Astrocytes in Motor Neuron Diseases.

Motor neuron disorders are highly debilitating and mostly fatal conditions for which only limited therapeutic options are available. To overcome this limitation and develop more effective therapeutic strategies, it is critical to discover the pathogenic mechanisms that trigger and sustain motor neuron degeneration with the greatest accuracy and detail. In the case of Amyotrophic Lateral Sclerosis (ALS), several genes have been associated with familial forms of the disease, whilst the vast majority of cases develop sporadically and no defined cause can be held responsible. On the contrary, the huge majority of Spinal Muscular Atrophy (SMA) occurrences are caused by loss-of-function mutations in a single gene, SMN1. Although the typical hallmark of both diseases is the loss of motor neurons, there is increasing awareness that pathological lesions are also present in the neighbouring glia, whose dysfunction clearly contributes to generating a toxic environment in the central nervous system. Here, ALS and SMA are sequentially presented, each disease section having a brief introduction, followed by a focussed discussion on the role of the astrocytes in the disease pathogenesis. Such a dissertation is substantiated by the findings that built awareness on the glial involvement and how the glial-neuronal interplay is perturbed, along with the appraisal of this new cellular site for possible therapeutic intervention.

Disruption of the astrocytic TNFR1-GDNF axis accelerates motor neuron degeneration and disease progression in amyotrophic lateral sclerosis.

Considerable evidence indicates that neurodegeneration in amyotrophic lateral sclerosis (ALS) can be conditioned by a deleterious interplay between motor neurons and astrocytes. Astrocytes are the major glial component in the central nervous system (CNS) and fulfill several activities that are essential to preserve CNS homeostasis. In physiological and pathological conditions, astrocytes secrete a wide range of factors by which they exert multimodal influences on their cellular neighbours. Among others, astrocytes can secrete glial cell line-derived neurotrophic factor (GDNF), one of the most potent protective agents for motor neurons. This suggests that the modulation of the endogenous mechanisms that control the production of astrocytic GDNF may have therapeutic implications in motor neuron diseases, particularly ALS. In this study, we identified TNF receptor 1 (TNFR1) signalling as a major promoter of GDNF synthesis/release from human and mouse spinal cord astrocytes in vitro and in vivo To determine whether endogenously produced TNFα can also trigger the synthesis of GDNF in the nervous system, we then focused on SOD1G93A ALS transgenic mice, whose affected tissues spontaneously exhibit high levels of TNFα and its receptor 1 at the onset and symptomatic stage of the disease. In SOD1G93A spinal cords, we verified a strict correlation in the expression of the TNFα, TNFR1 and GDNF triad at different stages of disease progression. Yet, ablation of TNFR1 completely abolished GDNF rises in both SOD1G93A astrocytes and spinal cords, a condition that accelerated motor neuron degeneration and disease progression. Our data suggest that the astrocytic TNFR1-GDNF axis represents a novel target for therapeutic intervention in ALS.

Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS.

Fused in sarcoma (FUS) is a nuclear protein that carries a proline-tyrosine nuclear localization signal (PY-NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY-NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN-binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN-mediated nuclear import of ALS-associated FUS mutants. The unmethylated arginine-glycine-glycine domain preceding the PY-NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS-FUS patients contain methylated FUS, while inclusions in FTLD-FUS patients are not methylated. Together with recent findings that FUS co-aggregates with two related proteins of the FET family and TRN in FTLD-FUS but not in ALS-FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.

PTEN depletion decreases disease severity and modestly prolongs survival in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the second most common genetic cause of death in childhood. However, no effective treatment is available to halt disease progression. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene. We previously reported that PTEN depletion leads to an increase in survival of SMN-deficient motor neurons. Here, we aimed to establish the impact of PTEN modulation in an SMA mouse model in vivo. Initial experiments using intramuscular delivery of adeno-associated vector serotype 6 (AAV6) expressing shRNA against PTEN in an established mouse model of severe SMA (SMNΔ7) demonstrated the ability to ameliorate the severity of neuromuscular junction pathology. Subsequently, we developed self-complementary AAV9 expressing siPTEN (scAAV9-siPTEN) to allow evaluation of the effect of systemic suppression of PTEN on the disease course of SMA in vivo. Treatment with a single injection of scAAV9-siPTEN at postnatal day 1 resulted in a modest threefold extension of the lifespan of SMNΔ7 mice, increasing mean survival to 30 days, compared to 10 days in untreated mice. Our data revealed that systemic PTEN depletion is an important disease modifier in SMNΔ7 mice, and therapies aimed at lowering PTEN expression may therefore offer a potential therapeutic strategy for SMA.

The multifaceted role of glial cells in amyotrophic lateral sclerosis.

Despite indisputable progress in the molecular and genetic aspects of amyotrophic lateral sclerosis (ALS), a mechanistic comprehension of the neurodegenerative processes typical of this disorder is still missing and no effective cures to halt the progression of this pathology have yet been developed. Therefore, it seems that a substantial improvement of the outcome of ALS treatments may depend on a better understanding of the molecular mechanisms underlying neuronal pathology and survival as well as on the establishment of novel etiological therapeutic strategies. Noteworthy, a convergence of recent data from multiple studies suggests that, in cellular and animal models of ALS, a complex pathological interplay subsists between motor neurons and their non-neuronal neighbours, particularly glial cells. These observations not only have drawn attention to the physiopathological changes glial cells undergo during ALS progression, but they have moved the focus of the investigations from intrinsic defects and weakening of motor neurons to glia-neuron interactions. In this review, we summarize the growing body of evidence supporting the concept that different glial populations are critically involved in the dreadful chain of events leading to motor neuron sufferance and death in various forms of ALS. The outlined observations strongly suggest that glial cells can be the targets for novel therapeutic interventions in ALS.

The BH4 domain of Bcl-X(L) rescues astrocyte degeneration in amyotrophic lateral sclerosis by modulating intracellular calcium signals.

Collective evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is non-cell-autonomous and requires the interaction with the neighboring astrocytes. Recently, we reported that a subpopulation of spinal cord astrocytes degenerates in the microenvironment of motor neurons in the hSOD1(G93A) mouse model of ALS. Mechanistic studies in vitro identified a role for the excitatory amino acid glutamate in the gliodegenerative process via the activation of its inositol 1,4,5-triphosphate (IP(3))-generating metabotropic receptor 5 (mGluR5). Since non-physiological formation of IP(3) can prompt IP(3) receptor (IP(3)R)-mediated Ca(2+) release from the intracellular stores and trigger various forms of cell death, here we investigated the intracellular Ca(2+) signaling that occurs downstream of mGluR5 in hSOD1(G93A)-expressing astrocytes. Contrary to wild-type cells, stimulation of mGluR5 causes aberrant and persistent elevations of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in the absence of spontaneous oscillations. The interaction of IP(3)Rs with the anti-apoptotic protein Bcl-X(L) was previously described to prevent cell death by modulating intracellular Ca(2+) signals. In mutant SOD1-expressing astrocytes, we found that the sole BH4 domain of Bcl-X(L), fused to the protein transduction domain of the HIV-1 TAT protein (TAT-BH4), is sufficient to restore sustained Ca(2+) oscillations and cell death resistance. Furthermore, chronic treatment of hSOD1(G93A) mice with the TAT-BH4 peptide reduces focal degeneration of astrocytes, slightly delays the onset of the disease and improves both motor performance and animal lifespan. Our results point at TAT-BH4 as a novel glioprotective agent with a therapeutic potential for ALS.

Viral delivery of antioxidant genes as a therapeutic strategy in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no effective treatment to date. Despite its multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of antioxidant genes that are downregulated in the presence of mutant SOD1 may serve as a therapeutic strategy for motor neuronal protection. Lentiviral vectors expressing either PRDX3 or NRF2 genes were tested in the motor neuronal-like NSC34 cell line, and in the ALS tissue culture model, NSC34 cells expressing the human SOD1(G93A) mutation. The NSC34 SOD1(G93A) cells overexpressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40 and 50% respectively compared with controls, whereas cell survival was increased by 30% in both cases. The neuroprotective potential of those two genes was further investigated in vivo in the SOD1(G93A) ALS mouse model, by administering intramuscular injections of adenoassociated virus serotype 6 (AAV6) expressing either of the target genes at a presymptomatic stage. Despite the absence of a significant effect in survival, disease onset or progression, which can be explained by the inefficient viral delivery, the promising in vitro data suggest that a more widespread CNS delivery is needed.

Astrocytes: Dissecting Their Diverse Roles in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders often co-occurring in the same patient, a feature that suggests a common origin of the two diseases. Consistently, pathological inclusions of the same proteins as well as mutations in the same genes can be identified in both ALS/FTD. Although many studies have described several disrupted pathways within neurons, glial cells are also regarded as crucial pathogenetic contributors in ALS/FTD. Here, we focus our attention on astrocytes, a heterogenous population of glial cells that perform several functions for optimal central nervous system homeostasis. Firstly, we discuss how post-mortem material from ALS/FTD patients supports astrocyte dysfunction around three pillars: neuroinflammation, abnormal protein aggregation, and atrophy/degeneration. Furthermore, we summarize current attempts at monitoring astrocyte functions in living patients using either novel imaging strategies or soluble biomarkers. We then address how astrocyte pathology is recapitulated in animal and cellular models of ALS/FTD and how we used these models both to understand the molecular mechanisms driving glial dysfunction and as platforms for pre-clinical testing of therapeutics. Finally, we present the current clinical trials for ALS/FTD, restricting our discussion to treatments that modulate astrocyte functions, directly or indirectly.

PTEN depletion rescues axonal growth defect and improves survival in SMN-deficient motor neurons.

Phosphatase and tensin homolog (PTEN), a negative regulator of the mammalian target of rapamycin (mTOR) pathway, is widely involved in the regulation of protein synthesis. Here we show that the PTEN protein is enriched in cell bodies and axon terminals of purified motor neurons. We explored the role of the PTEN pathway by manipulating PTEN expression in healthy and diseased motor neurons. PTEN depletion led to an increase in growth cone size, promotion of axonal elongation and increased survival of these cells. These changes were associated with alterations of downstream signaling pathways for local protein synthesis as revealed by an increase in pAKT and p70S6. Most notably, this treatment also restores beta-actin protein levels in axonal growth cones of SMN-deficient motor neurons. Furthermore, we report here that a single injection of adeno-associated virus serotype 6 (AAV6) expressing siPTEN into hind limb muscles at postnatal day 1 in SMNDelta7 mice leads to a significant PTEN depletion and robust improvement in motor neuron survival. Taken together, these data indicate that PTEN-mediated regulation of protein synthesis in motor neurons could represent a target for therapy in spinal muscular atrophy.

Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations.

Accumulation of the DNA/RNA binding protein fused in sarcoma (FUS) as inclusions in neurons and glia is the pathological hallmark of amyotrophic lateral sclerosis patients with mutations in FUS (ALS-FUS) as well as in several subtypes of frontotemporal lobar degeneration (FTLD-FUS), which are not associated with FUS mutations. Despite some overlap in the phenotype and neuropathology of FTLD-FUS and ALS-FUS, significant differences of potential pathomechanistic relevance were recently identified in the protein composition of inclusions in these conditions. While ALS-FUS showed only accumulation of FUS, inclusions in FTLD-FUS revealed co-accumulation of all members of the FET protein family, that include FUS, Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 15 (TAF15) suggesting a more complex disturbance of transportin-mediated nuclear import of proteins in FTLD-FUS compared to ALS-FUS. To gain more insight into the mechanisms of inclusion body formation, we investigated the role of Transportin 1 (Trn1) as well as 13 additional cargo proteins of Transportin in the spectrum of FUS-opathies by immunohistochemistry and biochemically. FUS-positive inclusions in six ALS-FUS cases including four different mutations did not label for Trn1. In sharp contrast, the FET-positive pathology in all FTLD-FUS subtypes was also strongly labeled for Trn1 and often associated with a reduction in the normal nuclear staining of Trn1 in inclusion bearing cells, while no biochemical changes of Trn1 were detectable in FTLD-FUS. Notably, despite the dramatic changes in the subcellular distribution of Trn1 in FTLD-FUS, alterations of its cargo proteins were restricted to FET proteins and no changes in the normal physiological staining of 13 additional Trn1 targets, such as hnRNPA1, PAPBN1 and Sam68, were observed in FTLD-FUS. These data imply a specific dysfunction in the interaction between Trn1 and FET proteins in the inclusion body formation in FTLD-FUS. Moreover, the absence of Trn1 in ALS-FUS provides further evidence that ALS-FUS and FTLD-FUS have different underlying pathomechanisms.

Phosphatase and tensin homologue/protein kinase B pathway linked to motor neuron survival in human superoxide dismutase 1-related amyotrophic lateral sclerosis.

Gene expression profiling has been used previously with spinal cord homogenates and laser capture microdissected motor neurons to determine the mechanisms involved in neurodegeneration in amyotrophic lateral sclerosis. However, while cellular and animal model work has focused on superoxide dismutase 1-related amyotrophic lateral sclerosis, the transcriptional profile of human mutant superoxide dismutase 1 motor neurons has remained undiscovered. The aim of this study was to apply gene expression profiling to laser captured motor neurons from human superoxide dismutase 1-related amyotrophic lateral sclerosis and neurologically normal control cases, in order to determine those pathways dysregulated in human superoxide dismutase 1-related neurodegeneration and to establish potential pathways suitable for therapeutic intervention. Identified targets were then validated in cultured cell models using lentiviral vectors to manipulate the expression of key genes. Microarray analysis identified 1170 differentially expressed genes in spinal cord motor neurons from superoxide dismutase 1-related amyotrophic lateral sclerosis, compared with controls. These genes encoded for proteins in multiple functional categories, including those involved in cell survival and cell death. Further analysis determined that multiple genes involved in the phosphatidylinositol-3 kinase signalling cascade were differentially expressed in motor neurons that survived the disease process. Functional experiments in cultured cells and primary motor neurons demonstrate that manipulating this pathway by reducing the expression of a single upstream target, the negative phosphatidylinositol-3 kinase regulator phosphatase and tensin homology, promotes a marked pro-survival effect. Therefore, these data indicate that proteins in the phosphatidylinositol-3 kinase pathway could represent a target for therapeutic manipulation in motor neuron degeneration.

Contribution of RNA/DNA Binding Protein Dysfunction in Oligodendrocytes in the Pathogenesis of the Amyotrophic Lateral Sclerosis/Frontotemporal Lobar Degeneration Spectrum Diseases.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders, often considered as the extreme manifestations of a disease spectrum, as they share similar pathomechanisms. In support of this, pathological aggregation of the RNA/DNA binding proteins trans-activation response element DNA-binding protein 43 (TDP-43) or fused in sarcoma (FUS) is the pathological hallmark found in neurons and glial cells of subsets of patients affected by either condition (i.e., ALS/FTLD-TDP-43 or ALS/FTLD-FUS, respectively). Among glia, oligodendrocytes are the most abundant population, designated to ensheath the axons with myelin and to provide them with metabolic and trophic support. In this minireview, we recapitulate the neuropathological evidence for oligodendroglia impairment in ALS/FTLD. We then debate how TDP-43 and FUS target oligodendrocyte transcripts, thereby controlling their homeostatic abilities toward the axons. Finally, we discuss cellular and animal models aimed at investigating the functional consequences of manipulating TDP-43 and FUS in oligodendrocytes in vivo. Taken together, current data provide increasing evidence for an important role of TDP-43 and FUS-mediated oligodendroglia dysfunction in the pathogenesis of ALS/FTLD. Thus, targeting disrupted oligodendroglial functions may represent a new treatment approach for these conditions.

Challenges and Opportunities of Targeting Astrocytes to Halt Neurodegenerative Disorders.

Neurodegenerative diseases are a heterogeneous group of disorders whose incidence is likely to duplicate in the next 30 years along with the progressive aging of the western population. Non-cell-specific therapeutics or therapeutics designed to tackle aberrant pathways within neurons failed to slow down or halt neurodegeneration. Yet, in the last few years, our knowledge of the importance of glial cells to maintain the central nervous system homeostasis in health conditions has increased exponentially, along with our awareness of their fundamental and multifaced role in pathological conditions. Among glial cells, astrocytes emerge as promising therapeutic targets in various neurodegenerative disorders. In this review, we present the latest evidence showing the astonishing level of specialization that astrocytes display to fulfill the demands of their neuronal partners as well as their plasticity upon injury. Then, we discuss the controversies that fuel the current debate on these cells. We tackle evidence of a potential beneficial effect of cell therapy, achieved by transplanting astrocytes or their precursors. Afterwards, we introduce the different strategies proposed to modulate astrocyte functions in neurodegeneration, ranging from lifestyle changes to environmental cues. Finally, we discuss the challenges and the recent advancements to develop astrocyte-specific delivery systems.

Deletion of the amino-terminal domain of the prion protein does not impair prion protein-dependent neuronal differentiation and neuritogenesis.

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein of unknown biological function. To gain insight into the physiological role of PrP(C), we generated a novel PrP knockout cell line, named PrP(o/o) ML, by immortalization of neuroepithelial precursor cells derived from the cerebellum of PrP-knockout mice using the temperature-sensitive simian virus 40 (SV40) large T antigen. We demonstrated that the PrP(o/o) ML cell line is a unipotent precursor line with glutamatergic properties, which can acquire neuronal features when cultivated under specific conditions. The role of the prion protein in the process of neuronal differentiation was then analyzed in the PrP(o/o) ML cells reconstituted with either the full-length or an amino-terminally deleted form of the prion protein. We show that the expression of PrP(C) facilitates the processes of neuronal differentiation and neuritogenesis and that the deletion of its amino-terminal domain reduces the efficiency, but does not suppress this activity. This cell line represents a useful tool for studying PrP-dependent signal transduction pathways during differentiation of neuronal stem/precursor cells.

Astrocytes: Emerging Therapeutic Targets in Neurological Disorders.

Astrocytes fulfil several functions that collectively contribute to maintain the optimal microenvironment for neuronal function and survival. The multiplicity and complexity of these activities clearly indicates that the correct performance of astrocytes is crucial for the physiological functioning of the nervous system, and its derangement may contribute to the occurrence and progression of many neurological disorders. Although rectifying astrocyte malfunction has successfully counteracted disease pathogenesis and outcome in many preclinical settings, the translation of this revolutionary approach into clinical practice urges the development of innovative, safe, and effective systems for both the selective delivery of therapeutics to the astrocytes and astrocyte replacement strategies.

Development and applications of non-HIV-based lentiviral vectors in neurological disorders.

Vectors based on non-HIV lentiviruses are opening up new approaches for the treatment of human disorders. These vectors efficiently deliver genes into many different types of cells from a broad range of species including man and the resulting gene expression is long-term. These features make them very attractive to be transformed into tools for gene therapy. HIV-1 based lentiviral vectors were initially developed, a process which provided valuable insights into the biology of these vectors allowing progressive improvement of non-HIV vectors. The latest vectors have been refined to a very high level and can be produced safely for the clinic. This review will describe the general features of lentiviral vectors with particular emphasis on vectors derived from the non-HIV lentiviruses such as equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). It will then describe some key examples of gene therapy applications in neurological diseases such as Parkinson's disease (PD), motor neuron diseases, lysosomal storage diseases and ocular disorders. Finally, the prospects for clinical application of non-HIV lentiviral vectors for these disorders will also be outlined.

Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy.

Spinal muscular atrophy is one of the most common genetic causes of death in childhood, and there is currently no effective treatment. The disease is caused by mutations in the survival motor neuron gene. Gene therapy aimed at restoring the protein encoded by this gene is a rational therapeutic approach to ameliorate the disease phenotype. We previously reported that intramuscular delivery of a lentiviral vector expressing survival motor neuron increased the life expectancy of transgenic mice with spinal muscular atrophy. The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant effect. Here, we report that a single injection of self-complementary adeno-associated virus serotype 9 expressing green fluorescent protein or of a codon-optimized version of the survival motor neuron protein into the facial vein 1 day after birth in mice carrying a defective survival motor neuron gene led to widespread gene transfer. Furthermore, this gene therapy resulted in a substantial extension of life span in these animals. These data demonstrate a significant increase in survival in a mouse model of spinal muscular atrophy and provide evidence for effective therapy.

Defective tumor necrosis factor-alpha-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease.

The cytokine tumor necrosis factor-alpha (TNFalpha) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFalpha production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702-710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFalpha is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFalpha-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous beta-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of beta-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFalpha evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFalpha-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFalpha-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.