Serotonin 2B receptor slows disease progression and prevents degeneration of spinal cord mononuclear phagocytes in amyotrophic lateral sclerosis.

Microglia are the resident mononuclear phagocytes of the central nervous system and have been implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). During neurodegeneration, microglial activation is accompanied by infiltration of circulating monocytes, leading to production of multiple inflammatory mediators in the spinal cord. Degenerative alterations in mononuclear phagocytes are commonly observed during neurodegenerative diseases, yet little is known concerning the mechanisms leading to their degeneration, or the consequences on disease progression. Here we observed that the serotonin 2B receptor (5-HT2B), a serotonin receptor expressed in microglia, is upregulated in the spinal cord of three different transgenic mouse models of ALS. In mutant SOD1 mice, this upregulation was restricted to cells positive for CD11b, a marker of mononuclear phagocytes. Ablation of 5-HT2B receptor in transgenic ALS mice expressing mutant SOD1 resulted in increased degeneration of mononuclear phagocytes, as evidenced by fragmentation of Iba1-positive cellular processes. This was accompanied by decreased expression of key neuroinflammatory genes but also loss of expression of homeostatic microglial genes. Importantly, the dramatic effect of 5-HT2B receptor ablation on mononuclear phagocytes was associated with acceleration of disease progression. To determine the translational relevance of these results, we studied polymorphisms in the human HTR2B gene, which encodes the 5-HT2B receptor, in a large cohort of ALS patients. In this cohort, the C allele of SNP rs10199752 in HTR2B was associated with longer survival. Moreover, patients carrying one copy of the C allele of SNP rs10199752 showed increased 5-HT2B mRNA in spinal cord and displayed less pronounced degeneration of Iba1 positive cells than patients carrying two copies of the more common A allele. Thus, the 5-HT2B receptor limits degeneration of spinal cord mononuclear phagocytes, most likely microglia, and slows disease progression in ALS. Targeting this receptor might be therapeutically useful.

Dynein mutations associated with hereditary motor neuropathies impair mitochondrial morphology and function with age.

Mutations in the DYNC1H1 gene encoding for dynein heavy chain cause two closely related human motor neuropathies, dominant spinal muscular atrophy with lower extremity predominance (SMA-LED) and axonal Charcot-Marie-Tooth (CMT) disease, and lead to sensory neuropathy and striatal atrophy in mutant mice. Dynein is the molecular motor carrying mitochondria retrogradely on microtubules, yet the consequences of dynein mutations on mitochondrial physiology have not been explored. Here, we show that mouse fibroblasts bearing heterozygous or homozygous point mutation in Dync1h1, similar to human mutations, show profoundly abnormal mitochondrial morphology associated with the loss of mitofusin 1. Furthermore, heterozygous Dync1h1 mutant mice display progressive mitochondrial dysfunction in muscle and mitochondria progressively increase in size and invade sarcomeres. As a likely consequence of systemic mitochondrial dysfunction, Dync1h1 mutant mice develop hyperinsulinemia and hyperglycemia and progress to glucose intolerance with age. Similar defects in mitochondrial morphology and mitofusin levels are observed in fibroblasts from patients with SMA-LED. Last, we show that Dync1h1 mutant fibroblasts show impaired perinuclear clustering of mitochondria in response to mitochondrial uncoupling. Our results show that dynein function is required for the maintenance of mitochondrial morphology and function with aging and suggest that mitochondrial dysfunction contributes to dynein-dependent neurological diseases, such as SMA-LED.

Mutations in cytoplasmic dynein lead to a Huntington's disease-like defect in energy metabolism of brown and white adipose tissues.

The molecular motor dynein is regulated by the huntingtin protein, and Huntington's disease (HD) mutations of huntingtin disrupt dynein motor activity. Besides abnormalities in the central nervous system, HD animal models develop prominent peripheral pathology, with defective brown tissue thermogenesis and dysfunctional white adipocytes, but whether this peripheral phenotype is recapitulated by dynein dysfunction is unknown. Here, we observed prominently increased adiposity in mice harboring the legs at odd angles (Loa/+) or the Cramping mutations (Cra/+) in the dynein heavy chain gene. In Cra/+ mice, hyperadiposity occurred in the absence of energy imbalance and was the result of impaired norepinephrine-stimulated lipolysis. A similar phenotype was observed in 3T3L1 adipocytes upon chemical inhibition of dynein showing that loss of functional dynein leads to impairment of lipolysis. Ex vivo, dynein mutant adipose tissue displayed increased reactive oxygen species production that was, at least partially, responsible for the decreased cellular responses to norepinephrine and subsequent defect in stimulated lipolysis. Dynein mutation also affected norepinephrine efficacy to elicit a thermogenic response and led to morphological abnormalities in brown adipose tissue and cold intolerance in dynein mutant mice. Interestingly, protein levels of huntingtin were decreased in dynein mutant adipose tissue. Collectively, our results provide genetic evidence that dynein plays a key role in lipid metabolism and thermogenesis through a modulation of oxidative stress elicited by norepinephrine. This peripheral phenotype of dynein mutant mice is similar to that observed in various animal models of HD, lending further support for a functional link between huntingtin and dynein.

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.

Increase in BDNF-mediated TrkB signaling promotes epileptogenesis in a mouse model of mesial temporal lobe epilepsy.

Mesio-temporal lobe epilepsy (MTLE), the most common drug-resistant epilepsy syndrome, is characterized by the recurrence of spontaneous focal seizures after a latent period that follows, in most patients, an initial insult during early childhood. Many of the mechanisms that have been associated with the pathophysiology of MTLE are known to be regulated by brain-derived neurotrophic factor (BDNF) in the healthy brain and an excess of this neurotrophin could therefore play a critical role in MTLE development. However, such a function remains controversial as other studies revealed that BDNF could, on the contrary, exert protective effects regarding epilepsy development. In the present study, we further addressed the role of increased BDNF/TrkB signaling on the progressive development of hippocampal seizures in the mouse model of MTLE obtained by intrahippocampal injection of kainate. We show that hippocampal seizures progressively developed in the injected hippocampus during the first two weeks following kainate treatment, within the same time-frame as a long-lasting and significant increase of BDNF expression in dentate granule cells. To determine whether such a BDNF increase could influence hippocampal epileptogenesis via its TrkB receptors, we examined the consequences of (i) increased or (ii) decreased TrkB signaling on epileptogenesis, in transgenic mice overexpressing the (i) TrkB full-length or (ii) truncated TrkB-T1 receptors of BDNF. Epileptogenesis was significantly facilitated in mice with increased TrkB signaling but delayed in mutants with reduced TrkB signaling. In contrast, TrkB signaling did not influence granule cell dispersion, an important feature of this mouse model which is also observed in most MTLE patients. These results suggest that an increase in TrkB signaling, mediated by a long-lasting BDNF overexpression in the hippocampus, promotes epileptogenesis in MTLE.

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.

A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. RESULTS: SOD1(G93A) mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice). Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A) mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. CONCLUSIONS: These findings suggest that the protection against SOD1(G93A) offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.

Oxidative stress in skeletal muscle stimulates early expression of Rad in a mouse model of amyotrophic lateral sclerosis.

Motor neuron degeneration and progressive muscle atrophy characterize amyotrophic lateral sclerosis (ALS) in humans and related mutant superoxide dismutase-1 (SOD1) transgenic mice. Our previous microarray studies on ALS muscle revealed strong up-regulation of Ras-related associated with diabetes (Rad), an inhibitor of voltage-gated calcium channels. The mechanisms controlling Rad expression in disease are unknown. We analyzed Rad expression in skeletal muscle from ALS patients and animal models and investigated whether it is regulated by oxidative stress. In mutant SOD1 mice, Rad up-regulation preceded motor symptoms and markedly increased as disease progressed. Increased Rad expression was also obtained in surgically denervated muscle. No clinical signs of denervation were seen in asymptomatic mice, however. We therefore suspected that muscular mutant SOD1 toxicity causes precocious Rad up-regulation. We confirmed the accumulation of reactive oxygen species (ROS) at asymptomatic stages, coincident with the rise in Rad expression. By subjecting muscle to ischemia-reperfusion, we observed ROS accumulation and Rad overexpression. The cell-permeative antioxidant Tempol inhibited the stimulatory effect of ischemia-reperfusion. Tempol also reduced Rad up-regulation after experimental denervation. Our study provides strong evidence for the implication of oxidative stress in modulating Rad expression, in association with the initiation and progression of ALS muscle atrophy.

Sp3 and sp4 transcription factor levels are increased in brains of patients with Alzheimer's disease.

BACKGROUND/AIMS: Alzheimer's disease (AD) is characterized by extracellular Abeta peptide deposition originating from amyloid precursor protein cleavage and intracellular neurofibrillary tangles resulting from pathological tau protein aggregation. These processes are accompanied by dramatic neuronal losses, further leading to different cognitive impairments. Neuronal death signalings involve gene expression modifications that rely on transcription factor alterations. Herein, we investigated the fate of the Sp family of transcription factors in postmortem brains from patients with AD disease and in different contexts of neuronal death. METHODS/RESULTS: By immunohistochemistry we found that the Sp3 and Sp4 levels were dramatically increased and associated with neurofibrillary tangles and pathological tau presence in neurons from the CA1 region of the hippocampus, as well as the entorhinal cortex of AD patient brains. The Sp transcription factor expression levels were further analyzed in cortical neurons in which death is induced by amyloid precursor protein signaling targeting. While the Sp1 levels remained constant, the Sp4 levels were slightly upregulated in response to the death signal. The Sp3 isoforms were rather degraded. Interestingly, when overexpressed by transfection experiments, the three Sp family members induced neuronal apoptosis, Sp3 and Sp4 being the most potent proapoptotic factors over Sp1. CONCLUSION: Our data evidence Sp3 and Sp4 as new hallmarks of AD in postmortem human brains and further point out that Sp proteins are potential triggers of neuronal death signaling cascades.

Unraveling in vivo functions of amyloid precursor protein: insights from knockout and knockdown studies.

The amyloid precursor protein (APP) is a widely expressed transmembrane protein that is cleaved to generate Abeta peptides in the central nervous system and is a key player in the pathogenesis of Alzheimer's disease. The precise biological functions of APP still remain unclear although various roles have been proposed. While a commonly accepted model argues that Abeta peptides are the cause of onset and early pathogenesis of Alzheimer's disease, recent discussions challenge this 'Abeta hypothesis' and suggest a direct role for APP in this neurodegenerative disease. Loss-of-function studies are an efficient way to elucidate the role of proteins and concurrently a variety of in vitro and in vivo studies has been performed for APP where protein levels have been downregulated and functional consequences monitored. Complete disruption of APP gene expression has been achieved by the generation of APP knockout animal models. Further knockdown studies using antisense and RNA interference have allowed scientists to reduce APP expression levels and have opened new avenues to explore the physiological roles of APP. In the present review, we focus on knockout and knockdown approaches that have provided insights into the physiological functions of APP and discuss their advantages and drawbacks.

Reticulons as markers of neurological diseases: focus on amyotrophic lateral sclerosis.

Reticulons (RTNs) are a family of proteins that are primarily associated with the endoplasmic reticulum. In mammals, four genes have been identified and referred as to rtn1, 2, 3 and the neurite outgrowth inhibitor rtn4/nogo. These genes generate multiple isoforms that contain a common C-terminal reticulon homology domain of 150-200 amino-acid residues. The N-terminal regions of RTNs are highly variable, and result from alternative splicing or differential promoter usage. Although widely distributed, the functions of RTNs are still poorly understood. Much interest has been focused on rtn4/nogo because of its activity as a potent inhibitor of axonal growth and repair. In the present study, we update recent knowledge on mammalian RTNs paying special attention to the involvement of these proteins as markers of neurological diseases. We also present recent data concerning RTN expression in amyotrophic lateral sclerosis, a fatal degenerative disorder characterized by loss of upper and lower motor neurons, and muscle atrophy. The rearrangement of RTN expression is regulated not only in suffering skeletal muscle but also preceding the onset of symptoms, and may relate to the disease process.

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.

Neuropeptide Y delays hippocampal kindling in the rat.

Chronic intrahippocampal infusion of the neurotrophin brain-derived neurotrophic factor (BDNF) has been shown to delay kindling epileptogenesis in the rat and several lines of evidence suggest that neuropeptide Y could mediate these inhibitory effects. Chronic infusion of BDNF leads to a sustained overexpression of neuropeptide Y in the hippocampus, which follows a time course similar to that of the suppressive effects of BDNF on kindling. In vivo, acute applications of neuropeptide Y or agonists of its receptors exert anticonvulsant properties, especially on seizures of hippocampal origin. In this study, we examined how chronic infusion of this neuropeptide in the hippocampus affected kindling epileptogenesis. A 7-day continuous infusion of neuropeptide Y in the hippocampus delayed the progression of hippocampal kindling in the rat, whereas anti-neuropeptide Y immunoglobulins had an aggravating effect. These results show that neuropeptide Y exerts anti-epileptogenic properties on seizures originating within the hippocampus and lend support to the hypothesis that BDNF delays kindling at least in part through upregulation of this neuropeptide. They also suggest that the seizure-induced upregulation of neuropeptide Y constitutes an endogenous mechanism counteracting excessive hippocampal excitability.