Cultured Motor Neurons Research Articles

“Preconditioning” with latrepirdine, an adenosine 5′-monophosphate-activated protein kinase activator, delays amyotrophic lateral sclerosis progression in SOD1G93A mice

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a master regulator of energy balance. As energy imbalance is documented as a key pathologic feature of amyotrophic lateral sclerosis (ALS), we investigated AMPK as a pharmacologic target in SOD1G93A mice. We noted a strong activation of AMPK in lumbar spinal cords of SOD1G93A mice. Pharmacologic activation of AMPK has shown protective effects in neuronal “preconditioning” models. We tested the hypothesis that “preconditioning” with a small molecule activator of AMPK, latrepirdine, exerts beneficial effects on disease progression. SOD1G93A mice (n = 24 animals per group; sex and litter matched) were treated with latrepirdine (1 μg/kg, intraperitoneal) or vehicle from postnatal day 70 to 120. Treatment with latrepirdine increased AMPK activity in primary mouse motor neuron cultures and in SOD1G93A lumbar spinal cords. Mice “preconditioned” with latrepirdine showed a delayed symptom onset and a significant increase in life span (p < 0.01). Our study suggests that “preconditioning” with latrepirdine may represent a possible therapeutic strategy for individuals harboring ALS-associated gene mutations who are at risk for developing ALS.

The BCL-2 family protein Bid is critical for pro-inflammatory signaling in astrocytes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons in the spinal cord, brainstem and motor cortex. Mutations in the superoxide dismutase 1 (SOD1) gene represent a frequent genetic determinant and recapitulate a disease phenotype similar to ALS when expressed in mice. Previous studies using SOD1G93A transgenic mice have suggested a paracrine mechanism of neuronal loss, in which cytokines and other toxic factors released from astroglia or microglia trigger motoneuron degeneration. Several pro-inflammatory cytokines activate death receptors and may downstream from this activate the Bcl-2 family protein, Bid. We here sought to investigate the role of Bid in astrocyte activation and non-cell autonomous motoneuron degeneration. We found that spinal cord Bid protein levels increased significantly during disease progression in SOD1G93A mice. Subsequent experiments in vitro indicated that Bid was expressed at relatively low levels in motoneurons, but was enriched in astrocytes and microglia. Bid was strongly induced in astrocytes in response to pro-inflammatory cytokines or exposure to lipopolysaccharide. Experiments in bid-deficient astrocytes or astrocytes treated with a small molecule Bid inhibitor demonstrated that Bid was required for the efficient activation of transcription factor nuclear factor-κB in response to these pro-inflammatory stimuli. Finally, we found that conditioned medium from wild-type astrocytes, but not from bid-deficient astrocytes, was toxic when applied to primary motoneuron cultures. Collectively, our data demonstrate a new role for the Bcl-2 family protein Bid as a mediator of astrocyte activation during neuroinflammation, and suggest that Bid activation may contribute to non-cell autonomous motoneuron degeneration in ALS.

Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross-talk that coordinates COPI vesicle formation and tubulin polymerization.

Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the Golgi-localized tubulin-binding cofactor E (TBCE). Loss of TBCE in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of COPI subunits, decreased recruitment of tethering factors p115/GM130 and impaired Golgi SNARE-mediated vesicle fusion. Conversely, ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. These data indicate an ARF1/TBCE-mediated cross-talk that coordinates COPI formation and tubulin polymerization at the Golgi. We conclude that interruption of this cross-talk causes Golgi fragmentation in pmn mice and hypothesize that similar mechanisms operate in human amyotrophic lateral sclerosis and spinal muscular atrophy.

Stem cell-derived motor neurons from spinal and bulbar muscular atrophy patients

Spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy.

A new method for generating high purity motoneurons from mouse embryonic stem cells.

A common problem with using embryonic stem (ES) cells as a source for analysis of gene expression, drug toxicity, or functional characterization studies is the heterogeneity that results from many differentiation protocols. The ability to generate large numbers of high purity differentiated cells from pluripotent stem cells could greatly enhance their utility for in vitro characterization studies and transplantation in pre-clinical injury models. Population heterogeneity is particularly troublesome for post-mitotic neurons, including motoneurons, because they do not proliferate and are quickly diluted in culture by proliferative phenotypes, such as glia. Studies of motoneuron biology and disease, in particular amyotrophic lateral sclerosis, can benefit from high purity motoneuron cultures. In this study, we engineered a transgenic-ES cell line where highly conserved enhancer elements for the motoneuron transcription factor Hb9 were used to drive puromycin N-acetyltransferase expression in ES cell-derived motoneurons. Antibiotic selection with puromycin was then used to obtain high purity motoneuron cultures following differentiation of mouse ES cells. Purity was maintained during maturation allowing the production of consistent, uniform populations of cholinergic ES cell-derived motoneurons. Appropriate functional properties of purified motoneurons were verified by acetylcholinesterase activity and electrophysiology. Antibiotic selection, therefore, can provide an inexpensive alternative to current methods for isolating ES cell-derived motoneurons at high purity that does not require specialized laboratory equipment and provides a unique platform for studies in motoneuron development and degeneration.

Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors.

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca(2+) homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca(2+) entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L- and T-type voltage-gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca(2+) and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca(2+) overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA-binding proteins transactive response DNA-binding protein 43 (TDP-43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP-43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

Purification and Culture of Corticospinal Motor Neurons

Corticospinal motor neurons (CSMNs) residing in cortical layer V of the mammalian brain project their axons to the spinal cord, where they connect with spinal motor neurons (SMNs) located in the ventral horn of the spinal cord. CSMNs and SMNs control voluntary movements, and their importance becomes obvious in situations where this network breaks down (i.e., in amyotrophic lateral sclerosis [ALS] and after spinal cord injury). Here we provide an overview of recent progress in the anatomical, morphological, and genetic characterization of developing CSMNs, as well as their survival requirements. We also describe model systems used to study CSMNs and introduce an immunopanning procedure for the purification and culture of CSMNs. Although these procedures have so far been used to purify only rodent CSMNs, in principle they should work to purify CSMNs from any vertebrate species, as well any type of central nervous system (CNS) or peripheral nervous system (PNS) neuron that can be retrograde labeled.

The Neuroprotection Exerted by Memantine, Minocycline and Lithium, against Neurotoxicity of CSF from Patients with Amyotrophic Lateral Sclerosis, Is Antagonized by Riluzole

In a recent study we found that cerebrospinal fluids (CSFs) from amyotrophic lateral sclerosis (ALS) patients caused 20-30% loss of cell viability in primary cultures of rat embryo motor cortex neurons. We also found that the antioxidant resveratrol protected against such damaging effects and that, surprisingly, riluzole antagonized its protecting effects. Here we have extended this study to the interactions of riluzole with 3 other recognized neuroprotective agents, namely memantine, minocycline and lithium. We found: (1) by itself riluzole exerted neurotoxic effects at concentrations of 3-30 µ<smlcap>M</smlcap>; this cell damage was similar to that elicited by 30 µ<smlcap>M</smlcap> glutamate and a 10% dilution of ALS/CSF; (2) memantine (0.1-30 µ<smlcap>M</smlcap>), minocycline (0.03-1 µ<smlcap>M</smlcap>) and lithium (1-80 µg/ml) afforded 10-30% protection against ALS/CSF-elicited neurotoxicity, and (3) at 1-10 µ<smlcap>M</smlcap>, riluzole antagonized the protection afforded by the 3 agents. These results strongly support the view that at the riluzole concentrations reached in the brain of patients, the neurotoxic effects of this drug could be masking the potential neuroprotective actions of new compounds being tested in clinical trials. Therefore, in the light of the present results, the inclusion of a group of patients free of riluzole treatment may be mandatory in future clinical trials performed in ALS patients with novel neuroprotective compounds.

Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72 Repeat Expansion

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice

BackgroundThe developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis.ResultsShh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture.ConclusionsShh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

Therapeutic Potential of Mesenchymal Stromal Cells and MSC Conditioned Medium in Amyotrophic Lateral Sclerosis (ALS) - In Vitro Evidence from Primary Motor Neuron Cultures, NSC-34 Cells, Astrocytes and Microglia

Administration of mesenchymal stromal cells (MSC) improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS) as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice), NSC-34 cells and glial cells (astrocytes, microglia) (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice) in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM) attenuated staurosporine (STS) - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF) in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1) was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

Prolonged Minocycline Treatment Impairs Motor Neuronal Survival and Glial Function in Organotypic Rat Spinal Cord Cultures

BackgroundMinocycline, a second-generation tetracycline antibiotic, exhibits anti-inflammatory and neuroprotective effects in various experimental models of neurological diseases, such as stroke, Alzheimer’s disease, amyotrophic lateral sclerosis and spinal cord injury. However, conflicting results have prompted a debate regarding the beneficial effects of minocycline.MethodsIn this study, we analyzed minocycline treatment in organotypic spinal cord cultures of neonatal rats as a model of motor neuron survival and regeneration after injury. Minocycline was administered in 2 different concentrations (10 and 100 µM) at various time points in culture and fixed after 1 week.ResultsProlonged minocycline administration decreased the survival of motor neurons in the organotypic cultures. This effect was strongly enhanced with higher concentrations of minocycline. High concentrations of minocycline reduced the number of DAPI-positive cell nuclei in organotypic cultures and simultaneously inhibited microglial activation. Astrocytes, which covered the surface of the control organotypic cultures, revealed a peripheral distribution after early minocycline treatment. Thus, we further analyzed the effects of 100 µM minocycline on the viability and migration ability of dispersed primary glial cell cultures. We found that minocycline reduced cell viability, delayed wound closure in a scratch migration assay and increased connexin 43 protein levels in these cultures.ConclusionsThe administration of high doses of minocycline was deleterious for motor neuron survival. In addition, it inhibited microglial activation and impaired glial viability and migration. These data suggest that especially high doses of minocycline might have undesired affects in treatment of spinal cord injury. Further experiments are required to determine the conditions for the safe clinical administration of minocycline in spinal cord injured patients.

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)-ATP. The P2X7 inhibitors BBG, oATP, and KN-62 prevented 2'(3')-O-(4-Benzoylbenzoyl)-ATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100μM). High concentrations of ATP (1mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons.

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor.

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1(G93A) transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1(G93A) by gene transfer or in presymptomatic SOD1(G93A) transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.

Frataxin Deficiency Leads to Reduced Expression and Impaired Translocation of NF-E2-Related Factor (Nrf2) in Cultured Motor Neurons

Oxidative stress has been implicated in the pathogenesis of Friedreich’s Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein responsible of iron homeostasis. Under conditions of oxidative stress, the activation of the transcription factor NF-E2-related factor (Nrf2) triggers the antioxidant cellular response by inducing antioxidant response element (ARE) driven genes. Increasing evidence supports a role for the Nrf2-ARE pathway in neurodegenerative diseases. In this study, we analyzed the expression and the distribution of Nrf2 in silenced neurons for frataxin gene. Decreased Nrf2 mRNA content and a defective activation after treatment with pro-oxidants have been evidenced in frataxin-silenced neurons by RT-PCR and confocal microscopy. The loss of Nrf2 in FRDA may greatly enhance the cellular susceptibility to oxidative stress and make FRDA neurons more vulnerable to injury. Our findings may help to focus on this promising target, especially in its emerging role in the neuroprotective response.

Purification and Culture of Spinal Motor Neurons

Motor neurons are responsible for voluntary movement. Lower motor neurons are characterized by large soma, the potential to form very long axons, and wide-ranging dendritic arborization. They receive direction from various neuronal cell types and induce movement of skeletal muscle fibers through acetylcholine release at the neuromuscular junction. Each lower motor neuron can communicate with 10 to several hundred muscle fibers at firing rates modulated by the balance of ongoing neurotransmitter signaling. Disease and trauma that affect lower motor neurons can cause paralysis and, in some cases, death. Studies using primary cultures of these cells have ongoing potential to facilitate a deeper understanding of their biology and function.

Purification and Culture of Spinal Motor Neurons from Rat Embryos

We describe an immunopanning protocol to isolate, enrich, and culture spinal motor neurons from rat embryonic spinal cords. The method takes advantage of several distinct properties of rat lower motor neurons to isolate them from neighboring cells. First, an ideal stage in development after motor neurons are born (embryonic day 14 during rat gestation), but prior to extensive axonal extension or developmental apoptosis, is exploited. Lower motor neurons cannot be viably isolated using this method after birth. After dissociating embryonic spinal cord tissue, which contains lower motor neurons among many other cell types, the uniquely large motor neurons are enriched using density gradient centrifugation. Finally, the collected cell population is further purified based on selective immunopanning for motor neurons, which express the low-affinity nerve growth factor (NGF) receptor often referred to as p75. The near-pure lower motor neuron cultures are plated and seeded in defined conditions optimal for survival and can be maintained for several weeks. The expected yield is approximately 70,000 cells per embryonic spinal cord.

Acidotoxicity and acid-sensing ion channels contribute to motoneuron degeneration

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition with no cure. Mitochondrial dysfunction, Ca(2+) overloading and local hypoxic/ischemic environments have been implicated in the pathophysiology of ALS and are conditions that may initiate metabolic acidosis in the affected tissue. We tested the hypothesis that acidotoxicity and acid-sensing ion channels (ASICs) are involved in the pathophysiology of ALS. We found that motoneurons were selectively vulnerable to acidotoxicity in vitro, and that acidotoxicity was partially reduced in asic1a-deficient motoneuron cultures. Cross-breeding of SOD1(G93A) ALS mice with asic1a-deficient mice delayed the onset and progression of motor dysfunction in SOD1 mice. Interestingly, we also noted a strong increase in ASIC2 expression in motoneurons of SOD1 mice and sporadic ALS patients during disease progression. Pharmacological pan-inhibition of ASIC channels with the lipophilic amiloride derivative, 5-(N,N-dimethyl)-amiloride hydrochloride, potently protected cultured motoneurons against acidotoxicity, and, given post-symptom onset, significantly improved lifespan, motor performance and motoneuron survival in SOD1 mice. Together, our data provide strong evidence for the involvement of acidotoxicity and ASIC channels in motoneuron degeneration, and highlight the potential of ASIC inhibitors as a new treatment approach for ALS.

Motor neurons and glia exhibit specific individualized responses to TDP-43 expression in a Drosophila model of amyotrophic lateral sclerosis

SUMMARYAmyotrophic lateral sclerosis (ALS) is a fatal disease characterized by complex neuronal and glial phenotypes. Recently, RNA-based mechanisms have been linked to ALS via RNA-binding proteins such as TDP-43, which has been studied in vivo using models ranging from yeast to rodents. We have developed a Drosophila model of ALS based on TDP-43 that recapitulates several aspects of pathology, including motor neuron loss, locomotor dysfunction and reduced survival. Here we report the phenotypic consequences of expressing wild-type and four different ALS-linked TDP-43 mutations in neurons and glia. We show that TDP-43-driven neurodegeneration phenotypes are dose- and age-dependent. In motor neurons, TDP-43 appears restricted to nuclei, which are significantly misshapen due to mutant but not wild-type protein expression. In glia and in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta. TDP-43-containing RNA granules are motile in cultured motor neurons, although wild-type and mutant variants exhibit different kinetic properties. At the neuromuscular junction, the expression of TDP-43 in motor neurons versus glia leads to seemingly opposite synaptic phenotypes that, surprisingly, translate into comparable locomotor defects. Finally, we explore sleep as a behavioral readout of TDP-43 expression and find evidence of sleep fragmentation consistent with hyperexcitability, a suggested mechanism in ALS. These findings support the notion that although motor neurons and glia are both involved in ALS pathology, at the cellular level they can exhibit different responses to TDP-43. In addition, our data suggest that individual TDP-43 alleles utilize distinct molecular mechanisms, which will be important for developing therapeutic strategies.