A53T Α-synuclein Transgenic Mice Research Articles

Cystatin C as a potential therapeutic mediator against Parkinson\u2019s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units

Cystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson’s disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T α-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal–vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways.

Expression signatures of long non-coding RNA in the substantia nigra of pre-symptomatic mouse model of Parkinson's disease

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that can be caused by a variety of factors. Growing evidence shows that prior to the motor phase of PD can express molecular or imaging markers. Many long non-coding RNAs (lncRNAs) have been identified in neurodegenerative disease. However, the biogenesis and function of lncRNAs in the pre-symptomatic stage of PD is poorly understood. Here, we profiled the expression of lncRNAs and mRNAs in the substantia nigra pars compacta (SNpc) of pre-symptomatic mice over-expressing human A30P*A53T α-synuclein by microarray analysis. Based on the Pearson correlation analysis, lncRNA/mRNA co-expression network was constructed. GO enrichment and pathway analysis of lncRNAs–coexpressed mRNAs was conducted to identify the related biological function and pathologic pathways. Real-time PCR was used to detect the expression pattern of lncRNAs. Approximately 756 lncRNAs were aberrantly expressed in the SNpc of early over-expressing human A30P*A53T α-synuclein transgenic mice, including 477 downregulated lncRNAs and 279 upregulated lncRNAs. GO analysis indicated that these lncRNAs–coexpressed mRNAs were targeted to regulation of transcription (ontology: biological process), membrane (ontology: cellular component), and protein binding (ontology: molecular function). Pathway analysis indicated that lncRNAs–coexpressed mRNAs were mostly enriched in axon guidance signaling pathway. In conclusion, the present study firstly identified a series of novel early PD-associated lncRNAs caused by mutant α-synuclein. Further study the function of these aberrantly expressed lncRNAs may provide insight into treatment of early PD.

Chronic mild stress accelerates the progression of Parkinson's disease in A53T α-synuclein transgenic mice

Daily stress is associated with increased risk for various diseases, and numerous studies have provided evidence that environmental stress leads to deleterious effects on the central nervous system. However, it remains unclear whether chronic stress exacerbates the progression of Parkinson's disease (PD). To investigate this hypothesis, we determined the effect of chronic mild stress (CMS) on the pathogenesis of PD in a transgenic mice line that overexpresses the human A53T mutant α-synuclein (A53T Tg mice). We show that when exposed to CMS, male, but not female, A53T Tg mice developed profound motor disabilities and exhibited olfactory sensitivity deficits. Pathological analysis also identified robust dopaminergic neuron degeneration and strong reduction of dopamine levels in A53T Tg male mice who underwent CMS treatment. Systematic examination of the abnormal aggregation of α-synuclein revealed a profound increase of inclusion in A53T Tg male mice subject to CMS resembling key pathological changes of PD. An insight into the mechanism underlying stress leading to the acceleration of neurodegeneration in those with genetic susceptibility, was revealed by evidence of microglia activation and elevated pro-inflammatory factor levels in A53T Tg male mice following CMS. Notably, these effects of CMS on the pathogenesis of PD showed a remarkable sexual dimorphism: only male A53T Tg mice exhibited exacerbation of the progression of PD. However, the molecular and cellular bases for this difference remains to be elucidated. Our results indicate a causative role for chronic mild stress using a PD animal model. Based on these findings, we propose that CMS acts as an environmental risk factor that leads to neuroinflammation and progressive neurodegeneration on a background of PD susceptibility.

Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy.

BackgroundSynucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy.MethodsWe determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches.ResultsAfter 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal.ConclusionTaken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy.

Neurodegenerative phenotypes in an A53T -synuclein transgenic mouse model are independent of LRRK2

Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain.

Transmission of Synucleinopathies in the Enteric Nervous System of A53T Alpha-Synuclein Transgenic Mice

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by abnormal deposition of α-synuclein aggregates in many regions of the central and peripheral nervous systems. Accumulating evidence suggests that the α-synuclein pathology initiates in a few discrete regions and spreads to larger areas in the nervous system. Recent pathological studies of PD patients have raised the possibility that the enteric nervous system is one of the initial sites of α-synuclein aggregation and propagation. Here, we evaluated the induction and propagation of α-synuclein aggregates in the enteric nervous system of the A53T α-synuclein transgenic mice after injection of human brain tissue extracts into the gastric walls of the mice. Western analysis of the brain extracts showed that the DLB extract contained detergent-stable α-synuclein aggregates, but the normal brain extract did not. Injection of the DLB extract resulted in an increased deposition of α-synuclein in the myenteric neurons, in which α-synuclein formed punctate aggregates over time up to 4 months. In these mice, inflammatory responses were increased transiently at early time points. None of these changes were observed in the A53T mice injected with saline or the normal brain extract, nor were these found in the wild type mice injected with the DLB extract. These results demonstrate that pathological α-synuclein aggregates present in the brain of DLB patient can induce the aggregation of endogenous α-synuclein in the myenteric neurons in A53T mice, suggesting the transmission of synucleinopathy lesions in the enteric nervous system.

Proteinase K-resistant α-synuclein is deposited in presynapses in human Lewy body disease and A53T α-synuclein transgenic mice

Abnormally modified alpha-synuclein is a pathological hallmark of Parkinson's disease and the other alpha-synucleinopathies. Since proteinase K (PK) treatment is known to enhance the immunoreactivity of abnormal alpha-synuclein, we immunohistochemically examined the brain of transgenic (Tg) mice expressing human mutant A53T alpha-synuclein using this retrieval method. PK treatment abolished the immunoreactivity of alpha-synuclein in abnormal inclusions as well as of endogenous alpha-synuclein in Tg mice, whereas PK-resistant alpha-synuclein was found in the presynaptic nerve terminals, especially in the hippocampus and temporal cortex. In human Lewy body disease, PK-resistant alpha-synuclein was deposited in Lewy bodies and Lewy neurites, as well as in the presynapses in distinct brain regions, including the hippocampus, temporal cortex and substantia nigra. Biochemical analysis revealed that PK-resistant alpha-synuclein was detected in the presynaptic fraction in Tg mice and human Lewy body disease. Although PK-resistant alpha-synuclein was found in the presynapse in Tg mice even at 1 week of age, it was not phosphorylated until at least 8 months of age. Moreover, PK-resistant alpha-synuclein in the presynapse was not phosphorylated in human Lewy body disease. These findings suggest that phosphorylation is not necessary to cause the conversion of soluble form to PK-resistant alpha-synuclein. Considering that native alpha-synuclein is a soluble protein localized to the presynaptic terminals, our findings suggest that PK-resistant alpha-synuclein may disturb the neurotransmission in alpha-synucleinopathies.

Locomotor hyperactivity and alterations in dopamine neurotransmission are associated with overexpression of A53T mutant human α-synuclein in mice

Genetic and biochemical abnormalities associated with alpha-synuclein are implicated in the etiology of Parkinson's disease (PD). In this study, altered locomotor behavior linked to the expression of mutant or wildtype human alpha-synuclein was investigated. A53T alpha-synuclein transgenic (A53T-tg) mice exhibited normal activity at 5 months of age; however, by 7 months, they developed marked hyperactivity that remained evident until 19 months. By contrast, mice expressing human wildtype or A30P mutant alpha-synuclein showed no locomotor alterations. Hyperactivity in A53T-tg mice was reversed by the D1 receptor antagonist SCH 23390. Furthermore, A53T-tg mice were supersensitive to the D1 receptor agonist SKF 81297 but not to the serotonin1B receptor agonist RU 24969. Hyperactivity in A53T-tg mice was also associated with increased D1 receptor expression in the substantia nigra and decreased dopamine transporter expression in the nucleus accumbens and striatum. Finally, striatal dopamine uptake measured by high-speed chronoamperometry was reduced by 40% in A53T-tg mice. Thus, expression of A53T mutant human alpha-synuclein in mice results in adult-onset hyperactivity associated with D1 receptor and dopamine transporter-mediated alterations in dopamine neurotransmission.