Decreased Neurite Outgrowth Research Articles

Dystrophin deficiency leads to dysfunctional glutamate clearance in iPSC derived astrocytes

Duchenne muscular dystrophy (DMD) results, beside muscle degeneration in cognitive defects. As neuronal function is supported by astrocytes, which express dystrophin, we hypothesized that loss of dystrophin from DMD astrocytes might contribute to these cognitive defects. We generated cortical neuronal and astrocytic progeny from induced pluripotent stem cells (PSC) from six DMD subjects carrying different mutations and several unaffected PSC lines. DMD astrocytes displayed cytoskeletal abnormalities, defects in Ca+2 homeostasis and nitric oxide signaling. In addition, defects in glutamate clearance were identified in DMD PSC-derived astrocytes; these deficits were related to a decreased neurite outgrowth and hyperexcitability of neurons derived from healthy PSC. Read-through molecule restored dystrophin expression in DMD PSC-derived astrocytes harboring a premature stop codon mutation, corrected the defective astrocyte glutamate clearance and prevented associated neurotoxicity. We propose a role for dystrophin deficiency in defective astroglial glutamate homeostasis which initiates defects in neuronal development.

Propionic Acid Induces Gliosis and Neuro-inflammation through Modulation of PTEN/AKT Pathway in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by glia over-proliferation, neuro-inflammation, perturbed neural circuitry, and gastrointestinal symptoms. The role of gut dys-biosis in ASD is intriguing and should be elucidated. We investigated the effect of Propionic acid (PPA), a short-chain fatty acid (SCFA) and a product of dys-biotic ASD gut, on human neural stem cells (hNSCs) proliferation, differentiation and inflammation. hNSCs proliferated to 66 neuropsheres when exposed to PPA versus 45 in control. The neurosphere diameter also increased at day 10 post PPA treatment to (Mean: 193.47 um ± SEM: 6.673 um) versus (154.16 um ± 9.95 um) in control, p < 0.001. Pre-treatment with β-HB, SCFA receptor inhibitor, hindered neurosphere expansion (p < 0.001). While hNSCs spontaneously differentiated to (48.38% ± 6.08%) neurons (Tubulin-IIIβ positive) and (46.63% ± 2.5%) glia (GFAP positive), PPA treatment drastically shifted differentiation to 80% GFAP cells (p < 0.05). Following 2 mM PPA exposure, TNF-α transcription increased 4.98 fold and the cytokine increased 3.29 fold compared to control (P < 0.001). Likewise, GPR41 (PPA receptor) and pro-survival p-Akt protein were elevated (p < 0.001). PTEN (Akt inhibitor) level decreased to (0.42 ug/ul ± 0.04 ug/ul) at 2 mM PPA compared to (0.83 ug/ul ± 0.09 ug/ul) in control (p < 0.001). PPA at 2 mM decreased neurite outgrowth to (80.70 um ± 5.5 um) compared to (194.93 um ± 19.7 um) in control. Clearly, the data supports a significant role for PPA in modulating hNSC patterning leading to gliosis, disturbed neuro-circuitry, and inflammatory response as seen in ASD.

Human Apolipoprotein E Isoforms Influence Neurite Outgrowth and Regeneration In Vitro and In Vivo

Translating spinal cord injury (SCI) therapies which promote axonal regeneration from preclinical animal models into the human population is challenging. One potential obstacle is that human genetic predispositions may limit the efficacy of such experimental treatments. The clinically relevant ApoE4 (E4) allele, present in about 14% of the human population, corresponds to an increased incidence of Alzheimer's disease—however, its role in recovery from SCI is poorly understood despite suggestive data implicating its involvement. Indeed, two clinical studies found that SCI individuals with the E4 allele had less motor recovery than individuals without the allele despite longer time in rehabilitation. ApoE4 may mediate this diminished recovery by limiting regeneration and sprouting. Robust regeneration is energy intensive and requires efficient mitochondria, and studies have shown that ApoE4 impairs mitochondrial function. Given these mitochondrial deficits, we hypothesize that ApoE4 can impair regeneration and sprouting. To test this hypothesis, we investigated the impact of ApoE4 on sprouting and neurite outgrowth. In our experiments, we cultured dorsal root ganglia neurons from mice expressing human ApoE isoforms—ApoE2 (E2), ApoE3 (E3), or ApoE4—under the control of the endogenous mouse ApoE promoter. We then analyzed differences in 1) neurite complexity and 2) robustness of outgrowth between genotypes. In two of three independent experiments, E4 neurons had decreased neurite outgrowth and decreased neurite branching compared to E2 and E3 neurons. Data from the Spot Assay, an in vitro model of the glial scar and CNS regeneration, also suggest that chondroitin sulfate proteoglycans may inhibit regeneration in E4 neurons to an even greater extent than in E3 neurons. In addition, with preliminary in vivo data, we are beginning to characterize serotonergic sprouting after lateral C2 hemisection in mice of each ApoE genotype. Since outgrowth, sprouting, and regeneration all partially mediate recovery after CNS injury, impairments in these processes can adversely affect recovery. These foundational studies address not only the possible genetic influence of ApoE4 on recovery from CNS injury, but also a critical gap in knowledge—whether there is a genetic contribution underlying responses to treatment in SCI individuals.Support or Funding InformationUniversity of Kentucky Startup FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Taurine Promotes Neuritic Growth of Dorsal Root Ganglion Cells Exposed to High Glucose in Vitro.

Diabetic neuropathy (DN) is the most common chronic complication of DM and its major pathological changes show axonal dysfunction, atrophy and loss. However, there are few reports that taurine promotes neurite growth of dorsal root ganglion (DRG) cells. In current study, DRG neurons were exposed to high glucose (HG) with or without taurine. The neurite outgrowth of DRG neurons was observed by fluorescent immunohistochemistry method. Expression of Gap-43, Akt, phosphorylated Akt, mTOR and phosphorylated mTOR was determined by Western blot assay. Our results showed that HG significantly decreased the neurite outgrowth and expression of Gap-43 in DRG neurons. Moreover, phosphorylated levels of Akt and mTOR were downregulated in DRG neurons exposed to HG. On the contrary, taurine supplementation significantly reversed the decreased neurite outgrowth and Gap-43 expression, and the downregulated phosphorylated levels of Akt and mTOR. However, the protective effects of taurine were blocked in the presence of PI3K antagonists LY294002 or Akt antagonists Perifosine. These results indicate that taurine promotes neurite outgrowth of DRG neurons exposed to HG via activating Akt/mTOR signal pathway.

Convergence of independent DISC1 mutations on impaired neurite growth via decreased UNC5D expression

The identification of convergent phenotypes in different models of psychiatric illness highlights robust phenotypes that are more likely to be implicated in disease pathophysiology. Here, we utilize human iPSCs harboring distinct mutations in DISC1 that have been found in families with major mental illness. One mutation was engineered to mimic the consequences on DISC1 protein of a balanced translocation linked to mental illness in a Scottish pedigree; the other mutation was identified in an American pedigree with a high incidence of mental illness. Directed differentiation of these iPSCs using NGN2 expression shows rapid conversion to a homogenous population of mature excitatory neurons. Both DISC1 mutations result in reduced DISC1 protein expression, and show subtle effects on certain presynaptic proteins. In addition, RNA sequencing and qPCR showed decreased expression of UNC5D, DPP10, PCDHA6, and ZNF506 in neurons with both DISC1 mutations. Longitudinal analysis of neurite outgrowth revealed decreased neurite outgrowth in neurons with each DISC1 mutation, which was mimicked by UNC5D knockdown and rescued by transient upregulation of endogenous UNC5D. This study shows a narrow range of convergent phenotypes of two mutations found in families with major mental illness, and implicates dysregulated netrin signaling in DISC1 biology.

The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation

BackgroundNeuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive.MethodsThe correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro.ResultsBased on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What’s more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest.ConclusionOur findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Novel synthetic chalcone-coumarin hybrid for Aβ aggregation reduction, antioxidation, and neuroprotection.

Aggregation of misfolded amyloid β (Aβ) in senile plaques causes oxidative stress and neuronal death in Alzheimer's disease (AD). Compounds possessing antiaggregation and antioxidant properties are promising candidate compounds for AD treatment. We examined the potential of synthetic derivatives of licochalcone A and coumarin for inhibiting Aβ aggregation, scavenging reactive oxygen species (ROS), and providing neuroprotection by using biochemical assays and Tet-On Aβ-GFP 293/SH-SY5Y cell models for AD. Among test compounds, LM-031, a novel chalcone-coumarin hybrid, inhibited Aβ aggregation and scavenged free oxygen radicals. LM-031 markedly reduced Aβ misfolding and ROS as well as promoted neurite outgrowth and inhibited acetylcholinesterase in Tet-On Aβ-GFP 293/SH-SY5Y cells. Mechanistic studies showed upregulation of the HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB/BDNF/BCL2 pathway. Decreased neurite outgrowth upon the induction of Aβ-GFP was rescued by LM-031, which was counteracted by knockdown of HSPB1, NRF2, or CREB. Taken together, these findings demonstrate that LM-031 exhibited antiaggregation, antioxidant, and neuroprotective effects against Aβ toxicity by enhancing HSPB1 and the NRF2-related antioxidant pathway as well as by activating the CREB-dependent survival and antiapoptosis pathway. These results imply that LM-031 may be a new therapeutic compound for AD.

Altered development of dopaminergic neurons differentiated from stem cells from human exfoliated deciduous teeth of a patient with Down syndrome

BackgroundDown syndrome (DS) is a common developmental disorder resulting from the presence of an additional copy of chromosome 21. Abnormalities in dopamine signaling are suggested to be involved in cognitive dysfunction, one of the symptoms of DS, but the pathophysiological mechanism has not been fully elucidated at the cellular level. Stem cells from human exfoliated deciduous teeth (SHED) can be prepared from the dental pulp of primary teeth. Importantly, SHED can be collected noninvasively, have multipotency, and differentiate into dopaminergic neurons (DN). Therefore, we examined dopamine signaling in DS at the cellular level by isolating SHED from a patient with DS, differentiating the cells into DN, and examining development and function of DN.MethodsHere, SHED were prepared from a normal participant (Ctrl-SHED) and a patient with DS (DS-SHED). Initial experiments were performed to confirm the morphological, chromosomal, and stem cell characteristics of both SHED populations. Next, Ctrl-SHED and DS-SHED were differentiated into DN and morphological analysis of DN was examined by immunostaining. Functional analysis of DN was performed by measuring extracellular dopamine levels under basal and glutamate-stimulated conditions. In addition, expression of molecules involved in dopamine homeostasis was examined by quantitative real-time polymerase chain reaction and immunostaining. Statistical analysis was performed using two-tailed Student’s t-tests.ResultsCompared with Ctrl-SHED, DS-SHED showed decreased expression of nestin, a neural stem-cell marker. Further, DS-SHED differentiated into DN (DS-DN) exhibiting decreased neurite outgrowth and branching compared with Ctrl-DN. In addition, DS-DN dopamine secretion was lower than Ctrl-DN dopamine secretion. Moreover, aberrant expression of molecules involved in dopaminergic homeostasis was observed in DS-DN.ConclusionsOur results suggest that there was developmental abnormality and DN malfunction in the DS-SHED donor in this study. In the future, to clarify the detailed mechanism of dopamine-signal abnormality due to DN developmental and functional abnormalities in DS, it is necessary to increase the number of patients for analysis. Non-invasively harvested SHED may be very useful in the analysis of DS pathology.

Compromised astrocyte function and survival negatively impact neurons in infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early localized glial activation occurs in these disorders, and accurately predicts where neuronal loss is most pronounced. Recent evidence suggests that glial dysfunction may contribute to neuron loss, and we have now explored this possibility in infantile NCL (INCL, CLN1 disease). We grew primary cultures of astrocytes, microglia, and neurons derived from Ppt1 deficient mice (Ppt1−/−) and assessed their properties compared to wildtype (WT) cultures, before co-culturing them in different combinations (astrocytes with microglia, astrocytes or microglia with neurons, all three cell types together). These studies revealed that both Ppt1−/− astrocytes and microglia exhibit a more activated phenotype under basal unstimulated conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1- /− astrocytes also displayed abnormal calcium signalling and an elevated cytoplasmic Ca2+ level, and a profound defect in their survival. Ppt1−/− neurons displayed decreased neurite outgrowth, altered complexity, a reduction in cell body size, and impaired neuron survival with prolonged time in culture. In co-cultures, the presence of both astrocytes and microglia from Ppt1−/− mice further impaired the morphology of both wild type and Ppt1−/− neurons. This negative influence was more pronounced for Ppt1−/− microglia, which appeared to trigger increased Ppt1−/− neuronal death. In contrast, wild type glial cells, especially astrocytes, ameliorated some of the morphological defects observed in Ppt1−/− neurons. These findings suggest that both Ppt1−/− microglia and astrocytes are dysfunctional and may contribute to the neurodegeneration observed in CLN1 disease. However, the dysfunctional phenotypes of Ppt1−/− glia are different from those present in CLN3 disease, suggesting that the pathogenic role of glia may differ between NCLs.

Neuronal-Specific TUBB3 Is Not Required for Normal Neuronal Function but Is Essential for Timely Axon Regeneration

SUMMARYWe generated a knockout mouse for the neuronalspecific β-tubulin isoform Tubb3 to investigate its role in nervous system formation and maintenance. Tubb3−/− mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of mRNA and protein of the remaining β-tubulin isotypes results in equivalent total b-tubulin levels in Tubb3−/− and wild-type mice. Despite similar levels of total β-tubulin, adult dorsal root ganglia lacking TUBB3 have decreased growth cone microtubule dynamics and a decreased neurite outgrowth rate of 22% in vitro and in vivo. The effect of the 22% slower growth rate is exacerbated for sensory recovery, where fibers must reinnervate the full volume of the skin to recover touch function. Overall, these data reveal that, while TUBB3 is not required for formation of the nervous system, it has a specific role in the rate of peripheral axon regeneration that cannot be replaced by other β-tubulins.

Amyloid Precursor Protein Dimerisation Reduces Neurite Outgrowth

The amyloid precursor protein (APP) undergoes extensive metabolism, and its transport and proteolytic processing can be modulated by its ability to form a homodimer. We have investigated the functional consequences of stabilised APP dimer expression in cells by studying the engineered dimerisation of the APPL17C (residue 17 in Aβ sequence) construct, which is associated with a 30% increase in APP dimer expression, on APP's neurite outgrowth promoting activity. Overexpression of APPL17C in SH-SY5Y cells decreased neurite outgrowth upon retinoic acid differentiation as compared to overexpressing APPWT cells. The APPL17C phenotype was rescued by replacing the APPL17C media with conditioned media from APPWT cells, indicating that the APPL17C mutant is impairing the secretion of a neuritogenic promoting factor. APPL17C had altered transport and was localised in the endoplasmic reticulum. Defining the molecular basis of the APPL17C phenotype showed that RhoA GTPase activity, a negative regulator of neurite outgrowth, was increased in APPL17C cells. RhoA activity was decreased after APPWT conditioned media rescue. Moreover, treatment with the RhoA inhibitor, Y27632, restored a wild-type morphology to the APPL17C cells. Small RNAseq analysis of APPL17C and APPWT cells identified several differentially expressed miRNAs relating to neurite outgrowth. Of these, miR-34a showed the greatest decrease in expression. Lentiviral-mediated overexpression of miR-34a rescued neurite outgrowth in APPL17C cells to APPWT levels and changed RhoA activation. This study has identified a novel link between APP dimerisation and its neuritogenic activity which is mediated by miR-34a expression.

Akt regulates neurite growth by phosphorylation-dependent inhibition of radixin proteasomal degradation

Neurite growth is controlled by a complex molecular signaling network that regulates filamentous actin (F-actin) dynamics at the growth cone. The evolutionarily conserved ezrin, radixin, and moesin family of proteins tether F-actin to the cell membrane when phosphorylated at a conserved threonine residue and modulate neurite outgrowth. Here we show that Akt binds to and phosphorylates a threonine 573 residue on radixin. Akt-mediated phosphorylation protects radixin from ubiquitin-dependent proteasomal degradation, thereby enhancing radixin protein stability, which permits proper neurite outgrowth and growth cone formation. Conversely, the inhibition of Akt kinase or disruption of Akt-dependent phosphorylation reduces the binding affinity of radixin to F-actin as well as lowers radixin protein levels, resulting in decreased neurite outgrowth and growth cone formation. Our findings suggest that Akt signaling regulates neurite outgrowth by stabilizing radixin interactions with F-actin, thus facilitating local F-actin dynamics.

α6 and β1 Integrin Heterodimer Mediates Schwann Cell Interactions with Axons and Facilitates Axonal Regeneration after Peripheral Nerve Injury

Several isoforms of integrin subunits are expressed in Schwann cells and mediate Schwann cell interactions with axons. Here, we identify α6 and β1 integrins as heterodimeric proteins expressed in Schwann cells and define their functions in axonal regeneration. α6 and β1 integrins are induced in Schwann cells in the sciatic nerve after a crush injury, and the blocking of integrin activity by siRNA expression and by treatment with anti-integrin antibodies attenuates Schwann cell contact with cultured neurons and decreases neurite outgrowth. After nerve transection, the levels of α6 and β1 integrins in the distal nerve stump are lower than those in the corresponding nerve area after a crush injury. Schwann cells prepared from the distal nerves 7 days after transection are less supportive of neurite outgrowth in co-cultured neurons than those prepared from the nerves 7 days after a crush injury. When the transected nerves are reconnected after a delay of 1 to 2 weeks, the induced levels of α6 and β1 integrins in the reconnected distal nerves are significantly reduced compared to those in the nerves after a crush injury. These changes correlate with retarded axonal regeneration in animals that have experienced nerve transections and delayed coaptation, which implies an attenuated Schwann cell capacity to support axonal regeneration due to delayed Schwann cell contact with axons. The present data suggest that α6 and β1 integrins induced in Schwann cells after nerve injury may play a role in mediating Schwann cell interactions with axons and promote axonal regeneration.

Neuroprotection by aripiprazole against β-amyloid-induced toxicity by P-CK2α activation via inhibition of GSK-3β.

Psychosis is reported over 30% of patients with Alzheimer's disease (AD) in clinics. Aripiprazole is an atypical antipsychotic drug with partial agonist activity at the D2 dopamine and 5-HT1A receptors with low side-effect profile. We identified aripiprazole is able to overcome the amyloid-β (Aβ)-evoked neurotoxicity and then increase the cell viability. This study elucidated the mechanism(s) by which aripiprazole ameliorates Aβ1-42-induced decreased neurite outgrowth and viability in neuronal cells. Pretreatment with aripiprazole increased Brain-derived neurotrophic factor (BDNF) mRNA and protein expressions in N2a cells. Additionally, phosphorylated casein kinase 2α at Y 255 (P-CK2α) was increased in time- and concentration-dependent manners. Furthermore, Aβ1-42-induced decreased BDNF and P-CK2α expression were increased over control level by aripiprazole. Subsequently, Aβ1-42-induced decreased levels of phosphorylated glycogen synthase-3β at Ser9 (P-GSK-3β) and nuclear P-β-catenin (Ser675) were elevated by aripiprazole, which were inhibited by K252A (inhibitor of BDNF receptor) and tetrabromocinnamic acid (TBCA, CK2 inhibitor), indicating that BDNF and P-CK2α activation are implicated in the aripiprazole effects. Expressions of cyclin D1 and insulin-like growth factor 2 (IGF2) mRNA were increased by aripiprazole; even in the presence of Aβ1-42, which was blocked by K252A and TBCA. In CK2α gene-silenced N2a cells, aripiprazole failed to increase P-GSK-3β and P-β-catenin expressions. Consequently, aripiprazole ameliorated Aβ1-42-induced attenuation of neurite elongation in HT22 cells, and this effect was blocked by both TBCA and imatinib. Decreased viability induced by Aβ1-42 was recovered by aripiprazole. These findings provide evidence supporting that aripiprazole can provide an effective therapeutic strategy against Aβ-induced neurotoxicity in AD-associated psychosis.

Regulation of RhoA activity by the cellular prion protein

The cellular prion protein (PrPC) is a highly conserved glycosylphosphatidylinositol (GPI)-anchored membrane protein that is involved in the signal transduction during the initial phase of neurite outgrowth. The Ras homolog gene family member A (RhoA) is a small GTPase that is known to have an essential role in regulating the development, differentiation, survival, and death of neurons in the central nervous system. Although recent studies have shown the dysregulation of RhoA in a variety of neurodegenerative diseases, the role of RhoA in prion pathogenesis remains unclear. Here, we investigated the regulation of RhoA-mediated signaling by PrPC using both in vitro and in vivo models and found that overexpression of PrPC significantly induced RhoA inactivation and RhoA phosphorylation in hippocampal neuronal cells and in the brains of transgenic mice. Using siRNA-mediated depletion of endogenous PrPC and overexpression of disease-associated mutants of PrPC, we confirmed that PrPC induced RhoA inactivation, which accompanied RhoA phosphorylation but reduced the phosphorylation levels of LIM kinase (LIMK), leading to cofilin activation. In addition, PrPC colocalized with RhoA, and the overexpression of PrPC significantly increased neurite outgrowth in nerve growth factor-treated PC12 cells through RhoA inactivation. However, the disease-associated mutants of PrPC decreased neurite outgrowth compared with wild-type PrPC. Moreover, inhibition of Rho-associated kinase (ROCK) substantially facilitated neurite outgrowth in NGF-treated PC12 cells, similar to the effect induced by PrPC. Interestingly, we found that the induction of RhoA inactivation occurred through the interaction of PrPC with RhoA and that PrPC enhanced the interaction between RhoA and p190RhoGAP (a GTPase-activating protein). These findings suggest that the interactions of PrPC with RhoA and p190RhoGAP contribute to neurite outgrowth by controlling RhoA inactivation and RhoA-mediated signaling and that disease-associated mutations of PrPC impair RhoA inactivation, which in turn leads to prion-related neurodegeneration.

Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth.

The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development.

Gallium containing composites as a tunable material to understand neuronal behavior under variable stiffness and radiation conditions

We report a composite biomaterial containing nanostructured GaOOH and Matrigel™ that can be modulated with respect to its stiffness and radiosensitization properties. A variety of concentrations of GaOOH were added to the composite to alter the mechanical properties of the material as well as to tune the radiosensitizing properties to the composite. PC-12 cells were used to study the combined effects of different stimuli on cell behavior. NGF was given to the cells to record their morphology as well as viability. An increase in the substrate stiffness caused an increase in neurite outgrowth but a decrease in cell viability. In addition, increasing the radiation dose decreased neurite outgrowth but increased cell viability when radiosensitizing particles were present. A subtractive effect between radiosensitizing and mechanical stimuli was observed when PC-12 cells were grown on the GaOOH containing composite.

Non-cytotoxic Concentration of Cisplatin Decreases Neuroplasticity-Related Proteins and Neurite Outgrowth Without Affecting the Expression of NGF in PC12 Cells.

Cisplatin is the most effective and neurotoxic platinum chemotherapeutic agent. It induces a peripheral neuropathy characterized by distal axonal degeneration that might progress to degeneration of cell bodies and apoptosis. Most symptoms occur nearby distal axonal branches and axonal degeneration might induce peripheral neuropathy regardless neuronal apoptosis. The toxic mechanism of cisplatin has been mainly associated with DNA damage, but cisplatin might also affect neurite outgrowth. Nevertheless, the neurotoxic mechanism of cisplatin remains unclear. We investigated the early effects of cisplatin on axonal plasticity by using non-cytotoxic concentrations of cisplatin and PC12 cells as a model of neurite outgrowth and differentiation. PC12 cells express NGF-receptors (trkA) and respond to NGF by forming neurites, branches and synaptic vesicles. For comparison, we used a neuronal model (SH-SY5Y cells) that does not express trkA nor responds to NGF. Cisplatin did not change NGF expression in PC12 cells and decreased neurite outgrowth in both models, suggesting a NGF/trkA independent mechanism. It also reduced axonal growth (GAP-43) and synaptic (synapsin I and synaptophysin) proteins in PC12 cells, without inducing mitochondrial damage or apoptosis. Therefore, cisplatin might affect axonal plasticity before DNA damage, NGF/trkA down-regulation, mitochondrial damage or neuronal apoptosis. This is the first study to show that neuroplasticity-related proteins might be early targets of the neurotoxic action of cisplatin and their role on cisplatin-induced peripheral neuropathy should be investigated in vivo.

Overexpression of the Fibroblast Growth Factor Receptor 1 (FGFR1) in a Model of Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a severe condition that affects many people and results in high health care costs. Therefore, it is essential to find new targets for treatment. The fibroblast growth factor receptor 1 (FGFR1) signalling pathway has a history of being explored for SCI treatment. Several groups have examined the effect of high availability of different FGFR1 ligands at the injury site and reported corticospinal tract (CST) regeneration as well as improved motor functions. In this study, we investigated overexpression of the FGFR1 in rat corticospinal neurons in vivo after injury (unilateral pyramidotomy) and in cerebellar granule neurons (CGNs) in vitro. We show that overexpression of FGFR1 using AAV1 intracortical injections did not increase sprouting of the treated corticospinal tract and did not improve dexterity or walking in a rat model of SCI. Furthermore, we show that overexpression of FGFR1 in vitro resulted in decreased neurite outgrowth compared to control. Thus, our results suggest that the FGFR1 is not a suitable therapeutic target after SCI.

Involvement of autophagy upregulation in 3,4-methylenedioxymethamphetamine (‘ecstasy’)-induced serotonergic neurotoxicity

It has been suggested that autophagy plays pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug that causes long-term serotonergic neurotoxicity in the brain. Apoptosis and necrosis have been implicated in MDMA-induced neurotoxicity, but the role of autophagy in MDMA-elicited serotonergic toxicity has not been investigated. The present study aimed to examine the contribution of autophagy to neurotoxicity in serotonergic neurons in in vitro and in vivo animal models challenged with MDMA. Here, we demonstrated that in cultured rat serotonergic neurons, MDMA exposure induced LC3B-densely stained autophagosome formation, accompanying by a decrease in neurite outgrowth. Autophagy inhibitor 3-methyladenine (3-MA) significantly attenuated MDMA-induced autophagosome accumulation, and ameliorated MDMA-triggered serotonergic neurite damage and neuron death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in serotonergic neurons and aggravated neurite degeneration. In addition, MDMA-induced autophagy activation in cultured serotonergic neurons might be mediated by serotonin transporter (SERT). In an in vivo animal model administered MDMA, neuroimaging showed that 3-MA protected the serotonin system against MDMA-induced downregulation of SERT evaluated by animal-PET with 4-[18F]-ADAM, a SERT radioligand. Taken together, our results demonstrated that MDMA triggers upregulation of autophagy in serotonergic neurons, which appears to be detrimental to neuronal growth.