Lycopene protects against trimethyltin-induced neurotoxicity in primary cultured rat hippocampal neurons by inhibiting the mitochondrial apoptotic pathway

Lycopene is a potent free radicals scavenger with demonstrated protective efficacy in several experimental models of oxidative damage. Trimethyltin (TMT) is an organotin compound with neurotoxic effects on the hippocampus and other limbic structures and is used to model neurodegenerative diseases targeting these brain areas. Oxidative stress is widely accepted as a central pathogenic mechanism of TMT-mediated neurotoxicity. The present study investigated whether the plant carotene lycopene protects against TMT-induced neurotoxicity in primary cultured rat hippocampal neurons. Lycopene pretreatment improved cell viability in TMT-treated hippocampal neurons and inhibited neuronal apoptosis. Microfluorometric imaging revealed that lycopene inhibited the accumulation of mitochondria-derived reactive oxygen species (ROS) during TMT exposure. Moreover, lycopene ameliorated TMT-induced activation of the mitochondrial permeability transition pore (mPTP) and the concomitant depolarization of the mitochondrial membrane potential (ΔΨ_m). Consequently, cytochrome c release from the mitochondria and ensuing caspase-3 activation were markedly reduced. These findings reveal that lycopene protects against TMT-induced neurotoxicity by inhibiting the mitochondrial apoptotic pathway. The anti-apoptotic effect of lycopene on hippocampal neurons highlights the therapeutic potential of plant-derived antioxidants against neurodegenerative diseases.     

Mice deficient in Epg5 exhibit selective neuronal vulnerability to degeneration

The molecular mechanism underlying the selective vulnerability of certain neuronal populations associated with neurodegenerative diseases remains poorly understood. Basal autophagy is important for maintaining axonal homeostasis and preventing neurodegeneration. In this paper, we demonstrate that mice deficient in the metazoan-specific autophagy gene Epg5/epg-5 exhibit selective damage of cortical layer 5 pyramidal neurons and spinal cord motor neurons. Pathologically, Epg5 knockout mice suffered muscle denervation, myofiber atrophy, late-onset progressive hindquarter paralysis, and dramatically reduced survival, recapitulating key features of amyotrophic lateral sclerosis (ALS). Epg5 deficiency impaired autophagic flux by blocking the maturation of autophagosomes into degradative autolysosomes, leading to accumulation of p62 aggregates and ubiquitin-positive inclusions in neurons and glial cells. Epg5 knockdown also impaired endocytic trafficking. Our study establishes Epg5-deficient mice as a model for investigating the pathogenesis of ALS and indicates that dysfunction of the autophagic–endolysosomal system causes selective damage of neurons associated with neurodegenerative diseases.     

Novel compounds for the modulation of mTOR and autophagy to treat neurodegenerative diseases

Most neurodegenerative diseases show a disruption of autophagic function and display abnormal accumulation of toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway, which promotes cellular survival, is the main modulator upstream of mTOR, and alterations in this pathway are common in neurodegenerative diseases, e.g. Alzheimer’s disease (AD) and Parkinson’s disease (PD). In the present work we revised mammalian target of rapamycin complex 1 (mTORC1) pathway and mTORC2 as a complementary an important element in mTORC1 signaling. In addition, we revised the extracellular signal regulated kinase (ERK) pathway, which has become relevant in the regulation of the autophagic process and cellular survival through mTORC2 signaling. Finally, we summarize novel compounds that promote autophagy and neuronal protection in the last five years.     

Metabolites of Cerebellar Neurons and Hippocampal Neurons Play Opposite Roles in Pathogenesis of Alzheimer's Disease

Metabolites of neural cells, is known to have a significant effect on the normal physiology and function of neurons in brain. However, whether they play a role in pathogenesis of neurodegenerative diseases is unknown. Here, we show that metabolites of neurons play essential role in the pathogenesis of Alzheimer''s disease (AD). Firstly, in vivo and in vitro metabolites of cerebellar neurons both significantly induced the expression of Aβ-degrading enzymes in the hippocampus and cerebral cortex and promoted Aβ clearance. Moreover, metabolites of cerebellar neurons significantly reduced brain Aβ levels and reversed cognitive impairments and other AD-like phenotypes of APP/PS1 transgenic mice, in both early and late stages of AD pathology. On the other hand, metabolites of hippocampal neurons reduced the expression of Aβ-degrading enzymes in the cerebellum and caused cerebellar neurodegeneration in APP/PS1 transgenic mice. Thus, we report, for the first time, that metabolites of neurons not only are required for maintaining the normal physiology of neurons but also play essential role in the pathogenesis of AD and may be responsible for the regional-specificity of Aβ deposition and AD pathology.     

Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons

The Wnt signaling pathway plays an important role in developmental processes, including embryonic patterning, cell specification, and cell polarity. Wnt components participate in the development of the central nervous system, and growing evidence indicates that this pathway also regulates the function of the adult nervous system. In this study, we report that Wnt-5a, a noncanonical Wnt ligand, is a potent activator of mitochondrial dynamics and induces acute fission and fusion events in the mitochondria of rat hippocampal neurons. The effect of Wnt-5a was inhibited in the presence of sFRP, a Wnt scavenger. Similarly, the canonical Wnt-3a ligand had no effect on mitochondrial fission-fusion events, suggesting that this effect is specific for Wnt-5a alone. We also show that the Wnt-5a effects on mitochondrial dynamics occur with an increase in both intracellular and mitochondrial calcium (Ca²⁺), which was correlated with an increased phosphorylation of Drp1(Ser-616) and a decrease of Ser-637 phosphorylation, both indicators of mitochondrial dynamics. Electron microscope analysis of hippocampal tissues in the CA1 region showed an increase in the number of mitochondria present in the postsynaptic region, and this finding correlated with a change in mitochondrial morphology. We conclude that Wnt-5a/Ca²⁺ signaling regulates the mitochondrial fission-fusion process in hippocampal neurons, a feature that might help to further understand the role of Wnt-related pathologies, including neurodegenerative diseases associated with mitochondrial dysfunction, and represents a potentially important link between impaired metabolic function and degenerative disorders.     

A brown alga Sargassum fulvellum facilitates neuronal maturation and synaptogenesis

Sargassum fulvellum (Turner) C. Agardh is an edible brown macroalgae having pharmacological importance. In previous reports, we described the screening of marine algae for their neuritogenic activity in developing hippocampal neurons and found that ethanol extract of S. fulvellum (SFE) possesses promising neurite-outgrowth-promoting activity. In this study, we evaluated whether the initial neurite promoting effect of SFE was followed on the further neuronal maturation and synapse formation. SFE exhibited dose-dependent effect on neurite maturation with an optimum concentration of 5???g/mL. The initial neuronal differentiation is significantly promoted by SFE. Subsequently, compared with control culture, SFE increased the indices of axonal and dendritic developments such as the number and the length of primary processes, and branching frequencies. In addition to its effect on neurite development, SFE significantly increased the number of puncta for postsynaptic density-95, synaptic vesicle 2, and synapse (about 35%, 67%, and 125%, respectively, of control). Moreover, SFE dose-dependently protects neurons from naturally occurring death in normal culture condition. Taken together, our data demonstrate that SFE can promote neuronal maturation and synaptogenesis and support neuronal survival, suggesting the beneficial effect of this alga in nervous system.     

Long-Term Potentiation Promotes Proliferation/Survival and Neuronal Differentiation of Neural Stem/Progenitor Cells

Neural stem cell (NSC) replacement therapy is considered a promising cell replacement therapy for various neurodegenerative diseases. However, the low rate of NSC survival and neurogenesis currently limits its clinical potential. Here, we examined if hippocampal long-term potentiation (LTP), one of the most well characterized forms of synaptic plasticity, promotes neurogenesis by facilitating proliferation/survival and neuronal differentiation of NSCs. We found that the induction of hippocampal LTP significantly facilitates proliferation/survival and neuronal differentiation of both endogenous neural progenitor cells (NPCs) and exogenously transplanted NSCs in the hippocampus in rats. These effects were eliminated by preventing LTP induction by pharmacological blockade of the N-methyl-D-aspartate glutamate receptor (NMDAR) via systemic application of the receptor antagonist, 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP). Moreover, using a NPC-neuron co-culture system, we were able to demonstrate that the LTP-promoted NPC neurogenesis is at least in part mediated by a LTP-increased neuronal release of brain-derived neurotrophic factor (BDNF) and its consequent activation of tropomysosin receptor kinase B (TrkB) receptors on NSCs. Our results indicate that LTP promotes the neurogenesis of both endogenous and exogenously transplanted NSCs in the brain. The study suggests that pre-conditioning of the host brain receiving area with a LTP-inducing deep brain stimulation protocol prior to NSC transplantation may increase the likelihood of success of using NSC transplantation as an effective cell therapy for various neurodegenerative diseases.     

Commentary: ZFYVE26/SPASTIZIN: A close link between complicated hereditary spastic paraparesis and autophagy

Defective autophagy is associated with neurodegenerative disorders including Alzheimer, Parkinson and Huntington diseases, amyotrophic lateral sclerosis and SCA (spinocerebellar ataxias). Autophagy defects were detected also in SPG49, a complicated form of hereditary spastic paraparesis (cHSP) associated with mutations in the TECPR2 gene, suggesting a role of autophagy also in this heterogeneous group of neurodegenerative diseases. We recently found defective autophagy in SPG15, another HSP subtype associated with mutations in the ZFYVE26/SPG15 gene. Patient-derived cells (fibroblasts/lymphoblasts) carrying different ZFYVE26 mutations show accumulation of immature autophagosomes and increased MAP1LC3B-II and SQSTM1/p62 levels. These findings indicate that ZFYVE26 is a key determinant of autophagosome maturation, which is impaired when the protein is defective or absent. Replication of these findings in primary neurons supports the relevance of defective autophagy in SPG15-related neurodegeneration.     

A New Target for Amyloid Beta Toxicity Validated by Standard and High-Throughput Electrophysiology

Background

Soluble oligomers of amyloid beta (Aβ) are considered to be one of the major contributing factors to the development of Alzheimer''s disease. Most therapeutic development studies have focused on toxicity directly at the synapse.

Methodology/Principal Findings

Patch clamp studies detailed here have demonstrated that soluble Aβ can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of the synapse as well, but may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of the temporal electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs) at low concentrations of Aβ (1–42). We have also shown that this functional deficit can be reversed through use of curcumin, an inhibitor of Aβ oligomerization, using both analysis methods.

Conclusions/Significance

The MEA recording method utilized here is non-invasive, thus long term chronic measurements are possible and it does not require precise positioning of electrodes, thus it is ideal for functional screens. Even more significantly, we believe we have now identified a new target for drug development for AD based on functional toxicity of hippocampal neurons that could treat neurodegenerative diseases prior to the development of mild cognitive impairment.     

Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1^S738/742E (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1^K612W (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.     

Zebrafish Tg(hb9:MTS-Kaede): a new in vivo tool for studying the axonal movement of mitochondria

ObjectivesDeregulation of axonal transport in neurons is emerging as the major cause of many neurodegenerative diseases in human, such as Charcot-Marie-Tooth (CMT) neuropathy. However, little is known about how mitochondria move in vivo and whether cell culture systems truly represent what happens in living animals. Here we describe the generation of a new zebrafish transgenic line that specifically allows to study mitochondrial dynamics in motor neurons and its application to analyse mitochondrial movement in zebrafish models expressing CMT2A causing mutations.MethodsThe Tol2 transposon system was used to generate a transgenic zebrafish line expressing the photoconvertible fluorescent protein Kaede in mitochondria of motor neurons. Mitochondrial shape and movement were monitored by time-lapse confocal live imaging and measured by kymograph analysis. The effects of two well-known CMT causing mutations, L76P and R94Q substitutions in MFN2, were then investigated with the same methods.ResultsWe generated the transgenic zebrafish Tg(hb9:MTS-Kaede) line with genetically labelled mitochondria in motor neurons. Kaede protein was correctly and stably targeted to mitochondrial matrix while retaining its photoconvertibility, thus qualifying this model for in vivo studies. Expression of the L76P and R94Q mutations reduced mitochondrial movement in axons and altered mitochondrial distribution in distinct ways.Conclusions and general significanceThese findings confirm previously published data obtained in cell cultures and strengthen the hypothesis of different mechanism of action of the two MFN2 mutations. Considering the number of neurodegenerative diseases associated to mitochondrial dynamics, the Tg(hb9:MTS-Kaede) zebrafish line is a promising model to study in vivo alterations of mitochondrial transport underlying human diseases.     

Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin

Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.     

Dynactin Subunit p150Glued Is a Neuron-Specific Anti-Catastrophe Factor

Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. We have recently shown that the p150^Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150^Glued may also modulate the dynamics of microtubules, we hypothesized that p150^Glued might promote cargo initiation by stabilizing the microtubule track. Here, we demonstrate in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150^Glued is a potent anti-catastrophe factor for microtubules. p150^Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150^Glued are required in tandem for this activity. p150^Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, we find that RNAi of p150^Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150^Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150^Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, we find that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease.     

Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo.     

Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons

Small-conductance Ca²⁺-activated K⁺ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson''s disease, where dopaminergic cell loss is associated with progression of the disease.     

Induced pluripotent stem cells (iPSCs) as game-changing tools in the treatment of neurodegenerative disease: Mirage or reality?

Based on investigations, there exist tight correlations between neurodegenerative diseases' incidence and progression and aberrant protein aggregreferates in nervous tissue. However, the pathology of these diseases is not well known, leading to an inability to find an appropriate therapeutic approach to delay occurrence or slow many neurodegenerative diseases' development. The accessibility of induced pluripotent stem cells (iPSCs) in mimicking the phenotypes of various late-onset neurodegenerative diseases presents a novel strategy for in vitro disease modeling. The iPSCs provide a valuable and well-identified resource to clarify neurodegenerative disease mechanisms, as well as prepare a promising human stem cell platform for drug screening. Undoubtedly, neurodegenerative disease modeling using iPSCs has established innovative opportunities for both mechanistic types of research and recognition of novel disease treatments. Most important, the iPSCs have been considered as a novel autologous cell origin for cell-based therapy of neurodegenerative diseases following differentiation to varied types of neural lineage cells (e.g. GABAergic neurons, dopamine neurons, cortical neurons, and motor neurons). In this review, we summarize iPSC-based disease modeling in neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Moreover, we discuss the efficacy of cell-replacement therapies for neurodegenerative disease.     

Fingolimod Phosphate Attenuates Oligomeric Amyloid β–Induced Neurotoxicity via Increased Brain-Derived Neurotrophic Factor Expression in Neurons

The neurodegenerative processes that underlie Alzheimer''s disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P) receptor (S1PR) agonist fingolimod phosphate (FTY720-P)-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF) in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer''s disease.