconi, C.; Renzulli, M.; Roselli, P.; Dell'Isola, S.; Ialungo, A. M.; Risso, D.; Marenco, S.; Sammito, G.; Bruzzone, L.; Bosco, G.; Grieco, A.; Pompili, M.; Rinninella, E.; Siciliano, M.; Chiaramonte, M.; Guarino, M.; Camma, C.; Maida, M.; Costantino, A.; Barcellona, M. R.; Schiada, L.; Gemini, S.; Lanzi, A.; Stefanini, G. F.; Dall'Aglio, A. C.; Cappa, F. M.; Suzzi, A.; Mussetto, A.; Treossi, O.; Missale, G.; Porro, E.; Mismas, V.; Vivaldi, C.; Bolondi, L.; Zoli, M.; Granito, A.; Malagotti, D.; Tovoli, F.; Trevisani, F.; Venerandi, L.; Brandi, G.; Cucchetti, A.; Bugianesi, E.; Vanni, E.; Mezzabotta, L.; Cabibbo, G.; Petta, S.; Fracanzani, A.; Fargion, S.; Marra, F.; Fani, B.; Biasini, E.; Sacco, R.; Morisco, F.; Caporaso, N.; Colombo, M.; D'Ambrosio, R.; Croce, L. S.; Patti, R.; Giannini, E. G.; Loria, P.; Lonardo, A.; Baldelli, E.; Miele, L.; Farinati, F.; Borzio, M.; Dionigi, E.; Soardo, G.; Caturelli, E.; Ciccarese, F.; Virdone, R.; Affronti, A.; Foschi, F. G.; Borzio, F

Regulation of Neurogenesis during Prion Disease

Neurogenesis is impaired during chronic progressive neurodegenerative diseases, with the potential to replace damaged neurons at lesion sites. These findings support the need for a better understanding of injury-induced neurogenesis in the adult brain undergoing neurodegeneration and suggest the potential of directing neural precursor regulation as a strategy for brain repair. Among the existing experimental models of neurodegeneration, prion disease models present many features in common with diseases such as Alzheimer’s or Parkinson’s and is an ideal tractable laboratory model to study chronic progressive neurodegenerative diseases. In the present work, using the ME7 murine model of prion disease, we studied the dynamics and regulation of neurogenesis since this would provide a basis for the therapeutical manipulation of neurogenesis which would impact both behaviour and pathology. Our results provide insights into the dynamics of hippocampal cell death and the generation of neural precursor cells and new neurons in the dentate gyrus (DG) region. Using retroviral vectors to label and trace proliferating cells, we found significant changes in the maturation and integration of newly generated neurons within the DG. In addition, using conditional ablation of the prion protein (PrP) in neurons we also investigated the role of differentiated neurons in the regulation of neurogenesis during the course of prion disease. Finally, we studied neurogenesis in hippocampi from variant Creutzfeldt-Jacob’s disease (vCJD) and Alzheimer’s disease (AD) patients, showing similarities with the results obtained in the murine model of prion disease. The present results describe for the first time the regulation of neurogenesis during prion disease and open a window for the full understanding of the regulation and role of neurogenesis during chronic progressive neurodegenerative diseases

Loss of lrrk2 impairs dopamine catabolism, cell proliferation, and neuronal regeneration in the zebrafish brain

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age)

Loss of lrrk2 impairs dopamine catabolism, cell proliferation, and neuronal regeneration in the zebrafish brain

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age)

Regulation of microglial proliferation during chronic neurodegeneration

An important component of chronic neurodegenerative diseases is the generation of an innate inflammatory response within the CNS. Microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and activation. We studied the time course and regulation of microglial proliferation, using a mouse model of prion disease. Our results show that the proliferation of resident microglial cells accounts for the expansion of the population during the development of the disease. We identify the pathway regulated by the activation of CSF1R and the transcription factors PU.1 and C/EBP? as the molecular regulators of the proliferative response, correlating with the chronic human neurodegenerative conditions variant Creutzfeldt-Jakob disease and Alzheimer's disease. We show that targeting the activity of CSF1R inhibits microglial proliferation and slows neuronal damage and disease progression. Our results demonstrate that microglial proliferation is a major component in the evolution of chronic neurodegeneration, with direct implications for understanding the contribution of the CNS innate immune response to disease progression