We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and ΔfosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ΔfosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled ΔfosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ΔfosB mRNA in living brains.

The involvement of matrix metalloproteinase-9 (MMP-9) activities in the development of abnormal water diffusion in the brain after cardiac arrest is not fully understood. We used magnetic resonance imaging to determine the correlation between MMP-9 activity and the mechanism of abnormal water diffusion after global cerebral ischemia (GCI)-induced brain damage in C57black6 mice. We induced GCI in mice by occluding both carotid arteries for 60 min, then allowing reperfusion. We labeled a short DNA that targets mmp-9 mRNA activity [phosphorothioate-modified oligodeoxynucleotide (sODN)-mmp9] or a control probe without intracellular target (sODN-Ran) with iron-based MR contrast agent [superparamagnetic iron oxide nanoparticle (SPION)-mmp9 or SPION-Ran] or fluorescein isothiocyanate (FITC)-sODN-mmp9 or FITC-sODN-Ran; we then delivered these probes by intracerebroventricular infusion or intraperitoneal injection with in 3 h of reperfusion. At low dose (120 pmol/kg) the SPION-mmp9 probe was retained at significant levels in the striatum and cortex of living brains 10 h after GCI. Probe retention was validated by similar elevation of mmp-9 mRNA and antigens in postmortem samples taken from regions that exhibited GCI-induced hyperintensity in diffusion-weighted imaging, and a significant reduction in apparent diffusion coefficient (rADC, p = 0.0006, n = 12). At a higher dose (120 nmol/kg), the FITC-sODN-mmp9 probe revealed significant knockdown of MMP-9 activity, per zymography, and a reversal of striatal rADC (p = 0.004, n = 6). These observations were not duplicated in the control group. We conclude that expression of mmp-9 mRNA is associated with abnormal ADC after GCI.

While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase (LAL) that hydrolyzes C esters and NPC1. In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1-/- mouse, HP-β-CD, a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a T½ of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of SREBP2 target genes, and activation of LXR controlled genes. These findings indicated that HP-β-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED50 for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted more than 7 days. Continuous infusion of HP-β-CD into the ventricular system of npc1-/- animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-β-CD into the CNS.

In many sensory systems, receptive fields (RFs) measured by spike responses undergo progressive refinement during development. It has been proposed that elimination of excitatory synaptic inputs underlies such functional refinement. However, despite many extracellular recording and anatomical studies, direct in vivo intracellular evidence has remained limited. In this study, by cell-attached recordings in the developing optic tectum of zebrafish, we found that during a short period after the initial formation of retinotectal synapses, spike visual RFs of tectal neurons underwent a two-stage developmental modulation: from an initial expansion to a later refinement. Whole-cell voltage-clamp recordings revealed that the underlying excitatory synaptic RF exhibited a similar developmental progression with its spatial extent first increased and then reduced, and its spatial tuning profile gradually sharpened. The inhibitory RF was initially larger than the excitatory RF, but became matched with the excitatory RF at later stages. Simulation with the integrate-and-fire neuron model suggested that the developmental changes of excitatory RFs primarily accounted for the initial enlargement and later refinement of spike RFs, while inhibitory inputs generally reduced the size of the spike RF without affecting its developmental progression. In addition, spike RF of individual retinal ganglion cells (RGCs) did not significantly change in size during the same period, and the spatial extent and tuning profile of the tectal excitatory RF barely changed after intratectal excitatory connections were silenced. Together, our results demonstrate that the functional refinement of tectal visual RFs results primarily from a selective elimination of feedforward retinotectal inputs.

Information processing in the CNS is controlled by the activity of neuronal networks composed of principal neurons and interneurons. Activity-dependent modification of synaptic transmission onto principal neurons is well studied, but little is known about the modulation of inhibitory transmission between interneurons. However synaptic plasticity at this level has clear implications for the generation of synchronized activity. We have investigated the molecular mechanism(s) and functional consequences of an activity-induced lasting increase in GABA release that occurs between inhibitory interneurons (stellate cells) in the cerebellum. Using whole cell recording and cerebellar slices, we found that stimulation of glutamatergic inputs (parallel fibres) with a physiological-like pattern of activity triggered a lasting increase in GABA release from stellate cells. This activity also potentiated inhibitory transmission between synaptically connected interneurons. Extracellular recording revealed that the enhanced inhibitory transmission reduced the firing frequency and altered the pattern of action potential activity in stellate cells. The induction of the sustained increase in GABA release required activation of NMDA receptors (NMDARs). Using pharmacological and genetic approaches we found that presynaptic cAMP/PKA signaling and RIM1α, an active zone protein, is the critical pathway that is required for the lasting enhancement of GABA release. Thus a common mechanism can underlie presynaptic plasticity of both excitatory and inhibitory transmission. This activity-dependent regulation of synaptic transmission between inhibitory interneurons may serve as an important mechanism for interneuronal network plasticity.

Here we show that conditional deletion of PTEN in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to the constitutive neurogenesis in the olfactory bulb. As a result, the Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, contrary to olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.

The rhombic lip (RL) is the neuroepithelium immediately adjacent to the roof plate of the fourth ventricle and it gives rise to various brainstem and cerebellar cell types. Our study shows that the basic helix-loop-helix (bHLH) transcription factor Olig3 is expressed in the progenitors of RL and ablation of Olig3 significantly affects the development of RL. In Olig3−/− caudal RL, the expression level of Math1 in the dI1 domain is reduced and the formation of four mossy-fiber nuclei is compromised; dI2–dI3 neurons are mis-specified to dI4 interneurons and the climbing-fiber neurons (inferior olive nucleus) are completely lost. In addition, the formation of brainstem (nor)adrenergic centers and first-order relay visceral sensory neurons is also dependent on Olig3. Therefore, Olig3 plays an important role in the fate specification and differentiation of caudal RL-derived neurons.

To circumvent the limitations of using postmortem brain in molecular assays, we used avidin– biotin binding to couple superparamagnetic iron oxide nanoparticles (SPIONs) (15–20 nm) to phosphorothioate-modified oligodeoxynucleotides (sODNs) with sequence complementary to c-fos and β-actin mRNA (SPION-cfos and SPION-βactin, respectively) (14 –22 nm). The Stern–Volmer constant for the complex of SPION and fluorescein isothiocyanate (FITC)-sODN is 3.1 × 106/m. We studied the feasibility of using the conjugates for in vivo magnetic resonance imaging (MRI) to monitor gene transcription, and demonstrated that these complexes at 40 μg of Fe per kilogram of body weight were retained at least 1 d after intracerebroventricular infusion into the left ventricle of C57Black6 mice. SPION retention measured by MRI as T2* or R2* maps (R2* = 1/T2*) was compared with histology of iron oxide (Prussian blue) and FITC-labeled sODN. We observed significant reduction in magnetic resonance (MR) T2* signal in the right cortex and striatum; retention of SPION-cfos and SPION-βactin positively correlated with c-fos and β-actin mRNA maps obtained from in situ hybridization. Histological examination showed that intracellular iron oxide and FITC-sODN correlated positively with in vivo MR signal reduction. Furthermore, in animals that were administered SPION-cfos and amphetamine (4 mg/kg, i.p.), retention was significantly elevated in the nucleus accumbens, striatum, and medial prefrontal cortex of the forebrain. Control groups that received SPION-cfos and saline or that received a SPION conjugate with a random-sequence probe and amphetamine showed no retention. These results demonstrated that SPION-sODN conjugates can detect active transcriptions of specific mRNA species in living animals with MRI.

Trimeric sodium channels of the DEG/ENaC family have important roles in neurons, but the specific functions of different subunits present in heteromeric channels are poorly understood. We previously reported that the Drosophila DEG/ENaC subunit Ppk25 is essential in a small subset of gustatory neurons for activation of male courtship behavior, likely through detection of female pheromones. Here we show that, like mutations in ppk25, mutations in another Drosophila DEG/ENaC subunit, nope, specifically impair male courtship of females. nope regulatory sequences drive reporter gene expression in gustatory neurons of the labellum, wings and legs, including all gustatory neurons in which ppk25 function is required for male courtship of females. In addition, gustatory-specific knockdown of nope impairs male courtship. Further, the impaired courtship response of nope mutant males to females is rescued by targeted expression of nope in the subset of gustatory neurons in which ppk25 functions. However, nope and ppk25 have non-redundant functions, as targeted expression of ppk25 does not compensate for the lack of nope and vice versa. Moreover, Nope and Ppk25 form specific complexes when co-expressed in cultured cells. Together, these data indicate that the Nope and Ppk25 subunits have specific, non-redundant functions in a subset of gustatory neurons required for activation of male courtship in response to females, and suggest the hypothesis that Nope and Ppk25 function as subunits of a heteromeric DEG/ENaC channel required for gustatory detection of female pheromones.

Acidification of synaptic vesicles relies on the Vacuolar-type ATPase (V-ATPase), which provides the electrochemical driving force for neurotransmitter exchange. The regulatory mechanisms that ensure assembly of the V-ATPase holoenzyme on synaptic vesicles are unknown. Rabconnectin3α (Rbc3α) is a potential candidate for regulation of V-ATPase activity because of its association with synaptic vesicles and its requirement for acidification of intracellular compartments. Here, we provide the first evidence for a role of Rbc3α in synaptic vesicle acidification and neurotransmission. In this study, we characterized mutant alleles of rbc3α isolated from a large-scale screen for zebrafish with auditory/vestibular defects. We show that Rbc3α is localized to basal regions of hair cells where synaptic vesicles are present. To determine whether Rbc3α regulates V-ATPase activity, we examined the acidification of synaptic vesicles and localization of the V-ATPase in hair cells. In contrast to wild-type hair cells, we observed that synaptic vesicles had elevated pH and a cytosolic subunit of the V-ATPase was no longer enriched in synaptic regions of mutant hair cells. As a consequence of defective acidification of synaptic vesicles, afferent neurons in rbc3α mutants had reduced firing rates and reduced accuracy of phase-locked action potentials in response to mechanical stimulation of hair cells. Collectively, our data suggest that Rbc3α modulates synaptic transmission in hair cells by promoting V-ATPase activity in synaptic vesicles.

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

Sox2 plays critical roles in cell fate specification during development and in stem cell formation; however, its role in postmitotic cells is largely unknown. Sox2 is highly expressed in supporting cells (SCs) of the postnatal mammalian auditory sensory epithelium, which unlike non-mammalian vertebrates remains quiescent even after sensory hair cell damage. Here, we induced the ablation of Sox2, specifically in SCs at three different postnatal ages (neonatal, juvenile and adult) in mice. In neonatal mice, Sox2-null inner pillar cells (IPCs, a subtype of SCs) proliferated and generated daughter cells, while other SC subtypes remained quiescent. Furthermore, p27Kip1, a cell cycle inhibitor, was absent in Sox2-null IPCs. Similarly, upon direct deletion of p27Kip1, p27Kip1-null IPCs also proliferated but retained Sox2 expression. Interestingly, cell cycle control of IPCs by Sox2-mediated expression of p27Kip1 gradually declined with age. In addition, deletion of Sox2 or p27Kip1 did not cause a cell fate change. Finally, chromatin immunoprecipitation with Sox2 antibodies and luciferase reporter assays with the p27Kip1 promoter support that Sox2 directly activates p27Kip1 transcription in postmitotic IPCs. Hence, in contrast to the well-known activity of Sox2 in promoting proliferation and cell fate determination, our data demonstrate that Sox2 plays a novel role as a key upstream regulator of p27Kip1 to maintain the quiescent state of postmitotic IPCs. Our studies suggest that manipulating Sox2 or p27Kip1 expression is an effective approach to inducing proliferation of neonatal auditory IPCs, an initial but necessary step toward restoring hearing in mammals.

How human beings integrate information from external sources and internal cognition to produce a coherent experience is still not well understood. During the past decades, anatomical, neurophysiological and neuroimaging research in multimodal integration have stood out in the effort to understand the perceptual binding properties of the brain. Areas in the human lateral occipito-temporal, prefrontal and posterior parietal cortices have been associated with sensory multimodal processing. Even though this, rather patchy, organization of brain regions gives us a glimpse of the perceptual convergence, the articulation of the flow of information from modality-related to the more parallel cognitive processing systems remains elusive. Using a method called Stepwise Functional Connectivity analysis, the present study analyzes the functional connectome and transitions from primary sensory cortices to higher-order brain systems. We identify the large-scale multimodal integration network and essential connectivity axes for perceptual integration in the human brain.

Phosphoinositide 3-kinase enhancer (PIKE) binds and enhances PI3K/Akt activities. However, its physiological functions in brain have never been explored. Here we show that PIKE is important in regulating the neuronal survival and development of neocortex. During development, enhanced apoptosis is observed in the ventricular zone of PIKE knockout (PIKE −/−) cortex. Moreover, PIKE −/− neurons show reduced dendritic complexity, dendritic branch length and soma size. These defects are due to the reduced PI3K/Akt activities in PIKE −/− neurons, as the impaired dendritic arborization can be rescued when PI3K/Akt cascade is augmented in vitro or in PIKE−/−PTEN−/− double knockout mice. Interestingly, PIKE −/− mice display behavioral abnormality in locomotion and spatial navigation. Because of the diminished PI3K/Akt activities, PIKE −/− neurons are more vulnerable to glutamate or stroke-induced neuronal cell death. Together, our data established the critical role of PIKE in regulating neuronal survival and development by substantiating the PI3K/Akt pathway.

Thioredoxin interacting protein (TXNIP) has recently been described as a key regulator of energy metabolism through pleiotropic actions that include nutrient sensing in the mediobasal hypothalamus (MBH). However, the role of TXNIP in neurochemically specific hypothalamic subpopulations, and the circuits downstream from MBH TXNIP engaged to regulate energy homeostasis remain unexplored. To evaluate the metabolic role of TXNIP activity specifically within arcuate Agrp neurons, we generated Agrp-specific TXNIP gain- and loss-of-function mouse models using Agrp-Ires-cre mice, TXNIPflox/flox mice and a lentivector expressing the human TXNIP isoform conditionally in the presence of Cre recombinase. Overexpression of TXNIP in Agrp neurons predisposed to diet-induced obesity and adipose tissue storage by decreasing energy expenditure and spontaneous locomotion, without affecting food intake. Conversely, Agrp neuronal TXNIP deletion protected against diet-induced obesity and adipose tissue storage by increasing energy expenditure and spontaneous locomotion, without affecting food intake. TXNIP overexpression in Agrp neurons did not primarily affect glycemic control, whereas deletion of TXNIP in AgRP neurons improved fasting glucose levels and glucose tolerance independently of its effects on body weight and adiposity. Bidirectional manipulation of TXNIP expression induced reciprocal changes in central leptin sensitivity and the neural regulation of lipolysis. Together these results identify a critical role for TXNIP in Agrp neurons in mediating diet-induced obesity through the regulation of energy expenditure and adipose tissue metabolism, independently of food intake. They also reveal a previously unidentified role for Agrp neurons in the brain-adipose axis.

α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Cuprizone inhibits mitochondrial function and induces demyelination in the corpus callosum which resembles pattern III lesions in multiple sclerosis (MS) patients. However, the molecular and cellular mechanism by which cuprizone induces demyelination remains unclear. Interleukin-17 (IL-17) secreted by T helper 17 (Th17) cells and γδT cells are essential in the development of experimental autoimmune encephalomyelitis (EAE). In this study, we examined the importance of IL-17 signaling in cuprizone-induced demyelination. We found that mice deficient in IL-17A, IL-17RC and adaptor protein Act1 (of IL-17R) all had reduced demyelination accompanied by lessened microglial and polydendrocyte cellular reactivity compared to that in wild-type mice in response to cuprizone feeding, demonstrating the essential role of IL-17-induced Act1-mediated signaling in cuprizone-induced demyelination. Importantly, specific deletion of Act1 in astrocytes reduced the severity of tissue injury in this model, indicating the critical role of CNS resident cells in the pathogenesis of cuprizone-induced demyelination. In cuprizone-fed mice IL-17 was produced by CNS CD3+ T cells suggesting a source of IL-17 in CNS upon cuprizone treatment.

Dystrobrevin is a major component of a dystrophin-associated protein complex (DAPC). It is widely expressed in mammalian tissues including the nervous system, where it is localized to the presynaptic nerve terminal with unknown function. In a genetic screen for suppressors of a lethargic phenotype caused by a gain-of-function (gf) isoform of SLO-1 in C. elegans, we isolated multiple loss-of-function (lf) mutants of the dystrobrevin gene dyb-1. dyb-1(lf) phenocopied slo-1(lf), causing increased neurotransmitter release at the neuromuscular junction, increased frequency of Ca2+ transients in body-wall muscle, and abnormal locomotion behavior. Neuron- and muscle-specific rescue experiments suggest that DYB-1 is required for SLO-1 function in both neurons and muscle cells. DYB-1 colocalized with SLO-1 at presynaptic sites in neurons and dense body regions in muscle cells, and dyb-1(lf) caused SLO-1 mislocalization in both types of cells without altering SLO-1 protein level. The neuronal phenotypes of dyb-1(lf) were partially rescued by mouse α-dystrobrevin-1 (αDB1). These observations revealed novel functions of the BK channel in regulating muscle Ca2+ transients, and of dystrobrevin in controlling neurotransmitter release and muscle Ca2+ transients by localizing the BK channel.

FMRI studies have revealed three scene-selective regions in human visual cortex (the Parahippocampal Place Area (PPA), Transverse Occipital Sulcus (TOS) and RetroSplenial Cortex (RSC)), which have been linked to higher-order functions such as navigation, scene perception/recognition, and contextual association.

Here, we document corresponding (presumptively homologous) scene-selective regions in the awake macaque monkey, based on direct comparison to human maps, using identical stimuli and largely overlapping fMRI procedures. In humans, our results showed that the three scene-selective regions are centered near - but distinct from - the gyri/sulci for which they were originally named. In addition, all these regions are located within or adjacent to known retinotopic areas. Human RSC and PPA are located adjacent to the peripheral representation of primary and secondary visual cortex, respectively. Human TOS is located immediately anterior/ventral to retinotopic area V3A, within retinotopic regions LO-1, V3B, and/or V7.

Mirroring the arrangement of human regions FFA and PPA (which are adjacent to each other in cortex), the presumptive monkey homologue of human PPA is located adjacent to the monkey homologue of human FFA, near the posterior superior temporal sulcus. Monkey TOS includes the region predicted from the human maps (macaque V4d), extending into retinotopically-defined V3A. A possible monkey homologue of human RSC lies in the medial bank, near peripheral V1.

Overall, our findings suggest a homologous neural architecture for scene-selective regions in visual cortex of humans and non-human primates, analogous to the face-selective regions demonstrated earlier in these two species.

Matrix metalloproteinases (MMPs) are widely hypothesized to regulate signaling events through processing of extracellular matrix (ECM) molecules. We previously demonstrated that membrane-associated Mmp2 is expressed in exit glia and contributes to motor axon targeting. To identify possible substrates, we undertook a yeast interaction screen for Mmp2-binding proteins and identified the novel ECM protein Faulty Attraction (Frac). Frac encodes a multi-domain extracellular protein rich in EGF and cbEGF domains, related to the vertebrate Fibrillin and Fibulin gene families. It is expressed in mesodermal domains flanking Mmp2-positive glia. The juxtaposition of Mmp2 and Frac proteins raises the possibility that Frac is a proteolytic target of Mmp2. Consistent with this hypothesis, levels of full-length Frac are increased in Mmp2 LOF and decreased in Mmp2 GOF embryos indicating that Frac cleavage is Mmp2-dependent. To test whether frac is necessary for axon targeting, we characterized guidance in frac LOF mutants. Motor axons in frac LOF embryos are loosely associated and project ectopically, a phenotype essentially equivalent to that of Mmp2 LOF. The phenotypic similarity between enzyme and substrate mutants argues that Mmp2 activates Frac. In addition, Mmp2 overexpression pathfinding phenotypes depend on frac activity—indicating that Mmp2 is genetically upstream of frac. Lastly, overexpression experiments suggest that Frac is unlikely to have intrinsic signaling activity, raising the possibility that an Mmp2-generated Frac fragment acts as a guidance cue cofactor. Indeed, we present genetic evidence that Frac regulates a non-canonical LIM kinase 1-dependent BMP signaling pathway in motorneurons necessary for axon pathfinding during embryogenesis.

The multiprotein complexes that receive and transmit axon pathfinding cues during development are essential to circuit generation. Here, we identify and characterize the Drosophila sterile α-motif (SAM) domain-containing protein Caskin, which shares homology with vertebrate Caskin, a CASK [calcium/calmodulin-(CaM)-activated serine-threonine kinase]-interacting protein. Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related (Lar) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of Ckn mediates its interaction with Lar. Like Caskin, Liprin-α is a neuronal adaptor protein that interacts with Lar via a SAM domain-mediated interaction. We present evidence that Lar does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on Lar. Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in Lar signal transduction is evolutionarily conserved. Last, we characterize several ckn mutants that retain Lar binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, we identify the SH2/SH3 adaptor protein Dock as a second Caskin-binding protein and find that Caskin binds Lar and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in Lar-dependent signaling during motor axon targeting, ckn and dock have overlapping roles in axon outgrowth in the CNS. Together, these studies identify caskin as a neuronal adaptor protein required for axon growth and guidance.

BDNF plays a critical role in the regulation of synaptic strength and is essential for long-term potentiation (LTP), a phenomenon that underlies learning and memory. However, whether BDNF acts in a diffuse manner, or is targeted to specific neuronal sub-compartments or synaptic sites to affect circuit function, remains unknown. Here, using photoactivation of BDNF or syt-IV (a regulator of exocytosis present on BDNF-containing vesicles) in transfected rat hippocampal neurons, we discovered that distinct subsets of BDNF vesicles are targeted to axons versus dendrites and are not shared between these compartments. Moreover, syt-IV and BDNF-harboring vesicles are recruited to both pre and post-synaptic sites in response to increased neuronal activity. Finally, using syt-IV knockout mouse neurons, we found that syt-IV is necessary for both pre- and post-synaptic scaling of synaptic strength in response to changes in network activity. These findings demonstrate that BDNF-containing vesicles can be targeted to specific sites in neurons, and suggest that syt-IV-regulated BDNF secretion is subject to spatial control to regulate synaptic function in a site-specific manner.

Accumulation of misfolded α-synuclein (αS) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other α-synucleinopathies. However, how αS causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in α-synucleinopathy. Using the A53T mutant human αS transgenic (A53TαS Tg) mouse model of α-synucleinopathy, we show that disease onset in the αS Tg model is coincident with induction of ER chaperone in neurons exhibiting αS pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2α, indicating that α-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated αS monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M αS than the control cases. Moreover, αS interacts with ER chaperones and overexpression of αS sensitizes neuronal cells to ERS-induced toxicity, suggesting that αS may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both the A53TαS Tg mouse model and the Adeno-associated Virus-transduced rat model of A53T α S-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation αS within ER leads to chronic ER stress conditions that contribute to neurodegeneration in α-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other α-synucleinopathies.

Serotonin (5-HT)-induced long-term facilitation (LTF) of the Aplysia sensorimotor synapse depends on enhanced gene expression and protein synthesis, but identification of the genes whose expression and regulation are necessary for LTF remains incomplete. In this study we found that one such gene is synapsin, which encodes a synaptic vesicle-associated protein known to regulate short-term synaptic plasticity. Both synapsin mRNA and protein levels were increased by 5-HT. Upregulation of synapsin protein occurred in presynaptic sensory neurons at neurotransmitter release sites. To investigate the molecular mechanisms underlying synapsin regulation, we cloned the promoter region of Aplysia synapsin, and found that the synapsin promoter contained a cAMP response element (CRE), raising the possibility that the transcriptional activator CREB1 mediates 5-HT-induced regulation of synapsin. Indeed, binding of CREB1 to the synapsin promoter was increased following treatment with 5-HT. Furthermore, increased acetylation of histones H3 and H4 and decreased association of histone deacetylase 5 near the CRE site is consistent with transcriptional activation by CREB1. RNA interference (RNAi) targeting synapsin mRNA blocked the 5-HT-induced increase in synapsin protein levels and LTF during a time window when basal synapsin levels were unaffected by RNAi. These results indicate that the 5-HT-induced regulation of synapsin levels is necessary for LTF and that this regulation is part of the cascade of synaptic events involved in the consolidation of memory.

Huntington’s disease, caused by an expanded triplet repeat in the huntingtin gene (Htt), results in extensive neuropathology but study of the Htt gene in CNS development through gene knockout is problematic as the knockout leads to embryonic lethality in mice. Here, we report that the knockdown of Htt expression in neuroepithelial cells of neocortex results in disturbed cell migration, reduced proliferation, and increased cell death that is relatively specific to early neural development. In the cerebellum, however, Htt knockdown results in cell death but not perturbed migration. The cell death phenotype in cortex can be partially reversed with co-knockdown of Casp9, indicating mitochondria-mediated cell apoptotic processes are involved in the neuronal death. The timing of knock-down during early development is also an important variable. These results indicate a spatial and temporal requirement for Htt expression in neural development. Although it is uncertain if the loss of wildtype huntingtin function contributes to pathogenesis in Huntington’s disease, these results clearly contraindicate the use of non-specific knockdown of Htt as a therapeutic measure in HD, particularly in utero.