Expression Of Human Α-synuclein Research Articles

A rapidly progressive multiple system atrophy-cerebellar variant model presenting marked glial reactions with inflammation and spreading of α-synuclein oligomers and phosphorylated α-synuclein aggregates

Multiple system atrophy (MSA) is a severe α-synucleinopathy facilitated by glial reactions; the cerebellar variant (MSA-C) preferentially involves olivopontocerebellar fibres with conspicuous demyelination. A lack of aggressive models that preferentially involve olivopontocerebellar tracts in adulthood has hindered our understanding of the mechanisms of demyelination and neuroaxonal loss, and thus the development of effective treatments for MSA. We therefore aimed to develop a rapidly progressive mouse model that recaptures MSA-C pathology. We crossed Plp1-tTA and tetO-SNCA*A53T mice to generate Plp1-tTA::tetO-SNCA*A53T bi-transgenic mice, in which human A53T α-synuclein—a mutant protein with enhanced aggregability—was specifically produced in the oligodendrocytes of adult mice using Tet-Off regulation. These bi-transgenic mice expressed mutant α-synuclein from 8 weeks of age, when doxycycline was removed from the diet. All bi-transgenic mice presented rapidly progressive motor deterioration, with wide-based ataxic gait around 22 weeks of age and death around 30 weeks of age. They also had prominent demyelination in the brainstem/cerebellum. Double immunostaining demonstrated that myelin basic protein was markedly decreased in areas in which SM132, an axonal marker, was relatively preserved. Demyelinating lesions exhibited marked ionised calcium-binding adaptor molecule 1-, arginase-1-, and toll-like receptor 2-positive microglial reactivity and glial fibrillary acidic protein-positive astrocytic reactivity. Microarray analysis revealed a strong inflammatory response and cytokine/chemokine production in bi-transgenic mice. Neuronal nuclei-positive neuronal loss and patchy microtubule-associated protein 2-positive dendritic loss became prominent at 30 weeks of age. However, a perceived decrease in tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta in bi-transgenic mice compared with wild-type mice was not significant, even at 30 weeks of age. Wild-type, Plp1-tTA, and tetO-SNCA*A53T mice developed neither motor deficits nor demyelination. In bi-transgenic mice, double immunostaining revealed human α-synuclein accumulation in neurite outgrowth inhibitor A (Nogo-A)-positive oligodendrocytes beginning at 9 weeks of age; its expression was further increased at 10 to 12 weeks, and these increased levels were maintained at 12, 24, and 30 weeks. In an α-synuclein-proximity ligation assay, α-synuclein oligomers first appeared in brainstem oligodendrocytes as early as 9 weeks of age; they then spread to astrocytes, neuropil, and neurons at 12 and 16 weeks of age. α-Synuclein oligomers in the brainstem neuropil were most abundant at 16 weeks of age and decreased thereafter; however, those in Purkinje cells successively increased until 30 weeks of age. Double immunostaining revealed the presence of phosphorylated α-synuclein in Nogo-A-positive oligodendrocytes in the brainstem/cerebellum as early as 9 weeks of age. In quantitative assessments, phosphorylated α-synuclein gradually and successively accumulated at 12, 24, and 30 weeks in bi-transgenic mice. By contrast, no phosphorylated α-synuclein was detected in wild-type, tetO-SNCA*A53T, or Plp1-tTA mice at any age examined. Pronounced demyelination and tubulin polymerisation, promoting protein-positive oligodendrocytic loss, was closely associated with phosphorylated α-synuclein aggregates at 24 and 30 weeks of age. Early inhibition of mutant α-synuclein expression by doxycycline diet at 23 weeks led to fully recovered demyelination; inhibition at 27 weeks led to persistent demyelination with glial reactions, despite resolving phosphorylated α-synuclein aggregates. In conclusion, our bi-transgenic mice exhibited progressively increasing demyelination and neuroaxonal loss in the brainstem/cerebellum, with rapidly progressive motor deterioration in adulthood. These mice showed marked microglial and astrocytic reactions with inflammation that was closely associated with phosphorylated α-synuclein aggregates. These features closely mimic human MSA-C pathology. Notably, our model is the first to suggest that α-synuclein oligomers may spread from oligodendrocytes to neurons in transgenic mice with human α-synuclein expression in oligodendrocytes. This model of MSA is therefore particularly useful for elucidating the in vivo mechanisms of α-synuclein spreading from glia to neurons, and for developing therapies that target glial reactions and/or α-synuclein oligomer spreading and aggregate formation in MSA.

Gut mucosal cells transfer α-synuclein to the vagus nerve.

Epidemiological and histopathological findings have raised the possibility that misfolded α-synuclein protein might spread from the gut to the brain and increase the risk of Parkinson's disease. Although past experimental studies in mouse models have relied on gut injections of exogenous recombinant α-synuclein fibrils to study gut-to-brain α-synuclein transfer, the possible origins of misfolded α-synuclein within the gut have remained elusive. We recently demonstrated that sensory cells of intestinal mucosa express α-synuclein. Here, we employed mouse intestinal organoids expressing human α-synuclein to observe the transfer of α-synuclein protein from epithelial cells in organoids to cocultured nodose neurons devoid of α-synuclein. In mice expressing human α-synuclein, but no mouse α-synuclein, α-synuclein fibril-templating activity emerged in α-synuclein-seeded fibril aggregation assays in intestine, vagus nerve, and dorsal motor nucleus. In newly engineered transgenic mice that restrict pathological human α-synuclein expression to intestinal epithelial cells, α-synuclein fibril-templating activity transfered to the vagus nerve and dorsal motor nucleus. Subdiaphragmatic vagotomy prior to induction of α-synuclein expression in intestinal epithelial cells effectively protected the hindbrain from emergence of α-synuclein fibril-templating activity. Overall, these findings highlight a potential non-neuronal source of fibrillar α-synuclein protein that might arise in gut mucosal cells.

Ca2+ administration prevents α-synuclein proteotoxicity by stimulating calcineurin-dependent lysosomal proteolysis.

The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson’s disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson’s disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.

Construction of model strain of yeast Saccharomyces cerevisiae with regulated expression of recombinant human alpha-synuclein

Background. Improper folding and accumulation of a-synuclein aggregates are among the causes of Parkinson’s disease. The most important factor influencing the process of α-synuclein aggregation is the level of this protein in neurons which depends on the balance between its synthesis, degradation and secretion. Under certain conditions, when α-synuclein is synthesized at a high level, monomers of this protein can aggregate on the lipid membrane, which leads to the formation of amyloids, fibrils and protofibrils unable to perform their physiological functions. Since it is virtually impossible to study the properties of α-synuclein in vivo, researchers are actively using model biological systems (single-celled microorganisms, human cell lines, animal models etc.). The aim of this study was to construct a recombinant strain of Saccharomyces cerevisiae with controlled expression of human α-synuclein to study the regulation and properties of this protein and for screening for new low molecular weight chemi­cal compounds which can induce α-synuclein aggregation and/or degradation. Materials and methods. A recombinant strain of S. cerevisiae with controlled expression of α-synuclein conjugated to a green fluorescent protein was isolated. Western blotting with specific anti-α-synuclein antibodies was used to detect recombinant α-synuclein in yeast cells. Intracellular localization of heterologous chimeric green fluorescent protein conjugated to α-synuclein was also examined by fluorescence microscopy. Results. To construct a recombinant strain of S. cerevisiae, the coding sequence of the human wild-type α-synuclein gene was expressed under the regulated promoter of the ScMET25 gene. Analysis of the effect of different concentrations of exogenous methionine as a factor regulating the expression of the ScMET25 promoter on the content of recombinant protein showed that the expression of the human α-synuclein gene in S. cerevisiae is repressed in the presence of methionine at a concentration of 10 mg/L and higher. During long-term cultivation of yeast cells, this effect decreased due to the depletion of methionine in the growth medium. As a result, recombinant protein synthesis was restored, and α-synuclein content in such cells approached that of cells grown in a medium with a low concentration of (5 mg/L), or without methionine. It was also found that overproduction of recombinant α-synuclein in S. cerevisiae cells had virtually no effect on culture growth, indicating the absence or a very weak toxic effect of human α-synuclein on yeast physiology. Conclusions. The obtained data indicate a concentration-dependent effect of methionine on the level of recombinant α-synuclein synthesis in S. cerevisiae yeast cells. Such controlled expression of the studied protein can be used to screen for compounds capable of promoting dose-dependent aggregation or degradation of α-synuclein in yeast cells and potentially in human cells as well.

α-synuclein impairs autophagosome maturation through abnormal actin stabilization.

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson's disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson's disease and related α-synucleinopathies.

Apoptosis, Induced by Human α-Synuclein in Yeast, Can Occur Independent of Functional Mitochondria.

Human α-synuclein expression in baker’s yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ−) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ0 or ρ− petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25/GAL1 promoter, gives rise to ρ− petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ− petites and also in ρ0 petites (produced by treating ρ+ cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ− petites expressing α-synuclein from fully-induced MET25/GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson’s disease.

Radicicol rescues yeast cell death triggered by expression of human α-synuclein and its A53T mutant, but not by human βA4 peptide and proapoptotic protein bax

Aggregation/misfolding of α-synuclein and βA4 proteins cause neuronal cell death (NCD) associated with Parkinson’s and Alzheimer’s disease. It has been suggested that a heat shock protein-90 (Hsp90) inhibitor can prevent NCD by activating the heat shock transcription factor-1 which, in turn, upregulates molecular chaperones such as Hsp70 that targets aggregated/misfolded proteins for refolding/degradation. We have isolated radicicol, an Hsp90 inhibitor, from a fungus occurring in the crevices of marble rocks of Central India. Radicicol, which was found to be a strong antioxidant, was tested for its ability to rescue yeast cells from death induced by expression of wild-type α-synuclein, its more toxic A53T mutant, and βA4. It effectively overcomes wild-type/mutant α-synuclein mediated yeast cell death, concomitantly diminishes ROS levels, reverses mitochondrial dysfunction and prevents nuclear DNA-fragmentation, a hallmark of apoptosis. Surprisingly however, radicicol is unable to rescue yeast cells from death triggered by expression of secreted βA4. Moreover, although radicicol acts as an antioxidant it fails to prevent yeast cell death inflicted by the proapoptotic protein, Bax. Our results indicate that radicicol specifically targets aggregated/misfolded α-synuclein’s toxicity and opens up the possibility of using multiple yeast assays to screen natural product libraries for compounds that would unambiguously target α-synuclein aggregation/misfolding.

Experimental Parkinson’s disease models

Parkinson's disease (PD) is a neurodegenerative disease that develops slowly; however, there is no efficient method of early diagnosis, nor is there a cure. It is characterized by the relatively selective loss of dopaminergic neuronal cells in the substantia nigra pars compacta and the presence of alpha-synuclein aggregation named as Lewy bodies and Lewy neurites in surviving affected neurons. Nigrostriatal dopaminergic neurodegeneration is shared with other parkinsonian disorders, including some genetic forms of parkinsonism, but many of these disorders do not have Lewy bodies. An ideal animal model for PD, therefore, should exhibit age-dependent and progressive dopaminergic neurodegeneration, motor and non-motor dysfunction, and abnormal alpha-synuclein pathology. A wide range of neurotoxic agents are used to induce PD, alterations that are similar with dose observed in human PD. These agents are classified mainly by administration route and the species involved. The toxins that are mainly used in present 6- hydroxydopamine, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine, rotenone, paraquat, reserpine, methamphetamine, 3-nitrotyrosine and isoquinoline derivatives (Tieu, 2011; McDowell and Chesselet, 2012; Bezard et al. 2013). In addition, viral mediated expression of human α-synuclein, as well as the inoculation of pathogenic α-synuclein species from Lewy bodies of PD patients, for accurately modelling progressive self-propagating neurodegeneration and genetic LRRK2 models (PARK8 gene mutation) has been used (Jiang and Dickson, 2018). In conclusion, these models are only approximations, each possibly holding a certain degree of relevance. Thus, researchers should select models whose characteristics are most suitable for addressing the experimental question.

Glucocerebrosidase Deficiency in Drosophila Results in α-Synuclein-Independent Protein Aggregation and Neurodegeneration.

Mutations in the glucosidase, beta, acid (GBA1) gene cause Gaucher’s disease, and are the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) excluding variants of low penetrance. Because α-synuclein-containing neuronal aggregates are a defining feature of PD and DLB, it is widely believed that mutations in GBA1 act by enhancing α-synuclein toxicity. To explore this hypothesis, we deleted the Drosophila GBA1 homolog, dGBA1b, and compared the phenotypes of dGBA1b mutants in the presence and absence of α-synuclein expression. Homozygous dGBA1b mutants exhibit shortened lifespan, locomotor and memory deficits, neurodegeneration, and dramatically increased accumulation of ubiquitinated protein aggregates that are normally degraded through an autophagic mechanism. Ectopic expression of human α-synuclein in dGBA1b mutants resulted in a mild enhancement of dopaminergic neuron loss and increased α-synuclein aggregation relative to controls. However, α-synuclein expression did not substantially enhance other dGBA1b mutant phenotypes. Our findings indicate that dGBA1b plays an important role in the metabolism of protein aggregates, but that the deleterious consequences of mutations in dGBA1b are largely independent of α-synuclein. Future work with dGBA1b mutants should reveal the mechanism by which mutations in dGBA1b lead to accumulation of protein aggregates, and the potential influence of this protein aggregation on neuronal integrity.

Brain propagation of transduced α-synuclein involves non-fibrillar protein species and is enhanced in α-synuclein null mice.

Aggregation and neuron-to-neuron transmission are attributes of α-synuclein relevant to its pathogenetic role in human synucleinopathies such as Parkinson's disease. Intraparenchymal injections of fibrillar α-synuclein trigger widespread propagation of amyloidogenic protein species via mechanisms that require expression of endogenous α-synuclein and, possibly, its structural corruption by misfolded conformers acting as pathological seeds. Here we describe another paradigm of long-distance brain diffusion of α-synuclein that involves inter-neuronal transfer of monomeric and/or oligomeric species and is independent of recruitment of the endogenous protein. Targeted expression of human α-synuclein was induced in the mouse medulla oblongata through an injection of viral vectors into the vagus nerve. Enhanced levels of intra-neuronal α-synuclein were sufficient to initiate its caudo-rostral diffusion that likely involved at least one synaptic transfer and progressively reached specific brain regions such as the locus coeruleus, dorsal raphae and amygdala in the pons, midbrain and forebrain. Transfer of human α-synuclein was compared in two separate lines of α-synuclein-deficient mice versus their respective wild-type controls and, interestingly, lack of endogenous α-synuclein expression did not counteract diffusion but actually resulted in a more pronounced and advanced propagation of exogenous α-synuclein. Self-interaction of adjacent molecules of human α-synuclein was detected in both wild-type and mutant mice. In the former, interaction of human α-synuclein with mouse α-synuclein was also observed and might have contributed to differences in protein transmission. In wild-type and α-synuclein-deficient mice, accumulation of human α-synuclein within recipient axons in the pons, midbrain and forebrain caused morphological evidence of neuritic pathology. Tissue sections from the medulla oblongata and pons were stained with different antibodies recognizing oligomeric, fibrillar and/or total (monomeric and aggregated) α-synuclein. Following viral vector transduction, monomeric, oligomeric and fibrillar protein was detected within donor neurons in the medulla oblongata. In contrast, recipient axons in the pons were devoid of immunoreactivity for fibrillar α-synuclein, indicating that non-fibrillar forms of α-synuclein were primarily transferred from one neuron to the other, diffused within the brain and led to initial neuronal injury. This study elucidates a paradigm of α-synuclein propagation that may play a particularly important role under pathophysiological conditions associated with enhanced α-synuclein expression. Rapid long-distance diffusion and accumulation of monomeric and oligomeric α-synuclein does not necessarily involve pathological seeding but could still result in a significant neuronal burden during the pathogenesis of neurodegenerative diseases.

Nurr1 and Retinoid X Receptor Ligands Stimulate Ret Signaling in Dopamine Neurons and Can Alleviate α-Synuclein Disrupted Gene Expression.

How α-synuclein, a protein enriched in Lewy bodies in Parkinson's disease, is causing neuropathology in dopamine neurons remains unclear. This study elucidated how α-synuclein is influencing gene expression and how Nurr1, a transcription factor known to protect dopamine neurons against α-synuclein toxicity, can counteract these effects. Moreover, given the protective role of Nurr1, this study also investigated how Nurr1 could be pharmacologically targeted via bexarotene, a ligand of Nurr1's heterodimerization partner retinoid X receptor (RXR). The results showed that RXR ligands could increase neurotrophic signaling, but provided a mixed picture of its potential in a Parkinson's disease rat model in vivo. However, this study clearly emphasized Nurr1's neuroprotective role and indicated that other RXR ligands could have therapeutic potential in Parkinson's disease.

α-Synuclein-induced dopaminergic neurodegeneration in a rat model of Parkinson's disease occurs independent of ATP13A2 (PARK9)

Mutations in the ATP13A2 (PARK9) gene cause early-onset, autosomal recessive Parkinson's disease (PD) and Kufor–Rakeb syndrome. ATP13A2 mRNA is spliced into three distinct isoforms encoding a P5-type ATPase involved in regulating heavy metal transport across vesicular membranes. Here, we demonstrate that three ATP13A2 mRNA isoforms are expressed in the normal human brain and are modestly increased in the cingulate cortex of PD cases. ATP13A2 can mediate protection toward a number of stressors in mammalian cells and can protect against α-synuclein-induced toxicity in cellular and invertebrate models of PD. Using a primary cortical neuronal model combined with lentiviral-mediated gene transfer, we demonstrate that human ATP13A2 isoforms 1 and 2 display selective neuroprotective effects toward toxicity induced by manganese and hydrogen peroxide exposure through an ATPase-independent mechanism. The familial PD mutations, F182L and G504R, abolish the neuroprotective effects of ATP13A2 consistent with a loss-of-function mechanism. We further demonstrate that the AAV-mediated overexpression of human ATP13A2 is not sufficient to attenuate dopaminergic neurodegeneration, neuropathology, and striatal dopamine and motoric deficits induced by human α-synuclein expression in a rat model of PD. Intriguingly, the delivery of an ATPase-deficient form of ATP13A2 (D513N) to the substantia nigra is sufficient to induce dopaminergic neuronal degeneration and motor deficits in rats, potentially suggesting a dominant-negative mechanism of action. Collectively, our data demonstrate a distinct lack of ATP13A2-mediated protection against α-synuclein-induced neurotoxicity in the rat nigrostriatal dopaminergic pathway, and limited neuroprotective capacity overall, and raise doubts about the potential of ATP13A2 as a therapeutic target for PD.

Differential Expression of Alpha-Synuclein in Hippocampal Neurons

α-Synuclein is the major pathological component of synucleinopathies including Parkinson's disease and dementia with Lewy bodies. Recent studies have demonstrated that α-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of α-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of α-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of α-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These α-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, α-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of α-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic α-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activity-dependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant α-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous α-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human α-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of α-synuclein between excitatory and inhibitory neurons. Furthermore, α-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.

Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease.

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

α-Synuclein expression is modulated at the translational level by iron

Several studies have suggested an interaction between α-synuclein protein and iron in Parkinson's disease. The presence of iron together with α-synuclein in Lewy bodies, the increase of iron in the substantia nigra and the correlation between polymorphism of the several genes implicated in iron metabolism and Parkinson's disease, support a role for iron in the neurodegeneration. Analysis of post mortem brains revealed increased amount of insoluble α-synuclein protein despite unchanged/reduced levels of α-synuclein mRNA in Parkinson's disease. Interestingly, on the basis of the presence of a putative iron responsive element in the 5'-UTR, it has been suggested that there is a possible iron-dependent translational control of human α-synuclein mRNA. Considering the similarity between the sequences present in human α-synuclein mRNA and the ferritin iron responsive element, we postulated that iron deficiency would decrease the translation of α-synuclein mRNA. Here we used HEK293 cells treated with iron chelator deferoxamine or ferric ammonium citrate to verify the possible iron-dependent translational control of human α-synuclein biosynthesis. We show that the amount of polysome-associated endogenous human α-synuclein mRNA decreases in presence of deferoxamine. Our data demonstrate that human α-synuclein expression is regulated by iron mainly at the translational level. This result not only supports a role for iron in the translational control of α-synuclein expression, but also suggests that iron chelation may be a valid approach to control α-synuclein levels in the brain.

Alpha-synuclein transgenic mice display age-related slowing of gastrointestinal motility associated with transgene expression in the vagal system

Gastrointestinal (GI) dysfunction is the one of the most common non-motor symptoms of Parkinson's disease (PD) and occurs in nearly every patient afflicted with this common neurodegenerative disorder. While parkinsonian motor symptoms are caused by degeneration of dopamine neurons in the midbrain substantia nigra, the neurological localization of non-motor symptoms in PD is not known. In this study, we examined a transgenic mouse model of PD in which mutant (A53T) human α-synuclein was expressed under control of the prion promoter (AS mice). We found that gastrointestinal expression of human α-synuclein in this transgenic line was limited to efferent fibers projecting from the dorsal motor nucleus of the vagus nerve (DMV) to the enteric nervous system (ENS). Older transgenic mice had a lower density of human α-synuclein expression in the GI tract, suggesting an age-related disruption of efferent vagal fibers in this model. At the same time, mice developed age-related declines in stool frequency and gastric emptying consistent with those seen in human PD. These behavioral and neuropathological patterns parallel those seen in PD patients and suggest the DMV as a target for further investigation into causes for GI neuropathology and symptomatology in parkinsonism.

Hypokinesia and Reduced Dopamine Levels in Zebrafish Lacking β- and γ1-Synucleins

α-Synuclein is strongly implicated in the pathogenesis of Parkinson disease. However, the normal functions of synucleins and how these relate to disease pathogenesis are uncertain. We characterized endogenous zebrafish synucleins in order to develop tractable models to elucidate the physiological roles of synucleins in neurons in vivo. Three zebrafish genes, sncb, sncg1, and sncg2 (encoding β-, γ1-, and γ2-synucleins respectively), show extensive phylogenetic conservation with respect to their human paralogues. A zebrafish α-synuclein orthologue was not found. Abundant 1.45-kb sncb and 2.7-kb sncg1 mRNAs were detected in the CNS from early development through adulthood and showed overlapping but distinct expression patterns. Both transcripts were detected in catecholaminergic neurons throughout the CNS. Zebrafish lacking β-, γ1-, or both synucleins during early development showed normal CNS and body morphology but exhibited decreased spontaneous motor activity that resolved as gene expression recovered. Zebrafish lacking both β- and γ1-synucleins were more severely hypokinetic than animals lacking one or the other synuclein and showed delayed differentiation of dopaminergic neurons and reduced dopamine levels. Phenotypic abnormalities resulting from loss of endogenous zebrafish synucleins were rescued by expression of human α-synuclein. These data demonstrate that synucleins have essential phylogenetically conserved neuronal functions that regulate dopamine homeostasis and spontaneous motor behavior. Zebrafish models will allow further elucidation of the molecular physiology and pathophysiology of synucleins in vivo.

Age and α-synuclein expression interact to reveal a dependence of dopaminergic axons on endogenous Akt/PKB signaling

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7 weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.

Resistance to MPTP-Neurotoxicity in α-Synuclein Knockout Mice Is Complemented by Human α-Synuclein and Associated with Increased β-Synuclein and Akt Activation

Genetic and biochemical abnormalities of α-synuclein are associated with the pathogenesis of Parkinson's disease. In the present study we investigated the in vivo interaction of mouse and human α-synuclein with the potent parkinsonian neurotoxin, MPTP. We find that while lack of mouse α-synuclein in mice is associated with reduced vulnerability to MPTP, increased levels of human α-synuclein expression is not associated with obvious changes in the vulnerability of dopaminergic neurons to MPTP. However, expressing human α-synuclein variants (human wild type or A53T) in the α-synuclein null mice completely restores the vulnerability of nigral dopaminergic neurons to MPTP. These results indicate that human α-synuclein can functionally replace mouse α-synuclein in regard to vulnerability of dopaminergic neurons to MPTP-toxicity. Significantly, α-synuclein null mice and wild type mice were equally sensitive to neurodegeneration induced by 2′NH2-MPTP, a MPTP analog that is selective for serotoninergic and noradrenergic neurons. These results suggest that effects of α-synuclein on MPTP like compounds are selective for nigral dopaminergic neurons. Immunoblot analysis of β-synuclein and Akt levels in the mice reveals selective increases in β-synuclein and phosphorylated Akt levels in ventral midbrain, but not in other brain regions, of α-synuclein null mice, implicating the α-synuclein-level dependent regulation of β-synuclein expression in modulation of MPTP-toxicity by α-synuclein. Together these findings provide new mechanistic insights on the role α-synuclein in modulating neurodegenerative phenotypes by regulation of Akt-mediated cell survival signaling in vivo.

Biochemical and morphological consequences of human α-synuclein expression in a mouse α-synuclein null background.

A consensus about the functions of human wild-type or mutated α-synuclein (αSYN) is lacking. Both forms of αSYN are implicated in Parkinson's disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild-type αSYN (hwαSYN) or a doubly mutated (A30P*A53T) αSYN (hm(2) αSYN) in a C57Bl/6J line spontaneously deleted in mouse αSYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites and the enzyme tyrosine hydroxylase compared with null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at 9 months of age. Human αSYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm(2) αSYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hwαSYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared with the levels of the hm(2) αSYN transgene. These observations suggest that mutations in αSYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between hαSYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.