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Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G-protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate a new optogenetic tool that disrupt Gαqsignaling through membrane recruitment of a minimal Regulator of G-protein signaling (RGS) domain. This approach,Photo-inducedModulation ofGα protein –Inhibition of Gαq(PiGM-Iq), exhibited potent and selective inhibition of Gαqsignaling. We alter the behavior ofC. elegansandDrosophilawith outcomes consistent with GPCR-Gαqdisruption. PiGM-Iq also changes axon guidance in culture dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. By altering the choice of minimal RGS domain, we also show that this approach is amenable to Gαisignaling.

The neurotransmitter serotonin has been implicated in a range of complex neurological disorders linked to alterations of neuronal circuitry. Serotonin is synthesized in the developing brain before most neuronal circuits become fully functional, suggesting that serotonin might play a distinct regulatory role in shaping circuits prior to its function as a classical neurotransmitter. In this study, we asked if serotonin acts as a guidance cue by examining how serotonin alters growth cone motility of rodent sensory neurons in vitro. Using a growth cone motility assay, we found that serotonin acted as both an attractive and repulsive guidance cue through a narrow concentration range. Extracellular gradients of 50 µM serotonin elicited attraction, mediated by the serotonin 5-HT2a receptor while 100 µM serotonin elicited repulsion mediated by the 5-HT1b receptor. Importantly, high resolution imaging of growth cones indicated that these receptors signalled through their canonical pathways of endoplasmic reticulum-mediated calcium release and cAMP depletion, respectively. This novel characterisation of growth cone motility in response to serotonin gradients provides compelling evidence that secreted serotonin acts at the molecular level as an axon guidance cue to shape neuronal circuit formation during development.

Accurate modelling type 2 diabetes and diabetic complications in rodents has proven a challenge, largely as a result of the long-time course of disease development in humans. In the present study, we aimed to develop and comprehensively characterise a new rodent model of type 2 diabetes. To do this, we fed Sprague-Dawley rats a high fat/high sugar diet (HFD) to induce obesity and dyslipidaemia. After 3 weeks, we s.c. implanted osmotic mini pumps to enable a 14 day, slow infusion of streptozotocin (STZ; lower dose = 100 mg kg

Diabetic retinopathy (DR), one of the leading causes of blindness, is mainly diagnosed based on the vascular pathology of the disease. Current treatment options largely focus on this aspect with mostly insufficient therapeutic long-term efficacy. Mounting evidence implicates mitochondrial dysfunction and oxidative stress in the central etiology of DR. Consequently, drug candidates that aim at normalizing mitochondrial function could be an attractive therapeutic approach. This study compared the mitoprotective compounds, idebenone and elamipretide, side-by-side against two novel short-chain quinones (SCQs) in a rat model of DR. The model effectively mimicked type 2 diabetes over 21 weeks. During this period, visual acuity was monitored by measuring optokinetic response (OKR). Vision loss occurred 5&ndash, 8 weeks after the onset of hyperglycemia. After 10 weeks of hyperglycemia, visual function was reduced by 65%. From this point, the right eyes of the animals were topically treated once daily with the test compounds. The left, untreated eye served as an internal control. Only three weeks of topical treatment significantly restored vision from 35% to 58&ndash, 80%, while visual acuity of the non-treated eyes continued to deteriorate. Interestingly, the two novel SCQs restored visual acuity better than idebenone or elamipretide. This was also reflected by protection of retinal pathology against oxidative damage, retinal ganglion cell loss, reactive gliosis, vascular leakage, and retinal thinning. Overall, mitoprotective and, in particular, SCQ-based compounds have the potential to be developed into effective and fast-acting drug candidates against DR.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a large, endocytic cell surface receptor that is highly expressed by oligodendrocyte progenitor cells (OPCs) and LRP1 expression is rapidly downregulated as OPCs differentiate into oligodendrocytes (OLs). We report that the conditional deletion of Lrp1 from adult mouse OPCs (Pdgfrα-CreER :: Lrp1fl/fl) increases the number of newborn, mature myelinating OLs added to the corpus callosum and motor cortex. As these additional OLs extend a normal number of internodes that are of a normal length, Lrp1-deletion increases adult myelination. OPC proliferation is also elevated following Lrp1 deletion in vivo, however, this may be a secondary, homeostatic response to increased OPC differentiation, as our in vitro experiments show that LRP1 is a direct negative regulator of OPC differentiation, not proliferation. Deleting Lrp1 from adult OPCs also increases the number of newborn mature OLs added to the corpus callosum in response to cuprizone-induced demyelination. These data suggest that the selective blockade of LRP1 function on adult OPCs may enhance myelin repair in demyelinating diseases such as multiple sclerosis., Graphical Abstract LRP1 regulates adult oligodendrogenesis and remyelination.

Background Previous studies have shown an association between prenatal exposure to particulate matter (PM) and adverse brain development. However, it is unclear whether gestational exposure to community-sampled residential PM has an impact on the developing brain. Objectives We aimed to test whether in utero exposure to PM from residential roof spaces (ceiling voids) alters critical foetal neurodevelopmental processes. Methods Pregnant C57BL/6 mice were intranasally exposed to 100 μg of roof space particles (~5 mg kg−1) in 50 μl of saline, or saline alone under light methoxyflurane anaesthesia, throughout mid-to-late gestation. At 2 weeks post-natal age, pups were sacrificed and assessed for body and brain growth. The brain tissue was collected and examined for a range of neurodevelopmental markers for synaptogenesis, synaptic plasticity, gliogenic events and myelination by immunohistochemistry. Results Gestational exposure to roof space PM reduced post-natal body and brain weights. There was no significant effect of roof space PM exposure on synaptogenesis, synaptic plasticity or astrocyte density. However, PM exposure caused increased myelin load in the white matter and elevated microglial density which was dependent on the PM sample. These effects were found to be consistent between male and female mice. Conclusions Our data suggest that exposure to residential roof space PM during pregnancy impairs somatic growth and causes neuropathological changes in the developing brain.

The precision with which neurons form connections is crucial for the normal development and function of the nervous system. The development of neuronal circuitry in the nervous system is accomplished by axon pathfinding: a process where growth cones guide axons through the embryonic environment to connect with their appropriate synaptic partners to form functional circuits. Despite intense efforts over many years to understand how this process is regulated, the complete repertoire of molecular mechanisms that govern the growth cone cytoskeleton and hence motility, remain unresolved. A central tenet in the axon guidance field is that calcium signals regulate growth cone behaviours such as extension, turning and pausing by regulating rearrangements of the growth cone cytoskeleton. Here, we provide evidence that not only the amplitude of a calcium signal is critical for growth cone motility but also the source of calcium mobilisation. We provide an example of this idea by demonstrating that manipulation of calcium signalling via L-type voltage gated calcium channels can perturb sensory neuron motility towards a source of netrin-1. Understanding how calcium signals can be transduced to initiate cytoskeletal changes represents a significant gap in our current knowledge of the mechanisms that govern axon guidance, and consequently the formation of functional neural circuits in the developing nervous system.

Our understanding of the impact of in utero exposure to PM on post-natal immune function and the subsequent response to PM exposure is limited. Similarly, very few studies have considered the effect of exposure to PM from different sources. Thus, the aim of this study was to examine how in utero exposure to PM from different sources effects the post-natal response to pro-inflammatory and immune stimuli. C56BL/6J pregnant mice were exposed intranasally on gestational day (E)7.5, E12.5 and E17.5-50 μg of diesel exhaust particles (DEP), silica or saline. At 4-weeks post-natal age, sub-groups of male and female mice were exposed intranasally to 50 μg of DEP or saline. Lung inflammatory responses were assessed 6 h later by quantifying inflammatory cells and cytokine production (MCP-1, MIP-2, IL-6). In separate groups of mice, the spleen was harvested to quantify B and T cell populations. Splenocytes were isolated and exposed to lipopolysaccharide or poly I:C for assessment of cytokine production. Exposure to DEP in utero decreased %CD1dhighCD5+ B cells in female mice and IFN-γ production by splenocytes in both sexes. Male mice had elevations in macrophage and lymphocyte numbers in response to DEP whereas female mice only had elevated IL-6, MCP-1 and MIP-2 levels. In utero exposure to silica had no effect on these measures. These data suggest that in utero exposure to PM alters immune development and post-natal immune function. This response is dependent on the source of PM, which has implications for understanding the community health effects of exposure to air pollution.

The amyloid-β precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU+ neural progenitor cells and the number of PSA-NCAM+ neuroblasts produced in the SVZ and dentate gyrus were not affected by the life-long absence of APP. However, by labelling newborn cells with EdU and then following their fate over-time, we determined that ~48% more newly generated EdU+ NeuN+ neurons accumulated in the granule cell layer of the olfactory bulb and ~57% more in the dentate gyrus of young adult APP knockout mice relative to C57bl6 controls. Furthermore, proportionally fewer of the adult-born olfactory bulb granule neurons were calretinin+. To determine whether APP was having an effect on neuronal maturation, we administered tamoxifen to young adult Nestin-CreERT2::Rosa26-YFP and Nestin-CreERT2::Rosa26-YFP::APP-knockout mice, fluorescently labelling ~80% of newborn (EdU+) NeuN+ dentate granule neurons formed between P75 and P105. Our analysis of their morphology revealed that neurons added to the hippocampus of APP knockout mice have shorter dendritic arbors and only half the number of branch points as those generated in C57bl6 mice. We conclude that APP reduces the survival of newborn neurons in the olfactory bulb and hippocampus, but that it does not influence all neuronal subtypes equally. Additionally, APP influences dentate granule neuron maturation, acting as a robust regulator of dendritic extension and arborisation.

Throughout life, oligodendrocyte progenitor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes. OPCs express cell surface receptors and channels that allow them to detect and respond to neuronal activity, including voltage‐gated calcium channel (VGCC)s. The major L‐type VGCC expressed by developmental OPCs, CaV1.2, regulates their differentiation. However, it is unclear whether CaV1.2 similarly influences OPC behavior in the healthy adult central nervous system (CNS). To examine the role of CaV1.2 in adulthood, we conditionally deleted this channel from OPCs by administering tamoxifen to P60 Cacna1c fl/fl (control) and Pdgfrα‐CreER:: Cacna1c fl/fl (CaV1.2‐deleted) mice. Whole cell patch clamp analysis revealed that CaV1.2 deletion reduced L‐type voltage‐gated calcium entry into adult OPCs by ~60%, confirming that it remains the major L‐type VGCC expressed by OPCs in adulthood. The conditional deletion of CaV1.2 from adult OPCs significantly increased their proliferation but did not affect the number of new oligodendrocytes produced or influence the length or number of internodes they elaborated. Unexpectedly, CaV1.2 deletion resulted in the dramatic loss of OPCs from the corpus callosum, such that 7 days after tamoxifen administration CaV1.2‐deleted mice had an OPC density ~42% that of control mice. OPC density recovered within 2 weeks of CaV1.2 deletion, as the lost OPCs were replaced by surviving CaV1.2‐deleted OPCs. As OPC density was not affected in the motor cortex or spinal cord, we conclude that calcium entry through CaV1.2 is a critical survival signal for a subpopulation of callosal OPCs but not for all OPCs in the mature CNS., Main points OPCs express CaV1.2 in development and adulthood.The conditional deletion of CaV1.2 from adult OPCs enhances their proliferation.Deleting CaV1.2 from adult OPCs has no effect on oligodendrogenesis or myelination.Loss of CaV1.2 kills OPCs in the corpus callosum but these cells are rapidly replaced by surviving parenchymal OPCs that are not reliant on CaV1.2 for survival.

The low-density lipoprotein receptor-related protein receptors 1 and 2 (LRP1 and LRP2) are emerging as important cell signaling mediators in modulating neuronal growth and repair. We examined whether LRP1 and LRP2 are able to mediate a specific aspect of neuronal growth: axon guidance. We sought to identify LRP1 and LRP2 ligands that could induce axonal chemoattraction, which might have therapeutic potential. Using embryonic sensory neurons (rat dorsal root ganglia) in a growth cone turning assay, we tested a range of LRP1 and LRP2 ligands for the ability to guide growth cone navigation. Three ligands were chemorepulsive: α-2-macroglobulin, tissue plasminogen activator, and metallothionein III. Conversely, only one LRP ligand, metallothionein II, was found to be chemoattractive. Chemoattraction toward a gradient of metallothionein II was calcium-dependent, required the expression of both LRP1 and LRP2, and likely involves further co-receptors such as the tropomyosin-related kinase A (TrkA) receptor. The potential for LRP-mediated chemoattraction to mediate axonal regeneration was examined in vivo in a model of chemical denervation in adult rats. In these in vivo studies, metallothionein II was shown to enhance epidermal nerve fiber regeneration so that it was complete within 7 days compared with 14 days in saline-treated animals. Our data demonstrate that both LRP1 and LRP2 are necessary for metallothionein II-mediated chemotactic signal transduction and that they may form part of a signaling complex. Furthermore, the data suggest that LRP-mediated chemoattraction represents a novel, non-classical signaling system that has therapeutic potential as a disease-modifying agent for the injured peripheral nervous system.

Type 2 diabetes is a chronic metabolic disorder that is becoming a leading cause of morbidity and mortality. The prolonged time-course of human type 2 diabetes makes modelling of the disease difficult and additional animal models and methodologies are needed. The goal of this study was to develop and characterise a new method that allows controlled, targeted and sustained induction of discrete stages of type 2 diabetes in rodents. Using adult, male rats, we employed a three-week high fat-diet regimen and confirmed development of obesity-associated glucose intolerance, a key feature of human type 2 diabetes. Next, we utilised osmotic mini-pumps to infuse streptozotocin (STZ; doses ranging 80–200 mg/kg) over the course of 14-days to decrease insulin-producing capacity thus promoting hyperglycemia. Using this new approach, we demonstrate a dose-dependent effect of STZ on circulating glucose and insulin levels as well as glucose tolerance, while retaining a state of obesity. Importantly, we found that insulin secretion in response to a glucose load was present, but reduced in a dose-dependent manner by increasing STZ. In conclusion, we demonstrate a novel method that enables induction of discrete stages of type 2 diabetes in rodents that closely mirrors the different stages of type 2 diabetes in humans.

The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity.

Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are key regulators of synaptic function and cognition. In Alzheimer's disease (AD), cell-surface AMPARs are downregulated, however the reason for this downregulation is not clear. In the present study, we found that Abeta significantly decreased levels of the cell-surface AMPA-type glutamate receptor subunit 2 (GluR2), and increased the concentration of free cytosolic calcium ion ([Ca2+]i) in hippocampal neurons. Ion channel blockers (nifedipine, tetrodotoxin, SKF96365) decreased [Ca2+ and increased the level of cell-surface GluR2, whereas Bay K 8644, an activator of L-type voltage-gated calcium channels increased [Ca2+]i and decreased cell-surface GluR2. Abeta and Bay K 8644 increased phosphorylation of serine-880 (S880) on GluR2, whereas the nifedipine. tetrodotoxin and SKF96365 decreased S880 phosphorylation. Finally, we found that bisindolylmeimide I (GF 109203X, GFX), an inhibitor of protein kinase C (PKC) blocked both the decrease in cell-surface GluR2 and the increase in phospho-S880 induced by Abeta and Bay K 8644. Taken together, these results demonstrate that Abeta decreases cell-surface GluR2 by increasing PKC-mediated phosphorylation of S880. Our study supports the view that a rise in cytosolic [Ca2+]i induced by Abeta could impair synaptic function by decreasing the availability of AMPARs at the synapse. This decrease in AMPARs may contribute to the decline in cognitive function seen in AD.

The b-site APP cleaving enzyme (BACE1) is responsible for the first step in the production of the b-amyloid protein of Alzheimer’s disease. BACE1 is synthesized as a partially active zymogen (proBACE1). We previously showed that the glycosaminoglycan (GAG) heparin can increase the enzyme activity of proBACE1. In this study, the structural requirements and the mechanism for the GAG-induced activation were examined. The effect of heparin on proBACE1 was influenced by the degree of sulfation and carboxylation of the GAG, as well as by the length of the sugar. Although low molecular weight heparin fragments did not strongly stimulate proBACE1, they inhibited heparin-induced activation of the enzyme. The structure of the zymogen was modeled using the known X-ray structures of the BACE1 catalytic domain and the homologous prodomain of porcine pepsinogen. The modeled structure suggested that a heparin-binding domain may reside close to the prodomain, and that movement of a loop region between residues 46–65, lying adjacent to the prodomain, may be needed to accommodate heparin binding. The presence of the loop domain adjacent to the active site may account for the lower activity of the zymogen relative to the mature enzyme. Movement of the loop region upon heparin binding could expose the active site region to allow for increased substrate binding. The results suggest a model in which conformational changes close to the prodomain may be involved in the mechanism of heparin-induced activation of proBACE1.

Acute axonal shear and stretch in the brain induces an evolving form of axonopathy and is a major cause of ongoing motor, cognitive and emotional dysfunction. We have utilized an in vitro model of mild axon bundle stretch injury, in cultured primary cortical neurons, to determine potential early critical cellular alterations leading to secondary axonal degeneration. We determined that transient axonal stretch injury induced an initial acute increase in intracellular calcium, principally derived from intracellular stores, which was followed by a delayed increase in calcium over 48 h post-injury (PI). This progressive and persistent increase in intracellular calcium was also associated with increased frequency of spontaneous calcium fluxes as well as cytoskeletal abnormalities. Additionally, at 48 h post-injury, stretch-injured axon bundles demonstrated filopodia-like sprout formation that preceded secondary axotomy and degeneration. Pharmacological inhibition of the calcium-activated phosphatase, calcineurin, resulted in reduced secondary axotomy (p < 0.05) and increased filopodial sprout length. In summary, these results demonstrate that stretch injury of axons induced an initial substantial release of calcium from intracellular stores with elevated intracellular calcium persisting over 2 days. These long-lasting calcium alterations may provide new insight into the earliest neuronal abnormalities that follow traumatic brain injury as well as the key cellular changes that lead to the development of diffuse axonal injury and secondary degeneration.

Although many of the biochemical mechanisms which regulate production or clearance of the amyloid-beta protein (Abeta) of Alzheimer's disease (AD) are now well understood, the mechanism of Abeta neurotoxicity remains unclear. A number of studies have shown that Abeta can disrupt neuronal Ca(2+) homeostasis by inducing influx of extracellular Ca(2+) into the neuronal cytoplasm. Ca(2+) is known to play an important role in neuronal excitability, synaptic plasticity and neurotoxicity. Therefore, Abeta-induced Ca(2+) dysregulation may contribute to many of the cognitive and neuropathologic features of AD. In vitro studies show that Abeta can increase ion permeability in lipid membranes. This increased permeability is reportedly associated with the formation of artificial ion pores formed from Abeta oligomers. However, a number of other studies show that Abeta can activate endogenous ion channels on the cell surface. There is also increasing evidence that presenilin mutations alter intracellular Ca(2+) stores. It is likely that elucidation of the mechanism by which Abeta and presenilin cause Ca(2+) dysregulation in neurons will help to identify new drug targets for the treatment of AD.

Homer proteins are best known as scaffold proteins at the post-synaptic density where they facilitate synaptic signalling and are thought to be required for learning and memory. Evidence implicating Homer proteins in the development of the nervous system is also steadily accumulating. Homer is highly conserved and is expressed at key developmental time points in the nervous system of several species. Homer regulates intracellular calcium homeostasis, clustering and trafficking of receptors and proteins at the cytosolic surface of the plasma membrane, transcription and translation, and cytoskeletal organization. Each of these functions has obvious potential to regulate neuronal development, and indeed Homer is implicated in several pathologies associated with the developing nervous system. Current data justify more critical experimental approaches to the role of Homer in the developing nervous system and related neurological disorders.

The central nervous system (CNS) is a highly organised structure. Many signalling systems work in concert to ensure that neural stem cells are appropriately directed to generate progenitor cells, which in turn mature into functional cell types including projection neurons, interneurons, astrocytes, and oligodendrocytes. Herein we explore the role of the low density lipoprotein (LDL) receptor family, in particular family members LRP1 and LRP2, in regulating the behaviour of neural stem and progenitor cells during development and adulthood. The ability of LRP1 and LRP2 to bind a diverse and extensive range of ligands, regulate ligand endocytosis, recruit nonreceptor tyrosine kinases for direct signal transduction and signal in conjunction with other receptors, enables them to modulate many crucial neural cell functions.

Stem cell therapy may be a suitable approach for the treatment of many neurodegenerative diseases. However, one major impediment to the development of successful cell-based therapies is our limited understanding of the mechanisms that instruct neural stem cell behaviour, such as proliferation and cell fate specification. The β-amyloid precursor protein (APP) of Alzheimer's disease (AD) may play an important role in neural stem cell proliferation and differentiation. Our recent work shows that in vitro, APP stimulates neural stem or progenitor cell proliferation and neuronal differentiation. The effect on proliferation is mediated by an autocrine factor that we have identified as cystatin C. As cystatin C expression is also reported to inhibit the development of amyloid pathology in APP transgenic mice, our finding has implications for the possible use of cystatin C for the therapy of AD.

Amyloid-β precursor protein (APP) is well studied for its role in Alzheimer disease, although its normal function remains uncertain. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem/progenitor cells (NSPCs). In this study we examined the role of APP in NSPC differentiation. To identify proteins that may mediate the effect of APP on NSPC differentiation, we used a gene array approach to find genes whose expression correlated with APP-induced neurogenesis. We found that the expression of neurogenin 2 (Ngn2), a basic helix-loop-helix transcription factor, was significantly down-regulated in NSPCs from APP knock-out mice (APPKO) and increased in APP transgenic (Tg2576) mice. Ngn2 overexpression in APPKO NSPCs promoted neuronal differentiation, whereas siRNA knockdown of Ngn2 expression in wild-type NSPCs decreased neuronal differentiation. The results demonstrate that APP-stimulated neuronal differentiation of NSPCs is mediated by Ngn2.

The function of the β-amyloid precursor protein (APP) of Alzheimer's disease is poorly understood. The secreted ectodomain fragment of APP (sAPPα) can be readily cleaved to produce a small N-terminal fragment (N-APP) that contains heparin-binding and metal-binding domains and that has been found to have biological activity. In the present study, we examined whether N-APP can bind to lipids. We found that N-APP binds selectively to phosphoinositides (PIPs) but poorly to most other lipids. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 )-rich microdomains were identified on the extracellular surface of neurons and glia in primary hippocampal cultures. N-APP bound to neurons and colocalized with PIPs on the cell surface. Furthermore, the binding of N-APP to neurons increased the level of cell-surface PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate. However, PIPs were not the principal cell-surface binding site for N-APP, because N-APP binding to neurons was not inhibited by a short-acyl-chain PIP analogue, and N-APP did not bind to glial cells which also possessed PI(4,5)P2 on the cell surface. The data are explained by a model in which N-APP binds to two distinct components on neurons, one of which is an unidentified receptor and the second of which is a PIP lipid, which binds more weakly to a distinct site within N-APP. Our data provide further support for the idea that N-APP may be an important mediator of APP's biological activity.

The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.

Background Familial amyloidotic polyneuropathy (FAP) is a peripheral neuropathy caused by the extracellular accumulation and deposition of insoluble transthyretin (TTR) aggregates. However the molecular mechanism that underlies TTR toxicity in peripheral nerves is unclear. Previous studies have suggested that amyloidogenic proteins can aggregate into oligomers which disrupt intracellular calcium homeostasis by increasing the permeability of the plasma membrane to extracellular calcium. The aim of the present study was to examine the effect of TTR on calcium influx in dorsal root ganglion neurons. Results Levels of intracellular cytosolic calcium were monitored in dorsal root ganglion (DRG) neurons isolated from embryonic rats using the calcium-sensitive fluorescent indicator Fluo4. An amyloidogenic mutant form of TTR, L55P, induced calcium influx into the growth cones of DRG neurons, whereas wild-type TTR had no significant effect. Atomic force microscopy and dynamic light scattering studies confirmed that the L55P TTR contained oligomeric species of TTR. The effect of L55P TTR was decreased by blockers of voltage-gated calcium channels (VGCC), as well as by blockers of Nav1.8 voltage-gated sodium channels and transient receptor potential M8 (TRPM8) channels. siRNA knockdown of TRPM8 channels using three different TRPM8 siRNAs strongly inhibited calcium influx in DRG growth cones. Conclusions These data suggest that activation of TRPM8 channels triggers the activation of Nav1.8 channels which leads to calcium influx through VGCC. We suggest that TTR-induced calcium influx into DRG neurons may contribute to the pathophysiology of FAP. Furthermore, we speculate that similar mechanisms may mediate the toxic effects of other amyloidogenic proteins such as the β-amyloid protein of Alzheimer's disease.

Alzheimer's disease (AD) is caused by the accumulation of amyloid-β (Aβ), which induces progressive decline in learning, memory, and other cognitive functions. Aβ is a neurotoxic protein that disrupts calcium signaling in neurons and alters synaptic plasticity. These effects lead to loss of synapses, neural network dysfunction, and inactivation of neuronal signaling. However, the precise mechanism by which Aβ causes neurodegeneration is still not clear, despite decades of intensive research. The role of astrocytes in early cognitive decline is a major component of disease pathology that has been poorly understood. Recent research suggests that astrocytes are not simply passive support cells for neurons, but are active participants in neural information processing in the brain. Aβ can disrupt astrocytic calcium signaling and gliotransmitter release, processes that are vital for astrocyte-neuron communication. Therefore, astrocyte dysfunction may contribute to the earliest neuronal deficits in AD. Here we discuss emerging concepts in glial biology and the implications of astrocyte dysfunction on neurodegeneration in AD.

Alzheimer's disease (AD) is characterized by the extracellular deposition of the β-amyloid protein (Aβ). Aβ is a fragment of a much larger precursor protein, the amyloid precursor protein (APP). Sequential proteolytic cleavage of APP by β-secretase and γ-secretase liberates Aβ from APP. The aspartyl protease BACE1 (β-siteAPP-cleavingenzyme 1) catalyses the rate-limiting step in the production of Aβ, and as such it is considered to be a major target for drug development in Alzheimer's disease. However, the development of a BACE1 inhibitor therapy is problematic for two reasons. First, BACE1 has been found to have important physiological roles. Therefore, inhibition of the enzyme could have toxic consequences. Second, the active site of BACE1 is relatively large, and many of the bulky compounds that are needed to inhibit BACE1 activity are unlikely to cross the blood-brain barrier. This review focuses on the structure BACE1, current therapeutic strategies based on developing active-site inhibitors, and new approaches to therapy involving targeting the expression or post-translational regulation of BACE1.

Background: The β-site APP cleaving enzyme 1 (BACE1) is a major target for drug design in Alzheimer’s disease. BACE1 binds strongly to heparin and other glycosaminoglycans, and there is evidence that the enzyme may interact with proteoglycans in vivo. Several studies suggest that heparin or heparan sulfate analogues may have value as therapeutic agents for the treatment of AD. Objective: To determine whether heparin can inhibit Aβ production in cortical neurons by inhibiting BACE1. Methods: Cortical neurons from APP (SW) Tg2576 mice were incubated with heparin and the amount of APP processing and Aβ production were measured by enzyme-linked immunosorbent assay and Western blotting. Results: Treatment of cortical neurons with heparin inhibited Aβ secretion. However, this effect was not mediated via inhibition of BACE1. Conclusions: Heparin or other glycosaminoglycans may have value for the treatment of Alzheimer’s disease. However, the data do not support the view that a heparin-induced decrease in Aβ secretion is due to inhibition of BACE1.

The presenilins form part of a complex of membrane proteins that are involved in the proteolytic cleavage of cell-surface molecules. This article reviews the history of the discovery of the presenilins, their role in the pathogenesis of Alzheimer's disease and in the metabolism of the amyloid-β precursor protein. Unanswered questions about their biochemical mechanism of action and their effects on Ca2+ homeostasis are examined.

Homer proteins are integral components of the postsynaptic density and are thought to function in synaptogenesis and plasticity. In addition, overexpression of Homer in the developing Xenopus retinotectal system results in axonal pathfinding errors. Here we report that Xenopus contains the homer1 gene, expressed as the isoform, xhomer1b, which is highly homologous to the mammalian homer1b. The mammalian homer1 gene is expressed as three isoforms, the truncated or short form homer1a and the long forms homer1b and -1c. For Xenopus, we cloned three very similar variants of homer1b, identified as Xenopus xhomer1b.1, xhomer1b.2, and xhomer1b.3, which display up to 98% homology with each other and 90% similarity to mammalian homer1b. Furthermore, we demonstrate that Xenopus also contains a truncated form of the Homer1 protein, which could be induced by kainic acid injection and is likely homologous to the mammalian Homer1a. xHomer1b expression was unaffected by neuronal activity levels but was developmentally regulated. Within the brain, the spatial and temporal distributions of both Homer isoforms were similar in the neuropil and cell body regions. Homer1 was detected in motor axons. Differential distribution of the two isoforms was apparent: Homer1b immunoreactivity was prominent at junctions between soma and the ventricular surface; in the retina, the Mueller radial glia were immunoreactive for Homer1, but not Homer1b, suggesting the retinal glia contain only the Homer1a isoform. Homer1b expression in muscle was prominent throughout development and was aligned with the actin striations in skeletal muscle. The high level of conservation of the xhomer1 gene and the protein expression in the developing nervous system suggest that Homer1 expression may be important for normal neuronal circuit development.

The zebrafish, (Danio rerio) is an important model organism for the analysis of molecular mechanisms that govern neuronal circuit development. The neuronal circuitry that mediates olfaction is crucial for the development and survival of all teleost fishes. In concert with other sensory systems, olfaction is functional at early stages in zebrafish development and mediates important behavioral and survival strategies in the developing larva. Odorant cues are transduced by an array of signaling molecules from receptors in olfactory sensory neurons. The scaffolding protein family known as Homer is well placed to orchestrate this signaling cascade by interacting with and coupling membrane bound receptors to cytosolic signaling partners. To date, Homer has not been demonstrated in the zebrafish. Here we report that the Homer 1b/c isoform was prominent in the olfactory system from the earliest stages of differentiation. We describe the spatial and temporal distribution of Homer in the zebrafish olfactory system. At 24 hours post fertilization (hpf), Homer expression delineated the boundary of the presumptive olfactory placode. Subsequent expression steadily increased throughout the developing olfactory placode, with a prominent localization to the dendritic knobs of the olfactory sensory neurons. Homer expression in the developing olfactory bulb was punctate and prominent in the glomeruli, displaying an apparent synaptic localization. This work supports the hypothesis that Homer is an important molecule in neuronal circuit development, necessary for crucial behaviors required for development and survival.

Electroporation is becoming more popular as a technique for transfecting neurons within intact tissues. One of the advantages of electroporation over other transfection techniques is the ability to precisely target an area for transfection. Here we highlight this advantage by describing methods to restrict transfection to either a single cell, clusters of cells, or to include large portions of the brain of the intact Xenopus tadpole. Electroporation is also an effective means of gene delivery in the retina. We have developed these techniques to examine the effects of regulated gene expression on various neuronal properties, including structural plasticity and synaptic transmission. Restriction of transfection to individual cells aids in imaging of neuronal morphology, while bulk cell transfection allows examination of the affects of gene expression on populations of cells by biochemical assays, imaging, and electrophysiological recording.

Homer proteins are a family of multidomain cytosolic proteins that have been postulated to serve as scaffold proteins that affect responses to extracellular signals by regulating protein-protein interactions. We tested whether Homer proteins are involved in axon pathfinding in vivo, by expressing both wild-type and mutant isoforms of Homer in Xenopus optic tectal neurons. Time-lapse imaging demonstrated that interfering with the ability of endogenous Homer to form protein-protein interactions resulted in axon pathfinding errors at stereotypical choice points. These data demonstrate a function for scaffold proteins such as Homer in axon guidance. Homer may facilitate signal transduction from cell-surface receptors to intracellular proteins that govern the establishment of axon trajectories.

The distribution and ontogeny of GABA- and glutamate-like immunoreactivity in embryos of the Australian freshwater crayfish Cherax destructor were investigated over the period from 30% development until hatching. GABA-like immunoreactive cells and fibres appeared first in the brain at 40–45% development. By 70% development, GABA-like immunoreactive cells were present in almost all ganglia, and GABA-like immunoreactive fibres were distributed extensively throughout the neuropil, commissures and connectives of the central nervous system, and were also found in peripheral nerve roots supplying the appendages and the abdominal musculature. In contrast, glutamate-like immunoreactivity did not appear in the central nervous system until 60–65% development. By the time of hatching, the distribution of glutamate-like immunoreactivity was restricted to discrete regions of neuropil and fibre staining in the thoracic and abdominal nerve cord, the abdominal musculature and the appendages. The precocious establishment of the extensive distribution of GABA-like immunoreactive neurons in the developing crayfish embryo is consistent with the possibility that these neurons play a trophic role in controlling or modulating the development of the nervous system.

Background Homer proteins are post-synaptic density proteins with known functions in receptor trafficking and calcium homeostasis. While they are key mediators of synaptic plasticity, they are also known to function in axon guidance, albeit by mechanisms that are yet to be elucidated. Homer proteins couple extracellular receptors – such as metabotropic glutamate receptors and the transient receptor potential canonical family of cation channels – to intracellular receptors such as inositol triphosphate and ryanodine receptors on intracellular calcium stores and, therefore, are well placed to regulate calcium dynamics within the neural growth cone. Here we used growth cones from dorsal root ganglia, a well established model in the field of axon guidance, and a growth cone turning assay to examine Homer1 function in axon guidance. Results Homer1 knockdown reversed growth cone turning from attraction to repulsion in response to the calcium-dependent guidance cues brain derived neurotrophic factor and netrin-1. Conversely, Homer1 knockdown had no effect on repulsion to the calcium-independent guidance cue Semaphorin-3A. This reversal of attractive turning suggested a requirement for Homer1 in a molecular switch. Pharmacological experiments confirmed that the operational state of a calcium-calmodulin dependent protein kinase II/calcineurin phosphatase molecular switch was dependent on Homer1 expression. Calcium imaging of motile growth cones revealed that Homer1 is required for guidance-cue-induced rise of cytosolic calcium and the attenuation of spontaneous cytosolic calcium transients. Homer1 knockdown-induced calcium transients and turning were inhibited by antagonists of store-operated channels. In addition, immunocytochemistry revealed the close association of Homer1 with the store-operated proteins TRPC1 and STIM1 within dorsal root ganglia growth cones. Conclusion These experiments provide evidence that Homer1 is an essential component of the calcium signalling repertoire within motile growth cones, regulating guidance-cue-induced calcium release and maintaining basal cytosolic calcium.