Nicole Hersmus

Astrocytes regulate GluR2 expression in motor neurons and their vulnerability to excitotoxicity

Influx of Ca2+ ions through α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca2+ permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca2+ ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.

Oligodendrocyte dysfunction in the pathogenesis of amyotrophic lateral sclerosis

Oligodendrocytes are well known targets for immune-mediated and infectious diseases, and have been suggested to play a role in neurodegeneration. Here, we report the involvement of oligodendrocytes and their progenitor cells in the ventral grey matter of the spinal cord in amyotrophic lateral sclerosis, a neurodegenerative disease of motor neurons. Degenerative changes in oligodendrocytes were abundantly present in human patients with amyotrophic lateral sclerosis and in an amyotrophic lateral sclerosis mouse model. In the mouse model, morphological changes in grey matter oligodendrocytes became apparent before disease onset, increasingly so during disease progression, and oligodendrocytes ultimately died. This loss was compensated by increased proliferation and differentiation of oligodendrocyte precursor cells. However, these newly differentiated oligodendrocytes were dysfunctional as suggested by their reduced myelin basic protein and monocarboxylate transporter 1 expression. Mutant superoxide dismutase 1 was found to directly affect monocarboxylate transporter 1 protein expression. Our data suggest that oligodendroglial dysfunction may be a contributor to motor neuron degeneration in amyotrophic lateral sclerosis.

VEGF protects motor neurons against excitotoxicity by upregulation of GluR2

Influx of Ca(2+) ions through the α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptors is toxic to neurons and contributes to motor neuron degeneration observed in amyotrophic lateral sclerosis (ALS). The Ca(2+) permeability of the AMPA receptor depends on its subunit composition. If the GluR2 subunit is present in the receptor complex, the AMPA receptor is impermeable to Ca(2+). In this study, we identified vascular endothelial growth factor-A (VEGF) as a GluR2 inducing molecule. Cultured motor neurons pretreated with VEGF displayed higher GluR2 levels. This resulted in AMPA receptor currents with a low relative Ca(2+) permeability and in motor neurons that were less vulnerable to AMPA receptor-mediated excitotoxicity. This effect of VEGF was mediated through the VEGFR2 present on the motor neurons and was due to stimulation of GluR2 transcription. Intracerebroventricular treatment with VEGF similarly induced GluR2 expression in the ventral spinal cord of rats and this mechanism contributes to the protective effect of VEGF on motor neurons.

Hdac6 deletion delays disease progression in the SOD1G93A mouse model of ALS

Defects in axonal transport are thought to contribute to the pathogenesis of neurodegenerative disease. Because α-tubulin acetylation facilitates axonal transport, inhibition of the α-tubulin deacetylating enzymes, histone deacetylase 6 (Hdac6) and silent information regulator 2 (Sirt2), is thought to be an interesting therapeutic strategy for these conditions. Amyotrophic lateral sclerosis (ALS) is a one such rapidly progressive and fatal neurodegenerative disorder, in which axonal transport defects have been found in vitro and in vivo. To establish whether the inhibition of Hdac6 or Sirt2 may be of interest for ALS treatment, we investigated whether deleting Hdac6 or Sirt2 from the superoxide dismutase 1, SOD1(G93A) mouse affects the motor neuron degeneration in this ALS model. Deletion of Hdac6 significantly extended the survival of SOD1(G93A) mice without affecting disease onset, and maintained motor axon integrity. This protective effect was associated with increased α-tubulin acetylation. Deletion of Sirt2 failed to affect the disease course, but also did not modify α-tubulin acetylation. These findings show that Hdac6, rather than Sirt2, is a therapeutic target for the treatment of ALS. Moreover, Sirt2 appears not to be a major α-tubulin deacetylase in the nervous system.

The occurrence of mutations in FUS in a Belgian cohort of patients with familial ALS.

Background and purpose:  Mutations in fused in sarcoma (FUS) were recently identified as a cause of familial amyotrophic lateral sclerosis (ALS). The frequency of occurrence of mutations in FUS in sets of patients with familial ALS remains to be established.

Melanocortin Peptides Stimulate Prolactin Gene Expression and Prolactin Accumulation in Rat Pituitary Aggregate Cell Cultures

Treatment for 40 h of reaggregate pituitary cell cultures from 14‐day‐old female rats with nanomolar concentrations of γ3‐melanocyte‐stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR). During the 40 h incubation, γ3‐MSH stimulated prolactin accumulation in the culture medium. α‐MSH, a potent agonist of the rat melanocortin‐3 receptor (MC3R) and Ala8‐γ2‐MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as γ3‐MSH. The effect of γ3‐MSH on prolactin mRNA expression was abolished when aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol. By contrast, oestradiol abolished the stimulatory effect of Ala8‐γ2‐MSH on prolactin mRNA expression. In GH3 cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3‐kb prolactin promoter fragment, a dose as low as 1 nMγ3‐MSH, added for 24 h, significantly increased eGFP fluorescence. Agouti‐related protein (AgRP83−132), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by γ3‐MSH or Ala8‐γ2‐MSH. On its own, AgRP83−132 significantly increased prolactin mRNA expression level and prolactin accumulation. Both γ2‐MSH and Ala8‐γ2‐MSH increased [S35]GTPγS binding in membrane preparations of 14‐day‐old rat pituitaries and of GH3 cells. Whereas MC3R and MC5R mRNA were detectable by RT‐PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 cells using various oligonucleotide primer sets. The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved. AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.

Neuronal overexpression of IP3 receptor 2 is detrimental in mutant SOD1 mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing progressive paralysis of the patient followed by death on average 3-5 years after diagnosis. Disease pathology is multi-factorial including the process of excitotoxicity that induces cell death by cytosolic Ca(2+) overload. In this study, we increased the neuronal expression of an endoplasmic reticulum (ER) Ca(2+) release channel, inositol 1,4,5-trisphosphate receptor 2 (IP(3)R2), to assess whether increased cytosolic Ca(2+) originating from the ER is detrimental for neurons. Overexpression of IP(3)R2 in N2a cells using a Thy1.2-IP(3)R2 construct increases cytosolic Ca(2+) concentrations evoked by bradykinin. In addition, mice generated from this construct have increased expression of IP(3)R2 in the spinal cord and brain. This overexpression of IP(3)R2 does not affect symptom onset, but decreases disease duration and shortens the lifespan of the ALS mice significantly. These data suggest that ER Ca(2+) released by IP(3) receptors may be detrimental in ALS and that motor neurons are vulnerable to impaired Ca(2+) metabolism.

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism

The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine–arginine and proline–arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD.

β1-Adrenoceptor Expression in Rat Anterior Pituitary Gonadotrophs and in Mouse αT3-1 and LβT2 Gonadotrophic Cell Lines

The rat anterior pituitary expresses beta(2)-adrenoceptors (ARs) on somatotrophs, lactotrophs, and corticotrophs. The present study investigates whether beta(1)-ARs exist in the anterior pituitary, in which cell type(s) they are found, and whether they are regulated by glucocorticoids. As determined by quantitative RT-PCR and Western immunoblotting, the rat anterior pituitary expressed beta(1)-AR mRNA and protein. Unlike the beta(2)-AR, expression decreased to very low levels after 5-d aggregate cell culture but was strongly up-regulated in a dose- and time-dependent manner by dexamethasone (DEX). Glucocorticoids attenuated isoproterenol-induced down-regulation of beta(1)-AR mRNA levels. As examined by immunofluorescence confocal microscopy, beta(1)-AR immunoreactivity was detected in a subpopulation of gonadotrophs, but not in somatotrophs, lactotrophs, corticotrophs, thyrotrophs, or folliculo-stellate cells. beta(1)-AR-immunoreactivity cells were often surrounded by cup-shaped lactotrophs. Consistent with these findings, beta(1)-AR mRNA was considerably more abundant in the gonadotrophic alphaT3-1 and LbetaT2 cell lines than in the GHFT, GH3, and TtT/GF cell lines. DEX did not affect expression level in the cell lines. DEX also failed to up-regulate beta(1)-AR mRNA levels in aggregates from a subpopulation enriched in large gonadotrophs obtained by gradient sedimentation. In contrast, excessive DEX-dependent up-regulation of beta(1)-AR mRNA was found in a subpopulation enriched in small nonhormonal cells. The present data indicate that beta(1)-AR is expressed in a subpopulation of gonadotrophs with a topographical relationship to lactotrophs. However, the glucocorticoid-induced up-regulation does not seem to occur directly in the gonadotrophs but within (an)other unidentified cell type(s), or is transduced by that cell type on gonadotrophs.

Molecular Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains in Toxicity

Summary RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.