Astrocytic Clearance Research Articles

Long-term inhibition of ODC1 in APP/PS1 mice rescues amyloid pathology and switches astrocytes from a reactive to active state

Alzheimer’s disease (AD) is characterized by the loss of memory due to aggregation of misphosphorylated tau and amyloid beta (Aβ) plaques in the brain, elevated release of inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and reactive oxygen species from astrocytes, and subsequent neurodegeneration. Recently, it was found that enzyme Ornithine Decarboxylase 1 (ODC1) acts as a bridge between the astrocytic urea cycle and the putrescine-to-GABA conversion pathway in the brain of AD mouse models as well as human patients. In this study, we show that the long-term knockdown of astrocytic Odc1 in APP/PS1 animals was sufficient to completely clear Aβ plaques in the hippocampus while simultaneously switching the astrocytes from a detrimental reactive state to a regenerative active state, characterized by proBDNF expression. Our experiments also reveal an effect of astrocytic ODC1 inhibition on the expression of genes involved in synapse pruning and organization, histone modification, apoptotic signaling and protein processing. These genes are previously known to be associated with astrocytic activation and together create a neuroregeneration-supportive environment in the brain. By inhibiting ODC1 for a long period of 3 months in AD mice, we demonstrate that the beneficial amyloid-clearing process of astrocytes can be completely segregated from the systemically harmful astrocytic response to insult. Our study reports an almost complete clearance of Aβ plaques by controlling an endogenous degradation process, which also modifies the astrocytic state to create a regeneration-supportive environment in the brain. These findings present the potential of modulating astrocytic clearance of Aβ as a powerful therapeutic strategy against AD.

Astrocytic clearance and fragmentation of toxic proteins in Alzheimer’s disease on large-scale brain networks

The human brain is the most complicated biological structure on the planet. A major challenge of brain network modelling lies in its multi-scale spatio-temporal nature, covering scales from synapses to the whole brain. The coupled multiphysics and biochemical activities which spread through such a complex system shape brain capacity inside a structure-function relationship that requires a particular mathematical framework. Next-generation coupled-based mathematical modelling approaches to brain networks and the analysis of data-driven dynamical systems are needed to advance state-of-the-art therapeutic strategies for treating neurodegenerative diseases (NDDs) that affect millions of people worldwide, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Importantly, AD is marked by the presence of amyloid-beta (Aβ) plaques and tau (τ) proteins. Some disease-specific misfolded proteins can interact with healthy proteins to form long chains and aggregates of different sizes that have different transport properties and toxicity. An improved large-scale brain network model is proposed here to understand the pathogenesis of AD, especially the role of astrocytes in the presence of misfolded proteins (Aβ and τ). The idea involves astrocytic clearance, which assists in eliminating toxic Aβ via fragmentation. We use the general Smoluchowski theory of nucleation, aggregation, and fragmentation to predict the development and propagation of aggregates of misfolded proteins in the brain. It has been shown that the developed model leads to different size distributions and propagation along the network. We predicted that astrocytic clearance varies with the aggregate size, which is key to slowing down AD progression. The clearance and fragmentation of toxic proteins span several spatial and temporal scales, and this research will potentially yield new insight into the associated processes and brain networks in health and disease. Detailed multi-scale brain modelling provides a promising approach for consolidating, organizing, and bridging the data sets of data-driven brain network models.

Transient Inward KCC2 Transport Balances Perisynaptic Potassium at Excitatory Synapses

Accumulation of extracellular potassium [K+]o during neuronal activity challenges neuronal function and, therefore, requires a tight control. Based on electrophysiological recordings of astrocytic /Kir as a sensor of [K+]o, we demonstrate that KCC2 clears perisynaptic [K+]o during synaptic excitation by transient and activity-dependent reversal of its canonical outward mode. This operational mode of KCC2 complements astrocytic clearance of [K+]o and thus contributes to an attenuation of presynaptic vesicle release and a shortening of LTP, preventing excess excitation. We thus provide experimental evidence in support of a longstanding hypothesis, ascribing an additional potential to KCC2 as a therapeutic target in CNS diseases.

Neuromodulation of Astrocytic K+ Clearance.

Potassium homeostasis is fundamental for brain function. Therefore, effective removal of excessive K+ from the synaptic cleft during neuronal activity is paramount. Astrocytes play a key role in K+ clearance from the extracellular milieu using various mechanisms, including uptake via Kir channels and the Na+-K+ ATPase, and spatial buffering through the astrocytic gap-junction coupled network. Recently we showed that alterations in the concentrations of extracellular potassium ([K+]o) or impairments of the astrocytic clearance mechanism affect the resonance and oscillatory behavior of both the individual and networks of neurons. These results indicate that astrocytes have the potential to modulate neuronal network activity, however, the cellular effectors that may affect the astrocytic K+ clearance process are still unknown. In this study, we have investigated the impact of neuromodulators, which are known to mediate changes in network oscillatory behavior, on the astrocytic clearance process. Our results suggest that while some neuromodulators (5-HT; NA) might affect astrocytic spatial buffering via gap-junctions, others (DA; Histamine) primarily affect the uptake mechanism via Kir channels. These results suggest that neuromodulators can affect network oscillatory activity through parallel activation of both neurons and astrocytes, establishing a synergistic mechanism to maximize the synchronous network activity.

Enhancing the Astrocytic Clearance of Extracellular α-Synuclein Aggregates by Ginkgolides Attenuates Neural Cell Injury.

The accumulation of aggregated forms of the α-Synuclein (α-Syn) is associated with the pathogenesis of Parkinson's disease (PD), and the efficient clearance of aggregated α-Syn represents a potential approach in PD therapy. Astrocytes are the most numerous glia cells in the brain and play an essential role in supporting brain functions in PD state. In the present study, we demonstrated that cultured primary astrocytes engulfed and degraded extracellular aggregated recombinant human α-Syn. Meanwhile, we observed that the clearance of α-Syn by astrocytes was abolished by proteasome inhibitor MG132 and autophagy inhibitor 3-methyladenine (3MA). We further showed that intracellular α-Syn was reduced after ginkgolide B (GB) and bilobalide (BB) treatment, and the decrease was reversed by MG132 and 3MA. More importantly, GB and BB reduced indirect neurotoxicity to neurons induced by α-Syn-stimulated astrocytic conditioned medium. Together, we firstly find that astrocytes can engulf and degrade α-Syn aggregates via the proteasome and autophagy pathways, and further show that GB and BB enhance astrocytic clearance of α-Syn, which gives us an insight into the novel therapy for PD in future.

Multifaceted roles for astrocytes in spreading depolarization: A target for limiting spreading depolarization in acute brain injury?

Spreading depolarizations (SDs) are coordinated waves of synchronous depolarization, involving large numbers of neurons and astrocytes as they spread slowly through brain tissue. The recent identification of SDs as likely contributors to pathophysiology in human subjects has led to a significant increase in interest in SD mechanisms, and possible approaches to limit the numbers of SDs or their deleterious consequences in injured brain. Astrocytes regulate many events associated with SD. SD initiation and propagation is dependent on extracellular accumulation of K(+) and glutamate, both of which involve astrocytic clearance. SDs are extremely metabolically demanding events, and signaling through astrocyte networks is likely central to the dramatic increase in regional blood flow that accompanies SD in otherwise healthy tissues. Astrocytes may provide metabolic support to neurons following SD, and may provide a source of adenosine that inhibits neuronal activity following SD. It is also possible that astrocytes contribute to the pathophysiology of SD, as a consequence of excessive glutamate release, facilitation of NMDA receptor activation, brain edema due to astrocyte swelling, or disrupted coupling to appropriate vascular responses after SD. Direct or indirect evidence has accumulated implicating astrocytes in many of these responses, but much remains unknown about their specific contributions, especially in the context of injury. Conversion of astrocytes to a reactive phenotype is a prominent feature of injured brain, and recent work suggests that the different functional properties of reactive astrocytes could be targeted to limit SDs in pathophysiological conditions.

Animal model of autism induced by prenatal exposure to valproate: Altered glutamate metabolism in the hippocampus

Autism spectrum disorders (ASD) are characterized by deficits in social interaction, language and communication impairments and repetitive and stereotyped behaviors, with involvement of several areas of the central nervous system (CNS), including hippocampus. Although neurons have been the target of most studies reported in the literature, recently, considerable attention has been centered upon the functionality and plasticity of glial cells, particularly astrocytes. These cells participate in normal brain development and also in neuropathological processes. The present work investigated hippocampi from 15 (P15) and 120 (P120) days old male rats prenatally exposed to valproic acid (VPA) as an animal model of autism. Herein, we analyzed astrocytic parameters such as glutamate transporters and glutamate uptake, glutamine synthetase (GS) activity and glutathione (GSH) content. In the VPA group glutamate uptake was unchanged at P15 and increased 160% at P120; the protein expression of GLAST did not change neither in P15 nor in P120, while GLT1 decreased 40% at P15 and increased 92% at P120; GS activity increased 43% at P15 and decreased 28% at P120; GSH content was unaltered at P15 and had a 27% increase at P120. These data highlight that the astrocytic clearance and destination of glutamate in the synaptic cleft might be altered in autism, pointing out important aspects to be considered from both pathophysiologic and pharmacological approaches in ASD.

Role of Endosomal Sodium/Hydrogen Exchangers NHE6 and NHE9 in a Neurobiological Model of Disease

Ionic and pH gradients must be tightly regulated for many aspects of neuronal signaling and synaptic cleft homeostasis, including neurotransmitter uptake and astrocytic clearance through transporters, receptor recycling, and communication through gap junctions. Recently, several independent studies have correlated a range of neurological disorders, including autism, X‐linked mental retardation, epilepsy, attention deficit hyperactivity disorder and addiction, to mutations in a subgroup of sodium‐hydrogen exchangers, NHE6 and NHE9. These transporters are widely expressed in the brain and localize to early (NHE6) and recycling endosomes (NHE9), where they are thought to mediate the electrically silent counter transport of cations with protons to regulate luminal pH and control cargo trafficking. We have tested the hypothesis that NHE6 and NHE9 share overlapping functions in regulating intravesicular pH to control the delivery and turnover of glial specific glutamate transporters at the synapse. We have shown that isoform‐specific overexpression of NHE9 alters trafficking with increased surface expression of general (transferrin) receptors and synapse specific (glutamate) transporters. These findings provide the first insight into the neurobiological basis of NHE dysfunction. We will further elucidate the molecular mechanisms by measuring the pH of the endosomes, as well as create single and double knock down lines of NHE6 and NHE9 to demonstrate their potential overlapping functions..

Kyotorphin transport and metabolism in rat and mouse neonatal astrocytes

Neuropeptide inactivation is generally thought to occur via peptidase-mediated degradation. However, a recent study found increased analgesia after L-kyotorphin (L-Tyr-L-Arg; L-KTP) administration in mice lacking an oligopeptide transporter, PEPT2. The current study examines the role of PEPT2 in L-KTP uptake by astrocytes and compares it to astrocytic L-KTP degradation. L-[ 3H]KTP uptake was measured in primary cultures of neonatal astrocytes from rats and from Pept2 +/+ and Pept2 −/− mice. Uptake was further characterized using potential inhibitors. L-[ 3H]KTP degradation was examined in primary astrocyte cultures from Pept2 −/− mice by following the formation of L-[ 3H]tyrosine. The uptake of L-[ 3H]KTP in both rat and Pept2 +/+ mouse neonatal astrocytes was inhibited by known PEPT2 inhibitors. L-[ 3H]KTP uptake was also reduced in Pept2 −/− astrocytes as compared to those from Pept2 +/+ mice. Kinetic analysis indicated the presence of a high affinity ( K m ∼ 50 μM) transporter for L-[ 3H]KTP, identified as Pept2, and a low affinity transporter ( K m ∼ 3–4 mM), inhibited by amastatin, bestatin and tyrosine. Astrocytes also degraded L-KTP through a low affinity peptidase ( K m ∼ 2 mM). Astrocytic clearance of L-KTP occurs via both peptidase activity and transport. These processes occur at similar rates and may be linked. This supports the contention that oligopeptide transport may have an impact on the extracellular clearance (and potentially activity) of certain neuropeptides.

Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition.

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia coli lipopolysaccharide, LPS) and the inflammatory cytokine, interferon-gamma (IFNgamma), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNgamma (LPS + IFNgammay) up-regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre-treatment with ascorbate-2-O-phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNgamma. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNy decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.

Dependence of neurones on astrocytes in a coculture system renders neurones sensitive to transforming growth factor beta1-induced glutamate toxicity.

Transforming growth factor beta1 (TGF-beta1) has been implicated in formation of astrocyte scars, which prevents axonal regeneration. A coculture system of astrocytes and cerebellar cells was used to investigate possible neurotoxic effects of TGF-beta1. Although not directly neurotoxic, TGF-beta1 was toxic to cerebellar cells in the presence of astrocytes. This toxicity is based on an effect of the cytokine on astrocytes, as conditioned medium from astrocyte cultures treated with TGF-beta1 was more toxic by a similar mechanism. This neurotoxicity was mediated by glutamate present in the culture medium as demonstrated by inhibition by MK-801. Astrocytic ability to metabolise glutamate was compromised by TGF-beta1, as this cytokine increased glutamate concentration. The astrocytes in the coculture system responded to the presence of neurones by secreting neuroprotective interleukin-6, which was partly protective against the TGF-beta1-induced toxicity. In the coculture system, neurones responded to the presence of astrocytes by a reduction in resistance to glutamate toxicity. On addition of TGF-beta1, which compromised astrocytic clearance of glutamate, this reduction in resistance to glutamate toxicity led to a reduction in neuronal survival. These results suggest that when neurones are cocultured with astrocytes they become dependent on astrocytes for survival. This dependence makes neurones susceptible to damage when astrocytes are activated by substances such as TGF-beta1.