Olga Baron - Academia.edu (original) (raw)

Papers by Olga Baron

Research paper thumbnail of Evaluating Baseline and Sensitised Heat Nociception in Adult Drosophila

BIO-PROTOCOL

Chronic pain is a complex disease that affects a large proportion of the population. With little ... more Chronic pain is a complex disease that affects a large proportion of the population. With little to no effective treatments currently available for patients, this malady presents a large burden to society. Drosophila melanogaster has been previously used to describe conserved molecular components of nociception in larvae and adults. However, adult assays tend to rely on avoidance behaviours, and whilst larval acute thermal avoidance assays exist, larvae are not best suited to a chronic pain scenario as the condition must be long-term. Therefore, an adult thermal nociception response assay was required to study injury-evoked changes in heat nociception threshold (allodynia and hyperalgesia) over time, and we describe such a protocol here. Following leg amputation, flies display increased thermal sensitivity (allodynia) to innocuous temperatures but not an increase in magnitude of response (hyperalgesia) to noxious heat. Our method allows for individualised analysis of both allodynia and hyperalgesia.

Research paper thumbnail of Role of basic fibroblast growth factor (FGF-2) during development of mesencephalic dopaminergic neurons of substantia nigra in mice

Progredient loss of mesencephalic dopaminergic neurons (mDA) in the substantia nigra pars compact... more Progredient loss of mesencephalic dopaminergic neurons (mDA) in the substantia nigra pars compacta (SNpc) is the main cause for characteristic symptoms in Parkinson’s disease. Insight in the regulation of the SNpc development may benefit to the understanding of disease pathophysiology and improvement of therapeutic approaches. Previous studies revealed in addition to a protecting function of FGF-2 in mature mDA neurons, a regulatory role of FGF-2 for proper development of substantia nigra pars compacta (SNpc). The increased numbers of tyrosine hydroxylase immunoreactive (THir) neurons in SNpc of adult FGF-2 deficient mice correlated with decreased numbers in FGF-2 overexpressing mice. However, with regard to the mitogenic and neuroprotective function of FGF-2 on dopaminergic precursors and adult neurons, respectively, the opposed outcome was anticipated. To elucidate the physiological function of FGF-2 in the nigrostriatal development, the present study concentrated on embryonic (E1...

Research paper thumbnail of The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy

Autophagy, 2021

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autoph... more Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. Additionally, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.

Research paper thumbnail of Fat cadherins in mouse models of degenerative ataxias

Scientific Reports, 2019

Autophagy is a lysosomal degradation pathway that plays an essential role in neuronal homeostasis... more Autophagy is a lysosomal degradation pathway that plays an essential role in neuronal homeostasis and is perturbed in many neurological diseases. Transcriptional downregulation of fat was previously observed in a Drosophila model of the polyglutamine disease Dentatorubral-pallidoluysian atrophy (DRPLA) and this was shown to be partially responsible for autophagy defects and neurodegeneration. However, it is still unclear whether a downregulation of mammalian Fat orthologues is associated with neurodegeneration in mice. We hereby show that all four Fat orthologues are transcriptionally downregulated in the cerebellum in a mouse model of DRPLA. To elucidate the possible roles of single Fat genes, this study concentrates on Fat3. This fat homologue is shown to be the most widely expressed in the brain. Conditional knockout (KO) of Fat3 in brains of adult mice was attempted using the inducible Thy1Cre(ERT2) SLICK H line. Behavioral and biochemical analysis revealed that mice with condit...

Research paper thumbnail of Intersections between Regulated Cell Death and Autophagy

Trends in Cell Biology, 2019

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to... more Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a b-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG. Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Research paper thumbnail of Karyoptosis: A novel type of cell death caused by chronic autophagy inhibition

Autophagy, 2018

Macroautophagy/autophagy influences onset and progression of several human neurodegenerative dise... more Macroautophagy/autophagy influences onset and progression of several human neurodegenerative diseases, because of its critical role as a regulator of neuronal proteostasis and organelle quality control. In many neurodegenerative diseases, impairment in autophagy is thought to play a fundamental part in the terminal phases of cellular degeneration and death. However, the ultimate mechanism of neuronal cell death remains elusive. In a recent study we have identified a new form of regulated cell death, which arises upon autophagy inhibition.

Research paper thumbnail of Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration

Current biology : CB, Jan 4, 2017

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusi... more The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activ...

Research paper thumbnail of Fibroblast growth factor 23 signaling in hippocampal cells:Impact on neuronal morphology and synaptic density

Journal of neurochemistry, Jan 20, 2016

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilit... more Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease (CKD), FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced n...

Research paper thumbnail of FGF-2 Deficiency Does Not Influence FGF Ligand and Receptor Expression during Development of the Nigrostriatal System

PLoS ONE, 2011

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during develo... more Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGFligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1,-2,-22, FgfR-2c,-3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.

Research paper thumbnail of Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Journal of Neurochemistry, 2012

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of prolif... more Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.

Research paper thumbnail of Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice

Journal of Comparative Neurology, 2012

Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of ... more Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinson's disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF-2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF-2-deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable

Research paper thumbnail of Cooperation of Nuclear Fibroblast Growth Factor Receptor 1 and Nurr1 Offers New Interactive Mechanism in Postmitotic Development of Mesencephalic Dopaminergic Neurons

Journal of Biological Chemistry, 2012

Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopamine... more Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopaminergic neuron development. Results: Both nuclear receptors show a functional interaction in co-immunoprecipitation, FRAP, ChIP, and luciferase gene reporter assay. Conclusion: Cooperation of nuclear FGFR1 and Nurr1 offers a new mechanism in transcriptional regulation and integration. Significance: This mechanism may channel diverse stimuli in developing and mature dopaminergic neurons, providing a potential therapeutic target. Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-2 23) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

Research paper thumbnail of The Colayer Method as an Efficient Way to Genetically Modify Mesencephalic Progenitor Cells Transplanted Into 6-OHDA Rat Model of Parkinson's Disease

Cell Transplantation, 2012

Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However,... more Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However, the low survival rate of transplanted dopaminergic neurons (DA) calls for methodological improvements. Here we evaluated a method to combine transient genetic modification of neuronal progenitor cells with an optimized cell culture protocol prior to intrastriatal transplantation into 6-hydroxydopamine (6-OHDA) unilateral lesioned rats. Plasmid-based delivery of brain-derived neurotrophic factor (BDNF) increases the number of DA neurons, identified by tyrosine hydroxylase immunoreactivity (TH-ir), by 25% in vitro, compared to enhanced green fluorescence protein (EGFP)-transfected controls. However, the nucleofection itself, especially the cell detachment and reseeding procedure, decreases the TH-ir neuron number to 40% compared with nontransfected control cultures. To circumvent this drawback we established the colayer method, which contains a mix of nucleofected cells reseeded on top of an adherent sister culture in a ratio 1:3. In this setup TH-ir neuron number remains high and could be further increased by 25% after BDNF transfection. Comparison of both cell culture procedures (standard and colayer) after intrastriatal transplantation revealed a similar DA neuron survival as seen in vitro. Two weeks after grafting TH-ir neuron number was strongly reduced in animals receiving the standard EGFP-transfected cells (271 ± 62) compared to 1,723 ± 199 TH-ir neurons in the colayer group. In contrast to the in vitro results, no differences in the number of grafted TH-ir neurons were observed between BDNF, EGFP, and nontransfected colayer groups, neither 2 nor 13 weeks after transplantation. Likewise, amphetamine and apomorphine-induced rotational behavior improved similarly over time in all groups. Nevertheless, the colayer protocol provides an efficient way for neurotrophic factor release by transplanted progenitor cells and will help to study the effects of candidate factors on survival and integration of transplanted DA neurons.

Research paper thumbnail of Evaluating Baseline and Sensitised Heat Nociception in Adult Drosophila

BIO-PROTOCOL

Chronic pain is a complex disease that affects a large proportion of the population. With little ... more Chronic pain is a complex disease that affects a large proportion of the population. With little to no effective treatments currently available for patients, this malady presents a large burden to society. Drosophila melanogaster has been previously used to describe conserved molecular components of nociception in larvae and adults. However, adult assays tend to rely on avoidance behaviours, and whilst larval acute thermal avoidance assays exist, larvae are not best suited to a chronic pain scenario as the condition must be long-term. Therefore, an adult thermal nociception response assay was required to study injury-evoked changes in heat nociception threshold (allodynia and hyperalgesia) over time, and we describe such a protocol here. Following leg amputation, flies display increased thermal sensitivity (allodynia) to innocuous temperatures but not an increase in magnitude of response (hyperalgesia) to noxious heat. Our method allows for individualised analysis of both allodynia and hyperalgesia.

Research paper thumbnail of Role of basic fibroblast growth factor (FGF-2) during development of mesencephalic dopaminergic neurons of substantia nigra in mice

Progredient loss of mesencephalic dopaminergic neurons (mDA) in the substantia nigra pars compact... more Progredient loss of mesencephalic dopaminergic neurons (mDA) in the substantia nigra pars compacta (SNpc) is the main cause for characteristic symptoms in Parkinson’s disease. Insight in the regulation of the SNpc development may benefit to the understanding of disease pathophysiology and improvement of therapeutic approaches. Previous studies revealed in addition to a protecting function of FGF-2 in mature mDA neurons, a regulatory role of FGF-2 for proper development of substantia nigra pars compacta (SNpc). The increased numbers of tyrosine hydroxylase immunoreactive (THir) neurons in SNpc of adult FGF-2 deficient mice correlated with decreased numbers in FGF-2 overexpressing mice. However, with regard to the mitogenic and neuroprotective function of FGF-2 on dopaminergic precursors and adult neurons, respectively, the opposed outcome was anticipated. To elucidate the physiological function of FGF-2 in the nigrostriatal development, the present study concentrated on embryonic (E1...

Research paper thumbnail of The spectrum of neurodevelopmental, neuromuscular and neurodegenerative disorders due to defective autophagy

Autophagy, 2021

Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autoph... more Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. Additionally, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.

Research paper thumbnail of Fat cadherins in mouse models of degenerative ataxias

Scientific Reports, 2019

Autophagy is a lysosomal degradation pathway that plays an essential role in neuronal homeostasis... more Autophagy is a lysosomal degradation pathway that plays an essential role in neuronal homeostasis and is perturbed in many neurological diseases. Transcriptional downregulation of fat was previously observed in a Drosophila model of the polyglutamine disease Dentatorubral-pallidoluysian atrophy (DRPLA) and this was shown to be partially responsible for autophagy defects and neurodegeneration. However, it is still unclear whether a downregulation of mammalian Fat orthologues is associated with neurodegeneration in mice. We hereby show that all four Fat orthologues are transcriptionally downregulated in the cerebellum in a mouse model of DRPLA. To elucidate the possible roles of single Fat genes, this study concentrates on Fat3. This fat homologue is shown to be the most widely expressed in the brain. Conditional knockout (KO) of Fat3 in brains of adult mice was attempted using the inducible Thy1Cre(ERT2) SLICK H line. Behavioral and biochemical analysis revealed that mice with condit...

Research paper thumbnail of Intersections between Regulated Cell Death and Autophagy

Trends in Cell Biology, 2019

Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to... more Mutations in Lef1 occur in human and mouse sebaceous gland (SG) tumors, but their contribution to carcinogenesis remains unclear. Since Gata6 controls lineage identity in SG, we investigated the link between these two transcription factors. Here, we show that Gata6 is a b-catenin-independent transcriptional target of mutant Lef1. During epidermal development, Gata6 is expressed in a subset of Sox9-positive Lef1-negative hair follicle progenitors that give rise to the upper SG. Overexpression of Gata6 by in utero lentiviral injection is sufficient to induce ectopic sebaceous gland elements. In mice overexpressing mutant Lef1, Gata6 ablation increases the total number of skin tumors yet decreases the proportion of SG tumors. The increased tumor burden correlates with impaired DNA mismatch repair and decreased expression of Mlh1 and Msh2 genes, defects frequently observed in human sebaceous neoplasia. Gata6 specifically marks human SG tumors and also defines tumors with elements of sebaceous differentiation, including a subset of basal cell carcinomas. Our findings reveal that Gata6 controls sebaceous gland development and cancer.

Research paper thumbnail of Karyoptosis: A novel type of cell death caused by chronic autophagy inhibition

Autophagy, 2018

Macroautophagy/autophagy influences onset and progression of several human neurodegenerative dise... more Macroautophagy/autophagy influences onset and progression of several human neurodegenerative diseases, because of its critical role as a regulator of neuronal proteostasis and organelle quality control. In many neurodegenerative diseases, impairment in autophagy is thought to play a fundamental part in the terminal phases of cellular degeneration and death. However, the ultimate mechanism of neuronal cell death remains elusive. In a recent study we have identified a new form of regulated cell death, which arises upon autophagy inhibition.

Research paper thumbnail of Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration

Current biology : CB, Jan 4, 2017

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusi... more The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activ...

Research paper thumbnail of Fibroblast growth factor 23 signaling in hippocampal cells:Impact on neuronal morphology and synaptic density

Journal of neurochemistry, Jan 20, 2016

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilit... more Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease (CKD), FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced n...

Research paper thumbnail of FGF-2 Deficiency Does Not Influence FGF Ligand and Receptor Expression during Development of the Nigrostriatal System

PLoS ONE, 2011

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during develo... more Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGFligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1,-2,-22, FgfR-2c,-3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.

Research paper thumbnail of Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Journal of Neurochemistry, 2012

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of prolif... more Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.

Research paper thumbnail of Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice

Journal of Comparative Neurology, 2012

Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of ... more Fibroblast growth factor 2 (FGF-2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinson's disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF-2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF-2-deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable

Research paper thumbnail of Cooperation of Nuclear Fibroblast Growth Factor Receptor 1 and Nurr1 Offers New Interactive Mechanism in Postmitotic Development of Mesencephalic Dopaminergic Neurons

Journal of Biological Chemistry, 2012

Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopamine... more Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopaminergic neuron development. Results: Both nuclear receptors show a functional interaction in co-immunoprecipitation, FRAP, ChIP, and luciferase gene reporter assay. Conclusion: Cooperation of nuclear FGFR1 and Nurr1 offers a new mechanism in transcriptional regulation and integration. Significance: This mechanism may channel diverse stimuli in developing and mature dopaminergic neurons, providing a potential therapeutic target. Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-2 23) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

Research paper thumbnail of The Colayer Method as an Efficient Way to Genetically Modify Mesencephalic Progenitor Cells Transplanted Into 6-OHDA Rat Model of Parkinson's Disease

Cell Transplantation, 2012

Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However,... more Exogenous cell replacement represents a potent treatment option for Parkinson's disease. However, the low survival rate of transplanted dopaminergic neurons (DA) calls for methodological improvements. Here we evaluated a method to combine transient genetic modification of neuronal progenitor cells with an optimized cell culture protocol prior to intrastriatal transplantation into 6-hydroxydopamine (6-OHDA) unilateral lesioned rats. Plasmid-based delivery of brain-derived neurotrophic factor (BDNF) increases the number of DA neurons, identified by tyrosine hydroxylase immunoreactivity (TH-ir), by 25% in vitro, compared to enhanced green fluorescence protein (EGFP)-transfected controls. However, the nucleofection itself, especially the cell detachment and reseeding procedure, decreases the TH-ir neuron number to 40% compared with nontransfected control cultures. To circumvent this drawback we established the colayer method, which contains a mix of nucleofected cells reseeded on top of an adherent sister culture in a ratio 1:3. In this setup TH-ir neuron number remains high and could be further increased by 25% after BDNF transfection. Comparison of both cell culture procedures (standard and colayer) after intrastriatal transplantation revealed a similar DA neuron survival as seen in vitro. Two weeks after grafting TH-ir neuron number was strongly reduced in animals receiving the standard EGFP-transfected cells (271 ± 62) compared to 1,723 ± 199 TH-ir neurons in the colayer group. In contrast to the in vitro results, no differences in the number of grafted TH-ir neurons were observed between BDNF, EGFP, and nontransfected colayer groups, neither 2 nor 13 weeks after transplantation. Likewise, amphetamine and apomorphine-induced rotational behavior improved similarly over time in all groups. Nevertheless, the colayer protocol provides an efficient way for neurotrophic factor release by transplanted progenitor cells and will help to study the effects of candidate factors on survival and integration of transplanted DA neurons.