Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Inhibition of ß-Glucocerebrosidase Activity Preserves Motor Unit Integrity in a Mouse Model of Amyotrophic Lateral Sclerosis.

Recent metabolomic reports connect dysregulation of glycosphingolipids, particularly ceramide and glucosylceramide, to neurodegeneration and to motor unit dismantling in amyotrophic lateral sclerosis at late disease stage. We report here altered levels of gangliosides in the cerebrospinal fluid of amyotrophic lateral sclerosis patients in early disease stage. Conduritol B epoxide is an inhibitor of acid beta-glucosidase, and lowers glucosylceramide degradation. Glucosylceramide is the precursor for all of the more complex glycosphingolipids. In SOD1G86R mice, an animal model of amyotrophic lateral sclerosis, conduritol B epoxide preserved ganglioside distribution at the neuromuscular junction, delayed disease onset, improved motor function and preserved motor neurons as well as neuromuscular junctions from degeneration. Conduritol B epoxide mitigated gene dysregulation in the spinal cord and restored the expression of genes involved in signal transduction and axonal elongation. Inhibition of acid beta-glucosidase promoted faster axonal elongation in an in vitro model of neuromuscular junctions and hastened recovery after peripheral nerve injury in wild type mice. Here, we provide evidence that glycosphingolipids play an important role in muscle innervation, which degenerates in amyotrophic lateral sclerosis from the early disease stage. This is a first proof of concept study showing that modulating the catabolism of glucosylceramide may be a therapeutic target for this devastating disease.

Autophagy in neuroinflammatory diseases.

Autophagy is a metabolically-central process that is crucial in diverse areas of cell physiology. It ensures a fair balance between life and death molecular and cellular flows, and any disruption in this vital intracellular pathway can have consequences leading to major diseases such as cancer, metabolic and neurodegenerative disorders, and cardiovascular and pulmonary diseases. Recent pharmacological studies have shown evidence that small molecules and peptides able to activate or inhibit autophagy might be valuable therapeutic agents by down- or up-regulating excessive or defective autophagy, or to modulate normal autophagy to allow other drugs to repair some cell alteration or destroy some cell subsets (e.g. in the case of cancer concurrent treatments). Here, we provide an overview of neuronal autophagy and of its potential implication in some inflammatory diseases of central and peripheral nervous systems. Based on our own studies centred on a peptide called P140 that targets autophagy, we highlight the validity of autophagy processes, and in particular of chaperone-mediated autophagy, as a particularly pertinent pathway for developing novel selective therapeutic approaches for treating some neuronal diseases. Our findings with the P140 peptide support a direct cross-talk between autophagy and certain central and peripheral neuronal diseases. They also illustrate the fact that autophagy alterations are not evenly distributed across all organs and tissues of the same individual, and can evolve in different stages along the disease course.

Late-Life Environmental Enrichment Induces Acetylation Events and Nuclear Factor κB-Dependent Regulations in the Hippocampus of Aged Rats Showing Improved Plasticity and Learning.

Aging weakens memory functions. Exposing healthy rodents or pathological rodent models to environmental enrichment (EE) housing improves their cognitive functions by changing neuronal levels of excitation, cellular signaling, and plasticity, notably in the hippocampus. At the molecular level, brain derived-neurotrophic factor (BDNF) represents an important player that supports EE-associated changes. EE facilitation of learning was also shown to correlate with chromatin acetylation in the hippocampus. It is not known, however, whether such mechanisms are still into play during aging. In this study, we exposed a cohort of aged rats (18-month-old) to either a 6 month period of EE or standard housing conditions and investigated chromatin acetylation-associated events [histone acetyltranferase activity, gene expression, and histone 3 (H3) acetylation] and epigenetic modulation of the Bdnf gene under rest conditions and during learning. We show that EE leads to upregulation of acetylation-dependent mechanisms in aged rats, whether at rest or following a learning challenge. We found an increased expression of Bdnf through Exon-I-dependent transcription, associated with an enrichment of acetylated H3 at several sites of Bdnf promoter I, more particularly on a proximal nuclear factor κB (NF-κB) site under learning conditions. We further evidenced p65/NF-κB binding to chromatin at promoters of genes important for plasticity and hippocampus-dependent learning (e.g., Bdnf, CamK2D). Altogether, our findings demonstrate that aged rats respond to a belated period of EE by increasing hippocampal plasticity, together with activating sustained acetylation-associated mechanisms recruiting NF-κB and promoting related gene transcription. These responses are likely to trigger beneficial effects associated with EE during aging. SIGNIFICANCE STATEMENT: Aging weakens memory functions. Optimizing the neuronal circuitry required for normal brain function can be achieved by increasing sensory, motor, and cognitive stimuli resulting from interactions with the environment (behavioral therapy). This can be experimentally modeled by exposing rodents to environmental enrichment (EE), as with large cages, numerous and varied toys, and interaction with other rodents. However, EE effects in aged rodents has been poorly studied, and it is not known whether beneficial mechanisms evidenced in the young adults can still be recruited during aging. Our study shows that aged rats respond to a belated period of EE by activating specific epigenetic and transcriptional signaling that promotes gene expression likely to facilitate plasticity and learning behaviors.

Induction of caspase 8 and reactive oxygen species by ruthenium-derived anticancer compounds with improved water solubility and cytotoxicity.

Organometallic compounds which contain metals, such as ruthenium or gold, have been investigated as a replacement for platinum-derived anticancer drugs. They often show good antitumor effects, but the identification of their precise mode of action or their pharmacological optimization is still challenging. We have previously described a class of ruthenium(II) compounds with interesting anticancer properties. In comparison to cisplatin, these molecules have lower side effects, a reduced ability to interact with DNA, and they induce cell death in absence of p53 through CHOP/DDIT3. We have now optimized these molecules by improving their cytotoxicity and their water solubility. In this article, we demonstrate that by changing the ligands around the ruthenium we modify the ability of the compounds to interact with DNA. We show that these optimized molecules reduce tumor growth in different mouse models and retain their ability to induce CHOP/DDIT3. However, they are more potent inducers of cancer cell death and trigger the production of reactive oxygen species and the activation of caspase 8. More importantly, we show that blocking reactive oxygen species production or caspase 8 activity reduces significantly the activity of the compounds. Altogether our data suggest that water-soluble ruthenium(II)-derived compounds represent an interesting class of molecules that, depending on their structures, can target several pro-apoptotic signaling pathways leading to reactive oxygen species production and caspase 8 activation.

Complex regulation of p73 isoforms after alteration of amyloid precursor polypeptide (APP) function and DNA damage in neurons.

Genetic ablations of p73 have shown its implication in the development of the nervous system. However, the relative contribution of ΔNp73 and TAp73 isoforms in neuronal functions is still unclear. In this study, we have analyzed the expression of these isoforms during neuronal death induced by alteration of the amyloid-ß precursor protein function or cisplatin. We observed a concomitant up-regulation of a TAp73 isoform and a down-regulation of a ΔNp73 isoform. The shift in favor of the pro-apoptotic isoform correlated with an induction of the p53/p73 target genes such as Noxa. At a functional level, we showed that TAp73 induced neuronal death and that ΔNp73 has a neuroprotective role toward amyloid-ß precursor protein alteration or cisplatin. We investigated the mechanisms of p73 expression and found that the TAp73 expression was regulated at the promoter level. In contrast, regulation of ΔNp73 protein levels was regulated by phosphorylation at residue 86 and multiple proteases. Thus, this study indicates that tight transcriptional and post-translational mechanisms regulate the p73 isoform ratios that play an important role in neuronal survival.

A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons.

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntington's disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150(Glued). However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD.

P-glycoprotein expression and function are increased in an animal model of amyotrophic lateral sclerosis.

The efflux pumps located at the blood-brain barrier (BBB) prevent drugs entering the brain. As such, efflux pumps are a major obstacle to drug brain distribution. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with little therapeutics available: riluzole is the only drug approved in its treatment. The lack of response to treatment in ALS may be, at least in part, due to increased activities of efflux pumps in relation to disease, leading to subtherapeutic brain concentrations of drugs. In the present study, we used a transgenic mouse model of ALS (G86R mSOD1 mice) to test this hypothesis. Expression and functionality of P-glycoprotein (ABCB1, P-gp) and Breast Cancer Resistance Protein (ABCG2, BCRP), two major efflux pumps, were studied. We observed an increased P-gp expression (1.5-fold) in presymptomatic mSOD1 mice compared to wild-type controls. Consistent with this, P-gp function was also increased by 1.5-fold and riluzole brain disposition was decreased by 1.7-fold in mSOD1 mice. Contrasting with this, BCRP expression and function were unaltered by the pathology. These results demonstrate that BBB transport proteins are modified in G86R mSOD1 mice ALS model. Such findings underline potential problems in extrapolating the results of animal studies to humans and developing clinical trials, especially for drugs transported by P-gp.

Impaired glucose tolerance in patients with amyotrophic lateral sclerosis.

Our objectives were to analyse carbohydrate metabolism in a series of ALS patients and to examine potential association with parameters of lipid metabolism and clinical features. Glucose tolerance was assessed by the oral glucose tolerance test in 21 non-diabetic ALS patients and compared with 21 age- and sex-matched normal subjects. Lipids and lactate/pyruvate ratio, levels of pro-inflammatory cytokines (tumour necrosis factor-alpha and interleukin-6) and adipocytokines (leptin and adiponectin) were also measured in ALS patients. Mann-Whitney U-tests analysed continuous data and Fisher's exact tests assessed categorical data. Blood glucose determined 120 min after the glucose bolus was significantly higher in patients with ALS (7.41 mmol/l+/-1.68) compared to controls (6.05+/-1.44, p=0.006). ALS patients with impaired glucose tolerance (IGT) according to WHO criteria (n=7, 33%) were more likely to have elevated free fatty acids (FFA) levels compared to patients with normal glucose tolerance (0.77 nmol/l+/-0.30 vs. 0.57+/-0.19, p=0.04). IGT was not associated with disease duration or severity. In conclusion, patients with ALS show abnormal glucose tolerance that could be associated with increased FFA levels, a key determinant of insulin resistance. The origin of glucose homeostasis abnormalities in ALS may be multifactorial and deserves further investigation.

A ruthenium-containing organometallic compound reduces tumor growth through induction of the endoplasmic reticulum stress gene CHOP.

Cisplatin-derived anticancer therapy has been used for three decades despite its side effects. Other types of organometallic complexes, namely, some ruthenium-derived compounds (RDC), which would display cytotoxicity through different modes of action, might represent alternative therapeutic agents. We have studied both in vitro and in vivo the biological properties of RDC11, one of the most active compounds of a new class of RDCs that contain a covalent bond between the ruthenium atom and a carbon. We showed that RDC11 inhibited the growth of various tumors implanted in mice more efficiently than cisplatin. Importantly, in striking contrast with cisplatin, RDC11 did not cause severe side effects on the liver, kidneys, or the neuronal sensory system. We analyzed the mode of action of RDC11 and showed that RDC11 interacted poorly with DNA and induced only limited DNA damages compared with cisplatin, suggesting alternative transduction pathways. Indeed, we found that target genes of the endoplasmic reticulum stress pathway, such as Bip, XBP1, PDI, and CHOP, were activated in RDC11-treated cells. Induction of the transcription factor CHOP, a crucial mediator of endoplasmic reticulum stress apoptosis, was also confirmed in tumors treated with RDC11. Activation of CHOP led to the expression of several of its target genes, including proapoptotic genes. In addition, the silencing of CHOP by RNA interference significantly reduced the cytotoxicity of RDC11. Altogether, our results led us to conclude that RDC11 acts by an atypical pathway involving CHOP and endoplasmic reticulum stress, and thus might provide an interesting alternative for anticancer therapy.

Electrophysiological studies in a mouse model of Schwartz-Jampel syndrome demonstrate muscle fiber hyperactivity of peripheral nerve origin.

Schwartz-Jampel syndrome (SJS) is an autosomal-recessive condition characterized by muscle stiffness and chondrodysplasia. It is due to loss-of-function hypomorphic mutations in the HSPG2 gene that encodes for perlecan, a proteoglycan secreted into the basement membrane. The origin of muscle stiffness in SJS is debated. To resolve this issue, we performed an electrophysiological investigation of an SJS mouse model with a missense mutation in the HSPG2 gene. Compound muscle action potential amplitudes, distal motor latencies, repetitive nerve stimulation tests, and sensory nerve conduction velocities of SJS mice were normal. On electromyography (EMG), neuromyotonic discharges, that is, bursts of motor unit action potentials firing at high rates (120-300 HZ), were constantly observed in SJS mice in all muscles, except in the diaphragm. Neuromyotonic discharges were not influenced by general anesthesia and disappeared with curare administration. They persisted after complete motor nerve section, terminating only with Wallerian degeneration. These results demonstrate that perlecan deficiency in SJS provokes a neuromyotonic syndrome. The findings further suggest a distal axonal localization of the generator of neuromyotonic discharges. SJS should now be considered as an inherited disorder with peripheral nerve hyperexcitability.

Mice with a mutation in the dynein heavy chain 1 gene display sensory neuropathy but lack motor neuron disease.

In neurons, cytoplasmic dynein functions as a molecular motor responsible for retrograde axonal transport. An impairment of axonal transport is thought to play a key role in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis, the most frequent motor neuron disease in the elderly. In this regard, previous studies described two heterozygous mouse strains bearing missense point mutations in the dynein heavy chain 1 gene that were reported to display late-onset progressive motor neuron degeneration. Here we show, however, that one of these mutant strains, the so-called Cra mice does not suffer from motor neuron loss, even in aged animals. Consistently, we did not observe electrophysiological or biochemical signs of muscle denervation, indicative of motor neuron disease. The "hindlimb clasping" phenotype of Cra mice could rather be due to the prominent degeneration of sensory neurons associated with a loss of muscle spindles. Altogether, these findings show that dynein heavy chain mutation triggers sensory neuropathy rather than motor neuron disease.

Diffusion-weighted MRI of denervated muscle: a clinical and experimental study.

OBJECTIVE: The aim of this study was to investigate skeletal muscle denervation using diffusion-weighted magnetic resonance imaging (DWMRI). MATERIALS AND METHODS: Sciatic nerve axotomy was performed in a group of nine New Zealand White rabbits, and electromyographic (EMG), pathological, and DWMRI studies were conducted on ipsilateral hamstring muscles 1 and 8 days after axotomy. In addition, DWMRI studies were carried out on leg muscles of ten patients with acute and subacute lumbosacral radiculopathy. RESULTS: High intensity signals on short tau inversion recovery (STIR) magnetic resonance imaging and an increased apparent diffusion coefficient (ADC) were observed in denervated muscles of the animals 1 and 8 days after axotomy as well as in denervated muscles of the patients with radiculopathy. In the clinical study, ADC was 1.26 +/- 0.18 x 10(-9) m(2)/s in normal muscle and increased to 1.56 +/- 0.23 x 10(-9) m(2)/s in denervated muscles (p = 0.0016). In animals, EMG and muscle pathological studies were normal 1 day after axotomy, and the muscles demonstrated spontaneous activity on EMG and neurogenic atrophy on histological studies 7 days later. CONCLUSION: This DWMRI study demonstrates that enlargement of extracellular fluid space in muscle denervation is an early phenomenon occurring several days before the appearance of EMG and histological abnormalities.

[Amyotrophic lateral sclerosis: role of energy deficiency in neuromuscular junction dismantlement]. / Sclérose latérale amyotrophique, jonction neuromusculaire et déficit énergétique.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult onset motor neuron disorder. A subset of ALS cases is linked to mutations in the copper/zinc superoxide dismutase (sod1) gene and detailed phenotypic analysis of transgenic mice overexpressing mutant forms of SOD1 (mSOD1) allowed a better understanding of the pathophysiological mechanisms leading to motor neuron death. The promising results obtained in these animal models however poorly translated into conclusive clinical trials. In this review, we summarize the main pathological mechanisms at work in mSOD1 mice. In particular, recent results showed that the key pathological event was the destruction of the neuromuscular junction rather than motor neuron death. Neuromuscular junction dismantlement is likely the result of a chronic energy deficiency at the level of the whole organism. These results, along with a comparative analysis between the phenotype of mSOD1 mice and ALS patients, suggest new therapeutic strategies and show the interests but also the limits of the animal models.

Sodium valproate exerts neuroprotective effects in vivo through CREB-binding protein-dependent mechanisms but does not improve survival in an amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis (ALS) is characterized by motoneuron (MN) degeneration, generalized weakness, and muscle atrophy. The premature death of MNs is thought to be a determinant in the onset of this disease. In a transgenic mouse model of ALS expressing the G86R mutant superoxide dismutase 1 (mSOD1), we demonstrated previously that CREB (cAMP response element-binding protein)-binding protein (CBP) and histone acetylation levels were specifically decreased in nuclei of degenerating MNs. We show here that oxidative stress and mSOD1 overexpression can both impinge on CBP levels by transcriptional repression, in an MN-derived cell line. Histone deacetylase inhibitor (HDACi) treatment was able to reset proper acetylation levels and displayed an efficient neuroprotective capacity against oxidative stress in vitro. Interestingly, HDACi also upregulated CBP transcriptional expression in MNs. Moreover, when injected to G86R mice in vivo, the HDACi sodium valproate (VPA) maintained normal acetylation levels in the spinal cord, efficiently restored CBP levels in MNs, and significantly prevented MN death in these animals. However, despite neuroprotection, mean survival of treated animals was not significantly improved (<5%), and they died presenting the classical ALS symptoms. VPA was not able to prevent disruption of neuromuscular junctions, although it slightly delayed the onset of motor decline and retarded muscular atrophy to some extent. Together, these data show that neuroprotection can improve disease onset, but clearly provide evidence that one can uncouple MN survival from whole-animal survival and point to the neuromuscular junction perturbation as a primary event of ALS onset.

Muscle Nogo-A expression is a prognostic marker in lower motor neuron syndromes.

OBJECTIVE: A proportion of patients with pure lower motor neuron syndrome (LMNS) progress to amyotrophic lateral sclerosis (ALS). Early detection of this progression is impossible, which delays the patient's access to treatment. Muscle expression of Nogo-A is a new candidate marker of ALS. We tested whether detection of Nogo-A in a muscle biopsy from patients with LMNS predicts progression to ALS. METHODS: Thirty-three patients who had undergone a muscle biopsy during the diagnostic workup of spinal LMNS were observed for 12 months. Nogo-A expression was measured by Western blot in muscle biopsy samples and compared with the final diagnosis. RESULTS: Nogo-A expression was detected in 17 patients and was absent in 16 patients. The detection of Nogo-A in muscle biopsy samples from LMNS patients correctly identified patients who further progressed to ALS with 91% accuracy, 94% sensitivity, and 88% specificity. In patients who later developed typical ALS, Nogo-A may be detected as early as 3 months after the onset of symptoms. INTERPRETATION: Nogo-A test is able to identify ALS early in the course of the disease when diagnosis is difficult, requiring further progression. Use of the test in clinical practice may shorten the delay before introduction of neuroprotective drugs or inclusion in clinical trials.

Muscular mitochondrial function in amyotrophic lateral sclerosis is progressively altered as the disease develops: a temporal study in man.

We performed repeated analysis of mitochondrial respiratory function in skeletal muscle (SM) of patients with early-stage sporadic amyotrophic lateral sclerosis (SALS) to determine whether mitochondrial function was altered as the disease advanced. SM biopsies were obtained from 7 patients with newly diagnosed SALS, the same 7 patients 3 months later, and 7 sedentary controls. Muscle fibers were permeabilized with saponin, then skinned and placed in an oxygraphic chamber to measure basal and maximal adenosine diphosphate (ADP)-stimulated respiration rates and to assess mitochondrial regulation by ADP. We found that the maximal oxidative phosphorylation capacity of muscular mitochondria significantly increased, and muscular mitochondrial respiratory complex IV activity significantly decreased as the disease advanced. This temporal study demonstrates for the first time that mitochondrial function in SM in human SALS is progressively altered as the disease develops.

Antibody-bound beta-amyloid precursor protein stimulates the production of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 by cortical neurons.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar beta-amyloid (A beta), which is derived from the alternative processing of beta-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun N-terminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.

Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types.

Nogo-A, -B, and -C are generated from the Nogo/RTN-4 gene and share a highly conserved C-terminal domain. They lack an N-terminal signal sequence and are predominantly localized to the endoplasmic reticulum (ER). We found the N terminus of endogenous Nogo-A exposed on the surface of fibroblasts, DRG neurons, and myoblasts. Surface-expressed Nogo-A was also present on presynaptic terminals of the neuromuscular junction and on DRG neurons in vivo. Surface biotinylations confirmed the presence of all Nogo isoforms on the surface. To search for proteins that interact with Nogo-A and suggest a function for the large intracellular pool of Nogo-A, immunoprecipitations were performed. Surprisingly, the most predominant proteins that interact with Nogo-A are Nogo-B and Nogo-C as seen with radiolabeled lysates and as confirmed by Western blotting in multiple cell lines. Nogo-A, -B, and -C share a 180-amino acid C-terminal domain with two highly conserved hydrophobic stretches that could form a channel or transporter in the ER and/or on the cell surface.

HDAC-3 participates in the repression of e2f-dependent gene transcription in primary differentiated neurons.

Activation of e2f-1 gene expression is an event that has been now established in many models of neuronal apoptosis. Accumulated E2F-1 protein has also been observed in post mortem brains obtained from patients suffering from different neurodegenerative diseases. We have previously shown in primary neuronal cultures that e2f-1 gene transcription was actively repressed in neuroprotective conditions through HDAC-dependent regulation on the E2F-responsive elements (E2F-REs) located in the e2f-1 gene promoter. Here, we further investigated the protein complex bound to these sites by gel shift analysis. We found that the specific protein binding to E2F-REs is altered in apoptotic conditions compared to neuroprotective conditions, suggesting that the proteic constituents of the complex are likely to be modified upon apoptosis onset. Indeed, Western blot analysis showed a time-dependent degradation of the Rb/E2F binding protein HDAC-3 during apoptosis, a degradation that is caspase-dependent. Altogether, these data point to HDAC-3 as a good candidate involved in the active e2f-1 repression necessary for neuroprotection.