Lilla Farkas

Reduction of endogenous transforming growth factors |[beta]| prevents ontogeneticneuron death

We show that following immunoneutralization of endogenous transforming growth factors β (TGF-β) in the chick embryo, ontogenetic neuron death of ciliary, dorsal root and spinal motor neurons was largely prevented, and neuron losses following limb bud ablation were greatly reduced. Likewise, preventing TGF-β signaling by treatment with a TβR-II fusion protein during the period of ontogenetic cell death in the ciliary ganglion rescued all neurons that normally die. TUNEL staining revealed decreased numbers of apoptotic cells following antibody treatment. Exogenous TGF-β rescued the TGF-β-deprived phenotype. We conclude that TGF-β is critical in regulating ontogenetic neuron death as well as cell death following neuronal target deprivation.

Neurodegeneration in Methylmalonic Aciduria Involves Inhibition of Complex II and the Tricarboxylic Acid Cycle, and Synergistically Acting Excitotoxicity

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonate (MMA) and alternative metabolites. There is growing evidence for basal ganglia degeneration in these patients. The pathomechanisms involved are still unknown, a contribution of toxic organic acids, in particular MMA, has been suggested. Here we report that MMA induces neuronal damage in cultures of embryonic rat striatal cells at a concentration range encountered in affected patients. MMA-induced cell damage was reduced by ionotropic glutamate receptor antagonists, antioxidants, and succinate. These results suggest the involvement of secondary excitotoxic mechanisms in MMA-induced cell damage. MMA has been implicated in inhibition of respiratory chain complex II. However, MMA failed to inhibit complex II activity in submitochondrial particles from bovine heart. To unravel the mechanism underlying neuronal MMA toxicity, we investigated the formation of intracellular metabolites in MMA-loaded striatal neurons. There was a time-dependent intracellular increase in malonate, an inhibitor of complex II, and 2-methylcitrate, a compound with multiple inhibitory effects on the tricarboxylic acid cycle, suggesting their putative implication in MMA neurotoxicity. We propose that neuropathogenesis of methylmalonic aciduria may involve an inhibition of complex II and the tricarboxylic acid cycle by accumulating toxic organic acids, and synergistic secondary excitotoxic mechanisms.

TGF-β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-beta expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-beta2 and -beta3 as well as the TGF-beta receptor TbetaR-II, but are not ir for TGF-beta1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-beta3 mRNA and TGF-beta biological activity in cultures of E8 CG neurons. None of the TGF-beta isoforms--beta1, beta2, or beta3--has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-beta significantly promotes CG neuron survival. However, TGF-beta does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-beta released from CG neurons using an antibody to TGF-beta1/-beta2/-beta3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti-pan-TGF-beta antibody could be rescued by adding exogenous TGF-beta. Together, these data suggest that para-/autocrine TGF-beta signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons.

GDNF‐related factor persephin is widely distributed throughout the nervous system

Persephin (PSP) is the most recently discovered member of the GDNF family of neurotrophic factors. We have used an RT‐PCR approach to start addressing the putative functional significance of PSP by determining sites of its synthesis in the neonatal rat brain. Generally, two transcripts were found. Sequence analysis of the transcripts identifies an 88 bp intronic sequence. Neural tissues analysed included cortex, hippocampus, striatum, diencephalon, mesencephalon, cerebellum, hindbrain and spinal cord as well as superior cervical, dorsal root ganglia, adrenal gland, and PC12 pheochromocytoma cells. As non‐neuronal tissues, sciatic nerve, optic nerve, primary astroglial, oligodendroglial, O2A progenitor, and glioma cells (C6, B49) were also included. All tissues/cells except oligodendrocytes and O2A progenitor cells were strongly positive for PSP mRNA. To test the hypothesis of whether PSP might act as a target‐derived factor, as suggested for GDNF, the motoneuron–muscle axis has been analysed. PSP is synthesized in skeletal muscle and, to a higher extent, in the spinal cord. Moreover, PSP is synthesized in purified embryonic motoneurons. Together, these data do not support a role for PSP as a typical target‐derived neurotrophic factor for motoneurons. We conclude that PSP is synthesized throughout the nervous system and that it is presumably of both astroglial and neuronal origin, in contrast to GDNF and neurturin, which seem to be predominantly of neuronal origin. J. Neurosci. Res. 53:494–501, 1998.

GDNF INDUCES THE CALRETININ PHENOTYPE IN CULTURES OF EMBRYONIC STRIATAL NEURONS

Glial cell line‐derived neurotrophic factor (GDNF), a member of the transforming growth factor‐β (TGF‐β) superfamily, is a potent neurotrophic factor for several neuron populations in the central and peripheral nervous system. Members of the neurotrophin, neurokine, and TGF‐β families of growth factors can affect neurons beyond their capacity to promote survival. They can play instructive roles including the determination of a particular transmitter phenotype. Here, we show that GDNF enhances the number of calretinin (CaR)‐positive neurons in serum‐free cultures of striatal cells isolated from embryonic rats. The effect is dose‐dependent, can be elicited with concentrations as low as 0.1 ng/ml, and is not accompanied by increased incorporation of 5‐bromo‐2′‐desoxyuridine and appearance of glial fibrillary acidic protein–positive cells. Similar, but weaker effects can be elicited by brain‐derived neurotrophic factor, neurotrophin‐3 and ‐4, fibroblast growth factor‐2. Ciliary neurotrophic factor, nerve growth factor, and TGF‐β1 do not affect striatal CaR expression. GDNF can augment CaR‐positive cells at any time point and with a minimal exposure of 18 hr, suggesting induction of the phenotype rather than increased survival. By reverse transcription polymerase chain reaction (RT‐PCR), we show that GDNF is expressed in the E16 striatum and in cultures derived from this tissue. GDNF also protected striatal CaR‐positive neurons against glutamate toxicity. We conclude that striatal GDNF, in addition to its retrograde trophic role for nigrostriatal dopaminergic neurons, may also act locally within the striatum (e.g., by inducing the CaR phenotype and protecting these cells against toxic insult). J. Neurosci. Res. 50:361–372, 1997.

Characterization of growth/differentiation factor 5 (GDF-5) as a neurotrophic factor for cultured neurons from chicken dorsal root ganglia

Growth/differentiation factor-5 (GDF-5), a morphogenetic protein, has previously been shown to act as a neurotrophic factor for midbrain dopaminergic neurons. To further elucidate the neurotrophic potential of GDF-5, serum free cultures of dorsal root ganglionic (DRG) neurons from developing chick embryos were treated with GDF-5 with or without the simultaneous addition of other trophic factors. Our results show that GDF-5 has a minor promoting effect on its own, but it can enhance the survival promoting effect of neurotrophin-3 (NT-3) and nerve growth factor (NGF) on cultured DRG neurons. Our finding fits well into the concept that neurotrophic factors may act synergistically in ensuring survival of different neuronal populations. The capacity of GDF-5 to reduce the requirement of a subpopulation of sensory neurons for NT-3 may have implications for the treatment of peripheral neuropathies.