Synphilin-1 exhibits trophic and protective effects against Rotenone toxicity

Synphilin-1 is a cytoplasmic protein with unclear function. Synphilin-1 has been identified as an interaction partner of α-synuclein. The interaction between synphilin-1 and α-synuclein has implications in Parkinson's disease. In this study, we stably overexpressed human synphilin-1 in mouse N1E-115 neuroblastoma cells. We found that overexpression of synphilin-1 shortened cell growth doubling time and increased neurite outgrowth. Knockdown of endogenous synphilin-1 caused neuronal toxicity and shortened neurite outgrowth. We further found that synphilin-1 increased activation of the extracellular signal-regulated kinases (ERK1/2) and mediated neurite outgrowth. Rotenone, mitochondrial complex I inhibitor, has been shown previously to induce dopaminergic neurodegeneration and Parkinsonism in rats and Drosophila. We found that Rotenone induced apoptotic cell death in N1E-115 cells via caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage. Overexpression of synphilin-1 significantly reduced Rotenone-induced cell death, caspase-3 activation and PARP cleavage. The results indicate that synphilin-1 displays trophic and protective effects in vitro, suggesting that synphilin-1 may play a protective role in Parkinson's disease (PD) pathogenesis and may lead to a potential therapeutic target for PD intervention.     

Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease

Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an alpha-synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.     

NUB1 suppresses the formation of Lewy body-like inclusions by proteasomal degradation of synphilin-1

NUB1 is a potent down-regulator of the ubiquitin-like protein NEDD8, because it targets NEDD8 to the proteasome for proteolytic degradation. From results in this study, we found that NUB1 physically interacts with synphilin-1 through its NEDD8-binding site, implying that NUB1 also targets synphilin-1 to the proteasome for degradation. Synphilin-1 is a major component of inclusion bodies found in the brains of patients with neurodegenerative alpha-synucleinopathies, including Parkinson's disease. In this study, we immunostained sections of brains from patients with Parkinson's disease and other alpha-synucleinopathies and demonstrated that NUB1, as well as synphilin-1, accumulates in the inclusion bodies. To define the role of NUB1 in the formation of these inclusion bodies, we performed a co-transfection assay using cultured HEK293 cells. This assay showed that NUB1 suppresses the formation of synphilin-1-positive inclusions. Further, biochemical assays revealed that NUB1 overexpression leads to the proteasomal degradation of synphilin-1. These results and our previous observations suggest that NUB1 indeed targets synphilin-1 to the proteasome for its efficient degradation, which, because of the resultant reduction in synphilin-1, suppresses the formation of synphilin-1-positive inclusions.     

AIF translocates to the nucleus in the spinal motor neurons in a mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the brain stem and the spinal cords. One of the causes for the familial ALS has been attributed to the mutations in copper-zinc superoxide dismutase (SOD1). Although the toxic function of the mutant enzyme has not been fully understood, the final cell death pathway has been suggested as caspase-dependent. In the present study, we present evidence that the activation of apoptosis inducing factor (AIF) may play a role to induce motor neuron death during ALS pathogenesis. In the spinal cord of SOD1 G93A transgenic mice, expression of AIF was detected in the motor neurons and astrocytes. The level of AIF expression increased as the disease progressed. In the symptomatic SOD1 G93A transgenic mice, AIF released from the mitochondria and translocated into the nucleus in the motor neurons as evidenced by confocal microscopy and biochemical analysis. These results suggest that AIF may play a role to induce motor neuron death in a mouse model of ALS.     

Expression of human apolipoprotein E4 in neurons causes hyperphosphorylation of protein tau in the brains of transgenic mice

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.     

Age-dependent axonal degeneration in an Alzheimer mouse model

Some neurodegenerative diseases including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) exhibit prominent defects in axonal transport. These defects can manifest as axonal swellings or spheroids, which correspond to axonal enlargements and aberrant accumulation of axonal cargoes, cytoskeletal proteins and lipids. Recently, a controversial scientific debate focussed on the issue whether Abeta serves as a trigger for aberrant axonal transport in the pathophysiology of AD. Prominent axonopathy has been shown to be induced by overexpression of proteins involved in several neurodegenerative diseases. Neurofilament, apolipoprotein E, Niemann-Pick protein and Tau transgenic mice with axonal trafficking deficits have been reported. Furthermore, motor deficits are frequently observed in patients with AD, which has been attributed to the typical tauopathy in post-mortem brain tissue. In the present report, we analyzed axonal neuropathology in the brain and spinal cord of a transgenic mouse model with abundant intraneuronal Abeta42 production and provide compelling evidence for axonal degeneration. The APP/PS1ki mice showed characteristic axonal swellings, spheroids, axonal demyelination and ovoids, which are myelin remnants of degenerated nerve fibers in an age-dependent manner. Abundant accumulation of intraneuronal N-modified Abeta, Thioflavin S-positive material and ubiquitin was found within the somatodendritic compartment of neurons. We conclude that the intraneuronal accumulation of Abeta-amyloid peptides is followed by axonal degeneration, and thus might be a causative factor for the axonal changes seen in AD.     

Transgenic Mice in the Study of ALS: The Role of Neurofilaments

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder of multiple etiologies that affects primarily motor neurons in the brain and spinal cord. Abnormal accumulations of neurofilaments (NFs) in motor neurons and a down-regulation of mRNA for the NF light subunit (NF-L) are associated with ALS, but it remains unclear to what extent these NF perturbations contribute to human disease. Transgenic mouse studies demonstrated that overexpression of normal and mutant NF proteins can sometimes provoke a motor neuronopathy characterized by the presence of abnormal NF accumulations resembling those found in ALS. Remarkably, the motor neuronopathy in transgenic mice overexpressing human NF heavy (NF-H) sub-units was rescued by the co- expression of a human NF-L transgene at levels that restored a correct stoichiometry of NF-L to NF-H subunits. Transgenic approaches have also been used to investigate the role of NFs in disease caused by Cu/Zn superoxide dismutase (SOD1) mutations, which is responsible for ˜2% cases of ALS. Studies with transgenic mice expressing low levels of a fusion NF-H/lacZ protein, in which NFs are withheld from the axonal compartment, suggested that axonal NFs are not toxic intermediates required for SOD1 -mediated disease. On the contrary, overexpression of human NF-H proteins was found to confer an effective protection against mutant SOD1 toxicity in transgenic mice, a phenomenon that may be due to the ability of NF proteins to chelate calcium. In conclusion, transgenic studies showed that disorganized NFs can sometimes have noxious effects resulting in neuronopathy. However, in the context of motor neuron disease caused by mutant SOD1, there is emerging evidence that NF proteins rather play a protective role.     

RNA-binding protein is involved in aggregation of light neurofilament protein and is implicated in the pathogenesis of motor neuron degeneration

Abnormal protein aggregation is emerging as a common theme in the pathogenesis of neurodegenerative disease. Our previous studies have shown that overexpression of untranslated light neurofilament (NF-L) RNA causes motor neuron degeneration in transgenic mice, leads to accumulation of ubiquitinated aggregates in degenerating cultured motor neurons and triggers aggregation of NF-L protein and co-aggregation of mutant SOD1 protein in neuronal cells. Here, we report that p190RhoGEF, an RNA-binding protein that binds to a destabilizing element in NF-L mRNA, is involved in aggregation of NF-L protein and is implicated in the pathogenesis of motor neuron degeneration. We show that p190RhoGEF co-aggregates with unassembled NF-L protein and that co-aggregation is associated with down-regulation of parent NF-L mRNA in neuronal cells. Co-expression of NF-M increases NF assembly and reduces RNA-triggered aggregation as well as loss of solubility of NF-L protein. siRNA-induced down-regulation of p190RhoGEF not only reduces aggregation and promotes assembly of NF-L and NF-M, but also causes reversal of aggregation and recovery of NF assembly in transfected cells. Examination of transgenic models of motor neuron disease shows that prominent aggregates of p190RhoGEF and NF-L and down-regulation of NF-L expression occur in degenerating motor neurons of mice expressing untranslated NF-L RNA or a G93A mutant SOD1 transgene. Moreover, aggregates of p190RhoGEF and NF-L appear as early pathological changes in presymptomatic G93A mutant SOD1 transgenic mice. Together, the findings indicate that p190RhoGEF is involved in aggregation of NF-L protein and support a working hypothesis that aggregation of p190RhoGEF and NF-L is an upstream event triggering neurotoxicity in motor neuron disease.     

Ubiquitous expression of human SCA2 gene under the regulation of the SCA2 self promoter cause specific Purkinje cell degeneration in transgenic mice

The objective of this work was the generation of an animal model of the SCA2 disease for future studies on the benefits of therapeutic molecules and neuropathological mechanisms that underline this human disorder. The transgenic fragment was microinjected into pronuclei of B6D2F1 X OF1 mouse hybrid strain. For Northern blots, RNAs were hybridized with a human cDNA fragment from the SCA2 gene and a mouse beta-actin cDNA fragment. Monoclonal antibody directed to the N-terminal of the ataxin 2 protein with 22Q was used for Western blot analysis. A rotating rod apparatus was utilized to measure motor coordination of mice. Immunohistochemical detection of Purkinje neurons was performed with anti-calbindin 28K as primary antibody. Ubiquitous expression of the SCA2 transgene with 75 CAG repeats regulated by the SCA2 self promoter was obtained after generation of our transgenic mice. Analysis of transgenic mice revealed significant differences of motor coordination compared with the wild type littermates. Specific degeneration of Purkinje neurons and transgene over-expression in the brain, liver and skeletal muscle, rather than in lungs and kidneys was also observed, resembling the expression pattern of the ataxin 2 in humans.     

Changes in human regional cerebral blood flow following hypertonic saline induced experimental muscle pain: a positron emission tomography study

Synphilin-1 interacts with alpha-synuclein, which has been implicated in the pathogenesis of Parkinson's disease (PD). By examination of their interactions quantitatively, with the use of the yeast two-hybrid beta-galactosidase assay, we find that the synuclein amino acid (aa) 1-65 region is sufficient for an interaction. A central domain of synphilin-1, aa 349-555, is both necessary and sufficient for an interaction with alpha-synuclein. We did not observe an effect of the synuclein A53T mutation, which causes one familial form of PD, on interactions with synphilin-1. However, the A30P mutation caused an increase in the interaction between the synuclein aa 1-65 fragment and the synphilin-1 central domain.     

Calcyclin binding protein and Siah-1 interacting protein in Alzheimer's disease pathology: neuronal localization and possible function

The calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein was shown to play a role in the organization of microtubules. In this work we have examined the neuronal distribution and possible function of CacyBP/SIP in cytoskeletal pathophysiology. We have used brain tissue from Alzheimer's disease (AD) patients and from transgenic mice modeling 2 different pathologies characteristic for AD: amyloid and tau. In the brain from AD patients, CacyBP/SIP was found to be almost exclusively present in neuronal somata, and in control patients it was seen in the somata and neuronal processes. In mice doubly transgenic for amyloid precursor protein and presenilin 1 there was no difference in CacyBP/SIP neuronal localization in comparison with the nontransgenic animals. By contrast in tau transgenic mice, localization of CacyBP/SIP was similar to that observed for AD patients. To find the relation between CacyBP/SIP and tau we examined dephosphorylation of tau by CacyBP/SIP. We found that indeed it exhibited phosphatase activity toward tau. Altogether, our results suggest that CacyBP/SIP might play a role in AD pathology.     

Prominent axonopathy and disruption of axonal transport in transgenic mice expressing human apolipoprotein E4 in neurons of brain and spinal cord

The epsilon 4 allele of the human apolipoprotein E gene (ApoE4) constitutes an important genetic risk factor for Alzheimer's disease. Recent experimental evidence suggests that human ApoE is expressed in neurons, in addition to being synthesized in glial cells. Moreover, brain regions in which neurons express ApoE seem to be most vulnerable to neurofibrillary pathology. The hypothesis that the expression pattern of human ApoE might be important for the pathogenesis of Alzheimer's disease was tested by generating transgenic mice that express human ApoE4 in neurons or in astrocytes of the central nervous system. Transgenic mice expressing human ApoE4 in neurons developed axonal degeneration and gliosis in brain and in spinal cord, resulting in reduced sensorimotor capacities. In these mice, axonal dilatations with accumulation of synaptophysin, neurofilaments, mitochondria, and vesicles were documented, suggesting impairment of axonal transport. In contrast, transgenic mice expressing human ApoE4 in astrocytes remained normal throughout life. These results suggest that expression of human ApoE in neurons of the central nervous system could contribute to impaired axonal transport and axonal degeneration. The possible contribution of hyperphosphorylation of protein Tau to the resulting phenotype is discussed.     

Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3′ untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.     

Creatine-supplemented diet extends Purkinje cell survival in spinocerebellar ataxia type 1 transgenic mice but does not prevent the ataxic phenotype

It is not known why expression of a protein with an expanded polyglutamine region is pathogenic in spinocerebellar ataxia, Huntington's disease and several other neurodegenerative diseases. Dietary supplementation with creatine improves survival and motor performance and delays neuronal atrophy in the R6/2 transgenic mouse model of Huntington's disease. These effects may be due to improved energy and calcium homeostasis, enhanced presynaptic glutamate uptake, or protection of mitochondria from the mitochondrial permeability transition. We tested the effects of a 2% creatine-supplemented diet and treatment with taurine-conjugated ursodeoxycholic acid, a bile constituent that can inhibit the mitochondrial permeability transition, on ataxia and Purkinje cell survival in a transgenic model of spinocerebellar ataxia type 1. After 24 weeks, transgenic mice on the 2% creatine diet had cerebellar phosphocreatine levels that were 72.5% of wildtype controls, compared to 26.8% in transgenic mice fed a control diet. The creatine diet resulted in maintenance of Purkinje cell numbers in these transgenic mice at levels comparable to wildtype controls, while transgenic mice fed a control diet lost over 25% of their Purkinje cell population. Nevertheless, the ataxic phenotype was neither improved nor delayed. Repeated s.c. ursodeoxycholic acid injections markedly elevated ursodeoxycholic acid levels in the brain without adverse effects, but provided no improvement in phenotype or cell survival in spinocerebellar ataxia type 1 mice.These results demonstrate that preserving neurons from degeneration is insufficient to prevent a behavioral phenotype in this transgenic model of polyglutamine disease. In addition, we suggest that the means by which creatine mitigates against the neurodegenerative effects of an ataxin-1 protein containing an expanded polyglutamine region is through mechanisms other than stabilization of mitochondrial membranes.     

The expression of PEA-15 (phosphoprotein enriched in astrocytes of 15 kDa) defines subpopulations of astrocytes and neurons throughout the adult mouse brain

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is an abundant phosphoprotein in primary cultures of mouse brain astrocytes. Its capability to interact with members of the apoptotic and mitogen activated protein (MAP) kinase cascades endows PEA-15 with anti-apoptotic and anti-proliferative properties. We analyzed the in vivo cellular sources of PEA-15 in the normal adult mouse brain using a novel polyclonal antibody. Immunohistochemical assays revealed numerous PEA-15-immunoreactive cells throughout the brain of wild-type adult mice while no immunoreactive signal was observed in the brain of PEA-15 -/- mice. Cell morphology and double immunofluorescent staining showed that both astrocytes and neurons could be cellular sources of PEA-15. Closer examination revealed that in a given area only part of the astrocytes expressed the protein. The hippocampus was the most striking example of this heterogeneity, a spatial segregation restricting PEA-15 positive astrocytes to the CA1 and CA3 regions. A PEA-15 immunoreactive signal was also observed in a few cells within the subventricular zone and the rostral migratory stream. In vivo analysis of an eventual PEA-15 regulation in astrocytes was performed using a model of astrogliosis occurring along motor neurons degeneration, the transgenic mouse expressing the mutant G93A human superoxyde-dismutase-1, a model of amyotrophic lateral sclerosis. We observed a marked up-regulation of PEA-15 in reactive astrocytes that had developed throughout the ventral horn of the lumbar spinal cord of the transgenic mice. The heterogeneous cellular expression of the protein and its increased expression in pathological situations, combined with the known properties of PEA-15, suggest that PEA-15 expression is associated with a particular metabolic status of cells challenged with potentially apoptotic and/or proliferative signals.