Primary neuronal culture

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Phosphorylation of tau at Y18, but not tau-fyn binding, is required for tau to modulate NMDA receptor-dependent excitotoxicity in primary neuronal culture

Phosphorylation of tau at Y18, but not tau-fyn binding, is required for tau to modulate NMDA receptor-dependent excitotoxicity in primary neuronal culture

Fig. 1 Glutamate-induced neurotoxicity depends on NMDARs. a-f Neurons from wildtype (WT) mice were exposed to different concentrations of glutamate for 15 min at DIV13. Relative levels of survival were quantified by alamarBlue assay 24 h later. Cultures were treated with vehicle or different antagonists starting 1 h before the glutamate exposure: a non-competitive NMDAR antagonist MK801 (20 μ M), b competitive NMDAR antagonist APV (50 μ M), non-competitive GluN2B-selective NMDAR antagonists c Ro 25 – 6981 (1 μ M) or d ifenprodil (10 μ M), e competitive AMPAR antagonist DNQX (20 μ M), or f sodium channel blocker tetrodotoxin (TTX, 1 μ M). The boxplots represent the distribution of the differences in mean fluorescence between antagonist- vs. vehicle-treated neurons at each dose across independent experiments. The lower and upper ends of the boxes represent the 25th and 75th quartile of each distribution, respectively. The horizontal line in each box represents the median. The ends of the whiskers terminate at the farthest points that are within 1.5 times the inter-quartile range (difference between upper and lower ends of the box). Individual dots shown in some of the panels represent outliers that fell outside the range defined by the whiskers. Consequently, the curve in a indicates that the survival-promoting effects of MK801 became more and more evident as glutamate concentrations increased, whereas the bell-shaped curves in b – d indicate that, at the particular concentrations used, the respective antagonists were protective at moderate, but not higher, concentrations of glutamate. Numbers of independent experiments (n) with cumulative well numbers per condition in parentheses: a 4 (24 – 32), b 10 (76 – 80), c 9 (68 – 72), d 8 (56 – 64), e 9 (70 – 72), and f 4 (30 – 32). When comparing mean differences across all doses within any given panel, a one-sided, one-sample t-test revealed significant differences between experimental and control conditions in (a, P < 0.05), (b, P < 0.0001), (c, P < 0.001), and (d, P < 0.001). g, h Neuronal cultures were treated with glutamate at DIV13 and fixed 24 h later, followed by immunostaining for the neuronal marker NeuN and the astroglial marker glutamine synthetase and nuclear staining with Hoechst33342.
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Injury induced plasticity in primary neuronal culture and the mature brain

Injury induced plasticity in primary neuronal culture and the mature brain

Paclitaxel, one of the taxanes, has been used in various animal models of nervous system trauma, and has been shown to promote axonal elongation and regeneration, reduce glial scar formation, and improve outcomes after nerve injury (Hellal et al., 2011, Sengottuvel et al., 2011). Despite these encouraging findings, Paclitaxel is not an ideal drug for the treatment of TBI due to poor blood–brain barrier (BBB) permeability, which prevents access of the drug to the CNS. However Epothilone D (Epo D), a microtubule-stabilizing drug that competes with Paclitaxel for the binding site on β-tubulin, (Brunden et al., 2011) may be an excellent alternative. Epo D readily crosses the BBB and is retained within the CNS for several days (Wang et al., 2005b, Andrieux et al., 2006, Cortes and Baselga, 2007, Brunden et al., 2012). It has been administered to mice carrying genetic mutations associated with familial Alzheimer’s disease and other related tauopathies, and was shown to compensate for the loss of tau function, improve axonal transport, reduce axonal dystrophy, decrease tau neuropathology, reduce neuronal loss and improve cognitive performance (Brunden et al., 2010, Barten et al., 2012, Lou et al., 2014). The beneficial effects of Epo D observed in these studies, coupled with the vital BBB penetration, suggest that this drug holds considerable promise as a potential therapeutic strategy for the treatment of TBI.
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Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium : a methodological approach to improve target protein quantification

Distortion of protein analysis in primary neuronal cultures by serum albumin from culture medium : a methodological approach to improve target protein quantification

Fig. 1. Bar charts of total protein extracted (mg/ml) from polystyrene-based plastic cell culture plates as measured by Bradford assay. N= 3 for each condition. (A) Total protein yield from samples extracted from plates incubated in the absence of cells, under the same media conditions utilised for primary neuronal culture (i.e. B27 supplemented Neurobasal medium with the initial addition of DMEM 10% serum medium at 0DIV). (B) Total protein yield from samples extracted from plates incubated in the absence of cells with a BSA/PBS solution mimicking the total albumin content of a typical incubation with culture medium. All plate were incubated for 14DIV, and wash steps were varied in terms of volume of PBS (µl), number of washes, and length of incubation. Data are presented as mean ± SEM.
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Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

Injections were performed in a laminar flow hood equipped with a microscope to ensure aseptic condi- tions. The pipette was loaded using Eppendorf microloa- der pipette tips (ThermoFisher), inserted into the holder, and the tip was cut with a pair of fine scissors under vis- ual guidance. Pressure pulse was applied to test whether the PFF suspension was expelled from the tip, appearing as a small droplet at the tip of the needle. Correct injec- tion was confirmed by temporary lifting of the surface of the tissue at the injection site. After injection, the needle was left in place for 20 s and then slowly removed. The volume injected in the slices was estimated by counting the number of shots with the adjusted Pulse Pal set up (10–12 shots/slice, 10 nL/shot, 0.1 μg/slice). The final volume was injected at the DG at two to three injection sites depending on the slice thickness at the site of nee- dle insertion. It is important to pay attention to tissue architecture under microscopic guidance during the in- jection procedure to avoid injection of a large volume at a single site, which will cause rupture of the tissue and release of the PFFs to the surface of the OHSC. The ses- sion lasted approximately 6 to 8 min for an insert hold- ing four slices. The final volume injected in each slice was about 0.1 μL of either WT PFFs (1 mg/mL), S129A PFFs (1 mg/mL), monomeric α-syn (1 mg/mL), or PBS. After injecting all slices on a culture insert, the medium was replaced with fresh, pre-heated medium.
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Scaffolds for 3D in vitro culture of neural lineage cells

Scaffolds for 3D in vitro culture of neural lineage cells

The ability to control and direct the differentiation of human pluripotent stem cells has huge potential impact for disease modelling and treatment strategies. As different neural cell types vary in their susceptibility to specific neurological diseases, patient-derived hiPSCs, and their derivative neuronal and glial lineage cells, may hold the key to accurately modelling an environment where these diseases can be studied in greater context and drug screening can identify effective therapeutics. This, coupled with the development of different types of scaffold materials in which to culture iPSC-derived neural precursor cells in a 3D format, represents a powerful technology platform for developing human neural tissue models, disease modelling and screening for new therapeutics. The majority of examples in this review explore the use of hPSC-derived NPCs and their progeny cultured in 3D formats.
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Long term culture of pluripotent stem cell derived human neurons on diamonds" A substrate for neurodegeneration research and therapy

Long term culture of pluripotent stem cell derived human neurons on diamonds" A substrate for neurodegeneration research and therapy

In the course of the neuronal differentiation protocol we observed that the polyornithine/laminin coating of the substrate was effective in maintaining cellular attachment to the substrate in the fi rst stages of the protocol, when cells were passaged relatively frequently. However, during fi nal neuronal differentiation it became dif fi cult to maintain the monolayer attached to diamond long term (over 30 days). This problem was solved by supple- menting the media with laminin (2 m g ml 1 ) once a week (Fig. 4b). Furthermore, N2B27-R medium has a formulation very similar to the original N2B27 developed over 10 years ago for the neural differentiation of mouse ES cells [36]. Since mouse neural pro- genitors are highly proliferating cells, able to undergo terminal neuronal differentiation in just two weeks, we inferred that this particular formulation might not be ideally suited for long-term maintenance of human neurons. For this reason we developed a related medium (N2B27-L) with reduced levels of insulin, non- essential amino acids and glutamine. Two weeks after passage onto the diamond substrate the media was changed from N2B27-R to N2B27-L (Fig. 4a). This media modi fi cation allowed us to consistently culture neuronal cells on diamond past 200 days of differentiation.
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Behavioural and neuronal correlates of sensory prioritization in the rat whisker system

Behavioural and neuronal correlates of sensory prioritization in the rat whisker system

attended condition. Admittedly, the paradigm was characterized by long-durations of prioritizing to a specific modality (minutes). Nevertheless, we did not observe an increase in spontaneous firing rate during the cue-stimulus period in the current experiment. Instead, at the onset of the auditory cue, we observed a decrease in activity in the somatosensory cortex, followed by a strong increase at the onset of the vibration stimulus. Temporal cueing was captured only by changes in firing rate that were specific to stimulus driven activity. A possible interpretation is that suppression plays an important role in the neural mechanism underlying temporal attention in the sensory cortex. Suppressing neuronal activity during the cue period can prevent premature responding and effectively improves the signal to noise ratio to the stimulus. Alternatively, previous research in multisensory interaction has shown that focal cortical activation can inhibit neuronal activity of neigbouring cortical sensory networks (Han et al., 2009; Shu et al., 2003). For example, in vivo, focal photostimulation in monkey neocortex is immediately followed by firing suppression in neighbour units (Han et al., 2009). Specifically on the interaction of auditory and somatosensory cortex, past research has demonstrated acoustic stimulation can cause widespread and near synchronous hyperpolarization in non-auditory related cortical areas. For example, an acoustic noise burst stimulus can cause hyperpolarization in layer 2/3 pyramidal neurons in the somatosensory cortex (peak amplitude: 5.2 +- 0.3mV; onset latency: 31.3 +- 2.2ms)(Iurilli et al., 2012). However, this explanation does not account for the increase in discrimination performance observed in our study. Whilst stimulus driven or focally driven excitation in the auditory cortex may lead to a decrease in activity in the somatosensory cortex, no studies have shown that this would lead to increases in neuronal and behavioral performance. Alternatively, the decrease in neuronal activity during the cue period and the increases in
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Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

The Gal4/UAS system [20] allows us to genetically iden- tify and manipulate particular neuronal lineages in the developing brain. Gal4 driven membrane tethered mCD8::GFP is used to visualize morphology and arbori- zations of neuronal cells to describe neuronal circuits. We tested whether the GAL4/UAS system can be applied to identify and trace particular neuronal lineages or neuron subtypes in primary cell culture. We used three specific Gal4 driver lines to induce mCD8::GFP in dis- tinct neuronal lineages in the larval brain. pdf-Gal4 is specifically expressed in the eight main pace maker neu- rons of the clock circuit [21,22] (Figure 2A). GH146-Gal4 is expressed in projection neurons of the antennal lobe and the developing optic ganglia [23,24] (Figure 2C) and MB247-Gal4 is expressed in the Kenyon cells of the mushroom bodies [25-27] (Figure 2E). As expected in primary cell cultures derived from pdf-Gal4/UAS-mCD8:: GFP animals, we found a small number of GFP positive cells (Figure 2B). Since only eight cells per brain are GFP- positive, this finding shows that even rare cell types can be studied in primary cell culture. In cultures from GH146-Gal4/UAS-mCD8::GFP and MB247-Gal4/UAS- m CD8 :: GFP animals, we found GFP expressing cells (Figure 2D, F) and neurite extensions can be observed in differentiating neurons (Figure 2D).
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MSK1 regulates transcriptional induction of Arc/Arg3 1 in response to neurotrophins

MSK1 regulates transcriptional induction of Arc/Arg3 1 in response to neurotrophins

The synaptic activity-responsive element also con- tained binding sites for the serum response complex and MEF2, and this MEF2 – CREB – SRE motif has subsequently been identified in other activity-regulated genes [61]. The identification of SREs in Arc/Arg3.1 promoter has been reported previously [57,62]. These studies, which were also performed in primary neu- ronal cultures, have shown that in addition to the proximal SRE shown in Fig. 4, at least two more dis- tal SREs exist in the Arc/Arg3.1 promoter [57,62]. In particular, a distal SRE 6.5 kb upstream of Arc/ Arg3.1 has been identified as being important to the maximal induction of Arc/Arg3.1 by BDNF [62]. Knockout of the serum response factor has also been found to reduce Arc/Arg3.1 induction following mGlu5 stimulation of striatal neurons or in the dentate gyrus following electroconvulsive shock [48,49]. In the A
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Defining the Role of Mechanical Signals During Nerve Root Compression in the Development of Sustained Pain and Neurophysiological Correlates that Develop in the Injured Tissue and Spinal Cord

Defining the Role of Mechanical Signals During Nerve Root Compression in the Development of Sustained Pain and Neurophysiological Correlates that Develop in the Injured Tissue and Spinal Cord

Hains et al. 2003, Pitcher et al. 2004, Shim et al. 2005). Spinal excitability also mediates behavioral responses in nerve root injury (Ramer et al. 2000, Terashima et al. 2011). Specifically, rodent models of nerve root trauma demonstrate that loss of sensation in the rat forepaw after crush injury of multiple cervical nerve roots is associated with reduced afferent activity in the spinal cord, while enhanced behavioral sensitivity is associated with an increase in the amplitude of excitatory post-synaptic currents (EPSCs) after a lumbar root constriction (Ramer et al. 2000, Terashima et al. 2011). EPSCs were only evaluated in lamina II of the superficial dorsal horn (Terashima et al. 2011); but, the amplitude increase does suggest that post-synaptic neurons in the spinal cord are more likely to propagate an action potential in response to excitatory input, thereby increasing the firing rate of second order neurons in the spinal cord (Cata et al. 2009, Inquimbert et al. 2012, Kuner 2009, Nguyen et al. 2009). However, it has not been determined whether mechanical trauma to the nerve root increases the frequency of evoked action potentials of dorsal horn neurons nor has it been established whether such responses occur in the superficial laminae, where the primary nociceptors synapse, and/or the deep laminae, where many low-threshold mechanoreceptors synapse to polysynaptic neurons that also respond to noxious stimuli (Basbaum et al. 2009, Todd 2010).
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Lifespan development: a social-cultural perspective

Lifespan development: a social-cultural perspective

variables, only on the supervariable) and also due to findings that Chinese individuals are more sensitive to contextual relationships and tend to process information holistically. Yoon et al. (2000) reasoned that, as there is a cultural bias for holistic processing that remains well-preserved across the lifespan among the Chinese, it is reasonable to expect smaller decrements in performance, compared with the Americans on memory tasks associated with this type of processing. Yoon et al. (2000) also used a composite variable for memory and results were consistent with Levy and Langer (1994); namely, significant effects for age and culture, and main effects were qualified by a significant interaction effect. Yoon et al. (2000) then replicated the analysis for each memory test and found that: a) old Chinese Canadian did perform better than old Anglo speakers on only two (immediate and delayed recall) out of four tests (there were no differences in memory for complex figures and abstract design); and b) the young Chinese Canadians outperformed the old Chinese Canadians (in Levy’s results both old and young Chinese performed equally well). Yoon et al. (2000), however, utilized different memory tests, but the measures on aging stereotypes were taken from Levy and Langer (1994). Consistent with Levy and Langer (1994), Chinese individuals hold more positive views of aging than their Canadian counterparts. However, two measures did not achieve
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Canalization of genetic and pharmacological perturbations in developing primary neuronal activity patterns

Canalization of genetic and pharmacological perturbations in developing primary neuronal activity patterns

A number of different mechanisms have been proposed to play a role in canalization. Phenotypes caused by SNPs affecting protein structure are canalized in Drosophila by the HSP90 chaperone protein (Rutherford and Lindquist, 1998). Another general mecha- nism is gene duplication, where redundant paralogs in gene fam- ilies obscure phenotypes (Koonin, 2005). A third mechanism is the robustness conferred by organization of molecular networks, such as gene regulatory networks (Bergman and Siegal, 2003) or protein interaction networks (Ideker and Sharan, 2008). Each of these mechanisms are present in the postsynaptic proteome of mammalian synapses (HSP90 (Collins et al., 2006), paralogs (Emes et al., 2008), molecular networks (Pocklington et al., 2006)) and upregulated prior to the onset of buffering in the stabilization phase in cultured neurons (Valor et al., 2007). Since mutations change the transcriptome of cells and hence their identity, it is possible that there are changes in the populations of cell types in the mixed cultures. Future studies manipulating these mechanisms at speci fi c times in development will be necessary to determine their role in neuronal activity.
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Lifespan development: a social-cultural perspective

Lifespan development: a social-cultural perspective

This thesis explores some of the social factors that may affect individuals as they age. A lifespan developmental perspective is employed in investigating the effects of societal aging stereotypes on will-to-live and risk-taking skills. Results suggest negative aging stereotypes may have deleterious effects on the elderly, but not young individuals in terms of will-to-live, but have no effect on risk-taking abilities. Furthermore, a cross-cultural analysis of Americans and Japanese reveals robust differences in self-concept between countries, which in turn partially mediate the effects of culture and age on control strategies. It appears culture and age may play important roles in determining individuals’ self-concept, motivation, and regulation of behavior.
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Iron accentuated reactive oxygen species release by NADPH oxidase in activated microglia contributes to oxidative stress in vitro

Iron accentuated reactive oxygen species release by NADPH oxidase in activated microglia contributes to oxidative stress in vitro

It is well established that iron deposition, oxidative stress, and activated microglia are present within CNS lesions. This triad is considered a major contributor to neural degeneration among many CNS diseases. The exact interactions between the components of the triad are unknown; however, multiple studies purport iron chelation or antioxidant therapies can reduce CNS in- flammation leading to improved functional outcomes, reduced lesion volumes, and altered neuronal morph- ology after injury [25–27]. Since it is generally accepted that improved neuronal survivability following injury may translate into improved long-term outcomes, it is impera- tive to evaluate the relationship between Fe 2+ , oxidative stress, microglial polarization, and their effect on neuronal survivability. Therefore, we hypothesized that Fe 2+ can ac- centuate a ROS-producing pro-inflammatory phenotype in activated microglia, including primary cells or micro- glial cell lines, in a NOX2- and/or a NOX4-dependent manner, which subsequently reduces neuronal viability in vitro. This experiment involved both primary microglial cells and the BV2 microglial cell line. While the microglial cell line BV2 has been shown to have some marked differ- ences from primary cell line responses [28], it has noted to be an acceptable alternative or adjunct to primary cells in many experimental lines of investigation [29–31], and we herein demonstrate that it responds similarly to pri- mary cells for all outcomes evaluated.
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Neuronal induction and regional identity by co culture of adherent human embryonic stem cells with chicken notochords and somites

Neuronal induction and regional identity by co culture of adherent human embryonic stem cells with chicken notochords and somites

The neural inductive role for notochord is not far from expecta- tion, since notochord secrets Shh and BMPs antagonists such as noggin, chordin, follistatin, and flik in the chicken among other vertebrate species (Rolf W. Stottmann, 2006) which have always been involved in neural induction in vivo and in vitro. Moreover, hESC-derived neuronal cells in the N group had a ventral anterior identity with OTX2, IRX3 and PAX6 expression, which suggests that the N influences neuronal specification in both the rostro- caudal and dorso-ventral axes. It has been reported that the competent ectoderm of Xenopus embryos (stage 9, late blastula) when wrapped around the anterior notochord of stage 12.5 ex- pressed higher En2, an early anterior neuroectoderm marker, like OTX2 that expresses in this domain compared to the ectoderm which wrapped posterior notochord (Brivanlou and Harland, 1989; Hemmati-Brivanlou et al., 1990). The creation of distinct classes of neurons within the dorso-ventral axis of the developing neuronal tube depends on differential exposure to BMP, Shh and RA from surrounding tissues and this exposure must be carried out at an appropriate time and duration (Ericson, 2001; Jessell, 2000). The reason for the lack of other specific gene expressions may arise from limited co-culture time and the lack of a suitable time window for the neuronal regional identity effect of secreted molecules during co-culture with hESCs or other unknown mechanisms by which these effects are induced.
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Primary culture and mRNA analysis of human ovarian cells

Primary culture and mRNA analysis of human ovarian cells

Primary human OC cells were isolated from patient ascites; we usually receive 2-4 litres of ascites fluid in sterile vacuum containers. For each flask of cells 20 ml of ascitic fluid was mixed with 20 ml growth medium (MCDB105/M199 supplemented with 10% FBS and 100 units/ml penicillin, 100 µg/ml streptomycin) and plated in T75 flasks (5-10 flasks per sample). Ascitic fluid usually contains a large number of red blood cells, however, they do not interfere with cell plating. Ovarian tumor cell clumps or grape-like clusters will be apparent in the ascitic fluid, which will eventually adhere to the cell culture plate surface. After 3-4 days the supernatant was removed and attached cells were re-fed growth medium. All experiments using primary OC cells were performed at culture passages 1-8.
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Culture and detection of primary cilia in endothelial cell models

Culture and detection of primary cilia in endothelial cell models

Previous studies have used human umbilical vein endothelial cells (HUVECs) to examine primary cilia in the vasculature. Geerts et  al.  [12] demonstrated that HUVECs cultured in  vitro beyond the point of confluence form a ‘cobblestone’ morphology, whereby HUVECs establish cell–cell contacts such as tight junc- tions. This condition is thought to mimic the in  vivo lining of umbilical veins, thus providing an appropri- ate human cell line model [12]. HUVECs have also been employed to demonstrate cilia disassembly in high fluid shear stress of greater than 1.5  Pa [6]. Beyond cilia studies, HUVECs are a common model used to study the interaction of ECs with fluid-induced WSS. Sev- eral studies have shown, using HUVECs, that WSS and WSS gradients affect EC morphology [13], alignment [14, 15] and transcription profile [16]. Furthermore, HUVECs have been used to investigate the mecha- nisms by which high WSS can lead to athero-protective EC states [17].
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The PI3K-Akt-mTOR pathway regulates Aβ oligomer induced neuronal cell cycle events

The PI3K-Akt-mTOR pathway regulates Aβ oligomer induced neuronal cell cycle events

Aβ treatment in N2a cell lines respectively, an increase in mTOR phosphorylation has been reported in the AD brain [22,25]. The discrepancy between increased phos- pho-mTOR levels in our study and reduced phospho- mTOR levels in Aβ treated N2a cells [23] may be due to different preparations of Aβ peptide used (oligomeric Aβ at < 4.0 μg/ml or in 1000 nM concentration-our study; versus fibrillar Aβ at 20 μM or equivalent of 80 μg/ml - [23]). Finally, direct evidence for the participation of the PI3K/Akt/mTOR pathway in the induction of neuronal CCEs upon Aβ oligomer exposure was provided by dem- onstration that inhibition of either any one component of this pathway significantly inhibited BrdU incorporation. Interestingly, earlier reports, using pharmacological and genetic means, have demonstrated that PI3K/Akt does play a crucial role in the proliferation of neural progenitor cells by transducing intracellular signals from multiple mitogens including fibroblast growth factor-2, sonic hedgehog, and insulin-like growth factor [49,50]. In addi- tion, inhibition of mTOR in cancerous tissue has been shown to decrease cell proliferation (reviewed in [51]). In summary, our results demonstrate that purified Aβ oli- gomers, but not Aβ monomers or fibrils, induce neuronal CCEs by altering components of the PI3K/Akt/mTOR pathway (Figure 8C). Further studies are required to examine the detailed molecular mechanisms underlying the various biological effects of Aβ oligomers and relate
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Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis

Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis

RHOA is a member of the Rho GTPase family, intracellular proteins that play an important role in neuronal cell survival and cell death by transducing extracellular signals to the cytoskeleton (Linseman and Loucks, 2008). Expression levels of RHOA are decreased 4.25-fold in the mtSOD1 motor neurons and this was confirmed by qPCR (P = 0.0017) (Supplementary Fig. 1A). The expression of RHOA is negatively regulated by the p110-delta catalytic subunit (p110/PIK3CD) of phosphatidylinositol-3 kinase (PI3K) (Papakonstanti et al., 2007), which is highly expressed in the nervous system, including in spinal cord motor neuron (Eickholt et al., 2007). This subunit is increased ( + 3.95) in mtSOD1 motor neurons and this was confirmed by qPCR (P = 0.0005) (Supplementary Fig. 1B). PI3K is a heterodimeric enzyme that phosphorylates phosphatidylinositol-4,5-P2 (PI[4,5]P2) to phosphatidylinositol-3,4,5-P3 (PI[3,4,5]P3) (Fig. 1A). PI[3,4,5]P3 then recruits protein kinase B (AKT) to the membrane where AKT is activated by phosphorylation (see below). The PIK3CD subunit also negatively regulates PTEN (Papakonstanti et al., 2007), which shows decreased expression in mtSOD1 motor neurons ( 2.07). This differential expression has been confirmed by qPCR (P = 0.0017) (Supplementary Fig. 1C). PTEN (phosphatase and tensin homologue) is a dual phosphatase, which negatively regulates the PI3K-AKT pathway by catalysing the conversion of PI[3,4,5]P3 to PI[4,5]P2. PI[4,5]P2 can then be catalysed to PI[4]P, by the action of inositol polyphosphate 5-phosphatases (IPP) and synaptojanin2, which is a ubiquitiously expressed inositol polyphosphate 5-phosphatase. Synaptojanin2 is decreased ( 3.50) in mtSOD1 motor neurons. PI[4,5]P2 can also be converted by phospholipase C to inositol 1,4,5 triphos- phate and diacylglycerol (Fig. 1B). Diacylglycerol activates protein kinase C epsilon (see below) (Basu and Sivaprasad, 2007), while inositol 1,4,5 triphosphate causes cytosolic Ca 2 + influx. Thus,
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Senescence of human vocal fold fibroblasts in primary culture

Senescence of human vocal fold fibroblasts in primary culture

Primary-culture vocal fold fibroblasts were obtained from the vocal ligament (intermediate and deep layers of the lamina propria) of a larynx excised from a 58-year- old Hispanic female who underwent total laryngectomy due to papillary columnar thyroid carcinoma. The la- ryngeal specimens were examined by an otolaryngolo- gist as well as by a pathologist, and were certified to be free of cancerous tissue, i.e., without any epithelial to mesenchymal transition [11]. The protocols for the pro- curement of the laryngeal specimens and the subsequent experimental procedures were approved by the Institu- tional Review Board of UT Southwestern Medical Cen- ter. Using primary explant techniques as described in Xu et al. the specimens were disinfected with 70% ethanol, washed twice with phosphate-buffered saline (PBS), dissected using phonomicrosurgical instruments and cut into 1- to 2-mm 3 sections [8]. The tissue sections were
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