Α-synuclein Transgenic Mouse Model Research Articles

MLKL deficiency alleviates neuroinflammation and motor deficits in the α-synuclein transgenic mouse model of Parkinson’s disease

Parkinson’s disease (PD), one of the most devastating neurodegenerative brain disorders, is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and deposits of α-synuclein aggregates. Currently, pharmacological interventions for PD remain inadequate. The cell necroptosis executor protein MLKL (Mixed-lineage kinase domain-like) is involved in various diseases, including inflammatory bowel disease and neurodegenerative diseases; however, its precise role in PD remains unclear. Here, we investigated the neuroprotective role of MLKL inhibition or ablation against primary neuronal cells and human iPSC-derived midbrain organoids induced by toxic α-Synuclein preformed fibrils (PFFs). Using a mouse model (Tg-Mlkl−/−) generated by crossbreeding the SNCA A53T synuclein transgenic mice with MLKL knockout (KO)mice, we assessed the impact of MLKL deficiency on the progression of Parkinsonian traits. Our findings demonstrate that Tg-Mlkl−/− mice exhibited a significant improvement in motor symptoms and reduced phosphorylated α-synuclein expression compared to the classic A53T transgenic mice. Furthermore, MLKL deficiency alleviated tyrosine hydroxylase (TH)-positive neuron loss and attenuated neuroinflammation by inhibiting the activation of microglia and astrocytes. Single-cell RNA-seq (scRNA-seq) analysis of the SN of Tg-Mlkl−/− mice revealed a unique cell type-specific transcriptome profile, including downregulated prostaglandin D synthase (PTGDS) expression, indicating reduced microglial cells and dampened neuron death. Thus, MLKL represents a critical therapeutic target for reducing neuroinflammation and preventing motor deficits in PD.

Environmental Enrichment Attenuates Oxidative Stress and Alters Detoxifying Enzymes in an A53T α-Synuclein Transgenic Mouse Model of Parkinson's Disease.

Although environmental enrichment (EE) is known to reduce oxidative stress in Parkinson’s disease (PD), the metabolic alternations for detoxifying endogenous and xenobiotic compounds according to various brain regions are not fully elucidated yet. This study aimed to further understand the role of EE on detoxifying enzymes, especially those participating in phase I of metabolism, by investigating the levels of enzymes in various brain regions such as the olfactory bulb, brain stem, frontal cortex, and striatum. Eight-month-old transgenic PD mice with the overexpression of human A53T α-synuclein and wild-type mice were randomly allocated to either standard cage condition or EE for 2 months. At 10 months of age, the expression of detoxifying enzymes was evaluated and compared with wild-type of the same age raised in standard cages. EE improved neurobehavioral outcomes such as olfactory and motor function in PD mice. EE-treated mice showed that oxidative stress was attenuated in the olfactory bulb, brain stem, and frontal cortex. EE also reduced apoptosis and induced cell proliferation in the subventricular zone of PD mice. The overexpression of detoxifying enzymes was observed in the olfactory bulb and brain stem of PD mice, which was ameliorated by EE. These findings were not apparent in the other experimental regions. These results suggest the stage of PD pathogenesis may differ according to brain region, and that EE has a protective effect on the PD pathogenesis by decreasing oxidative stress.

Developmental Changes in Dendritic Spine Morphology in the Striatum and Their Alteration in an A53T α-Synuclein Transgenic Mouse Model of Parkinson's Disease.

The aging process is accompanied by various neurophysiological changes, and the severity of neurodegenerative disorders such as Parkinson’s disease (PD) increases with aging. However, the precise neuroanatomical changes that accompany the aging process in both normal and pathologic conditions remain unknown. This is in part because there is a lack of high-resolution imaging tool that has the capacity to image a desired volume of neurons in a high-throughput and automated manner. In the present study, focused ion beam/scanning electron microscopy (FIB/SEM) was used to image striatal neuropil in both wild-type (WT) mice and an A53T bacterial artificial chromosome (BAC) human α-synuclein (A53T-BAC-SNCA) transgenic (Tg) mouse model of PD, at 1, 3, 6, and 22 months of age. We demonstrated that spine density gradually decreases, and average spine head volume gradually increases with age in WT mice, suggesting a homeostatic balance between spine head volume and spine density. However, this inverse relationship between spine head volume and spine density was not observed in A53T-BAC-SNCA Tg mice. Taken together, our data suggest that PD is accompanied by an abnormality in the mechanisms that control synapse growth and maturity.

Reduced cerebral blood flow in an α-synuclein transgenic mouse model of Parkinson’s disease

There is increasing evidence that widespread cortical cerebral blood flow deficits occur early in the course of Parkinson’s disease. Although cerebral blood flow measurement has been suggested as a potential biomarker for early diagnosis of Parkinson’s disease, as well as a means for tracking response to treatment, the relationship of cerebral blood flow to α-synucleinopathy, a major pathological hallmark of Parkinson’s disease, remains unclear. Therefore, we performed arterial spin-labeling magnetic resonance imaging and diffusion tensor imaging on transgenic mice overexpressing human wild-type α-synuclein and age-matched controls to measure cerebral blood flow and degenerative changes. As reported for early-stage Parkinson’s disease, α-synuclein mice exhibited a significant reduction in cortical cerebral blood flow, which was accompanied by motor coordination deficits and olfactory dysfunction. Although no overt degenerative changes were apparent in diffusion tensor imaging images, magnetic resonance imaging volumetric analysis revealed a significant reduction in olfactory bulb volume, similar to that seen in Parkinson’s disease patients. Our data, representing the first report of cerebral blood flow deficit in an animal model of Parkinson’s disease, suggest a causative role for α-synucleinopathy in cerebral blood flow deficits in Parkinson’s disease. Thus, α-synuclein transgenic mice comprise a promising model to study Parkinson’s disease-related mechanisms of cerebral blood flow deficits and to investigate further its utility as a potential biomarker for Parkinson’s disease.

Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model.

Parkinson’s disease is one of the most common neurodegenerative disorders of the elderly and ageing hence described to be a major risk factor. Telomere shortening as a result of the inability to fully replicate the ends of linear chromosomes is one of the hallmarks of ageing. The role of telomere dysfunction in neurological diseases and the ageing brain is not clarified and there is an ongoing discussion whether telomere shortening is linked to Parkinson’s disease. Here we studied a mouse model of Parkinson’s disease (Thy-1 [A30P] α-synuclein transgenic mouse model) in the background of telomere shortening (Terc knockout mouse model). α-synuclein transgenic mice with short telomeres (αSYNtg/tg G3Terc-/-) developed an accelerated disease with significantly decreased survival. This accelerated phenotype of mice with short telomeres was characterized by a declined motor performance and an increased formation of α-synuclein aggregates. Immunohistochemical analysis and mRNA expression studies revealed that the disease end-stage brain stem microglia showed an impaired response in αSYNtg/tg G3Terc-/- microglia animals. These results provide the first experimental data that telomere shortening accelerates α-synuclein pathology that is linked to limited microglia function in the brainstem.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0364-x) contains supplementary material, which is available to authorized users.

Cdk5-Dependent Activation of Neuronal Inflammasomes in Parkinson's Disease.

Inflammasomes, which mediate the activation of caspase-1 and maturation of IL-1β and IL-18, have been unambiguously verified to participate in many diseases, such as lung diseases, infectious diseases and Alzheimer's disease, but the relation between Parkinson's disease and inflammasomes is poorly understood. The expression, maturation, and secretion of inflammasomes in neurons were measured. The activation of inflammasomes in the substantia nigra of the brain was tested in acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and an α-synuclein transgenic mouse model. The levels of IL-1β and IL-18 in cerebrospinal fluid and serum samples of Parkinson's disease (PD) patients and control subjects were measured. The role of cyclin-dependent kinase 5 (Cdk5) in neuronal inflammasome activation was evaluated using the pharmacological Cdk5 inhibitor roscovitine or Cdk5-targeted deletion. Here, we observed the expression of core molecules of inflammasomes, including NALP3, ASC, caspase-1, and IL-1β, in neuronal cells. The PD inducer rotenone could activate neuronal inflammasomes and promote the maturation and secretion of the cleaved IL-1β and IL-18 in a dose- and time-dependent manner. We also detected the activation of inflammasomes in the substantia nigra of a PD mouse model and in cerebrospinal fluid of PD patients. Furthermore, Cdk5 is required for the activation of inflammasomes, and both inhibition and deletion of Cdk5 could efficiently block inflammasome activation in PD models. Together, our results indicated that Cdk5-dependent activation of neuronal inflammasomes was involved in the progression of PD.

Immunotherapy targeting α-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P] α-synuclein mice

Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event in Parkinson's disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. In support of this, (Thy-1)-h[A30P] α-synuclein transgenic mice with motor dysfunction symptoms were found to display increased levels of α-synuclein protofibrils in the CNS. An α-synuclein protofibril-selective monoclonal antibody (mAb47) was evaluated in this α-synuclein transgenic mouse model. As measured by ELISA, 14month old mice treated for 14weeks with weekly intraperitoneal injections of mAb47 displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. Besides the lower levels of pathogenic α-synuclein demonstrated, a reduction of motor dysfunction in transgenic mice upon peripheral administration of mAb47 was indicated. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson's disease and related disorders.

α-Synuclein Overexpression Represses 14-3-3θ Transcription

Previous gene microarray studies have shown that expression of 14-3-3θ is significantly decreased in an α-synuclein transgenic mouse model. In this study, we tested whether α-synuclein can regulate 14-3-3θ transcription. We demonstrate that the 14-3-3θ mRNA level is decreased in SH-SY5Y cells overexpressing α-synuclein. Luciferase activity under the control of the 14-3-3θ promoter is reduced both in stable SH-SY5Y cells constitutively overexpressing α-synuclein and in doxycycline-inducible SH-SY5Y cells upon α-synuclein induction, suggesting that the regulation of 14-3-3θ by α-synuclein occurs at the transcriptional level. Knockdown of α-synuclein by RNA interference does not increase the 14-3-3θ mRNA level. These findings suggest that α-synuclein represses 14-3-3θ transcription under pathologic conditions, but that regulation of 14-3-3θ expression is not a function of endogenous α-synuclein at baseline.

A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.

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.

Increased Calcium Influx and Decreased Buffering Capacity of Intracellular Stores Underlie Neuropathology Induced by Over-Expression of α-Synuclein

Abnormal accumulation of α-synuclein is centrally involved in the pathogenesis of many disorders with Parkinsonism and dementia. Although the mechanism through which α-synuclein triggers neuronal vulnerability is not entirely clear, in vitro studies suggest that neuronal accumulation of α-synuclein might lead to increase in intracellular calcium levels. In this study, we investigated in vivo calcium dynamics in an α-synuclein transgenic mouse model of Parkinson's Disease and dementia with Lewy bodies using two photon microscopy. We imaged spontaneous and stimulus-induced neuronal activity in the barrel cortex. Transgenic mice exhibited abnormal calcium activity characterized by augmented, long-lasting transients with considerable deviation from the exponential decay. The most evident pathology was observed in response to a repetitive stimulation when subsequent stimuli were presented before relaxation of calcium signal to the baseline. Augmented calcium entry can result from an increase in neuronal spiking or a decrease in a cell buffering capacity. To address the former possibility, we performed in vivo electrophysiological recordings of population spiking activity. Our measurements revealed no significant increase in neuronal spiking response compared to age-matched controls. To investigate potential impairment in calcium buffering, we conducted experiments in a neuronal cell line expressing α-synuclein. Inhibitors of the calcium influx into the intracellular calcium stores (mitochondria and endoplasmic reticulum) rescued cytosolic calcium levels, elevated by α-synuclein, while inhibitors of the calcium influx via channels located on the plasma membrane were ineffective. These studies support the notion that α-synuclein dysregulates calcium buffering capacity in vivo. Moreover, the characteristic shape of calcium decay and augmentation of the response amplitude during repetitive stimulation can serve as in vivo imaging biomarkers for screening of potential therapeutic agents in this model of neurodegeneration.