RICERCA ITALIANA     

Pathogenetic role of Brain-derived neurotrophic factor (BDNF) in the aetioloogy of neurodegenerative disorders: from animal models to human study.

Università degli Studi di Brescia

Abstract

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are a unique family of polypeptide growth factors that influence differentiation and survival of neurons in the developing nervous system, thus being likely involved in neurodegenerative disorders. Neurotrophins might represent a breakthrough in the treatment of Alzheimer Disease (AD) and other neurodegenerative disorders, as recent clinical trials shown benefit from their exogenous application.
Among others, BDNF has been widely studied both in human genetic studies and in in vitro studies.
A functional polymorphism within BDNF gene consisting of a missense change (G196A) has been described. BDNF Val/Met polymorphism has been previously suggested as a genetic risk factor for Alzheimer Disease (AD), even if with conflicting results, and no data on other neurodegenerative disorders are available yet. Its role in neurodegeneration has also been supported by the evidence that in the AD brain, decreased BDNF protein levels were reported in hippocampus, entorhinal cortex, and temporal neocortex while no changes were observed in areas less affected by the disease, such as the frontal, parietal, and cerebellar cortices.
These observations, taken together, underline the need of a deeper knowledge of BDNF functions and its modifications in neurodegenerative process. The study of genetic, epigenetic and posttrasductional BDNF mechanisms are of interest for their crucial role in neuronal loss and consequent decline of cognitive functions.
These may be of help for disentangling the issue of the role of BDNF in cognitive decline and for establishing new therapeutic targets.
Up to now, literature data lack of comparative studies across different neurodegenerative disorders compared to controls, to test the specific role of BDNF in defining risk to AD or in increasing risk to neurodegenerative processes aspecifically. It is not known whether BDNF Val/Met genetic variation influences disease phenotype in AD or in neurodegenerative disorders, such as Frontotemporal Dementia (FTD) or PD. Further, despite these studies on BDNF Val/Met polymorphism, no work on other polymorphisms recognised within BDNF sequence and on their interactions in haplotypes have been performed, either as risk factor for neurodegenerative disorders or as modulators of cognitive performances. Finally, the relationship between BDNF genetic variations and BDNF expression in plasma across neurodegenerative diseases is not known. It is noteworthy to say that processes occurring in neurodegeneration, such as inflammation, may profoundly impaired BDNF signalling. Thus, the disruption of the pro-inflammatory cytokine/neurotrophin balance, proposed as an associated factor in AD and PD, should be taken into account and studied in determining BDNF expression in patients with neurodegenerative disorders.

All these observations prompted the present project aimed at evaluating the cross-sectional effect of BDNF from genetics to biology in neurodegenerative disorders. We will study in a wide sample of subjects with neurodegenerative disorders and age-matched healthy controls (n=320) i) the risk conferred by BDNF genetic variations, i.e. BDNF haplotypes, to AD and other neurodegenerative diseases, ii) the risk conferred by post-traslational modification of BDNF, i.e. BDNF methylation, on neurodegenerative disorders; iii) BDNF plasma levels and their correlations with genetic variations within BDNF gene; iv) the potential relationship existing between inflammation and BDNF in neurodegenerative pathogenesis, and v) the influence of BDNF genetic variations and BDNF plasma levels on cognitive performances in each patient group.
Moreover, in vitro and animal model approaches will be applied. In two different animal models of pre-clinical AD (obtained by cell-permeable peptides that manipulate ADAM10/SP97 complex) and AD (obtained by saporine-mediated damage of nucleus basalis magnocellularis), the influence of BDNF expression on BDNF receptor (trkB), on parameters of amyloid cascade, and on brain morphological changes will be tested. The effect of enviroment enrichment performed in a subgroup of these animals on BDNF expression will be also evaluated. Finally, the neuroblastoma cell line, SH-SY5Y, widely used as dopaminergic neuron, will be used to verify a possible role of BDNF in alpha-synuclein level regulation.
The identification of a role of the neurotrophines might not only contribute to the understanding of AD pathogenesis and neurodegeneration, but also open new ways to more targeted therapeutical approaches directed to enhance neuroprotection. The opportunity to test neurotrophine-modulating drugs in combination with current available therapy, might be supported by the results of this study. <<<

Principal Investigator

Alessandro Padovani Università degli Studi di BRESCIA

Research Objectives

The aim of this project will be to evaluate the role of neurotrophin, such as BDNF, in the pathogenesis of neurodegenerative disorders.
The work will be divided into two different steps, the first will be aimed at evaluating the
role of BDNF in vivo, in patients affected by different neurodegenerative disorders, such as AD, FTD or PD.
The second step will be dedicated at in vitro and in animal studies for establishing the role of BDNF in models of neurodegeneration.

In particular, the objectives of this study will be the followings:


- In human study
1) To investigate in a wide sample of subjects with neurodegenerative disorders and age-matched healthy controls the role of genetic predisposing variations within BDNF gene in determining the risk of AD and other neurodegenerative diseases, i.e. FTD and PD.
Three-hundred twenty subjects (110 AD, 110 age-matched controls and 100 patients with neurodegenerative disorders other than AD) will be enrolled. To this aim, four different BDNF polymorphisms will be evaluated such as BDNF Val66Met, BDNF C270T, BDNF G11757C, and BDNF – T633A. The single SNPs analysis and the haplotype analysis will be performed across patient groups and controls.

2) To investigate the role of epigenetic variations, i.e. BDNF gene promoter methylation, in determining the risk of AD and other neurodegenerative diseases, i.e. FTD and PD.
The correlations between BDNF gene promoter methylation and BDNF polymorphisms/haplotypes will be further analysed.

3) To investigate plasma BDNF levels in the different neurodegenerative disorders compared to controls, and their correlations with BDNF polymorphisms/haplotypes.

4) To identify the potential relationship existing between inflammation and BDNF in neurodegenerative pathogenesis. <br />To this aim, pro-inflammatory cytokines such as such Interleukin (IL)-1 beta, Tumor Necrosis Factor (TNF) alpha, IL-18 will be tested in plasma of AD patients and controls. Pro-inflammatory cytochines will be further tested in supernatants of stimulated blood cells.

5) To investigate the influence of BDNF genetic variations and BDNF plasma levels on cognitive performances in each patient group.
To this aim a standardised experimental neuropsychological battery will be designed.
According to previous literature data regarding the role of BDNF in cognition, decision-making and executive functions, memory, apathy, depression and anhedonia will be considered.
To this, the most recognised BDNF Val/Met polymorphism will be considered for comparisons.



- In vitro and in animal model study
1) To evaluate BDNF expression in two animal models of pre-clinical AD (obtained by cell-permeable peptides that manipulate ADAM10/SP97 complex) and AD (obtained by saporine-mediated damage of nucleus basalis magnocellularis).
In these models, the influence of BDNF expression on BDNF receptor (trkB), on parameters of amyloid cascade, and on brain morphological changes will be tested. The effect of enviroment enrichment performed in a subgroup of these animals on BDNF expression will be also evaluated.

2) To verify a possible role of BDNF in alpha-synuclein level regulation in a cellular model of PD.
The neuroblastoma cell line, SH-SY5Y, widely used as dopaminergic neuron model because they express the tyrosine hydroxylase, the dopamine decarboxylase and the dopamine transporters, will be tested. <<<

First Results

We strongly believe that the proposed research program may represent an important contribution to the definition of the role of neurotrophines, such as BDNF, in AD and other neurodegenerative disorders.
This project will be divided into two steps.
The first step will be mainly devoted to the enrolment of normal controls, patients with AD and patients with other neurodegenerative diseases, such as FTD, PD or PD with dementia, and to the preparation and standardisation of protocols shared by all the Units for neuropsychological assessment and blood sampling. The research protocol, including the inclusion/exclusion criteria, will allow to select a relatively large sample of patients where to test the effect of BDNF, compared with previous studies reported in literature. The single SNP analysis and the haplotype analysis will be conducted in AD patients, in other neurodegenerative disorders and in aged-matched healthy control subjects. This will represent the first study on the issue (1nd result). Indeed, the methodological approach based on the use of haplotypes can allow to get more information on the influence of a gene on the pathogenesis of a disease, with respect to a classical model based on the analysis of a single polymorphism. To circumvent the difficulty of molecular haplotyping, several methods based on statistical inference will be proposed to estimate haplotype frequencies from genotypic data and their correlations with clinical and biological variables (2nd result).
Correlations between polymorphisms/haplotypes and cognitive patterns (3rd result), BDNF epigenetic modification, i.e. methylation (4th result), BDNF plasma levels (5th result), and inflammatory pathway (6th result) will be performed across study groups.
The second step will be devoted to verify in two animal models of AD the expression of BDNF (7th result), the interaction between BDNF expression and its receptor extression, i.e. trkB (8th result), the interaction between BDNF and amyloid cascade (9th result), the interaction between BDNF and the brain morphology changes (10th result), and the effect of environment enrichment on BDNF levels (11th result). In a cell model od PD, interaction between BDNF and alpha-synuclin will be tested (12th result); in this model, the evaluation of the role of estrogen, involved in neurodegeneration, on BDNF levels will be further analysed as an adjunctive aim (13th result)
No previous work have been performed to answer to these questions, and no data on results from 1 to 13 are available yet. Thus, this study represents a break-through in the field. The most important feature of the proposed study is the merge between basic and clinical aspects.
The identification of a role of the neurotrophines and their modulation by genetic and epigenetic processes, their interaction with pro-inflammatory immune-mediated mechanisms, might not only contribute to the understanding of AD pathogenesis and neurodegeneration, but also open new ways to more targeted therapeutical approaches directed to enhance neuroprotection. The opportunity to test neurotrophine-modulating drugs in combination with current available therapy, might be supported by the results of this study. <<<

Timescale

24 months

National and international background

Establishing the targets of neurodegeneration and neuroprotection is crucial for more effective treatment strategies.
Previous studies have suggested that neurodegenerative diseases are the results of a complex interaction of genetic predisposing factors, defective neuroprotective strategies and damage due to inflammation, involving specific cerebral pathways. In this view, the role of neurotrophic factors, which are demonstrated to be involved in modulation of learning mechanisms, are critical.
Among these, the brain-derived neurotrophic factor (BDNF) is a polypeptide fundamental in survival, differentiation and growth of the peripheral and central neurons both during and after development. The BDNF mature protein derives from uncleaved precursor BDNF protein, pro-BDNF. It binds to a specific receptor and selectively interacts with protein-tyrosine kinase receptors type B (trkB), that are present both in pre- and post-synaptic membranes. The BDNF promotes cell survival and it is involved in neuronal homeostasis and brain plasticity-related processes such as memory and learning. Animal studies have observed that in the brain, BDNF mRNA and protein expression becomes detectable during embryonic development, reaching the highest levels by days10-14 postnatally and decreasing thereafter. In the adult animal brain, BDNF is expressed throughout the brain, with the highest levels in hippocampus; a similar distribution with high levels of BDNF in cortical areas, such as insular and temporal cortex, has been also observed in the adult human brain. Further, increased BDNF expression has been observed in normal animals reared in enriched environment displaying improved performances in learning and memory functions and explorative activities.
These evidences have contributed to an increase interest for BDNF and to look at its possible role in the pathogenetic mechanisms of several neurological diseases. Some authors have observed an altered BDNF expression in depression, epilepsy, Huntington’s and Parkinson’s diseases (PD). Furthermore, a decreased expression of BDNF in hippocampus, temporal and frontal cortex has been described in Alzheimer’s disease (AD). In this disease, a possible relationship between presenilin-1 gene mutations and deficit in BDNF receptor phosphorilation has been reported, suggesting a likely direct interaction with amyloid cascade.
These findings support the hypothesis of a BDNF role in the AD pathogenesis, but the causes of its decreased expression are unclear as well its role on amyloid cascade and on AD-related brain structural changes. A causative role of BDNF has also been postulated in PD, as BDNF affected the expression of alpha-synuclein, the pathogenetic key-player in PD, but no clear-cut studies are available yet.
The role of BDNF in AD has also been evaluated by genetic studies, as the evidence of common single-nucleotide polymorphism in the human BDNF gene, resulting in a valine to methionine substitution in the prodomain (Val66Met). It has been shown that this polymorphism alters BDNF expression, influencing BDNF pathway. Thus, several work have been made to understand a possible role in conferring risk to AD, with contrasting results. Some studies supported an increased risk to AD in BDNF mutation carriers, others found no differences. Moreover, previous studies claimed for a role of BDNF functional polymorphism in modulating cognitive functions in healthy volunteers, depending on specific BDNF expression in the brain. Prefrontal task, long term memory and mood disturbances seem to be influenced by BDNF Val/Met polymorphism.
Up to now, literature data lack of comparative studies across different neurodegenerative disorders compared to controls, to test the specific role of BDNF in defining risk to AD or in increasing risk to neurodegenerative processes aspecifically. It is not known whether this BDNF genetic variation influences disease phenotype in AD or in neurodegenerative disorders, such as Frontotemporal Dementia (FTD) or PD. Further, despite these studies on BDNF Val/Met polymorphism, no work on the other polymorphisms recognised within BDNF sequence and on their interactions in haplotypes have been performed, either as risk factor for neurodegenerative disorders or as modulators of cognitive performances. Finally, the relationship between BDNF genetic variations and BDNF expression in plasma across neurodegenerative diseases is not known. It is noteworthy to say that processes occurring in neurodegeneration, such as inflammation, may profoundly impaired BDNF signalling. Thus, the disruption of the pro-inflammatory cytokine/neurotrophin balance, proposed as an associated factor in AD and PD, should be taken into account and studied in determining BDNF expression in patients with neurodegenerative disorders.
Not only genetic variations within BDNF are matter of interest for researchers, but also the epigenetic mechanisms of BDNF transcription should be further analysed. BDNF gene transcriptional process is regulated by several factors such as the methylation of DNA promoter within specific regions and the binding of specific proteins (methyl CpG-binding domain, MBD) to the methylated DNA. Recently, the DNA methylation and the histone deacetylation have been confirmed as important factors for the regulation of BDNF gene expression. Following the membrane depolarization and the calcium influx, there is a dysfunction of DNA methylation and a phosphorylation of the MeCP2, a MBD protein, that is unable to bind the promoter changing the gene transcriptional process. The effective role of BDNF gene in cerebral degenerative diseases, and the value of the DNA methylation of the BDNF gene in AD, and in other neurodegenerative diseases, is still to be known.
These observations, taken together, underlin the need of a deeper knowledge of BDNF functions and its modifications in neurodegenerative process. The study of genetic, epigenetic and posttrasductional BDNF mechanisms are of interest for their crucial role in neuronal loss and consequent decline of cognitive functions.
The study of BDNF signaling pathway influencing neural plasticity and neural network needed different methodological approaches, by in vitro experiments, the use of animal models of neurodegeneration and in vivo studies. These may be of help for disentangling the issue of the role of BDNF in cognitive decline and for establishing new therapeutic targets. <<<