Sensory information is often mapped systematically in the brain with neighboring neurons responding to similar stimulus features. The olfactory system represents chemical information as spatial and temporal activity patterns across glomeruli in the olfactory bulb. However, the degree to which chemical features are mapped systematically in the glomerular array has remained controversial. Here, we test the hypothesis that the dual roles of odorant receptors, in axon guidance and odor detection, can serve as a mechanism to map olfactory inputs with respect to their function. We compared the relationship between response specificity and glomerular formation in genetically-defined olfactory sensory neurons expressing variant odorant receptors. We find that sensory neurons with the same odor response profile can be mapped to different regions of the bulb, and that neurons with different response profiles can be mapped to the same glomeruli. Our data demonstrate that the two functions of odorant receptors can be uncoupled, indicating that the mechanisms that map olfactory sensory inputs to glomeruli do so without regard to stimulus specificity.
Olfactory; odorant receptors; mapping; glomeruli; olfactory sensory neuron; electrophysiology
Neuroligins are postsynaptic neural cell adhesion molecules that mediate synaptic maturation and function in vertebrates and invertebrates, but their mechanisms of action and regulation are not well understood. At the Drosophila larval neuromuscular junction (NMJ), previous analysis demonstrated a requirement for Drosophila neuroligin 1 (dnlg1) in synaptic growth and maturation. The goal of the present study was to better understand the effects and mechanisms of loss-of-function and overexpression of dnlg1 on synapse size and function, and to identify signaling pathways that control dnlg1 expression. Consistent with a reduced synapse size, evoked excitatory junctional currents (EJCs) were diminished in dnlg1 mutants but displayed normal Ca2+ sensitivity and short-term plasticity. However, postsynaptic function was also perturbed, in that glutamate receptor staining and the distribution of amplitudes of miniature excitatory junctional currents (mEJCs) were abnormal in mutants. All the above phenotypes were rescued by a genomic transgene. Overexpression of dnlg1 in muscle resulted in synaptic overgrowth, but reduced the amplitudes of EJCs and mEJCs. Overgrowth and reduced EJC amplitude required dnrx1 function, suggesting that increased dnlg1/dnrx1 signaling attenuates synaptic transmission and regulates growth through a retrograde mechanism. In contrast, reduced mEJC amplitude was independent of dnrx1. Synaptic overgrowth, triggered by neuronal hyperactivity, absence of the E3 ubiquitin ligase highwire, and increased phosphoinositide-3-kinase (PI3K) signaling in motor neurons reduced synaptic DNlg1 levels. Likewise, postsynaptic attenuation of PI3K, which increases synaptic strength, was associated with reduced DNlg1 levels. These observations suggest that activity and PI3K signaling pathways modulate growth and synaptic transmission through dnlg1-dependent mechanisms.
Drosophila; neuroligin; neurexin; synapse; activity; phosphoinositide-3-kinase
At the mammalian neuromuscular junction (NMJ), the CT (cytotoxic T cell) carbohydrate antigen [GalNAcβ1,4[Neu5Ac/Gcα2,3]Galβ1,4GlcNAc-] is a uniquely synaptic cell surface carbohydrate present in both the presynaptic and postsynaptic membrane. Here we show that Galgt1, which synthesizes the β1,4GalNAc linkage of the CT carbohydrate on gangliosides, is required for presynaptic expression of the CT carbohydrate at the NMJ, while Galgt2, which can synthesize the β1,4GalNAc of the CT carbohydrate on glycoproteins, is required for postsynaptic expression. Proper postsynaptic localization of the CT carbohydrate also required muscle expression of dystroglycan, a known muscle substrate for Galgt2. Transgenic overexpression of Galgt2 in skeletal myofibers altered the expression of synaptic muscle proteins and altered neuromuscular topography, which was partially NCAM-dependent, while an increase in postsynaptic AChR-rich domains was observed in both neuron- and skeletal muscle-specific Galgt2 transgenic mice. By contrast, overexpression of Galgt1 in muscle did not allow for increased expression of CT carbohydrate on the sarcolemmal membrane and instead caused muscle histopathology. Loss of Galgt2 increased intracellular accumulation of acetylcholine receptors and acetylcholinesterase within skeletal myofibers, suggesting an additional role for Galgt2 in neuromuscular stability. These experiments demonstrate that Galgt1 and Galgt2 contribute in distinct ways to the expression and function of synaptic βGalNAc-containing carbohydrates at the NMJ.
Neuromuscular junction; synapse; ganglioside; dystroglycan; muscular dystrophy; acetylcholine receptor
One of the most common types of epilepsy in adults is temporal lobe epilepsy. Temporal lobe epilepsy is often resistant to pharmacological treatment, requiring urgent understanding of its molecular and cellular mechanisms. It is generally accepted that an imbalance between excitatory and inhibitory inputs is related to epileptogenesis. We have recently identified that fibroblast growth factor (FGF) 7 is critical for inhibitory synapse formation in the developing hippocampus. Remarkably, FGF7 knockout mice are prone to epileptic seizures induced by chemical kindling (Terauchi et al., 2010). Here we show that FGF7 knockout mice exhibit epileptogenesis-related changes in the hippocampus even without kindling induction. FGF7 knockout mice show mossy fiber sprouting and enhanced dentate neurogenesis by 2 months of age, without apparent spontaneous seizures. These results suggest that FGF7-deficiency impairs inhibitory synapse formation, which results in mossy fiber sprouting and enhanced neurogenesis during development, making FGF7 knockout mice vulnerable to epilepsy.
Hippocampus; Fibroblast growth factor 7; Inhibitory synapse formation; Mossy fiber sprouting; Neurogenesis; Epilepsy
Subcellular localization of protein synthesis provides a means to regulate the protein composition in far reaches of a cell. This localized protein synthesis gives neuronal processes autonomy to rapidly respond to extracellular stimuli. Locally synthesized axonal proteins enable neurons to respond to guidance cues and can help to initiate regeneration after injury. Most studies of axonal mRNA translation have concentrated on cytoplasmic proteins. While ultrastructural studies suggest that axons do not have rough endoplasmic reticulum or Golgi apparatus, mRNAs for transmembrane and secreted proteins localize to axons. Here, we show that growing axons with protein synthetic activity contain ER and Golgi components needed for classical protein synthesis and secretion. Isolated axons have the capacity to traffic locally synthesized proteins into secretory pathways and inhibition of Golgi function attenuates translation-dependent axonal growth responses. Finally, the capacity for secreting locally synthesized proteins in axons appears to be increased by injury.
Axonal protein synthesis; Secretory pathway; Rough endoplasmic reticulum; Golgi apparatus; mRNA localization; Axon guidance
Glucocorticoid receptor (GR) ontogeny and distribution in postnatal rat brain have been demonstrated, but onset and distribution of GR gene expression during fetal life has not been reported. This study focuses on the distribution of GR-mRNA in the fetal and postnatal rat fore-brain, with emphasis on hypothalamic and limbic structures. Time pregnant rats were decapitated at 8:30–9:30 AM on Gestational Days 14 (F14), F16, F17, F18, and F19. Postnatally, rats were sacrificed on Days 1, 4, 6,10, and 16. Cryostat sections were subjected to in situ hybridization, using a cRNA probe directed to the GR-mRNA. GR-mRNA was detectible in the hippocampo-septal formation as early as F14. By F16, GR gene expression was evident in the hypothalamic paraventricular nucleus (PVN) as well. During late gestation (F17–F19), GR-mRNA was localized also in the thalamus, hippocampus, amygdala, and discrete cortical regions. Postnatally, GR-mRNA abundance was high in the PVN, CA1/CA2 hippocampal field, piriform cortex and dorsal endopiriform nucleus, specific amygdaloid nuclei, and the suprachiasmatic nucleus. In PVN, GR-mRNA was present prior to the onset of CRH gene expression (F17), which may suggest a role for GR in neuronal differentiation.
The neocortex represents the brain region that has undergone a major increase in its relative size during the course of mammalian evolution. The larger cortex results from a corresponding increase in progenitor cell number. The progenitors giving rise to neocortex are located in the ventricular zone of the dorsal telencephalon and highly express Lhx2, a LIM-homeodomain transcription factor. The neocortex fails to form in the Lhx2 constitutive knockout, indicating a role for Lhx2 in corticogenesis, but this outcome, and mid-embryonic lethality, requires use of conditional strategies for further study. Therefore, to explore Lhx2 function in neocortical progenitors, we generated mice with Lhx2 conditionally deleted from cortical progenitors at the onset of neurogenesis. We find that Lhx2 is critical for maintaining the proliferative state of neocortical progenitors during corticogenesis. In the conditional knockouts, the neocortex is formed but is significantly smaller than wild type. We find that deletion of Lhx2 leads to significantly decreased numbers of cortical progenitors and premature neuronal differentiation. A likely mechanism is indicated by our findings that Lhx2 is required for the expression of Hes1 in cortical progenitors, a key effector in the Notch signaling pathway that maintains the proliferative progenitor state. We conclude that Lhx2 regulates the balance between proliferation and differentiation in cortical progenitors and through this mechanism controls cortical size.
cortical neurogenesis; Lhx2; Hes1
Huntington’s disease (HD) is a fatal, autosomal dominant neurodegenerative disorder caused by an expanded trinucleotide (CAG) repeat in exon 1 of the huntingtin gene (Htt). This expansion creates a toxic polyglutamine tract in the huntingtin protein (HTT). Currently, there is no treatment for either the progression or prevention of the disease. RNA interference (RNAi) technology has shown promise in transgenic mouse models of HD by reducing expression of mutant HTT and slowing disease progression. The advancement of RNAi therapies to human clinical trials is hampered by problems delivering RNAi to affected neurons in a robust and sustainable manner. Mesenchymal stem cells (MSC) have demonstrated a strong safety profile in both completed and numerous ongoing clinical trials. MSC exhibit a number of innate therapeutic effects, such as immune system modulation, homing to injury, and cytokine release into damaged microenvironments. The ability of MSC to transfer larger molecules and even organelles suggested their potential usefulness as delivery vehicles for therapeutic RNA inhibition. In a series of model systems we have found evidence that MSC can transfer RNAi targeting both reporter genes and mutant huntingtin in neural cell lines. MSC expressing shRNA antisense to GFP were found to decrease expression of GFP in SH-SY5Y cells after co-culture when assayed by flow cytometry. Additionally MSC expressing shRNA antisense to HTT were able to decrease levels of mutant HTT expressed in both U87 and SH-SY5Y target cells when assayed by Western blot and densitometry. These results are encouraging for expanding the therapeutic abilities of both RNAi and MSC for future treatments of Huntington’s disease.
Mesenchymal stem cell; Huntington’s disease; RNAi; Cellular therapy
Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision.
2′OMePS, 2′-O-methyl phosphorothioate; 2′MOE-PS, 2′-O-methoxyethyl phosphorothioate; AON, antisense oligonucleotide; CPP, cell-penetrating peptide; DMD, Duchenne muscular dystrophy; PMO, phosphorodiamidate morpholino; PPMO, peptide-conjugated phosphorodiamidate morpholino; SMA, spinal muscular atrophy; DMD; SMA; Antisense; Splicing; Exon skipping; Exon inclusion
Dendritic spines are major sites of excitatory synaptic transmission and changes in their numbers and morphology have been associated with neurodevelopmental and neurodegenerative disorders. Brain-derived Neurotrophic Factor (BDNF) is a secreted growth factor that influences hippocampal, striatal and neocortical pyramidal neuron dendritic spine density. However, the mechanisms by which BDNF regulates dendritic spines and how BDNF interacts with other regulators of spines remain unclear. We propose that one mechanism by which BDNF promotes dendritic spine formation is through an interaction with Wnt signaling. Here, we show that Wnt signaling inhibition in cultured cortical neurons disrupts dendritic spine development, reduces dendritic arbor size and complexity, and blocks BDNF-induced dendritic spine formation and maturation. Additionally, we show that BDNF regulates expression of Wnt2, and that Wnt2 is sufficient to promote cortical dendrite growth and dendritic spine formation. Together, these data suggest that BDNF and Wnt signaling cooperatively regulate dendritic spine formation.
Wnt signaling; BDNF; Dendritic spine; Dendrite branching; Synapse
The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration.
CLIP; Neurodegeneration; FUS; Muscleblind; RNA binding proteins
Odorant receptor (OR) genes constitute with 1200 members the largest gene family in the mouse genome. A mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and from one allele. The cell bodies of OSNs that express a given OR gene display a mosaic pattern within a particular region of the main olfactory epithelium. The mechanisms and cis-acting DNA elements that regulate the expression of one OR gene per OSN – OR gene choice – remain poorly understood. Here, we describe a reporter assay to identify minimal promoters for OR genes in transgenic mice, which are produced by the conventional method of pronuclear injection of DNA. The promoter transgenes are devoid of an OR coding sequence, and instead drive expression of the axonal marker tau-βgalactosidase. For four mouse OR genes (M71, M72, MOR23, and P3) and one human OR gene (hM72), a mosaic, OSN-specific pattern of reporter expression can be obtained in transgenic mice with contiguous DNA segments of only ∼300 bp that are centered around the transcription start site (TSS). The ∼150 bp region upstream of the TSS contains three conserved sequence motifs, including homeodomain (HD) binding sites. Such HD binding sites are also present in the H and P elements, DNA sequences that are known to strongly influence OR gene expression. When a 19mer encompassing a HD binding site from the P element is multimerized nine times and added upstream of a MOR23 minigene that contains the MOR23 coding region, we observe a dramatic increase in the number of transgene-expressing founders and lines and in the number of labeled OSNs. By contrast, a nine-times multimerized 19mer with a mutant HD binding site does not have these effects. We hypothesize that HD binding sites in the H and P elements and in OR promoters modulate the probability of OR gene choice.
The amyloid precursor protein (APP) plays a critical role in Alzheimer’s disease (AD) pathogenesis. APP is proteolytically cleaved by β- and γ-secretases to generate the amyloid β-protein (Aβ), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aβ peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP−/−) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP−/− mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP−/− mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP−/− mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15 months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to Alzheimer’s changes independent of Aβ-mediated synaptic toxicity.
Alzheimer’s disease; amyloid precursor protein; knock-out mice; extracellular domain; soluble amyloid β; synapse
The Bcl-2 family is responsible for regulating cell death pathways in neurons during development, after injury and in disease. The activation of the pro-death family member BAX is often the final step before cell death in neurons. Pro-survival family members such as BCL-X (BCL2L1) act to inhibit BAX activation. Overexpression studies have suggested that BCL-X could play an important physiological role in mediating neuronal viability. Loss-of-function studies performed in vivo have implicated BCL-X as a mediator of neuronal survival during the early stages of neurodevelopment. To assess whether BCL-X is needed to promote the survival of neurons in the central nervous system throughout life, Bcl-x was conditionally removed from the optic cup or throughout the adult mouse. During development BCL-X was required for the survival of differentiating retinal ganglion cells (RGCs) leading up to their normal window of developmental death. Despite its expression in adult RGCs, BCL-X was not required for maintaining RGC viability in adult retinas. However, the loss of BCL-X in adult RGCs did significantly increase the rate of death of RGCs after axonal injury. Thus, in developing and injured RGCs there appears to be an active cell survival program preventing neuronal death.
Purkinje cell (PC) dysfunction or death have been implicated in a number of disorders including ataxia, autism and multiple sclerosis. Plasma membrane calcium ATPase 2 (PMCA2), an important calcium (Ca2+) extrusion pump that interacts with synaptic signaling complexes, is most abundantly expressed in PCs compared to other neurons. Using the PMCA2 heterozygous mouse as a model, we investigated whether a reduction in PMCA2 levels affects PC function. We focused on Ca2+ signaling and the expression of glutamate receptors which play a key role in PC function including synaptic plasticity. We found that the amplitude of depolarization and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor (AMPAR)-mediated Ca2+ transients is significantly higher in cultured PMCA2+/− PCs than in PMCA2+/+ PCs. This is due to increased Ca2+ influx, since P/Q type voltage-gated Ca2+ channel (VGCC) expression was more pronounced in PCs and cerebella of PMCA2+/− mice and VGCC blockade prevented the elevation in amplitude. Neuronal nitric oxide synthase (nNOS) activity was higher in PMCA2+/−cerebella and inhibition of nNOS or the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway, which mediates nitric oxide (NO) signaling, reduced the amplitude of Ca2+ transients in PMCA2+/− PCs, in vitro. In addition, there was an age-dependent decrease in metabotropic glutamate receptor 1 (mGluR1) and AMPA receptor subunit GluR2/3 transcript and protein levels at 8 weeks of age. These changes were followed by PC loss in the 20-week-old PMCA2+/− mice. Our studies highlight the importance of PMCA2 in Ca2+ signaling, glutamate receptor expression and survival of Purkinje cells.
Ataxia; autism; neurodegeneration; glutamate; AMPA; calcium channel; ion pump; nitric oxide
The N-methyl-D-aspartate receptor (NMDAR) is critically involved in learning and memory, neuronal survival, as well as neuroexcitotoxicity and seizures. We hypothesize that even mild reductions in the numbers of hippocampal NMDARs could impair learning and memory, whereas increasing receptor activity would facilitate learning but reduce seizure threshold. We developed novel gene transfer strategies assisted by an adeno-associated viral vector 1/2 to bi-directionally modulate expression levels of the NR1 protein in rat hippocampus. Functional consequences of the altered NR1 expression were examined in the acute seizure model, and on normal processes of fear memory and neurogenesis. We found that knocking down NR1 protected against seizures at the expense of impaired learning, as predicted. Paradoxically, NR1 overexpression not only increased fear memory and neurogenesis, but also delayed onset of more severe seizures. In conclusion, the observed consequences of NR1 knockdown and overexpression underscore NMDAR requirement for neuronal plasticity, and are in agreement with its dichotomous functions.
Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axons terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.
development; glomeruli; olfactory sensory neuron; synaptosomes; tetraspanin; tetraspanin-enriched microdomain
MicroRNAs (miRNAs) are critical regulators of nervous system function, and in vivo knockout studies have demonstrated that miRNAs are necessary for multiple aspects of neuronal development and survival. However, the requirements of miRNA biogenesis in the formation and function of synapses in the cerebral cortex are only minimally understood. Here, we have generated and characterized a mouse line with a conditional neuronal deletion of Dgcr8, a miRNA biogenesis protein predicted to process miRNAs exclusively. Loss of Dgcr8 in pyramidal neurons of the cortex results in a non-cell-autonomous reduction in parvalbumin interneurons in the prefrontal cortex, accompanied by a severe deficit in inhibitory synaptic transmission and a corresponding reduction of inhibitory synapses. Together, these results suggest a vital role for miRNAs in governing essential aspects of inhibitory transmission and interneuron development in the mammalian nervous system. These results may be relevant to human diseases such as schizophrenia, where both altered Dgcr8 levels as well as aberrant inhibitory transmission in the prefrontal cortex have been postulated to contribute to the pathophysiology of the disease.
microRNA; Dgcr8; inhibitory synaptic transmission; parvalbumin interneurons; prefrontal cortex
Retinal ganglion cells (RGCs) project their long axons, composing the optic nerve, to the brain, transmitting the visual information gathered by the retina, ultimately leading to formed vision in the visual cortex. The RGC cellular system, representing the anterior part of the visual pathway, is vulnerable to mitochondrial dysfunction and optic atrophy is a very frequent feature of mitochondrial and neurodegenerative diseases. The start of the molecular era of mitochondrial medicine, the year 1988, was marked by the identification of a maternally inherited form of optic atrophy, Leber's hereditary optic neuropathy, as the first disease due to mitochondrial DNA point mutations. The field of mitochondrial medicine has expanded enormously over the last two decades and many neurodegenerative diseases are now known to have a primary mitochondrial etiology or mitochondrial dysfunction plays a relevant role in their pathogenic mechanism. Recent technical advancements in neuro-ophthalmology, such as optical coherence tomography, prompted a still ongoing systematic re-investigation of retinal and optic nerve involvement in neurodegenerative disorders. In addition to inherited optic neuropathies, such as Leber's hereditary optic neuropathy and dominant optic atrophy, and in addition to the syndromic mitochondrial encephalomyopathies or mitochondrial neurodegenerative disorders such as some spinocerebellar ataxias or familial spastic paraparesis and other disorders, we draw attention to the involvement of the optic nerve in classic age-related neurodegenerative disorders such as Parkinson and Alzheimer disease. We here provide an overview of optic nerve pathology in these different clinical settings, and we review the possible mechanisms involved in the pathogenesis of optic atrophy. This may be a model of general value for the field of neurodegeneration. This article is part of a Special Issue entitled ‘Mitochondrial function and dysfunction in neurodegeneration’.
Mitochondrial disease; Optic atrophy; Mitochondrial functions; LHON; DOA; Retinal ganglion cells
In the developing neocortex, brain-derived neurotrophic factor (BDNF) exerts a trophic activity to increase the expression and channel activity of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor subunits. Here, we demonstrate that the epidermal growth factor (EGF) receptor (ErbB1) ligands exert the opposite biological activity in cultured neocortical neurons. Subchronic stimulation of ErbB1 with transforming growth factor α (TGFα), EGF, or heparin-binding EGF (HB-EGF) down-regulated protein expression of the GluR1 AMPA receptor subunit in cultured neocortical neurons. In agreement, TGFα treatment decreased the Bmax of [3H] AMPA binding and GluR1 mRNA levels. Immunocytochemistry revealed that the decrease in GluR1 was most pronounced in multipolar GABAergic neurons. To examine the physiological consequences, we recorded AMPA-evoked currents as well as miniature excitatory postsynaptic currents in morphologically identified putative GABAergic neurons in culture. Subchronic TGFα treatment decreased AMPA-triggered currents as well as the amplitude and frequency of miniature excitatory postsynaptic currents. An ErbB1 tyrosine kinase inhibitor, PD153035, inhibited the TGFα effect. Moreover, TGFα counteracted the neurotrophic activity of BDNF on AMPA receptor expression. Co-application of TGFα with BDNF blocked the BDNF-triggered up-regulation of AMPA receptor expression and currents. These observations reveal a negative regulatory activity of the ErbB1 ligand, TGFα, which reduces the input sensitivity of cortical GABAergic neurons to attenuate their inhibitory function.
EGF; ErbB1; Her1; GluR1; BDNF; GABA; Neocortex
Semaphorins are a large family of secreted and transmembrane glycoproteins. Sema III, a member of the Class III semaphorins is a potent chemorepulsive signal for subsets of sensory axons and steers them away from tissue regions with high levels of expression. Previous studies in mutant mice lacking sema III gene showed various neural and nonneural abnormalities. In this study, we focused on the developing trigeminal pathway of sema III knockout mice. We show that the peripheral and central trigeminal projections are impaired during initial pathway formation when they develop into distinct nerves or tracts. These axons defasciculate and compromise the normal bundling of nerves and restricted alignment of the central tract. In contrast to trigeminal projections, thalamocortical projections to the barrel cortex appear normal. Furthermore, sema III receptor, neuropilin, is expressed during a short period of development when the tract is laid down, but not in the developing thalamocortical pathway. Peripherally, trigeminal axons express neuropilin for longer duration than their central counterparts. In spite of projection errors, whisker follicle innervation appears normal and whisker-related patterns form in the trigeminal nuclei and upstream thalamic and cortical centers. Our observations suggest that sema III plays a limited role during restriction of developing trigeminal axons to proper pathways and tracts. Other molecular and cellular mechanisms must act in concert with semaphorins in ensuring target recognition, topographic order of projections, and patterning of neural connections.
Dipeptidyl peptidase-like proteins (DPLs) and Kv-channel interacting proteins (KChIPs) join Kv4 pore-forming subunits to form multi-protein complexes that underlie subthreshold A-type currents (ISA) in neuronal somatodendritic compartments. Here, we characterize the functional effects and brain distributions of N-terminal variants belonging to the DPL dipeptidyl peptidase 10 (DPP10). In the Kv4.2+KChIP3+DPP10 channel complex, all DPP10 variants accelerate channel gating kinetics; however, the splice variant DPP10a produces uniquely fast inactivation kinetics that accelerates with increasing depolarization. This DPP10a-specific inactivation dominates in co-expression studies with KChIP4a and other DPP10 isoforms. Real-time qRT-PCR and in situ hybridization analyses reveal differential expression of DPP10 variants in rat brain. DPP10a transcripts are prominently expressed in the cortex, whereas DPP10c and DPP10d mRNAs exhibit more diffuse distributions. Our results suggest that DPP10a underlies rapid inactivation of cortical ISA, and the regulation of isoform expression may contribute to the variable inactivation properties of ISA across different brain regions.
Axonal growth from both intact and severed fibers is limited after adult mammalian CNS injury. Myelin proteins contribute to inhibition of axonal growth. Semaphorin6A protein inhibits the extension of developing axons and is highly expressed in adult oligodendrocytes. This expression pattern suggests that a developmental axon guidance cue contributes to the restriction of adult CNS growth. Here, we assessed the role of a Sema6A receptor, PlexinA2, in recovery from adult trauma. Adult sensory neuron inhibition by Sema6A requires PlexinA2, with complete protection in PlexinA2-/- cultures. Mice lacking another myelin inhibitor receptor, NgR1, are known to exhibit greater axonal sprouting and functional recovery after lesions of the corticospinal tract at the medullary pyramid, so we investigated PlexinA2 in this lesion. Without injury, the corticofugal projection into the cervical spinal cord is normal in adult PlexinA2 null mice. After unilateral pyramidotomy, unlesioned PlexinA2-/- corticospinal fibers sprout across the midline to innervate the contralateral gray matter of the spinal cord to a significantly greater extent than do fibers in wild type mice. Sprouted fibers display frequent synaptophysin-positive synaptic puncta. The increased axonal growth in PlexinA2-/- mice after injury is accompanied by improved behavioral recovery in a pellet retrieval task using the impaired forelimb, and in a tape removal task. Thus, PlexinA2, as a receptor for oligodendrocyte-derived Sema6A and for secreted class 3 Semaphorins, plays a role in limiting adult axon growth and recovery after trauma.
The vertebrate neural retina contains seven major cell types, which arise from a common multipotent progenitor pool. During neurogenesis, these cells stop cycling, commit to a single fate, and differentiate. The mechanism and order of these steps remain unclear. The first-born type of retinal neurons, ganglion cells (RGCs), develop through the actions of Math5 (Atoh7), Brn3b (Pou4f2) and Islet1 (Isl1) factors, whereas inhibitory amacrine and horizontal precursors require Ptf1a for differentiation. We have examined the link between these markers, and the timing of their expression during the terminal cell cycle, by nucleoside pulse-chase analysis in the mouse retina. We show that G2 phase lasts 1–2 hours at embryonic (E) 13.5 and E15.5 stages. Surprisingly, we found that cells expressing Brn3b and/or Isl1 were frequently co-labeled with EdU after a short chase (<1 hr) in early embryos (E15), Brn3b and Isl1 were exclusively expressed in post-mitotic cells, even as new RGCs are still generated. In contrast, Ptf1a and amacrine marker AP2α were detected only after terminal mitosis, at all developmental stages. Using a retroviral tracer in embryonic retinal explants (E12–E13), we identified two-cell clones containing paired ganglion cells, consistent with RGC fate commitment prior to terminal mitosis. Thus, although cell cycle exit and fate determination are temporally correlated during retinal neurogenesis, the order of these events varies according to developmental stage and final cell type.
retinal ganglion cell (RGC); clonal analysis; optic nerve; retinal cell cycle; cell fate specification; Brn3b; Pou4f2; Isl1; cell cycle exit; Ptf1a
Injury to the central nervous system (CNS) can result in lifelong loss of function due in part to the regenerative failure of CNS neurons. Inhibitory proteins derived from myelin and the astroglial scar are major barriers for the successful regeneration of injured CNS neurons. Previously, we described the identification of a novel compound, F05, which promotes neurite growth from neurons challenged with inhibitory substrates in vitro, and promotes axonal regeneration in vivo (Usher et al., 2010). To identify additional regeneration-promoting compounds, we used F05-induced gene expression profiles to query the Broad Institute Connectivity Map, a gene expression database of cells treated with >1,300 compounds. Despite no shared chemical similarity, F05-induced changes in gene expression were remarkably similar to those seen with a group of piperazine phenothiazine antipsychotics (PhAPs). In contrast to antipsychotics of other structural classes, PhAPs promoted neurite growth of CNS neurons challenged with two different glial derived inhibitory substrates. Our pharmacological studies suggest a mechanism whereby PhAPs promote growth through antagonism of calmodulin signaling, independent of dopamine receptor antagonism. These findings shed light on mechanisms underlying neurite-inhibitory signaling, and suggest that clinically approved antipsychotic compounds may be repurposed for use in CNS injured patients.
piperazine; phenothiazine; antipsychotics; chondroitin sulfate proteoglycans; myelin; regeneration; trifluoperazine; prochlorperazine