Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions. The molecular hallmark of ...synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson's disease and dementia with Lewy bodies. The discovery of α-SYN assemblies with different structural characteristics or 'strains' has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson's disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood-brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.
In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not ...known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.
Background The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different ...neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly.
Methods We studied the effect of acetylcholine (ACh), serotonin (5‐HT), and adenosine triphosphate (ATP) on intracellular Ca2+ signaling using aequorin‐expressing and Fluo‐4 AM‐loaded CRL‐2690 rat and human enteric glial cell cultures devoid of neurons. The influence of these neurotransmitters on the proliferation of glia was measured and their effect on the expression of c‐Fos as well as glial fibrillary acidic protein (GFAP), Sox10, and S100 was examined by immunohistochemistry and quantitative RT‐PCR.
Key Results Apart from ATP, also ACh and 5‐HT induced a dose‐dependent increase in intracellular Ca2+ concentration in CRL‐2690 cells. Similarly, these neurotransmitters also evoked Ca2+ transients in human primary enteric glial cells obtained from mucosal biopsies. In contrast with ATP, stimulation with ACh and 5‐HT induced early gene expression in CRL‐2690 cells. The proliferation of enteric glia and their expression of GFAP, Sox10, and S100 were not affected following stimulation with these neurotransmitters.
Conclusions & Inferences We provide evidence that enteric glial cells respond to fast excitatory neurotransmitters by changes in intracellular Ca2+. On the basis of our experimental in vitro setting, we show that enteric glia are not only directly responsive to purinergic but also to serotonergic and cholinergic signaling mechanisms.
ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb Syndrome (KRS), a form of ...autosomal Parkinson's disease (PD). In spite of a growing interest in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their nature remains incompletely understood. To this end, we generated a set of melanoma and neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N) and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress conditions, evoked by the proteasome inhibitor Bortezomib, endo-/lysosomal associated full-length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-proteins, a pattern also observed in patient-derived fibroblasts harbouring ATP13A2 loss-of function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve, compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity-independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in response to proteotoxic stress.
Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene ...therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.
Alzheimer's disease (AD) is the most common cause of dementia. An early feature of the AD pathology is the dysregulation of intracellular Ca2+ signaling in neurons. In particular, increased Ca2+ ...release from endoplasmic reticulum-located Ca2+ channels, including inositol-1,4,5-trisphosphate type 1 receptors (IP3R1) and ryanodine receptors type 2 (RyR2), have been extensively reported. Known for its anti-apoptotic properties, Bcl-2 also has the ability to bind to and inhibit the Ca2+-flux properties of IP3Rs and RyRs. In this study, the hypothesis that the expression of Bcl-2 proteins can normalize dysregulated Ca2+ signaling in a mouse model of AD (5xFAD) and thereby prevent or slow the progression of AD was examined. Therefore, stereotactic injections of adeno-associated viral vectors expressing Bcl-2 proteins were performed in the CA1 region of the 5xFAD mouse hippocampus. In order to assess the importance of the association with IP3R1, the Bcl-2K17D mutant was also included in these experiments. This K17D mutation has been previously shown to decrease the association of Bcl-2 with IP3R1, thereby impairing its ability to inhibit IP3R1 while not affecting Bcl-2′s ability to inhibit RyRs. Here, we demonstrate that Bcl-2 protein expression leads to synaptoprotective and amyloid-protective effects in the 5xFAD animal model. Several of these neuroprotective features are also observed by Bcl-2K17D protein expression, suggesting that these effects are not associated with Bcl-2-mediated inhibition of IP3R1. Potential mechanisms for this Bcl-2 synaptoprotective action may be related to its ability to inhibit RyR2 activity as Bcl-2 and Bcl-2K17D are equally potent in inhibiting RyR2-mediated Ca2+ fluxes. This work indicates that Bcl-2-based strategies hold neuroprotective potential in AD models, though the underlying mechanisms requires further investigation.
Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, ...differential glutamatergic
responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated
in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as
3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal âstubs,â and Aβ(40) and Aβ(42) peptides
in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected,
the Aβ(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid
plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually
signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits
and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models
for both early and late neuropathological and clinical aspects of Alzheimerâs disease.
Deposition of amyloid β-peptide (Aβ) in cerebral vessel walls (cerebral amyloid angiopathy, CAA) is very frequent in Alzheimer’s disease and occurs also as a sporadic disorder. Here, we describe ...significant CAA in addition to amyloid plaques, in aging APP/Ld transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP) exclusively in neurons. The number of amyloid-bearing vessels increased with age, from ∼10 to >50 per coronal brain section in APP/Ld transgenic mice, aged 13 to 24 months. Vascular amyloid was preferentially deposited in arterioles and ranged from small focal to large circumferential depositions. Ultrastructural analysis allowed us to identify specific features contributing to weakening of the vessel wall and aneurysm formation, ie, disruption of the external elastic lamina, thinning of the internal elastic lamina, interruption of the smooth muscle layer, and loss of smooth muscle cells. Biochemically, the much lower Aβ42:Aβ40 ratio evident in vascular relative to plaque amyloid, demonstrated that in blood vessel walls Aβ40 was the more abundant amyloid peptide. The exclusive neuronal origin of transgenic APP, the high levels of Aβ in cerebrospinal fluid compared to plasma, and the specific neuroanatomical localization of vascular amyloid strongly suggest specific drainage pathways, rather than local production or blood uptake of Aβ as the primary mechanism underlying CAA. The demonstration in APP/Ld mice of rare vascular amyloid deposits that immunostained only for Aβ42, suggests that, similar to senile plaque formation, Aβ42 may be the first amyloid to be deposited in the vessel walls and that it entraps the more soluble Aβ40. Its ability to diffuse for larger distances along perivascular drainage pathways would also explain the abundance of Aβ40 in vascular amyloid. Consistent with this hypothesis, incorporation of mutant presenilin-1 in APP/Ld mice, which resulted in selectively higher levels of Aβ42, caused an increase in CAA and senile plaques. This mouse model will be useful in further elucidating the pathogenesis of CAA and Alzheimer’s disease, and will allow testing of diagnostic and therapeutic strategies.
Epidemiological studies have established that the epsilon 4 allele of the
ApoE gene (
ApoE4) constitutes an important risk factor for Alzheimer's disease and might influence the outcome of central ...nervous system injury. The mechanism by which
ApoE4 contributes to the development of neurodegeneration remains unknown. To test one hypothesis or mode of action of
ApoE, we generated transgenic mice that overexpressed human ApoE4 in different cell types in the brain, using four distinct gene promoter constructs. Many transgenic mice expressing ApoE4 in neurons developed motor problems accompanied by muscle wasting, loss of body weight, and premature death. Overexpression of human ApoE4 in neurons resulted in hyperphosphorylation of the microtubule-associated protein tau. In three independent transgenic lines from two different promoter constructs, increased phosphorylation of protein tau was correlated with ApoE4 expression levels. Hyperphosphorylation of protein tau increased with age. In the hippocampus, astrogliosis and ubiquitin-positive inclusions were demonstrated. These findings demonstrate that expression of ApoE in neurons results in hyperphosphorylation of protein tau and suggests a role for ApoE in neuronal cytoskeletal stability and metabolism.
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