Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.     

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.     

Zonisamide reduces nigrostriatal dopaminergic neurodegeneration in a mouse genetic model of Parkinson's disease

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of nigrostriatal dopaminergic neurons and consequent motor dysfunction. Zonisamide (1,2‐benzisoxazole‐3‐methanesulfonamide), which was originally developed as an antiepileptic drug, has been found to have therapeutic benefits for PD. However, the pharmacological mechanisms behind the beneficial actions of zonisamide in PD are not fully understood. Here, we investigated the neuroprotective effects of zonisamide on nigrostriatal dopaminergic neurons of the Engrailed mutant mouse, a genetic model of PD. Chronic administration of zonisamide in Engrailed mutant mice was shown to improve the survival of nigrostriatal dopaminergic neurons compared with that under saline treatment. In addition, dopaminergic terminals in the striatum and the motor function were improved in zonisamide‐treated Engrailed mutant mice to the levels of those in control mice. To clarify the mechanism behind the neuroprotective effects of zonisamide, the contents of neurotrophic factors were determined after chronic administration of zonisamide. Brain‐derived neurotrophic factor content was increased in the striatum and ventral midbrain of the zonisamide‐treated mice compared to saline‐treated mice. These findings imply that zonisamide reduces nigrostriatal dopaminergic cell death through brain‐derived neurotrophic factor signaling and may have similar beneficial effects in human parkinsonian patients as well.

     

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin–stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin–stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca²⁺/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin– and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 633–641, 1998     

Regulation of neurite growth by tumour necrosis superfamily member RANKL

RANKL (receptor-activator of NF-κB ligand, TNFSF11) is a member of the TNF superfamily that regulates bone remodelling and the development of the thymus, lymph nodes and mammary glands. While RANKL and its membrane bound receptor RANK (TNFRSF11A) are expressed in the adult central nervous system and have been implicated in thermoregulation, the potential function of RANK signalling in the developing nervous system remains unexplored. Here, we show that RANK is expressed by sympathetic and sensory neurons of the developing mouse peripheral nervous system and that activating RANK signalling in these neurons during perinatal development by either treating cultured neurons with soluble RANKL or overexpressing RANK in the neurons inhibited neurotrophin-promoted neurite growth without affecting neurotrophin-promoted neuronal survival. RANKL is expressed in tissues innervated by these neurons, and studies in compartment cultures demonstrated that RANKL is capable of acting directly on neurites to inhibit growth locally. Enhancing RANK signalling in cultured neurons resulted in NF-κB activation and phosphorylation of the p65 NF-κB subunit on serine 536. Transfecting neurons with a series of mutated signalling proteins showed that NF-κB activation and p65 phosphorylation occurred by an IKKβ-dependent mechanism and that blockade of this signalling pathway prevented neurite growth inhibition by RANKL. These findings reveal that RANKL is a novel negative regulator of neurite growth from developing PNS neurons and that it exerts its effects by IKKβ-dependent activation of NF-κB.     

Dendritic synchrony and transient dynamics in a coupled oscillator model of the dopaminergic neuron

Transient increases in spontaneous firing rate of mesencephalic dopaminergic neurons have been suggested to act as a reward prediction error signal. A mechanism previously proposed involves subthreshold calcium-dependent oscillations in all parts of the neuron. In that mechanism, the natural frequency of oscillation varies with diameter of cell processes, so there is a wide variation of natural frequencies on the cell, but strong voltage coupling enforces a single frequency of oscillation under resting conditions. In previous work, mathematical analysis of a simpler system of oscillators showed that the chain of oscillators could produce transient dynamics in which the frequency of the coupled system increased temporarily, as seen in a biophysical model of the dopaminergic neuron. The transient dynamics was shown to be consequence of a slow drift along an invariant subset of phase space, with rate of drift given by a Lyapunov function. In this paper, we show that the same mathematical structure exists for the full biophysical model, giving physiological meaning to the slow drift and the Lyapunov function, which is shown to describe differences in intracellular calcium concentration in different parts of the cell. The duration of transients was long, being comparable to the time constant of calcium disposition. These results indicate that brief changes in input to the dopaminergic neuron can produce long lasting firing rate transients whose form is determined by intrinsic cell properties.     

Motor neuron degeneration in a mouse model of seipinopathy

Heterozygosity for missense mutations (N88S/S90L) in BSCL2 (Berardinelli–Seip congenital lipodystrophy type 2)/Seipin is associated with a broad spectrum of motoneuron diseases. To understand the underlying mechanisms how the mutations lead to motor neuropathy, we generated transgenic mice with neuron-specific expression of wild-type (tgWT) or N88S/S90L mutant (tgMT) human Seipin. Transgenes led to the broad expression of WT or mutant Seipin in the brain and spinal cord. TgMT, but not tgWT, mice exhibited late-onset altered locomotor activities and gait abnormalities that recapitulate symptoms of seipinopathy patients. We found loss of alpha motor neurons in tgMT spinal cord. Mild endoreticular stress was present in both tgMT and tgWT neurons; however, only tgMT mice exhibited protein aggregates and disrupted Golgi apparatus. Furthermore, autophagosomes were significantly increased, along with elevated light chain 3 (LC3)-II level in tgMT spinal cord, consistent with the activation of autophagy pathway in response to mutant Seipin expression and protein aggregation. These results suggest that induction of autophagy pathway is involved in the cellular response to mutant Seipin in seipinopathy and that motoneuron loss is a key pathogenic process underlying the development of locomotor abnormalities.     

Midbrain dopamine neuron differentiation: factors and fates

Dopaminergic neurons in the midbrain (mDNs) play a central role in the regulation of voluntary movement as well as other complex behaviors, and their loss is associated with Parkinson's disease (PD). The development of functional mDNs from multipotent progenitors is orchestrated by cell-intrinsic factors and cell-extrinsic environmental cues in a series of stages: early midbrain patterning, specification of mitotic precursors, postmitotic mDN development, and functional maturation. Of particular interest is how extracellular information is integrated with cell-intrinsic developmental programs. Cell fate mapping studies suggest that the stem-like progenitors for mDNs reside at the ventral midline floor plate, a region that also serves as a source of inductive signals for mDN specification such as Sonic Hedgehog (SHH). Cell replacement therapies, and in particular the use of embryonic or adult stem cell-derived dopaminergic neurons, offer potential novel treatment venues for PD, but such strategies require a detailed understanding of mDN development.     

Constitutively expressed catalytic proteasomal subunits are up-regulated during neuronal differentiation and required for axon initiation, elongation and maintenance

Inhibition of the proteasome by lactacystin, a specific blocker of the catalytic beta-subunits, results in transient neurite outgrowth by neuronal cell lines. Vice versa, as demonstrated in this study, treatment of pheochromocytoma (PC12) cells with nerve growth factor (NGF) or other differentiating agents reduces proteasomal activity. This is accompanied by an increase in mRNA and protein levels of the catalytically active subunits beta1, beta2 and beta5, but not of their inducible counterparts, indicating changes in subunit composition of the proteasome during neuronal differentiation. In contrast to neuronal cell lines, however, pre-treatment of primary neurons with proteasome inhibitors completely prevents axon formation, and lower concentrations of lactacystin (0.5-5 microm) significantly reduce axonal elongation and branching in vitro. Furthermore, established axonal networks degenerate rapidly and long-term survival of peripheral neurons is impaired in the presence of proteasome inhibitors. Axonal pathology is reminiscent of the morphological changes observed in neurodegenerative disorders and supports a crucial role of the constitutive catalytic subunits in axon initiation, maintenance and regeneration.     

Neuronal Galectin‐4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin‐4 (Gal‐4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule‐ and sulfatide‐dependent manner. Gal‐4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal‐4 addition. This Gal‐4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth‐promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal‐4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N‐glycans. Impairing the maturation of these N‐glycans precludes Gal‐4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal‐4 sorts to axon plasma membrane segments by binding to sulfatide‐containing microtubule‐associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.     

Initial axon growth rate from embryonic sensory neurons is correlated with birth date

Axon growth rate from different populations of sensory neurons is correlated with the distance they have to grow to reach their targets in development: neurons with more distant targets extend axons at intrinsically faster rates. With growth of the embryo, later‐born neurons within each population have further to extend their axons to reach their targets than early‐born neurons. Here we examined whether the axon growth rate is related to birth date by studying the axon growth from neurons that differentiate in vitro from precursor cells isolated throughout the period of neurogenesis. We first showed that neurons that differentiated in vitro from different precursor cell populations exhibited differences in axon growth rate related to in vivo target distance. We then examined the axon growth rate from neurons that differentiate from the same precursor population at different stages throughout the period of neurogenesis. We studied the epibranchial placode precursors that give rise to nodose ganglion neurons in the chicken embryo. We observed a highly significant, threefold difference in axon growth rate from neurons that differentiate from precursor cells cultured early and late during the period of neurogenesis. Our findings suggest that intrinsic differences in axon growth rate are correlated with the neuronal birth date.     

Ventricular injection of nerve growth factor increases dopamine content in the striata of MPTP-treated mice

Experimental depletion of dopaminergic striatal neurons was induced in mice with the neurotoxin MPTP. To investigate a possible effect of nerve growth factor on the damaged neurons, we injected 4 g into the right cerebral ventricle of mice three days after the last administration of MPTP. We found a significant increase of dopamine and homovanillic acid in the striatum of MPTP treated mice after NGF administration when compared with dopamine and HVA levels in MPTP-treated control mice (p<0.001). The increase of dopamine and homovanillic acid seems to be related to a partial restorative effect of NGF on the damaged dopaminergic cells, since ventricular administration of NGF to normal mice did not increase dopamine or homovanillic acid contents above the levels measured in untreated controls. It appears that administration of nerve growth factor prcduces a beneficial effect on damaged dopaminergic neurons; this effect could be due to stimulation of neuron sprouting from neurons that survived the toxic effect of MPTP. The increase of dopamine levels was seen 8 days after injection of nerve growth factor and was maintained at least until day 25, showing a lasting persistence of the restorative effect.