Molecular signatures in exosomes as diagnostic markers for neurodegenerative disorders

Exosomes are small membrane-bound entities of endocytic origin. These membrane-derived, extracellular vesicles have been shown to be secreted by a number of cell types such as adipocytes, platelets, cardiac progenitor cells, muscle cells, mesenchymal stem cells,  lymphocytes, tumor cells, embryonic stem cells, umbilical cord blood-derived cells and cells in the central nervous system including neurons, neuroglial cells etc., These extracellular vesicles contain various protein, lipid, proinfl ammatory cytokines and RNA species whose content is altered under pathological diseased conditions of the CNS. Currently, the techniques available to diagnose neurodegenerative disorders involve analysing the physiological levels of certain proteins in the Cerebrospinal Fluid (CSF) and checking for extracellular senile plaque formation (protein aggregation and accumulation) in the brain using MRI/CT scans. These techniques are quite expensive, invasive and painful in nature as collecting the CSF and accessing the brain area are diffi cult. In the past few years, there is a growing interest on using exosomes for diagnosis of neurodegenerative disorders due to their easy availability from most of the biological fl uids including the blood, urine, saliva, breast milk, semen etc., their extremely high disease-specifi c bio-molecular signature/profi le, the ability of exosomes to cargo a variety of biomolecules in between cells and their capacity to cross the blood-brain barrier. These make them a potential biomarker for neurodegenerative disorder. This review begins with a brief introduction about exosomes and focuses mainly on using exosomes for diagnosing major neurodegenerative diseases like Prion disease, Amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease. Review Article Molecular signatures in exosomes as diagnostic markers for neurodegenerative disorders Palaniswamy Rani*, Sevugan Karthik and Sampathkumar Srisharnitha A Department of Biotechnology, PSG College of Technology, Coimbatore, Tamil Nadu 641004, India Received: 06 March, 2020 Accepted: 05 June, 2020 Published: 06 June, 2020 *Corresponding author: Dr. P Rani, Professor, Department of Biotechnology, PSG College of Technology, Avinashi Rd, Peelamedu, Coimbatore, Tamil Nadu 641004, India, Tel: 9443161653; Fax: 91-0422-2573833; E-mail:


Introduction
Exosomes are small cell-derived vesicles whose secretion has been reported for a number of cell types and are present in various biological fl uids and Central Nervous System (CNS) tissues. These are the extracellular vesicles that are released from cells due to the fusion of the Multivesicular Body (MVB), an intermediate endocytic compartment, with the plasma membrane. This releases the Intraluminal Vesicles (ILVs) into the extracellular milieu, known as exosomes [1 ]. Exosomes are known to have various important functions under both physiological and pathological conditions in the CNS, such as the cell-to-cell communication which is done by the transfer of exosomes between neurons [2 ].
Neurons make up the nervous system which comprises the brain and spinal cord. Neurons lack the ability to reproduce or renew themselves, as a result of which the body is unable to replace them if dead or damaged. Neurodegenerative disorders are chronic and fatigue conditions resulting in the death of neurons causing ataxias or dementias. Dementia refers to a syndrome that is characterized by progressive deterioration of cognitive functions. Few examples of neurodegenerative diseases include Prion disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), Parkinson's Disease (PD), Alzheimer's Disease (AD) [3] .
Evidence is accumulating that exosomes play a key role in processes such as coagulation and intercellular signalling. Consequently, there is a growing interest in the clinical applications of exosomes as they can potentially be used for diagnosis, prognosis, therapy and as biomarkers for health and a variety of diseases. First of all, Exosomes carry signifi cant amounts of molecular constituents including proteins, lipids and nucleic acids whose content and composition solely depend and vary with their tissue of origin under pathological conditions [4,5] . Second, exosomes can be concentrated to signifi cantly increase the detection sensitivity and their ability to cross the blood-brain barrier in both the directions [6] , helps to overcome the problem of the circulating proteins and nucleic acids getting diluted in the bloodstream (most of them originate from other tissues besides neurons due to the tight regulation of the blood-brain barrier in molecule transport). Third, isolation of exosomes can be done using painlessinvasive methods from easily available biological fl uids like blood, saliva, urine etc [7][8][9] , making it a potential biomarker for the diagnosis of neurodegenerative disorders.
This review is focused on using exosomes as biomarkers for diagnosing various neurodegenerative disorders. Here, we fi rst provide an overview of exosomes and in the later part; we discuss using exosomes as a diagnostic tool in the context of Prion disease, ALS, HD, PD, and AD.

Exosomes: Origin, composition and functions
Exosomes are lipid bi-membranous entities having a diameter of 30-120 nm and are of endocytic origin that is present in almost all biological fl uids including blood, saliva, semen, urine, breast milk, the culture medium of cell cultures and extracellular matrix bioscaffolds (non-fl uid). Invagination of a cell's plasma membrane forms small intracellular vesicles which upon fusion results in early endosomes [10]. O n maturation, these early endosomes give raise to the intraluminal vesicles (ILVs) [10, 1 1] . The accumulation of cytoplasmic molecules such as proteins, messenger RNA (mRNA) and small non-coding microRNAs (miRNAs) inside the ILVs is aided by a subset of molecules called endosomal sorting complex required for transport (ESCRT). Accumulation of ILVs in the late endosomes forms a Multivesicular body (MVB).
Exosomes released from the CNS cells such as neurons, oligodendrocytes etc., play a signifi cant role in the communication between neuron-neuron, neuron-glia and in the regeneration of damaged axons. These endosome-derived vesicles carry specifi c RNA and protein cargo that regulates certain signal transduction pathways of the recipient cells [2].

E xosomes in disease diagnostics
Prion disease: Prions, meaning proteinaceous infectious particles are composed of prion proteins (PrP), which causes transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease (CJD) in humans [15]. N ormally PrPc, a protein with a well-defi ned 3D structure is present on the surface membrane of many cells. This protein has been reported to play a signifi cant role in cell-cell adhesion and intracellular signalling [16]. U nder pathological conditions, this protein abnormally folds and clumps in the brain forming PrP Sc , leading to the damage of brain tissues [15]. T his infectious isoform of PrP (PrP Sc ) has the ability to convert normal PrP (PrP c ) into infectious isoforms by causing conformation changes in them [17], m aking prion disease a progressive and a fatal neurodegenerative disorder.
Dissemination of prions is found to be mediated through exosomal pathways. Exosomes were identifi ed as intercellular carriers of both PrP Sc and PrP c [18,19]. The ESCRT machinery and the need for ceramide, is signifi cant for exosome biogenesis.
Hence, the spread of infectious prions can be controlled by altering the ESCRT complex and decreasing ceramide levels [20]. H owever, data from certain studies showed that in some cases, the prion infection might not necessarily be associated Citation: Rani  with PrP Sc (i.e.) Transmission of prion infection has occurred even in extremely low levels or in the absence of detectable abnormal Prion Proteins, thereby eliminating the possibility for considering the presence of PrP Sc in exosomes as a potential biomarker for diagnosis of the disease [21, 2 2] . Generally, under pathological conditions, the miRNA expression levels are altered. In case of prion infection, 15 miRNAs were found to be de-regulated; miR-342-3p, miR-320, let-7b, miR-328, miR-128 and miR-139-5p were up-regulated over 2.5 folds and miR-338-3p, miR-337-3p were down-regulated over 2.5 folds.
Hence, the prion-induced neurodegeneration is found to be associated with a highly conserved, disease specifi c deregulation and differential expression pattern of a unique set of miRNAs.
Deregulation of certain miRNA seen in prion disease might be a response to the abnormal accumulation of PrP Sc [23, 2 4] .
Exosomes that were released from prion-infected neural cells were observed to contain a deregulated miRNA cargo (i.e.) the levels of let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 were increased and the levels of miR-146a was decreased when compared to the miRNA content of exosomes released from normal brain tissues. Therefore, the circulating exosomes released from prion-infected brain tissues have extremely specifi c, conserved and a distinct miRNA signature that can be exploited for disease diagnostics in an important fi nding that SOD1, being a cytoplasmic protein that lacks a signal peptide, depends on exosomes for its extracellular export [31]. I n order to identify distinct RNA profi les from exosomes secreted by mutant cells, a study was conducted, which assessed a set of infl ammatory-associated miRNAs in NSC-34 MN-like cells, which were transfected with mutant SOD1(G93A) and the effects produced by their derived exosomes (mSOD1 exosomes) in the activation and polarization of the recipient N9 microglial cells. The results showed that the expression level of infl ammatory-associated miR-124 was highly increased in mSOD1 NSC-34 cells and mSOD1 exosomes.
Thus, this data suggests that the miR-124 is translocated to the exosomes from mSOD1 MNs and this modulation in mSOD1 exosomal cargo, in turn determines the early and late phenotypic alterations in the recipient N9-microglial cells, making them a promising therapeutic agent in halting microglia activation and associated effects in motor neuron degeneration [32]. E xosomes which were obtained from the blood plasma samples of ALS patients were analysed to identify ALS-associated, specifi c miRNAsignatures. On subjecting the exosomes obtained from ALS patients and healthy controls to next generation sequencing, differentially expressed miRNAs were identifi ed. This data was subsequently validated by droplet digital PCR which showed elevated levels of 5 miRNAs and reduced levels of 22 miRNAs in the exosomes collected from patients with ALS as compared with healthy control subjects. Exosomes that were secreted by ALS brain tissues were found to have a deregulated miRNA profi le including miR-9-5p, miR-183-5p, miR-338-3p and miR-1246. MiR-15a-5p and miR-193a-5p. Therefore, exosomes released from ALS brain tissues, which can cross the blood-brain barrier and enter the circulatory system, have highly specifi c and distinct miRNA signatures making them a potential biomarker for disease diagnostics [33]. inclusions of aggregated -synuclein (deposits of ubiquitinated protein in neural cytosol) termed Lewy bodies. Mitochondrial dysfunction has also been proposed to play an important role in the pathogenesis of PD [40][41][42].
On evaluating the levels of -synuclein in exosomes, which are relatively more specifi c to the CNS, it was found that the concentration of cerebrospinal fl uid (CSF) exosomal -synuclein was lower in PD patients when compared to healthy controls. In contrast, it has also been reported that the concentrations of plasma exosomal -synuclein are higher in PD patients compared to the controls [43]. Exo so mes have also been found to be present in the saliva of PD patients and the levels of -synOlig, -synOlig/-synTotal in salivary exosomes are comparatively higher in PD patients than in controls [44]. Blo od plasma levels of exosomes derived from various sources such as neurons, astrocytes and oligodendrocytes were quantifi ed in diseased patients and healthy controls to formulate a correlation between the exosome levels and PD. Results showed that the plasma levels of neuron-derived exosomes (characterized using the biomarkers CD81 and SNAP25) were signifi cantly higher in PD compared to controls [45]. On an alysing the proteomic data of exosomes isolated from serum samples of PD patients, it was found that 23 proteins including Syntenin 1 were differentially abundant in Parkinson's patients [46]. Micr oR NA profi ling was performed on exosomalmiRNA isolated from CSF of Parkinson's patients and the data showed that there was a signifi cant up-regulation in 16 exosomalmiRNAs including miR-10a-5p, let-7g-3p, miR-153 and miR-409-3p and 11 exosomalmiRNAs including miR-1 and miR-19b-3p were under regulated in PD CSF when compared to healthy controls [47]. Globa l microRNA expression profi les were obtained from circulating plasma exosomalmiRNA in diseased patients and healthy controls, as a result 13 most differentially-expressed miRNAs in PD, namely, miR-548b-3p, miR-1307, miR-647, miR-505, miR-192*, miR-626, miR-506, miR-1826, miR-222, miR-572, miR-671-5p, miR-1225-5p and miR-9* were identifi ed. Data show that under pathological conditions, there is a signifi cant over expression of exosomal miRNA-331-5p and the levels of exosomal miR-505 were signifi cantly lower in PD Plasma compared to controls. k-TSP algorithm has been used to identify 9 pairs of PD-predictive miRNA classifi ers, namely, miR-1307/miR-632, miR-1225-5p/miR-891b, miR-579/miR-708*, miR-647/miR-99a*, miR-1826/miR-450b-3p, miR-506/miR-1253, miR-488/ miR-518c*, miR-192*/miR-485-5p and miR-200a/miR-455-3p. The above-mentioned pairs can be interpreted as, for the fi rst pair miR-1307/miR-632, if the expression of miR-1307 is signifi cantly higher than that of miR-632, then the patient has PD else they are normal. A similar interpretation pattern can be applied for the rest of the miRNA k-TSP pairs as well [48,49]. Th is data as a whole suggests that exosomes play a signifi cant role in the pathogenesis of PD by acting as an intercellular carrier of -synuclein and various miRNAs, thus can be used as a potential diagnostic biomarker for PD.
Alzheimer's disease: Alzheimer's is a chronic, progressive and most common neurodegenerative disorder which causes irreversible damage and death of neurons, mainly in the cortex and hippocampus. On average, two thirds of all dementia cases (42 to 81 percentage) are Alzheimer's types [40]. The path ol ogical hallmarks of AD usually include the deposition of -amyloid fi brils (encoded by APP) and neurofi brillary tangles, composed of abnormally hyperphosphorylated microtubule binding protein tau (encoded by MAPT). The deposits of the Amyloid- peptide (A) in the form of extracellular senile plaques is the cleavage product of  -amyloid precursor protein (APP) by -, and secretases. In particular, the secretase cleaves APP into A 42 (Amyloid- peptide which is 42 amino acids long) which has pathogenetic signifi cance as it forms the toxic insoluble fi brils that deposit and accumulates as extracellular senile plaques causing AD. In short, Alzheimer's is associated with mutations in either one of the three proteins namely, APP, presenilin-1 (PS1) or presenilin-2 (PS2) (the catalytic subunit of secretase is composed either of PS1 or PS2) [50,51].
Quantifi c ation of the levels of various AD related proteins in plasma neuronal derived exosomes (NDEs) led to a fi nding that the levels of P-S396-tau, P-T181-tau and A1-42 in plasma NDEs were extremely high whereas, the levels of neurogranin (NRGN) and the Repressor Element 1-Silencing Transcription factor (REST) were signifi cantly reduced in Alzheimer's patients and patients with Mild Cognitive Impairment (MCI) converting to AD when compared to those in cognitively normal controls and patients with a stable MCI [52,53]. Also, the p lasma NDE levels of cathepsin D, ubiquitinylated proteins and lysosome-associated membrane protein 1 (LAMP-1) were signifi cantly higher and the levels of heat-shock factor-1, heat-shock protein 70, and low-density lipoprotein receptorrelated protein 6 were signifi cantly lower in AD patients when compared to healthy controls [54]. On measuring t he expression levels of Micro RNA in plasma exosomes, it was found that there was a signifi cant difference in the expression levels of 20 miRNAs namely, miR-185-5p, miR-23b-3p, miR-29b-3p, miR-125b-5p, miR-138-5p, miR-24-3p, miR-139-5p, miR-150-5p, miR-152-3p, miR-338-3p, miR-342-3p, miR-342-5p, miR-548at-5p, miR-141-3p, miR-659-5p, miR-3613-3p, miR-4772-3p,miR-3916,miR-5001-3p and miR-3065-5p among the AD group, especially, miR-342-3p was signifi cantly down-regulated in Alzheimer's patients [55]. Studies indic at e that the overexpression of microRNA -miR-193b signifi cantly represses the mRNA and protein expression of APP. Thus, the level of exosomal miR-193b in blood and CSF was found to be signifi cantly lowered in Alzheimer's patients as compared to the controls [56]. Release of the a bo ve-mentioned Alzheimer's related proteins and miRNAs in plasma NDEs occur approximately 10 years prior to the onset of the disease, making neuronal derived exosomes a novel biomarker for Alzheimer's diagnosis.

Conclusion
In the last decade, there is a growing interest in using exosomes as a potential disease diagnostic tool. Initially, waste material disposal from cells was considered as the only known Citation: Rani