Selective activation and down-regulation of Trk receptors by neurotrophins in human neurons co-expressing TrkB and TrkC.

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.

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library.

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.

Brain-derived Neurotrophic Factor in Megakaryocytes.

The biosynthesis of endogenous brain-derived neurotrophic factor (BDNF) has thus far been examined in neurons where it is expressed at very low levels, in an activity-dependent fashion. In humans, BDNF has long been known to accumulate in circulating platelets, at levels far higher than in the brain. During the process of blood coagulation, BDNF is released from platelets, which has led to its extensive use as a readily accessible biomarker, under the assumption that serum levels may somehow reflect brain levels. To identify the cellular origin of BDNF in platelets, we established primary cultures of megakaryocytes, the progenitors of platelets, and we found that human and rat megakaryocytes express the BDNF gene. Surprisingly, the pattern of mRNA transcripts is similar to neurons. In the presence of thapsigargin and external calcium, the levels of the mRNA species leading to efficient BDNF translation rapidly increase. Under these conditions, pro-BDNF, the obligatory precursor of biologically active BDNF, becomes readily detectable. Megakaryocytes store BDNF in α-granules, with more than 80% of them also containing platelet factor 4. By contrast, BDNF is undetectable in mouse megakaryocytes, in line with the absence of BDNF in mouse serum. These findings suggest that alterations of BDNF levels in human serum as reported in studies dealing with depression or physical exercise may primarily reflect changes occurring in megakaryocytes and platelets, including the ability of the latter to retain and release BDNF.

Brain-derived neurotrophic factor prevents dendritic retraction of adult mouse retinal ganglion cells.

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.

Neurotrophin receptors TrkA and TrkC cause neuronal death whereas TrkB does not.

Neurons of the peripheral nervous system have long been known to require survival factors to prevent their death during development. But why they selectively become dependent on secretory molecules has remained a mystery, as is the observation that in the central nervous system, most neurons do not show this dependency. Using engineered embryonic stem cells, we show here that the neurotrophin receptors TrkA and TrkC (tropomyosin receptor kinase A and C, also known as Ntrk1 and Ntrk3, respectively) instruct developing neurons to die, both in vitro and in vivo. By contrast, TrkB (also known as Ntrk2), a closely related receptor primarily expressed in the central nervous system, does not. These results indicate that TrkA and TrkC behave as dependence receptors, explaining why developing sympathetic and sensory neurons become trophic-factor-dependent for survival. We suggest that the expansion of the Trk gene family that accompanied the segregation of the peripheral from the central nervous system generated a novel mechanism of cell number control.

Astrocyte reactivity after brain injury-: The role of galectins 1 and 3.

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.

Fingolimod, a sphingosine-1 phosphate receptor modulator, increases BDNF levels and improves symptoms of a mouse model of Rett syndrome.

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.

Target genes of Topoisomerase IIß regulate neuronal survival and are defined by their chromatin state.

Topoisomerases are essential for DNA replication in dividing cells, but their genomic targets and function in postmitotic cells remain poorly understood. Here we show that a switch in the expression from Topoisomerases IIα (Top2α) to IIß (Top2ß) occurs during neuronal differentiation in vitro and in vivo. Genome-scale location analysis in stem cell-derived postmitotic neurons reveals Top2ß binding to chromosomal sites that are methylated at lysine 4 of histone H3, a feature of regulatory regions. Indeed Top2ß-bound sites are preferentially promoters and become targets during the transition from neuronal progenitors to neurons, at a time when cells exit the cell cycle. Absence of Top2ß protein or its activity leads to changes in transcription and chromatin accessibility at many target genes. Top2ß deficiency does not impair stem cell properties and early steps of neuronal differentiation but causes premature death of postmitotic neurons. This neuronal degeneration is caused by up-regulation of Ngfr p75, a gene bound and repressed by Top2ß. These findings suggest a chromatin-based targeting of Top2ß to regulatory regions in the genome to govern the transcriptional program associated with neuronal differentiation and longevity.

Seizure-induced neuronal death is suppressed in the absence of the endogenous lectin Galectin-1.

Pilocarpine injection induces epileptic seizures in rodents, an experimental paradigm extensively used to model temporal lobe epilepsy in humans. It includes conspicuous neuronal death in the forebrain and previous work has demonstrated an involvement of the neurotrophin receptor p75(NTR) in this process. Following the identification of Galectin-1 (Gal-1) as a downstream effector of p75(NTR), we examine here the role of this endogenous lectin in pilocarpine-induced cell death in adult mice. We found that most somatostatin-positive neurons also express Gal-1 and that in mice lacking the corresponding gene Lgals1, pilocarpine-induced neuronal death was essentially abolished in the forebrain. We also found that the related lectin Galectin-3 (Gal-3) was strongly upregulated by pilocarpine in microglial cells. This upregulation was absent in Lgals1 mutants and our results with Lgals3-null animals show that Gal-3 is not required for neuronal death in the hippocampus. These findings provide new insights into the roles and regulation of endogenous lectins in the adult CNS and a surprisingly selective proapoptotic role of Gal-1 for a subpopulation of GABAergic interneurons.

Disease modeling using embryonic stem cells: MeCP2 regulates nuclear size and RNA synthesis in neurons.

Mutations in the gene encoding the methyl-CpG-binding protein MECP2 are the major cause of Rett syndrome, an autism spectrum disorder mainly affecting young females. MeCP2 is an abundant chromatin-associated protein, but how and when its absence begins to alter brain function is still far from clear. Using a stem cell-based system allowing the synchronous differentiation of neuronal progenitors, we found that in the absence of MeCP2, the size of neuronal nuclei fails to increase at normal rates during differentiation. This is accompanied by a marked decrease in the rate of ribonucleotide incorporation, indicating an early role of MeCP2 in regulating total gene transcription, not restricted to selected mRNAs. We also found that the levels of brain-derived neurotrophic factor (BDNF) were decreased in mutant neurons, while those of the presynaptic protein synaptophysin increased at similar rates in wild-type and mutant neurons. By contrast, nuclear size, transcription rates, and BDNF levels remained unchanged in astrocytes lacking MeCP2. Re-expressing MeCP2 in mutant neurons rescued the nuclear size phenotype as well as BDNF levels. These results reveal a new role of MeCP2 in regulating overall RNA synthesis in neurons during the course of their maturation, in line with recent findings indicating a reduced nucleolar size in neurons of the developing brain of mice lacking Mecp2.

Neurotrophin signalling in the human nervous system.

This review focuses on neurotrophins and their tyrosine kinase receptors, with an emphasis on their relevance to the function and dysfunction in the human nervous system. It also deals with measurements of BDNF levels and highlights recent findings from our laboratory on TrkB and TrkC signalling in human neurons. These include ligand selectivity and Trk activation by neurotrophins and non-neurotrophin ligands. The ligand-induced down-regulation and re-activation of Trk receptors is also discussed.

Brain-derived neurotrophic factor measurements in mouse serum and plasma using a sensitive and specific enzyme-linked immunosorbent assay.

This study is about the quantification and validation of BDNF levels in mouse serum and plasma using a sensitive immunoassay. While BDNF levels are readily detectable in human serum, the functional implications of these measurements are unclear as BDNF released from human blood platelets is the main contributor to the serum levels of BDNF. As mouse platelets do not contain BDNF, this confounding factor is absent in the mouse. Accordingly, BDNF levels in mouse serum and plasma were found to be indistinguishable at 9.92 ± 1.97 pg/mL for serum and 10.58 ± 2.43 pg/mL for plasma (p = 0.473). These levels are approximately a thousand times lower than those measured in human serum and pre-adsorption with anti-BDNF, but not with anti-NGF or anti-NT3 monoclonal antibodies, markedly reduced the BDNF signal. These results open the possibility to explore the relevance of BDNF levels as a biomarker in accessible body fluids using existing mouse models mimicking human pathological conditions.

Brain-derived neurotrophic factor released from blood platelets prevents dendritic atrophy of lesioned adult central nervous system neurons.

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.

Heterozygous NTF4 mutations impairing neurotrophin-4 signaling in patients with primary open-angle glaucoma.

Glaucoma, a main cause of blindness in the developed world, is characterized by progressive degeneration of retinal ganglion cells (RGCs), resulting in irreversible loss of vision. Although members of the neurotrophin gene family in various species are known to support the survival of numerous neuronal populations, including RGCs, it is less clear whether they are also required for survival and maintenance of adult neurons in humans. Here, we report seven different heterozygous mutations in the Neurotrophin-4 (NTF4) gene accounting for about 1.7% of primary open-angle glaucoma patients of European origin. Molecular modeling predicted a decreased affinity of neurotrophin 4 protein (NT-4) mutants with its specific tyrosine kinase receptor B (TrkB). Expression of recombinant NT-4 carrying the most frequent mutation was demonstrated to lead to decreased activation of TrkB. These findings suggest a pathway in the pathophysiology of glaucoma through loss of neurotrophic function and may eventually open the possibility of using ligands activating TrkB to prevent the progression of the disease.

Biosynthesis and processing of endogenous BDNF: CNS neurons store and secrete BDNF, not pro-BDNF.

Pro- and mature BDNF activate very different receptors and intracellular pathways, potentially leading to either neuronal death or survival. Here we examined the biochemistry of endogenous BDNF in mouse neurons using sensitive reagents and found that pro-BDNF is rapidly converted intracellularly to mature BDNF, the latter being stored and released by excitatory input.

Global deprivation of brain-derived neurotrophic factor in the CNS reveals an area-specific requirement for dendritic growth.

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 the bdnf-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 these in vivo observations, 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.

Identification of a lectin causing the degeneration of neuronal processes using engineered embryonic stem cells.

Unlike the mechanisms involved in the death of neuronal cell bodies, those causing the elimination of processes are not well understood owing to the lack of suitable experimental systems. As the neurotrophin receptor p75(NTR) is known to restrict the growth of neuronal processes, we engineered mouse embryonic stem (ES) cells to express an Ngfr (p75(NTR)) cDNA under the control of the Mapt locus (the gene encoding tau), which begins to be active when ES cell-derived progenitors start elongating processes. This caused a progressive, synchronous degeneration of all processes, and a prospective proteomic analysis showed increased levels of the sugar-binding protein galectin-1 in the p75(NTR)-engineered cells. Function-blocking galectin-1 antibodies prevented the degeneration of processes, and recombinant galectin-1 caused the processes of wild-type neurons to degenerate first, followed by the cell bodies. In vivo, the application of a glutamate receptor agonist, a maneuver known to upregulate p75(NTR), led to an increase in the amount of galectin-1 and to the degeneration of neurons and their processes in a galectin-1-dependent fashion. Section of the sciatic nerve also rapidly upregulated levels of p75(NTR) and galectin-1 in terminal Schwann cells, and the elimination of nerve endings was delayed at the neuromuscular junction of mice lacking Lgals1 (the gene encoding galectin-1). These results indicate that galectin-1 actively participates in the elimination of neuronal processes after lesion, and that engineered ES cells are a useful tool for studying relevant aspects of neuronal degeneration that have been hitherto difficult to analyze.

Fingolimod in children with Rett syndrome: the FINGORETT study.

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 < 0.01/daily living: estimate 0.03, mult.effect 1.03, CI [1.02; 1.04], p < 0.01/social skills: estimate 0.07, mult.effect 1.08, CI [1.05; 1.11], p < 0.01/motoric skills: estimate 0.04, mult.effect 1.04, CI [1.03; 1.06], p = 0.02). 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 .

The placenta protects the fetal circulation from anxiety-driven elevations in maternal serum levels of brain-derived neurotrophic factor.

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.

A New Mouse Line Reporting the Translation of Brain-Derived Neurotrophic Factor Using Green Fluorescent Protein.

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 <10% of the neurons do not translate the Bdnf mRNA at detectable levels, with the highest proportion of strongly labeled neurons found in CA3.