The dopamine transporter (DAT) is the primary protein responsible for the uptake of dopamine from the extracellular space back into presynaptic neurons. As such, it plays an important role in the cessation of dopaminergic neurotransmission and in the maintenance of extracellular dopamine homeostasis. Here, we report the development of a new BAC transgenic mouse line that expresses DAT with an N-terminal HA-epitope (HAD-Tg). In this line, two copies of the HA-DAT BAC are incorporated into the genome, increasing DAT mRNA levels by 47%. Despite the increase in mRNA levels, HAD-Tg mice show no significant increase in the level of DAT protein in the striatum, indicating a defect in protein trafficking or stability. By crossing HAD-Tg mice with DAT knockout mice (DAT-KO), we engineered mice that exclusively express HA-tagged DAT in the absence of endogenous DAT (DAT-KO/HAD-Tg). We show that DAT-KO/HAD-Tg mice express only 8.5% of WT DAT levels in the striatum. Importantly, the HA-tagged DAT that is present in DAT-KO/HAD-Tg mice is functional, as it is able to partially rescue the DAT-KO hyperactive phenotype. Finally, we provide evidence that the HA-tagged DAT is retained in the cell body based on a reduction in the striatum:midbrain protein ratio. These results demonstrate that the presence of the N-terminal tag leads to impaired DAT protein expression in vivo due in part to improper trafficking of the tagged transporter, and highlight the importance of the N-terminus in the transport of DAT to striatal terminals.
PMID: 24886986 CAMSID: cams4546
Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed innumerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling.
Anaphase-Promoting Complex; Ubiquitin ligase; GABA; NMJ; Synapse
The disruption of neurotransmitter and neurotrophic factor signaling in the central nervous system (CNS) is implicated as the root cause of neuropsychiatric disorders, including schizophrenia, epilepsy, chronic pain, and depression. Therefore, identifying the underlying molecular mechanisms by which neurotransmitter and neurotrophic factor signaling regulates neuronal survival or growth may facilitate identification of more effective therapies for these disorders. Previously, our lab found that the heterotrimeric G protein, Gz, mediates crosstalk between G protein-coupled receptors and neurotrophin signaling in the neural cell line PC12. These data, combined with Gαz expression profiles - predominantly in neuronal cells with higher expression levels corresponding to developmental times of target tissue innervation - suggested that Gαz may play an important role in neurotrophin signaling and neuronal development. Here, we provide evidence in cortical neurons, both manipulated ex vivo and those cultured from Gz knockout mice, that Gαz is localized to axonal growth cones and plays a significant role in the development of axons of cortical neurons in the CNS. Our findings indicate that Gαz inhibits BDNF-stimulated axon growth in cortical neurons, establishing an endogenous role for Gαz in regulating neurotrophin signaling in the CNS.
BDNF; GNAZ; G proteins; Neurotrophin
The dystrobrevin binding protein (DTNBP) 1 gene has emerged over the last decade as a potential susceptibility locus for schizophrenia. While no causative mutations have been found, reduced expression of the encoded protein, dysbindin, was reported in patients. Dysbindin likely plays a role in the neuronal trafficking of proteins including receptors. One important pathway suspected to be affected in schizophrenia is the fast excitatory glutamatergic transmission mediated by AMPA receptors. Here, we investigated excitatory synaptic transmission and plasticity in hippocampal neurons from dysbindin-deficient sandy mice bred on the DBA/2J strain. In cultured neurons an enhancement of AMPAR responses was observed. The enhancement of AMPAR-mediated transmission was confirmed in hippocampal CA3-CA1 synapses, and was not associated with changes in the expression of GluA1–4 subunits nor an increase in GluR2-lacking receptor complexes. Lastly, an enhancement in LTP was also found in these mice. These data provide compelling evidence that dysbindin, a widely suspected susceptibility protein in schizophrenia, is important for AMPAR-mediated synaptic transmission and plasticity in the developing hippocampus.
DTNBP1; dysbindin; schizophrenia; AMPAR; LTP
The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicerflox/flox). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicerflox/flox mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, is essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
During development and after birth neural stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to populate the olfactory bulb (OB) with neurons. Multiple factors promote neuroblast migration, but the contribution that many of these make to guidance within the intact RMS is not known. In the present study we have characterised in detail how endocannabinoid (eCB), BDNF and FGF receptor (FGFR) signalling regulates motility and guidance, and also determined whether any of these receptors operate in a regionally restricted manner. We used in vivo electroporation in postnatal mice to fluorescently label neuroblasts, and live cell imaging to detail their migratory properties. Cannabinoid receptor antagonists rendered neuroblasts less mobile, and when they did move guidance was lost. Similar results were obtained when eCB synthesis was blocked with diacylglycerol lipase (DAGL) inhibitors, and importantly eCB function is required for directed migration at both ends of the RMS. Likewise, inhibition of BDNF signalling disrupted motility and guidance in a similar manner along the entire RMS. In contrast, altering FGFR signalling inhibits motility and perturbs guidance, but only at the beginning of the stream. Inhibition of FGFR signalling in vivo also reduces the length of the leading process on migratory neuroblasts in a graded manner along the RMS. These results provide evidence for a guidance function for all three of the above receptor systems in the intact RMS, but show that FGFR signalling is unique as it is required in a regionally specific manner.
•Live imaging is used to characterise neuroblast migration in the intact RMS.•Endocannabinoids are required for motility and guidance throughout the RMS.•BDNF regulates guidance in a similar manner.•FGFR signalling is also required for guidance, but only at the beginning of the stream.•FGFR regulates neuroblasts morphology in a graded manner along the RMS in vivo.
Neuroblast migration rostral migratory stream; Endocannabinoid; BDNF; FGF
HspB1 is a small heat shock protein implicated in neuronal survival and neurite growth; mutations in HspB1 have been identified in hereditary motor neuronopathies and Charcot Marie Tooth Type 2 neuropathies. In cortical neurons we found that expression of HspB1 decreased RhoA activity and RhoA-GTP protein, and reversed the inhibition of neurite extension induced by NogoA. HspB1 decreased PDZ-RhoGEF, a RhoA specific guanine nucleotide exchange factor, while other regulators of RhoA activity were unchanged. The decrease in PDZ-RhoGEF was independent of proteasomal or lysosomal degradation pathways and was not associated with changes in PDZ-RhoGEF mRNA. We sequenced the 3’UTR of rat PDZ-RhoGEF and found binding sites for miRNAs miR-20a, miR-128 and miR-132. Expression of these microRNAs was substantially increased in cortical neurons transfected with HspB1. Co-transfection of HspB1 with specific inhibitors of miR-20a or miR-128 prevented the decrease in PDZ-RhoGEF and blocked the neurite growth promoting effects of HspB1. Using the 3'UTR of PDZ-RhoGEF mRNA in a luciferase reporter construct we observed that HspB1, miR-20a and miR-128 each inhibited luciferase expression. We conclude that HspB1 regulates RhoA activity through modulation of PDZ-RhoGEF levels achieved by translational control through enhanced expression of specific miRNAs (miR-20a and miR-128). Regulation of RhoA activity by translational silencing of PDZ-RhoGEF may be the mechanism through which HspB1 is involved in regulation of neurite growth. As RhoA-GTPase plays a regulatory role in the organization and stability of cytoskeletal networks through its downstream effectors, the results suggest a possible mechanism linking HspB1 mutations and axonal cytoskeletal pathology.
heat shock protein; guanine nucleotide exchange factor (GEF); microRNA; neurite outgrowth; RhoA
Neuropathic pain is associated with hyperexcitability of DRG neurons. Despite the importance of leakage potassium channels for neuronal excitability, little is known about their cell-specific expression in DRGs and possible modulation in neuropathic pain. Multiple leakage channels are expressed in DRG neurons, including TASK1, TASK3, TRESK, TRAAK, TWIK1, TREK1 and TREK2 but little is known about their distribution among different cell types. Our immunohistochemical studies show robust TWIK1 expression in large and medium size neurons, without overlap with TRPV1 or IB4 staining. TASK1 and TASK3, on the contrary, are selectively expressed in small cells; TASK1 expression closely overlaps TRPV1-positive cells, while TASK3 is expressed in TRPV1- and IB4-negative cells. We also studied mRNA expression of these channels in L4-L5 DRGs in control conditions and up to 4 weeks after spared nerve injury lesion. We found that TWIK1 expression is much higher than TASK1 and TASK3 and is strongly decreased 1, 2 and 4 weeks after neuropathic injury. TASK3 expression, on the other hand, decreases 1 week after surgery but reverts to baseline by 2 weeks; TASK1 shows no significant change at any time point. These data suggest an involvement of TWIK1 in the maintenance of the pain condition.
KCNK; PCR; immunohistochemistry; nerve injury, TWIK1
Plasma gelsolin (pGSN), a secreted form of gelsolin, is constitutively expressed throughout the central nervous system (CNS). Neurons, astrocytes and oligodendrocytes are the major sources of pGSN in the CNS. It has been shown that levels of pGSN in cerebrospinal fluid (CSF) are decreased in several neurological conditions including HIV-1-associated neurocognitive disorders (HAND). Although there is no direct evidence that a decreased level of pGSN in CSF is causally related to the pathogenesis of neurological disorders, neural cells, if lacking pGSN, are more vulnerable to cell death. To understand how GSN levels relate to neuronal injury in HAND, we studied the effects of pGSN on HIV-1 gp120-activated outward K+ currents in primary rat cortical neuronal cultures. Incubation of rat cortical neurons with gp120 enhanced the outward K+ currents induced by voltage steps and resulted in neuronal apoptosis. Treatment with pGSN suppressed the gp120-induced increase of delayed rectifier current (IK) and reduced vulnerability to gp120-induced neuronal apoptosis. Application of Guangxitoxin-1E (GxTx), a Kv2.1 specific channel inhibitor, inhibited gp120 enhancement of IK and associated neuronal apoptosis, similar effects to pGSN. Western blot and PCR analysis revealed gp120 exposure to up-regulate Kv2.1 channel expression, which was also inhibited by treatment with pGSN. Taken together, these results indicate pGSN protects neurons by suppressing gp120 enhancement of IK through Kv2.1 channels and reduction of pGSN in HIV-1-infected brain may contribute to HIV-1-associated neuropathy.
Cortical neurons; Kv channels; HIV-1gp120; Neuronal injury; Plasma gelsolin; Neuroprotection
Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we also demonstrated have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration. Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.
Calpain; caspase; HIV-associated neurocognitive disorder; MDMx; neuron; neuroprotection
While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.
Sema4D; GABAergic; proteolytic cleavage; inhibitory synapse
Mitochondrial dysfunction leading to deficits in energy production, Ca2+ uptake capacity, and free radical generation has been implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) caused by mutations in Cu, Zn superoxide dismutase (SOD1). Numerous studies link UCP2, a member of the uncoupling protein family, to protection of neurons from mitochondrial dysfunction and oxidative damage in various mouse models of acute stress and neurodegeneration, including Parkinson’s disease. Here, we tested the potential neuroprotective effects of UCP2 and its ability to modulate mitochondrial function, in the G93A mutant SOD1 mouse model of familial ALS. Disease phenotype, mitochondrial bioenergetics, and Ca2+ uptake capacity were investigated in the central nervous system of double transgenic mice, expressing both human mutant G93A SOD1 and human UCP2 (hUCP2). Unexpectedly, hUCP2 expression accelerated the disease course of SOD1 mutant mice. In addition, we did not observe a classical uncoupling effect of hUCP2 in G93A brain mitochondria, although we did detect a decrease in reactive oxygen species (ROS) production from mitochondria challenged with the respiratory chain inhibitors rotenone and antimycin A. We also found that mitochondrial Ca2+ uptake capacity was decreased in the double transgenic mice, as compared to G93A mice. Taken together our results indicate that the neuroprotective role of UCP2 in neurodegeneration is disease-specific and that, while a mild uncoupling by UCP2 in brain mitochondria may protect against neurodegeneration in some injury paradigms, the mitochondrial damage and the disease caused by mutant SOD1 cannot be ameliorated by UCP2 overexpression.
ALS; mitochondria; UCP2; SOD1
γ amino-butyric acid type-A receptors (GABARs) containing γ2 or δ subunits form separate pools of receptors in vivo, with distinct localization and function. We determined the rate of surface membrane insertion of native and recombinant γ2 and δ subunit-containing GABARs (γ2-GABARs and δ-GABARs). Insertion of the α-bungarotoxin binding site (BBS) tagged γ2 subunit (t-γ2)-containing GABARs in the surface membrane of HEK293 cells occurred within minutes and reached a peak by 30 min. In contrast, insertion of the BBS-tagged δ subunit (t-δ)-containing receptors required longer incubation and peaked in 120 min. Insertion of the t-γ2 subunit-containing receptors was not influenced by assembling α1 or α4 subunits. In contrast, insertion of the α4β3t-δ subunit-containing receptors was faster than those containing α1β3t-δ subunits. The rate of insertion of native GABARs in the surface membrane of cultured hippocampal neurons, determined by an antibody saturation assay, was similar to that of the recombinant receptors expressed in HEK293 cells. Insertion of the γ2-GABARs was rapid and new γ2-GABARs were detected on the surface membrane of cell soma and dendrites within minutes. In contrast, insertion of the δ-GABARs was slow and newly inserted receptors were initially present only in the surface membrane of cell soma and later also appeared over the dendrites. Thus the rate of insertion of GABARs was dependent on their subunit composition.
The transcription factor STAT3 has been implicated in axon regeneration. Here we investigate a role for STAT3 in sympathetic nerve sprouting after myocardial infarction (MI) - a common injury in humans. We show that NGF stimulates serine phosphorylation (S727) of STAT3 in sympathetic neurons via ERK1/2, in contrast to cytokine phosphorylation of Y705. Maximal sympathetic axon regeneration in vitro requires phosphorylation of both S727 and Y705. Furthermore, cytokine signaling is necessary for NGF-induced sympathetic nerve sprouting in the heart after MI. Transfection studies in neurons lacking STAT3 suggest two independent pools of STAT3, phosphorylated on either S727 or Y705, that regulate sympathetic regeneration via both transcriptional and non-transcriptional means. Additional data identify STAT3-microtubule interactions that may complement the well-characterized role of STAT3 stimulating regeneration associated genes. These data show that STAT3 is critical for sympathetic axon regeneration in vitro and in vivo, and identify a novel non-transcriptional mode of action.
ciliary neurotrophic factor; myocardial infarction; nerve growth factor; signal transducer and activator of transcription 3; sympathetic axon regeneration
The axon initial segment (AIS) is highly enriched in the structural proteins ankyrin G and βIV-spectrin, the pore-forming (α) subunits of voltage-gated sodium (Nav) channels, and functional Nav channels, and is critical for the initiation of action potentials. We previously reported that FGF14, a member of the intracellular FGF (iFGF) sub-family, is expressed in cerebellar Purkinje neurons and that the targeted inactivation of Fgf14 in mice (Fgf14−/−) results in markedly reduced Purkinje neuron excitability. Here, we demonstrate that FGF14 immunoreactivity is high in the AIS of Purkinje neurons and is distributed in a decreasing, proximal to distal, gradient. This pattern is evident early in the postnatal development of Purkinje neurons and is also observed in many other types of central neurons. In ( Scn8amed) mice, which are deficient in expression of the Nav1.6 α subunit, FGF14 immunoreactivity is markedly increased and expanded in the Purkinje neuron AIS, in parallel with increased expression of the Nav1.1 (Scn1a) α subunit and expanded expression of βIV-spectrin. Although Nav1.1, FGF14, and βIV-spectrin are affected, ankyrin G immunoreactivity at the AIS of Scn8amed and wild type (WT) Purkinje neurons was not significantly different. In Fgf14−/− Purkinje neurons, βIV-spectrin and ankyrin G immunoreactivity at the AIS were also similar to WT Purkinje neurons, although both the Nav1.1 and Nav1.6 α subunits are modestly, but significantly (P<0.005), reduced within sub-domains of the AIS, changes that may contribute to the reduced excitability of Fgf14−/− Purkinje neurons.
Purkinje neuron; axon initial segment; AIS; iFGF; FGF14; voltage-gated sodium channel; Nav1.1; Scn1a; Nav1.6; Scn8a; ankyrin G; βIV-spectrin
Huntington’s disease (HD) causes severe motor dysfunction, behavioral abnormalities, cognitive impairment and death. Investigations into its molecular pathology have primarily relied on murine tissues; however, the recent discovery of induced pluripotent stem cells (iPSCs) has opened new possibilities to model neurodegenerative disease using cells derived directly from patients, and therefore may provide a human-cell-based platform for unique insights into the pathogenesis of HD. Here, we will examine the practical implementation of iPSCs to study HD, such as approaches to differentiate embryonic stem cells (ESCs) or iPSCs into medium spiny neurons, the cell type most susceptible in HD. We will explore the HD-related phenotypes identified in iPSCs and ESCs and review how brain development and neurogenesis may actually be altered early, before the onset of HD symptoms, which could inform the search for drugs that delay disease onset. Finally, we will speculate on the exciting possibility that ESCs or iPSCs might be used as therapeutics to restore or replace dying neurons in HD brains.
Huntington’s disease; induced pluripotent stem cells; IPSC; MSN; stem cell models; Neurodegenerative disease
Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cav), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of Caveolin isoforms in microglia physiology is debatable. We propose caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CV) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.
caveolin; cytoskeleton; mitochondria; microglial activation; neuroinflammation
The COMPLEXIN (CPX) proteins play a critical role in synaptic vesicle fusion and neurotransmitter release. Previous studies demonstrated that CPX functions in both activation of evoked neurotransmitter release and inhibition/clamping of spontaneous synaptic vesicle fusion. Here we report a new cpx mutant in Drosophila melanogaster, cpx1257, revealing spatially defined and separable pools of CPX which make distinct contributions to the activation and clamping functions. In cpx1257, lack of only the last C-terminal amino acid of CPX is predicted to disrupt prenylation and membrane targeting of CPX. Immunocytochemical analysis established localization of wild-type CPX to active zone (AZ) regions containing neurotransmitter release sites as well as broader presynaptic membrane compartments including synaptic vesicles. Parallel biochemical studies confirmed CPX membrane association and demonstrated robust binding interactions of CPX with all three SNAREs. This is in contrast to the cpx1257 mutant, in which AZ localization of CPX persists but general membrane localization and, surprisingly, the bulk of CPX-SNARE protein interactions are abolished. Furthermore, electrophysiological analysis of neuromuscular synapses revealed interesting differences between cpx1257 and a cpx null mutant. The cpx null exhibited a marked decrease in the EPSC amplitude, slowed EPSC rise and decay times and an increased mEPSC frequency with respect to wild-type. In contrast, cpx1257 exhibited a wild-type EPSC with an increased mEPSC frequency and thus a selective failure to clamp spontaneous release. These results indicate that spatially distinct and separable interactions of CPX with presynaptic membranes and SNARE proteins mediate separable activation and clamping functions of CPX in neurotransmitter release.
Neurotransmitter release; Farnesylation; Neuromuscular; SNARE; DLM
Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.
Chromogranin B (CgB); Inositol 1,4,5 trisphosphate receptor (InsP3R); Intracellular calcium signaling; Multiple Sclerosis (MS)
Cellular viability depends upon the well-orchestrated functions carried out by numerous protein-coding and non-coding RNAs, as well as RNA-binding proteins. During the last decade, it has become increasingly evident that abnormalities in RNA processing represent a common feature among many neurodegenerative diseases. In “RNAopathies”, which include diseases caused by non-coding repeat expansions, RNAs exert toxicity via diverse mechanisms: RNA foci formation, bidirectional transcription, and the production of toxic RNAs and proteins by repeat associated non-ATG translation. The mechanisms of toxicity in “RNA-binding proteinopathies”, diseases in which RNA-binding proteins like TDP-43 and FUS play a prominent role, have yet to be fully elucidated. Nonetheless, both loss of function of the RNA binding protein, and a toxic gain of function resulting from its aggregation, are thought to be involved in disease pathogenesis. As part of the special issue on RNA and Splicing Regulation in Neurodegeneration, this review intends to explore the diverse RNA-related mechanisms contributing to neurodegeneration, with a special emphasis on findings emerging from animal models.
RNA processing; Bidirectional transcription; RAN translation; RNA foci; Neurodegenerative diseases; Mouse models
Neuronal connectivity and synaptic remodeling are fundamental substrates for higher brain functions. Understanding their dynamics in the mammalian allocortex emerges as a critical step to tackle the cellular basis of cognitive decline that occurs during normal aging and in neurodegenerative disorders. In this work we have designed a novel approach to assess alterations in the dynamics of functional and structural connectivity elicited by chronic cell-autonomous overexpression of the human amyloid precursor protein (hAPP). We have taken advantage of the fact that the hippocampus continuously generates new dentate granule cells (GCs) to probe morphofunctional development of GCs expressing different variants of hAPP in a healthy background. hAPP was expressed together with a fluorescent reporter in neural progenitor cells of the dentate gyrus of juvenile mice by retroviral delivery. Neuronal progeny was analyzed several days post infection (dpi). Amyloidogenic cleavage products of hAPP such as the β-C terminal fragment (β-CTF) induced a substantial reduction in glutamatergic connectivity at 21 dpi, at which time new GCs undergo active growth and synaptogenesis. Interestingly, this effect was transient, since the strength of glutamatergic inputs was normal by 35 dpi. This delay in glutamatergic synaptogenesis was paralleled by a decrease in dendritic length with no changes in spine density, consistent with a protracted dendritic development without alterations in synapse formation. Finally, similar defects in newborn GC development were observed by overexpression of α-CTF, a non-amyloidogenic cleavage product of hAPP. These results indicate that hAPP can elicit protracted dendritic development independently of the amyloidogenic processing pathway.
Synaptogenesis; Neuronal maturation; Neurodegeneration; Dentate gyrus; Synaptic transmission
C-terminal binding proteins (CtBPs) are transcriptional co-repressors that are subject to proteasome-dependent downregulation during apoptosis. Alternative mechanisms that regulate CtBP expression are currently under investigation and the role of CtBPs in neuronal survival is largely unexplored. Here, we show that CtBPs are downregulated in cerebellar granule neurons (CGNs) induced to undergo apoptosis by a variety of stressors. Moreover, antisense-mediated downregulation of CtBP1 is sufficient to cause CGN apoptosis. Similarly, the CtBP inhibitor, 4-methylthio-2-oxobutyric acid, induces expression of the CtBP target Noxa and causes actinomycin-sensitive CGN apoptosis. Unexpectedly, we found that the mechanism of CtBP downregulation in CGNs undergoing apoptosis varies in a stimulus-specific manner involving either the proteasome or caspases. In the case of CGNs deprived of depolarizing potassium (5K apoptotic condition), caspases appear to play a dominant role in CtBP downregulation. However, incubation in 5K does not enhance the kinetics of CtBP1 degradation and recombinant CtBP1 is not cleaved in vitro by caspase-3. In addition, 5K has no significant effect on CtBP transcript expression. Finally, mouse embryonic stem cells display caspase-dependent downregulation of CtBP1 following exposure to staurosporine, an effect that is not observed in DGCR8 knockout cells which are deficient in miRNA processing. These data identify caspase-dependent downregulation of CtBPs as an alternative mechanism to the proteasome for regulation of these transcriptional co-repressors in neurons undergoing apoptosis. Moreover, caspases appear to regulate CtBP expression indirectly, at a post-transcriptional level, and via a mechanism that is dependent upon miRNA processing. We conclude that CtBPs are essential pro-survival proteins in neurons and their downregulation contributes significantly to neuronal apoptosis via the de-repression of pro-apoptotic genes.
C-terminal binding proteins (CtBPs); caspase-3; miRNA; neuronal apoptosis
Fragile X syndrome (FXS) is caused by lack of expression of fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. In many cases FXS is associated with abnormalities in CNS myelination. Although FMRP is expressed in oligodendrocyte progenitor cells and immature oligodendrocytes (OLGs) previous studies have not detected it in mature, myelin-producing OLGs. FMRP represses translation of myelin basic protein (MBP) RNA in vitro and is believed to prevent premature MBP expression in immature OLGs. Lack of FMRP in FXS could lead to premature myelination and/or myelin abnormalities. Here we show that FMRP is expressed in mature, MBP-positive OLGs of rodents and in MBP-positive human OLGs. We confirm that FMRP is a translational repressor of MBP mRNA in vitro, but at concentrations likely too high to be physiologically relevant in vivo. We find MBP expression in cultured Fmr1 KO OLGs to be similar to wild type, and expression of MBP and other myelin proteins in brain homogenates of the Fmr1 KO mouse to be similar to wild type before, during, and after the period of active myelination. These results suggest that while FMRP is expressed in mature OLGs, myelin abnormalities caused by lack of FMRP expression in FXS are not recapitulated in rodents.
On retinal ganglion cells (RGCs) transmit light encoded information to the brain and receive excitatory input from On cone bipolar cells (CBPs). The synaptic CBP input onto On RGCs is mediated by AMPA-type glutamate receptors (AMPARs) that include both those lacking a GluA2 subunit, and are therefore permeable to Ca2+, and those that possess at least one GluA2 subunit and are Ca2+-impermeable. We have previously demonstrated in electrophysiological studies that periods of low synaptic activity, brought about by housing animals in darkness, enhances the proportion of GluA2-lacking AMPARs at the On CBP-On RGC synapse by mobilizing surface GluA2 containing receptors into a receptor pool that rapidly cycles in and out of the membrane. AMPAR cycling induction by reduced synaptic activity takes several hours. This delay suggests that changes in expression of proteins which regulate AMPAR trafficking may mediate the altered mobility of GluA2 AMPARs in RGCs. In this study, we test the hypothesis that AMPAR trafficking proteins couple synaptic activity to AMPAR cycling in RGCs. Immunocytochemical and biochemical analysis confirmed that darkness decreases surface GluA2 in RGCs and changed the expression levels of three proteins associated with GluA2 trafficking. GRIP was decreased, while PICK1 and Arc were increased. Knockdown of GRIP with siRNA elevated constitutive AMPAR cycling, mimicking effects of reduced synaptic activity, while knockdown of PICK1 and ARC blocked increases in constitutive GluA2 trafficking. Our results support a role for correlated, activity-driven changes in multiple AMPAR trafficking proteins that modulate GluA2 cycling which can in turn affect synaptic AMPAR composition in RGCs.
AMPA receptor; retina; plasticity; PICK1; GRIP; Arc
SYD-2/liprin-α is a multi-domain protein that associates with and recruits multiple active zone molecules to form presynaptic specializations. Given SYD-2's critical role in synapse formation, its synaptogenic ability is likely tightly regulated. However, mechanisms that regulate SYD-2 function are poorly understood. In this study, we provide evidence that SYD-2's function may be regulated by interactions between its coiled-coil (CC) domains and sterile α-motif (SAM) domains. We show that the N-terminal CC domains are necessary and sufficient to assemble functional synapses while C-terminal SAM domains are not, suggesting that the CC domains are responsible for the synaptogenic activity of SYD-2. Surprisingly, syd-2 alleles with single amino acid mutations in the SAM domain show strong loss of function phenotypes, suggesting that SAM domains also play an important role in SYD-2's function. A previously characterized syd-2 gain-of-function mutation within the CC domains is epistatic to the loss-of-function mutations in the SAM domain. In addition, yeast two-hybrid analysis showed interactions between the CC and SAM domains. Thus, the data is consistent with a model where the SAM domains regulate the CC domain-dependent synaptogenic activity of SYD-2. Taken together, our study provides new mechanistic insights into how SYD-2's activity may be modulated to regulate synapse formation during development.
Synapse formation; Negative regulation; SYD-2; Liprin-α; Structure–function