Gabriele Baj

Dendritic trafficking of BDNF mRNA is mediated by translin and blocked by the G196A (Val66Met) mutation

Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.

BDNF mRNA splice variants display activity-dependent targeting to distinct hippocampal laminae

Brain-derived neurotrophic factor (BDNF) may exert contrasting effects depending on its different subcellular sites of action (soma, dendrites, axons). These contrasting effects may explain contradictory findings, for example that BDNF may favour or oppose epileptogenesis. We determined the distribution of five BDNF splice variants in the soma and dendrites of rat hippocampal principal neurons, after application of stimuli that prompt BDNF mRNA accumulation in dendrites (epileptogenic seizures). Under basal conditions, no BDNF mRNA splice variant was detectable in dendrites, while specific splice variants were found in dendrites in response to epileptogenic seizures. Three hours after pilocarpine administration, exon VI and exon II splice variants were found in dendrites, while exons I and IV transcripts displayed a strictly somatic localization. Three hours after kainate administration, only exon VI was found in dendrites. These data suggest that the regulated expression of different splice variants may provide a spatial code to ensure the delivery of BDNF to precise destinations in the cell soma or along the dendrites.

Spatial segregation of BDNF transcripts enables BDNF to differentially shape distinct dendritic compartments

BDNF is produced from many transcripts that display distinct subcellular localization, suggesting that spatially restricted effects occur as a function of genetic and physiological regulation. Different BDNF 5′ splice variants give a restricted localization in the cell body or the proximal and distal compartments of dendrites; however, the functional consequences are not known. Silencing individual endogenous transcripts or overexpressing BDNF-GFP transcripts in cultured neurons demonstrated that whereas some transcripts (1 and 4) selectively affected proximal dendrites, others (2C and 6) affected distal dendrites. Moreover, segregation of BDNF transcripts resulted in a highly selective activation of the BDNF TrkB receptor. These studies indicate that spatial segregation of BDNF transcripts enables BDNF to differentially shape distinct dendritic compartments.

Low serum truncated-BDNF isoform correlates with higher cognitive impairment in schizophrenia

Brain-derived neurotrophic factor (BDNF) is a key factor in learning and memory. Altered BDNF-signalling is thought to contribute to the pathogenesis of schizophrenia (SZ) especially in relation to cognitive deficits. However, analysis of serum BDNF as a potential biomarker in schizophrenia has provided controversial data. We hypothesized that these confounding results might be due to a differential regulation of BDNF precursor pro-BDNF (32 KDa) and proteolytic products mature (mat-BDNF; 14 KDa), and truncated-BDNF (28 KDa). Accordingly, we investigated the serum abundance of these BDNF isoforms and its relationship with cognitive impairment in schizophrenia. Schizophrenia was diagnosed with PANSS test. Abbreviated cognitive assessment included tests for attention, perceptual-motor skills, processing speed and memory. Using an ELISA assay, we found a slight reduction in serum BDNF levels in SZ patients (n = 40) with respect to healthy controls (HC, n = 40; p = 0.018). Western-blot analysis revealed increased serum pro-BDNF and mat-BDNF and reduced truncated-BDNF (p < 0.001) in SZ with respect to HC. Patients with an increase in pro-BDNF (n = 15/40) or mat-BDNF (n = 9/40) higher than the HC mean + 2 Standard Deviations (SD) also had >2SD reduction of truncated-BDNF (n = 27/40). Reduced truncated-BDNF correlated significantly with higher positive and lower negative PANNS scores and a worst performance in all cognitive assays but not with antipsychotic type. Measurement of serum truncated-BDNF abundance predicted for high cognitive deficits with sensitivity = 67.5%, specificity = 97.5%, Negative Predictive Value = 75% and Positive Predictive Value = 96.4%. These results suggest deficiency in pro-BDNF processing as a possible biological mechanism underlying schizophrenia with cognitive impairment.

Physical Exercise and Antidepressants Enhance BDNF Targeting in Hippocampal CA3 Dendrites: Further Evidence of a Spatial Code for BDNF Splice Variants

Brain-derived neurotrophic factor (BDNF) is encoded by multiple BDNF transcripts, whose function is unclear. We recently showed that a subset of BDNF transcripts can traffic into distal dendrites in response to electrical activity, while others are segregated into the somatoproximal domains. Physical exercise and antidepressant treatments exert their beneficial effects through upregulation of BDNF, which is required to support survival and differentiation of newborn dentate gyrus (DG) neurons. While these DG processes are required for the antidepressant effect, a role for CA1 in antidepressant action has been excluded, and the effect on CA3 neurons remains unclear. Here, we show for the first time that physical exercise and antidepressants induce local increase of BDNF in CA3. Voluntary physical exercise for 28 consecutive days, or 2-week treatment with 10 mg/kg per day fluoxetine or reboxetine, produced a global increase of BDNF mRNA and protein in the neuronal somata of the whole hippocampus and a specific increase of BDNF in dendrites of CA3 neurons. This increase was accounted for by BDNF exon 6 variant. In cultured hippocampal neurons, application of serotonin or norepinephrine (10–50 μM) induced increase in synaptic transmission and targeting of BDNF mRNA in dendrites. The increased expression of BDNF in CA3 dendrites following antidepressants or exercise further supports the neurotrophin hypothesis of antidepressants action and confirms that the differential subcellular localization of BDNF mRNA splice variants provides a spatial code for a selective expression of BDNF in specific subcellular districts. This selective expression may be exploited to design more specific antidepressants.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

Crigler‐Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin‐induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno‐associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV‐treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene‐addition‐based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin‐induced neonatal lethality in a mouse model of Crigler‐Najjar syndrome type I by AAV9‐mediated gene transfer. FASEB J. 26, 1052‐1063 (2012). www.fasebj.org

ACTN1-related thrombocytopenia: identification of novel families for phenotypic characterization

Inherited thrombocytopenias (ITs) are a heterogeneous group of syndromic and nonsyndromic diseases caused by mutations affecting different genes. Alterations of ACTN1, the gene encoding for α-actinin 1, have recently been identified in a few families as being responsible for a mild form of IT (ACTN1-related thrombocytopenia; ACTN1-RT). To better characterize this disease, we screened ACTN1 in 128 probands and found 10 (8 novel) missense heterozygous variants in 11 families. Combining bioinformatics, segregation, and functional studies, we demonstrated that all but 1 amino acid substitution had deleterious effects. The clinical and laboratory findings of 31 affected individuals confirmed that ACTN1-RT is a mild macrothrombocytopenia with low risk for bleeding. Low reticulated platelet counts and only slightly increased serum thrombopoietin levels indicated that the latest phases of megakaryopoiesis were affected. Given its relatively high frequency in our cohort (4.2%), ACTN1-RT has to be taken into consideration in the differential diagnosis of ITs.

Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro

Rett Syndrome (RTT) is a neurodevelopmental disorder associated with intellectual disability, mainly caused by loss-of-function mutations in the MECP2 gene. RTT brains display decreased neuronal size and dendritic arborization possibly caused by either a developmental failure or a deficit in the maintenance of dendritic arbor structure. To distinguish between these two hypotheses, the development of Mecp2-knockout mouse hippocampal neurons was analyzed in vitro. Since a staging system for the in vitro development of mouse neurons was lacking, mouse and rat hippocampal neurons development was compared between 1–15 days in vitro (DIV) leading to a 6-stage model for both species. Mecp2-knockout hippocampal neurons displayed reduced growth of dendritic branches from stage 4 (DIV4) onwards. At stages 5–6 (DIV9-15), synapse number was lowered in Mecp2-knockout neurons, suggesting increased synapse elimination. These results point to both a developmental and a maintenance setback affecting the final shape and function of neurons in RTT.

Regulation of the spatial code for BDNF mRNA isoforms in the rat hippocampus following pilocarpine-treatment: A systematic analysis using laser microdissection and quantitative real-time PCR

Brain‐derived neurotrophic factor (BDNF) is essential for neuronal survival, differentiation, and plasticity and is one of those genes that generate multiple mRNAs with different alternatively spliced 5′UTRs. The functional significance of many BDNF transcripts, each producing the same protein, is emerging. On the basis of the analysis of the four most abundant brain BDNF transcripts, we recently proposed the “spatial code hypothesis of BDNF splice variants” according to which the BDNF transcripts, through their differential subcellular localization in soma or dendrites, represent a mechanism to synthesize the protein at distinct locations and produce local effects. In this study, using laser microdissection of hippocampal laminae and reverse transcription‐quantitative real‐time PCR (RT‐qPCR), we analyzed all known BDNF mRNA variants at resting conditions or following 3 h pilocarpine‐induced status epilepticus. In untreated rats, we found dendritic enrichment of BDNF transcripts encoding exons 6 and 7 in CA1; exons 1, 6, and 9a in CA3; and exons 5, 6, 7, and 8 in DG. Considering the low abundance of the other transcripts, exon 6 was the main transcript in dendrites under resting conditions. Pilocarpine treatment induced an increase of BDNF transcripts encoding exons 4 and 6 in all dendritic laminae and, additionally, of exon 2 in CA1 stratum radiatum and exons 2, 3, 9a in DG molecular layer while the other transcripts were decreased in dendrites, suggesting restriction to the soma. These results support the hypothesis of a spatial code to differentially regulate BDNF in the somatic or dendritic compartment under conditions of pilocarpine‐induced status epilepticus and, furthermore, highlight the existence of subfield‐specific differences.

A method for reproducible measurements of serum BDNF: comparison of the performance of six commercial assays

Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKineTM, Promega-Emax®, R&D-System-Quantikine®) and one multiplexing assay (Millipore-Milliplex®). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.