Research Online Research Online Neurotrophin signaling and Alzheimer's Disease neurodegeneration - Focus Neurotrophin signaling and Alzheimer's Disease neurodegeneration - Focus on BDNF/TrkB signaling on BDNF/TrkB signaling

Abstract Neurotrophins are small proteins vital for neuronal growth, differentiation, survival, and plasticity. Members of the mammalian neurotrophin family include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin- 4/5 (NT-4/5). Their neurotrophic effects are mediated by the tropomyosin receptor kinase (Trk) receptors, membrane-bound receptor tyrosine kinases (NGF for TrkA, BDNF and NT4/5 for TrkB, and NT-3 for TrkC) which activate various cell signaling pathways linked to growth, differentiation, and survival. The importance of neurotrophin signaling in brain development is highlighted by findings showing that knockout mice for any one of the neurotrophins or their receptors are fatal or exhibit severe neural defects.


Neurotrophins
Neurotrophins are small proteins vital for neuronal growth, differentiation, survival, and plasticity [1]. Members of the mammalian neurotrophin family include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5). Their neurotrophic effects are mediated by the tropomyosin receptor kinase (Trk) receptors, membrane-bound receptor tyrosine kinases (NGF for TrkA, BDNF and NT4/5 for TrkB, and NT-3 for TrkC) which activate various cell signaling pathways linked to growth, differentiation, and survival [2]. The importance of neurotrophin signaling in brain development is highlighted by findings showing that knockout mice for any one of the neurotrophins or their receptors are fatal or exhibit severe neural defects [3].

BDNF and Alzheimer's disease
The neurotrophin, NGF, is reduced in the nucleus basalis, a region concentrated in basal forebrain cholinergic neurons, which show substantial degeneration in Alzheimer's disease (AD) [4,5]. However, there is conflicting evidence demonstrating that NGF levels are unchanged or even increased in other brain regions including the frontal cortex and hippocampus, two major brain regions affected in AD [6][7][8][9]. In contrast, BDNF is more highly expressed and widely distributed in the brain compared to NGF, and its expression and growth promoting actions are critical for survival and plasticity of a variety of neurons throughout the brain, particularly in brain regions heavily affected in AD such as hippocampal, cortical, and cholinergic neurons [10][11][12][13][14]. Moreover, in cell culture and animal models, functioning of the BDNF signaling pathway has been repeatedly demonstrated to be critical for neuronal differentiation, survival, plasticity, and cognition [3,11,13,[15][16][17][18][19][20][21]. Independent lines of evidence suggest that dysfunction in BDNF signaling may contribute to the neurodegeneration in AD. Brain regions associated with reduced BDNF expression are those displaying the highest levels of neurodegeneration (eg. hippocampus). The role of BDNF in AD has been studied extensively. In AD brains, BDNF mRNA and protein levels have been found to be reduced in the hippocampus and neocortex [8,[22][23][24][25][26][27]. With findings of reduced BDNF expression in AD, interest emerged in the role of the TrkB receptor, as reductions in BDNF signaling may also occur through alterations in and/or through decreased expression of this BDNF receptor.

The BDNF receptor − TrkB
The TrkB receptor is the principal component of the BDNF signaling pathway. In the human brain, multiple isoforms of TrkB are expressed. There are three major isoforms of the TrkB receptor characterized to date: the full-length (TrkB-TK+) and two C-terminal truncated TrkB receptors (TrkB-TK-and TrkB-Shc) that are generated by alternative splicing of the TrkB pre-mRNA [28]. The full-length TrkB receptor, TrkB-TK+, is the principal mediator of the neurotrophic effects of BDNF. Upon ligand binding, monomeric TrkB-TK+ homodimerizes and undergoes trans-phosphorylation at key tyrosine residues in the C-terminal domain that couple it to downstream signaling pathways that promote neuronal survival, growth, differentiation, and plasticity including mitogen-activated protein kinase kinase (MEK), phosphatidyl inositol 3 kinase (PI3K), and phospholipase C-gamma (PLCγ) [29][30][31][32] ( Figure  1). The two truncated TrkB receptor isoforms include TrkB-TK-and TrkB-Shc. Both truncated isoforms are generated from alternatively spliced transcripts and are truncated at the Cterminus, thus lacking the tyrosine kinase domain [28,33]. However, the TrkB-TK-and TrkB-Shc receptors differ in that each contain unique amino acid sequences at their C-terminus. The TrkB-Shc isoform includes the sarc homology containing (Shc) binding domain that is absent in TrkB-TK- [28].

TrkB and Alzheimer's disease
Previous reports on TrkB-TK+ and TrkB-TK-expression levels in AD have been variable due to brain cohort differences and the variable techniques used to measure their expression. In general, reductions in TrkB-TK+ in neurons have mostly been found in the hippocampus and the frontal and temporal cortices in AD [27,34]. Conversely, up-regulation of TrkB-TKhas been found in association with senile plaques in AD, and is suggested to be linked to increases in reactive glial cells [9,27,34]. Furthermore, increases in TrkB-TK+ have also been found in glial cells in the hippocampus [27]. Surprisingly, while the existence of TrkB-Shc has been known for some time, its role in AD has not been defined.

Importance of TrkB
Neuron viability and function is dependent upon BDNF-stimulated TrkB-TK+ signaling. In AD, much evidence suggests that BDNF/TrkB-TK+ signaling is reduced [8,[22][23][24][25][26][27][34][35][36]. In addition to changes in BDNF expression, neuronal BDNF/TrkB-TK+ signaling can also be modulated by alterations in the ratio of full-length (TrkB-TK+) to truncated (TrkB-TK-and TrkB-Shc) TrkB expressed [31,37]. Homodimers of TrkB-TK+ receptors bind to BDNF and initiates intracellular second messenger signaling ( Figure 1). Changes in TrkB alternative transcript expression or protein stability, such as increased TrkB-TK-and/or TrkB-Shc, will have a profound negative impact on BDNF/TrkB-TK+ signaling as homodimers of truncated receptors and heterodimers of full-length and truncated receptors can not initiate BDNFstimulated second messenger signaling ( Figure 2). This is important because changes in the ratio of full-length to truncated TrkB expression in neurons may underlie reductions in neurotrophic support in AD, which ultimately lead to neurodegeneration and profound neuron and brain volume loss. Considering that TrkB-Shc is a brain-and neuron-specific TrkB isoform that has been demonstrated to inhibit BDNF/TrkB-TK+ signaling, it is important to establish what role TrkB-Shc plays in AD development and progression.

Evidence that TrkB-Shc alternative transcripts are selectively increased in the hippocampus during severe, late stage AD
In Wong et al. (2012) [38], we measured changes in TrkB alternate transcript levels in control and AD postmortem human brain tissue derived from the hippocampus, temporal cortex, occipital cortex, and cerebellum (Braak stages V and VI) [38]. By quantitative real-time PCR, using primers specific for each TrkB alternative transcript, we found significant increases in TrkB-Shc mRNA expression in the hippocampus but not in any other brain region ( Figure 3).
Considering that brain homogenates contain a mixed population of cells, we determined whether the changes found in TrkB transcript expression using the hippocampal tissue homogenates also occur in neurons exposed to an amyloidogenic environment. Here, changes in TrkB transcripts were assessed by incubating differentiated SHSY5Y cells (a human neuroblastoma cell-line which express TrkB) with different species of amyloid beta 1-42 (Aβ 42 ) peptides at various stages of aggregation. Oligomers and fibrils were prepared as described in Ryan et al. [39] and characterized by western blotting and atomic force microscopy imaging [38]. A significant increase in TrkB-Shc mRNA levels was found when cells were incubated with preparations of Aβ 42 containing fibrils compared to controls ( Figure 4). The small magnitude of change was expected as the Aβ 42 fibril preparation contained mixed Aβ 42 spe-cies and the absolute amount of fibrils would be low (fibrils were absent in the monomer and oligomer Aβ 42 preparations). Further, in comparison to the Aβ 42 monomer and oligomer preparations, the Aβ 42 fibril preparations would be most representative of all Aβ 42 species present in the AD hippocampus as this preparation comprises a mix of all three species [38]. These results were consistent with findings of increased TrkB-Shc mRNA levels in the AD hippocampus (Figures 3 and 4). In regards to the brain regions examined, AD brain pathology from most severe to least affected is: hippocampus>temporal cortex>occipital cortex>cerebellum. The selective increase in TrkB-Shc transcripts observed only in the hippocampus suggested that the elevated TrkB-Shc transcript levels were occurring in brain regions that are most severely affected in the diseased state and that the observed increase may likely be influenced by the neuronal cell population present. This was supported by our in vitro AD cell culture model showing that TrkB-Shc mRNA levels in the differentiated SHSY5Y neuronal cells can be increased by exposure to preparations of Aβ 42 containing fibrils. Aβ 42 fibril species are increased in the advanced stages of AD [40]. While it is widely accepted that soluble oligomers are the more neurotoxic of the Aβ 42 species, evidence also suggest that the neurotoxicity of Aβ 42 requires its aggregation in the fibrillar form, particularly in the form of protofibrils [41,42]. In our Aβ 42 preparations, we detected various sizes of fibrils in the Aβ 42 fibril preparations, includ-ing protofibrils which can be ~100 nm in length. If we consider that Aβ 42 fibrils are predominant in the advanced stages of AD [40], that the hippocampus is the most affected brain region, and that the CA1 subregion (the hippocampal region assessed) is the most severely impacted subregion of the hippocampus in AD, altogether, our findings suggest that the selective increase in the TrkB-Shc alternative splice transcript in the AD hippocampus may be specific to severe, late stage pathology.  duced but little change was observed for phosphorylated TrkB:total TrkB-TK+ and phosphorylated AKT:total AKT ratios ( Figure 5). In a parallel study [43], we determined how TrkB-Shc exerts its dominant negative effect on BDNF-stimulated TrkB-TK+ signaling. Using a non-neuronal cell-line, Chinese Hamster Ovary K1 (CHOK1) cells (which do not express endogenous TrkB receptors), we transiently overexpressed TrkB-Shc protein and examined its effect on TrkB-TK+ protein stability. We used cycloheximide to block protein synthesis as this would prevent newly synthesized protein from replacing protein that has been degraded. From this, we found that TrkB-Shc protein levels were rapidly decreased when cells were exposed to exogenous BDNF. Moreover, in co-expression experiments where TrkB-Shc and TrkB-TK+ were co-expressed, cycloheximide treatment revealed increased protein degradation of phosphorylated TrkB-TK+ protein, a process that is accelerated by BDNF exposure (Figure 6A and B) [43]. Interestingly, while the reduction of phosphorylated TrkB-TK+ protein was more pronounced in the presence of TrkB-Shc following BDNF exposure, the stability of TrkB-Shc protein itself was increased ( Figure 6C).

Discussion
Our recent findings have important implications in regards to the role of TrkB-Shc and its impact on BDNF-mediated TrkB-TK+ signaling in neurons in AD. MEK signaling through ERK1/2 phosphorylation is increased in vulnerable neurons in AD and is implicated in the abnormal phosphorylation of tau and neurofilament proteins [44]. Our finding of a selective attenuation in MEK signaling activity in neurons with elevated levels of cellular TrkB-Shc implicates that elevated levels of this neuron-specific truncated TrkB receptor in AD may occur as a response to the disease. However, our finding that cells may increase TrkB-Shc protein levels in response to BDNF stimulation to regulate TrkB-TK+ activity by increasing degradation of activated receptor complexes also has ramifications for BDNF/TrkB-TK+ signaling in AD. In the non-diseased or control state, this process is akin to feedback regulatory loops observed in metabolic pathways (Figure 7). While BDNF/TrkB-TK+ signaling is important in multiple aspects related to neuronal viability and differentiation, overactivation or inappropriate temporal and spatial activation of BDNF/TrkB-TK+ signaling during brain development or "leakage" of BDNF to adjacent neurons or brain regions can negatively impact brain function. However, in AD, elevated levels of TrkB-Shc in association with reduced BDNF protein levels (which is well documented) and no change in TrkB-TK+ expression may also result in an overall increase in the degradation of phosphorylated TrkB-TK+ receptors, and thus, reduce overall BDNF/TrkB-TK+ activity in neurons in AD.  In the normal, non-diseased state, binding of BDNF to TrkB leads to autophosphorylation of TrkB-TK+ homodimers (but not other TrkB dimer combinations). This leads to activation of downstream second messenger signaling pathways including PLCγ (phospholipase C-gamma), MEK (mitogen-activated protein kinase kinase), and PI3K (phosphatidyl inositol 3 kinase), which are critical for neuronal viability and function. TrkB-TK+ homodimers can also be auto-phosphorylated in the absence of BDNF, although activation of downstream signaling pathways are less intense. BDNF-stimulation of TrkB receptors also leads to their degradation. The arrow thickness indicates the magnitude of effect. The magnitude of decrease in protein levels are greatest for the following receptor combinations when stimulated with exogenous BDNF: TrkB-TK+/TrkB-Shc>TrkB-TK+/TrkB-TK+>TrkB-Shc/ TrkB-Shc. Lower panel: In AD, there is Aβ plaque accumulation. Neurons are exposed to mixed species of Aβ, including Aβ 42 fibrils and oligomers at various stages of aggregation. BDNF protein expression has been reported to be reduced in AD (indicated by red arrow) and TrkB-Shc levels have been reported by us to be increased in AD (hippocampus, CA1) (indicated by green arrow) and in response to Aβ 42 fibril exposure. The increase in TrkB-Shc is predicted to increase heterodimer combinations of TrkB-TK+/TrkB-Shc and homodimer combinations of TrkB-Shc, which would lead to an overall reduction in downstream signaling. Moreover, the increase in TrkB-Shc dimer combinations also increases TrkB receptor degradation. Thus, the combination of reduced BDNF expression and increased TrkB-Shc expression in the AD hippocampus would likely result in an overall decrease in BDNF/TrkB-TK+ signaling. Figure utilizes

Conclusion
Neurotrophin signaling via BDNF/TrkB-TK+ is critical for neuronal viability and function. Further research into this topic area is required to determine when changes in BDNF/TrkB-TK+ signaling begin to occur and whether changes in TrkB-Shc expression, function, and interaction with TrkB-TK+ at different stages of the disease process, in response to Aβ, or in response to other amyloidogenic factors may be protective or deleterious.