Perisynaptic Astrocytes Research Articles

Astrocyte TrkB.T1 deficiency disrupts glutamatergic synaptogenesis and astrocyte-synapse interactions.

Perisynaptic astrocyte processes (PAPs) contact pre- and post-synaptic elements to provide structural and functional support to synapses. Accumulating research demonstrates that the cradling of synapses by PAPs is critical for synapse formation, stabilization, and plasticity. The specific signaling pathways that govern these astrocyte-synapse interactions, however, remain to be elucidated. Herein, we demonstrate a role for the astrocyte TrkB.T1 receptor, a truncated isoform of the canonical receptor for brain derived neurotrophic factor (BDNF), in modulating astrocyte-synapse interactions and excitatory synapse development. Neuron-astrocyte co-culture studies revealed that loss of astrocyte TrkB.T1 disrupts the formation of PAPs. To elucidate the role of TrkB.T1 in synapse development, we conditionally deleted TrkB.T1 in astrocytes in mice. Synaptosome preparations were employed to probe for TrkB.T1 localization at the PAP, and confocal three-dimensional microscopy revealed a significant reduction in synapse density and astrocyte-synapse interactions across development in the absence of astrocytic TrkB.T1. These findings suggest that BDNF/TrkB.T1 signaling in astrocytes is critical for normal excitatory synapse formation in the cortex and that astrocyte TrkB.T1 serves a requisite role in astrocyte synapse interactions. Overall, this work provides new insights into the molecular mechanisms of astrocyte-mediated synaptogenesis and may have implications for understanding neurodevelopmental disorders and developing potential therapeutic targets.

Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.

Astrocytes, a major glial cell type of the brain, regulate synapse numbers and function. However, whether astrocyte dysfunction can cause synaptic pathologies in neurological disorders such as Parkinson's Disease (PD) is unknown. Here, we investigated the impact of the most common PD-linked mutation in the leucine-rich repeat kinase 2 (LRRK2) gene (G2019S) on the synaptic functions of astrocytes. We found that both in human and mouse cortex, the LRRK2 G2019S mutation causes astrocyte morphology deficits and enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), which are important components of perisynaptic astrocyte processes. Reducing ERM phosphorylation in LRRK2 G2019S mouse astrocytes restored astrocyte morphology and corrected excitatory synaptic deficits. Using an in vivo BioID proteomic approach, we found Ezrin, the most abundant astrocytic ERM protein, interacts with the Autophagy-Related 7 (Atg7), a master regulator of catabolic processes. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in the LRRK2 G2019S astrocytes. Importantly, Atg7 function is required to maintain proper astrocyte morphology. These studies reveal an astrocytic molecular mechanism that could serve as a therapeutic target in PD.

Morphological changes in perisynaptic astrocytes induced by dopamine neuronal degeneration in the striatum of rats

IntroductionThe typical functionality of astrocytes was previously shown to be disrupted by Parkinson's disease (PD), which actively regulates synaptic neurotransmission. However, the morphological changes in astrocytes wrapping glutamatergic synapses in the striatum after dopamine (DA) neuronal degeneration is unclear. MethodsWe utilized a range of methodologies, encompassing the 6-hydroxydopamine (6OHDA)-induced PD model, as well as techniques such as immunohistochemistry, Western blotting, immunofluorescence and immunoelectron microscopy (IEM) to delve into the consequences of DA neuronal degeneration on the morphological attributes of perisynaptic astrocytes. ResultsOur findings demonstrated a notable rise in glial fibrillary acidic protein (GFAP) + astrocyte density and an upregulation in GFAP protein expression within the striatum due to DA neuronal degeneration, coincided with the enlargement, elongation, and thickening of astrocyte protuberances. However, the expression levels of glutamate transporter 1 (GLT1) and glutamine synthetase (GS), which are related to glutamate–glutamine cycle, were significantly reduced. Double immunofluorescence and IEM results indicated that different proportions of vesicular glutamate transporter 1 (VGlut1)+ and vesicular glutamate transporter 2 (VGlut2) + terminals were wrapped by astrocytes. Additionally, DA neuronal degeneration increased the percentage and area of VGlut1+ and VGlut2+ terminals wrapped by GFAP + astrocytes in the striatum. Furthermore, we noted that DA neuronal degeneration increased the percentage of VGlut1+ and VGlut2+ axo-spinous synapses wrapped by astrocytes but had no effect on axo-dendritic synapses. ConclusionHence, perisynaptic astrocytes wrapping striatal glutamatergic synapses exhibit substantial morphological and functional alterations following DA neuronal degeneration making them a potential target for therapeutic interventions in PD.

A Closer Look at the Perivascular Unit in the Development of Enlarged Perivascular Spaces in Obesity, Metabolic Syndrome, and Type 2 Diabetes Mellitus.

The recently described perivascular unit (PVU) resides immediately adjacent to the true capillary neurovascular unit (NVU) in the postcapillary venule and contains the normal-benign perivascular spaces (PVS) and pathological enlarged perivascular spaces (EPVS). The PVS are important in that they have recently been identified to be the construct and the conduit responsible for the delivery of metabolic waste from the interstitial fluid to the ventricular cerebrospinal fluid for disposal into the systemic circulation, termed the glymphatic system. Importantly, the outermost boundary of the PVS is lined by protoplasmic perivascular astrocyte endfeet (pvACef) that communicate with regional neurons. As compared to the well-recognized and described neurovascular unit (NVU) and NVU coupling, the PVU is less well understood and remains an emerging concept. The primary focus of this narrative review is to compare the similarities and differences between these two units and discuss each of their structural and functional relationships and how they relate not only to brain homeostasis but also how they may relate to the development of multiple clinical neurological disease states and specifically how they may relate to obesity, metabolic syndrome, and type 2 diabetes mellitus. Additionally, the concept and importance of a perisynaptic astrocyte coupling to the neuronal synapses with pre- and postsynaptic neurons will also be considered as a perisynaptic unit to provide for the creation of the information transfer in the brain via synaptic transmission and brain homeostasis. Multiple electron microscopic images and illustrations will be utilized in order to help explain these complex units.

Protoplasmic Perivascular Astrocytes Play a Crucial Role in the Development of Enlarged Perivascular Spaces in Obesity, Metabolic Syndrome, and Type 2 Diabetes Mellitus

Astrocytes (ACs) are the most abundant cells in the brain and, importantly, are the master connecting and communicating cells that provide structural and functional support for brain cells at all levels of organization. Further, they are recognized as the guardians and housekeepers of the brain. Protoplasmic perivascular astrocyte endfeet and their basal lamina form the delimiting outermost barrier (glia limitans) of the perivascular spaces in postcapillary venules and are important for the clearance of metabolic waste. They comprise the glymphatic system, which is critically dependent on proper waste removal by the pvACef polarized aquaporin-4 water channels. Also, the protoplasmic perisynaptic astrocyte endfeet (psACef) are important in cradling the neuronal synapses that serve to maintain homeostasis and serve a functional and supportive role in synaptic transmission. Enlarged perivascular spaces (EPVS) are emerging as important aberrant findings on magnetic resonance imaging (MRI), and are associated with white matter hyperintensities, lacunes, and aging, and are accepted as biomarkers for cerebral small vessel disease, increased obesity, metabolic syndrome, and type 2 diabetes. Knowledge is exponentially expanding regarding EPVS along with the glymphatic system, since EPVS are closely associated with impaired glymphatic function and waste removal from the brain to the cerebrospinal fluid and systemic circulation. This review intends to focus on how the pvACef play a crucial role in the development of EPVS.

A novel role for MLC1 in regulating astrocyte-synapse interactions.

Loss of function of the astrocyte membrane protein MLC1 is the primary genetic cause of the rare white matter disease Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), which is characterized by disrupted brain ion and water homeostasis. MLC1 is prominently present around fluid barriers in the brain, such as in astrocyte endfeet contacting blood vessels and in processes contacting the meninges. Whether the protein plays a role in other astrocyte domains is unknown. Here, we show that MLC1 is present in distal astrocyte processes, also known as perisynaptic astrocyte processes (PAPs) or astrocyte leaflets, which closely interact with excitatory synapses in the CA1 region of the hippocampus. We find that the PAP tip extending toward excitatory synapses is shortened in Mlc1-null mice. This affects glutamatergic synaptic transmission, resulting in a reduced rate of spontaneous release events and slower glutamate re-uptake under challenging conditions. Moreover, while PAPs in wildtype mice retract from the synapse upon fear conditioning, we reveal that this structural plasticity is disturbed in Mlc1-null mice, where PAPs are already shorter. Finally, Mlc1-null mice show reduced contextual fear memory. In conclusion, our study uncovers an unexpected role for the astrocyte protein MLC1 in regulating the structure of PAPs. Loss of MLC1 alters excitatory synaptic transmission, prevents normal PAP remodeling induced by fear conditioning and disrupts contextual fear memory expression. Thus, MLC1 is a new player in the regulation of astrocyte-synapse interactions.

Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

The Effect of Inhibition of Perisynaptic Astrocyte Glycogen Utilization on Depression-Like Behavior.

Under physiological conditions, astrocytes produce lactate to meet the increased synaptic energy demand due to neuronal activity. In the light of the findings showing that this process is disrupted in the pathophysiology of major depression, the aim of this study is to investigate the effect of pharmacological inhibition of perisynaptic astrocyte glycogen utilization on anxiety-like behavior and depression-like behavior in female and male mice. In this study, DAB (1,4-dideoxy-1,4-imino-D-arabinitol), which is an inhibitor of glycogen breaking enzyme glycogen phosphorylase, was intrahippocampally administered to 15 female and 14 male Swiss albino mice, while 15 female and 12 male Swiss albino mice received intrahippocampal saline injections. Three and five days after the injections, the anxiety-like and depression-like behaviors of the mice were assessed by locomotor activity, open-field test, light-dark box test, tail suspension test and sucrose preference test. Three days after injection, neither depression-like nor anxietylike significant behavioral changes were detected in the male experimental group mice compared to the control group; but an increase in locomotor activity (p=0.05) and time spent in the open-field (p=0.01) were observed on the fifth day. In evaluations of the female experimental group mice on the third and fifth days, depression-like and anxiety-like behaviors were found similar to the control group, as seen in the male mice. The only significant difference in the experimental group female mice was found in the sucrose preference test, which revealed an increased tendency to prefer sucrose (p=0.003) compared to the control group. The inhibition of glycogen use in the hippocampus by DAB did not affect anxiety-like and depression-like behaviors 3 and 5 days after injection in both female and male mice. The increase in the time spent in the open-field by male experimental group mice was associated not with anxiety, but with increase in the locomotor activity. The fact that no significant difference was observed in the light-dark box test, which is another test used to evaluate anxiety, supported this opinion. The increase seen in the sucrose preference test in female experimental group mice was not interpreted as an increase in hedonic behavior because prevention of glycogen breakdown in the hypothalamus might have homeostatically increased sugar-craving and therefore resulted in an increase in sucrose preference. Different set of tests better targeting the energy and glucose metabolism and applied at farther time points than surgery are recommended for future studies.

Activity-dependent translation dynamically alters the proteome of the perisynaptic astrocyte process.

Within eukaryotic cells, translation is regulated independent of transcription, enabling nuanced, localized, and rapid responses to stimuli. Neurons respond transcriptionally and translationally to synaptic activity. Although transcriptional responses are documented in astrocytes, here we test whether astrocytes have programmed translational responses. We show that seizure activity rapidly changes the transcripts on astrocyte ribosomes, some predicted to be downstream of BDNF signaling. In acute slices, we quantify the extent to which cues of neuronal activity activate translation in astrocytes and show that this translational response requires the presence of neurons, indicating that the response is non-cell autonomous. We also show that this induction of new translation extends into the periphery of astrocytes. Finally, synaptic proteomics show that new translation is required for changes that occur in perisynaptic astrocyte protein composition after fear conditioning. Regulation of translation in astrocytes by neuronal activity suggests an additional mechanism by which astrocytes may dynamically modulate nervous system functioning.

Perirhinal Cortex LTP Does Not Require Astrocyte BDNF-TrkB Signaling.

Neurons release and respond to brain-derived neurotrophic factor (BDNF) with bursts of brain activity. BDNF action is known to extend to peri-synaptic astrocytes, contributing to synaptic strengthening. This implies that astrocytes have a set of dynamic responses, some of which might be secondary to activation of the tropomyosin tyrosine kinase B (TrkB) receptor. Here, we assessed the contribution of BDNF to long-term synaptic potentiation (LTP), by specifically deleting TrkB in cortical astrocytes. TrkB deletion had no effect on LTP induction, stabilization and maintenance, indicating that TrkB signaling in astrocytes is extraneous to transducing BDNF activity for synaptic strengthening.

Uncoupling the Excitatory Amino Acid Transporter 2 From Its C-Terminal Interactome Restores Synaptic Glutamate Clearance at Corticostriatal Synapses and Alleviates Mutant Huntingtin-Induced Hypokinesia

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. However, despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have not been fully uncovered. This limits the options for pharmacological treatment of glutamate-related motor disorders, including Huntington’s disease (HD). We therefore investigated the possible binding partners of transgenic EAAT2 and their alterations under the influence of mutant huntingtin (mHTT). Mass spectrometry analysis after pull-down of striatal YFP-EAAT2 from wild-type (WT) mice and heterozygote (HET) Q175 mHTT-knock-in mice identified a total of 148 significant (FDR < 0.05) binders to full-length EAAT2. Of them 58 proteins exhibited mHTT-related differences. Most important, in 26 of the 58 mHTT-sensitive cases, protein abundance changed back toward WT levels when the mice expressed a C-terminal-truncated instead of full-length variant of EAAT2. These findings motivated new attempts to clarify the role of astrocytic EAAT2 regulation in cortico-basal movement control. Striatal astrocytes of Q175 HET mice were targeted by a PHP.B vector encoding EAAT2 with different degree of C-terminal modification, i.e., EAAT2-S506X (truncation at S506), EAAT2-4KR (4 lysine to arginine substitutions) or EAAT2 (full-length). The results were compared to HET and WT injected with a tag-only vector (CTRL). It was found that the presence of a C-terminal-modified EAAT2 transgene (i) increased the level of native EAAT2 protein in striatal lysates and perisynaptic astrocyte processes, (ii) enhanced the glutamate uptake of transduced astrocytes, (iii) stimulated glutamate clearance at individual corticostriatal synapses, (iv) increased the glutamate uptake of striatal astrocytes and (iv) alleviated the mHTT-related hypokinesia (open field indicators of movement initiation). In contrast, over-expression of full-length EAAT2 neither facilitated glutamate uptake nor locomotion. Together, our results support the new hypothesis that preventing abnormal protein-protein interactions at the C-terminal of EAAT2 could eliminate the mHTT-related deficits in corticostriatal synaptic glutamate clearance and movement initiation.

Neuronal contact upregulates astrocytic sphingosine-1-phosphate receptor 1 to coordinate astrocyte-neuron cross communication.

Astrocytes, the most abundant glial cells in the mammalian brain, directly associate with and regulate neuronal processes and synapses and are important regulators of brain development. Yet little is known of the molecular mechanisms that control the establishment of astrocyte morphology and the bi-directional communication between astrocytes and neurons. Here we show that neuronal contact stimulates expression of S1PR1, the receptor for the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P), on perisynaptic astrocyte processes and that S1PR1 drives astrocyte morphological complexity and morphogenesis. Moreover, the S1P/S1PR1 axis increases neuronal contact-induced expression of astrocyte secreted synaptogenic factors SPARCL1 and thrombospondin 4 that are involved in neural circuit assembly. Our findings have uncovered new functions for astrocytic S1PR1 signaling in regulation of bi-directional astrocyte-neuron crosstalk at the nexus of astrocyte morphogenesis and synaptogenesis.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy.

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.

Cell adhesion molecules regulating astrocyte-neuron interactions.

A tripartite synapse comprises a neuronal presynaptic axon and a postsynaptic dendrite, which are closely ensheathed by a perisynaptic astrocyte process. Through their structural and functional association with thousands of neuronal synapses, astrocytes regulate synapse formation and function. Recent work revealed a diverse range of cell adhesion-based mechanisms that mediate astrocyte-synapse interactions at tripartite synapses. Here, we will review some of these findings unveiling a highly dynamic bidirectional signaling between astrocytes and synapses, which orchestrates astrocyte morphological maturation and synapse development. Moreover, we will discuss the roles of these newly discovered molecular pathways in brain physiology and function both in health and disease.

Characterization of Subcellular Organelles in Cortical Perisynaptic Astrocytes.

Perisynaptic astrocytic processes (PAPs) carry out several different functions, from metabolite clearing to control of neuronal excitability and synaptic plasticity. All these functions are likely orchestrated by complex cellular machinery that resides within the PAPs and relies on a fine interplay between multiple subcellular components. However, traditional transmission electron microscopy (EM) studies have found that PAPs are remarkably poor of intracellular organelles, failing to explain how such a variety of PAP functions are achieved in the absence of a proportional complex network of intracellular structures. Here, we use serial block-face scanning EM to reconstruct and describe in three dimensions PAPs and their intracellular organelles in two different mouse cortical regions. We described five distinct organelles, which included empty and full endosomes, phagosomes, mitochondria, and endoplasmic reticulum (ER) cisternae, distributed within three PAPs categories (branches, branchlets, and leaflets). The majority of PAPs belonged to the leaflets category (~60%), with branchlets representing a minority (~37%). Branches were rarely in contact with synapses (<3%). Branches had a higher density of mitochondria and ER cisternae than branchlets and leaflets. Also, branches and branchlets displayed organelles more frequently than leaflets. Endosomes and phagosomes, which accounted for more than 60% of all the organelles detected, were often associated with the same PAP. Likewise, mitochondria and ER cisternae, representing ~40% of all organelles were usually associated. No differences were noted between the organelle distribution of the somatosensory and the anterior cingulate cortex. Finally, the organelle distribution in PAPs did not largely depend on the presence of a spine apparatus or a pre-synaptic mitochondrion in the synapse that PAPs were enwrapping, with some exceptions regarding the presence of phagosomes and ER cisternae, which were slightly more represented around synapses lacking a spine apparatus and a presynaptic mitochondrion, respectively. Thus, PAPs contain several subcellular organelles that could underlie the diverse astrocytic functions carried out at central synapses.

Comparison of the Tripartite Organization of Synaptic Terminals in Intraocular Septal Transplants and in the Septal Area of the Brain

The septal area of the brain is functionally connected to other limbic structures and is involved in cognitive processes. Intraocular neural transplants provide an experimental model for studying the endogenous morphofunctional properties of the septum. The aim of the present work was to compare the structure of synaptic contacts in septal intraocular neural transplants and in the septal area of the brain in situ. Neurons are regarded as thee-component complexes including not only pre- and postsynaptic components, but also the astrocyte processes encircling them. Ultrastructural studies showed that despite the absence of normal afferent and efferent connections in intraocular neural transplants, their synapses reproduced the tripartite organization. Morphometric analysis of their parameters showed a decrease in the statistical mean area and perimeter of the presynaptic compartment as compared with normal. At the same time, the extent of encirclement by perisynaptic astrocyte processes in transplants was, conversely, significantly greater than in the septum in situ. The mean extents of active zones in synaptic profiles in transplanted neurons was greater than that in controls. The morphometric data obtained here indicate coordinated regulation of the sizes of the three compartments of synapses depending on the functional state of the synaptic apparatus overall.

Local Efficacy of Glutamate Uptake Decreases with Synapse Size

Synaptically released glutamate is largely cleared by glutamate transporters localized on perisynaptic astrocyte processes. Therefore, the substantial variability of astrocyte coverage of individual hippocampal synapses implies that the efficacy of local glutamate uptake and thus the spatial fidelity of synaptic transmission is synapse dependent. By visualization of sub-diffraction-limit perisynaptic astrocytic processes and adjacent postsynaptic spines, we show that, relative to their size, small spines display a stronger coverage by astroglial transporters than bigger neighboring spines. Similarly, glutamate transients evoked by synaptic stimulation are more sensitive to pharmacological inhibition of glutamate uptake at smaller spines, whose high-affinity N-methyl-D-aspartate receptors (NMDARs) are better shielded from remotely released glutamate. At small spines, glutamate-induced and NMDAR-dependent Ca2+ entry is also more strongly increased by uptake inhibition. These findings indicate that spine size inversely correlates with the efficacy of local glutamate uptake and thereby likely determines the probability of synaptic crosstalk.

Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis.

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS.

Ultrastructural and functional changes at the tripartite synapse during epileptogenesis in a model of temporal lobe epilepsy

The persistent unresponsiveness of many of the acquired epilepsies to traditional antiseizure medications has motivated the search for prophylactic drug therapies that could reduce the incidence of epilepsy in this at risk population. These studies are based on the idea of a period of epileptogenesis that can follow a wide variety of brain injuries. Epileptogenesis is hypothesized to involve changes in the brain not initially associated with seizures, but which result finally in seizure prone networks. Understanding these changes will provide crucial clues for the development of prophylactic drugs. Using the repeated low-dose kainate rat model of epilepsy, we have studied the period of epileptogenesis following status epilepticus, verifying the latent period with continuous EEG monitoring. Focusing on ultrastructural properties of the tripartite synapse in the CA1 region of hippocampus we found increased astrocyte ensheathment around both the presynaptic and postsynaptic elements, reduced synaptic AMPA receptor subunit and perisynaptic astrocyte GLT-1 expression, and increased number of docked vesicles at the presynaptic terminal. These findings were associated with an increase in frequency of the mEPSCs observed in patch clamp recordings of CA1 pyramidal cells. The results suggest a complex set of changes, some of which have been associated with increasingly excitable networks such as increased vesicles and mEPSC frequency, and some associated with compensatory mechanisms, such as increased astrocyte ensheathment. The diversity of ultrastructural and electrophysiological changes observed during epileptogeneiss suggests that potential drug targets for this period should be broadened to include all components of the tripartite synapse.

Altered glucose catabolism in the presynaptic and perisynaptic compartments of SOD1G93A mouse spinal cord and motor cortex indicates that mitochondria are the site of bioenergetic imbalance in ALS.

Amyotrophic lateral sclerosis is an adult-onset neurodegenerative disease that develops because of motor neuron death. Several mechanisms occur supporting neurodegeneration, including mitochondrial dysfunction. Recently, we demonstrated that the synaptosomes from the spinal cord of SOD1G93A mice, an in vitro model of presynapses, displayed impaired mitochondrial metabolism at early pre-symptomatic stages of the disease, whereas perisynaptic astrocyte particles, or gliosomes, were characterized by mild energy impairment only at symptomatic stages. This work aimed to understand whether mitochondrial impairment is a consequence of upstream metabolic damage. We analyzed the critical pathways involved in glucose catabolism at presynaptic and perisynaptic compartments. Spinal cord and motor cortex synaptosomes from SOD1G93A mice displayed high activity of hexokinase and phosphofructokinase, key glycolysis enzymes, and of citrate synthase and malate dehydrogenase, key Krebs cycle enzymes, but did not display high lactate dehydrogenase activity, the key enzyme in lactate fermentation. This enhancement was evident in the spinal cord from the early stages of the disease and in the motor cortex at only symptomatic stages. Conversely, an increase in glycolysis and lactate fermentation activity, but not Krebs cycle activity, was observed in gliosomes from the spinal cord and motor cortex of SOD1G93A mice although only at the symptomatic stages of the disease. The cited enzymatic activities were enhanced in spinal cord and motor cortex homogenates, paralleling the time-course of the effect observed in synaptosomes and gliosomes. The observed metabolic modifications might be considered an attempt to restore altered energetic balance and indicate that mitochondria represent the ultimate site of bioenergetic impairment.