Synaptic Structural Integrity Research Articles

Genome-wide analysis reveals novel regulators of synaptic maintenance in Drosophila.

Maintaining synaptic communication is required to preserve nervous system function as an organism ages. While much work has been accomplished to understand synapse formation and development, we understand relatively little regarding maintaining synaptic integrity throughout aging. To better understand the mechanisms responsible for maintaining synaptic structure and function, we performed an unbiased forward genetic screen to identify genes required for synapse maintenance of adult Drosophila neuromuscular junctions. Using flight behavior as a screening tool, we evaluated flight ability in 198 lines from the Drosophila Genetic Reference Panel to identify single nucleotide polymorphisms (SNPs) that are associated with a progressive loss of flight ability with age. Among the many candidate genes identified from this screen, we focus here on 10 genes with clear human homologs harboring SNPs that are most highly associated with synaptic maintenance. Functional validation of these genes using mutant alleles revealed a progressive loss of synaptic structural integrity. Tissue-specific knockdown of these genes using RNA interference (RNAi) uncovered important roles for these genes in either presynaptic motor neurons, postsynaptic muscles, or associated glial cells, highlighting the importance of each component of tripartite synapses. These results offer greater insight into the mechanisms responsible for maintaining structural and functional integrity of synapses with age.

Theta-burst transcranial magnetic stimulation promotes stroke recovery by vascular protection and neovascularization.

Rationale: The integrity and function of the blood-brain barrier (BBB) is compromised after stroke. The current study was performed to examine potential beneficial effects and underlying mechanisms of repetitive transcranial magnetic stimulation (rTMS) on angiogenesis and vascular protection, function, and repair following stroke, which are largely unknown.Methods: Using a rat photothrombotic (PT) stroke model, continuous theta-burst rTMS was administered once daily to the infarcted hemisphere for 5 min, beginning 3 h after PT stroke. This treatment was applied for 6 days. BBB integrity, blood flow, vascular associated proteins, angiogenesis, integrity of neuronal morphology and structure, and behavioral outcome were measured and analyzed at 6 and/or 22 days after PT stroke.Results: We report that rTMS significantly mitigated BBB permeabilization and preserved important BBB components ZO-1, claudin-5, occludin, and caveolin-1 from PT-induced degradation. Damage to vascular structure, morphology, and perfusion was ameliorated by rTMS, resulting in improved local tissue oxygenation. This was accompanied with robust protection of critical vascular components and upregulation of regulatory factors. A complex cytokine response was induced by PT, particularly at the late phase. Application of rTMS modulated this response, ameliorating levels of cytokines related to peripheral immune cell infiltration. Further investigation revealed that rTMS promoted and sustained post-ischemic angiogenesis long-term and reduced apoptosis of newborn and existing vascular endothelial cells. Application of rTMS also inhibited PT-induced excessive astrocyte-vasculature interactions and stimulated an A1 to A2 shift in vessel-associated astrocytes. Mechanistic studies revealed that rTMS dramatically increased levels of PDGFRβ associated with A2 astrocytes and their adjacent vasculature. As well, A2 astrocytes displayed marked amplification of the angiogenesis-related factors VEGF and TGFβ. PT induced a rise in vessel-associated expression of HIF-1α that was starkly intensified by rTMS treatment. Finally, rTMS preserved neuronal morphology, synaptic structure integrity and behavioral outcome.Conclusions: These results indicate that rTMS can exert powerful protective and restorative effects on the peri-infarct microvasculature after PT stroke by, in part, promoting HIF-1α signaling and shifting vessel-associated astrocytic polarization to the A2 phenotype. This study provides further support for the potent protective effects of rTMS in the context of ischemic stroke, and these findings implicate vascular repair and protection as an important underlying phenomenon.

Synaptic structure and alterations in the hippocampus in neonatal rats exposed to lipopolysaccharide

Synaptic structure integrity plays a key role in learning and memory. Previous studies have shown that there is cognitive dysfunction in septic neonates in later life. In this study, intraperitoneal injection of lipopolysaccharide (LPS) in the developing rats was used as a sepsis model to determine whether hippocampal synapses would be affected. Expression of synaptophysin (Syn), synaptosomal associated protein of 25 kD (SNAP-25), and N-methyl d-aspartate receptor (NMDAR) in the hippocampus in septic brain were significantly reduced. Consistent with this, the number of dendritic spines associated with the pyramidal neurons in the CA1 region of hippocampus at 28d after LPS administration was decreased. Additionally, the number of synapse and synaptic vesicles were reduced and appeared swollen. The number of neurons in the CA1 and CA3 of hippocampus at 14, and 28d after LPS injection remained unchanged. Coupled with the above was upregulated expression of interleukin-1β (IL-1β), IL-1 receptor 1 (IL-R1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) at 1-3d after LPS injection. IL-1β expression was specifically detected in activated microglia. The plasma corticosterone (CORT) concentration in the LPS treatment rats was increased; but the glucocorticoid receptor (GR) expression in the hippocampus was decreased. We conclude that LPS injection in neonatal rats can cause synaptic disruption in the hippocampus which may be attributed to inflammatory response due to excess production of proinflammatory cytokines e.g., IL-1β derived from activated microglia. The significance of increased plasma CORT concentration and decreased GR expression in the hippocampus is discussed.

Human neural progenitor cells decrease photoreceptor degeneration, normalize opsin distribution and support synapse structure in cultured porcine retina

Retinal neurodegenerative disorders like retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy and retinal detachment decrease retinal functionality leading to visual impairment. The pathological events are characterized by photoreceptor degeneration, synaptic disassembly, remodeling of postsynaptic neurons and activation of glial cells. Despite intense research, no effective treatment has been found for these disorders. The current study explores the potential of human neural progenitor cell (hNPC) derived factors to slow the degenerative processes in adult porcine retinal explants. Retinas were cultured for 3 days with or without hNPCs as a feeder layer and investigated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), immunohistochemical, western blot and quantitative real time-polymerase chain reaction (qRT-PCR) techniques. TUNEL showed that hNPCs had the capacity to limit photoreceptor cell death. Among cone photoreceptors, hNPC coculture resulted in better maintenance of cone outer segments and reduced opsin mislocalization. Additionally, maintained synaptic structural integrity and preservation of second order calbindin positive horizontal cells was also observed. However, Müller cell gliosis only seemed to be alleviated in terms of reduced Müller cell density. Our observations indicate that at 3 days of coculture, hNPC derived factors had the capacity to protect photoreceptors, maintain synaptic integrity and support horizontal cell survival. Human neural progenitor cell applied treatment modalities may be an effective strategy to help maintain retinal functionality in neurodegenerative pathologies. Whether hNPCs can independently hinder Müller cell gliosis by utilizing higher concentrations or by combination with other pharmacological agents still needs to be determined.

The Neuronal Kinesin UNC-104/KIF1A Is a Key Regulator of Synaptic Aging and Insulin Signaling-Regulated Memory

Aging is the greatest risk factor for a number of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Furthermore, normal aging is associated with a decline in sensory, motor, and cognitive functions. Emerging evidence suggests that synapse alterations, rather than neuronal cell death, are the causes of neuronal dysfunctions in normal aging and in early stages of neurodegenerative diseases. However, little is known about the mechanisms underlying age-related synaptic decline. Here, we uncover a surprising role of the anterograde molecular motor UNC-104/KIF1A as a key regulator of neural circuit deterioration in aging C. elegans. Through analyses of synapse protein localization, synaptic transmission, and animal behaviors, we find that reduced function of UNC-104 accelerates motor circuit dysfunction with age, whereas upregulation of UNC-104 significantly improves motor function at advanced ages and also mildly extends lifespan. In addition, UNC-104-overexpressing animals outperform wild-type controls in associative learning and memory tests. Further genetic analyses suggest that UNC-104 functions downstream of the DAF-2-signaling pathway and is regulated by the FOXO transcription factor DAF-16, which contributes to the effects of DAF-2 in neuronal aging. Together, our cellular, electrophysiological, and behavioral analyses highlight the importance of axonal transport in the maintenance of synaptic structural integrity and function during aging and raise the possibility of targeting kinesins to slow age-related neural circuit dysfunction.

Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs∗71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.

Chaperoning the synapse—NMNAT protects Bruchpilot from crashing

Maintaining active zone structure is crucial for synaptic function. In this issue of EMBO reports, NMNAT is shown to act as a chaperone that protects the active zone structural protein Bruchpilot from degradation.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

Drebrin A expression is altered after pilocarpine‐induced seizures: Time course of changes is consistent for a role in the integrity and stability of dendritic spines of hippocampal granule cells

We used a pathophysiological model of temporal lobe epilepsy induced by pilocarpine in adult rats in order to assess the in vivo role of drebrin A (DA), one of the major regulators of F-actin. This model displays a dynamic reorganization of the glutamatergic network including neo-spinogenesis, morphogenesis, and neo-synaptogenesis associated with an aberrant sprouting of granule cell axons in the dentate gyrus (DG). This reactive plasticity contributes in dentate granule-cell hyperexcitability that could lead to the emergence of recurrent spontaneous seizures. We investigated the hippocampal DA expression changes in pilocarpine animals using immunohistochemical, Western blot, and in situ hybridization analyses. We showed that DA immunoreactivity was decreased in the inner molecular layer (IML) and in the hilus (H) of the DG, at latent stage, when spinogenesis and morphogenesis occur. Western blot analysis confirmed these overall hippocampal decreases of DA protein expression. At chronic stage, when newly formed glutamatergic synapses are being established, the levels of immunolabeling for DA in the H and the IML were similar to control rats. This recovery is likely due to the increase of DA mRNA in perikarya of hilar and granule cells. Interestingly, our data showed that the changes pattern of labeling for Bassoon, a specific marker for presynaptic active zone, in the IML of pilocarpine-treated animals paralleled those found for DA at all time points examined. Furthermore, our double and triple immunofluorescence studies showed that the recovery in DA levels in the IML occurred within the dendritic spines involved in glutamatergic active synapses of presumed granule cells. Altogether, our results indicate that in vivo DA is not critical for spinogenesis and morphogenesis but instead is consistent with an involvement in synaptic structural integrity, stabilization, and function. Thus, DA appears as a novel modulator of reactive synaptic plasticity associated with epilepsy.

Impaired Spine Stability Underlies Plaque-Related Spine Loss in an Alzheimer's Disease Mouse Model

Dendritic spines, the site of most excitatory synapses in the brain, are lost in Alzheimer's disease and in related mouse models, undoubtedly contributing to cognitive dysfunction. We hypothesized that spine loss results from plaque-associated alterations of spine stability, causing an imbalance in spine formation and elimination. To investigate effects of plaques on spine stability in vivo, we observed cortical neurons using multiphoton microscopy in a mouse model of amyloid pathology before and after extensive plaque deposition. We also observed age-matched nontransgenic mice to study normal effects of aging on spine plasticity. We found that spine density and structural plasticity are maintained during normal aging. Tg2576 mice had normal spine density and plasticity before plaques appeared, but after amyloid pathology is established, severe disruptions were observed. In control animals, spine formation and elimination were equivalent over 1 hour of observation ( approximately 5% of observed spines), resulting in stable spine density. However, in aged Tg2576 mice spine elimination increased, specifically in the immediate vicinity of plaques. Spine formation was unchanged, resulting in spine loss. These data show a small population of rapidly changing spines in adult and even elderly mouse cortex; further, in the vicinity of amyloid plaques, spine stability is markedly impaired leading to loss of synaptic structural integrity.