The BDNF val-66-met Polymorphism Affects Neuronal Morphology and Synaptic Transmission in Cultured Hippocampal Neurons from Rett Syndrome Mice

Brain-derived neurotrophic factor (Bdnf) has been implicated in several neurological disorders including Rett syndrome (RTT), an X-linked neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2). The human BDNF gene has a single nucleotide polymorphism (SNP)—a methionine (met) substitution for valine (val) at codon 66—that affects BDNF’s trafficking and activity-dependent release and results in cognitive dysfunction. Humans that are carriers of the met-BDNF allele have subclinical memory deficits and reduced hippocampal volume and activation. It is still unclear whether this BDNF SNP affects the clinical outcome of RTT individuals. To evaluate whether this BDNF SNP contributes to RTT pathophysiology, we examined the consequences of expression of either val-BDNF or met-BDNF on dendrite and dendritic spine morphology, and synaptic function in cultured hippocampal neurons from wildtype (WT) and Mecp2 knockout (KO) mice. Our findings revealed that met-BDNF does not increase dendritic growth and branching, dendritic spine density and individual spine volume, and the number of excitatory synapses in WT neurons, as val-BDNF does. Furthermore, met-BDNF reduces dendritic complexity, dendritic spine volume and quantal excitatory synaptic transmission in Mecp2 KO neurons. These results suggest that the val-BDNF variant contributes to RTT pathophysiology, and that BDNF-based therapies should take into consideration the BDNF genotype of the RTT individuals.


SIGNIFICANCE STATEMENT
The neuroprotective effects of BDNF have been demonstrated in various animal models of neurological and psychiatric disorders. BDNF dysfunction has been implicated in pathophysiological mechanisms of Rett syndrome, the most common intellectual disability in women after Down syndrome (1:10,000 incidence). The BDNF val66met polymorphism is carried by approximately 30% of people worldwide and has been associated with cognitive deficits. Whether this BDNF single nucleotide polymorphism (SNP) contributes to RTT and how it affects the clinical outcome of RTT individuals remain unclear. Our findings help to understand the impact INTRODUCTION Brain-derived neurotrophic factor (Bdnf ) has been implicated in several neurological disorders due to its widespread function in neuronal development, plasticity, differentiation and survival (Poo, 2001;Fahnestock et al., 2002;Gines et al., 2010;Hartmann et al., 2012). The main function of BDNF in the adult brain is to regulate synaptic strength, promote synaptic growth and participate in plasticity-related processes underlying learning and memory (Tyler et al., 2002;Yamada et al., 2002;Lu, 2003a;Lu et al., 2013). A reduction of BDNF levels can cause impaired synaptic transmission and plasticity, reduced number of synapses and deficits in learning and memory in various pathological conditions (Mu et al., 1999;Durany et al., 2000;Ferrer et al., 2000;Lu et al., 2013).
BDNF has been implicated in Rett syndrome (RTT), an X-linked neurological disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2; Amir et al., 1999;Percy and Lane, 2005;Bienvenu and Chelly, 2006;Chahrour and Zoghbi, 2007). MeCP2 binds to the Bdnf promoter and directly regulates Bdnf expression in an activity-dependent manner (Chen et al., 2003;Martinowich et al., 2003;Zhou et al., 2006). Bdnf mRNA and protein levels are lower in MeCP2-deficient models and RTT individuals (Chang et al., 2006;Wang et al., 2006;Ogier et al., 2007;, and its overexpression rescues cellular and behavioral deficits (Chang et al., 2006;Chahrour and Zoghbi, 2007;Larimore et al., 2009). Dysfunctional BDNF signaling has been demonstrated in several pathophysiological mechanisms of RTT disease progression (Katz, 2014;Li and Pozzo-Miller, 2014), but the contribution of the BDNF val66met SNP to RTT symptoms remains unclear: one study reported that the met BDNF allele is protective for seizure onset in RTT individuals (Nectoux et al., 2008), while another described that it leads to earlier seizure onset (Zeev et al., 2009). Therefore, it is highly relevant to characterize the role of this BDNF SNP in synaptic and cellular features of Mecp2 deficient neurons from RTT mice. Our findings revealed that while met-BDNF does not promote dendritic growth and excitatory synapse formation in wildtype (WT) neurons, it actually reduces dendritic complexity, dendritic spine volume and quantal excitatory synaptic transmission in Mecp2 knockout (KO) neurons.

Animals
Breeding pairs of mice lacking exon 3 of the X chromosomelinked Mecp2 gene (B6.Cg-Mecp2 tm1.1Jae , ''Jaenisch'' strain in a pure C57BL/6 background; Chen et al., 2001) were purchased from the Mutant Mouse Regional Resource Center at the University of California, Davis. A colony was established at the University of Alabama at Birmingham (UAB) by mating WT males with heterozygous Mecp2 tm1.1Jae mutant females, as recommended by the supplier. Genotyping was performed by PCR of DNA sample from tail clips. Hemizygous Mecp2 tm1.1Jae mutant males (called KOs) are healthy until 5-6 weeks of age, when they begin to show RTT-like symptoms, such as hypoactivity, hind limb clasping, reflex impairments and irregular breathing (Chen et al., 2001). Animals were handled and housed according to the Committee on Laboratory Animal Resources of the National Institutes of Health; all experimental protocols were reviewed annually and approved by the Institutional Animals Care and Use Committee of the UAB.

Primary Culture of Hippocampal Neurons and Transfections
Both hippocampi were dissected from anesthetized postnatal day 0 or 1 (P0-1) male Mecp2 KO mice and WT littermates, and dissociated in papain (20 U/ml) plus DNase I (Worthington) for 20-30 min at 37 • C, as described (Amaral and Pozzo-Miller, 2007). The tissue was then triturated to obtain a single-cell suspension, and the cells were plated at a density of 40,000 cells/cm 2 on 18 mm poly-L-lysine/laminin coated glass coverslips, and immersed in Neurobasal medium (Life technologies) supplemented with 2% B27 (Life technologies) and 0.5 mM glutamine (Life technologies). Neurons were grown in 37 • C, 5% CO 2 , 90% relative humidity incubators (Thermo-Forma), with half of the fresh medium changed every 3-4 days. After 7-8 days in vitro (DIV), cDNA plasmids encoding either human val-BDNF or met-BDNF (tagged with green fluorescent protein GFP for their localization; 1.6 µg DNA; a gift from Dr. Masami Kojima) were transfected alone or in combination with soluble GFP (for imaging neuronal morphology) using Lipofectamine 2000 (Life technologies) according to the protocol of the manufacturer.

Image Analysis
The morphology of neurons was analyzed with the Filament Tracing and Surface Rendering modules of Imaris software (Bitplane). The fraction of cell body or dendrites filled with either val-BDNF-GFP or met-BDNF-GFP relative to the total territory filled with either val-BDNF-GFP or met-BDNF-GFP was calculated using ImageJ using the same threshold for fluorescence intensity. To trace spines, a region of interest (ROI) was selected and a new filament was created using the Autopath mode, as previously described (Swanger et al., 2011). The minimum dendrite end diameter was set at 0.75 µm, and automatic thresholds were used for dendrite surface rendering. The maximum spine length was set at 5 µm. Protrusions longer than 5 µm were rarely observed in neurons at DIV 10 and were not considered as spines. Spines were manually counted and spine density was calculated by quantifying the number of spines per dendritic segment, and normalized to 10 µm of dendrite length. Sholl analysis and branch order analyses were performed using NeuronStudio software (Wearne et al., 2005). The density of excitatory synapses was determined by the number of VGLUT1/GluA1 co-localized puncta per length of GFP-positive dendrite, and normalized to 10 µm of dendrite length. Three randomly selected segments of primary or secondary dendrites (30-40 µm for each segment) were analyzed for spine density and synaptic density; these dendritic segments were located at least one soma diameter away from the soma, and were void of crossing dendrites and axons from other neurons.

Statistical Analyses
Data were presented as mean ± standard error of the mean (SEM), and were compared using unpaired Student's t-test, one-way ANOVA, or Kolmogorov-Smirnov (K-S) test using Prism software (GraphPad Software, San Diego, CA, USA). Statistical Power was calculated using G * Power (Faul et al., 2007); p < 0.05 was considered significant.

DISCUSSION
The val66met SNP in the human BDNF gene is carried by approximately 30% of people worldwide and has been associated with cognitive deficits Hariri et al., 2003;Harris et al., 2006;Liu et al., 2012). In this SNP, val at position 66 is changed to met in the pro region of proBDNF (Lu, 2003b;Hartmann et al., 2012). This SNP does not affect the expression levels or intracellular signaling triggered by the mature BDNF protein. However, the intracellular distribution and activity-dependent secretion are significantly impaired in neurons expressing met-BDNF, resulting in met-BDNF-GFP clustered in the perinuclear regions rather than in synaptic regions of dendrites and axons (Lu, 2003b;Hartmann et al., 2012). The inability of met-BDNF-GFP to be transported to neuronal processes and localized to synapses is thought to be due to impaired binding of met-BDNF to the sorting protein sortilin, which interacts with the pro region of BDNF and directs it from the trans-Golgi network into the regulated secretory pathway (Hartmann et al., 2012;Baj et al., 2013). The results presented here confirm that met-BDNF distribution is restricted to somata and only partially transported to the proximal area of primary dendrites in hippocampal neurons of both WT and Mecp2 KO mice.
BDNF plays a critical role in activity-dependent dendritic and synaptic development (Chapleau et al., 2009b). Consisted with previous studies, our findings show that val-BDNF promotes dendritic growth and branching, and increases dendritic spine density and the volume of individual spines in hippocampal neurons. On the other hand, met-BDNF reduces dendritic length and branching, and fails to increase dendritic spine density. The function of BDNF relies on its proper trafficking to axons and dendrites, as well as sorting to the regulated secretory pathway, which allows Ca 2+ -dependent release. met-BDNF reduces the intracellular trafficking of BDNF messenger RNA (mRNA) to dendrites (Chiaruttini et al., 2009;Liu et al., 2012), and impairs the regulated BDNF secretion at synaptic sites Chen et al., 2004), thus affects the dendritic growth. Therefore, met-BDNF hijacks val-BDNF, producing an overall deficit in BDNF trafficking into release-ready dense core vesicles. We found met-BDNF caused a decreased spine density, but not statistically different from control. Since we only analyzed spines from proximal (primary and secondary) dendrites, we cannot exclude that spines from distal dendrites may show larger differences, as Liu et al. (2012) reported in the prefrontal cortex.
Reduction in the size and complexity of dendritic arbors are common in disorders associated with intellectual disability, such as Rett syndrome (Kaufmann and Moser, 2000). Consistent with previous studies in autopsy brains from RTT individuals and symptomatic Mecp2 KO mice (Armstrong et al., 1995;Fukuda et al., 2005;Schüle et al., 2008;Belichenko et al., 2009;Chapleau et al., 2009a), hippocampal neurons from newborn Mecp2 KO mice maintained in primary culture have reduced dendritic complexity and lower spine density than WT neurons. These deficits are due to both a failure of the formation as well as of the maintenance of dendritic arbors (Baj et al., 2014). Expression of val-BDNF fully rescues dendritic growth and spine density in Mecp2 KO neurons, as shown previously (Larimore et al., 2009). On the other hand, met-BDNF reduces dendritic complexity in Mecp2 KO neurons, which correlates well with the observations of that smaller hippocampal volumes in humans and rodents carrying the met-BDNF allele Hariri et al., 2003;Szeszko et al., 2005;Chen et al., 2006;Baj et al., 2013).
The number of bona fide dendritic spines (i.e., with a well-defined head) is a consistent estimate of excitatory synapses, while their volume is correlated with the strength of the particular synapse they receive (Bourne and Harris, 2008;Swanger et al., 2011). Even though Mecp2 KO neurons have fewer excitatory spine synapses (identified by the co-localization of presynaptic VGLUT1 and postsynaptic GluA1), the volume of each individual spine is larger than in WT neurons. Fewer excitatory synapses may reflect delayed neuronal maturation, since adult newborn neurons in the dentate gyrus have lower dendritic spine density than their mature neighboring neurons (Smrt et al., 2007). However, the surface levels of synaptic GluA1 is higher in Mecp2 KO neurons, consistent with stronger synapses that saturate long-term synaptic plasticity (Li et al., 2016), a consistent impairment of several mouse models of RTT (Chahrour and Zoghbi, 2007).
Consistent with numerous studies of the effects of bath-applied recombinant BDNF and expression of the rodent Bdnf gene, expression of the human val-BDNF increased dendritic spine density and the volume of individual dendritic spines in neurons from both WT and Mecp2 KO mice, which provides support that BDNF-based therapies might be beneficial for RTT individuals. However, expression of met-BDNF failed to affect spine density in WT neurons, and in fact resulted in a reduction of dendritic spine volume in Mecp2 KO neurons, which raises caution about the consequences of therapies aimed at increasing BDNF expression in individuals carrying the BDNF met allele, at least those that also harbor MECP2 mutations.
The enhancement of glutamatergic synaptic transmission by BDNF is a consistent observation in hippocampal neurons (Lessman et al., 1994;Levine et al., 1998;Li et al., 1998). For example, the increase in mEPSC frequency in neurons expressing val-BDNF (without changes in mEPSC amplitude) is consistent with the presynaptic effects of bath-applied recombinant BDNF to CA1 pyramidal neurons (Tyler and Pozzo-Miller, 2001;Amaral and Pozzo-Miller, 2012). Previously, we described more frequent and larger mEPSCs in CA1 pyramidal neurons of adult symptomatic Mecp2 KO mice compared to age-matched WT littermates, together with higher synaptic GluA1 levels and larger spine volumes, indicating stronger synaptic strength (Li et al., 2016). Here, we found that mEPSC amplitude is not affected in cultured pyramidal neurons from neonatal Mecp2 KO mice. This apparent inconsistency could be due to the different developmental ages (neonatal vs. postnatal-day 60), or the expression of soluble GFP in cultured neurons, which was used for identification of transfected neurons in this study. Interestingly, the expression of val-BDNF decreased mEPSC frequency in Mecp2 KO neurons, which would be beneficial by reducing the atypically enhanced synaptic strength in Mecp2 KO neurons. On the other hand, met-BDNF decreased mEPSC amplitude in both WT and Mecp2 KO neurons, suggesting that glutamatergic transmission is impaired. Consistently, the BDNF Val66Met polymorphism impairs NMDA receptor-dependent synaptic plasticity in various brain regions of BDNF met/met mice (Ninan et al., 2010;Liu et al., 2012;Pattwell et al., 2012;Galvin et al., 2015;Jing et al., 2017), although some studies have described normal basal glutamatergic neurotransmission (Ninan et al., 2010;Pattwell et al., 2012) or increased (Jing et al., 2017).
In conclusion, expression of the human BDNF val-66-met SNP has deleterious consequences on dendritic complexity, the density and morphology of excitatory synapses on dendritic spines, as well as synaptic transmission in Mecp2 KO neurons, suggesting that the met-BDNF variant contributes negatively to RTT pathophysiology. The outcome of therapies aimed at increasing BDNF expression may depend on the BDNF val-66met SNP genotype, at least in those RTT individuals that also harbor MECP2 mutations.

AUTHOR CONTRIBUTIONS
XX designed and performed experiments, analyzed data and wrote the manuscript; JG performed experiments and analyzed data; RE performed experiments and analyzed data; SN performed experiments and analyzed data; LP-M designed experiments, analyzed data and wrote the manuscript.