Fatiha Nothias

Microtubule-associated protein 1B controls directionality of growth cone migration and axonal branching in regeneration of adult dorsal root ganglia neurons.

During development, microtubule-associated protein 1B (MAP1B) is one of the earliest MAPs, preferentially localized in axons and growth cones, and plays a role in axonal outgrowth. Although generally downregulated in the adult, we have shown that MAP1B is constitutively highly expressed in adult dorsal root ganglia (DRGs) and associated with central sprouting and peripheral regeneration of these neurons. Mutant mice with a complete MAP1B null allele that survive until adulthood exhibit a reduced myelin sheath diameter and conductance velocity of peripheral axons and lack of the corpus callosum. Here, to determine the function of MAP1B in axonal regeneration, we used cultures of adult DRG explants and/or dissociated neurons derived from this map1b-/- mouse line. Whereas the overall length of regenerating neurites lacking MAP1B was similar to wild-type controls, our analysis revealed two main defects. First, map1b-/- neurites exhibited significantly (twofold) higher terminal and collateral branching. Second, the turning capacity of growth cones (i.e., “choice” of a proper orientation) was impaired. In addition, lack of MAP1B may affect the post-translational modification of tubulin polymers: quantitative analysis showed a reduced amount of acetylated microtubules within growth cones, whereas the distribution of tyrosinated or detyrosinated microtubules was normal. Both growth cone turning and axonal branch formation are known to involve local regulation of the microtubule network. Our results demonstrate that MAP1B plays a role in these processes during plastic changes in the adult. In particular, the data suggest MAP1B implication in the locally coordinated assembly of cytoskeletal components required for branching and straight directional axon growth.

Distinct Roles of c-Jun N-Terminal Kinase Isoforms in Neurite Initiation and Elongation during Axonal Regeneration

c-Jun N-terminal kinases (JNKs) (comprising JNK1–3 isoforms) are members of the MAPK (mitogen-activated protein kinase) family, activated in response to various stimuli including growth factors and inflammatory cytokines. Their activation is facilitated by scaffold proteins, notably JNK-interacting protein-1 (JIP1). Originally considered to be mediators of neuronal degeneration in response to stress and injury, recent studies support a role of JNKs in early stages of neurite outgrowth, including adult axonal regeneration. However, the function of individual JNK isoforms, and their potential effector molecules, remained unknown. Here, we analyzed the role of JNK signaling during axonal regeneration from adult mouse dorsal root ganglion (DRG) neurons, combining pharmacological JNK inhibition and mice deficient for each JNK isoform and for JIP1. We demonstrate that neuritogenesis is delayed by lack of JNK2 and JNK3, but not JNK1. JNK signaling is further required for sustained neurite elongation, as pharmacological JNK inhibition resulted in massive neurite retraction. This function relies on JNK1 and JNK2. Neurite regeneration of jip1−/− DRG neurons is affected at both initiation and extension stages. Interestingly, activated JNKs (phospho-JNKs), as well as JIP1, are also present in the cytoplasm of sprouting or regenerating axons, suggesting a local action on cytoskeleton proteins. Indeed, we have shown that JNK1 and JNK2 regulate the phosphorylation state of microtubule-associated protein MAP1B, whose role in axonal regeneration was previously characterized. Moreover, lack of MAP1B prevents neurite retraction induced by JNK inhibition. Thus, signaling by individual JNKs is differentially implicated in the reorganization of the cytoskeleton, and neurite regeneration.

Expression of a phosphorylated isoform of MAP1B is maintained in adult central nervous system areas that retain capacity for structural plasticity

Microtubule‐associated protein IB (MAP1B) is the first MAP to be detected in the developing nervous system, and it becomes markedly down‐regulated postnatally. Its expression, particularly that of its phosphorylated isoform, is associated with axonal growth. To determine whether adult central nervous system (CNS) areas that retain immunoreactivity for MAP1B are associated with morphological plasticity, we compared the distribution of a phosphorylated MAP1B isoform (MAP1B‐P) to the distribution of total MAP1B protein and MAP1B‐mRNA. Although they were present only at very low levels, both protein and message were found ubiquitously in almost all adult CNS neurons. The intensity of staining, however, varied markedly among different regions, with only a few nuclei retaining relatively high levels. MAP1B‐P was restricted to axons, whereas total MAP1B was present in cell bodies and processes. Relatively to total MAP1B protein and its mRNA, MAP1B‐P levels decreased more dramatically with maturation, and they were detectable in only a few specific areas that underwent structural modifications. These included primary afferents and motor neurons, olfactory tubercles, habenular and raphe projections to interpeduncular nuclei, septum, and the hypothalamus. The distribution pattern of MAP1B‐P was compared to that of the embryonic N‐CAM rich in polysialic acid (PSA‐NCAM). We found that the PSA‐NCAM immunostaining was largely overlapped with that of MAP1B‐P in the adult CNS. These results suggest that, like PSA‐NCAM, MAP1B may be one of the molecules expressed during brain development that also plays a role in structural remodeling in the adult.

Modulation of NCAM Polvsialvlation is Associated with Morphofunctional Modifkations in the Hypothalamo‐neurohypophysial System During Lactation

Post‐transcriptional modification of the neural cell adhesion molecule (NCAM) by polysialic acid significantly decreases NCAM adhesiveness and more generally modifies cell‐cell interactions. Polysialic acid‐NCAM (PSA‐NCAM) is mainly expressed in the developing nervous system. In the adult, its expression is restricted to regions that retain morphological plasticity, such as the hypothalamo‐neurohypophysial system during lactation in rats. Since cell‐cell interactions and synaptic contacts in the hypothalamo‐neurohypophysial system are greatly increased during lactation, we examined whether PSA‐NCAM expression is modified during this period. lmmunohistochemistry and immunoblotting showed that, compared with virgin rats, PSA‐NCAM dramatically decreased during lactation in both the supraoptic nuclei and the neurohypophysis, and returned to its initial level only after weaning. This decrease was progressive and became significant only at the end of the first week of lactation. By contrast, modifications in the level of NCAM protein or changes in the splicing pattern of NCAM mRNAs could not be detected. The decline in polysialic acid on the NCAM molecule could strengthen membrane appositions, thereby stabilizing the newly established synapses and neurohaemal contacts in the hypothalamo‐neurohypophysial system that accompany the increased neuronal activity that occurs during lactation. We also studied the regulation of the phosphorylated microtubule‐associated protein‐1B (MAP1B‐P), whose distribution pattern largely overlaps with that of PSA‐NCAM in the adult brain. Expression of MAP1B‐P was greatly increased during lactation in the hypothalamic axons projecting into the neurohypophysis. Thus, the expression patterns of both PSA‐NCAM and MAP1B‐P may reflect the permanent structural plasticity characterizing the hypothalamo‐neurohypophysial system in the adult.

Loss of MAP Function Leads to Hippocampal Synapse Loss and Deficits in the Morris Water Maze with Aging

Hyperphosphorylation and accumulation of tau aggregates are prominent features in tauopathies, including Alzheimers disease, but the impact of loss of tau function on synaptic and cognitive deficits remains poorly understood. We report that old (19–20 months; OKO) but not middle-aged (8–9 months; MKO) tau knock-out mice develop Morris Water Maze (MWM) deficits and loss of hippocampal acetylated α-tubulin and excitatory synaptic proteins. Mild motor deficits and reduction in tyrosine hydroxylase (TH) in the substantia nigra were present by middle age, but did not affect MWM performance, whereas OKO mice showed MWM deficits paralleling hippocampal deficits. Deletion of tau, a microtubule-associated protein (MAP), resulted in increased levels of MAP1A, MAP1B, and MAP2 in MKO, followed by loss of MAP2 and MAP1B in OKO. Hippocampal synaptic deficits in OKO mice were partially corrected with dietary supplementation with docosahexaenoic acid (DHA) and both MWM and synaptic deficits were fully corrected by combining DHA with α-lipoic acid (ALA), which also prevented TH loss. DHA or DHA/ALA restored phosphorylated and total GSK3β and attenuated hyperactivation of the tau C-Jun N-terminal kinases (JNKs) while increasing MAP1B, dephosphorylated (active) MAP2, and acetylated α-tubulin, suggesting improved microtubule stability and maintenance of active compensatory MAPs. Our results implicate the loss of MAP function in age-associated hippocampal deficits and identify a safe dietary intervention, rescuing both MAP function and TH in OKO mice. Therefore, in addition to microtubule-stabilizing therapeutic drugs, preserving or restoring compensatory MAP function may be a useful new prevention strategy.

S-nitrosylation of microtubule-associated protein 1B mediates nitric-oxide-induced axon retraction

Treatment of cultured vertebrate neurons with nitric oxide leads to growth-cone collapse, axon retraction and the reconfiguration of axonal microtubules. We show that the light chain of microtubule-associated protein (MAP) 1B is a substrate for S-nitrosylation in vivo, in cultured cells and in vitro. S-nitrosylation occurs at Cys 2457 in the COOH terminus. Nitrosylation of MAP1B leads to enhanced interaction with microtubules and correlates with the inhibition of neuroblastoma cell differentiation. We further show, in dorsal root ganglion neurons, that MAP1B is necessary for neuronal nitric oxide synthase control of growth-cone size, growth-cone collapse and axon retraction. These results reveal an S-nitrosylation-dependent signal-transduction pathway that is involved in regulation of the axonal cytoskeleton and identify MAP1B as a major component of this pathway. We propose that MAP1B acts by inhibiting a microtubule- and dynein-based mechanism that normally prevents axon retraction.

Somatotopic reciprocal connections between the somatosensory cortex and the thalamic Po nucleus in the rat

Retrogradely labeled neurons are observed in the posterior group of the thalamus (Po) after injection of wheatgerm agglutinin-horseradish peroxidase in the rat somatosensory cortex. These neurons are organized in rods elongated rostrocaudally, defining a clear somatotopic map. Injections of tritiated leucine in the somatosensory cortex indicate that these somatotopically organized connections are reciprocal. Injections of tritiated leucine in the dorsal column nuclei label afferent fibers in a small area dorsal to Po but not in the core of the nucleus. Po does not receive direct projections of ascending somatosensory afferents. It is hypothesized that this thalamic area participates in a thalamo-cortico-thalamic loop.

Raf‐1 and B‐Raf proteins have similar regional distributions but differential subcellular localization in adult rat brain

The Raf kinases play an important and specific role in the activation of extracellular signal‐regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron‐specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B‐Raf and Raf‐1 proteins are present in most brain areas, whereas A‐Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro‐caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf‐1 is localized mainly in the cytosolic fraction around the nucleus, whereas B‐Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B‐Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton‐insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization.

Vascularization of fetal cell suspension grafts in the excitotoxically lesioned adult rat thalamus.

Several studies have considered the establishment of vascularization in intracerebral solid transplants of neural tissue. The widely supported interpretation of the results is that the vascular network of the solid grafts is already present before implantation into the host brain. The situation is different when dissociated fetal tissue is transplanted as a cell suspension because in these conditions the fetal vascular network is disrupted. The present study has, therefore, been undertaken to follow the angiogenesis in a transplant of dissociated fetal cells implanted into the excitotoxically neuron-depleted thalamus. The vascular network is compared to that observed in the intact and in the lesioned thalamus both in terms of morphology of the capillaries and of the function of the blood-brain barrier (BBB). In the transplant, capillaries, stained by Indian ink, are very few in number and have very fine calibers during the first 20 days after grafting. Some structures can be identified as immature blood vessels at the electron microscopic level. The blood vessels are progressively more numerous in the graft and they demonstrate mature ultrastructural features 2 months after grafting. Last, there is no leakage of the BBB for peroxidase. The vascularization seems to follow a pattern of maturation comparable to that described during development in the literature. In contrast, in the lesioned area, there is a reactive angiogenesis: 10 days after the excitotoxic injection (shortest time studied), there are many wide caliber vessels with expanded perivascular spaces engorged with mesodermal cells. A microvascularization also develops transiently during the first two months. Capillaries are abnormal from the functional point of view, since there is a leakage of the BBB to macromolecules. The use of an experimental model in which transplant had to grow in a lesioned area permits to determine two types of vascularization: an apparently normal developmental timetable, normal morphological and functional characteristics, in the transplant; a reactive angiogenesis, in the lesioned area.

Stage- and Region-Specific Expression of Estrogen Receptor α Isoforms during Ontogeny of the Pituitary Gland1

The expression time course of estrogen receptor α (ERα) was analyzed by RT-PCR in fetal and newborn rat pituitaries. In addition to the classical ERα messenger RNA (mRNA), three shorter transcripts were detected and subsequently cloned. Sequence analysis showed that they corresponded to ERα mRNAs lacking exon 3 (which encodes a zinc finger in the DNA-binding domain), exon 4 (which encodes the nuclear localization signal and part of the steroid-binding domain), or both exons 3 and 4. As analyzed by RT-PCR and ribonuclease protection assay, the respective expression levels of the different transcripts varied dramatically during pituitary development; short forms appeared 4 days before full-length ERα mRNA. On Western blots from rat pituitaries of different ages, an ERα-specific antiserum labeled four protein bands of the expected molecular weights, revealing that all four ERα mRNAs are translated in vivo. Immunocytochemistry, using the same antiserum, showed the ERα to be present first in the cytosol of int...