ROS networks: designs, aging, Parkinson's disease and precision therapies.

How the network around ROS protects against oxidative stress and Parkinson's disease (PD), and how processes at the minutes timescale cause disease and aging after decades, remains enigmatic. Challenging whether the ROS network is as complex as it seems, we built a fairly comprehensive version thereof which we disentangled into a hierarchy of only five simpler subnetworks each delivering one type of robustness. The comprehensive dynamic model described in vitro data sets from two independent laboratories. Notwithstanding its five-fold robustness, it exhibited a relatively sudden breakdown, after some 80 years of virtually steady performance: it predicted aging. PD-related conditions such as lack of DJ-1 protein or increased α-synuclein accelerated the collapse, while antioxidants or caffeine retarded it. Introducing a new concept (aging-time-control coefficient), we found that as many as 25 out of 57 molecular processes controlled aging. We identified new targets for "life-extending interventions": mitochondrial synthesis, KEAP1 degradation, and p62 metabolism.

ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

Differential Modulation of NF-κB in Neurons and Astrocytes Underlies Neuroprotection and Antigliosis Activity of Natural Antioxidant Molecules.

Neuroinflammation, a hallmark of chronic neurodegenerative disorders, is characterized by sustained glial activation and the generation of an inflammatory loop, through the release of cytokines and other neurotoxic mediators that cause oxidative stress and limit functional repair of brain parenchyma. Dietary antioxidants may protect against neurodegenerative diseases by counteracting chronic neuroinflammation and reducing oxidative stress. Here, we describe the effects of a number of natural antioxidants (polyphenols, carotenoids, and thiolic molecules) in rescuing astrocytic function and neuronal viability following glial activation by reducing astrocyte proliferation and restoring astrocytic and neuronal survival and basal levels of reactive oxygen species (ROS). All antioxidant molecules are also effective under conditions of oxidative stress and glutamate toxicity, two maladaptive components of neuroinflammatory processes. Moreover, it is remarkable that their antioxidant and anti-inflammatory activity occurs through differential modulation of NF-κB binding activity in neurons and astrocytes. In fact, we show that inflammatory stimuli promote a significant induction of NF-κB binding activity in astrocytes and its concomitant reduction in neurons. These changes are prevented in astrocytes and neurons pretreated with the antioxidant molecules, suggesting that NF-κB plays a key role in the modulation of survival and anti-inflammatory responses. Finally, we newly demonstrate that effective antigliosis and neuroprotective activity is achieved with a defined cocktail of four natural antioxidants at very low concentrations, suggesting a promising strategy to reduce inflammatory and oxidative damage in neurodegenerative diseases with limited side effects.

Dysregulation of Astrocytic HMGB1 Signaling in Amyotrophic Lateral Sclerosis.

Astrocytes have emerged as critical elements for the maintenance and function of the central nervous system. The expression on their cell membrane of RAGE and TLR4 receptors makes astrocytes susceptible to High-mobility group box 1 (HMGB1), a nuclear protein typically released in the extracellular milieu by living cells experiencing physiological stress conditions or by damaged cells. Here, we show that the interaction of HMGB1 with normal spinal cord astrocytes induces the astrocytic production of neurotrophic factors, particularly brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Multiple investigations suggest a role for HMGB1 in amyotrophic lateral sclerosis (ALS). Yet, no mechanistic information on the implication of HMGB1 signaling in this disorder is currently available. We demonstrate that non-transgenic and transgenic SOD1WT spinal motor neurons exhibit only a basal nucleus-to-cytoplasm shuttling of the HMGB1 protein. Conversely, in SOD1G93A ALS mouse spinal cords, HMGB1 significantly translocates from the nucleus to the cytoplasm of motor neurons, thereby suggesting that it may be eventually released in the extracellular environment during the progression of the disease. We postulate that extracellular HMGB1 can paracrinally interact with the neighboring astrocytes in an attempt to counteract the neurodegenerative process. Yet, at variance with normal cells, SOD1G93A-expressing astrocytes show impaired capacity to raise BDNF and GDNF levels upon HMGB1 stimulation. Our data suggest that HMGB1 have a potential to promote neuroprotective actions by healthy astrocytes. However, this neurotrophic response is disrupted in ALS astrocytes. This indicates that diseased astroglial cells may exacerbate motor neuron degeneration in ALS because of the loss of their neurosupportive functions.

Differentiation by nerve growth factor (NGF) involves mechanisms of crosstalk between energy homeostasis and mitochondrial remodeling.

Neuronal differentiation involves extensive modification of biochemical and morphological properties to meet novel functional requirements. Reorganization of the mitochondrial network to match the higher energy demand plays a pivotal role in this process. Mechanisms of neuronal differentiation in response to nerve growth factor (NGF) have been largely characterized in terms of signaling, however, little is known about its impact on mitochondrial remodeling and metabolic function. In this work, we show that NGF-induced differentiation requires the activation of autophagy mediated by Atg9b and Ambra1, as it is disrupted by their genetic knockdown and by autophagy blockers. NGF differentiation involves the induction of P-AMPK and P-CaMK, and is prevented by their pharmacological inhibition. These molecular events correlate with modifications of energy and redox homeostasis, as determined by ATP and NADPH changes, higher oxygen consumption (OCR) and ROS production. Our data indicate that autophagy aims to clear out exhausted mitochondria, as determined by enhanced localization of p62 and Lysotracker-red to mitochondria. In addition, we newly demonstrate that NGF differentiation is accompanied by increased mitochondrial remodeling involving higher levels of fission (P-Drp1) and fusion proteins (Opa1 and Mfn2), as well as induction of Sirt3 and the transcription factors mtTFA and PPARγ, which regulate mitochondria biogenesis and metabolism to sustain increased mitochondrial mass, potential, and bioenergetics. Overall, our data indicate a new NGF-dependent mechanism involving mitophagy and extensive mitochondrial remodeling, which plays a key role in both neurogenesis and nerve regeneration.

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.

Withaferin A Inhibits Nuclear Factor-κB-Dependent Pro-Inflammatory and Stress Response Pathways in the Astrocytes.

Several lines of evidence suggest that astrocytes play a key role in modulating the immune responses of the central nervous system (CNS) to infections, injuries, or pathologies. Yet, their contribution to these processes remains mostly elusive. Astroglia are endowed with a wide range of toll-like receptors (TLR) by which they can sense infectious agents as well as endogenous danger signals released by damaged cells. Here we demonstrate that the activation of astrocytic TLR4 by bacterial lipopolysaccharide (LPS) challenge can promote nuclear factor κB (NF-κB)-dependent induction of pro-inflammatory and stress response mediators, particularly Tumor Necrosis Factor α (TNFα), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS). Since the steroid lactone Withaferin A was described to inhibit NF-κB activity in different cell types, we next determined the impact of this natural compound towards the identified astrocytic signalling pathway. Innate immune activation was induced by stimulation of the LPS/TLR4 axis in spinal cord astrocytes. We provide evidence that both pre-treating and post-treating the cells with Withaferin A attenuate astrocytic NF-κB activity as well as the consequent production of TNFα, COX-2, and iNOS induced by stimulation of the LPS/TLR4 pathway. This study suggests that Withaferin A may be an eligible candidate for the treatment of neuroinflammatory and stress conditions characterized by an important astrocytic input.

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.

Astrocyte signaling and neurodegeneration: new insights into CNS disorders.

Growing evidence indicates that astrocytes cannot be just considered as passive supportive cells deputed to preserve neuronal activity and survival, but rather they are involved in a striking number of active functions that are critical to the performance of the central nervous system (CNS). As a consequence, it is becoming more and more evident that the peculiar properties of these cells can actively contribute to the extraordinary functional complexity of the brain and spinal cord. This new perception of the functioning of the CNS opens up a wide range of new possibilities to interpret various physiological and pathological events, and moves the focus beyond the neuronal compartment toward astrocyte-neuron interactions. With this in mind, here we provide a synopsis of the activities astrocytes perform in normal conditions, and we try to discuss what goes wrong with these cells in specific pathological conditions, such as Alzheimer Disease, prion diseases and amyotrophic lateral sclerosis.

The peroxisome proliferator-activated receptor γ (PPARγ) controls natural protective mechanisms against lipid peroxidation in amyotrophic lateral sclerosis.

Recent evidence highlights the peroxisome proliferator-activated receptors (PPARs) as critical neuroprotective factors in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To gain new mechanistic insights into the role of these receptors in the context of ALS, here we investigated how PPAR transcriptional activity varies in hSOD1(G93A) ALS transgenic mice. We demonstrate that PPARγ-driven transcription selectively increases in the spinal cord of symptomatic hSOD1(G93A) mice. This phenomenon correlates with the up-regulation of target genes, such as lipoprotein lipase and glutathione S-transferase α-2, which are implicated in scavenging lipid peroxidation by-products. Such events are associated with enhanced PPARγ immunoreactivity within motor neuronal nuclei. This observation, and the fact that PPARγ displays increased responsiveness in cultured hSOD1(G93A) motor neurons, points to a role for this receptor in neutralizing deleterious lipoperoxidation derivatives within the motor cells. Consistently, in both motor neuron-like cultures and animal models, we report that PPARγ is activated by lipid peroxidation end products, such as 4-hydroxynonenal, whose levels are elevated in the cerebrospinal fluid and spinal cord from ALS patients. We propose that the accumulation of critical concentrations of lipid peroxidation adducts during ALS progression leads to the activation of PPARγ in motor neurons. This in turn triggers self-protective mechanisms that involve the up-regulation of lipid detoxification enzymes, such as lipoprotein lipase and glutathione S-transferase α-2. Our findings indicate that anticipating natural protective reactions by pharmacologically modulating PPARγ transcriptional activity may attenuate neurodegeneration by limiting the damage induced by lipid peroxidation derivatives.

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.

Implications of gliotransmission for the pharmacotherapy of CNS disorders.

The seminal discovery that glial cells, particularly astrocytes, can release a number of gliotransmitters that serve as signalling molecules for the cross-talk with neighbouring cellular populations has recently changed our perception of brain functioning, as well as our view of the pathogenesis of several disorders of the CNS. Since glutamate was one of the first gliotransmitters to be identified and characterized, we tackle the mechanisms that underlie its release from astrocytes, including the Ca2+ signals underlying its efflux from astroglia, and we discuss the involvement of these events in a number of relevant physiological processes, from the modulatory control of neighbouring synapses to the regulation of blood supply to cerebral tissues. The relevance of these mechanisms strongly indicates that the contribution of glial cells and gliotransmission to the activities of the brain cannot be overlooked, and any study of CNS physiopathology needs to consider glial biology to have a comprehensive overview of brain function and dysfunction. Abnormalites in the signalling that controls the astrocytic release of glutamate are described in several experimental models of neurological disorders, for example, AIDS dementia complex, Alzheimer's disease and cerebral ischaemia. While the modalities of glutamate release from astrocytes remain poorly understood, and this represents a major impediment to the definition of novel therapeutic strategies targeting this process at the molecular level, some key mediators deputed to the control of the glial release of this excitatory amino acid have been identified. Among these, we can mention, for instance, proinflammatory cytokines, such as tumour necrosis factor-α, and prostaglandins. Agents that are able to block the major steps of tumour necrosis factor-α and prostaglandin production and/or signalling can be proposed as novel therapeutic targets for the treatment of these disorders.