Leigh J. Hsu

α-Synuclein Promotes Mitochondrial Deficit and Oxidative Stress

Abnormal accumulation of the presynaptic protein α-synuclein has recently been implicated in the pathogenesis of Alzheimer’s and Parkinson’s diseases. Because neurodegeneration in these conditions might be associated with mitochondrial dysfunction and oxidative stress, the effects of α-synuclein were investigated in a hypothalamic neuronal cell line (GT1-7). α-Synuclein overexpression in these cells resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations accompanied by increased levels of free radicals and decreased secretion of gonadotropin-releasing hormone. These alterations were ameliorated by pretreatment with anti-oxidants such as vitamin E. Taken together these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that may result in oxidative stress and, eventually, cell death.

Oxidative stress induces amyloid-like aggregate formation of Nacp/α-synuclein in vitro

The precursor of non-amyloid beta protein component of Alzheimers disease amyloid (NACP/alpha-synuclein), found in Lewy bodies of Parkinsons disease (PD), is a presynaptic protein genetically linked to some familial types PD. Mechanisms of abnormal NACP/alpha-synuclein aggregation in neurodegenerative diseases are unclear. Since oxidative stress might play a role in PD pathogenesis, we investigated the role of iron and peroxide in NACP/alpha-synuclein aggregation. Immunoblot analysis showed that human NACP/alpha-synuclein (but not beta-synuclein) aggregated in the presence of ferric ion and was inhibited by the iron chelator deferoxamine. Ferrous ion was not effective by itself, but it potentially aggregated NACP/alpha-synuclein in the presence of hydrogen peroxide. NACP/ alpha-synuclein aggregates displayed strong thioflavine-S and congo-red reactivity, reminiscent of amyloid. This study suggests that NACP/alpha-synuclein aggregation might be closely related to oxidative reactions which may play a critical role in neurodegeneration in disorders with Lewy bodies.

Human recombinant NACP/α-synuclein is aggregated and fibrillated in vitro: Relevance for Lewy body disease

The precursor of non-amyloid beta protein component of Alzheimers disease amyloid (NACP/alpha-synuclein) is aggregated and fibrillated under certain conditions, i.e., increasing time lag, high temperature and low pH. These in vitro aggregates form Thioflavine-S-positive filamentous structures, reminiscent of amyloid-like fibrils. Since some Lewy bodies in Parkinsons disease display Thioflavine-S reactivity, our results may suggest that amyloidogenic properties of NACP/alpha-synuclein may play a crucial role in pathogenesis of disorders with Lewy bodies such as Parkinsons disease.

ROLE OF CYTOCHROME C AS A STIMULATOR OF ALPHA -SYNUCLEIN AGGREGATION IN LEWY BODY DISEASE

α-Synuclein is a major component of aggregates forming amyloid-like fibrils in diseases with Lewy bodies and other neurodegenerative disorders, yet the mechanism by which α-synuclein is intracellularly aggregated during neurodegeneration is poorly understood. Recent studies suggest that oxidative stress reactions might contribute to abnormal aggregation of this molecule. In this context, the main objective of the present study was to determine the potential role of the heme protein cytochrome c in α-synuclein aggregation. When recombinant α-synuclein was coincubated with cytochrome c/hydrogen peroxide, α-synuclein was concomitantly induced to be aggregated. This process was blocked by antioxidant agents such asN-acetyl-l-cysteine. Hemin/hydrogen peroxide similarly induced aggregation of α-synuclein, and both cytochromec/hydrogen peroxide- and hemin/hydrogen peroxide-induced aggregation of α-synuclein was partially inhibited by treatment with iron chelator deferoxisamine. This indicates that iron-catalyzed oxidative reaction mediated by cytochrome c/hydrogen peroxide might be critically involved in promoting α-synuclein aggregation. Furthermore, double labeling studies for cytochromec/α-synuclein showed that they were colocalized in Lewy bodies of patients with Parkinson’s disease. Taken together, these results suggest that cytochrome c, a well known electron transfer, and mediator of apoptotic cell death may be involved in the oxidative stress-induced aggregation of α-synuclein in Parkinson’s disease and related disorders.

α-Synuclein Protects against Oxidative Stress via Inactivation of the c-Jun N-terminal Kinase Stress-signaling Pathway in Neuronal Cells

The expression of α-synuclein, a synaptic molecule implicated in the pathogenesis of neurodegenerative disorders such as Parkinsons disease and Lewy body disease is increased upon injury to the nervous system, indicating that it might play a role in regeneration and plasticity; however, the mechanisms are unclear. Because c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, plays an important role in stress response, the main objective of the present study was to better understand the involvement of this pathway in the signaling responses associated with resistance to injury in cells expressing α-synuclein. For this purpose, the JNK-signaling pathway was investigated in α-synuclein-transfected neuronal cell line glucose transporter (GT) 1–7 under oxidative stress conditions. Although hydrogen peroxide challenge resulted in JNK activation and cell death in cells transfected with vector control or β-synuclein, α-synuclein-transfected cells were resistant to hydrogen peroxide, and JNK was not activated. The inactivation of JNK in the α-synuclein-transfected cells was associated with increased expression and activity of JNK-interacting protein (JIP)-1b/islet-brain (IB)1, the scaffold protein for the JNK pathway. Similarly, cells transfected with JIP-1b/IB1 were resistant to hydrogen peroxide associated with inactivation of the JNK pathway. In these cells, expression of endogenous α-synuclein was significantly increased at the protein level. Furthermore, α-synuclein was co-localized with JIP-1b/IB1 in the growth cones. Taken together, these results suggest that increased α-synuclein expression might protect cells from oxidative stress by inactivation of JNK via increased expression of JIP-1b/IB1. Furthermore, interactions between α-synuclein and JIP-1b/IB1 may play a mutual role in the neuronal response to injury and neurodegeneration.

Expression Pattern of Synucleins (Non‐Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α‐Synuclein) During Murine Brain Development

Abstract: The non‐Aβ component of Alzheimers disease amyloid precursor protein (NACP) is predominantly a neuron‐specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimers disease amyloid and a mutation in the NACP gene is associated with familial Parkinsons disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.

Reduced Neuritic Outgrowth and Cell Adhesion in Neuronal Cells Transfected with Human α-Synuclein

Since recent reports have suggested that alpha-synuclein might play a role in neuronal plasticity, the main objective of this study was to determine the effects of alpha-synuclein on neuritic outgrowth. We stably transfected either human (h) alpha- or beta-synuclein cDNA in B103 rat neuronal cells. Expression of h(alpha)-synuclein resulted in reduced neurite extension and weak adhesion compared to vector-transfected and h(beta)-synuclein expressing cells. To investigate the potential pathways involved, we studied the effects of reagents known to modulate B103 proliferation and differentiation. Neither phorbol 12-myristate 13-acetate nor forskolin or antioxidants (catalase, superoxide dismutase, or vitamin E) were able to restore the reduced length of neurites in h(alpha)-synuclein-expressing cells. These results suggest that reduced neuritic activity in the h(alpha)-synuclein-expressing cells might be due, in part, to alterations in cell adhesion capacity. This might be attributed to alpha-synuclein affecting a signal transduction pathway distinct from protein kinase C and protein kinase A.

In vitro synaptotrophic effects of Cerebrolysin in NT2N cells

Abstract Recent studies have shown that Cerebrolysin can enhance synaptic function and ameliorate synapto-dendritic alterations in animal models of neurodegeneration, suggesting a synaptotrophic effect. We hypothesize that Cerebrolysin might exert this effect, in part, by regulating the expression of amyloid precursor protein (APP). We studied the patterns of expression of synaptic proteins during differentiation of human teratocarcinoma cell line NTera 2 (NT2) in the presence or absence of Cerebrolysin. This study showed that the terminally differentiated neurons (NT2N) expressed a wide variety of synaptic markers and that expression of these synaptic-associated proteins coincided with the shift in expression from APP770/751 to APP695. Furthermore, APP immunoreactivity was colocalized with synaptophysin-immunoreactive neuritic varicosities in NT2N neurites, and Cerebrolysin treatment of NT2N cells resulted in an augmented and earlier expression of synaptic-associated proteins. This increased synaptic protein expression coincided with an increase in APP695 over APP770/751. These results support the possibility that synaptotrophic effects of Cerebrolysin might be mediated via regulation of APP expression.

Altered expression of glutamate transporters under hypoxic conditions in vitro

Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1–7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO2, 95% N2) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1–7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D‐aspartate uptake. Finally, although the protein kinase C stimulator phorbol‐12‐myristate‐13‐acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C‐dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction. J. Neurosci. Res. 64:193–202, 2001.