Benoit I. Giasson

Neuronal α-Synucleinopathy with Severe Movement Disorder in Mice Expressing A53T Human α-Synuclein

Abstract α-Synucleinopathies are neurodegenerative disorders that range pathologically from the demise of select groups of nuclei to pervasive degeneration throughout the neuraxis. Although mounting evidence suggests that α-synuclein lesions lead to neurodegeneration, this remains controversial. To explore this issue, we generated transgenic mice expressing wild-type and A53T human α-synuclein in CNS neurons. Mice expressing mutant, but not wild-type, α-synuclein developed a severe and complex motor impairment leading to paralysis and death. These animals developed age-dependent intracytoplasmic neuronal α-synuclein inclusions paralleling disease onset, and the α-synuclein inclusions recapitulated features of human counterparts. Moreover, immunoelectron microscopy revealed that the α-synuclein inclusions contained 10–16 nm wide fibrils similar to human pathological inclusions. These mice demonstrate that A53T α-synuclein leads to the formation of toxic filamentous α-synuclein neuronal inclusions that cause neurodegeneration.

MUTANT AND WILD TYPE HUMAN ALPHA -SYNUCLEINS ASSEMBLE INTO ELONGATED FILAMENTS WITH DISTINCT MORPHOLOGIES IN VITRO

α-Synuclein is a soluble presynaptic protein which is pathologically redistributed within intracellular lesions characteristic of several neurodegenerative diseases. Here we demonstrate that wild type and two mutant forms of α-synuclein linked to familial Parkinson’s disease (Ala30 → Pro and Ala53 → Thr) self-aggregate and assemble into 10–19-nm-wide filaments with distinct morphologies under definedin vitro conditions. Immunogold labeling demonstrates that the central region of all these filaments are more robustly labeled than the N-terminal or C-terminal regions, suggesting that the latter regions are buried within the filaments. Since in vitrogenerated α-synuclein filaments resemble the major ultrastructural elements of authentic Lewy bodies that are hallmark lesions of Parkinson’s disease, we propose that self-aggregating α-synuclein is the major subunit protein of these filamentous lesions.

Lewy Bodies Contain Altered α-Synuclein in Brains of Many Familial Alzheimer’s Disease Patients with Mutations in Presenilin and Amyloid Precursor Protein Genes

Missense mutations in the alpha-synuclein gene cause familial Parkinsons disease (PD), and alpha-synuclein is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimers disease (AD). To determine whether alpha-synuclein is a component of LBs in familial AD (FAD) patients with known mutations in presenilin (n = 65) or amyloid precursor protein (n = 9) genes, studies were conducted with antibodies to alpha-, beta-, and gamma-synuclein. LBs were detected with alpha- but not beta- or gamma-synuclein antibodies in 22% of FAD brains, and alpha-synuclein-positive LBs were most numerous in amygdala where some LBs co-localized with tau-positive neurofibrillary tangles. As 12 (63%) of 19 FAD amygdala samples contained alpha-synuclein-positive LBs, these inclusions may be more common in FAD brains than previously reported. Furthermore, alpha-synuclein antibodies decorated LB filaments by immunoelectron microscopy, and Western blots revealed that the solubility of alpha-synuclein was reduced compared with control brains. The presence of alpha-synuclein-positive LBs was not associated with any specific FAD mutation. These studies suggest that insoluble alpha-synuclein aggregates into filaments that form LBs in many FAD patients, and we speculate that these inclusions may compromise the function and/or viability of affected neurons in the FAD brain.

Induction of α-Synuclein Aggregation by Intracellular Nitrative Insult

Brain lesions containing filamentous and aggregated α-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant α-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of α-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of α-synuclein. Light microscopy using antibodies against α-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. α-Synuclein aggregates were observed in 20–30% of cells expressing wild-type or A53T mutant α-synuclein and in 5% of cells expressing A30P mutant α-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing β-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing α-synuclein. However, α-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote α-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of α-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of α-synuclein, indicating that lysosomes are involved in degradation of α-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of α-synuclein aggregates.

Misfolded proteinase K–resistant hyperphosphorylated α-synuclein in aged transgenic mice with locomotor deterioration and in human α-synucleinopathies

The pathological modifications of α-synuclein (αS) in Parkinson disease and related diseases are poorly understood. We have detected misfolded αS in situ based on the proteinase K resistance (PK resistance) of αS fibrils, and using specific antibodies against S129-phosphorylated αS as well as oxidized αS. Unexpectedly massive neuritic pathology was found in affected human brain regions, in addition to classical αS pathology. PK resistance and abnormal phosphorylation of αS developed with increasing age in (Thy1)-h[A30P] αS transgenic mice, concomitant with formation of argyrophilic, thioflavin S-positive, and electron-dense inclusions that were occasionally ubiquitinated. αS pathology in the transgenic mice was predominantly in the brainstem and spinal cord. Astrogliosis was found in these heavily affected tissues. Homozygous mice showed the same pathology approximately one year earlier. The transgenic mice showed a progressive deterioration of locomotor function.

Novel antibodies to synuclein show abundant striatal pathology in Lewy body diseases

Intracytoplasmic inclusions composed of α‐synuclein (α‐syn) are characteristic of neurodegenerative Lewy body disorders. Using novel monoclonal antibodies raised against altered α‐syn, we uncovered an unprecedented and extensive burden of α‐syn pathology in the striatum of Lewy body disorders. The highest density of striatal pathology was observed in patients with a combination of Alzheimers disease and dementia with Lewy bodies or pure dementia with Lewy bodies, and these α‐syn aggregates may contribute to the parkinsonism seen in these disorders.

The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer's and Parkinson's diseases.

Alzheimers (AD) and Parkinsons diseases (PD) are late-onset neurodegenerative diseases that have tremendous impact on the lives of affected individuals, their families, and society as a whole. Remarkable efforts are being made to elucidate the dominant factors that result in the pathogenesis of these disorders. Extensive postmortem studies suggest that oxidative/nitrative stresses are prominent features of these diseases, and several animal models support this notion. Furthermore, it is likely that protein modifications resulting from oxidative/nitrative damage contribute to the formation of intracytoplasmic inclusions characteristic of each disease. The frequent presentation of both AD and PD in individuals and the co-occurrence of inclusions characteristic of AD and PD in several other neurodegenerative diseases suggests the involvement of a common underlying aberrant process. It can be surmised that oxidative/nitrative stress, which is cooperatively influenced by environmental factors, genetic predisposition, and senescence, may be a link between these disorders.

Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis.

Abstract : Mutations in the Cu/Zn‐superoxidedismutase (SOD‐1) gene underlie some familial cases of amytotrophic lateral sclerosis, a neurodegenerative disorder charactreized by loss of cortical, brainstem, and spinal motor nrurons. We present evidence that SOD‐1 mutants alter the activity of molecular chaperones that aid in proper protein folding and targeting of abnormal proteins for degradation. In a cultured cell line (NOH 3T3), resistance to mutant SOD‐1 toxicity correlated with increased overall chaperoning activity (measured by the ability of cytosolic extracts to prevent heat denaturation of catalase) as well as with up‐regulation of individual chaperones/stress proteins. In transgenic mice expressing human SOD‐1 with the G93A mutation, chaperoning activity was decreased in lumbar spinal cord but increased or unchanged in clinically unaffected tissues. Increasing the level of the stress‐inducible chaperone 70‐kDa heat shock protein by gene transfer reduced formation of mutant SOD‐containing proteinaceous aggregates in cultured primary motor neurons expressing G93A SOG‐1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.

Concurrence of α-synuclein and tau brain pathology in the Contursi kindred

Abstract. Previous genetic analysis of the familial Parkinsons disease Contursi kindred led to the identification of an Ala53Thr pathogenic mutation in the α-synuclein gene. We have re-examined one of the original brains from this kindred using new immunohistochemical reagents, thioflavin S staining and immunoelectron microscopy. Surprisingly, we uncovered a dense burden of α-synuclein neuritic pathology and rare Lewy bodies. Immunoelectron microscopy demonstrated fibrillar α-synuclein-immunoreactive aggregates. Unexpected tau neuritic and less frequent perikaryal inclusions were also observed. Some inclusions were comprised of both proteins with almost complete spatial disparity. We suggest that it is important to recognize that the neurodegenerative process caused by the Ala53Thr mutation in α-synuclein is not identical to that seen in typical idiopathic Parkinsons disease brains.

Widespread Nitration of Pathological Inclusions in Neurodegenerative Synucleinopathies

Reactive nitrogen species may play a mechanistic role in neurodegenerative diseases by posttranslationally altering normal brain proteins. In support of this hypothesis, we demonstrate that an anti-3-nitrotyrosine polyclonal antibody stains all of the major hallmark lesions of synucleinopathies including Lewy bodies, Lewy neurites and neuraxonal spheroids in dementia with Lewy bodies, the Lewy body variant of Alzheimers disease, and neurodegeneration with brain iron accumulation type 1, as well as glial and neuronal cytoplasmic inclusions in multiple system atrophy. This antibody predominantly recognized nitrated alpha-synuclein when compared to other in vitro nitrated constituents of these pathological lesions, such as neurofilament subunits and microtubules. Collectively, these findings imply that alpha-synuclein is nitrated in pathological lesions. The widespread presence of nitrated alpha-synuclein in diverse intracellular inclusions suggests that oxidation/nitration is involved in the onset and/or progression of neurodegenerative diseases.