Motor Neuron Cell Line Research Articles

The D2 Dopamine Receptor Interferes With the Protective Effect of the A2A Adenosine Receptor on TDP-43 Mislocalization in Experimental Models of Motor Neuron Degeneration.

The A2A adenosine receptor (A2AR) and D2 dopamine receptor (D2R) are two G-protein-coupled receptors that can form dimers and negatively regulate their partners. TAR DNA-binding protein (TDP-43) is a nuclear protein that has been implicated in amyotrophic lateral sclerosis (ALS). Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early step of TDP-43 proteinopathy. Our previous studies indicated that A2AR is a potential drug target for ALS because treatment with an A2AR agonist (JMF1907; a T1-11 analog) prevents reactive oxygen species (ROS)-induced TDP-43 mislocalization in a motor neuron cell line (NSC34) and delays motor impairment in a TDP-43 transgenic ALS mouse model. Here, we set out to assess whether activation of D2R interferes with the beneficial effects of an A2AR agonist on motor neurons. We first demonstrated that A2AR and D2R are both located in motor neurons of mouse and human spinal cords and human iPSC-derived motor neurons. Expression of A2AR and D2R in NSC34 cells led to dimer formation without affecting the binding affinity of A2AR toward T1-11. Importantly, activation of D2R reduced T1-11-mediated activation of cAMP/PKA signaling and subsequent inhibition of TDP-43 mislocalization in NSC34 cells. Treatment with quinpirole (a D2 agonist) blunted the rescuing effect of T1-11 on TDP-43 mislocalization and impaired grip strength in a mouse model of ALS. Our findings suggest that D2R activation may limit the beneficial responses of an A2AR agonist in motor neurons and may have an important role in ALS pathogenesis.

Inhibition of p70 S6 kinase activity by A77 1726 induces autophagy and enhances the degradation of superoxide dismutase 1 (SOD1) protein aggregates

Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.

Focus on the Role of D-serine and D-amino Acid Oxidase in Amyotrophic Lateral Sclerosis/Motor Neuron Disease (ALS).

We have investigated a pathogenic mutation in D-amino acid oxidase (DAO), DAOR199W, associated with familial Amyotrophic Lateral Sclerosis (ALS) that impairs D-serine metabolism and causes protein aggregation, autophagy and cell death in motor neuron cell lines. These features are consistent with the pathogenic processes occurring in ALS but most importantly, we have demonstrated that activation of the formation of ubiquitinated protein inclusions, increased autophagosome production and apoptotic cell death caused by the mutation in cell lines are attenuated by 5,7-dichlorokynurenic acid (DCKA), a selective inhibitor of the glycine/D-serine binding site of the NMDA receptor. D-serine is an essential co-agonist at this glutamate receptor. This data provides insight into potential upstream mechanisms that involve the action of D-serine at the NMDA receptor and might contribute to neurodegeneration. This is highly relevant to sporadic ALS (SALS), familial ALS, as well as ALS models, where elevated levels of D-serine have been reported and hence has broader clinical therapeutic implications. In order to investigate this further, we have generated a transgenic line expressing the pathogenic mutation, in order to determine whether mice expressing DAOR199W develop a motor phenotype and whether crossing the SOD1G93A model of ALS with mice expressing DAOR199W affects disease progression. We found that heterozygous expression of DAOR199W led to a significant loss of spinal cord motor neurons at 14 months, which is similar to that found in homozygous mice expressing DAOG181R. We hypothesize that DAO has potential for development as a therapeutic agent in ALS.

661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively

A photoreceptor cell line, 661W, derived from a mouse retinal tumor that expresses several markers of cone photoreceptor cells has been described earlier. However, these cells can be differentiated into neuronal cells. Here, we report that this cell line expressed certain markers specific to retinal ganglion cells such as Rbpms, Brn3b (Pou4f2), Brn3c (Pou4f3), Thy1 and γ-synuclein (Sncg), and some other markers of neuronal cells (beta-III tubulin, NeuN and MAP2). These cells also expressed Opn1mw, a cone-specific marker and nestin, a marker for neural precursor cells. Two glaucoma-associated mutants of OPTN, E50K and M98K, but not an amyotrophic lateral sclerosis-associated mutant, E478G, induced cell death selectively in 661W cells. However, in a motor neuron cell line, NSC34, E478G mutant of OPTN but not E50K and M98K induced cell death. We conclude that 661W is a retinal ganglion precursor-like cell line, which shows properties of both retinal ganglion and photoreceptor cells. We suggest that these cells could be utilized for exploring the mechanisms of cell death induction and cytoprotection relevant for glaucoma pathogenesis. RGC-5 cell line which probably arose from 661W cells showed expression of essentially the same markers of retinal ganglion cells and neuronal cells as seen in 661W cells.

Characterisation of the pathogenic effects of the in vivo expression of an ALS-linked mutation in D-amino acid oxidase: Phenotype and loss of spinal cord motor neurons.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disorder characterised by selective loss of motor neurons leading to fatal paralysis. Current therapeutic approaches are limited in their effectiveness. Substantial advances in understanding ALS disease mechanisms has come from the identification of pathogenic mutations in dominantly inherited familial ALS (FALS). We previously reported a coding mutation in D-amino acid oxidase (DAOR199W) associated with FALS. DAO metabolises D-serine, an essential co-agonist at the N-Methyl-D-aspartic acid glutamate receptor subtype (NMDAR). Using primary motor neuron cultures or motor neuron cell lines we demonstrated that expression of DAOR199W, promoted the formation of ubiquitinated protein aggregates, activated autophagy and increased apoptosis. The aim of this study was to characterise the effects of DAOR199W in vivo, using transgenic mice overexpressing DAOR199W. Marked abnormal motor features, e.g. kyphosis, were evident in mice expressing DAOR199W, which were associated with a significant loss (19%) of lumbar spinal cord motor neurons, analysed at 14 months. When separated by gender, this effect was greater in females (26%; p< 0.0132). In addition, we crossed the DAOR199W transgenic mouse line with the SOD1G93A mouse model of ALS to determine whether the effects of SOD1G93A were potentiated in the double transgenic line (DAOR199W/SOD1G93A). Although overall survival was not affected, onset of neurological signs was significantly earlier in female double transgenic animals than their female SOD1G93A littermates (125 days vs 131 days, P = 0.0239). In summary, some significant in vivo effects of DAOR199W on motor neuron function (i.e. kyphosis and loss of motor neurons) were detected which were most marked in females and could contribute to the earlier onset of neurological signs in double transgenic females compared to SOD1G93A littermates, highlighting the importance of recognizing gender effects present in animal models of ALS.

Activation of the D2 dopamine receptor hampers the protective effect of the A2A adenosine receptor on TDP‐43 mislocalization

TAR DNA‐binding protein (TDP‐43) is an RNA‐binding protein that exists mostly in the nucleus and regulates transcription and RNA splicing. Mislocalization of TDP‐43 to the cytoplasm has been observed in several neurodegeneration diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and is believed to initiate the pathogenesis of TDP‐43 proteinopathy. We have previously published that several adenosine analogues (T1‐11 and its analogues) prevented the reactive oxygen species (ROS)‐induced TDP‐43 mislocalization in a mouse motor neuron cell line (NSC34) via the A2A adenosine receptor (A2AR), and prevented the degenerated motor functions in a TDP‐43 transgenic ALS mouse model. In the striatum, previous studies suggest that A2AR and the D2 dopamine receptor (D2R) formed dimmer and negatively cross‐regulated each other's functions. In the present study, we first demonstrated that A2AR and D2R both exist in the motor neurons of the spinal cord of normal subjects and ALS patients by using immunofluorescence assay. Expression of both A2AR and D2R in NSC34 cells led to dimer formation as assessed using the proximity ligation assay (in situ PLA). No change in the binding affinity of T1‐11 was observed when A2AR formed dimmer with D2R. Nonetheless, activation of D2R reversed the A2AR‐mediated prevention of ROS‐induced AMPK activation and TDP‐43 mislocalization in NSC34 cells. Given that D2R agonists have been commonly used in clinical settings, the impact of D2R activation on the normalization of TDP‐43 mislocalization by A2AR in ALS warrants further investigation.

TDP-43 mutations causing amyotrophic lateral sclerosis are associated with altered expression of RNA-binding protein hnRNP K and affect the Nrf2 antioxidant pathway.

TAR DNA binding protein 43 (TDP-43) is a major disease-associated protein involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Our previous studies found a direct association between TDP-43 and heterogeneous nuclear ribonucleoprotein K (hnRNP K). In this study, utilizing ALS patient fibroblasts harboring a TDP-43M337V mutation and NSC-34 motor neuronal cell line expressing TDP-43Q331K mutation, we show that hnRNP K expression is impaired in urea soluble extracts from mutant TDP-43 cell models. This was confirmed in vivo using TDP-43Q331K and inducible TDP-43A315T murine ALS models. We further investigated the potential pathological effects of mutant TDP-43-mediated changes to hnRNP K metabolism by RNA binding immunoprecipitation analysis. hnRNP K protein was bound to antioxidant NFE2L2 transcripts encoding Nrf2 antioxidant transcription factor, with greater enrichment in TDP-43M337V patient fibroblasts compared to healthy controls. Subsequent gene expression profiling revealed an increase in downstream antioxidant transcript expression of Nrf2 signaling in the spinal cord of TDP-43Q331K mice compared to control counterparts, yet the corresponding protein expression was not up-regulated in transgenic mice. Despite the elevated expression of antioxidant transcripts, we observed impaired levels of glutathione (downstream Nrf2 antioxidant) in TDP-43M337V patient fibroblasts and astrocyte cultures from TDP-43Q331K mice, indicative of elevated oxidative stress and failure of some upregulated antioxidant genes to be translated into protein. Our findings indicate that further exploration of the interplay between hnRNP K (or other hnRNPs) and Nrf2-mediated antioxidant signaling is warranted and may be an important driver for motor neuron degeneration in ALS.

VEGF alleviates ALS-CSF induced cytoplasmic accumulations of TDP-43 and FUS/TLS in NSC-34 cells

Cytoplasmic mislocalisation and aggregation of TDP-43 and FUS/TLS proteins in spinal motor neurons contribute to the pathogenesis of the highly fatal disorder amyotrophic lateral sclerosis (ALS). We investigated the neuroprotective effect of VEGF on expression of these proteins in the motor neuronal cell line NSC-34 modelled to reminisce sporadic form of ALS. We studied the expression of TDP-43 and FUS/TLS proteins after exposure to ALS-CSF and following VEGF supplementation by quantitative confocal microscopy and electron microscopy. ALS-CSF caused cytoplasmic overexpression of both the proteins and stress-granule formation in the cells. These alterations were alleviated by VEGF supplementation. The related ultrastructural changes like nuclear membrane dysmorphism and p-bodies associated changes were also reversed. However the protein expression did not completely translocate to the nucleus, as some cells continued to show to cytoplasmic mislocalisation. Thus, the present findings indicate that VEGF alleviates TDP43 and FUS pathology by complimenting its role in controlling apoptosis and reversing choline acetyl transferase expression. Hence, VEGF appears to target multiple pathogenic processes in the neurodegenerative cascade of ALS.

The alteration of serine transporter activity in a cell line model of amyotrophic lateral sclerosis (ALS)

The alteration of d-serine levels is associated with the pathogenesis of sporadic ALS and mutant SOD1 (G93A) animal model of ALS. However, the exact mechanism of d-serine transport is not known in ALS. To better understand the distribution of d-serine in ALS, we determined the activity and the expression of serine transporter in a motor neuronal cell line model of ALS (NSC-34/hSOD1G93A cells). The uptake of [3H]d-serine was significantly lower in NSC-34/hSOD1G93A cells than in control NSC-34 and NSC-34/hSOD1wt cells. In contrast, the uptake of [3H]l-serine, precursor of d-serine, was markedly increased in NSC-34/hSOD1G93A cells compared to control NSC-34 and NSC-34/hSOD1wt cells. Both [3H]d-serine and [3H]l-serine uptake were saturable in these cells. The estimated Michaelis-Menten constant, Km, for d-serine uptakes was higher in NSC-34/hSOD1G93A cells than in NSC-34/hSOD1wt cells while the Km for l-serine uptake was 2 fold lower in NSC-34/hSOD1G93A cells than in control cells. [3H]d-serine and [3H]l-serine uptakes took place in a Na+-dependent manner, and both uptakes were significantly inhibited by system ASC (alanine-serine-cysteine) substrates. As a result of small interfering RNA experiments, we found that ASCT2 (SLC1A5) and ASCT1 (SLC1A4) are involved in [3H]d-serine and [3H]l-serine uptake in NSC-34/hSOD1G93A cells, respectively. The level of SLC1A4 mRNA was significantly increased in NSC-34/hSOD1G93A compared to NSC-34 and NSC-34/hSOD1wt cells. In contrast, the level of SLC7A10 mRNA was relatively lower in NSC-34/hSOD1G93A cells than the control cells. Together, these data suggest that the pathological alteration of d- and l-serine uptakes in ALS is driven by the affinity change of d-and l-serine uptake system.

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway.

Enterovirus 71 (EV71) causing Hand, Foot and Mouth Disease, is regarded as the most important neurotropic virus worldwide. EV71 is believed to replicate in muscles and infect motor neurons to reach the central nervous system (CNS). To further investigate the mechanisms involved, we have employed the motor neuron cell line NSC-34. NSC-34 cells were permissive to EV71 and virus production yields were strain-dependent with differential efficacy at the entry, replication and egress steps. Furthermore, unlike all the other cell lines previously reported, EV71-infected NSC-34 cells neither displayed cytopathic effect nor underwent apoptosis. Instead, autophagy was markedly up-regulated and virus-containing autophagic vacuoles were isolated from the culture supernatant, providing the first experimental evidence that EV71 can adopt a non-lytic exit pathway. Finally, the ability of EV71 to infect productively NSC-34 cells correlated with its ability to invade the CNS in vivo, supporting the relevance of NSC-34 cells to study the intrinsic neurovirulence of EV71 strains.

Anti-Sulfoglucuronosyl Paragloboside Antibody: A Potential Serologic Marker of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Although the etiology of ALS is obscure, genetic studies of familiar ALS suggest a multifactorial etiology for this condition. Similarly, there probably are multiple causes for sporadic ALS. Autoimmune-mediated motor neuron dysfunction is one proposed etiology for sporadic ALS. In the present study, anti-glycolipid antibodies including GM1, GD1b, GD3, and sulfoglucuronosyl paragloboside (SGPG) were investigated in the sera of a large number of patient samples, including 113 ALS patients and 50 healthy controls, by means of enzyme-linked immunosorbent assay with affinity parametric complex criterion evaluation and thin-layer chromatography immunooverlay (immuno-TLC). Anti-SGPG antibodies were found in the sera of 13.3% ALS patients (15 out of 113). The highest titer reached 1:1600. The presence of anti-SGPG antibodies in the serum samples was also confirmed by immuno-TLC. Importantly, a multiple logistic regression analysis showed that the presence of anti-SGPG antibody was positively correlated with age (p < .01) and negatively correlated with ALS Functional Rating Scale score (p < .05). Moreover, the localization of SGPG-immunoreactivity on the motor neurons of rat spinal cord and a mouse motor neuronal cell line, NSC-34 was observed by an immunofluorescence method. These data suggest that SGPG could represent a specific pathogenic antigen in those ALS patients. The presence of anti-SGPG antibodies in the serum of ALS patients should represent a diagnostic biomarker of ALS, and it could reflect the severity of the disease.

Brain-Derived Neurotrophic Factor Facilitates Functional Recovery from ALS-Cerebral Spinal Fluid-Induced Neurodegenerative Changes in the NSC-34 Motor Neuron Cell Line

Background: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. Objective: To evaluate the protective role of BDNF in a model of sporadic ALS patients. Methods: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. Results: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca²⁺ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. Conclusion: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.

Astroglia acquires a toxic neuroinflammatory role in response to the cerebrospinal fluid from amyotrophic lateral sclerosis patients.

BackgroundNon-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF).MethodsVarious inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test, or Student’s t test, as applicable.ResultsHere, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells.ConclusionsOur study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0698-0) contains supplementary material, which is available to authorized users.

Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway

Trophic factors (TFs) play important role during development and adult tissue maintenance. In neurodegenerative diseases (ND) TF supplementation provides protection. Stromal cells (HUMS) derived from the human umbilical cord matrix provide neuroprotection in the ND models of mice. Though TF mediated protection is known, the exact mechanism of protection is not clear. So, here the essential TFs (secreted by HUMS cells) and the pathway of induction of neurite extension, differentiation and networking is addressed. The HUMS cells from the human umbilical cord matrix were derived and the mouse spinal cord motor neuron cell line, NSC-34 was extensively used. Flow cytometry, immunohistochemistry, RT- PCR, western blot, ELISA and antibody/inhibitor treatment were carried out to figure out the TF pathway. The HUMS cells secrete six neurotrophic factors (sTFs), namely, NT-3, NGF, BDNF, VEGF, IGF-1 and GDNF (TFs). These TFs are sufficient to induce differentiation, neurite extension and neural networking in a motor neuron cell line, NSC34. All the 5 TFs need to be neutralized simultaneously with their antibodies to abrogate neurite extension. These motor neurons express the concomitant receptors, which are either receptor tyrosine kinase (TrK) coupled or to the receptor followed by the TrKs, for the above trophic factors (except for BDNF). The tyrosine kinase inhibitor, K252a, drastically reduces neurite extension. In NSC34, the TFs are coupled to the PI3K-Akt-pathway and the RAS-MAP kinase signaling through phosphorylation of ERK1 and ERK2. PI3K inhibitor, Ly 294002, abolishes neural differentiation and neurite extension. Thus, differentiation, neurite extension and networking could be achieved through the PI3K pathway. Intriguingly, the cAMP second messenger system coupling was not required. H89, PKA-inhibitor caused extensive cell death. But, had no effect in the presence of HUMS-secreted-TFs(HSTFs) suggesting a pathway switch for cell survival itself. HUMS cells and their secreted factors could be of great use in regenerative medicine (RM). The activators of PI3K pathway, the major route of these HUMS-TFs action could be explored in RM and in the neurobiology of neural differentiation and extension.

DJ-1 knockout augments disease severity and shortens survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder, characterized by the degeneration of motor neurons. Oxidative stress plays a central role in the disease progression, in concert with an enhanced glutamate excitotoxicity and neuroinflammation. DJ-1 mutations, leading to the loss of functional protein, cause familial Parkinson’s disease and motor neuron disease in several patients. DJ-1 responds to oxidative stress and plays an important role in the cellular defense mechanisms. We aimed to investigate whether loss of functional DJ-1 alters the disease course and severity in an ALS mouse model. To this end we used mice that express the human SOD1G93A mutation, the commonly used model of ALS and knockout of DJ-1 mice to generate SOD1 DJ-1 KO mice. We found that knocking out DJ-1in the ALS model led to an accelerated disease course and shortened survival time. DJ-1 deficiency was found to increase neuronal loss in the spinal cord associated with increased gliosis in the spinal cord and reduced antioxidant response that was regulated by the Nrf2 mechanism.The importance of DJ-1 in ALS was also illustrated in a motor neuron cell line that was exposed to glutamate toxicity and oxidative stress. Addition of the DJ-1 derived peptide, ND-13, enhanced the resistance to glutamate and SIN-1 induced toxicity. Thus, our results maintain that DJ-1 plays a role in the disease process and promotes the necessity of further investigation of DJ-1 as a therapeutic target for ALS.

PTEN as a therapeutic target in motor neuron diseases (ALS/SMA)

The tumor suppressor protein Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN) is a member of the protein tyrosine phosphatase family that can negatively regulate the serine/threonine kinase Akt to exert its tumour suppressor function. In addition to its normal functions such as neuronal migration and neuronal size control, PTEN protein is involved in pathological processes surrounding neuronal injury such as those associated with brain ischemia, neurological and mental disorders. It has been shown that modulation of the PTEN/mTOR pathway promotes axon regeneration in the adult CNS. We have previously shown that down-regulating the expression of PTEN protects against ischaemic neuronal death in vitro and in vivo (Ning et al. 2014). Recently, we showed that PTEN knockdown via siRNA increases motor neuron survival in Amyotrophic lateral sclerosis (ALS) (Kirby et al. 2011) in vitro and spinal muscular atrophy (SMA) in vivo (Ning et al. 2010, Little et al., unpublished). Our preliminary data show that the PTEN inhibitor, bpV, promotes cell survival in NSC34 G93A motor neuronal cell line. We have also showed that PTEN silencing increases cell survival in iPS-derived motor neurons from human fibroblasts (D-J Yang et al., 2014). Taken together, PTEN inhibition results in neuroprotective effects on motor neuron survival in vitro and in vivo. The outcome of our studies provide evidence that PTEN is potential therapeutic target for neuroprotection in ALS or SMA patients and other neurodegenerative disorders.

Aberrant activation of AMP-activated protein kinase contributes to the abnormal distribution of HuR in amyotrophic lateral sclerosis

Distorted mRNA metabolism contributes to amyotrophic lateral sclerosis (ALS). The human antigen R (HuR) is a major mRNA stabilizer. We report that abnormal localization of HuR was associated with enhanced AMP-activated protein kinase (AMPK) activity in the motor neurons of ALS patients. Activation of AMPK changed the location of HuR in mouse motor neurons and in a motor neuron cell line via phosphorylation of importin-α1. Stimulation of the A2A adenosine receptor normalized the AMPK-evoked redistribution of HuR. This suggests that aberrant activation of AMPK in motor neurons disrupts the normal distribution of HuR, which might imbalance RNA metabolism and contribute to ALS pathogenesis.

Activation of AMP-activated protein kinase α1 mediates mislocalization of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein-43 (TDP-43) is a nuclear RNA-binding protein involved in many cellular pathways. TDP-43-positive inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). The major clinical presentation of ALS is muscle weakness due to the degeneration of motor neurons. Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early event of ALS. In this study, we demonstrate that cytoplasmic mislocalization of TDP-43 was accompanied by increased activation of AMP-activated protein kinase (AMPK) in motor neurons of ALS patients. The activation of AMPK in a motor neuron cell line (NSC34) or mouse spinal cords induced the mislocalization of TDP-43, recapitulating this characteristic of ALS. Down-regulation of AMPK-α1 or exogenous expression of a dominant-negative AMPK-α1 mutant reduced TDP-43 mislocalization. Suppression of AMPK activity using cAMP-simulating agents rescued the mislocalization of TDP-43 in NSC34 cells and delayed disease progression in TDP-43 transgenic mice. Our findings demonstrate that activation of AMPK-α1 plays a critical role in TDP-43 mislocalization and the development of ALS; thus, AMPK-α1 may be a potential drug target for this devastating disease.

SUMO3 modification accelerates the aggregation of ALS-linked SOD1 mutants.

Mutations in superoxide dismutase 1 (SOD1) are a major cause of familial amyotrophic lateral sclerosis (ALS), whereby the mutant proteins misfold and aggregate to form intracellular inclusions. We report that both small ubiquitin-like modifier (SUMO) 1 and SUMO2/3 modify ALS-linked SOD1 mutant proteins at lysine 75 in a motoneuronal cell line, the cell type affected in ALS. In these cells, SUMO1 modification occurred on both lysine 75 and lysine 9 of SOD1, and modification of ALS-linked SOD1 mutant proteins by SUMO3, rather than by SUMO1, significantly increased the stability of the proteins and accelerated intracellular aggregate formation. These findings suggest the contribution of sumoylation, particularly by SUMO3, to the protein aggregation process underlying the pathogenesis of ALS.

Role of VEGF and VEGFR2 Receptor in Reversal of ALS-CSF Induced Degeneration of NSC-34 Motor Neuron Cell Line.

Vascular endothelial growth factor (VEGF), the well-known angiogenic factor is both neurotrophic and neuroprotective. Altered VEGF signalling is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal degenerative disease of motor neurons. We have shown earlier that VEGF protects NSC-34 motor neuronal cell line, when exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF). Here, we have investigated the consequences of ALS-CSF and VEGF supplementation on the VEGFR2 receptor and endogenous VEGF expression. ALS-CSF caused significant down-regulation of VEGFR2 as well as the Calbindin-D28K levels, but not endogenous VEGF. Exogenous supplementation restored the depletion of VEGFR2 and Calbindin-D28K with a concomitant up-regulation of endogenous VEGF. The up-regulated caspase 3 in the ALS-CSF group was reinstated to basal levels along with a significant reduction in the number of TUNEL-positive cells. Electron photomicrographs of ALS-CSF-exposed cells divulged presence of cytoplasmic vacuoles alongside severe damage to organelles like mitochondria, endoplasmic reticulum, etc. Substantial recovery of most of the damaged organelles was noted in response to VEGF supplementation. While the enhancement in endogenous VEGF levels highlights the autocrine functions, the up-regulation of VEGFR2 receptor emphasizes the paracrine functions of VEGF in modulating its neuroprotective effect against ALS-CSF. The revival of cellular organellar structure, increased calbindin expression and enhanced survival in response to VEGF supplementation consolidates the opinion that VEGF indeed has a therapeutic potential in sporadic ALS.