Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral
sclerosis, and prion disease are representative neurodegenerative diseases that share
common sub-cellular features, the most obvious of which is a strong association with
the accumulation of misfolded, aggregated, and insoluble forms of proteins in the
brain, as evidenced in post-mortem brain tissues of patients.
Diagnosis of neurodegenerative diseases is often made by exclusion, and it is made
when individuals are at the advanced stage of disease with a brain that is markedly
damaged. Also, neurodegenerative diseases are not timely and effectively treated,
and current treatments are limited to a small number of drugs that control some of
the symptoms of early disease. This scenario emphasizes the need for alternative diagnostic
and therapeutic approaches.
Profiles of altered microRNAs (miRNAs) (reflected by increase or decrease of miRNAs
expression level with respect to the normal level) isolated from blood exosomes associated
with neurodegenerative disorders suggest the possibility of discovering new potential
candidate biomarkers for early diagnosis of neurodegenerative diseases (Van Giau and
An, 2016). miRNAs, which are small non-coding RNAs that can post-transcriptionally
regulate gene expression, are highly abundant in exosomes. In clinical setting, exosomes
are frequently proposed as therapeutic drug carriers, but, since the release of exosomes
and their molecular cargo are cell type specific, exosomes have been also proposed
as potential biomarkers for disease diagnosis and monitoring (Lin et al., 2015).
Thus, exosome-based approaches aimed at targeting dysregulated miRNAs or identifying
them in blood may be adopted as alternative strategies that might facilitate and anticipate
the therapy and diagnosis, respectively, of neurodegenerative diseases.
Figure 1
summarizes the potential fields of clinical application of blood exosomes and miRNA
to the neurodegenerative disorders.
Figure 1
Potential fields of clinical application of exosomes and microRNA (miRNA) to the neurodegenerative
diseases.
miRNAs as potential therapeutic agents for treating neurodegenerative diseases: Recently,
together with a colleague, I explored the feasibility of the therapy of neurodegenerative
diseases with miRNA through an extensive review of the literature concerning this
specific topic (Ridolfi and Abdel-Haq, 2017). First, we performed a rapid overview
of miRNAs involved in the pathogenesis of the above mentioned neurodegenerative diseases
to underline the extent of this involvement and its crucial role. We found that multiple
miRNAs are usually involved in the pathogenesis of neurodegeneration, and they often
concern different signal pathways, but; sometimes they act on the same pathway modulating
different down or upstream targets. On the other side, a single miRNA sequence can
simultaneously affect multiple pathways related to a single pathology, such as miR-196a
and miR-22 in Huntington’s disease [for details see Ridolfi and Abdel-Haq (2017)].
After this brief overview, we analyzed the experimental models performed till now
using miRNAs (miRNA mimics, miRNA inhibitors or artificial miRNAs for miR-155, miR-146a,
miR-124 or miR-7) to modulate the endogenous levels of individual miRNAs in Alzheimer’s
disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease,
thereby evaluating the pharmacologic feasibility of this approach for treating neurodegenerative
diseases. Our analysis led to the identification of the inflammatory pathway as a
potential target for the therapy of neurodegenerative diseases. In fact, the presence
of an immune and inflammatory response modulated by key inflammatory miRNAs (for example,
miRs 155, 146a and 124) has been observed in all the examined neurodegenerative diseases.
Besides, this response was present throughout the disease but with a different extent
and role during the different disease stages, suggesting the existence of a neuroinflammatory
response modulated by distinct disease-stage miRNAs. The analysis also highlighted
that, similarly to conventional therapeutics, miRNAs effect can be modulated by a
structure-activity relationship, which could negatively influence the miRNA pharmacokinetic
and pharmacodynamic profiles.
The most critical points that emerged during this analysis are mainly represented
by the lack of adequate characterization of the secondary effects produced by miRNA-based
therapies, and by the lack of basic data on the pharmacological properties of miRNA
mimics and antagonists. Suitable pharmaco- and toxico-kinetics that focus on the tissue
distribution and pharmacology safety are also needed to determine the superiority
of miRNAs in terms of efficacy and safety relative to analogous conventional therapies.
A careful optimization and worldwide standardization of the miRNA detection methods
and protocols would overcome many of the contradictions and inconsistencies observed
in the literature data concerning the identification of dysregulated miRNAs related
to neurodegenerations. Also, disease-stage studies for assessing miRNAs levels, as
well as the availability and the choice of appropriate animal/cellular models for
validating the efficacy of miRNA drugs, might appreciably facilitate the clinical
application of miRNA to the therapy of neurodegenerative diseases.
Thus, there are challenges in the clinical application of miRNA-based therapy to neurodegenerative
diseases. One more challenge is the availability of an effective drug delivery system
to the brain that can guarantee high treatment efficacy and can increase patient compliance.
Exosomes, which are small extracellular vesicles that have the intrinsic ability to
traverse biological barriers and to transport functional small RNAs between cells
naturally, are considered as an optimal delivery vehicle for miRNA. Efficient exosome-mediated
delivery of miRNA and the feasibility of loading miRNA mimics, miRNA inhibitors, and
shRNA expressing plasmids into exosomes are well-supported by proof-of-concept studies
(El-Andaloussi et al., 2012; Lee et al., 2017).
Are exosomes a possible key step toward the clinical application of miRNAs to the
therapy of neurodegenerative diseases? Exosomes are attractive for the delivery of
miRNA for several reasons (Ridolfi and Abdel-Haq, 2017) but especially because they
can prevent miRNAs degradation by RNase, thus allowing their stable circulation in
the bloodstream and other fluids and tissues. Free miRNAs are degraded immediately
and completely by RNase. Nevertheless, there are some challenges that can influence
the loading efficiency of exosomes in miRNA, as well as the quantity of miRNA that
reaches the brain (Ridolfi and Abdel-Haq, 2017).
Therefore, for an efficient treatment of neurodegenerative diseases, improved or innovative
approaches/strategies are needed that enable a better implementation of exosomes and
further increase and optimize their loading efficiency for efficient delivery of miRNAs
to the central nervous system.
In this respect, System Biosciences (SBI) has developed a new exosome-loading system
in which a specific RNA sequence tag “XMotif” has been fused to a miRNA (XMIRs) or
anti-miRNA (AXMIRs) sequence by using a designed oligo (SBI: http://www.systembio.com).
After transfection into cells, the XMotif sequence drives the selective encapsulation
of the small RNA into exosome. This latter can be delivered to target cells after
isolation. Such an approach would significantly increase the amount of miRNA incorporated
and delivered to the site of action.
miRNA stability and, consequently, duration of its activity and efficacy can be increased
by binding of miRNA to specific miRNA-binding proteins [such as nucleophosmin 1 (NPM1)]
at the level of the adenylate/uridylate-rich elements (AREs) structure; this protects
the miRNA from degradation before loading into exosomes. Empty synthetic exosomes
or engineered exosomes packaged with miRNA-binding protein complex can be delivered
to target cells. This approach would significantly improve the quantity and quality
of miRNA reaching the site of action due to the double protection offered by the miRNA-binding
protein and the exosomes themselves.
Delivering miRNA-containing exosomes secreted by differentiated stem cells is another
powerful approach that would increase and improve the delivery of miRNA to target
cells. After transfection of stem cells with the therapeutic miRNA and differentiation
into the desired precursor cells, exosomes secreted by miRNA-overexpressing precursor
cells can be delivered to target cells where they transfer their content, including
the therapeutic miRNA. This approach has several advantages over transplantation of
stem cells and can mitigate or even eliminate many of the safety concerns and limitations
associated with transplantation. However, for technical, ethical, and legal issues,
this promising approach will not be immediate and might advance to clinical applications
within the next few years.
miRNA-based therapy should be optimized to achieve the highest efficacy but avoiding
many of the side effects caused by both primary pharmacodynamics (action on the pharmacologic
target) and secondary pharmacodynamics (off-target effects), where these latter represent
the main limitation of miRNA-based therapy. Such an optimization could be performed
by monitoring treatment efficacy and disease progression. The possibility to monitor
treatment efficacy offers the opportunity to adjust treatment protocol or change therapeutic
strategy as early as possible, when necessary.
Thus, as a next step or in parallel to miRNA-based therapy, efforts should focus on
providing a valid means/tool for monitoring response to treatment/disease progression.
Exosomes are the elective candidates for this purpose since they are now largely known
as an enriched source of functional biomarkers for several diseases, including brain
disorders and neurodegenerative diseases (Lin et al., 2015).
Future prospects: blood exosomes as biomarkers for monitoring treatment outcome and
disease progression: Monitoring the response to therapy for brain disorders is hampered
by the fact that the core pathology lies in the brain, hidden from a direct study
in living patients. Thus, monitoring therapy outcome by using brain-derived exosomes
from tissues or biofluids outside the brain would be a great advance for the therapy
of brain disorders.
Exosomes are secreted by most cell types, including neuronal cells in vivo and in
vitro and have been found in various biofluids including blood, urine, amniotic fluid,
breast milk, malignant ascites fluid, and cerebrospinal fluid (Lin et al., 2015).
Exosomes contain distinct subsets of RNAs and proteins depending upon the cell type
from which they are secreted, making them useful as an enriched source of molecules
for biomarker discovery and profiling. Furthermore, since a drug target is part of
a signal pathway and, in many cases, it is also related to the biomarker of the disease,
exosomes also represent a valuable means for monitoring therapy efficacy and disease
progression.
Additionally, toxic forms of α-synuclein, amyloid beta, and prion protein, which are
hallmarks of Parkinson’s disease, Alzheimer’s disease, and prion diseases, respectively,
have been shown to be effectively packaged into exosomes that had been isolated from
the respective disease-associated material (Quek and Hill, 2017).
Using biofluids as a source of brain-derived exosomes for monitoring the response
to treatment for neurodegenerative diseases is preferred, and the cerebrospinal fluid
is the best biofluid for this purpose due to its proximity to the brain. However,
cerebrospinal fluid is not an ideal specimen for routine monitoring due to the invasive
nature of its collection by lumbar puncture, and therefore blood is preferred for
such purposes.
Blood, besides being routinely available and much less invasive than cerebrospinal
fluid, is similarly rich in brain-derived exosomes. Additionally, the feasibility
and reliability of using blood exosomes as a source of brain-derived biomarkers for
Alzheimer’s and other neurodegenerative diseases have already been assessed by several
proof-of-concept studies (Fiandaca et al., 2015; Kapogiannis et al., 2015; Winston
et al., 2016; Mustapic et al., 2017).
Using blood, exosomes can be collected noninvasively over a long period, allowing
for continuous monitoring of disease progression and response to therapy. Besides,
blood exosomes are abundant and can be preserved through several freeze-and-thaw cycles.
Growing evidence indicates that the content of exosomes secreted by neuronal cells
in blood can reflect the health-state of their originating cells/tissues. Indeed,
several Alzheimer’s disease-related proteins such as amyloid beta, tau and phosphorylated
tau, and amyloid precursor protein and its cleavage products have been shown to integrate
and secrete within blood exosomes from neuronal cells (Fiandaca et al., 2015; Quek
and Hill, 2017). Additionally, it has been found that blood exosomes contain proteins
such as cathepsin D, lysosome-associated membrane protein 1, ubiquitinylated proteins,
and heat shock protein 70, which levels are altered in preclinical Alzheimer’s disease
years before disease onset (Goetzl et al., 2015). Interestingly, Kapogiannis et al.
(2015) found that the ratio of phospho (P)-serine-type 1 insulin receptor substrate
(P-serine312-IRS-1) to P-pan-tyrosine-IRS-1 in neural-derived blood exosomes could
predict the development of Alzheimer’s disease (up to 10 years before Alzheimer’s
disease onset) in type 2 diabetic patients. While, plasma exosomes from Parkinson’s
disease patients contained alpha-synuclein (Shi et al., 2014).
Monitoring action can be performed by assessing the extent of the level of restoration
of distinct molecules after treatment. For example, it is possible to monitor the
levels of the dysregulated miRNA since it has been reported that transferred miRNA
might be encoded in the cargo of the exosomes of the recipient (target) cells. Otherwise,
it is possible to monitor the levels of molecules regulated by the dysregulated miRNA
or molecules closely correlated with the pathological mechanism (hallmark biomarkers).
The fact that exosomes express markers that allow their tracking to the cell of origin
makes the use of exosomes for diagnosis and monitoring purposes particularly appealing.
Conclusions: Optimization of miRNA-based therapy for neurodegenerative diseases by
using blood exosomes as a tool for monitoring both clinical and treatment outcomes
might represent a considerable challenge but offers opportunities for a wide range
of brain diseases (e.g., neurodegenerative diseases, such as Alzheimer’s disease,
Parkinson’s disease, amyotrophic lateral sclerosis, and prions) to be timely and effectively
treated with the most suitable treatment and approach. This could represent a key
step toward a correct and efficient clinical application of miRNAs.
Furthermore, this approach would reduce the global burden and the costs of care for
these diseases. Moreover, this approach might allow for studying the underlying mechanisms
involved, but not previously associated with neurodegenerative diseases. Finally,
neural-derived blood exosomes would make brain content (of limited accessibility)
available within the readily accessible blood.