Slit promotes branching and elongation of neurites of interneurons but not projection neurons from the developing telencephalon

Proper neuronal migration and establishment of circuitry are key processes for laying down the functional network of cortical neurons. A variety of environmental guidance cues, attractive or repulsive, have been shown to guide cell migration and axon arborization. One of these, Slit, appears to possess contrarian properties; it can either inhibit axon outgrowth or promote branching and elongation. The object of the present study was to assess the effect of Slit on MGE and neocortical neurons in culture and in the developing ventricle. When cocultured with a Slit source, E13.5 MGE explants displayed inhibited neurite outgrowth while GABA neuron dispersion away from Slit was increased. Similar inhibition of neurite outgrowth was seen in dissociated cells from E13.5 MGE, these cells were identified to be interneurons based upon their GABA staining. In contrast, E13.5 interneurons, after culture for another 5 days, were responsive to Slit by neurite branching and elongation. Projection neurons, identified by lack of GABA staining, did not respond to Slit, either by branching or elongation. Furthermore, GABA interneurons but not pyramidal neurons, appeared to avoid neocortical areas close to an implanted source of Slit in the ventricular wall. These results lead us to suggest that interneurons but not projection neurons are responsive to the chemorepellant effect of Slit. However, more mature interneurons appear to respond to Slit by neurite arborization. These results demonstrate a selective response to Slit by GABAergic neurons during neocortical development.     

Dopamine and serotonin inhibition of neurite elongation of different identified neurons

This study demonstrates that a second classical neurotransmitter, dopamine, can act to suppress regenerative neurite outgrowth. Single identified neurons were dissected from two central ganglia of the snail Helisoma, and growth cone motility was studied as neurites regenerated in cell culture. Both dopamine and serotonin inhibited growth cone motility and elongation of neurites. Outgrowth inhibition ranged from sustained arrest to a similar but transient response. The effects of dopamine and serotonin are neuron-selective. Specific neurons affected by dopamine and serotonin represent distinct sets. One neuron was found that responds to both agents. The implications of neurotransmitter regulation of the dynamics of neuronal morphology are discussed.     

GDNF enhances the synaptic efficacy of dopaminergic neurons in culture

Glial cell line-derived neurotrophic factor (GDNF) is known to promote the survival and differentiation of dopaminergic neurons of the midbrain. GDNF also causes an enhancement of dopamine release by a mechanism which is presently unclear. Using isolated dopaminergic neurons of the rat ventral tegmental area in culture, we have tested the hypothesis that GDNF regulates the establishment and functional properties of synaptic terminals. Previous studies have shown that single dopaminergic neurons in culture can co-release glutamate in addition to dopamine, leading to the generation of a fast excitatory autaptic current via glutamate receptors. Using excitatory autaptic currents as an assay for the activity of synapses established by identified dopaminergic neurons, we found that chronically applied GDNF produced a threefold increase in the amplitude of excitatory autaptic currents. This action was specific for dopaminergic neurons because GDNF had no such effect on ventral tegmental area GABAergic neurons. The enhancement of excitatory autaptic current amplitude caused by GDNF was accompanied by an increase in the frequency of spontaneous miniature excitatory autaptic currents. These observations confirmed a presynaptic locus of change. We identified synaptic terminals by using synapsin-1 immunofluorescence. In single tyrosine hydroxylase-positive neurons, the number of synapsin-positive puncta which represent putative synaptic terminals was found to be approximately doubled in GDNF-treated cells at 5, 10 and 15 days in culture. The number of such morphologically identified terminals in isolated GABAergic neurons was unchanged by GDNF. These results suggest that one mechanism through which GDNF may enhance dopamine release is through promoting the establishment of new functional synaptic terminals.     

Expressions and possible roles of GDNF receptors in the developing dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) has an essential role in the survival and maturation of the dopaminergic (DA) neurons in the substantia nigra (SN) of mammalian embryonic brain. In addition to Ret, cell adhesion molecules (CAMs) were also proposed to function as transmembrane signaling receptors of GDNF. The present study was to investigate whether these transmembrane receptors of GDNF were correlated with the tyrosine hydroxylase (TH) expression of SN DA neurons during early developmental stage. RT-PCR and Western blot were performed to detect TH expression in SN of perinatal rats at mRNA and protein level respectively; meanwhile, Western blot was performed to detect the expressions of the transmembrane proteins including Ret, neural cell adhesion molecule-140 (NCAM-140), integrin β1 and N-cadherin. The results showed that TH mRNA expression was positively correlated with both Ret and N-cadherin protein, while there was no correlation with NCAM-140 and integrin β1; TH protein expression was correlated with all of these transmembrane molecules. These data suggested that the expression of either TH mRNA or TH protein was subject to the mediation of different transmembrane receptor combinations of GDNF.     

GDNF and GFRalpha: a versatile molecular complex for developing neurons

The GDNF family ligands (GFLs) signal through the canonical signaling receptor Ret and a glycosyl-phosphatidylinositol-anchored co-receptor, GFRalpha. In recent years, signaling by GFLs has been shown to be more complex than originally assumed. The discrepant expression between GFRalphas and Ret has suggested the existence of additional signal-transducing GDNF receptors, such as NCAM. Here we summarize novel functions and Ret-independent signaling mechanisms for GDNF and GFRalpha, focusing on developing neurons. Emerging evidence indicates a prominent role of GDNF and GFRalpha in the control of neuroblast migration and chemoattraction and in the formation of neuronal synapses by a new mechanism of ligand-induced cell adhesion. Therefore, these data highlight the importance of this versatile molecular complex for nervous system development, function and regeneration.     

Integrin activation and neurotrophin signaling cooperate to enhance neurite outgrowth in sensory neurons

Neurite growth is influenced by many factors, including the availability of trophic support as well as the extracellular environment. In this study, we have investigated whether attachment to a permissive culture substrate such as laminin is sufficient to promote neurite outgrowth from dorsal root ganglion neurons in the absence of added nerve growth factor (NGF) and whether this attachment can enhance the response of these neurons to NGF. Adult dorsal root ganglia neurons plated on surfaces coated with a thin film of laminin exhibited increased neurite outgrowth. This effect was integrin-dependent as it was attenuated by treatment with RGD (arginine-glycine-aspartate) peptides and by a beta1-integrin blocking antibody. The addition of NGF resulted in a significant increase in the integrin-dependent outgrowth. We have correlated this increase in growth with increased expression of integrin subunits and activation of known downstream signaling intermediates such as focal adhesion kinase, Src, and Akt. We have also examined pathway cooperation through the use of an Src-specific inhibitor, PP2, and a beta1-integrin blocking antibody, beta1i, by observing downstream signaling intermediates in both integrin and growth factor signaling pathways. These results are among the first to detail the importance of interactions between neurotrophin- and integrin-activated signaling in adult primary neurons.     

Non-FK506-binding protein-12 neuroimmunophilin ligands increase neurite elongation and accelerate nerve regeneration

Neurotrophic activity of neuroimmunophilin ligands (FK506 and its nonimmunosuppressant derivatives) has been assumed to be mediated by the FK506-binding protein-12 (FKBP-12). We recently showed that activity is retained in hippocampal neurons from FKBP-12 knockout mice, indicating that binding to FKBP-12 is not necessary. Here we show that three nonimmunosuppressant FK506 derivatives (V-13,450, V-13,629, and V-13,670) that do not bind FKBP-12 (>12.5 mM affinity) are equipotent to FKBP-12 ligands (FK506, V-10,367, and V-13,449) for increasing neurite elongation in SH-SY5Y cells. One non-FKBP-12 ligand (V-13,670) is also shown to accelerate functional recovery and nerve regeneration in the rat sciatic nerve crush model. Surprisingly, it exhibited an unusual dose-response effect upon oral administration, showing a novel bimodal dose-response for behavioral functional recovery and myelination, but not for axonal size, suggesting both Schwann cell and neuronal targets. Orally active non-FKBP-12 neuroimmunophilin ligands may be useful for the treatment of human neurological disorders without any potential side effects resulting from FKBP-12 binding.     

GDNF family ligands activate multiple events during axonal growth in mature sensory neurons

The need for medical treatment of neuronal trauma motivates the search for new agents to stimulate posttraumatic axonal regrowth, as well as improving understanding of signaling cascades regulating this process. GDNF stimulates axonal regeneration in the peripheral nervous system, but little is known about the mechanism of this effect. Neurturin, artemin and persephin are homologs of GDNF, and their impact on axonal regeneration in adults has not been studied yet. Here we show that neurturin, artemin and GDNF, but not persephin, promote axonal initiation in cultured dorsal root ganglion neurons from young adult mice. This effect requires Src-family kinase activity as it was blocked by SU6656. In neurons from GFRalpha2-deficient mice, neurturin does not significantly promote axonal initiation. We also show that neurturin and GDNF induce extensive lamellipodia formation on neuronal somata and growth cones. GDNF, when applied after the time of axonal initiation in culture, also promotes axonal elongation.     

Metallothionein-III inhibits initial neurite formation in developing neurons as well as postinjury,regenerative neurite sprouting

Human metallothionein-III (MT-III) is an inhibitory factor deficient in the Alzheimer's disease (AD) brain. MT-III has been identified as an inhibitor of neurite sprouting, and its deficiency has been proposed to be involved in the formation of neurofibrillary tangles (NFT) in the neuropathology of AD. However, there has been limited investigation of the proposed neurite growth inhibitory properties of MT-III. We have applied recombinant human MT-III to both single cell embryonic cortical neurons (to investigate initial neurite formation), as well as mature (21 days postplating) clusters of cortical neurons (to investigate the regenerative sprouting response following injury). We report that MT-III inhibited the initial formation of neurites by rat embryonic (E18) cortical neurons. This was based on both the percentage of neurite positive neurons and the number of neurites per neuron (45 and 30% inhibition, respectively). Neurite inhibition was only observed in the presence of adult rat brain extract, and was also reversible following replacement of MT-III-containing medium. MT-III inhibited the formation and growth of both axons and dendrites. Of more physiological significance, MT-III also inhibited the regenerative neurite sprouting response following axonal transection. The morphology of sprouting neurites was also altered, with the distal tip often ending in bulb-like structures. Based on these results, we propose that MT-III, in the presence of brain extract, is a potent inhibitor of neurite sprouting, and may be involved in abnormal sprouting potentially underlying both AD and epilepsy.     

Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.     

Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine

The anatomy of peripheral serotonin-containing fibers (5-HT fibers) in the gut of wild-type Drosophila larvae was compared to mutants deficient in the gene that encodes the enzyme dopa decarboxylase (DfDdc mutants). The 5-HT fibers, located in the proventriculus and midgut, were visualized immunocytochemically by using a monoclonal antibody against 5-HT. Since DfDdc larvae are devoid of 5-HT and dopamine in the nervous system, the highly selective uptake capability of 5-HT neurons was used to visualize the 5-HT fibers. We found that the absence of 5-HT and dopamine in the nervous system of DfDdc animals does not prevent 5-HT fibers from reaching their appropriate targets. However, these fibers in the mutant show a 2-fold increase in the extent of branching. This effect is specific to 5-HT fibers, since glutamate-like and FMRFamide-like immunoreactive fibers of the proventriculus and midgut remain unaffected in the mutant. Low but detectable levels of dopamine and 5-HT in the CNS are sufficient to prevent the increase in arborization, as indicated by analyses of a temperature-sensitive Ddc allele (Ddcts2), which has very low dopa decarboxylase activity. The abnormally extensive branching of 5-HT fibers also can be partially rescued by feeding DfDdc larvae with dopamine. In contrast, feeding with a 5-HT-containing diet had no effect on the mutant phenotype. Hypotheses that could explain the mutant phenotype are proposed.     

GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.     

Glial process elongation and branching in the developing murine neocortex: a qualitative and quantitative immunohistochemical analysis

Cells of astroglial lineage in the murine cerebrum undergo a succession of transformations during prenatal and early postnatal development. The bipolar radial cell, the earliest astroglial form to appear, provides a radially aligned, parallel array of fibers that serves as a guide to neuronal migration. The multipolar astrocyte is the representative of this lineage that persists in the adult cerebrum. The processes of the multipolar astrocytes form a complex reticulum, which is considered critical to the development, function, and maintenance of neural circuits. A monopolar radial cell appears to be transitional between the two. The shift from the radial glial fiber system to a diffuse glial network is achieved largely in the E17-P2 interval in the mouse. This phenomenon has been studied qualitatively and quantitatively by staining cerebral tissue with monoclonal antibody RC2, a specific and sensitive ligand for cells of astroglial lineage in the mouse. Elongation and branching of glial processes contribute to the glial transformation. Elongation of radial fibers occurs under the guidance of other radial glial fibers (fasciculated elongation) or independently of other fibers (nonfasciculated elongation). Fasciculated elongation results in an increase in the density of radial glial fibers that span the cortical layers. Nonfasciculated elongation appears to be associated with process branching. This is the initial event in transformation of the bipolar radial cells to monopolar radial or multipolar cells. Only nonfasciculated elongation is characteristic of processes of the monopolar radial cells and multipolar astrocytes. Branching of the processes of all three cell forms appears to occur both by bifurcation at the elongating tip and by sprouting from the fiber shaft. Elongating fibers are tipped by growth cones that are relatively simple in shape as compared to those observed at the tips of elongating axons. Growth cones at the tips of nonfasciculated fibers are more complex in form than those at the tips of radial fibers elongating in contact with other radial fibers.     

Heat shock protein 27 is involved in neurite extension and branching of dorsal root ganglion neurons in vitro

Alteration of the cytoskeleton in response to growth factors and extracellular matrix proteins is necessary for neurite growth. The cytoskeletal components, such as actin and tubulin, can be modified through interaction with other cellular proteins, including the small heat shock protein Hsp27. Our previous work suggested that Hsp27 influences neurite growth, potentially via its phosphorylation state interactions with actin. To investigate further the role of Hsp27 in neurite outgrowth of adult dorsal root ganglion (DRG) neurons, we have both down-regulated endogenous Hsp27 and expressed exogenous Hsp27. Down-regulation of Hsp27 with Hsp27 siRNA resulted in a decrease of neuritic tree length and complexity. In contrast, expression of exogenous Hsp27 in these neurons resulted in an increase in neuritic tree length and branching. Collectively, these results demonstrate that Hsp27 may play a role in neuritic growth via modulation of the actin cytoskeleton.     

Toxicity of 6-hydroxydopamine and dopamine for dopaminergic neurons in culture

Toxicity of 6-hydroxydopamine (6-OHDA) and dopamine were studied in cultures of dissociated fetal rat mesencephalic cells. To assess survival and function of dopaminergic cells we quantified the number of tyrosine hydroxylase-positive cells and measured dopamine uptake. Non-dopaminergic cells were monitored by counting the number of cells visible with phase-contrast microscopy and measuring GABA uptake. 6-OHDA, in contrast to MPP+, which selectively destroyed dopaminergic neurons, was found to be a non-selective neurotoxin in this culture system. Between 10 and 100 microM, dopaminergic and non-dopaminergic cells were destroyed. At concentrations higher than 100 microM, i.e., concentrations frequently used to lesion catecholaminergic neurons in vivo, 6-OHDA resulted in structural fixation and loss of viability of dopaminergic and non-dopaminergic cells. Dopamine produced the same actions at slightly higher concentrations. One hundred to 300 microM was toxic for all cell types, and concentrations above 300 microM resulted in fixation. The findings suggest that 6-OHDA cannot be considered a selective toxin for catecholaminergic neurons in vitro. The demonstrated toxicity of dopamine tends to support speculations that processes related to dopamine metabolism may play a role in the pathogenesis of Parkinson's disease.     

Glia protect fetal midbrain dopamine neurons in culture from L-DOPA toxicity through multiple mechanisms

Summary Mesencephalic glia produce soluble factors that protect dopamine neurons from L-DOPA toxicity. The chemical composition of these soluble factors is unknown. We investigated the protective effect against L-DOPA neurotoxicity in midbrain dopamine neurons of fractions of different molecular size of glia conditioned medium and candidate neuroprotective agents produced by glia including neurotrophic factors and antioxidants. Protective effects were evaluated according to the number of tyrosine hydroxylase immunoreactive cells, high affinity dopamine uptake and levels of quinones. Both fractions of glia conditioned medium, smaller and larger than 10kD, protected against L-DOPA, but the fraction of smaller molecular size, that contains small free radical scanvenger molecules, was more effective than the fraction of larger molecular size, that contains large neurotrophic peptides. Among the neurotrophic factors GDNF and BDNF totally prevented L-DOPA neurotoxicity, while NGF and bFGF were less effective. However, only NGF significantly reduced the elevation of quinones induced by L-DOPA. Ascorbic acid, at the concentration found in glia conditioned medium, provided partial protective effect against L-DOPA toxicity. Glutathione, had neurotrophic effects on untreated midbrain dopamine neurons and prevented the effect of L-DOPA. In conclusion, the protective effect against L-DOPA neurotoxicity by glia conditioned medium is mediated by several compounds including neurotrophic factors and small antioxidants.     

Hepatocytes enhance neurite regeneration and survival from transected nerve terminals.

The culture of hepatocytes dissociated from adult mice by the collagenase perfusion method enables neurons or neuronal tissues to be cultured with hepatocytes or in hepatocyte-conditioned media. Co-culture with hepatocytes or hepatocyte-conditioned media enhances neurite regeneration and their survival from nerve-transected terminals of dorsal root ganglia with nerve fibers dissected from adult and aged mice. Hepatocytes secrete a factor which enhances not only neurite regeneration but also neurite survival. Activities of other known neurotrophic factors were not as crucial as those of the hepatocyte-conditioned medium, suggesting that this factor may differ from other trophic ones.     

NGF but not GDNF or neurturin enhance acetylcholine tissue levels in striatal organotypic brain slices

Trophic factors play important roles in survival and nerve fiber growth of cholinergic interneurons in the striatum in vivo and in vitro. In this study an organotypic slice model was used to investigate the effects of nerve growth factor and the novel factors glial cell line-derived neurotrophic factor and neurturin as well as other trophic factors on the striatal acetylcholine tissue levels. During culturing over 2 weeks acetylcholine tissue levels markedly decreased, representing degeneration of cholinergic neurons. When striatal slices were cultured for 2 weeks in the presence of 100 ng\ml nerve growth factor tissue levels of acetylcholine and the expression of choline acetyltransferase-like immunoreactivity and mRNA, as well as the muscarinic M2 autoreceptor mRNA were markedly enhanced compared to slices cultured without or with 10 ng\ml nerve growth factor. A single administration of nerve growth factor had no effect on acetylcholine tissue levels suggesting that nerve growth factor does not directly increase acetylcholine synthesis. All other trophic factors (glial cell line-derived neurotrophic factor, neurturin, brain-derived neurotrophic factor, neurotrophin-3 and -4\5, fibroblast growth factor-2, insulin like growth factor-I) had no effects on acetylcholine tissue levels. Thus, the organotypic slice model is a suitable system to study the effects of trophic factors and it is concluded that nerve growth factor selectively enhances acetylcholine tissue levels, indicating protection of cholinergic interneurons in the dorsal striatum.     

Identified postnatal mesolimbic dopamine neurons in culture: morphology and electrophysiology.

To examine the intrinsic properties of postnatal mesolimbic dopamine (DA) neurons, we dissociated the ventral tegmental area (VTA) from postnatal rats, enriched for DA neurons by microdissection or gradient purification, and grew the cells in culture. In these cultures, up to 50% of neurons were dopaminergic. DA neurons resembled their in vivo counterparts in soma shapes, and in showing two levels of tyrosine hydroxylase (TH) expression, axodendritic differentiation, two sizes of synaptic vesicles, nest-like synaptic arrangements with non-DA cells, and synaptic specializations. Electrophysiologically, however, they could not be distinguished from non-DA cells, which could be consistent with heterogeneity in cell properties. To examine a functional subset of VTA DA neurons, we retrogradely labeled VTA neurons projecting to the nucleus accumbens. These mesoaccumbens neurons were 86% TH positive, 56% cholecystokinin positive, and 0% neurotensin positive; they also displayed the soma shapes characteristic of DA neurons more generally and two levels of TH expression. Like their in vivo counterparts, mesoaccumbens cells generally fired single broad spikes that were triggered by slow depolarizations and had robust spike afterhyperpolarizations, low- and high-threshold Ca2+ spikes, rapid accommodation of firing, time-dependent anomalous rectification, and hyperpolarizing autoreceptor responses. Strikingly, the expression of these active properties did not change with time in culture. Mesoaccumbens DA cells could be identified by a distinctive subset of properties that made up an electrophysiological signature; however, unlike their in vivo counterparts, they were less often spontaneously active and never fired in bursts. These results suggest that most DA cell properties are intrinsic to the cells, including a significant heterogeneity that is maintained in postnatal culture; their level and mode of activity, however, appear to require afferent input. Culturing identified postnatal VTA DA neurons now makes possible examination of the impact of their individual properties on synaptic function.     

Action of thyroxine on the survival and neurite maintenance of cerebellar granule neurons in culture

Developmental hypothyroidism causes severe impairments in the cerebellum. To understand the role of thyroid hormones (THs) in cerebellar development, we examined the effect of three different THs, thyroxine (T4), 3,5,3′‐triidothyronine (T3), and 3,3′,5′‐triiodothyronine (reverse T3; rT3), on the survival and morphology of cerebellar granule neurons (CGNs) in culture and found novel actions specific to T4. Rat CGNs obtained at postnatal day 6 were first cultured for 2 days in serum‐containing medium with 25 mM K⁺ (K25), then switched to serum‐free medium with physiological 5 mM K⁺ (K5) or with K25 and cultured for an additional 2 or 4 days. CGNs underwent apoptosis in K5 but survived in K25. Addition of T4 at concentrations of 100–200 nM but not T3 or rT3 rescued CGNs from cell death in K5 in a dose‐dependent manner. Furthermore, 200 nM T4 was also effective in maintaining the neurites of CGNs in K5. In K5, T4 suppressed tau phosphorylation at two developmentally regulated sites as well as phosphorylation of c‐jun N‐terminal kinase (JNK) necessary for its activation and localization to axons. These results suggest that, during cerebellar development, T4 exerts its activity in cell survival and neurite maintenance in a manner distinct from the other two thyroid hormones through regulating the activity and localization of JNK. © 2014 Wiley Periodicals, Inc.