Dataset Information

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]?Syn transgenic mice.

ABSTRACT: Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (?Syn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of ?Syn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of ?Syn in vivo by generating two opposing mouse models. We crossed into human [A30P]?Syn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of ?Syn-positive aggregates, whereas loss of calpastatin led to increased truncation of ?Syn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]?Syn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of ?Syn, indicating a therapeutic potential of calpain inhibition in PD.

SUBMITTER: Diepenbroek M

PROVIDER: S-EPMC4110482 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

Diepenbroek Meike M Casadei Nicolas N Esmer Hakan H Saido Takaomi C TC Takano Jiro J Kahle Philipp J PJ Nixon Ralph A RA Rao Mala V MV Melki Ronald R Pieri Laura L Helling Stefan S Marcus Katrin K Krueger Rejko R Masliah Eliezer E Riess Olaf O Nuber Silke S

Human molecular genetics 20140311 15

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleav ...[more]

PMID: 24619358

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Project description:Synucleinopathies are characterized by the accumulation of insoluble ?-synuclein (?Syn). To test whether ?Syn aggregates modulate synaptic activity, we used a recently developed model in primary neurons for inducing ?Syn pathology. We demonstrated that preformed fibrils (PFFs) generated with recombinant human ?Syn compromised synaptic activity in a time- and dose-dependent manner and that the magnitude of these deficits correlated with the formation of ?Syn pathology in cultured excitatory hippocampal neurons from both sexes of mice. Remarkably, acute passive infusion of ?Syn PFFs from whole-cell patch-clamp pipette decreased mEPSC frequency within 10 min followed by induction of ?Syn pathology within 1 d. Moreover, by direct addition of ?Syn PFFs into culture medium, the formation of misfolded ?Syn inclusions dramatically compromised the colocalization of synaptic markers and altered dynamic changes of dendritic spines, but the viability of neurons was not affected up to 7 d post-treatment with ?Syn PFFs. Our data indicate that intraneuronal ?Syn fibrils impaired the initiation of synaptogenesis and their physiological functions, thereby suggesting that targeting synaptic dysfunction in synucleinopathies may provide a promising therapeutic direction.SIGNIFICANCE STATEMENT Under pathological conditions, the presynaptic protein ?-synuclein (?Syn) aggregates to form intraneuronal inclusions. To understand how and to what extent ?Syn aggregates modulate synaptic activity before neuron loss, we demonstrate that ?Syn preformed fibrils (PFFs) reduced synaptic activity in a dose- and time-dependent manner. The magnitude of these deficits correlated with the deposition of ?Syn pathology, which dramatically compromised the colocalization of synaptic markers and altered the dendritic spine dynamics. The present work further highlights the impact of ?Syn PFFs on synaptogenesis and physiological function, which may be applicable to other types of synucleinopathies.

Project description:The synucleinopathy underlying multiple system atrophy (MSA) is characterized by the presence of abundant amyloid inclusions containing fibrillar ?-synuclein (?-syn) aggregates in the brains of the patients and is associated with an extensive neurodegeneration. In contrast to Parkinson's disease (PD) where the pathological ?-syn aggregates are almost exclusively neuronal, the ?-syn inclusions in MSA are principally observed in oligodendrocytes (OLs) where they form glial cytoplasmic inclusions (GCIs). This is intriguing because differentiated OLs express low levels of ?-syn, yet pathogenic amyloid ?-syn seeds require significant amounts of ?-syn monomers to feed their fibrillar growth and to eventually cause the buildup of cytopathological inclusions. One of the transgenic mouse models of this disease is based on the targeted overexpression of human ?-syn in OLs using the PLP promoter. In these mice, the histopathological images showing a rapid emergence of S129-phosphorylated ?-syn inside OLs are considered as equivalent to GCIs. Instead, we report here that they correspond to the accumulation of phosphorylated ?-syn monomers/oligomers and not to the appearance of the distinctive fibrillar ?-syn aggregates that are present in the brains of MSA or PD patients. In spite of a propensity to co-sediment with myelin sheath contaminants, the phosphorylated forms found in the brains of the transgenic animals are soluble (>80%). In clear contrast, the phosphorylated species present in the brains of MSA and PD patients are insoluble fibrils (>95%). Using primary cultures of OLs from PLP-?Syn mice we observed a variable association of S129-phosphorylated ?-syn with the cytoplasmic compartment, the nucleus and with membrane domains suggesting that OLs functionally accommodate the phospho-?-syn deriving from experimental overexpression. Yet and while not taking place spontaneously, fibrillization can be seeded in these primary cultures by challenging the OLs with ?-syn preformed fibrils (PFFs). This indicates that a targeted overexpression of ?-syn does not model GCIs in mice but that it can provide a basis for seeding aggregation using PFFs. This approach could help establishing a link between ?-syn aggregation and the development of a clinical phenotype in these transgenic animals.

Dataset Information

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]?Syn transgenic mice.

Publications

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

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