Profiling the genes affected by pathogenic TDP‐43 in astrocytes

Mutation in TAR DNA binding protein 43 (TDP‐43) is a causative factor of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurodegeneration may not require the presence of pathogenic TDP‐43 in all types of relevant cells. Rather, expression of pathogenic TDP‐43 in neurons or astrocytes alone is sufficient to cause cell‐autonomous or non‐cell‐autonomous neuron death in transgenic rats. How pathogenic TDP‐43 in astrocytes causes non‐cell‐autonomous neuron death, however, is not clear. Here, we examined the effect of pathogenic TDP‐43 on gene expression in astrocytes. Microarray assay revealed that pathogenic TDP‐43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down‐regulated, neurotoxic genes were up‐regulated. Representative genes Lcn2 and chitinase‐3‐like protein 1 were markedly up‐regulated in astrocytes from primary culture and intact transgenic rats. Furthermore, synthetic chitinase‐3‐like protein 1 induced neuron death in a dose‐dependent manner. Our results suggest that TDP‐43 pathogenesis is associated with the simultaneous induction of multiple neurotoxic genes in astrocytes, which may synergistically produce adverse effects on neuronal survival and contribute to non‐cell‐autonomous neuron death.

     

Expression of ALS‐linked TDP‐43 mutant in astrocytes causes non‐cell‐autonomous motor neuron death in rats

Mutation of Tar DNA‐binding protein 43 (TDP‐43) is linked to amyotrophic lateral sclerosis. Although astrocytes have important roles in neuron function and survival, their potential contribution to TDP‐43 pathogenesis is unclear. Here, we created novel lines of transgenic rats that express a mutant form of human TDP‐43 (M337V substitution) restricted to astrocytes. Selective expression of mutant TDP‐43 in astrocytes caused a progressive loss of motor neurons and the denervation atrophy of skeletal muscles, resulting in progressive paralysis. The spinal cord of transgenic rats also exhibited a progressive depletion of the astroglial glutamate transporters GLT‐1 and GLAST. Astrocytic expression of mutant TDP‐43 led to activation of astrocytes and microglia, with an induction of the neurotoxic factor Lcn2 in reactive astrocytes that was independent of TDP‐43 expression. These results indicate that mutant TDP‐43 in astrocytes is sufficient to cause non‐cell‐autonomous death of motor neurons. This motor neuron death likely involves deficiency in neuroprotective genes and induction of neurotoxic genes in astrocytes.     

TDP‐43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA‐binding protein 43 (TDP‐43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP‐43 is a multi‐functional protein involved in RNA processing and a large number of TDP‐43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP‐43‐linked neurodegeneration remain elusive. In this study, we found that loss of TDP‐43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy–lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP‐43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP‐43‐depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP‐43‐mediated neurodegeneration.     

Lack of TNF‐alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression

Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1‐G93A co‐cultures. Deleting TNFR2 from SOD1‐G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1‐G93A/TNFR2?/? mice showed high phospho‐TAR DNA‐binding protein 43 (TDP‐43) accumulation and low levels of acetyl‐tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane‐bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology.

     

MicroRNA‐183‐5p is stress‐inducible and protects neurons against cell death in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. A fundamental pathogenesis of ALS is the prolonged cell stress in neurons, which is caused by either accumulation of protein aggregates or reactive oxygen species. However, the mechanistic link between stress sensing and cell death is unsettled. Here, we identify that miR‐183‐5p, a neuron‐enriched miRNA, couples stress sensing and cell death programming in ALS. miR‐183‐5p is immediately induced by hydrogen peroxide, tunicamycin or TNF‐α in neurons. The overexpression of miR‐183‐5p increases neuron survival under stress conditions, whereas its knockdown causes neuron death. miR‐183‐5p coordinates apoptosis and necroptosis pathways by directly targeting PDCD4 and RIPK3, and thus protects neurons against cell death under stress conditions. The consistent reduction of miR‐183‐5p in ALS patients and mouse models enhances the notion that miR‐183‐5p is a central regulator of motor neuron survival under stress conditions. Our study supplements current understanding of the mechanistic link between cell stress and death/survival, and provides novel targets for clinical interventions of ALS.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Substantially elevating the levels of αB‐crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation

There has been great interest in enhancing endogenous protein maintenance pathways such as the heat‐shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in amyotrophic lateral sclerosis and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co‐expression of αB‐crystallin (αB‐crys) has been shown to inhibit the formation of detergent‐insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB‐crys at > 6‐fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB‐crys alone contained 20% more motor neurons per section than littermate controls. Raising αB‐crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent‐insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB‐crys in spinal motor neurons by 6‐fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation.

     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

Exposure to predator odor and resulting anxiety enhances the expression of the α2δ subunit of voltage‐sensitive calcium channels in the amygdala

The α₂δ subunit of voltage‐sensitive calcium channels (VSCCs) is the molecular target of pregabalin and gabapentin, two drugs marked for the treatment of focal epilepsy, neuropathic pain, and anxiety disorders. Expression of the α₂δ subunit is up‐regulated in the dorsal horns of the spinal cord in models of neuropathic pain, suggesting that plastic changes in the α₂δ subunit are associated with pathological states. Here, we examined the expression of the α₂δ‐1 subunit in the amygdala, hippocampus, and frontal cortex in the trimethyltiazoline (TMT) mouse model of innate anxiety. TMT is a volatile molecule present in the feces of the rodent predator, red fox. Mice that show a high defensive behavior during TMT exposure developed anxiety‐like behavior in the following 72 h, as shown by the light–dark test. Anxiety was associated with an increased expression of the α₂δ‐1 subunit of VSCCs in the amygdaloid complex at all times following TMT exposure (4, 24, and 72 h). No changes in the α₂δ‐1 protein levels were seen in the hippocampus and frontal cortex of mice exposed to TMT. Pregabalin (30 mg/kg, i.p.) reduced anxiety‐like behavior in TMT‐exposed mice, but not in control mice. These data offer the first demonstration that the α₂δ‐1 subunit of VSCCs undergoes plastic changes in a model of innate anxiety, and supports the use of pregabalin as a disease‐dependent drug in the treatment of anxiety disorders.     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

Transport of the alpha subunit of the voltage gated L‐type calcium channel through the sarcoplasmic reticulum occurs prior to localization to triads and requires the beta subunit but not Stac3 in skeletal muscles

Contraction of skeletal muscle is initiated by excitation‐contraction (EC) coupling during which membrane voltage is transduced to intracellular Ca²⁺ release. EC coupling requires L‐type voltage gated Ca₂₊ channels (the dihydropyridine receptor or DHPR) located at triads, which are junctions between the transverse (T) tubule and sarcoplasmic reticulum (SR) membranes, that sense membrane depolarization in the T tubule membrane. Reduced EC coupling is associated with ageing, and disruptions of EC coupling result in congenital myopathies for which there are few therapies. The precise localization of DHPRs to triads is critical for EC coupling, yet trafficking of the DHPR to triads is not well understood. Using dynamic imaging of zebrafish muscle fibers, we find that DHPR is transported along the longitudinal SR in a microtubule‐independent mechanism. Furthermore, transport of DHPR in the SR membrane is differentially affected in null mutants of Stac3 or DHPRβ, two essential components of EC coupling. These findings reveal previously unappreciated features of DHPR motility within the SR prior to assembly at triads.      

The ROS‐induced cytotoxicity of ascorbate is attenuated by hypoxia and HIF‐1alpha in the NCI60 cancer cell lines

Intravenous application of high‐dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate‐mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC₅₀ values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride‐induced hypoxia‐inducible factor‐1α (HIF‐1α) and the glucose transporter 1 (GLUT‐1) expression (a pro‐survival HIF‐1α‐downstream‐target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O₂) globally increased the IC₅₀ of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM (2.2‐fold increase, P < 0.001, Mann–Whitney t‐test), thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF‐1α‐signalling, but did not correlate with cell line‐specific expression of the ascorbate transporter GLUT‐1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC₅₀ reduced the expression of GLUT‐1 in the cancer cells. Our data show a ROS‐induced, HIF‐1α‐ and O₂‐dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.