Lecture 8: GLIA

Glial cell types

gray matter: astrocyte, microglia white matter: oligodendrocyte

astrocytes at synapses

looks like it wrapped around the post-synapse

Schwann cells

peripheral nervous system glia

The reaction of the three major classes of glia in the CNS to local tissue damage

In each case, there is growth and change in expression of molecules normally associated with each cell class. (Top) Astrocytes labeled to visualize glial fibrillary acidic protein (GFAP) both before and after injury. (Center) The molecule NG2, notably present in glial scar tissue, is visualized here in oligodendroglial precursors and immature oligodendrocytes. (Bottom) CD1-1b, a marker for microglia. (Top from McGraw et al., 2001; center from Tan et al., 2005; bottom from Ladeby et al., 2005)

Astrocytes and neurotransmission

In some neurotransmitter systems, including glutamate, secreted transmitters are cleared from the synaptic cleft by neurotransmitter transporters on the plasma membrane of astrocytes, which wrap around many synapses. After being released into the synaptic cleft excess transmitters are taken up by plasma membrane transporters (PMTs) on the presynaptic or astrocytic plasma membrane; both are symporters that utilize energy from Na+ entry.

Astrocytes can clear neurotransmitters from the synaptic cleft

slide 13 for picture PMT channel takes in sodium ions

Astrocyte functions

• Most abundant glial cell in CNS • Multiple functions: • play a role in synapse formation and elimination • forms a supportive framework of nervous tissue • extensions (perivascular feet) that contact blood capillaries that form a tight seal called the blood-brain barrier • regulate chemical composition of tissue fluid by absorbing excess neurotransmitters and ions • convert blood glucose to lactate and supply this to the neurons for energy metabolism and synaptic plasticity • adult neural stem cells • glial scar- when neuron is damaged, astrocytes form scar tissue and fill space formerly occupied by the neuron

DAB Impairs In Vivo Hippocampal LTP and the Impairment Is Rescued by L-Lactate

(A) Average Field EPSP data recorded for 120 min posttetanus shows that DAB injection (bar) before high frequency stimulation (arrow) blocks LTP (p < 0.05, versus controls at 120 min). LTP is abolished in animals receiving an intraperitoneal injection of the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (3mg/kg) 30 min prior to tetanus (open triangles). Inset: Representative EPSP traces were recorded before and 120 min after (indicated by arrows) LTP induction. Left panel, control; right panel, in the presence of DAB. (E) L-lactate reversed the blocking effect of DAB on LTP.

L-Lactate Rescues the DAB-Induced Memory Impairment

(A) Dorsal hippocampal extracellular lactate in freely moving rats infused with either vehicle or DAB. Baseline was collected for 20 min before training (0 min, ) and continued for 50 min. Training resulted in a significant increase in lactate levels compared to baseline (*p < 0.05) that was completely blocked by DAB (# p <0.05). Data are expressed as % of baseline ± SEM (mean of the first 2 samples set 100%). (C) Acquisition (Acq) and retention are expressed as mean latency ± SEM (in seconds, sec). Hippocampal injection of DAB or vehicle in combination with 100 nmol L-lactate or vehicle were performed 15 min before training and memory was tested at 24 hr. 100 nmol of L-lactate rescued the memory impairment by DAB (Test 1). The effect persisted at 7 days after training (Test 2).

Astrocyte

- Most numerous glia in the brain - Fills spaces between neurons - Influences neurite and synapse growth - Regulates chemical content in extracellular space

Astroctyes

- Numerous in the CNS - One astrocyte can contact 1000s synapses - Enwraps 4-8 neuronal somata (cell bodies) and 300 -600 dendrites. - Express Glial fibrillary acidic protein (GFAP) - Regulate synaptic function, as well as formation and elimination of synapses). found in grey and white matter

Student Presenations

03/15/21: Haydon, P.G. (2017) Astrocytes and the modulation of sleep. Current Opinion in Neurobiology 44:28-33. 03/15/21: Wilton DK, Dissing-Olesen L, Stevens B. (2019) Neuron-Glia Signaling in Synapse Elimination. Annu Rev Neurosci. 42:107-127. 03/15/21: Adamsky A, Goshen I. (2018) Astrocytes in Memory Function: Pioneering Findings and Future Directions. Neuroscience. 370:14-26.

Neural progenitors (radial glia) express astrocytic marker GFAP

33-34

Cellular response to injury in the CNS

55 Local cellular changes at or near an injured site include the degeneration of myelin and other cellular elements; the clearing of this debris by microglia, which act as phagocytic cells in the CNS; local production of inhibitory factors by oligodendrocytes and other glia. Oligodendroglial cells and other glia either provide (on their cell surfaces) or secrete inhibitory signals that limit axon growth. Receptors for these signals are expressed on the newly generated axons of neurons whose original axons have been severed. NgR1 and 2 are receptors for the integral membrane protein NogoA. Lingo1 and PirB/LILRB2 are receptors for the myelin-associated proteins MAG (myelin-associated glycoprotein) and OMgp (oligodendrocyte myelin glycoprotein). Tissue necrosis factor receptors (TNFRs) bind the inflammatory cytokine TNF-a. Integrins bind several extracellular matrix proteins, and two classes of cytokine receptors bind pro- and anti-inflammatory cytokines released by astrocytes, oligodendroglia, or macrophages at sites of injury. (B after Kolodkin and Tessier-Lavigne, 2011.)

Immune-mediated inflammatory responses that drive glial responses and resistance to neuronal regrowth and repair

58 (A) The distribution and state of glial cells in an undamaged brain in which the blood-brain barrier is intact. Quiescent microglia, a few activated microglia, astrocytes, oligodendrocytes, and presumed oligodendrocyte precursors (Ng2 cells) are distributed throughout the tissue. (B) When local damage occurs in brain tissue (middle) or when the blood-brain barrier is disrupted (right), cytokines and other signaling molecules activate microglia and astrocytes. In turn, astrocytic processes as well as the astrocytes themselves increase in frequency, providing the scaffold for a local glial scar. Once the blood-brain barrier is compromised, neutrophils and other monocytes rapidly infiltrate the brain tissue and release additional pro-inflammatory cytokines that elicit a more robust astrocyte response and reinforce local inflammation. This leads to an additional decrease in the potential for preserving neuronal survival, tissue integrity, as well as the possibility of even modest regrowth and repair. The timing of immune responses and activation, proliferation or invasion of cells that mediate inflammation in CNS tissue following acute damage to the CNS and disruption of the blood/brain barrier. This process begins with the release of damage associated proteins, local cytokines and chemokines (produced by activated glia). Damage to brain tissue disrupts the blood brain barrier by disrupting the tight junctions that link the endothelial cells and thus prevent large molecules and circulating blood cells from directly entering the CNS. This allows for invasion of neutrophils and monocytes, the subsequent activation of microglia and astrocytes, and finally the invasion of T and B cells. This inflammatory response is initiated by the release of damage-associated proteins (contents of lysed neurons, reactive oxygen species). These proteins elicit a cascade of cellular responses that elicit secretion of proinflammatory cytokines, including pro-inflammatory cytokine IL-1. This results in glial scarring that encapsulates the site of inflammation and forms a protective barrier for adjacent health brain tissue. The astrocytes within the glial scar produce molecules that inhibit axon growth such as semaphoring 3A (which causes growth cone collapse during development), ephrins and slit. As a result growth cones turn away from the glial scar.

Astrocytic glycogenolysis affects memory retention, the inhibitor of glycogen phosphorylation 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) was injected into the hippocampus before Inhibitory Avoidance (IA) training

A) Hippocampal injections of DAB 15 min before IA training had no effect on short-term memory tested at 1 hr after training. (B) Hippocampal injections of DAB 15 min before training disrupted long-term memory at 24 hr (Test 1). The disruption persisted 7 days after training (Test 2), and memory did not recover after a reminder shock (Test 3). DAB-injected rats had normal retention after retraining (Test 4).

Model of the primary mechanism for neuronal apoptosis after injury.

Apoptosis can be elicited by excitotoxicity via excess glutamate, or by the binding of inflammatory cytokines to receptors in the neuronal membrane. In addition, loss of neuronal connections to a target and resultant deprivation of trophic support can initiate apoptosis. Any or all of these stimuli result in the removal of the anti-apoptotic gene Bcl2. Cytochrome c is then released from mitochondria, activating caspase-3 and obligating the cell to apoptotic death as caspase-3 stimulates destructive changes in downstream molecules. (A from Manabat et al., 2003.)

Astrocytes and memory function

Astrocyte-neuron lactate coupling plays a role in long-term memory formation. Glucose is taken up by astrocytes from surrounding capillaries via glucose transporters (GLUT1). Glucose can then be stored as glycogen in astrocytes or undergo glycolysis to become pyruvate. In astrocytes, pyruvate can be transported into the mitochondria or converted to lactate, which can be exported out of the astrocyte by the monocarboxylate transporter 1 or 4 (MCT1/4) and transported into neurons via MCT2. In neurons, astrocytic-derived lactate is converted back into pyruvate and transported into the mitochondria to generate ATP. Astrocytic-derived lactate from glycogenolysis may be important for long-term memory formation in rats and for the underlying regulation of molecular changes required for long-term memory formation. These changes include the phosphorylation of the transcription factor CREB, the expression of target genes and immediate early genes. slide 24 and 25

astrocytes, oligodendrocytes, microglia, ependymal cells

Central nervous system glia

Glial cell and brain injury

For repair of nerve cells regrowth of cell bodies, axons and dendrites would be needed. To achieve this, several developmental mechanisms must be reengaged, including appropriate regulation of cell polarity to distinguish dendrites and axons, adhesion signals to direct process extension and trophic signals to support growth. This type of repair requires the cooperative regrowth of existing neuronal and glial elements in a complex environment than that of the peripheral nervous system. This generally fails in the central nervous system (CNS) except over limited distances, most likely because of the overgrowth of glial cells and their production of signals that inhibit neuronal growth. A possible reason for glial overgrowth is the delicate balance of immune-system mediated clearance of damaged tissue versus maintenance of a small number of surviving neurons. This glial growth insulates surrounding intact tissue from further damage due to inflammation. The subsequent loss of trophic support to damaged axons and dendrites due to inaccessibility of targets obscured by glial overgrowth, along with the action of inflammatory cytokines (released by macrophages and other immune cells in response to tissue damage) may suppress reactivation of cellular mechanisms for dendritic or axonal growth as well as those for synapse formation.

Glial cells are different from neurons. There are about 10x-50x more glia in the nervous system than neurons. Glia do not participate directly in synaptic transmission or in electrical signaling, albeit that their supportive functions help define synaptic contacts and maintain the signaling properties of neurons. Glial cells have complex processes but these do not serve the same purposes as axons and dendrites. Cells with glial characteristics appear to be the only stem cells retained in the mature brain, and can give rise to new glia, and in certain locations (the dentate gyrus and subventricular zone) to new neurons.

Glial cells

Glial cell stems

Glial stem cells in the mature nervous system include stem cells with properties of astrocytes that can give rise to neurons, astrocytes, and oligodendrocytes.

Casualties of War and Sports

John Grimsley (right) makes a head on hit to a Buffalo Bills player. Grimsley's brain on postmortem analysis after his death at age 45 from an accidental gunshot wound. Brown label indicates deposits of tau protein, usually associated with Alzheimer's pathology. Damage to brain tissue leads to necrotic and apoptotic cell death for nearby neurons whose processes have been severed. The neuronal changes at the site of injury do not recapitulate developmental signaling that supports the initial establishment of brain and spinal cord circuits. Instead, a combination of glial growth and proliferation along with microglial activity (microglia have immune functions that lead to local inflammation) actively inhibits growth. Furthermore, there is an upregulation of growth inhibiting molecules related to chemorepellent factors that influence axon trajectories during development

Regional specificity: Spinal cord versus dentate gyrus astrocytes

Mature astrocytes from adult hippocampus, but not adult spinal cord, promote neurogenesis from adult stem cells. (a, b) Differentiation of GFP. adult neural stem cells in co-culture with astrocytes derived from adult hippocampus (a) or adult spinal cord (b). Cells were stained for MAP2ab and GFAP. (c) Quantification of the percentage of MAP2ab and GFP neurons.

microlgial cell

Microglial cells are derived primarily from hematopoietic precursor cells. Microglia share many properties with macrophages found in other tissues: They are primarily scavenger cells that remove cellular debris from sites of injury or normal cell turnover. In addition, microglia, like their macrophage counterparts, secrete signaling molecules - particularly a wide range of cytokines that are also produced by cells of the immune system - that can modulate local inflammation and influence whether other cells survive or die. Following brain damage the number of microglia at the site of injury increases dramatically. Some of these cells proliferate from microglia resident in the brain while others come from macrophages that migrate to the injured area and enter the brain via local disruptions in the cerebral vasculature (the blood brain barrier)

Glial cell functions

Modulating the rate of of nerve signal propagation Modulation synaptic activity by controlling the uptake and metabolism of neurotransmitters Providing a scaffold for some aspects of neural development Aiding (and impeding) recovery from neural injury Providing an interface between the brain and the immune system There are three types of differentiated glial cells in the mature nervous system: - Astrocytes - Oligodendrocytes - Microglial cells

Oligodendrocytes

Oligodendrocytes are restricted to the Central Nervous System, lay down a lipid-rich wrapping around some but not all axons, called myelin. Myelin affects the speed of neural transmission. Form myelin sheaths in the CNS. Each arm-like process wraps around a nerve fiber forming an insulating layer that speeds up signal conduction. Another class of glial stem cell, the oligodendrocyte precursor, has a more restricted potential, giving rise primarily to differentiated oligodendrocytes.

Neuron - Astrocyte Signaling

One of the hallmarks of the astrocytic response is the generation of calcium elevations, which further affect downstream cellular processes. However, the impact on brain function remains poorly understood. 30

Inhibitory avoidance test

Schema of inhibitory avoidance (IA) task. On the day of IA training, rats were placed in the light side of the box. When rats moved into the dark side box, researchers closed the door and applied electrical foot shock (1.6 mA) for 2 sec. The rats were returned to the home cage soon after the shock. Even after one training, rats remember the episode, spending much longer time in the light box after training.

Astrocytes stimulate synapse formation and maturation

Secreted factors from astrocytes promote synapse formation and maturation. Whole-cell recording of spontaneous postsynaptic currents from a retinal ganglion cell (RGC) cultured with other purified RGCs (left) or from an RGC co-cultured with purified RGCs and astrocytes (right). The presence of astrocytes increases the frequency and magnitude of spontaneous postsynaptic currents. Thrombospondins (TSPs), a class of astrocyte-derived factors, were found to enhance synapse number; adding TSP1 to the RGC culture mimicked the the synaptogenic effects of astrocytes or astrocyte-conditioned media, as measured by RGC synapse numbers. However, although TSP-induced synapses appeared structurally normal, they were functionally silent, as measured by spontaneous postsynaptic currents. Co-culture with astrocytes (astros) causes a sixfold increase in RGC synapse number as quantified from electron micrographs. This effect is mimicked by astrocyte-conditioned media (ACM) or purified thrombospondin1 (TSP1). Another class of astrocyte-derived factor, glypicans, glycosylated extracellular proteins anchored to the cell-surface was necessary to produce functional synapses. Further studies indicated that TSPs use a post-synaptic CA++ channel subunit called a2d-1 as a receptor to promote synapse formation. Glypican binds binds to a receptor protein tyrosine phosphatase (RTTPd) on the presynaptic membrane, triggering release of neuronal pentraxin 1 (NP1). NP1 enhances synaptic efficacy by binding to the AMPA receptor GluA1 and promoting its concentration on the postsynaptic surface. TSP is insufficient to enhance synaptic strength, as measured by postsynaptic currents (top). Addition of media from COS-7 cells expressing glypican4 enhances synaptic strength (bottom).

Glial Cells and Brain Injury

Section through the brain of a 7-day-old mouse in which the carotid artery was transiently constricted. Nissl stain was used to visualize cell bodies. The lighter region (i.e., little or no staining) shows the extent of cell damage and loss caused by this brief deprivation of oxygen. Cells in the higher-magnification image were stained for the neuronal marker Neu-N (red) and for activated caspase-3 (yellow), indicative of neurons undergoing apoptosis. Brain injury elicits responses from all three glial classes, astrocytes, oligodendrocytes and microglia, that actively oppose neuronal regrowth. Such cells are referred to as activated glia, are less susceptible to stimuli that result in neuronal apoptosis after injury. Thus, local growth of glial cells is preserved while neighboring neurons die. Most brain lesions cause local proliferation of otherwise quiescent glial precursors as well as extensive growth of processes from existing glial cells in and around the site of injury. These reactions lead to glial scarring which reflects a local overgrowth and sustained concentration of astrocytes and oligodendrocytes and their processes. Glial scars are thought to be a major barrier for axon and dendrite regrowth in the CNS. Several inhibitory molecules, most of which are related to brain myelin, prevent axon growth in the CNS. Most of these molecules are produced by central oligodendrocytes that contribute to glial scars. The protein components of brain myelin produced by oligodendrocytes inhibit axon growth, including the diminished ability of axons on substrates enriched in myelin-associated proteins such as myelin-associated glycoprotein (MAG). It is not clear as this factor does not affect peripheral nerve regeneration. Several transmembrane receptors expressed on injured CNS axons interact with myelin proteins including MAG, as well as ECM molecules, secreted signals for cytokines, including tumor necrosis factor (TNF and other cytokines.

Tripartite synapse

The idea that a synapse includes not only the pre- and postsynaptic neurons involved but also encompasses many connections with glial cells called astrocytes.

Astrocytes and development of neurotransmission

The roles of astrocytes in synapse development have been examined in cultured retinal ganglion cells (RGCs). Purified RGCs cultured in vitro extend axons and dendrites, but exhibit little synaptic activity as measured by spontaneous postsynaptic currents (in this culture system the RGCs provide the pre- and post-synaptic cells even though they do not form synapses with each other in vivo). When RGCs were co-cultured with astrocytes, postsynaptic currents increased dramatically in both magnitude and frequency. This was due to both increased synapse number and enhanced synaptic strength. Conditioned media from astrocytes could mimic the effect of astrocytes; this finding provided a way to biochemically identify astrocyte-derived factors that could promote synapse number and strength.

An ancient Egyptian papyrus acknowledges the difficulty of repairing the brain and spinal cord

The symbols in brown translate to: When you examine a man with a dislocation of a vertebra of his neck, and you find him unable to move his arms, and his legs... Then you have to say: a disease one cannot treat. (From Case and Tessier-Lavigne, 2005; courtesy of N. Y. Academy of Medicine Library.) With the exception of spinal cord and brain stem motor neurons, whose axons project to peripheral muscles and therefore have access to instructions for peripheral regeneration, there is very little long-distance axon growth or reestablishment of functional connections within the CNS following injury. The limited regrowth of damaged CNS axons, even those whose cell bodies remain intact, largely accounts for the relatively poor prognosis following brain or spinal cord damage. Damage to the CNS can occur in several ways. Damage can be a result of disease, such as stroke. The brain or spinal cord can be injured acutely by physical trauma. For example, injuries during war time in the battlefield and in an accident. Another way is by repeated force to the head that can cause concussive injuries (blast waves, hard blows during contact sports) that result in traumatic brain injury (TBI). Effects of TBI: loss of consciousness, disorientation, followed by possible lethargy, headache. In the long-term, depending on the severity of the injury there is risk of recurrent headaches, depression, early-onset cognitive impairment, dementia.