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In the central nervous system, most neurons co-express TrkB and TrkC, the tyrosine kinase receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). As NT3 can also activate TrkB, it has been difficult to understand how NT3 and TrkC can exert unique roles in the assembly of neuronal circuits. Using neurons differentiated from human embryonic stem cells expressing both TrkB and TrkC, we compared Trk activation by BDNF and NT3. To avoid the complications resulting from TrkB activation by NT3, we also generated neurons from stem cells engineered to lack TrkB. We found that NT3 activates TrkC at concentrations lower than those of BDNF needed to activate TrkB. Downstream of Trk activation, the changes in gene expression caused by TrkC activation were found to be similar to those resulting from TrkB activation by BDNF, including a number of genes involved in synaptic plasticity. At high NT3 concentrations, receptor selectivity was lost as a result of TrkB activation. In addition, TrkC was down-regulated, as was also the case with TrkB at high BDNF concentrations. By contrast, receptor selectivity as well as reactivation were preserved when neurons were exposed to low neurotrophin concentrations. These results indicate that the selectivity of NT3/TrkC signalling can be explained by the ability of NT3 to activate TrkC at concentrations lower than those needed to activate TrkB. They also suggest that in a therapeutic perspective, the dosage of Trk receptor agonists will need to be taken into account if prolonged receptor activation is to be achieved.

In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using ‘humanized’ mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group (P ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

Author(s): Naegelin, Yvonne; Saeuberli, Katharina; Schaedelin, Sabine; Dingsdale, Hayley; Magon, Stefano; Baranzini, Sergio; Amann, Michael; Parmar, Katrin; Tsagkas, Charidimos; Calabrese, Pasquale; Penner, Iris Katharina; Kappos, Ludwig; Barde, Yves-Alain | Abstract: ObjectiveTo determine the levels of brain-derived neurotrophic factor (BDNF) in the serum of patients suffering from multiple sclerosis (MS) to evaluate the potential of serum BDNF as a biomarker for MS.MethodsUsing a recently validated enzyme-linked immunoassay (ELISA) we measured BDNF in patients with MS (pwMS), diagnosed according to the 2001 McDonald criteria and aged between 18 and 70nyears, participating in a long-term cohort study with annual clinical visits, including blood sampling, neuropsychological testing, and brain magnetic resonance imaging (MRI). The results were compared with an age- and sex-matched cohort of healthy controls (HC). Correlations between BDNF levels and a range of clinical and magnetic resonance imaging variables were assessed using an adjusted linear model.ResultsIn total, 259 pwMS and 259 HC were included, with a mean age of 44.42n±n11.06 and 44.31n±n11.26nyears respectively. Eleven had a clinically isolated syndrome (CIS), 178 relapsing remitting MS (RRMS), 56 secondary progressive MS (SPMS), and 14 primary progressive MS (PPMS). Compared with controls, mean BDNF levels were lower by 8 % (p˂0.001) in pwMS. The level of BDNF in patients with SPMS was lower than in RRMS (pn=n0.004).InterpretationWe conclude that while the use of comparatively large cohorts enables the detection of a significant difference in BDNF levels between pwMS and HC, the difference is small and unlikely to usefully inform decision-making processes at an individual patient level.

Funder: European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement 663830, Funder: Welsh Government’s Sêr Cymru programme, Funder: NHS Blood and Transplant, Brain-derived neurotrophic factor (BDNF) plays crucial roles in brain function. Numerous studies report alterations in BDNF levels in human serum in various neurological conditions, including mood disorders such as depression. However, little is known about BDNF levels in the blood during pregnancy. We asked whether maternal depression and/or anxiety during pregnancy were associated with altered serum BDNF levels in mothers (n = 251) and their new-born infants (n = 212). As prenatal exposure to maternal mood disorders significantly increases the risk of neurological conditions in later life, we also examined the possibility of placental BDNF transfer by developing a new mouse model. We found no association between maternal symptoms of depression and either maternal or infant cord blood serum BDNF. However, maternal symptoms of anxiety correlated with significantly raised maternal serum BDNF exclusively in mothers of boys (r = 0.281; P = 0.005; n = 99). Serum BDNF was significantly lower in male infants than female infants but neither correlated with maternal anxiety symptoms. Consistent with this observation, we found no evidence for BDNF transfer across the placenta. We conclude that the placenta protects the developing fetus from maternal changes in serum BDNF that could otherwise have adverse consequences for fetal development.

Background Rett syndrome (RS) is a severe neurodevelopmental disorder for which there is no approved therapy. This study aimed to assess safety and efficacy of oral fingolimod in children with RS using a pre-post and case–control design. Methods At the University of Basel Children’s Hospital, Basel, Switzerland, children with RS were included if they were older than 6 years and met the established diagnostic criteria of RS, including a positive MeCP2 mutation. Participants were observed 6 months before and after treatment and received 12 months of fingolimod treatment. Serum samples of 50 children without RS served as reference for brain-derived neurotrophic factor (BDNF) measurements. Primary outcome measures were safety and efficacy, the latter measured by change in levels of BDNF in serum/CSF (cerebrospinal fluid) and change in deep gray matter volumes measured by magnetic resonance imaging (MRI). Secondary outcome measure was efficacy measured by change in clinical scores [Vineland Adaptive Behaviour Scale (VABS), Rett Severity Scale (RSSS) and Hand Apraxia Scale (HAS)]. Results Six children with RS (all girls, mean and SD age 11.3 ± 3.1 years) were included. Serum samples of 50 children without RS (25 females, mean and SD age 13.5 ± 3.9 years) served as reference for BDNF measurements. No serious adverse events occurred. Primary and secondary outcome measures were not met. CSF BDNF levels were associated with all clinical scores: RSSS (estimate − 0.04, mult.effect 0.96, CI [0.94; 0.98], p = 0.03), HAS (estimate − 0.09, mult.effect 0.91, CI [0.89; 0.94], p < 0.01) and VABS (communication: estimate 0.03, mult.effect 1.03, CI [1.02; 1.04], p Conclusions In children with RS, treatment with fingolimod was safe. The study did not provide supportive evidence for an effect of fingolimod on clinical, laboratory, and imaging measures. CSF BDNF levels were associated with clinical scores, indicating a need to further evaluate its potential as a biomarker for RS. This finding should be further validated in independent patient groups. Trial Registration Clinical Trials.gov NCT02061137, registered on August 27th 2013, https://clinicaltrials.gov/ct2/show/study/NCT02061137.

Additional file 8. Liver enzymes over the study period. A: ASAT, B: ALAT, C: GGT.

Additional file 1. Overview of visits during the study period. V = Visit; SC = Screening; BL = Baseline; FD = Fist Dose; M = Month; X = BDNF sapling and all endpoint variable measurements.

Additional file 3. Baseline characteristics of healthy control children.

Additional file 2. Modified Grand Total of EEG Score (GTE-Score).

Additional file 6. Changes of clinical scores over time. Change is indicated by absolute numbers of scores, eg. BL 7 and change over time -1, 3, -6: score changed from 7 to 6,9 and 3 respectively within the given time period. Changes over time in all scales and patients were not different in the intervals before during and after treatment. VABS = Vineland Adaptive Behaviour Scale; RSSS = Rett Severity Scale; HAS = Hand Apraxia Scale; M = Month of Study.

Additional file 4. BDNF(A) and NfL (B) in HC plotted by age and gender. Data of patients aged >15 years are shaded.

Visual Abstract, While BDNF is receiving considerable attention for its role in synaptic plasticity and in nervous system dysfunction, identifying brain circuits involving BDNF-expressing neurons has been challenging. BDNF levels are very low in most brain areas, except for the large mossy fiber terminals in the hippocampus where BDNF accumulates at readily detectable levels. This report describes the generation of a mouse line allowing the detection of single brain cells synthesizing BDNF. A bicistronic construct encoding BDNF tagged with a P2A sequence preceding GFP allows the translation of BDNF and GFP as separate proteins. Following its validation with transfected cells, this construct was used to replace the endogenous Bdnf gene. Viable and fertile homozygote animals were generated, with the GFP signal marking neuronal cell bodies translating the Bdnf mRNA. Importantly, the distribution of immunoreactive BDNF remained unchanged, as exemplified by its accumulation in mossy fiber terminals in the transgenic animals. GFP-labeled neurons could be readily visualized in distinct layers in the cerebral cortex where BDNF has been difficult to detect with currently available reagents. In the hippocampal formation, quantification of the GFP signal revealed that

Significance Neurotrophin receptors are a class of receptor tyrosine kinases that couple to signaling pathways critical for neuronal survival and growth. One member, TrkB, is particularly interesting because it plays a role in many severe degenerative neurological diseases. The TrkB natural ligand brain-derived neurotrophic factor (BDNF) is not suitable to be developed as a drug or therapy as proved by previous unsuccessful clinical trials. Here we report a selection method that produced potent full agonist antibodies that mimic BDNF function, yet with better biophysical properties. This study paves the road for the development of agonist antibodies for other receptor tyrosine kinases., The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.

Visual Abstract, Brain-derived neurotrophic factor (BDNF) secreted by neurons is a significant component of synaptic plasticity. In humans, it is also present in blood platelets where it accumulates following its biosynthesis in megakaryocytes. BDNF levels are thus readily detectable in human serum and it has been abundantly speculated that they may somehow serve as an indicator of brain function. However, there is a great deal of uncertainty with regard to the range of BDNF levels that can be considered normal, how stable these values are over time and even whether BDNF levels can be reliably measured in serum. Using monoclonal antibodies and a sandwich ELISA, this study reports on BDNF levels in the serum of 259 volunteers with a mean value of 32.69 ± 8.33 ng/ml (SD). The mean value for the same cohort after 12 months was not significantly different (N = 226, 32.97 ± 8.36 ng/ml SD, p = 0.19). Power analysis of these values indicates that relatively large cohorts are necessary to identify significant differences, requiring a group size of 60 to detect a 20% change. The levels determined by ELISA could be validated by Western blot analyses using a BDNF monoclonal antibody. While no association was observed with gender, a weak, positive correlation was found with age. The overall conclusions are that BDNF levels can be reliably measured in human serum, that these levels are quite stable over one year, and that comparisons between two populations may only be meaningful if cohorts of sufficient sizes are assembled.

Pain is not something universally enjoyed, especially chronic pain involving nerve damage, referred to as neuropathic pain ( 1 ). Pain perception can be modulated in a variety of ways—for example, by focusing attention on the painful stimulus. On page 1168 of this issue, Peng et al. ( 2 ) report that pain threshold in dorsal root ganglion neurons (DRG), which relay peripheral sensory information to the central nervous system, can be modulated by mechanisms involving a specific cluster of microRNAs (miRNAs). Remarkably, the same cluster also regulates the threshold of neuropathic pain in DRG neurons that would not be involved in pain under normal conditions. The study provides important new insights into molecular mechanisms that control pain threshold, both in normal and pathological conditions.

The concept that target tissues determine the survival of neurons has inspired much of the thinking on neuronal development in vertebrates, not least because it is supported by decades of research on nerve growth factor (NGF) in the peripheral nervous system (PNS). Recent discoveries now help to understand why only some developing neurons selectively depend on NGF. They also indicate that the survival of most neurons in the central nervous system (CNS) is not simply regulated by single growth factors like in the PNS. Additionally, components of the cell death machinery have begun to be recognized as regulators of selective axonal degeneration and synaptic function, thus playing a critical role in wiring up the nervous system.

Contrasting with the long-established retrograde model for neurotrophin function, specific immunohistochemical localization of brain-derived neurotrophic factor in the central nervous system supports the alternative model of presynaptic localization and anterograde function., Although brain-derived neurotrophic factor (BDNF) regulates numerous and complex biological processes including memory retention, its extremely low levels in the mature central nervous system have greatly complicated attempts to reliably localize it. Using rigorous specificity controls, we found that antibodies reacting either with BDNF or its pro-peptide both stained large dense core vesicles in excitatory presynaptic terminals of the adult mouse hippocampus. Both moieties were ∼10-fold more abundant than pro-BDNF. The lack of postsynaptic localization was confirmed in Bassoon mutants, a seizure-prone mouse line exhibiting markedly elevated levels of BDNF. These findings challenge previous conclusions based on work with cultured neurons, which suggested activity-dependent dendritic synthesis and release of BDNF. They instead provide an ultrastructural basis for an anterograde mode of action of BDNF, contrasting with the long-established retrograde model derived from experiments with nerve growth factor in the peripheral nervous system.

The functional relevance of brain-derived neurotrophic factor (BDNF) is beginning to be well appreciated not only in mice, but also in humans. Because reduced levels typically correlate with impaired neuronal function, increasing BDNF levels with well-tolerated drugs diffusing into the central nervous system may help in ameliorating functional deficits. With this objective in mind, we used the sphingosine-1 phosphate receptor agonist fingolimod, a drug that crosses the blood–brain barrier. In addition, fingolimod has recently been introduced as the first oral treatment for multiple sclerosis. In cultured neurons, fingolimod increases BDNF levels and counteracts NMDA-induced neuronal death in a BDNF-dependent manner. Ongoing synaptic activity and MAPK signaling is required for fingolimod-induced BDNF increase, a pathway that can also be activated in vivo by systemic fingolimod administration. Mice lacking Mecp2, a gene frequently mutated in Rett syndrome, show decreased levels of BDNF, and fingolimod administration was found to partially rescue these levels as well as the size of the striatum, a volumetric sensor of BDNF signaling in rodents. These changes correlate with increased locomotor activity of the Mecp2 -deficient animals, suggesting that fingolimod may improve the functional output of the nervous system, in addition to its well-documented effects on lymphocyte egress from lymph nodes.

Although brain-derived neurotrophic factor (BDNF) is linked with an increasing number of conditions causing brain dysfunction, its role in the postnatal CNS has remained difficult to assess. This is because thebdnf-null mutation causes the death of the animals before BDNF levels have reached adult levels. In addition, the anterograde axonal transport of BDNF complicates the interpretation of area-specific gene deletion. The present study describes the generation of a new conditional mouse mutant essentially lacking BDNF throughout the CNS. It shows that BDNF is not essential for prolonged postnatal survival, but that the behavior of such mutant animals is markedly altered. It also reveals that BDNF is not a major survival factor for most CNS neurons and for myelination of their axons. However, it is required for the postnatal growth of the striatum, and single-cell analyses revealed a marked decreased in dendritic complexity and spine density. In contrast, BDNF is dispensable for the growth of the hippocampus and only minimal changes were observed in the dendrites of CA1 pyramidal neurons in mutant animals. Spine density remained unchanged, whereas the proportion of the mushroom-type spine was moderately decreased. In line with thesein vivoobservations, we found that BDNF markedly promotes the growth of cultured striatal neurons and of their dendrites, but not of those of hippocampal neurons, suggesting that the differential responsiveness to BDNF is part of a neuron-intrinsic program.

Although brain-derived neurotrophic factor is the most abundant and widely distributed neurotrophin in the nervous system, reproducible determinations of its levels have been hampered by difficulties in raising suitable monoclonal antibodies. Following immunization of mice with recombinant fish and mammalian brain-derived neurotrophic factor, monoclonal antibodies were generated and used in an immunoassay based on the recognition of two different epitopes. Neither antibody crossreacts with neurotrophin homodimers other than brain-derived neurotrophic factor, although reactivity was detected with brain-derived neurotrophic factor/neurotrophin-3 heterodimers. As both nerve growth factor and neurotrophin-3 are known to affect the development of a variety of neurons expressing the brain-derived neurotrophic factor (bdnf) gene, this assay was used to determine levels in tissues isolated from newborn mice carrying a null mutation in the nerve growth factor (ngf) or the neurotrophin-3 (nt3) gene. Marked differences were observed between mutants and wild-type littermates in the PNS, but not in the CNS, suggesting that neither nerve growth factor nor neurotrophin-3 is a unique regulator of brain-derived neurotrophic factor levels in the newborn mouse CNS.

The in vitro generation of uniform populations of neurons from mouse embryonic stem cells (ESCs) provides a novel opportunity to study gene function in neurons. This is of particular interest when mutations lead to lethal in vivo phenotypes. Although the amyloid precursor protein (APP) and its proteolysis are regarded as key elements of the pathology of Alzheimer's disease, the physiological function of APP is not well understood and mice lacking App and the related gene Aplp2 die early postnatally without any obvious histopathological abnormalities. Here we show that glutamatergic neurons differentiated from ESCs lacking both genes reveal a decreased expression of the vesicular glutamate transporter 2 (VGLUT2) both at the mRNA and protein level, as well as a reduced uptake and/or release of glutamate. Blocking γ-secretase cleavage of APP in wild-type neurons resulted in a similar decrease of VGLUT2 expression, whereas VGLUT2 levels could be restored in App−/−Aplp2−/− neurons by a construct encompassing the C-terminal intracellular domain of APP. Electrophysiological recordings of hippocampal organotypic slice cultures prepared from corresponding mutant mice corroborated these observations. Gene expression profiling and pathway analysis of the differentiated App−/−Aplp2−/− neurons identified dysregulation of additional genes involved in synaptic transmission pathways. Our results indicate a significant functional role of APP and amyloid precursor-like protein 2 (APLP2) in the development of synaptic function by the regulation of glutamatergic neurotransmission. Differentiation of ESCs into homogeneous populations thus represents a new opportunity to explore gene function and to dissect signaling pathways in neurons. Disclosure of potential conflicts of interest is found at the end of this article.

SummaryPax6-positive radial glial (RG) cells are the progenitors of most glutamatergic neurons in the cortex, a lineage that can be recapitulated in vitro using embryonic stem (ES) cells. We show here that ES cells lacking Pax6, a transcription factor long known to be essential for cortical development, generate Mash1-positive RG cells that differentiate in GABAergic neurons. These neurons express high levels of the neurotrophin receptor p75NTR causing their rapid death. Pax6 function was also investigated following transplantation of ES cells in the developing chick telencephalon and in mice lacking both Pax6 and p75NTR. Taken together, our results indicate that reliable predictions can be made with cultured ES cells when used to explore the role of genes impacting early aspects of mammalian neurogenesis. They also provide a novel opportunity to compare the molecular constituents of glutamatergic with those of GABA-ergic neurons and to explore the mechanisms of their generation.

In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.

Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins—Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere‐forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes. GLIA 2015;63:2340–2361

We used cultured adult mouse retinae as a model system to follow and quantify the retraction of dendrites using diolistic labelling of retinal ganglion cells (RGCs) following explantation. Cell death was monitored in parallel by nuclear staining as ‘labelling’ with RGC and apoptotic markers was inconsistent and exceedingly difficult to quantify reliably. Nuclear staining allowed us to delineate a lengthy time window during which dendrite retraction can be monitored in the absence of RGC death. The addition of brain‐derived neurotrophic factor (BDNF) produced a marked reduction in dendritic degeneration, even when application was delayed for 3 days after retinal explantation. These results suggest that the delayed addition of trophic factors may be functionally beneficial before the loss of cell bodies in the course of conditions such as glaucoma.

The correlation between functional and structural neuronal plasticity is by now well documented. However, the molecular mechanisms translating patterns of neuronal activity into specific changes in the structure of neurons remain unclear. Neurotrophins can be released in an activity-dependent manner, and they are capable of controlling both neuronal morphology and functional synaptic changes. They are thus attractive molecules to be studied in the context of synaptic plasticity. In the CNS, most of the work so far has focused on the role of BDNF and of its tyrosine kinase B receptor (TrkB), but relatively little is known about the function of the pan-neurotrophin receptor p75NTR. In this study, we show in loss-of-function experiments that postnatal hippocampal pyramidal cells in two mutant lines ofp75NTRhave a higher spine density and greater dendritic complexity than wild-type (WT) mice. Conversely, in a gain-of-function approach,p75NTRoverexpression in WT neurons significantly reduces dendritic complexity, as well as spine density in all dendritic compartments. These results show that p75NTRnegatively modulates dendritic morphology in adult hippocampal pyramidal neurons and documents a new case of functional antagonism between Trk and p75NTRsignaling.

Neurotrophins are involved in the modulation of synaptic transmission, including the induction of long-term potentiation (LTP) through the receptor TrkB. Because previous studies have revealed a bidirectional mode of neurotrophin action by virtue of signaling through either the neurotrophin receptor p75 NTR or the Trk receptors, we tested the hypothesis that p75 NTR is important for longterm depression (LTD) to occur. Although LTP was found to be unaffected in hippocampal slices of two different strains of mice carrying mutations of the p75 NTR gene, hippocampal LTD was impaired in both p75 NTR -deficient mouse strains. Furthermore, the expression levels of two ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, GluR2 and GluR3, but not GluR1 or GluR4, were found to be significantly altered in the hippocampus of p75 NTR -deficient mice. These results implicate p75 NTR in activity-dependent synaptic plasticity and extend the concept of functional antagonism of the neurotrophin signaling system.

Radial glial cells have been identified as a major source of neurons during development. Here, we review the evidence for the distinct "glial" nature of radial glial cells and contrast these cells with their progenitors, the neuroepithelial cells. Recent results also suggest that not only during neurogenesis in vivo, but also during the differentiation of cultured embryonic stem cells toward neurons, progenitors with clear glial antigenic characteristics act as cellular intermediates.

Programmed cell death, or apoptosis, accompanies the development of many tissues, including the vertebrate nervous system. Most neurons are eliminated soon after synaptic contacts have been made between the neurons and their targets. This inspired the neurotrophic theory, which proposes that neurons compete for limited quantities of target-derived survival factors (1–3). Work on nerve growth factor (NGF) in the peripheral nervous system (PNS) (4) gave strong support for this theory: Not only is NGF essential for the survival of specific populations of neurons, but it is also localized in tissues innervated by NGF-responsive neurons in amounts that parallel the density of innervation (5). However, the finding by Southwell et al. (6) that programmed cell death in a major population of neurons in the central nervous system (CNS) is caused by an intrinsic program independent of external cues cannot be readily accommodated by the neurotrophic theory.

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG)cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.

Neurofibrillary tangles are composed of insoluble aggregates of the microtubule-associated protein tau. In Alzheimer's disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non-mutant human tau, in the absence of other exogenous factors, including β-amyloid. The pathology in these mice is Alzheimer-like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.

Hans Thoenen passed away on June 23, 2012, a few months after being diagnosed with lung cancer. He left us grateful for what he had been able to accomplish in his life as scientist, but he was neither exuberant nor proud. Hans remained extraordinarily modest about his achievements—he felt far more comfortable by understating his contributions and never liked receiving compliments from colleagues he did not know well. Given the choice, he preferred to have critics than adulators around him. “At least the former are honest,” he would say.

Pluripotency and the capacity for continuous self-renewal make embryonic stem (ES) cells an attractive donor source for cell-replacement strategies. A key prerequisite for a therapeutic application of ES cells is the generation of defined somatic cell populations. Here we demonstrate that a targeted insertion of the EGFP gene into the tau locus permits efficient fluorescence-activated cell sorting (FACS)-based lineage selection of ES cell-derived neurons. After in vitro differentiation of heterozygous tau EGFP ES cells into multipotent neural precursors, EGFP is selectively induced in postmitotic neurons of various neurotransmitter phenotypes. By using FACS, ES cell-derived neurons can be enriched to purities of more than 90%. Because neuron-specific EGFP fluorescence is also observed upon transplantation of ES cell-derived neural precursors, the tau EGFP mutant represents a useful tool for the in vivo analysis of grafted ES cell-derived neurons.

In response to injury and inflammation of the CNS, brain cells including microglia and astrocytes secrete tumor necrosis factor-alpha (TNF). This pro-inflammatory cytokine has been implicated in both neuronal cell death and survival. We now provide evidence that TNF affects the formation of neurites. Neurons cultured on astrocytic glial cells exhibited reduced outgrowth and branching of neurites after addition of recombinant TNF or prestimulation of glial cells to secrete TNF. This effect was absent in neurons of TNF receptor-deficient mice cultured on prestimulated glia of wild-type mice and was reverted by blocking TNF with soluble TNF receptor IgG fusion protein. TNF activated in neurons the small GTPase RhoA. By inactivating Rho with C3 transferase, the inhibitory effect of TNF on neurite outgrowth and branching was abolished. These results suggest that glia-derived TNF, as part of an injury or inflammatory process, can inhibit neurite elongation and branching during development and regeneration.

The zinc finger protein NRIF (neurotrophin receptor interacting factor) was originally identified by virtue of its interaction with the neurotrophin receptor p75NTR and its participation in embryonic apoptosis. Targeted deletion of the nrif gene in mice is embryonically lethal in the C57BL6 genetic background, where it blocks cell cycle progression, but not in the Sv129 strain. We have now identified a second, highly homologous nrif gene, designated nrif2, encoding a protein with similar structural and biochemical properties as well as subcellular distribution as NRIF1, and whose over-expression in transfected fibroblasts also correlates with impaired BrdU incorporation. Unexpectedly, the nrif2 transcript becomes significantly upregulated in nrif1-/- mice only in Sv129, the genetic background where the mutants are viable, suggesting that the functional complementation of the two nrif genes may be strain-specific.

This study reports on the full-length cDNA cloning of a gene identified on the basis of its preferential expression in nerve growth factor, compared with neurotrophin-3-dependent neurons. It encodes a putative 7-transmembrane polypeptide that is distantly related to other members of the G protein-coupled receptor superfamily. Unique features of this receptor include a very long carboxy-terminal tail of 360 amino acids and a specific expression pattern in the chick peripheral nervous system, including nerve growth factor-dependent sensory and sympathetic neurons, as well as enteric neurons. In the central nervous system, the receptor is strongly developmentally regulated and is expressed at high levels in the external granule cell layer of the cerebellum, as well as in motoneurons of the spinal cord, and in retinal ganglion cells.

Here, we review progress in our understanding of neuronal and glial cell biology during the past ten years, with an emphasis on glial cell fate specification, apoptosis, the cytoskeleton, neuronal polarity, synaptic vesicle recycling and targeting, regulation of the cytoskeleton by extracellular signals, and neuron-glia interactions.

This study examines the mechanisms by which the tyrosine kinase receptor TrkB is down-regulated following binding of brain-derived neurotrophic factor (BDNF). In primary cultures of cerebellar granule neurons, BDNF-induced reduction of TrkB receptors was largely prevented by the addition of specific proteasome inhibitors. HN10 cells, a neuronal cell line that can be readily transfected, also showed a marked down-regulation of cell surface TrkB following BDNF exposure. In addition, we observed that prolonged exposure to nerve growth factor of TrkA-transfected cells did not lead to the down-regulation seen with BDNF and TrkB. TrkA and TrkB chimeric molecules were therefore expressed in HN10 cells and tested for ligand-induced regulation. These experiments led to the conclusion that the motives responsible for down-regulation are contained in the cytoplasmic domain of TrkB, and a short sequence in the juxtamembrane domain of TrkB was identified that confers nerve growth factor-induced down-regulation when inserted into TrkA.

The role of nerve growth factor (NGF) and of the neurotrophin receptor p75 (p75NTR) in programmed cell death was investigated in the retina and the spinal cord of mouse embryos. Large numbers of cells express p75NTR in and along the developing optic nerve and in the mantle zone of the spinal cord. In embryos carrying deletions in the ngf or the p75NTR gene, cell death was reduced in the retina and in the spinal cord. Increased numbers of Islet-1-immunoreactive cells were detected in the dorsal spinal cord, and the mantle zone was enlarged in both mutants. These results indicate that NGF/p75NTR-dependent mechanisms are used to remove cells when axonal tracts elongate in developing neuroepithelia.

Nerve growth factor (NGF) was characterized over 4 decades ago, and like the other neurotrophins subsequently discovered, it is best known for its trophic role, including the prevention of programmed cell death in specific populations of neurones in the peripheral nervous system. This property can be accounted for by the activation of a tyrosine kinase receptor. NGF also regulates neuronal function, as illustrated by its role in pain and inflammation, and in synaptic plasticity. Finally, NGF recently was shown to activate the neurotrophin receptor p75 (p75NTR), a receptor with no intrinsic catalytic activity and with similarities to members of the tumor necrosis factor receptor family. During normal development, the activation of p75NTR by NGF actually kills cells in the central nervous system. One remarkable property of NGF is then that it controls cell numbers in opposite ways in the developing nervous system, a result of its unique ability to activate two different receptor types.

While nerve growth factor (NGF) is best known for its trophic functions, recent experiments indicate that it can also cause cell death during development by activating the neurotrophin receptor p75. We now identify microglial cells as the source of NGF as a killing agent in the developing eye. When the retina is separated from the surrounding tissue before colonization by microglial cells, no NGF can be detected, and cell death is dramatically reduced. It is restored by the addition of microglial cells, an effect that is blocked by NGF antibodies. NGF adsorbed at the surface of beads, but not soluble NGF, mimics the killing action of microglial cells. These results indicate an active role for macrophages in neuronal death.

High-affinity neurotrophin-3 (NT3) receptors have been identified on nerve growth factor (NGF)-dependent sympathetic neurons, but their occupancy by NT3 does not lead to neuronal survival. The molecular nature of these NT3 binding sites was investigated in this study. With freshly dissociated embryonic day 11 (E11) chick sympathetic neurons, cross-linking experiments revealed that the main receptor responsible for high-affinity specific binding was the neurotrophin receptor p75 (p75NTR), with only a small fraction corresponding to trkC. When E11 sympathetic neurons were cultured in the presence of NGF,trkCtranscripts became undetectable, but high-affinity specific NT3 binding persisted. Cross-linking and antibody inhibition experiments indicated that p75NTRwas the only detectable NT3 receptor protein. These characteristics were not observed when p75NTRwas expressed in transformed cells. We conclude that p75NTRcan exist in neurons in a confirmation conferring hitherto unrecognized properties to this receptor.

While brain-derived neurotrophic factor (BDNF) delays the death of axotomized retinal ganglion cells in rodents, it is unclear if it affects any aspect of the normal development of these cells. Here we examined the optic nerve of bdnf-/- mice. Axonal numbers were normal, but their diameter, as well as the proportion of myelinated axons, was reduced at postnatal day 20 (P20). In contrast, the facial nerve was not hypomyelinated. Expression levels of mRNAs coding for the myelin proteins PLP and MBP were substantially reduced in the hippocampus and cortex at P20, but not in the sciatic nerve. Intraventricular injections of BDNF into the ventricles of wild-type mice at P10 and P12 up-regulated expression of PLP in the hippocampus at P14. These results indicate a role of BDNF, discussed as indirect, in the control of myelination in the central nervous system.