Mice overexpressing wild‐type human alpha‐synuclein display alterations in colonic myenteric ganglia and defecation

Background Prevalent non‐motor symptoms of Parkinson’s disease (PD) include gastrointestinal motor impairments and advanced stage PD displays pathological aggregates of α‐synuclein in colonic enteric neurons. We previously showed that 12 months old mice overexpressing human wild type (WT) α‐synuclein under the Thy1 promoter (Thy1‐aSyn) displayed colonic motor dysfunction. We investigated functional gut alterations at earlier ages and histological correlates. Methods Defecation, gastric emptying (GE), and immunostaining for α‐synuclein, peripheral choline acetyltransferase (pChAT), tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), and vasoactive intestinal peptide (VIP) in distal colon myenteric plexuses were assessed in male Thy1‐aSyn compared to littermate WT mice. Key Results Thy1‐aSyn mice aged 2.5–3 or 7–8 months old had 81% and 55% reduction in fecal pellet output, respectively, in the first 15 min of exposure to a novel environment. The reduction remained significant in the older group for 2‐h, and subsequent refeeding resulted also in a 60% and 69% reduction of defecation in the first hour, respectively. Thy1‐aSyn mice (8–10 months) displayed increased α‐synuclein in the myenteric plexuses with abundant varicose terminals surrounding pChAT‐immunoreactive (ir) neurons, and only a few, nNOS‐ir neurons. There were no conspicuous changes in pChAT‐ and nNOS‐ir neurons, or TH‐ and VIP‐ir nerve fibers. Thy1‐aSyn mice aged 4–18 months had normal GE. Conclusions & Inferences The occurrence of over‐production of pre‐synaptic α‐synuclein in colonic myenteric ganglia several months before the loss of striatal dopamine may provide an anatomical basis for interference with cholinergic neuronal activation, causing an early impairment in defecation to stimuli.     

The Lewy body in Parkinson's disease: Molecules implicated in the formation and degradation of α‐synuclein aggregates

The histological hallmark of Parkinson's disease (PD) is the presence of fibrillar aggregates called Lewy bodies (LBs). LB formation has been considered to be a marker for neuronal degeneration, because neuronal loss is found in the predilection sites for LBs. To date, more than 70 molecules have been identified in LBs, in which α‐synuclein is a major constituent of LB fibrils. α‐synuclein immunohistochemistry reveals that diffuse cytoplasmic staining develops into pale bodies via compaction, and that LBs arise from the peripheral portion of pale bodies. This α‐synuclein abnormality is found in 10% of pigmented neurons in the substantia nigra and more than 50% of those in the locus ceruleus in PD. Recent studies have suggested that oligomers and protofibrils of α‐synuclein are cytotoxic, and that LBs may represent a cytoprotective mechanism in PD.     

Widespread and abundant α‐synuclein pathology in a neurologically unimpaired subject

The intracytoplasmic aggregation of α‐synuclein (αS) protein is a common denominator for a group of neurodegenerative disorders currently known as synucleinopathies. It is generally assumed that the incorporation of αS protein into compact inclusions compromises the function and viability of its host cell via mechanical disruption. Herein, we report a widespread and abundant αS pathology in an elderly subject, whose medical history gave no indication of any neurodegenerative disease. We compared neuronal and glial components in this neurologically unimpaired subject with a patient with a clinical syndrome of dementia with Lewy bodies (DLB) by using a range of antigenic determinants and an in situ end‐labeling technique. We detected no differences in vascular pathologies, in gliosis, or in apoptosis that would have explained the incompatible clinical end‐points. With respect to the Alzheimer's disease‐related changes, the only differences noted were the β‐amyloid aggregates in the putamen found in the DLB patient alone. Our findings suggest that there must be some currently unidentified factors rather than αS‐positive inclusions that are responsible for the neuronal dysfunction. The αS‐positive inclusions may well represent detoxified reserves that cells can tolerate for years, and thus prevention of their development could actually accelerate the diseases process.     

Effects of antiparkinsonian agents on β‐amyloid and α‐synuclein oligomer formation in vitro

The aggregation of β‐amyloid protein (Aβ) and α‐synuclein (αS) are hypothesized to be the key pathogenic event in Alzheimer's disease (AD) and Lewy body diseases (LBD), with oligomeric assemblies thought to be the most neurotoxic. Inhibitors of oligomer formation, therefore, could be valuable therapeutics for patients with AD and LBD. Here, we examined the effects of antiparkinsonian agents (dopamine, levodopa, trihexyphenidyl, selegiline, zonisamide, bromocriptine, peroxide, ropinirole, pramipexole, and entacapone) on the in vitro oligomer formation of Aβ40, Aβ42, and αS using a method of photo‐induced cross‐linking of unmodified proteins (PICUP), electron microscopy, and atomic force microscopy. The antiparkinsonian agents except for trihexyphenidyl inhibited both Aβ and αS oligomer formations, and, among them, dopamine, levodopa, pramipexole, and entacapone had the stronger in vitro activity. Circular dichroism and thioflavin T(S) assays showed that secondary structures of Aβ and αS assemblies inhibited by antiparkinsonian agents were statistical coil state and that their seeding activities had disappeared. The antiparkinsonian agents could be potential therapeutic agents to prevent or delay AD and LBD progression. © 2013 Wiley Periodicals, Inc.     

Alternations of central insulin‐like growth factor‐1 sensitivity in APP/PS1 transgenic mice and neuronal models

Although many post‐mortem studies have found evidence of central insulin resistance in Alzheimer's disease (AD) patients, results on changes of central insulin‐like growth factor‐1 (IGF‐1) signaling in the pathological process of AD remain controversial. In the present study, we observed the activation states of IGF‐1 downstream signaling in brain slices of transgenic mice carrying APPswe/PS1dE9 mutations (APP/PS1 mice) at both early and late stages (ex vivo) and further investigated the involvement of oligomeric β‐amyloid (Aβ) and Aβ‐enriched culture medium (CM) on IGF‐1 sensitivity employing neuronal models (in vitro). In 6‐ and 18‐month‐old APP/PS1 mice, the phosphorylations of IGF‐1 receptor (IGF‐1R) and Akt in response to IGF‐1 stimulation were significantly reduced in the hippocampal and cortical slices, whereas IGF‐1R protein expression and mRNA levels of IGF‐1 and IGF‐1R in the hippocampal slices were significantly higher than that in wild‐type mice. In agreement with these results, reduced IGF‐1 sensitivity was verified in APP and PS1 double stably transfected CHO cells; moreover, IGF‐1 stimulated phosphorylations of IGF‐1R and Akt were also markedly weakened by oligomeric Aβ or Aβ‐enriched CM posttreatment in CHO cells without APP/PS1‐transfected (K1 cells) and primary hippocampal neurons. These observations indicate that the impaired central IGF‐1 sensitivity at early and late stages of APP/PS1 transgenic mice might be attributable, at least partially, to the overproduced Aβ, especially the oligomeric Aβ. These findings may shed new light on the mechanisms underlying the defective IGF‐1 signaling in AD pathogenesis and provide important clues for AD drug discovery. © 2013 Wiley Periodicals, Inc.     

Phosphorylated α‐synuclein immunoreactivity in the posterior pituitary lobe

Parkinson's disease is now recognized as a major form of α‐synucleinopathy involving both the central and peripheral nervous systems. However, no research has focused on the posterior pituitary lobe (PPL), despite the fact that this organ also plays an important role in systemic homeostasis. In the present study, we aimed to distinguish phosphorylated α‐synuclein (pαSyn)‐positive deposits in the PPL, as is observed in Lewy body‐ and non‐Lewy body‐related disorders. PαSyn deposits were immunohistochemically analyzed using formalin‐fixed, paraffin‐embedded PPL specimens obtained from 60 autopsy cases. Among the cases with Lewy body‐related disorders, PPL pαSyn deposits were observed in almost all cases of Parkinson's disease (22/23), and in one case of dementia with Lewy bodies (1/1). On the other hand, only 3/36 cases of non‐Lewy body‐related disorders had pαSyn immunoreactivity in the PPL. The present study confirms the presence of pαSyn‐positive deposits, as demonstrated by high specificity (97.1%) and sensitivity (88.5%), in both Parkinson's disease and dementia with Lewy bodies, suggesting that this finding can be a useful hallmark of Lewy body‐related disorders.     

α-Synuclein transgenic mice exhibit reduced anxiety-like behaviour

Up to 40% of Parkinson's disease patients suffer from anxiety, but little is known about the mechanisms involved. We used the elevated plus maze and open field test to evaluate groups of young adult mice expressing different levels of α-synuclein, including mice transgenic for human α-synuclein with the A53T mutation. Compared to α-synuclein knock-out mice and wild-type controls, α-synuclein A53T transgenic mice exhibited reduced anxiety-like behaviour by spending markedly greater amounts of time on the maze open arms and by a higher proportion of entries to the open arms. In the open field, transgenic mice showed a trend towards reduced locomotor habituation and increased thigmotaxis. These results indicate a possible role for α-synuclein in anxiety-like behaviours.     

Dopaminergic lesioning impairs adult hippocampal neurogenesis by distinct modification of α‐synuclein

Nonmotor symptoms of cognitive and affective nature are present in premotor and motor stages of Parkinson's disease (PD). Neurogenesis, the generation of new neurons, persists throughout the mammalian life span in the hippocampal dentate gyrus. Adult hippocampal neurogenesis may be severely affected in the course of PD, accounting for some of the neuropsychiatric symptoms such as depression and cognitive impairment. Two important PD‐related pathogenic factors have separately been attributed to contribute to both PD and adult hippocampal neurogenesis: dopamine depletion and accumulation of α‐synuclein (α‐syn). In the acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine model, altered neurogenesis has been linked merely to a reduced dopamine level. Here, we seek to determine whether a distinct endogenous α‐syn expression pattern is associated, possibly contributing to the hippocampal neurogenic deficit. We observed a persistent reduction of striatal dopamine and a loss of tyrosine hydroxylase‐expressing neurons in the substantia nigra pars compacta in contrast to a complete recovery of tyrosine hydroxylase‐immunoreactive dopaminergic fibers within the striatum. However, dopamine levels in the hippocampus were significantly decreased. Survival of newly generated neurons was significantly reduced and paralleled by an accumulation of truncated, membrane‐associated, insoluble α‐syn within the hippocampus. Specifically, the presence of truncated α‐syn species was accompanied by increased activity of calpain‐1, a calcium‐dependent protease. Our results further substantiate the broad effects of dopamine loss in PD‐susceptible brain nuclei, gradually involved in the PD course. Our findings also indicate a detrimental synergistic interplay between dopamine depletion and posttranslational modification of α‐syn, contributing to impaired hippocampal plasticity in PD. © 2015 Wiley Periodicals, Inc.     

Utilization of myoblasts from transgenic mice to evaluate the efficacy of myoblast transplantation

A possible treatment for Duchenne muscular dystrophy is the injection of normal myoblasts into dystrophic muscles to induce the formation of new, healthy, and dystrophin-positive muscle fibers. To develop this therapy, it is important to identify the muscle fibers formed by the injected myoblasts in the host muscles. In this study, we used myoblasts from transgenic mice which have a gene expressing β-galactosidase under the control of the promoter of quail fast skeletal muscle troponin I. This transgene is expressed in myotubes and muscle fibers, but not in myoblasts. Twenty-eight days after myoblast transplantation in nude and in mdx mice, muscle fibers containing of β-galactosidase were identified by x-gal staining. In mdx mice, most of the β-galactosidase-positive muscle fibers resulting from the myoblast transplantation were also dystrophin positive. This technique could make it possible to follow the success of myoblast transplantation even in mice that are not depleted of dystrophin. © 1994 John Wiley & Sons, Inc.     

Alpha‐synuclein in colonic submucosa in early untreated Parkinson's disease

The diagnosis of Parkinson's disease rests on motor signs of advanced central dopamine deficiency. There is an urgent need for disease biomarkers. Clinicopathological evidence suggests that α‐synuclein aggregation, the pathological signature of Parkinson's disease, can be detected in gastrointestinal tract neurons in Parkinson's disease. We studied whether we could demonstrate α‐synuclein pathology in specimens from unprepped flexible sigmoidoscopy of the distal sigmoid colon in early subjects with Parkinson's disease. We also looked for 3‐nitrotyrosine, a marker of oxidative stress. Ten subjects with early Parkinson's disease not treated with dopaminergic agents (7 men; median age, 58.5 years; median disease duration, 1.5 years) underwent unprepped flexible sigmoidoscopy with biopsy of the distal sigmoid colon. Immunohistochemistry studies for α‐synuclein and 3‐nitrotyrosine were performed on biopsy specimens and control specimens from a tissue repository (23 healthy subjects and 23 subjects with inflammatory bowel disease). Nine of 10 Parkinson's disease samples were adequate for study. All showed staining for α‐synuclein in nerve fibers in colonic submucosa. No control sample showed this pattern. A few showed light α‐synuclein staining in round cells. 3‐Nitrotyrosine staining was seen in 87% of Parkinson's disease cases but was not specific for Parkinson's disease. This study suggests a pattern of α‐synuclein staining in Parkinson's disease that was distinct from healthy subjects and those with inflammatory bowel disease. The absence of this pattern in subjects with inflammatory bowel disease suggests it is not a sequel of inflammation or oxidative stress. 3‐Nitrotyrosine immunostaining was common in all groups studied, suggesting oxidative stress in the colonic submucosa. © 2011 Movement Disorder Society.     

5′-flanking sequences of the human HPRT gene direct neuronal expression in the brain of transgenic mice

Total deficiency of hypoxanthine phosphoribosyl-transferase (HPRT) in humans causes the neurological disorder Lesch-Nyhan syndrome. The HPRT gene is expressed at basal levels in all tissues but at higher levels in the brain, the relevance and mechanism of which is unknown. To determine if cis-acting DNA elements play a role in the tissue-differential pattern of expression, we generated transgenic mice carrying different sequences of the human HPRT (hHPRT) promoter fused to the bacterial lacZ gene. We show that a 1.6 kb fragment of the hHPRT promoter contains essential information to direct β-galactosidase expression preferentially to the basal ganglia, cerebral cortex, hippocampus, and several other areas of the forebrain. At least two elements within the 1.6 kb fragment appear to be required for neuronal expression. A 182 bp element (hHPRT-NE) represents one of those sequences and is involved not only in confering neuronal specificity but also in repressing transgene expression in non-neuronal tissues. These studies provide molecular insight into the mechanism of increased HPRT expression in the brain. © 1994 Wiley-Liss, Inc.     

Staging of α‐synuclein in the olfactory bulb in a model of Parkinson's disease: Cell types involved

Impaired olfaction is an early symptom of Parkinson's disease. The underlying neuropathology likely includes α‐synucleinopathy in the olfactory bulb at an earlier stage (Braak's stage1) than pathology in the substantia nigra, which is not observed until stage 3. In this report, we investigated the distribution and cell types affected by α‐synuclein in the olfactory bulb of transgenic mice (2–8 months of age) expressing the human A53T variant of α‐synuclein. α‐Synuclein immunostaining progressively affects interneurons and mitral cells. Double labeling studies demonstrate that dopaminergic cells are hardly involved, whereas glutamatergic‐ and calcium binding protein‐positive cells are severely affected. This temporal evolution and the cell types expressing α‐synuclein are reminiscent of idiopathic Parkinson's disease and support the usefulness of this model to address specific topics in the premotor phase of the disease. © 2010 Movement Disorder Society     

Altered levels and distribution of amyloid precursor protein and its processing enzymes in Niemann‐Pick type C1‐deficient mouse brains

Niemann‐Pick type C (NPC) disease is an autosomal recessive neurodegenerative disorder characterized by intracellular accumulation of cholesterol and glycosphingolipids in many tissues including the brain. The disease is caused by mutations of either NPC1 or NPC2 gene and is accompanied by a severe loss of neurons in the cerebellum, but not in the hippocampus. NPC pathology exhibits some similarities with Alzheimer's disease, including increased levels of amyloid β (Aβ)‐related peptides in vulnerable brain regions, but very little is known about the expression of amyloid precursor protein (APP) or APP secretases in NPC disease. In this article, we evaluated age‐related alterations in the level/distribution of APP and its processing enzymes, β‐ and γ‐secretases, in the hippocampus and cerebellum of Npc1^−/− mice, a well‐established model of NPC pathology. Our results show that levels and expression of APP and β‐secretase are elevated in the cerebellum prior to changes in the hippocampus, whereas γ‐secretase components are enhanced in both brain regions at the same time in Npc1^−/− mice. Interestingly, a subset of reactive astrocytes in Npc1^−/− mouse brains expresses high levels of APP as well as β‐ and γ‐secretase components. Additionally, the activity of β‐secretase is enhanced in both the hippocampus and cerebellum of Npc1^−/− mice at all ages, while the level of C‐terminal APP fragments is increased in the cerebellum of 10‐week‐old Npc1^−/− mice. These results, taken together, suggest that increased level and processing of APP may be associated with the development of pathology and/or degenerative events observed in Npc1^−/− mouse brains. © 2010 Wiley‐Liss, Inc.     

X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

Targeting α‐synuclein: Therapeutic options

The discovery of the central role of α‐synuclein (αSyn) in the pathogenesis of Parkinson's disease (PD) has powered, in the last decade, the emergence of novel relevant models of this condition based on viral vector‐mediated expression of the disease‐causing protein or inoculation of toxic species of αSyn. Although the development of these powerful tools and models has provided considerable insights into the mechanisms underlying neurodegeneration in PD, it has also been translated into the expansion of the landscape of preclinical therapeutic strategies. Much attention is now brought to the proteotoxic mechanisms induced by αSyn and how to block them using strategies inspired by intrinsic cellular pathways such as the enhancement of cellular clearance by the lysosomal‐autophagic system, through proteasome‐mediated degradation or through immunization. The important effort undertaken by several laboratories and consortia to tackle these issues and identify novel targets warrants great promise for the discovery not only of neuroprotective approaches but also of restorative strategies for PD and other synucleinopathies. In this viewpoint, we summarize the latest advances in this new area of PD research and will discuss promising approaches and ongoing challenges. © 2016 International Parkinson and Movement Disorder Society