Michael Mante

β-Synuclein Inhibits α-Synuclein Aggregation

Abstract We characterized β-synuclein, the non-amyloidogenic homolog of α-synuclein, as an inhibitor of aggregation of α-synuclein, a molecule implicated in Parkinsons disease. For this, doubly transgenic mice expressing human (h) α- and β-synuclein were generated. In doubly transgenic mice, β-synuclein ameliorated motor deficits, neurodegenerative alterations, and neuronal α-synuclein accumulation seen in hα-synuclein transgenic mice. Similarly, cell lines transfected with β-synuclein were resistant to α-synuclein accumulation. hα-synuclein was coimmunoprecipitated with hβ-synuclein in the brains of doubly transgenic mice and in the double-transfected cell lines. Our results raise the possibility that β-synuclein might be a natural negative regulator of α-synuclein aggregation and that a similar class of endogenous factors might regulate the aggregation state of other molecules involved in neurodegeneration. Such an anti-amyloidogenic property of β-synuclein might also provide a novel strategy for the treatment of neurodegenerative disorders.

Passive Immunization Reduces Behavioral and Neuropathological Deficits in an Alpha-Synuclein Transgenic Model of Lewy Body Disease

Dementia with Lewy bodies (DLB) and Parkinsons Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (α-syn). This study investigated whether passive immunization with a novel monoclonal α-syn antibody (9E4) against the C-terminus (CT) of α-syn was able to cross into the CNS and ameliorate the deficits associated with α-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved α-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display α-syn accumulation and promotes α-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of α-syn may be of therapeutic relevance in patients with PD and DLB.

Neuroprotective Effects of Regulators of the Glycogen Synthase Kinase-3β Signaling Pathway in a Transgenic Model of Alzheimer's Disease Are Associated with Reduced Amyloid Precursor Protein Phosphorylation

The glycogen synthase kinase-3β (GSK3β) pathway plays an important role in mediating neuronal fate and synaptic plasticity. In Alzheimers disease (AD), abnormal activation of this pathway might play an important role in neurodegeneration, and compounds such as lithium that modulate GSK3β activity have been shown to reduce amyloid production and tau phosphorylation in amyloid precursor protein (APP) transgenic (tg) mice. However, it is unclear whether regulation of GSK3β is neuroprotective in APP tg mice. In this context, the main objective of the present study was to determine whether pharmacological or genetic manipulations that block the GSK3β pathway might ameliorate the neurodegenerative alterations in APP tg mice and to better understand the mechanisms involved. For this purpose, two sets of experiments were performed. First, tg mice expressing mutant human APP under the Thy1 promoter (hAPP tg) were treated with either lithium chloride or saline alone. Second, hAPP tg mice were crossed with GSK3β tg mice, in which overexpression of this signaling molecule results in a dominant-negative (DN) effect with inhibition of activity. hAPP tg mice that were treated with lithium or that were crossed with DN–GSK3β tg mice displayed improved performance in the water maze, preservation of the dendritic structure in the frontal cortex and hippocampus, and decreased tau phosphorylation. Moreover, reduced activation of GSK3β was associated with decreased levels of APP phosphorylation that resulted in decreased amyloid-β production. In conclusion, the present study showed that modulation of the GSK3β signaling pathway might also have neuroprotective effects in tg mice by regulating APP maturation and processing and further supports the notion that GSK3β might be a suitable target for the treatment of AD.

Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of Abeta(1-42).

The main objective of the present study was to develop an alternative singly‐transgenic (tg) hAPP model where amyloid deposition will occur at an earlier age. For this purpose, we generated lines of tg mice expressing hAPP751 cDNA containing the London (V717I) and Swedish (K670M/N671L) mutations under the regulatory control of the murine (m)Thy‐1 gene (mThy1‐hAPP751). In the brains of the highest (line 41) and intermediate (lines 16 and 11) expressers, high levels of hAPP expression were found in neurons in layers 4–5 of the neocortex, hippocampal CA1 and olfactory bulb. As early as 3–4 months of age, line 41 mice developed mature plaques in the frontal cortex, whereas at 5–7 months plaque formation extended to the hippocampus, thalamus and olfactory region. Ultrastructural and double‐immunolabeling analysis confirmed that most plaques were mature and contained dystrophic neurites immunoreactive with antibodies against APP, synaptophysin, neurofilament and tau. In addition, a decrease in the number of synaptophysin‐immunoreactive terminals was most prominent in the frontal cortex of mice from line 41. Mice from line 11 developed diffuse amyloid deposits at 11 months of age, whereas mice from line 16 did not show evidence of amyloid deposition. Analysis of Aβ by ELISA showed that levels of Aβ1–40 were higher in mice that did not show any amyloid deposits (line 16), whereas Aβ1–42 was the predominant species in tg animals from the lines showing plaque formation (lines 41 and 11). Taken together this study indicates that early onset plaque formation depends on levels of Aβ1–42. J. Neurosci. Res. 66:573–582, 2001.

Neurological and Neurodegenerative Alterations in a Transgenic Mouse Model Expressing Human α-Synuclein under Oligodendrocyte Promoter: Implications for Multiple System Atrophy

Multiple system atrophy (MSA) is a progressive, neurodegenerative disease characterized by parkinsonism, ataxia, autonomic dysfunction, and accumulation of α-synuclein (α-syn) in oligodendrocytes. To better understand the mechanisms of neurodegeneration and the role of α-syn accumulation in oligodendrocytes in the pathogenesis of MSA, we generated transgenic mouse lines expressing human (h) α-syn under the control of the murine myelin basic protein promoter. Transgenic mice expressing high levels of hα-syn displayed severe neurological alterations and died prematurely at 6 months of age. Furthermore, mice developed progressive accumulation of hα-syn-immunoreactive inclusions in oligodendrocytes along the axonal tracts in the brainstem, basal ganglia, cerebellum, corpus callosum, and neocortex. The inclusions also reacted with antibodies against phospho-serine (129) hα-syn and ubiquitin, and hα-syn was found in the detergent-insoluble fraction. In high-expresser lines, the white matter tracts displayed intense astrogliosis, myelin pallor, and decreased neurofilament immunostaining. Accumulation of hα-syn in oligodendrocytes also leads to prominent neurodegenerative changes in the neocortex with decreased dendritic density and to loss of dopaminergic fibers in the basal ganglia. The oligodendrocytic inclusions were composed of fibrils and accompanied by mitochondrial alterations and disruption of the myelin lamina in the axons. Together, these studies support the contention that accumulation of α-syn in oligodendrocytes promotes neurodegeneration and recapitulates several of the key functional and neuropathological features of MSA.

Neurodegeneration and cognitive impairment in apoE-deficient mice is ameliorated by infusion of recombinant apoE.

Recent studies suggest that apolipoprotein E (apoE) might play a neurotrophic function in the central nervous system and that altered functioning of this molecule could result in neurodegeneration. The main objective of this study was to determine if neurodegenerative and cognitive alterations in apoE-deficient mice are reversible by infusion of recombinant apoE into the lateral ventricles. ApoE-deficient mice treated with either apoE3 or apoE4 showed a significant improvement in their learning capacity in the Morris water maze compared to saline-infused apoE-deficient mice. While this improved performance was associated with restoration of neuronal structure, the poor learning ability of apoE-deficient mice treated with saline correlated with the disrupted synapto-dendritic structure. This study supports the contention that apoE might play a neurotrophic effect in vivo and suggests that apoE might have a potential therapeutic role.

Reducing C-Terminal-Truncated Alpha-Synuclein by Immunotherapy Attenuates Neurodegeneration and Propagation in Parkinson's Disease-Like Models

Parkinsons disease (PD) and dementia with Lewy bodies (DLB) are common neurodegenerative disorders of the aging population, characterized by progressive and abnormal accumulation of α-synuclein (α-syn). Recent studies have shown that C-terminus (CT) truncation and propagation of α-syn play a role in the pathogenesis of PD/DLB. Therefore, we explored the effect of passive immunization against the CT of α-syn in the mThy1-α-syn transgenic (tg) mouse model, which resembles the striato-nigral and motor deficits of PD. Mice were immunized with the new monoclonal antibodies 1H7, 5C1, or 5D12, all directed against the CT of α-syn. CT α-syn antibodies attenuated synaptic and axonal pathology, reduced the accumulation of CT-truncated α-syn (CT-α-syn) in axons, rescued the loss of tyrosine hydroxylase fibers in striatum, and improved motor and memory deficits. Among them, 1H7 and 5C1 were most effective at decreasing levels of CT-α-syn and higher-molecular-weight aggregates. Furthermore, in vitro studies showed that preincubation of recombinant α-syn with 1H7 and 5C1 prevented CT cleavage of α-syn. In a cell-based system, CT antibodies reduced cell-to-cell propagation of full-length α-syn, but not of the CT-α-syn that lacked the 118–126 aa recognition site needed for antibody binding. Furthermore, the results obtained after lentiviral expression of α-syn suggest that antibodies might be blocking the extracellular truncation of α-syn by calpain-1. Together, these results demonstrate that antibodies against the CT of α-syn reduce levels of CT-truncated fragments of the protein and its propagation, thus ameliorating PD-like pathology and improving behavioral and motor functions in a mouse model of this disease.

Mutant α-synuclein exacerbates age-related decrease of neurogenesis

In Parkinson disease, wild-type α-synuclein accumulates during aging, whereas α-synuclein mutations lead to an early onset and accelerated course of the disease. The generation of new neurons is decreased in regions of neurogenesis in adult mice overexpressing wild-type human α-synuclein. We examined the subventricular zone/olfactory bulb neurogenesis in aged mice expressing either wild-type human or A53T mutant α-synuclein. Aging wild-type and mutant α-synuclein-expressing animals generated significantly fewer new neurons than their non-transgenic littermates. This decreased neurogenesis was caused by a reduction in cell proliferation within the subventricular zone of mutant α-synuclein mice. In contrast, no difference was detected in mice overexpressing the wild-type allele. Also, more TUNEL-positive profiles were detected in the subventricular zone, following mutant α-synuclein expression and in the olfactory bulb, following wild-type and mutant α-synuclein expression. The impaired neurogenesis in the olfactory bulb of different transgenic α-synuclein mice during aging highlights the need to further explore the interplay between olfactory dysfunction and neurogenesis in Parkinson disease.

Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

In Parkinsons disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinsons disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.

Neurodegeneration in a Transgenic Mouse Model of Multiple System Atrophy Is Associated with Altered Expression of Oligodendroglial-Derived Neurotrophic Factors

Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by striatonigral degeneration and olivo-pontocerebellar atrophy. Neuronal degeneration is accompanied by primarily oligodendrocytic accumulation of α-synuclein (αsyn) as opposed to the neuronal inclusions more commonly found in other α-synucleinopathies such as Parkinsons disease. It is unclear how αsyn accumulation in oligodendrocytes may lead to the extensive neurodegeneration observed in MSA; we hypothesize that the altered expression of oligodendrocyte-derived neurotrophic factors by αsyn may be involved. In this context, the expression of a number neurotrophic factors reportedly expressed by oligodendrocytes [glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor 1 (IGF-1), as well as basic fibroblast growth factor 2 (bFGF2), reportedly astrocyte derived] were examined in transgenic mouse models expressing human αsyn (hαsyn) under the control of either neuronal (PDGFβ or mThy1) or oligodendrocytic (MBP) promoters. Although protein levels of BDNF and IGF-1 were altered in all the αsyn transgenic mice regardless of promoter type, a specific decrease in GDNF protein expression was observed in the MBP-hαsyn transgenic mice. Intracerebroventricular infusion of GDNF improved behavioral deficits and ameliorated neurodegenerative pathology in the MBP-hαsyn transgenic mice. Consistent with the studies in the MBP-hαsyn transgenic mice, analysis of GDNF expression levels in human MSA samples demonstrated a decrease in the white frontal cortex and to a lesser degree in the cerebellum compared with controls. These results suggest a mechanism in which αsyn expression in oligodendrocytes impacts on the trophic support provided by these cells for neurons, perhaps contributing to neurodegeneration.