Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses.

Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 "latches" synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

Specific factors in blood from young but not old mice directly promote synapse formation and NMDA-receptor recruitment.

Aging drives a progressive decline in cognition and decreases synapse numbers and synaptic function in the brain, thereby increasing the risk for neurodegenerative disease. Pioneering studies showed that introduction of blood from young mice into aged mice reversed age-associated cognitive impairments and increased synaptic connectivity in brain, suggesting that young blood contains specific factors that remediate age-associated decreases in brain function. However, whether such factors in blood from young animals act directly on neurons to enhance synaptic connectivity, or whether they act by an indirect mechanism remains unknown. Moreover, which factors in young blood mediate cognitive improvements in old mice is incompletely understood. Here, we show that serum extracted from the blood of young but not old mice, when applied to neurons transdifferentiated from human embryonic stem cells, directly increased dendritic arborization, augmented synapse numbers, doubled dendritic spine-like structures, and elevated synaptic N-methyl-d-aspartate (NMDA) receptors, thereby increasing synaptic connectivity. Mass spectrometry revealed that thrombospondin-4 (THBS4) and SPARC-like protein 1 (SPARCL1) were enriched in serum from young mice. Strikingly, recombinant THBS4 and SPARCL1 both increased dendritic arborization and doubled synapse numbers in cultured neurons. In addition, SPARCL1 but not THBS4 tripled NMDA receptor-mediated synaptic responses. Thus, at least two proteins enriched in young blood, THBS4 and SPARCL1, directly act on neurons as synaptogenic factors. These proteins may represent rejuvenation factors that enhance synaptic connectivity by increasing dendritic arborization, synapse formation, and synaptic transmission.

SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins.

Emerging evidence supports roles for secreted extracellular matrix proteins in boosting synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (also known as Hevin), a secreted non-neuronal protein, was reported to increase synaptogenesis by simultaneously binding to presynaptic neurexin-1α and to postsynaptic neuroligin-1B, thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, neurexins and neuroligins do not themselves mediate synaptogenesis, raising the question of how SPARCL1 enhances synapse formation by binding to these molecules. Moreover, it remained unclear whether SPARCL1 acts on all synapses containing neurexins and neuroligins or only on a subset of synapses, and whether it enhances synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. To explore these questions, we examined the synaptic effects of SPARCL1 and their dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory synaptic transmission, and augments NMDAR-mediated synaptic responses more than AMPAR-mediated synaptic responses. None of these effects were mediated by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or from quadruple neuroligin-1/2/3/4 conditional KO mice that lacked all neurexins or all neuroligins were fully responsive to SPARCL1. Together, our results reveal that SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.SIGNIFICANCE STATEMENT Emerging evidence supports roles for extracellular matrix proteins in boosting synapse formation and function. Previous studies demonstrated that SPARCL1, a secreted non-neuronal protein, promotes synapse formation in rodent and human neurons. However, it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, induces functional or largely inactive synapses, and generates synapses by bridging presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 selectively induces excitatory synapses, increases their efficacy, and enhances their NMDAR content. Moreover, using rigorous genetic manipulations, we show that SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.

Atlas stumbled: kinesin light chain-1 variant E triggers a vicious cycle of axonal transport disruption and amyloid-ß generation in Alzheimer's disease.

Substantial evidence implicates fast axonal transport (FAT) defects in neurodegeneration. In Alzheimer's disease (AD), it is controversial whether transport defects cause or arise from amyloid-ß (Aß)-induced toxicity. Using a novel, unbiased genetic screen, Morihara et al. identified kinesin light chain-1 splice variant E (KLC1vE) as a modifier of Aß accumulation. Here, we propose three mechanisms to explain this causal role. First, KLC1vE reduces APP transport, leading to Aß accumulation. Second, reduced transport of APP by KLC1vE triggers an ER stress response that activates the amyloidogenic pathway. Third, KLC1vE impairs transport of other KLC1 cargos that regulate amyloidogenesis, promoting Aß retention within the secretory pathway. Collectively, KLC1vE perpetuates a vicious cycle of Aß generation, kinase dysregulation, and global FAT impairment that inevitably leads to cellular toxicity. These concepts implicate alternative splicing of KLC1 in AD and suggest that the reciprocal influence of transport mechanisms on disease states contributes to neurodegeneration.

Thyroid hormone accelerates opsin expression during early photoreceptor differentiation and induces opsin switching in differentiated TRα-expressing cones of the salmonid retina.

Thyroid hormone and its receptors (TRs) regulate photoreceptor differentiation and visual pigment protein (opsin) expression in the retinas of several vertebrates, including rodents and fish. In some of these animals, opsin expression can arise through switches within differentiated cone photoreceptors. In salmonid fishes, single cones express ultraviolet (SWS1) opsin during embryonic development and switch to blue (SWS2) opsin as the fishes grow. It is unknown whether thyroid hormone regulates opsin expression during early cone differentiation and acts through TRs to induce opsin switches in differentiated cones of the salmonid retina. Using in situ hybridization, we characterized the spatiotemporal dynamics of opsin expression and switching in embryos treated with exogenous thyroid hormone or propylthiouracil (PTU), a pharmacological inhibitor of thyroid hormone synthesis. We combined immunohistochemistry with in situ hybridization to map TRα expression with respect to cones undergoing the opsin switch in older juvenile fish. Thyroid hormone accelerated opsin expression in differentiating cones and induced the opsin switch in differentiated single cones, whereas PTU repressed the opsin switch. TRα was not detected in differentiating photoreceptors as opsin expression initiated, but was later expressed in differentiated single cones before the onset of the opsin switch. TRα expression exhibited a dynamic dorsoventral distribution that paralleled the progression of the opsin switch. Together, our results show that thyroid hormone is required for opsin switching in the retina of salmonid fishes and suggest that TRα may be involved in regulating this phenomenon.

The ultraviolet opsin is the first opsin expressed during retinal development of salmonid fishes.

PURPOSE: To determine the spatial and temporal progression of opsin appearance during retinal development of salmonid fishes (genus Oncorhynchus and Salmo). METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization with riboprobes against the five classes of opsins present in salmonids (UV, blue, green, red, and rhodopsin) were used to establish the sequence of opsin appearance and the localization of opsins to specific morphologic photoreceptor types. RESULTS: Both detection methods revealed that UV opsin mRNA was expressed first and was followed closely by red opsin mRNA. In situ hybridization results indicated the following opsin sequence: UV, red, rhodopsin, green, and blue. The UV opsin riboprobe labeled single cones, whereas the red and green riboprobes labeled opposite members of double cones. The blue riboprobe started labeling single center cones approximately 1 month after initial UV riboprobe labeling, confirming a switch in opsin expression of these cones from UV to blue. All probes first labeled a small patch of cells in the centrotemporal retina, and expression then expanded primarily toward the temporal and dorsal retina, with the exception of the blue opsin which expanded ventrally at first. CONCLUSIONS: The sequence of cone opsin appearance in salmonid fishes is similar to that in mammals, in which a violet-blue (SWS1) opsin is expressed first followed by a red (M/LWS) opsin. This sequence is different from that in zebrafish, goldfish, and chick, in which red and green opsins are expressed first. As in mammals, rhodopsin expression in salmonid fishes arises after the first cone opsin. The findings show similarity in the sequence of opsin expression between a group of lower vertebrates, the salmonid fishes, and mammals.

Imaging organelle transport in primary hippocampal neurons treated with amyloid-ß oligomers.

We describe a strategy for fluorescent imaging of organelle transport in primary hippocampal neurons treated with amyloid-ß (Aß) peptides that cause Alzheimer's disease (AD). This method enables careful, rigorous analyses of axonal transport defects, which are implicated in AD and other neurodegenerative diseases. Moreover, we present and emphasize guidelines for investigating Aß-induced mechanisms of axonal transport disruption in the absence of nonspecific, irreversible cellular toxicity. This approach should be accessible to most laboratories equipped with cell culture facilities and a standard fluorescent microscope and may be adapted to other cell types.

Dendritic and axonal mechanisms of Ca2+ elevation impair BDNF transport in Aß oligomer-treated hippocampal neurons.

Disruption of fast axonal transport (FAT) and intracellular Ca(2+) dysregulation are early pathological events in Alzheimer's disease (AD). Amyloid-ß oligomers (AßOs), a causative agent of AD, impair transport of BDNF independent of tau by nonexcitotoxic activation of calcineurin (CaN). Ca(2+)-dependent mechanisms that regulate the onset, severity, and spatiotemporal progression of BDNF transport defects from dendritic and axonal AßO binding sites are unknown. Here we show that BDNF transport defects in dendrites and axons are induced simultaneously but exhibit different rates of decline. The spatiotemporal progression of FAT impairment correlates with Ca(2+) elevation and CaN activation first in dendrites and subsequently in axons. Although many axonal pathologies have been described in AD, studies have primarily focused only on the dendritic effects of AßOs despite compelling reports of presynaptic AßOs in AD models and patients. Indeed, we observe that dendritic CaN activation converges on Ca(2+) influx through axonal voltage-gated Ca(2+) channels to impair FAT. Finally, FAT defects are prevented by dantrolene, a clinical compound that reduces Ca(2+) release from the ER. This work establishes a novel role for Ca(2+) dysregulation in BDNF transport disruption and tau-independent Aß toxicity in early AD.

Amyloid-ß oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons.

Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AßOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AßOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3ß, downstream targets of CaN, prevents BDNF transport defects induced by AßOs. We further show that AßOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AßO-induced BDNF transport disruption.

Thyroid hormone induces a time-dependent opsin switch in the retina of salmonid fishes.

PURPOSE: To determine the role of thyroid hormone in inducing the UV (SWS1)-to-blue (SWS2) opsin switch in the retina of two salmonid fishes, the coho salmon (Oncorhynchus kisutch) and the rainbow trout (O. mykiss). METHODS: Fish were treated with thyroid hormone (T(4)) or the vehicle solution (0.1 M NaOH, control), exogenously or by intraocular injection, at different life history stages. Microspectrophotometry and in situ hybridization with riboprobes against the SWS1 and SWS2 opsins were used to reveal the dynamics of opsin expression in treated and control animals. To assess whether thyroid hormone induced differentiation of retinal progenitor cells into cones, treated and control fish were injected intraocularly with bromodeoxyuridine (BrdU) and the number of proliferating cells in the outer nuclear layer (ONL) determined. These observations were accompanied by histologic counts of cone densities. RESULTS: Thyroid hormone induced a reversible UV-to-blue opsin switch in differentiated single cones of juvenile salmonids (alevin and parr stages), but failed to exert any effect in the retina of older fish (smolt stage). The switch progressed from the ventral to the dorsal retina in clockwise fashion. Thyroid hormone did not induce cone density changes or alterations in the number of BrdU-labeled cells, which were the same in control and treated animals. CONCLUSIONS: Thyroid hormone induces a UV (SWS1)-to-blue (SWS2) opsin switch in the retina of young salmonid fishes that is identical with that occurring during natural development. The switch occurs in differentiated photoreceptors, is reversible (maintained by thyroid hormone exposure), and can be induced only before its natural onset. Thyroid hormone did not cause changes in the number of proliferating cells in the ONL. These results conform to the dynamics of thyroid hormone-induced opsin expression in the mouse and are consistent with the opsin plasticity found in differentiated photoreceptors of the fruit fly, Drosophila melanogaster. This work establishes a role for thyroid hormone in triggering opsin switches in the vertebrate retina.