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- Calpain activation and CaMKIV reduction in spinal cords from hSOD1G93A mouse model. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jul 22.
Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.
- Multiple EphB receptors mediate dorsal-ventral retinotopic mapping via similar bi-functional responses to ephrin-B1. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jul 19.
The projection from the retina to the superior colliculus in mice is organized in a retinotopic map that develops through the formation and guidance of interstitial branches extended by retinal ganglion cell axons. Bidirectional branch guidance along the lateral-medial collicular axis is critical to mapping the dorsal-ventral retinal axis. EphB receptor tyrosine kinases expressed in an overall low to high dorsal-ventral retinal gradient have been implicated in this mapping in response to the graded low to high lateral-medial expression of a ligand, ephrin-B1, in the superior colliculus. However, the relative contributions of EphBs and ephrin-B1 are not well understood. We examined EphB1, EphB2, and EphB3 mutant mice and find that each has ectopic arborizations of retinal axon branches lateral to their appropriate termination zone, with no qualitative differences in aberrant mapping, suggesting a similar role for each EphB. However, the frequency of cases with map defects progressively rises in compound EphB mutants coincident with the number of EphB null alleles from one to five of the six total alleles indicating that EphB level is critical. We analyzed branch extension in vitro and find that dorsal branches, with low EphB levels, exhibit a negative response to ephrin-B1, whereas ventral branches, with high EphB levels, exhibit a positive response to ephrin-B1. Using EphB mutant retina, we show that both of these differential branch extension responses are dependent on EphB level. Our findings show a bifunctional action of ephrin-B1 regulated by EphB levels that can account for the bidirectional extension of interstitial branches required to establish a retinotopic map.
- LOTUS suppresses axon growth inhibition by blocking interaction between Nogo receptor-1 and all four types of its ligand. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jul 14.
Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and B lymphocyte stimulator (BLyS) commonly bind to Nogo receptor-1 (NgR1), leading to enormous restriction of functional recovery after damage to the adult central nervous system. Recently, we found that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated Nogo signaling. However, whether LOTUS exerts antagonism of NgR1 when bound by the other three ligands has not been determined. Overexpression of LOTUS together with NgR1 in COS7 cells blocked the binding of MAG, OMgp, and BLyS to NgR1. In cultured dorsal root ganglion neurons in which endogenous LOTUS is only weakly expressed, overexpression of LOTUS suppressed growth cone collapse and neurite outgrowth inhibition induced by these three NgR1 ligands. LOTUS suppressed NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons, which endogenously express LOTUS. Growth cone collapse was induced by NgR1 ligands in lotus-deficient mice. These data suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all four known NgR1 ligands, raising the possibility that LOTUS may protect neurons from NgR1-mediated axonal growth inhibition and thereby may be useful for promoting neuronal regeneration as a potent inhibitor of NgR1.
- Intracellular alpha-synuclein affects early maturation of primary oligodendrocyte progenitor cells. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jul 11.
Myelin loss is a widespread neuropathological hallmark of the atypical parkinsonian disorder multiple system atrophy (MSA). On a cellular level, MSA is characterized by alpha-synuclein (aSyn)-positive glial cytoplasmatic inclusions (GCIs) within mature oligodendrocytes leading to demyelination as well as axonal and neuronal loss. Oligodendrocyte progenitor cells (OPCs) represent a proliferative cell population distributed throughout the adult mammalian central nervous system. During remyelination, OPCs are recruited to sites of demyelination, differentiate, and finally replace dysfunctional mature oligodendrocytes. However, comprehensive studies investigating OPCs and remyelination processes in MSA are lacking. In the present study, we therefore investigate the effect of human aSyn (h-aSyn) on early primary rat OPC maturation. Upon lentiviral transduction, h-aSyn expressing OPCs exhibit fewer and shorter primary processes at the initiation of differentiation. Until day 4 of a 6day differentiation paradigm, h-aSyn expressing OPCs further show a severely delayed maturation evidenced by reduced myelin gene expression and increased levels of the progenitor marker platelet derived growth factor receptor-alpha (PDGFRα). Matching these results, OPCs that take up extracellular recombinant h-aSyn exhibit a similar delayed differentiation. In both experimental setups however, myelin gene expression is restored at day 6 of differentiation paralleled by decreased intracellular h-aSyn levels indicating a reverse correlation of h-aSyn and the differentiation potential of OPCs. Taken together, these findings suggest a tight link between the intracellular level of h-aSyn and maturation capacity of primary OPCs.
- Differential role of APP and APLPs for neuromuscular synaptic morphology and function. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jul 4.
The analysis of mouse models indicated that APP and the related APLPs are important for synapse formation and function. The synaptic role of APP is, however, complex due to partially overlapping functions within the gene family. APP/APLPs are proteolytically cleaved and have both adhesive and signaling properties. Mice lacking individual APP family members are viable, whereas APP/APLP2 and APLP1/APLP2 double knockout (DKO) mice die shortly after birth. Here, we analyzed the morphology of the neuromuscular junction (NMJ) of lethal APLP1/APLP2-DKO mice in comparison to lethal APP/APLP2-DKO mutants and viable single KO mice. We report that, surprisingly, the NMJ phenotype of APLP1/APLP2-DKO mice shows striking differences as compared to APP/APLP2-DKO mice. Unexpectedly, APLP1/APLP2-DKO mice exhibit normal endplate patterning and lack presynaptic nerve terminal sprouting. However, at the level of individual synapses we show that APLP1/APLP2-DKO mice exhibit reduced size of pre- and postsynaptic compartments and reduced co-localization. As APP/APLP2-DKO and APLP1/APLP2-DKO mice show similar penetrance of early postnatal lethality, this suggests that deficits at the level of individual synapses due to impaired synaptic apposition and/or deficits in transmitter release may cause lethality. Using an in vitro cell-adhesion assay, we observed that APP trans-dimerization is considerably less efficient than APLP2 trans-interaction. Thus, differences between APP/APLP2 and APP/APLP1 NMJ formation may be in part explained by differences in APP/APLP2 trans-dimerization properties. Collectively, our study further highlights the distinct and essential role of APLP2 at NMJ synapses that cannot be compensated by APP.
- Cortical parvalbumin GABAergic deficits with α7 nicotinic acetylcholine receptor deletion: Implications for schizophrenia. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (α7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of α7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the α7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. α7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, Glutamic Acid Decarboxylase 65/67 (GAD65/67) and the α1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of α7 nAChR null mice and α7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking α7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in α7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in α7 nAChR gene deletion models. This implicates crucial roles of α7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.
- Erythropoietin Attenuates Loss of Potassium Chloride Co-Transporters Following Prenatal Brain Injury. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Therapeutic agents that restore the inhibitory actions of γ-amino butyric acid (GABA) by modulating intracellular chloride concentrations will provide novel avenues to treat stroke, chronic pain, epilepsy, autism, neurodegenerative and cognitive disorders. During development upregulation of the potassium-chloride co-transporter KCC2, and the resultant switch from excitatory to inhibitory responses to GABA guides the formation of essential inhibitory circuits. Importantly, maturation of inhibitory mechanisms is also central to the development of excitatory circuits and proper balance between excitatory and inhibitory networks in the developing brain. Loss of KCC2 expression occurs in postmortem samples from human preterm infant brains with white matter lesions. Here we show late gestation brain injury in a rat model of extreme prematurity impairs the developmental upregulation of potassium chloride co-transporters during a critical postnatal period of circuit maturation in CA3 hippocampus by inducing a sustained loss of oligomeric KCC2 via a calpain-dependent mechanism. Further, administration of erythropoietin (EPO) in a clinically relevant postnatal dosing regimen following the prenatal injury protects the developing brain by reducing calpain activity, restoring oligomeric KCC2 expression and attenuating KCC2 fragmentation, thus providing the first report of a safe therapy to address deficits in KCC2 expression. Together, these data indicate it is possible to reverse abnormalities in KCC2 expression during the postnatal period, and potentially reverse deficits in inhibitory circuit formation central to cognitive impairment and epileptogenesis.
- The histone lysine demethylase Kdm6b is required for activity-dependent preconditioning of hippocampal neuronal survival. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Enzymes that regulate histone lysine methylation play important roles in neuronal differentiation, but little is known about their contributions to activity-regulated gene transcription in differentiated neurons. We characterized activity-regulated expression of lysine demethylases and lysine methyltransferases in the hippocampus of adult male mice following pilocarpine-induced seizure. Pilocarpine drove a 20-fold increase in mRNA encoding the histone H3 lysine27-specific demethylase Kdm6b selectively in granule neurons of the dentate gyrus, and this induction was recapitulated in cultured hippocampal neurons by bicuculline and 4-aminopyridine (Bic+4AP) stimulation of synaptic activity. Because activity-regulated gene expression is highly correlated with neuronal survival, we tested the requirement for Kdm6b expression in Bic+4AP induced preconditioning of neuronal survival. Prior exposure to Bic+4AP promoted neuronal survival in control neurons upon growth factor withdrawal, however this effect was ablated when we knocked down Kdm6b expression. Loss of Kdm6b did not disrupt activity-induced expression of most genes, including that of a gene set previously established to promote neuronal survival in this assay. However using bioinformatic analysis of RNA sequencing data, we discovered that Kdm6b knockdown neurons showed impaired inducibility of a discrete set of genes annotated for their function in inflammation. These data reveal a novel function for Kdm6b in activity-regulated neuronal survival, and they suggest that activity- and Kdm6b-dependent regulation of inflammatory gene pathways may serve as an adaptive pro-survival response to increased neuronal activity.
- Autophagy dysregulation in cell culture and animals models of Spinal Muscular Atrophy. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Abnormal autophagy has become a central thread linking neurodegenerative diseases, particularly of the motor neuron. One such disease is spinal muscular atrophy (SMA), a genetic neuromuscular disorder caused by mutations in the SMN1 gene resulting in low levels of Survival Motor Neuron (SMN) protein. Despite knowing the causal protein, the exact intracellular processes that are involved in the selective loss of motor neurons remains unclear. Autophagy induction can be helpful or harmful depending on the situation, and we sought to understand the state of the autophagic response in SMA. We show that cell culture and animal models demonstrate induction of autophagy accompanied by attenuated autophagic flux, resulting in the accumulation of autophagosomes and their associated cargo. Expression of the SMN-binding protein a-COP, a known modulator of autophagic flux, can ameliorate this autophagic traffic jam.
- In vitro growth conditions and development affect differential distributions of RNA in axonal growth cones and shafts of cultured rat hippocampal neurons. [JOURNAL ARTICLE]
- Mol Cell Neurosci 2014 Jun 28.
Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establish appropriate synaptic connections and confer plasticity. To study the transcripts present in the growth cones and axonal shafts of cultured rat hippocampal neurons, two chip devices, differing in their abilities to support axonal growth and branching, are designed and employed here to isolate large quantities of axonal materials. Cone-, shaft- and axon-residing transcripts with amounts higher than that of a somatodendritic transcript, Actg1 (γ-actin), are selected and classified. Since the chips are optically transparent, distribution of transcripts over axons can be studied by fluorescence in situ hybridization. Three transcripts, Cadm1 (cell adhesion molecule 1), Nefl (neurofilament light polypeptide), and Cfl1 (non-muscle cofilin) are confirmed to be preferentially localized to the growth cones, while Pfn2 (profilin2) is preferentially localized to the shafts of those axons growing on the chip that restricts axonal growth. The different growing conditions of axons on chips and on conventional coverslips do not affect the cone-preferred localization of Cadm1 and shaft-preferred localization of Pfn2, but affect the distributions of Nefl and Cfl1 over the axons at 14th day in vitro. Furthermore, the distributions of Cadm1 and Nefl over the axons growing on conventional coverslips undergo changes during in vitro development. Our results suggest a dynamic nature of the mechanisms regulating the distributions of transcripts in axonal substructures in a manner dependent upon both growth conditions and neuronal maturation.