Inhibiting proBDNF to mature BDNF conversion leads to ASD-like phenotypes in vivo.

Autism Spectrum Disorders (ASD) comprise a range of early age-onset neurodevelopment disorders with genetic heterogeneity. Most ASD related genes are involved in synaptic function, which is regulated by mature brain-derived neurotrophic factor (mBDNF) and its precursor proBDNF in a diametrically opposite manner: proBDNF inhibits while mBDNF potentiates synapses. Here we generated a knock-in mouse line (BDNFmet/leu) in which the conversion of proBDNF to mBDNF is attenuated. Biochemical experiments revealed residual mBDNF but excessive proBDNF in the brain. Similar to other ASD mouse models, the BDNFmet/leu mice showed reduced dendritic arborization, altered spines, and impaired synaptic transmission and plasticity in the hippocampus. They also exhibited ASD-like phenotypes, including stereotypical behaviors and deficits in social interaction. Moreover, the plasma proBDNF/mBDNF ratio was significantly increased in ASD patients compared to normal children in a case-control study. Thus, deficits in proBDNF to mBDNF conversion in the brain may contribute to ASD-like behaviors, and plasma proBDNF/mBDNF ratio may be a potential biomarker for ASD.

Emerging Insights into the Role of BDNF on Health and Disease in Periphery.

Brain-derived neurotrophic factor (BDNF) is a growth factor that promotes the survival and growth of developing neurons. It also enhances circuit formation to synaptic transmission for mature neurons in the brain. However, reduced BDNF expression and single nucleotide polymorphisms (SNP) are reported to be associated with functional deficit and disease development in the brain, suggesting that BDNF is a crucial molecule for brain health. Interestingly, BDNF is also expressed in the hypothalamus in appetite and energy metabolism. Previous reports demonstrated that BDNF knockout mice exhibited overeating and obesity phenotypes remarkably. Therefore, we could raise a hypothesis that the loss of function of BDNF may be associated with metabolic syndrome and peripheral diseases. In this review, we describe our recent finding that BDNF knockout mice develop metabolic dysfunction-associated steatohepatitis and recent reports demonstrating the role of one of the BDNF receptors, TrkB-T1, in some peripheral organ functions and diseases, and would provide an insight into the role of BDNF beyond the brain.

Brain-derived neurotrophic factor knock-out mice develop non-alcoholic steatohepatitis.

While brain-derived neurotrophic factor (BDNF), which is a growth factor associated with cognitive improvement and the alleviation of depression symptoms, is known to regulate food intake and body weight, the role of BDNF in peripheral disease is not fully understood. Here, we show that reduced BDNF expression is associated with weight gain and the chronic liver disease non-alcoholic steatohepatitis (NASH). At 10 months of age, BDNF-heterozygous (BDNF+/- ) mice developed symptoms of NASH: centrilobular/perivenular steatosis, lobular inflammation with infiltration of neutrophils, ballooning hepatocytes, and fibrosis of the liver. Obesity and higher serum levels of glucose and insulin - major pathologic features in human NASH - were dramatic. Dying adipocytes were surrounded by macrophages in visceral fat, suggesting that chronic inflammation occurs in peripheral organs. RNA sequencing (RNA-seq) studies of the liver revealed that the most significantly enriched Gene Ontology term involved fatty acid metabolic processes and the modulation of neutrophil aggregation, pathologies that well characterise NASH. Gene expression analysis by RNA-seq also support the notion that BDNF+/- mice are under oxidative stress, as indicated by alterations in the expression of the cytochrome P450 family and a reduction in glutathione S-transferase p, an antioxidant enzyme. Histopathologic phenotypes of NASH were also observed in a knock-in mouse (BDNF+/pro ), in which the precursor BDNF is inefficiently converted into the mature form of BDNF. Lastly, as BDNF reduction causes overeating and subsequent obesity, a food restriction study was conducted in BDNF+/pro mice. Pair-fed BDNF+/pro mice developed hepatocellular damage and showed infiltration of inflammatory cells, including neutrophils in the liver, despite having body weights and blood parameters that were comparable to those of controls. This is the first report demonstrating that reduced BDNF expression plays a role in the pathogenic mechanism of NASH, which is a hepatic manifestation of metabolic syndrome. © 2023 The Pathological Society of Great Britain and Ireland.

Threshold Ocular Exposure to Near Infrared Radiation for Causing Acute Opacification in the Rabbit Lens.

Surveys and epidemiological studies have reported an increased prevalence of cataracts in the glass and steel industries, which are associated with exposure to intense infrared radiation (IR). Indeed, animal studies have demonstrated that IR exposure can produce acute changes in the crystalline lens that are likely to lead to cataract formation. However, little is known about threshold IR exposure for causing acute cataractous changes, which is important in the prevention of IR-induced cataract, especially as a basis for short-term IR exposure limits. Previously, we exposed rabbit eyes to 808 nm wavelength IR at different irradiances for 6 min to determine the threshold irradiance to cause acute lens opacification. Presently, we similarly determined the threshold irradiance for exposure durations of 3 min, 1 min, 30 s, 10 s and 4 s. The threshold irradiance increased steadily with decreasing exposure duration, from 1.1 W cm-2 at 6 min to 4.1 W·cm-2 at 4 s. These threshold values are consistent with ICNIRP exposure limits to avoid IR-induced cataract formation in the tested range of exposure duration, but suggest that it may be necessary to lower the exposure limits for shorter exposure durations.

Involvement of Denervated Midbrain-Derived Factors in the Formation of Ectopic Cortico-Mesencephalic Projection after Hemispherectomy.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit ß three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.

Dielectric property measurements of corneal tissues for computational dosimetry of the eye in terahertz band in vivo and in vitro.

The dielectric constant of the normal corneal tissue of a rabbit eye was obtained in vitro in the range from approximately 0.1 to 1 THz, and the drying process on the eye surface exposed to high-power terahertz waves was investigated by in vivo reflectance measurement using terahertz time-domain spectroscopy. When the rabbit eye was exposed to terahertz waves at 162 GHz for 6 min with an irradiation power of 360 or 480 mW/cm2, the reflectance temporally increased and then decreased with a temperature increase. Based on multiple-reflection calculation using the dielectric constant and anterior segment optical coherence tomography images, those changes in reflectance were attributed to drying of the tear and epithelium of the cornea, respectively. Furthermore, the drying progressed over a temperature increase of around 5°C under our exposure conditions. These findings suggest that the possibility of eye damage increases with the progress of drying and that the setting of the eye surface conditions can be a cause of disagreement between computational and experimental data of absorbed energy under high-level irradiation because reflectance is related to terahertz wave penetration in the eye tissue. The time-domain spectroscopic measurements were useful for the acquisition of the dielectric constant as well as for the real-time monitoring of the eye conditions during exposure measurement.

Cataract Formation by Near-infrared Radiation in Rabbits.

Surveys and epidemiological studies have shown an increased prevalence of cataracts in workers in the glass and steel industries. These cataracts are associated with exposure to intense infrared radiation (IR) emitted from heated materials and industrial furnaces. Thermal model calculations predicted that near and far IR would cause cataract with different mechanisms. The present study investigated cataract formation by near IR. Eyes of pigmented rabbits were exposed to IR at a wavelength of 808 nm. Morphological changes in the anterior segment of the eye were assessed by slit-lamp microscopy, and temperature distributions in the anterior chamber of the eye were observed during IR exposure using microencapsulated thermochromic liquid crystals. Cortical cataract appeared below the exposed area of the iris in eyes that had been exposed for 6 min to an irradiance of 1.27 W cm-2 or higher. The monitored temperature in the anterior chamber began to increase in the region adjacent to the exposed area of the iris with the onset of IR exposure. These results demonstrate that 808-nm IR is absorbed and converted to heat within the iris, which is then conducted to the lens and produce a cataract, as Goldmann theory states.

Journey of brain-derived neurotrophic factor: from intracellular trafficking to secretion.

Brain-derived neurotrophic factor (BDNF) is known to control a wide variety of brain functions, ranging from memory formation to food intake. However, since the BDNF levels are extremely low in the nervous system, the dynamics in neurons from intracellular trafficking to secretion is absolutely complicated; the understanding is not fully promoted. We here review the findings of those critical mechanisms from intracellular trafficking to the secretion of BDNF. Furthermore, to solve this issue, technological advances for the detection, measurement, and imaging of this growth factor are essential. We believe that this review helps the study of these complex but critical mechanisms of BDNF.

Reduction in BDNF from Inefficient Precursor Conversion Influences Nest Building and Promotes Depressive-Like Behavior in Mice.

We generated a knock-in mouse line in which the gene encoding brain-derived neurotrophic factor (Bdnf) was replaced with a sequence for proBDNF containing human single nucleotide polymorphisms encoding arginines proximal to the cleavage site (R125M and R127L). The ratio of the mature form of BDNF (mBDNF) to precursor BDNF (proBDNF) in hippocampal tissue lysates was decreased in a manner dependent on the number of copies of the mutant gene, indicating that the mutations inhibited proteolytic conversion of proBDNF into mBDNF. Although homozygous mice had a proBDNF/mBDNF ratio of ~9:1, they survived until adulthood. The levels of mBDNF were reduced by 57% in heterozygous mutant mice, which exhibited a depressive-like behavior in the tail suspension test and weight gain when housed in social isolation, showing that impaired proBDNF cleavage contributes to stress-induced depressive-like phenotypes. Furthermore, socially isolated heterozygous mice displayed a pronounced deficit in daily nest-building behaviors. These findings suggest that the decreased production of mBDNF by impaired proBDNF cleavage disturbs daily activities in mice.

Creating a Stable Short-term Housing Environment for Rabbits in a Cargo Van.

A stable environment is a prerequisite for animal research. The absence of a suitable laboratory rabbit environment at the gyrotron facility, with the nearest housing being 6.3 miles away, made it challenging to investigate ocular damage induced in rabbit eyes due to exposure to high-frequency millimeter waves. Because rabbits are prone to transportation stress, it was vital to keep them on-site during research. Here we describe the creation of the stable environment necessary to perform reliable and reproducible animal experiments, by using a cargo van parked at the gyrotron facility. To control the interior environment, we placed a window air conditioner, humidifier, dehumidifier, and photocatalyst deodorizer inside the cargo area without altering the original configuration of the vehicle. Rabbits were housed in individual cages for a maximum of 6 d. Microbial contamination in the air was evaluated by using a passive sampling method. The average numbers of bacterial and fungal colony forming units per dish were 0.2 and 4.7, respectively, indicating that the van was as clean as a nonbarrier animal facility. The average temperature was 20.5 °C (range, 17.8 to 22.6 °C), and the average relative humidity was 49.4% (range, 36.2% to 63.2%). The concentration of ammonia was consistently below the detection limit of 0.5 ppm. Other environmental conditions were within appropriate levels. Rabbits lost 6.4% ± 2.2% (n =52) of their initial body weight during the 13- to 14-h transport but recovered the lost weight within 48 h after arrival.

BDNF pro-peptide: physiological mechanisms and implications for depression.

Most growth factors are synthesized as precursors and biologically active forms are generated by proteolytic cleavage of the pro-domain. However, the biological functions of pro-domains are ill-defined. New roles were recently reported for the pro-domain of brain-derived neurotrophic factor (BDNF), a well-known growth factor in the brain. Interestingly, the pro-domain of BDNF (BDNF pro-peptide) is localized at presynaptic termini, where it facilitates long-term depression (LTD) in hippocampal slices, implicating it as a novel synaptic modulator. BDNF binds its pro-peptide with high affinity in a pH-dependent manner and when bound to BDNF, the BDNF pro-peptide cannot facilitate hippocampal LTD, representing a new mechanism of regulation. The BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and levels were significantly lower in patients with major depressive disorder (MDD) than in controls. Notably, male MDD patients exhibit significantly lower levels of CSF pro-peptide than females. These findings demonstrate that the BDNF pro-peptide is a biologically important synaptic modulator and is associated with MDD, particularly in males.

Cerebrospinal fluid BDNF pro-peptide levels in major depressive disorder and schizophrenia.

The role of brain-derived neurotrophic factor (BDNF) and its related molecules has been extensively studied in the context of psychiatric disorders. In the present study, we focused on the newly identified BDNF pro-peptide, which is generated together with mature BDNF by proteolytic processing of their precursor, proBDNF. Here, we report, for the first time, that BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and quantifiable by western blotting. We measured CSF BDNF pro-peptide levels in 27 patients with schizophrenia, 18 patients with major depressive disorder (MDD), and 27 healthy controls matched for age, sex, and ethnicity (Japanese). The ratio of the BDNF pro-peptide level to the total protein level in MDD patients was significantly lower than that in controls (Kruskal-Wallis with Dunn's multiple comparisons test; p = 0.046). When men and women were examined separately, males with MDD had a significantly lower BDNF pro-peptide/protein ratio than male controls (p = 0.047); this difference was not found in female subjects. The ratio tended to be lower in male schizophrenia patients (p = 0.10). Although we tried to measure the levels of mature BDNF in CSF, they were below the limit of detection of the ELISA and multiple analyte profiling technology. Taken together, the results suggest that reduced CSF BDNF pro-peptide levels are associated with MDD, particularly in males. Further studies involving a larger sample size are warranted.

Ultraviolet B-induced Otx2 expression in lens epithelial cells promotes epithelial-mesenchymal transition.

Ultraviolet (UV) radiation of eyes is a major risk factor for cataractogenesis, although the molecular mechanisms underlying this process remain poorly understood and genes that are affected by UV radiation have not been fully identified. In this study, we examined the UV-related gene regulation in lens epithelial cells (LECs) of mouse eyes and investigated the molecular mechanisms of UV-triggered cataractogenesis. Forty-one genes were significantly upregulated in LECs following UVB exposure in vivo in two independent experiments. Among these, Otx2 was strongly upregulated in LECs, suggesting that it may act as an upstream regulator of UVB-induced changes in gene expression. Accordingly, Otx2 overexpression in LECs in vitro induced morphological changes in cell shapes. Epithelial-mesenchymal transition (EMT)-related molecules, such as TGFß2, αSMA and fibronectin were upregulated in Otx2-overexpressing LECs, concomitant with suppression of lens fiber cell marker genes, such as CRYAA and DNASEIIB. In vitro experiments suggested that UVB upregulated Otx2 through hydrogen peroxide generation. Aberrant upregulation of Otx2 in LECs following UV irradiation induces the EMT and alteration of the lens cell characteristics, likely contributing to cataractogenesis.

BDNF pro-peptide: a novel synaptic modulator generated as an N-terminal fragment from the BDNF precursor by proteolytic processing.

Most growth factors are initially synthesized as precursors and it was cleaved into bioactive mature domain and pro-domain. However, compared with the expression and function of bioactive mature domain, the biological role of the pro-domain is poorly understood. Unexpectedly, we found that the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which is well-known neurotrophic factor in brain, has a potential ability to facilitate hippocampal long-term depression. Furthermore, a BDNF polymorphism Val66Met, which substitute valine into methionine at 66 amino acid, impacted the biological activity of the BDNF pro-peptide. We lastly discuss the possible roles of BDNF and its pro-peptide in the generation of neural stem cells and progress of ischemia.

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.

Brain-derived neurotrophic factor in mood disorders and antidepressant treatments.

Levels of brain-derived neurotrophic factor (BDNF) are reduced in the brain and serum of depressed patients and at least the reduction in serum levels is reversible upon successful treatment. These data, together with a wealth of reports using different animal models with depression-like behavior or manipulation of expression of BDNF or its receptor TrkB have implicated BDNF in the pathophysiology of depression as well as in the mechanism of action of antidepressant treatments. Recent findings have shown that posttranslational processing of BDNF gene product can yield different molecular entities that differently influence signaling through BNDF receptor TrkB and the pan-neurotrophin receptor p75NTR. We will here review these data and discuss new insights into the possible pathophysiological roles of those new BDNF subtypes as well as recent findings on the role of BDNF mediated neuronal plasticity in mood disorders and their treatments.

Neurobiological actions by three distinct subtypes of brain-derived neurotrophic factor: Multi-ligand model of growth factor signaling.

Brain-derived neurotrophic factor (BDNF) is one of the most active members of the neurotrophin family. BDNF not only regulates neuronal survival and differentiation, but also functions in activity-dependent plasticity processes such as long-term potentiation (LTP), long-term depression (LTD), learning, and memory. Like other growth factors, BDNF is produced by molecular and cellular mechanisms including transcription and translation, and functions as a bioactive molecule in the nervous system. Among these mechanisms, a particular post-translational mechanism, namely the conversion of precursor BDNF into mature BDNF by proteolytic cleavage, was not fully understood. In this review, we discuss the manner through which this post-translational mechanism alters the biological actions of BDNF protein. In addition to the initially elucidated findings on BDNF, the biological roles of precursor BDNF and the BDNF pro-peptide, especially synaptic plasticity, will be extensively discussed. Recent findings on the BDNF pro-peptide will provide new insights for understanding the mechanisms of action of the pro-peptides of growth factors.

BDNF pro-peptide actions facilitate hippocampal LTD and are altered by the common BDNF polymorphism Val66Met.

Most growth factors are initially synthesized as precursor proteins and subsequently processed into their mature form by proteolytic cleavage, resulting in simultaneous removal of a pro-peptide. However, compared with that of mature form, the biological role of the pro-peptide is poorly understood. Here, we investigated the biological role of the pro-peptide of brain-derived neurotrophic factor (BDNF) and first showed that the pro-peptide is expressed and secreted in hippocampal tissues and cultures, respectively. Interestingly, we found that the BDNF pro-peptide directly facilitates hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA receptors and the pan-neurotrophin receptor p75(NTR). The BDNF pro-peptide also enhances NMDA-induced α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor endocytosis, a mechanism crucial for LTD expression. Thus, the BDNF pro-peptide is involved in synaptic plasticity that regulates a mechanism responsible for promoting LTD. The well-known BDNF polymorphism valine for methionine at amino acid position 66 (Val66Met) affects human memory function. Here, the BDNF pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings demonstrate functional roles for the BDNF pro-peptide and a naturally occurring human BDNF polymorphism in hippocampal synaptic depression.

Lack of stress responses to long-term effects of corticosterone in Caps2 knockout mice.

Chronic stress is associated with anxiety and depressive disorders, and can cause weight gain. Ca(2+)-dependent activator protein for secretion 2 (CAPS2) is involved in insulin release. Caps2 knockout (KO) mice exhibit decreased body weight, reduced glucose-induced insulin release, and abnormal psychiatric behaviors. We chronically administered the stress hormone corticosterone (CORT), which induces anxiety/depressive-like behavior and normally increases plasma insulin levels, via the drinking water for 10 weeks, and we examined the stress response in KO mice. Chronic CORT exposure inhibited stress-induced serum CORT elevation in wild-type (WT) mice, but not in KO mice. Poor weight gain in CORT-treated animals was observed until week 6 in WT mice, but persisted for the entire duration of the experiment in KO mice, although there is no difference in drug*genotype interaction. Among KO mice, food consumption was unchanged, while water consumption was higher, over the duration of the experiment in CORT-treated animals, compared with untreated animals. Moreover, serum insulin and leptin levels were increased in CORT-treated WT mice, but not in KO mice. Lastly, both WT and KO mice displayed anxiety/depressive-like behavior after CORT administration. These results suggest that Caps2 KO mice have altered endocrine responses to CORT administration, while maintaining CORT-induced anxiety/depressive-like behavior.

Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.