Project description:As the second most frequent neurodegenerative disorder of old age, ParkinsonM-bM-^@M-^Ys disease (PD) can result from autosomal dominant causes like increased alpha-synuclein (SNCA) dosage, or from autosomal recessive causes like PINK1 loss-of-function. Interactions between these triggers and their potential convergence onto shared pathways are crucial to understand, but currently conflicting evidence exists. Here, we crossed previously characterized mice with A53T-SNCA overexpression and mice with PINK1 deletion to generate double mutants (DM). We studied their lifespan and behavior, together with histological and molecular anomalies at late and early ages, respectively. DM animals showed potentiated phenotypes in comparison to both single mutants (SM), with markedly reduced survival after age 450 days and strongly reduced spontaneous movements from age 3 months onwards. A considerable part of DM animals manifested progressive paralysis at ages >1 year and also exhibited protein aggregates with immunoreactivity for pSer129-SNCA, p62, and ubiquitin in spinal cord and basal brain, contrasting with absence of such features from SM. A brain proteome quantification of ubiquitination sites documented altered degradation of SNCA and the DNA-damage marker H2AX at age 18 months. Global brain transcriptome profiles and qPCR validation experiments identified many consistent transcriptional dysregulations already at age 6 weeks, which were absent from SM. The observed downregulations for Dapk1, Dcaf17, Rab42 and upregulations for Dctn5, Mrpl9, Tmem181a, Xaf1 reflect changes in ubiquitination, mitochondrial / synaptic / microtubular dynamics, and DNA damage. Thus, our study confirmed that SNCA-triggered neurotoxicity is exacerbated by the absence of PINK1, and identified a novel molecular signature that is detectable early in the course of this double pathology. Factorial design comparing Pink1 knock-out/A53T-SNCA double transgenic mice with appropriate wild-type controls (129SvEv+FVB/N) in three different tissues (cerebellum, midbrain, striatum)
Project description:Parkinsons's Diseases causes early pre-motor somatosensory manifestations of the disease, leading to a sensory polyneuropathy with sensory loss and chronic pain. We assessed gene gene regulation in the dorsal root ganglia (DRGs) of aged-old Pink1SNCA double mutant mice by comparing RNA-seq expression profiles. Pink1SNCA mice are a model for Parkinsons's Disease (PD) carrying a Pink1 deletion plus transgene overexpression of human mutant alpha-synuclein. Homozygous Pink1-/-,SNCA A53T double mutant mice were generated by crossing Pink1-/- mice (background: 129/SvEv) with A53T-SNCA-overexpressing mice (background: FVB/N) and then, interbreeding the littermates. They contain 129/SvEv and FVB/N genetic backgrounds approximately in a 50:50 distribution. Wildtype control mice (FVBSv) are hybrids from a crossbreeding of 129/SvEv and FVB/N mice, which were descended from littermates of double mutant mice. RNAseq was performed in two sequential experiments with 4/4 old and 4/5 aged wildytpe and Pink1SNCA mice, respectively. The alignment was done consecutively. For analysis both sets were combined. A total number of 25702 genes was reliably detected in both datasets, and 22784 passed the filtering criteria of >7 valid samples in total. GSEA gene set enrichment analysis was used for gene ranking and identication of top up- and downregulated genes and evaluation of their functions based on P-value, Q-value, fold change and abundance. Apart from the knockout of Pink1, a prominent down-regulated gene was acidic fibroblast growth factor, Fgf1, which is known to restore the survival of dopaminergic neurons in PD models. Up-regulated genes included DNase1l3, CCL27 and CCL25, IFI44 and IL31ra, and pointed to increased DNAse activity and activation of the immune system.
Project description:As the second most frequent neurodegenerative disorder of old age, Parkinson’s disease (PD) can result from autosomal dominant causes like increased alpha-synuclein (SNCA) dosage, or from autosomal recessive causes like PINK1 loss-of-function. Interactions between these triggers and their potential convergence onto shared pathways are crucial to understand, but currently conflicting evidence exists. Here, we crossed previously characterized mice with A53T-SNCA overexpression and mice with PINK1 deletion to generate double mutants (DM). We studied their lifespan and behavior, together with histological and molecular anomalies at late and early ages, respectively. DM animals showed potentiated phenotypes in comparison to both single mutants (SM), with markedly reduced survival after age 450 days and strongly reduced spontaneous movements from age 3 months onwards. A considerable part of DM animals manifested progressive paralysis at ages >1 year and also exhibited protein aggregates with immunoreactivity for pSer129-SNCA, p62, and ubiquitin in spinal cord and basal brain, contrasting with absence of such features from SM. A brain proteome quantification of ubiquitination sites documented altered degradation of SNCA and the DNA-damage marker H2AX at age 18 months. Global brain transcriptome profiles and qPCR validation experiments identified many consistent transcriptional dysregulations already at age 6 weeks, which were absent from SM. The observed downregulations for Dapk1, Dcaf17, Rab42 and upregulations for Dctn5, Mrpl9, Tmem181a, Xaf1 reflect changes in ubiquitination, mitochondrial / synaptic / microtubular dynamics, and DNA damage. Thus, our study confirmed that SNCA-triggered neurotoxicity is exacerbated by the absence of PINK1, and identified a novel molecular signature that is detectable early in the course of this double pathology.
Project description:Parkinsonâs disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse. Parkinsonâs disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse. Tissue was dissected from the brain of 6 months old (2 WT / 2 TgA / 2 TgB striata, 2 WT / 2 TgA / 2 TgB brainstems/midbrains, 2 WT / 2 TgA / 2 TgB cerebella) and of 18+ months old mice (4 WT / 2 TgA / 2 TgB striata, 6 WT / 4 TgA / 3 TgB brainstems/midbrains, 6 WT / 5 TgA / 4 TgB cerebella). Tissues from individual, particularly old mice up to 28 months age were included here to strengthen the definition of progression markers reflecting old age.
Project description:Parkinson’s disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse. Parkinson’s disease (PD), the second most frequent neurodegenerative disorder at old age, can be caused by elevated expression, or the A53T mutation, of the presynaptic protein alpha-synuclein (SNCA). PD is characterized pathologically by the preferential vulnerability of the dopaminergic nigrostriatal projection neurons. Here, we used two mouse lines overexpressing human A53T-SNCA around ages 6 and 18 months and studied striatal dysfunction in the absence of neurodegeneration to understand early disease mechanisms. High pressure liquid chromatography analysis of striatal neurotransmitter content demonstrated that dopamine (DA) levels correlated directly with the level of expression of SNCA, an observation also observed in SNCA deficient mice. In the striatum of aged A53TSNCA overexpressing mice, where DA levels were elevated, a paradoxical upregulation of dopamine receptors DRD1A and DRD2 was detected by immunoblots and autoradiography, findings compatible with the notion of abnormal vesicle release. Extensive transcriptome studies via microarrays and quantitative real-time RT-PCR validation of altered Homer1, Cb1, Atf2 and Pde7b transcript levels indicated a progressive reduction in the postsynaptic DA response. As functional consequences, long term depression was absent in corticostriatal slices from aged transgenic mice and an insidious decrease of spontaneous locomotor activity of these animals was found in open field tests. Taken together, the dysfunctional neurotransmission and decreased synaptic plasticity seen in the A53T-SNCA overexpressing mice reflects early functional changes within the basal ganglia resulting from synucleinopathy prior to frank neurodegeneration. Thus, preclinical stages of PD may be modeled in this mouse.
Project description:Several genetic and environmental risk factors for Parkinson’s disease have been identified that converge on mitochondria as central elements in the disease process. However, the mechanisms by which mitochondrial dysfunction contributes to neurodegeneration remain incompletely understood. Non-bioenergetic pathways of the mitochondria are increasingly appreciated, but confounding bioenergetic defects are a major barrier to experimental validation. Here, we describe a novel bioenergetics-independent mechanism by which mild mitochondrial protein import stress augments neurodegeneration. We induced this mitochondrial protein import stress in an established mouse model of Parkinson’s disease expressing the A53T mutated form of human alpha-synuclein (SNCA). Mice with import stress in addition to the A53T mutation demonstrated increased size of SNCA aggregates, co-aggregation of mitochondrial preproteins with SNCA protein, worsened neurodegeneration and changes in gene expression, as determined by whole-transcriptome RNA-sequencing analysis of the forebrain (striatum), cerebellum, spinal cord, as well as heart and skeletal muscle.
Project description:Alpha synuclein (SNCA) has been linked to neurodegenerative diseases (synucleinopathies) that include Parkinson’s disease (PD). Although the primary neurodegeneration in PD involves nigrostriatal dopaminergic neurons, more extensive yet regionally selective neurodegeneration is observed in other synucleinopathies. Furthermore, SNCA is ubiquitously expressed in neurons and numerous neuronal systems are dysfunctional in PD. Therefore it is of interest to understand how overexpression of SNCA affects neuronal function in regions not directly targeted for neurodegeneration in PD. To gain a better understanding of the consequences of excessive SNCA expression on basal ganglia function, we performed transcriptome analysis of striatal tissue from male Thy1-aSyn-mice and wt littermates. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis. The present study investigated the consequences of SNCA overexpression on cellular processes and functions in the striatum of mice overexpressing wild-type, human SNCA under the Thy1 promoter (Thy1-aSyn mice) by transcriptome analysis. The analysis revealed alterations in multiple biological processes in the striatum of Thy1-aSyn mice, including synaptic plasticity, signaling, transcription, apoptosis, and neurogenesis.
Project description:Parkinson's disease (PD) is a neurodegenerative disorder that mainly affects the elder population, and its etiology is enigmatic. Both environmental risks and genetics may influence the development of PD. Excess copper causes neurotoxicity and accelerates the progression of neurodegenerative diseases. However, the underlying mechanisms of copper-induced neurotoxicity remain controversial. In this study, A53T transgenic α-synuclein (A53T) mice and their matching wild-type (WT) mice were treated with a low dose of copper (0.13 PPM copper chlorinated drinking water) for 4 months, and copper poisoning was performed on human A53T mutant SHSY5Y cells overexpressed with α-synuclein (dose of 1/4 IC50), to test the effects of copper exposure on the body. The results of the open field test showed that the moto function of Cu-treated mice was impaired. Proteomics revealed changes in neurodevelopment, transport function, and mitochondrial membrane-related function in Cu-treated WT mice, which were associated with reduced expression of mitochondrial complex (NDUFA10, NDUFS1, COX5A, ATP5A), dopamine neurons (TH), and dopamine transporter (DAT). Mitochondrial function, nervous system development, synaptic function, and immune response were altered in Cu-treated A53T mice. These changes were associated with increased mitochondrial splitting protein (Drp1), decreased mitochondrial fusion protein (OPA1, Mfn1), abnormalities in mitochondrial autophagy protein (LC3BII/I, P62), decreased dopamine neuron (TH) expression, increased α-synuclein expression, inflammatory factors (IL-6, IL-1β, and TNF-α) release and microglia (Iba1) activation.
Project description:Parkinson’s disease (PD) is characterized by the aggregation of α-synuclein into Lewy bodies and Lewy neurites in the brain. Microglia-driven neuroinflammation may contribute to neuronal death in PD, however the exact role of microglia remains unclear and has been understudied. The A53T mutation in the gene coding for α-synuclein has been linked to early-onset PD, and exposure to A53T-mutant human α-synuclein increases the potential for inflammation of murine microglia. To date, its effect has not been studied in human microglia. We aimed to study the impact of the A53T mutation on human microglia developed in a physiologically relevant context Here, we used 2-dimensional cultures of human iPSC-derived microglia and transplantation of these cells into the mouse brain to assess the effects of the A53T mutation on human microglia. We found that A53T-mutant human microglia had an intrinsically increased propensity towards pro-inflammatory activation upon inflammatory stimulus. Additionally, A53T mutant microglia showed increased oxidative stress, with a strong decrease in catalase expression in non-inflammatory conditions. Together, these results indicate that A53T mutant human microglia display cell-autonomous phenotypes that may worsen neuronal damage in early-onset PD.
Project description:RNA-seq analysis was used to investigate the transcriptional profile to reflect the changes in the parental vs engineered iPSC-derived macrophages. The RNA seq analysis provided an insight to uncover underlying mechanisms that might be triggered by perturbations in SNCA, Progranulin, and MeCP2 genes in iPSC derived macrophages. RNA seq data revealed an upregulation of 431 genes and concomitant downregulation of 276 in SNCA A53T Macrophages, an upregulation of 549 genes and concomitant downregulation of 356 in GRN R493X macrophages, an upregulation of 466 genes and concomitant downregulation of 406 genes in MeCP2HM macrophages when compared to the parental unengineered macrophages. These genes affected pathways regulating cell metabolism, exocytosis, transport, degranulation, immune activation, vascular circulation and neural differentiation and provided a comprehensive analysis of all cellular pathways perturbed by genetic engineering in SNCA, Progranulin, and MeCP2 genes.