Núria Gavaldà

Reduced expression of the TrkB receptor in Huntington's disease mouse models and in human brain

Deficits of neurotrophic support caused by reduced levels of brain‐derived neurotrophic factor (BDNF) have been implicated in the selective vulnerability of striatal neurones in Huntingtons disease (HD). Therapeutic strategies based on BDNF administration have been proposed to slow or prevent the disease progression. However, the effectiveness of BDNF may depend on the proper expression of its receptor TrkB. In this study, we analysed the expression of TrkB in several HD models and in postmortem HD brains. We found a specific reduction of TrkB receptors in transgenic exon‐1 and full‐length knock‐in HD mouse models and also in the motor cortex and caudate nucleus of HD brains. Our findings also demonstrated that continuous expression of mutant huntingtin is required to down‐regulate TrkB levels. This was shown by findings in an inducible HD mouse model showing rescue of TrkB by turning off mutant huntingtin expression. Interestingly, the length of the polyglutamine tract in huntingtin appears to modulate the reduction of TrkB. Finally, to analyse the effect of BDNF in TrkB we compared TrkB expression in mutant huntingtin R6/1 and double mutant (R6/1 : BDNF+/–) mice. Similar TrkB expression was found in both transgenic mice suggesting that reduced TrkB is not a direct consequence of decreased BDNF. Therefore, taken together our findings identify TrkB as an additional component that potentially might contribute to the altered neurotrophic support in HD.

Nuclear factor kappa B signaling either stimulates or inhibits neurite growth depending on the phosphorylation status of p65/RelA.

Nuclear factor κB (NF-κB) signaling is known to promote neurite growth from developing sensory neurons and to enhance the size and complexity of pyramidal neuron dendritic arbors in the developing cerebral cortex. In marked contrast, here we show that NF-κB signaling can also exert a potent inhibitory influence on neurite growth in certain neurons, and can either promote or inhibit neurite growth in the same neurons depending on the mechanism of NF-κB activation. In neonatal superior cervical ganglion sympathetic neurons, enhancing NF-κB transcriptional activity by overexpressing either the p65 NF-κB subunit or the IκB kinase-β (IKKβ) subunit of the IκB kinase complex, or by tumor necrosis factor α (TNFα) treatment, strongly inhibits neurite growth. Paradoxically in neonatal nodose ganglion sensory neurons, enhancing NF-κB transcriptional activity by p65/p50 overexpression increases neurite growth, whereas enhancing NF-κB transcriptional activity by IKKβ overexpression inhibits neurite growth. In addition to activating NF-κB, IKKβ overexpression leads to phosphorylation of p65 on serine 536. Blockade of serine 536 phosphorylation by a S536A-p65 mutant protein prevents the growth-inhibitory effects of IKKβ overexpression in both sensory and sympathetic neurons and the growth-inhibitory effects of TNFα on sympathetic neurons. Furthermore, expression of a p65 S536D phosphomimetic mutant inhibits neurite growth from sensory neurons. These results demonstrate that NF-κB can either stimulate or inhibit neurite growth in developing neurons depending on the phosphorylation status of p65.

Brain‐derived neurotrophic factor prevents changes in Bcl‐2 family members and caspase‐3 activation induced by excitotoxicity in the striatum

Brain‐derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl‐2 family members. We first analyzed the involvement of the phosphatidylinositol 3‐kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p‐Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non‐NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl‐2, Bcl‐xL and Bax protein levels and heterodimerization are selectively regulated by NMDA and non‐NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl‐2 protein levels, leading to reduced levels of Bax:Bcl‐2 heterodimers. In contrast, changes in Bax protein levels are not required for KA‐induced apoptotic cell death, but decreased levels of both Bax:Bcl‐2 and Bax:Bcl‐xL heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase‐3. Intrastriatal grafting of a BDNF‐secreting cell line counter‐regulated p‐AKT, Bcl‐2, Bcl‐xL and Bax protein levels, prevented changes in the heterodimerization between Bax and pro‐survival proteins, and blocked caspase‐3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti‐apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl‐2 family members, which is probably mediated by Akt activation.

Nuclear factor-kappaB activation via tyrosine phosphorylation of inhibitor kappaB-alpha is crucial for ciliary neurotrophic factor-promoted neurite growth from developing neurons

The cytokine ciliary neurotrophic factor (CNTF) promotes the growth of neural processes from many kinds of neurons in the developing and regenerating adult nervous system, but the intracellular signaling mechanisms mediating this important function of CNTF are poorly understood. Here, we show that CNTF activates the nuclear factor-κB (NF-κB) transcriptional system in neonatal sensory neurons and that blocking NF-κB-dependent transcription inhibits CNTF-promoted neurite growth. Selectively blocking NF-κB activation by the noncanonical pathway that requires tyrosine phosphorylation of inhibitor κB-α (IκB-α), but not by the canonical pathway that requires serine phosphorylation of IκB-α, also effectively inhibits CNTF-promoted neurite growth. CNTF treatment activates spleen tyrosine kinase (SYK) whose substrates include IκB-α. CNTF-induced SYK phosphorylation is rapidly followed by increased tyrosine phosphorylation of IκB-α, and blocking SYK activation or tyrosine phosphorylation of IκB-α prevents CNTF-induced NF-κB activation and CNTF-promoted neurite growth. These findings demonstrate that NF-κB signaling by an unusual activation mechanism is essential for the ability of CNTF to promote the growth of neural processes in the developing nervous system.

Excitatory Amino Acids Differentially Regulate the Expression of GDNF, Neurturin, and Their Receptors in the Adult Rat Striatum

Glial cell line-derived neurotrophic factor (GDNF) family ligands are important regulators of neuronal development and maintenance of the connectivity in the basal ganglia and show neuroprotective activities in several paradigms of brain injury. The mRNAs of two members of this family, GDNF and neurturin, and also their receptors have been detected in the basal ganglia. In the present work, we analyzed the time course changes in the expression of these neurotrophic factors and receptors in the adult rat striatum, induced by quinolinate or kainate excitotoxicity. Our results show that stimulation of NMDA or non-NMDA receptors induced different effects on the mRNA levels analyzed. Expression of GDNF and its preferred receptor, GDNF family receptor-alpha1 (GFRalpha1), was transiently up-regulated by quinolinate and kainate, but with differing intensity and temporal pattern. Immunohistochemical analysis showed that, although GDNF and GFRalpha1 were initially localized in neurons, excitotoxicity induced the expression of these proteins in astrocyte-like cells. Neurturin mRNA levels were only up-regulated after quinolinate injection, whereas quinolinate or kainate injection did not modify GFRalpha2 mRNA. The mRNA for the common receptor, c-Ret, was up-regulated by both agonists with similar temporal pattern but with differing intensity. Immunohistochemical analysis showed that c-Ret protein was located on neurons. These changes in mRNA levels and protein localization of GDNF family components could reflect an endogenous trophic response of striatal cells to different excitotoxic insults.

Developmental switch in NF-κB signalling required for neurite growth

For a given cell type, particular extracellular signals generate characteristic patterns of activity in intracellular signalling networks that lead to distinctive cell-type specific responses. Here, we report the first known occurrence of a developmental switch in the intracellular signalling network required for an identical cellular response to the same extracellular signal in the same cell type. We show that although NF-κB signalling is required for BDNF-promoted neurite growth from both foetal and postnatal mouse sensory neurons, there is a developmental switch between these stages in the NF-κB activation mechanism and the phosphorylation status of the p65 NF-κB subunit required for neurite growth. Shortly before birth, BDNF activates NF-κB by an atypical mechanism that involves tyrosine phosphorylation of IκBα by Src family kinases, and dephosphorylates p65 at serine 536. Immediately after birth, BDNF-independent constitutive activation of NF-κB signalling by serine phosphorylation of IκBα and constitutive dephosphorylation of p65 at serine 536 are required for BDNF-promoted neurite growth. This abrupt developmental switch in NF-κB signalling in a highly differentiated cell type illustrates an unsuspected plasticity in signalling networks in the generation of identical cellular responses to the same extracellular signal.

Differential involvement of phosphatidylinositol 3-kinase and p42/p44 mitogen activated protein kinase pathways in brain-derived neurotrophic factor-induced trophic effects on cultured striatal neurons.

Brain-derived neurotrophic factor (BDNF) is a potent trophic factor for striatal cells that promotes survival and/or differentiation of GABAergic neurons in vitro. In the present study, we show that the stimulation of cultured striatal cells with BDNF increased the phosphorylation of Akt and p42/p44. This effect was specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) pathways (LY294002 and wortmannin) or p42/p44 mitogen-activated protein (MAP) kinase (PD98059 and U0126). BDNF treatment induced an increase in the number of calbindin-positive neurons but not in the number of GABAergic or total cells. Furthermore, BDNF increased the degree of dendritic arborization, soma area and axon length of striatal neurons. However, PD98059 was more effective blocking BDNF effects on calbindin- than on GABA-positive neurons, whereas LY294002 inhibited morphological differentiation in both neuronal populations. Moreover, BDNF induced neuronal survival only through the activation of the PI3-K pathway.

Developmental Regulation of Sensory Neurite Growth by the Tumor Necrosis Factor Superfamily Member LIGHT

In a PCR screen to identify novel cytokine candidates involved in neuronal development, we identified transcripts for the tumor necrosis factor superfamily member 14 (TNFSF14), generally known as LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells), together with its receptors, lymphotoxin-β receptor (LTβR) and TNF family receptor herpesvirus entry mediator (HVEM), in the experimentally tractable sensory neurons of the mouse nodose ganglion. Immunocytochemistry revealed coexpression of LIGHT and its receptors in all nodose ganglion neurons in neonates. Enhancing LIGHT signaling in these neurons by overexpressing LIGHT inhibited BDNF-promoted neurite growth during a narrow window of development in the immediate perinatal period without affecting neuronal survival. Overexpressing a LIGHT mutant that selectively activates HVEM, but not one that selectively activates LTβR, also inhibited BDNF-promoted growth, suggesting that neurite growth inhibition is mediated via HVEM. Blocking HVEM signaling by a function-blocking anti-HVEM antibody significantly enhanced neurite growth from nodose neurons grown both with and without BDNF. Likewise, neurons from LIGHT-deficient neonates exhibited significantly greater neurite growth than neurons from wild-type littermates in both the presence and absence of BDNF. LIGHT overexpression significantly inhibited NF-κB activity, while preventing LIGHT-induced NF-κB inhibition by overexpressing the p65 and p50 NF-κB subunits prevented LIGHT-mediated growth inhibition. Together, these findings show that LIGHT/HVEM signaling negatively regulates neurite growth from developing sensory neurons via NF-κB inhibition.

Bone morphogenetic protein-6 is a neurotrophic factor for calbindin-positive striatal neurons.

Bone morphogenetic proteins (BMPs) are a set of members of the transforming growth factor‐β superfamily recently described as promoting the differentiation of several neuronal populations within the basal ganglia. This study examined whether a member of this family, BMP‐6, could exert neurotrophic effects on the neurons of the striatum, in which BMP‐6 mRNA had been previously detected during development. Here we show that BMP‐6 increases the number and differentiation of calbindin‐positive neurons in vitro. Indeed, BMP‐6 increased the total area, the perimeter, and the degree of arborization of this neuronal population. This trophic factor promoted dendritic growth without modifying axonal length or soma area. Furthermore, BMP‐6 increased the number of glial fibrillary acidic protein‐positive cells while decreasing the number of nestin‐positive cells. The suppression of cell proliferation or glial development by the antimitotic fluorodeoxyuridine removed the effects on striatal neurons, suggesting the involvement of astroglial cells in the differentiation induced by BMP‐6. The current results confirm the relevance of BMPs in the development of the striatum and emphasize the crucial importance of the trophic interaction between glial and neuronal cells.

Age-dependent maintenance of motor control and corticostriatal innervation by death receptor 3.

Death receptor 3 is a proinflammatory member of the immunomodulatory tumor necrosis factor receptor superfamily, which has been implicated in several inflammatory diseases such as arthritis and inflammatory bowel disease. Intriguingly however, constitutive DR3 expression has been detected in the brains of mice, rats, and humans, although its neurological function remains unknown. By mapping the normal brain expression pattern of DR3, we found that DR3 is expressed specifically by cells of the neuron lineage in a developmentally regulated and region-specific pattern. Behavioral studies on DR3-deficient (DR3ko) mice showed that constitutive neuronal DR3 expression was required for stable motor control function in the aging adult. DR3ko mice progressively developed behavioral defects characterized by altered gait, dyskinesia, and hyperactivity, which were associated with elevated dopamine and lower serotonin levels in the striatum. Importantly, retrograde tracing showed that absence of DR3 expression led to the loss of corticostriatal innervation without significant neuronal loss in aged DR3ko mice. These studies indicate that DR3 plays a key nonredundant role in the retention of normal motor control function during aging in mice and implicate DR3 in progressive neurological disease.