Behind Closed Doors: The Emerging Role of Focused Ultrasound in Alzheimer’s Disease

Justin Mendoza

Alzheimer’s Disease (AD) has become recognized as a neurodegenerative condition in which declination in cognitive function progressively worsens over time. Hallmarks of AD include basal forebrain cholinergic neuron (BFCN) dysfunction, leading to the loss of post-synaptic connections associated with memory functioning. Another known defect that has been correlated with the early onset of this particular condition is the aberrant decline in the levels of nerve growth factor (NGF), a selective neurotrophin that propagates survival of mature neurons, once bound to its cognate receptor, tropomyosin-related kinase A (TrkA). The NGF-mediated TrkA pathway has been elucidated to support survival mechanisms, as well as the mediation of apoptotic signaling, depending on the bound ligand. With the available information of the pathophysiology and intracellular mechanisms of AD, current research aims to mitigate the severity of early hallmarks; however, a major limitation that prevents the delivery and efficacy of therapeutic regimens is the tightly regulated barrier that is the blood-brain barrier (BBB). As a result, current research has adapted to this limitation, proposing the use of non-invasive methods, such as magnetic resonance imaging-guided focused ultrasound (MRIgFUS) to increase the permeability of the BBB, as a means to deliver therapeutic compounds to elicit the TrkA cascade pathway. Through the work of Xhima et al., the TrkA agonist known as D3, had rescued cholinergic functioning within AD murine models, as opposed to monotherapy involving NGF alone, using MRIgFUS to deliver the agonist. As a result, the use of protective neurotrophins, along with the use of MRIgFUS to circumvent the BBB holds promising results, which can be used to further clinical trials in AD patients. Further research is warranted to elucidate the mechanisms of NGF-mediated therapy, as NGF alone is unable to stimulate TrkA-dependent signaling in an AD murine model.

Key words: Alzheimer’s disease, nerve growth factor, blood-brain barrier, magnetic resonance imagingguided focused ultrasound, TrkA, D3

Introduction

The neurodegenerative disease that is Alzheimer’s Disease (AD), is commonly associated with a decline in cognitive function, being a major factor that plays a role in the progression of dementia [1]. With many pathways leading to neuronal deficits in AD, such as amyloid-b (Ab) plaques, with subsequent tau aggregation, a susceptible brain region is the basal forebrain cholinergic nuclei (BFCN), which has been associated for learning and memory [2]. The reason as to why the BFCN exhibit a decline in activity in AD is due to its dependence on the neurotrophin nerve growth factor (NGF), a biomolecule associated with neuronal survival [3]. Dysfunction of this particular neurotrophinreceptor axis is a common hallmark of AD, and begins to appear during the prodromal period of AD. During the preclinical stages of AD, occurring as early as 20 years prior to onset of clinical symptoms, there is a decrease in neuroprotective capacity, as cholinergic neurons consist of altered NGF levels, accompanied with defects in the processing pathways [4]. The role of NGF has been elucidated to induce neuronal survival and synaptic plasticity, when bound to its cognate receptors, p75 neurotrophin receptor (p75 NTR ) and tropomyosinrelated kinase A (TrkA). In response to the formation of the NGF/ TrkA axis at the BFCN terminals, NGF signals elicit a survival response through downstream molecules, such as the phosphoinositide 3 kinase (PI3K)/Akt pathway. As a result, BFCN have enhanced cholinergic activity, which occurs through increased transcription of an enzyme known as choline acetyltransferase (ChAT), essential for the biosynthesis of acetylcholine. However, in AD, where a declination of both NGF and TrkA is observed, the other cognate receptor p75 NTR has a higher affinity for the Ab plaques, and the precursor of NGF, known as proNGF. Binding of this receptor signals a neurodegenerative pathway, and is the reason for selective degradation of BFCN in AD [5]. With the available information on the intracellular mechanisms of AD, current research aims to develop a therapeutic regimen centralizing on NGF-mediated therapy. However, a limitation that is commonly addressed is the seemingly impermeable blood-brain barrier (BBB). Moreover, NGF is unable to cross the BBB. To address this issue, current research aims to increase the permeability of the BBB, through non-invasive techniques such as focused ultrasound. Writing in Science Advances, Xhima et al. [6] proposes the use of magnetic resonance imagingguided focused ultrasound (MRIgFUS) to deliver a selective TrkA agonist into the BFCN, as a means of rescuing cholinergic functioning within a TgCRND8 murine model that encapsulates the phenotype of human AD. With the known mechanism of action of MRIgFUS, which centralizes on increasing the permeability of the bloodbrain barrier [7], the authors aimed to deliver D3 to the basal forebrain, a site of interest that is associated with cognitive decline in AD. Through an appropriate murine model of AD, combined with the non-invasive mechanism of MRIgFUS, the researchers were able to efficiently deliver D3, a TrkA agonist that elicits a survival mechanism, similar to that of NGF, when bound to TrkA. MRIgFUS facilitated D3 delivery to the basal forebrain of a TgCRND8 murine model. D3 activity, not NGF, led to rescuing of cholinergic neuron functioning. The results of this study provide evidence of a potential therapeutic regimen to reverse the neurodegenerative effect that AD entails.

Results: TgCRND8 Murine Models as an acceptable model of human Alzheimer’s

First, a phenotypic analysis of the TgCRND8 murine model was conducted, to determine its eligibility as a model for human AD. This was elucidated through the measurement of NGF, TrkA and p75 NTR mRNA and protein levels through Western blot. By studying comparisons of age-matched TgCRND8 murine models to their wild type counterparts, a noticeable decline in NGF and TrkA mRNA levels within the Tg mice, keeping consistent with the human phenotype of AD [5]. Thus, this result verified the possibility of representing the human phenotype of AD with TgCRND8 murine models.

D3 stimulates TrkA signaling cascade, as opposed to NGF alone

Another finding centralized on the roles of D3 and NGF. Within this particular experiment, an in vivo analysis of these neurotrophins was conducted in Tg mice by examining the signaling cascades elicited upon binding of the cognate TrkA receptor. Upon intraparenchymal injection into the basal forebrain, D3, as opposed to the neurotrophin NGF, had stimulated the TrkAdependent signaling cascade, which was measured through levels of phosphorylated TrkA (pTrkA), pMAPK, and pCREB. This particular result had provided significant evidence of a potential therapeutic regimen designed to reverse cholinergic neuron degradation in AD, thus furthering the existing evidence of rescuing cholinergic function through a selective TrkA agonist [8].

Delivering D3 to the Basal Forebrain through MRIgFUS

With evidence of a therapeutic regimen that may prove to be optimal in treating cholinergic neuron degradation in AD, the only obstacle yet to be overcome was the tightly regulated BBB. The non-invasive method of MRIgFUS was employed to ensure an efficient delivery of the selective TrkA agonist to the basal forebrain. To measure the efficacy of this delivery method, highperformance liquid chromatography was used to measure D3 concentrations in both the Tg mice and the non-Tg control mice upon intravenous administration of D3. The hypothesis of MRIgFUS as an efficient non-invasive method to deliver neuroprotective agents was confirmed by an increase in D3 concentration within Tg mice, as opposed to non-Tg mice. This result was consistent with other findings using focused ultrasound to deliver therapeutic agents in patients with AD [9,10].

MRIgFUS D3 delivery rescues cholinergic functioning through TrkA-dependent signaling

Examining the functional effects of MRIgFUS-mediated delivery

of the TrkA agonist was done through Western blot, in which pTrkA was measured. There was an observed increase in pTrkA expression within the D3-FUS mice, as opposed to the D3 nonFUS mice. Furthermore, this indirectly led to the increased activity of cholinergic neurons within the Tg model. Normally, the enzymatic activity of ChAT was reduced in Tg mice, ultimately leading to decreased biosynthesis of ACh. 90 days after sonication, there was an observed increase of ChAT activity within D3-FUS treatment. Overall, this study provides evidence of a possible therapeutic regimen with an efficient delivery method, which may be implemented into future clinical trials as a means of dealing with the neurodegenerative effects of AD.

Discussion/Critical Appraisal (Talk about dismissing Ab, NGF selectivity, and impaired memory)

In this study, significant evidence was observed to propose a NGF. To measure the efficacy of NGF-based therapy, an immunoprecipitation and Western blot of the downstream media

possible treatment to AD, through the use of MRIgFUS and the TrkA agonist, D3. Moreover, it further validated the use of a TgCRND8 murine model as a potential model for human AD. The importance of these new findings centralizes on the ability to recapitulate human circumstances of AD, and develop possible mechanisms to increase the efficacy of the delivery of therapeutic agents. Generally, the common route of treatment for AD is the use of pharmaceutical compounds, such as acetylcholinesterase inhibitors (AChEIs), which prevent the breakdown of acetylcholine within the cholinergic neurons, maintaining some cholinergic function. However, the authors conclude that through MRIgFUS and a selective TrkA antagonist would maintain the morphology of the BFCN, as it would lead to a more profound effect through increased enzymatic activity of ChAT, which would compensate for the loss of ACh, whilst increasing neuronal survival, which cannot be achieved through AChEI alone.

While the work of Xhima et al. provides evidence of a potential therapeutic regimen to reverse the neurodegenerative effects of AD, there are some unanswered questions that need to be addressed. For instance, the study finds a mechanism of rescuing cholinergic neurons by enhancing the survival pathway (ie. D3 activating TrkA, leading to the Akt pathway), but does not address a method for eliminating the Ab pathology, which is known to associate with the p75 NTR receptor, in an apoptotic signaling cascade. Within the study, a major finding centralized on the efficacy of D3 administration, accompanied with the inefficacy of native NGF administration. The authors had hypothesized that NGF-mediated therapies within previous trials were limited to the Ab and tau pathologies, ultimately leading to dysregulation of NGF metabolism. Moreover, due to the invasive method of surgery to administer NGF-based agents, the authors conclude that the risks of NGF-based theracan be run. However, rather than the sole administration of D3, a combination therapy can be run, in which anti-Ab antibodies and NGF administration occurs. Through the use of noninvasive MRIgFUS, the elimination of Ab pathology is now possible. Moreover, while TrkA expression is compromised within AD, a quantitative analysis through radiolabeling prior to therapy can provide an accurate measurement of the concentration of TrkA receptors, which can determine the optimal concentration of NGF needed to engage TrkA receptors. Moreover, through MRIgFUS, larger antibodies targeting Ab plaques can cross through the BBB, targeting Ab plaques, which have been associated with dysregulation of the metabolic processing of

pies outweigh the benefits. tors relevant in TrkA signaling can be used. If there is increased levels of pCREB, pAKT, or pMAPK, this provides evidence of an efficient NGF-based therapy.

Future Directions

Proceeding with the findings of Xhima et al., to elucidate the efficacy that NGF-based therapy entails, a similar experiment

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