A Small Molecule TrkB Neurotrophin Receptor Partial Agonist as Possible Treatment for Experimental Nonarteritic Anterior Ischemic Optic Neuropathy.

PURPOSE: Brain-derived neurotrophic factor (BDNF) and activation of its high affinity receptor tropomyosin kinase (Trk) B promote retinal ganglion cells (RGCs) survival following injury. In this study, we tested the effects of LM22A-4, a small molecule TrkB receptor-specific partial agonist, on RGC survival in vitro and in experimental nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in those older than 50 years. METHODS: We assessed drug effects on immunopanned, cultured RGCs and calculated RGC survival and assessed TrkB receptor activation by mitogen-activated protein (MAP) kinase translocation. To assess effects in vivo, we induced murine AION and treated the animals with one intravitreal injection and three-week systemic treatment. We measured drug effects using serial spectral-domain optical coherence tomography (OCT) and quantified retinal Brn3A+ RGC density three weeks after ischemia. RESULTS: In vitro, LM22A-4 significantly increased the survival of cultured RGCs at day 2 (95% CI control: 8.4-13.6; LM22A-4: 23.7-30.3; BDNF: 24.3-29.9; P ≤ 0.0001), similar to the effect of the endogenous TrkB receptor ligand BDNF. There was also significant nuclear and cytoplasmic translocation of MAP kinase (95% CI control: 0.9-6.8; LM22A-4: 38.8-84.4; BDNF: 64.0-93.0; P = 0.0002), a known downstream event of TrkB receptor activation. Following AION, LM22A-4 treatment led to significant preservation of the ganglion cell complex (95% CI: AION-PBS: 66.8-70.7%; AION-LM22A-4: 70.0-73.1; P = 0.03) and total retinal thickness (95% CI: AION-PBS: 185-196%; AION-LM22A-4: 195-203; P = 0.002) as measured by OCT compared with non-treated eyes. There was also significant rescue of the Brn3A+ RGC density on morphometric analysis of whole mount retinae (95% CI control: 2360-2629; AION-PBS: 1647-2008 cells/mm2; AION-LM22A-4: 1958-2216 cells/mm2; P = 0.02). CONCLUSIONS: TrkB receptor partial agonist LM22A-4 promoted survival of cultured RGCs in vitro by TrkB receptor activation, and treatment in vivo led to increased survival of RGCs after optic nerve ischemia, providing support that LM22A-4 may be effective therapy to treat ischemic optic neuropathy. ABBREVIATIONS: AION: anterior ischemic optic neuropathy, BDNF: Brain-derived neurotrophic factor, GCC: ganglion cell complex, MAP: mitogen-activated protein, OCT: spectral-domain optical coherence tomography, OD: right eye, ON: optic nerve, ONH: optic nerve head, OS: left eye, RGC: retinal ganglion cell; Trk: tropomyosin kinase.

Normal aging induces A1-like astrocyte reactivity.

The decline of cognitive function occurs with aging, but the mechanisms responsible are unknown. Astrocytes instruct the formation, maturation, and elimination of synapses, and impairment of these functions has been implicated in many diseases. These findings raise the question of whether astrocyte dysfunction could contribute to cognitive decline in aging. We used the Bac-Trap method to perform RNA sequencing of astrocytes from different brain regions across the lifespan of the mouse. We found that astrocytes have region-specific transcriptional identities that change with age in a region-dependent manner. We validated our findings using fluorescence in situ hybridization and quantitative PCR. Detailed analysis of the differentially expressed genes in aging revealed that aged astrocytes take on a reactive phenotype of neuroinflammatory A1-like reactive astrocytes. Hippocampal and striatal astrocytes up-regulated a greater number of reactive astrocyte genes compared with cortical astrocytes. Moreover, aged brains formed many more A1 reactive astrocytes in response to the neuroinflammation inducer lipopolysaccharide. We found that the aging-induced up-regulation of reactive astrocyte genes was significantly reduced in mice lacking the microglial-secreted cytokines (IL-1α, TNF, and C1q) known to induce A1 reactive astrocyte formation, indicating that microglia promote astrocyte activation in aging. Since A1 reactive astrocytes lose the ability to carry out their normal functions, produce complement components, and release a toxic factor which kills neurons and oligodendrocytes, the aging-induced up-regulation of reactive genes by astrocytes could contribute to the cognitive decline in vulnerable brain regions in normal aging and contribute to the greater vulnerability of the aged brain to injury.

Novel allele-dependent role for APOE in controlling the rate of synapse pruning by astrocytes.

The strongest genetic risk factor influencing susceptibility to late-onset Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype. APOE has three common isoforms in humans, E2, E3, and E4. The presence of two copies of the E4 allele increases risk by ∼12-fold whereas E2 allele is associated with an ∼twofold decreased risk for AD. These data put APOE central to AD pathophysiology, but it is not yet clear how APOE alleles modify AD risk. Recently we found that astrocytes, a major central nervous system cell type that produces APOE, are highly phagocytic and participate in normal synapse pruning and turnover. Here, we report a novel role for APOE in controlling the phagocytic capacity of astrocytes that is highly dependent on APOE isoform. APOE2 enhances the rate of phagocytosis of synapses by astrocytes, whereas APO4 decreases it. We also found that the amount of C1q protein accumulation in hippocampus, which may represent the accumulation of senescent synapses with enhanced vulnerability to complement-mediated degeneration, is highly dependent on APOE alleles: C1q accumulation was significantly reduced in APOE2 knock-in (KI) animals and was significantly increased in APOE4 KI animals compared with APOE3 KI animals. These studies reveal a novel allele-dependent role for APOE in regulating the rate of synapse pruning by astrocytes. They also suggest the hypothesis that AD susceptibility of APOE4 may originate in part from defective phagocytic capacity of astrocytes which accelerates the rate of accumulation of C1q-coated senescent synapses, enhancing synaptic vulnerability to classical-complement-cascade mediated neurodegeneration.

Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways.

To achieve its precise neural connectivity, the developing mammalian nervous system undergoes extensive activity-dependent synapse remodelling. Recently, microglial cells have been shown to be responsible for a portion of synaptic pruning, but the remaining mechanisms remain unknown. Here we report a new role for astrocytes in actively engulfing central nervous system synapses. This process helps to mediate synapse elimination, requires the MEGF10 and MERTK phagocytic pathways, and is strongly dependent on neuronal activity. Developing mice deficient in both astrocyte pathways fail to refine their retinogeniculate connections normally and retain excess functional synapses. Finally, we show that in the adult mouse brain, astrocytes continuously engulf both excitatory and inhibitory synapses. These studies reveal a novel role for astrocytes in mediating synapse elimination in the developing and adult brain, identify MEGF10 and MERTK as critical proteins in the synapse remodelling underlying neural circuit refinement, and have important implications for understanding learning and memory as well as neurological disease processes.

Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors.

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC.

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.

Synapses are asymmetric cellular adhesions that are critical for nervous system development and function, but the mechanisms that induce their formation are not well understood. We have previously identified thrombospondin as an astrocyte-secreted protein that promotes central nervous system (CNS) synaptogenesis. Here, we identify the neuronal thrombospondin receptor involved in CNS synapse formation as alpha2delta-1, the receptor for the anti-epileptic and analgesic drug gabapentin. We show that the VWF-A domain of alpha2delta-1 interacts with the epidermal growth factor-like repeats common to all thrombospondins. alpha2delta-1 overexpression increases synaptogenesis in vitro and in vivo and is required postsynaptically for thrombospondin- and astrocyte-induced synapse formation in vitro. Gabapentin antagonizes thrombospondin binding to alpha2delta-1 and powerfully inhibits excitatory synapse formation in vitro and in vivo. These findings identify alpha2delta-1 as a receptor involved in excitatory synapse formation and suggest that gabapentin may function therapeutically by blocking new synapse formation.