**1. Introduction**

#### **1.1. 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 neurotro‐ phin-4/5 (NT-4/5). Their neurotrophic effects are mediated by the tropomyosin receptor kin‐ ase (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 neu‐ rotrophins or their receptors are fatal or exhibit severe neural defects [3].

#### **1.2. 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 un‐ changed or even increased in other brain regions including the frontal cortex and hippocam‐ pus, two major brain regions affected in AD [6-9]. 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 hippocam‐ pal, cortical, and cholinergic neurons [10-14]. 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, 15-21]. Independent

lines of evidence suggest that dysfunction in BDNF signaling may contribute to the neuro‐ degeneration in AD. Brain regions associated with reduced BDNF expression are those dis‐ playing 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, 22-27]. 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.

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 neu‐ rotrophic 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 iso‐ form that has been demonstrated to inhibit BDNF/TrkB-TK+ signaling, it is important to es‐

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**Figure 1. BDNF/TrkB-TK+ signaling pathway.** Activation of TrkB-TK+ by BDNF leads to auto-phosphorylation of tyro‐ sine residues in the intracellular C-terminal domain. This leads to the activation of TrkB-TK+-linked second messenger signaling pathways including PLCγ (phospholipase C-gamma), MEK (mitogen-activated protein kinase kinase), and PI3K (phosphatidyl inositol 3 kinase), which are linked to downstream processes involved in cell adhesion/migration, cells survival, synaptic plasticity, neurogenesis, and neuronal differentiation. This figure is modified from the BDNF

Pathway Figure (Protein Lounge Pathway Templates) from ProteinLounge using Pathway Builder Tool.

tablish what role TrkB-Shc plays in AD development and progression.

#### **1.3. 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+ homodimer‐ izes 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) [29-32] (Figure 1). The two truncated TrkB receptor isoforms include TrkB-TK- and TrkB-Shc. Both truncat‐ ed 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-termi‐ nus. The TrkB-Shc isoform includes the sarc homology containing (Shc) binding domain that is absent in TrkB-TK- [28].

#### **1.4. 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.

#### **1.5. 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, 22-27, 34-36]. 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 neu‐ rotrophic 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 iso‐ form that has been demonstrated to inhibit BDNF/TrkB-TK+ signaling, it is important to es‐ tablish what role TrkB-Shc plays in AD development and progression.

lines of evidence suggest that dysfunction in BDNF signaling may contribute to the neuro‐ degeneration in AD. Brain regions associated with reduced BDNF expression are those dis‐ playing 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, 22-27]. 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

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+ homodimer‐ izes 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) [29-32] (Figure 1). The two truncated TrkB receptor isoforms include TrkB-TK- and TrkB-Shc. Both truncat‐ ed 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-termi‐ nus. The TrkB-Shc isoform includes the sarc homology containing (Shc) binding domain that

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

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, 22-27, 34-36]. 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

has been known for some time, its role in AD has not been defined.

of this BDNF receptor.

**1.3. The BDNF receptor − TrkB**

182 Trends in Cell Signaling Pathways in Neuronal Fate Decision

is absent in TrkB-TK- [28].

**1.5. Importance of TrkB**

**1.4. TrkB and Alzheimer's disease**

**Figure 1. BDNF/TrkB-TK+ signaling pathway.** Activation of TrkB-TK+ by BDNF leads to auto-phosphorylation of tyro‐ sine residues in the intracellular C-terminal domain. This leads to the activation of TrkB-TK+-linked second messenger signaling pathways including PLCγ (phospholipase C-gamma), MEK (mitogen-activated protein kinase kinase), and PI3K (phosphatidyl inositol 3 kinase), which are linked to downstream processes involved in cell adhesion/migration, cells survival, synaptic plasticity, neurogenesis, and neuronal differentiation. This figure is modified from the BDNF Pathway Figure (Protein Lounge Pathway Templates) from ProteinLounge using Pathway Builder Tool.

cies and the absolute amount of fibrils would be low (fibrils were absent in the monomer and oligomer Aβ<sup>42</sup> preparations). Further, in comparison to the Aβ42 monomer and oligomer preparations, the Aβ42 fibril preparations would be most representative of all Aβ42 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

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**Figure 3. TrkB alternative transcript expression in various control and AD brain regions.** Expression of TrkB alter‐ native transcripts in the (A) hippocampus, (B) temporal cortex, (C) occipital cortex, and (D) cerebellum of control (CON) (white) and Alzheimer's disease (AD) (black) postmortem human brain tissue were measured by qPCR. \*\* P=0.004; # P=0.07. Figure is from Wong J, et al. Amyloid beta selectively modulates neuronal TrkB alternative transcript expres‐

In regards to the brain regions examined, AD brain pathology from most severe to least af‐ fected 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 ex‐ posure to preparations of Aβ42 containing fibrils. Aβ42 fibril species are increased in the ad‐ vanced stages of AD [40]. While it is widely accepted that soluble oligomers are the more neurotoxic of the Aβ42 species, evidence also suggest that the neurotoxicity of Aβ42 requires its aggregation in the fibrillar form, particularly in the form of protofibrils [41, 42]. In our Aβ42 preparations, we detected various sizes of fibrils in the Aβ<sup>42</sup> fibril preparations, includ‐

sion with implications for Alzheimer's disease. Neuroscience 2012;210 363-374.

hippocampus (Figures 3 and 4).

**Figure 2. TrkB receptor dimer combinations.** All dimer combinations of TrkB receptors can bind to BDNF. However, only a homodimer of TrkB-TK+ can initiate second messenger signaling. This figure utilizes modified ProteinLounge graphics created using the Pathway Builder Tool (www.proteinlounge.com).
