Small But Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration Part 4

What facilitates the incorporation of α-synuclein in exosomes? Alpha-synuclein is found to be associated with lysosomes [210] and endosomes extracted from mouse brains [207], suggesting that α-synuclein may be recruited to early or late endosomes. 

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Late endosomes may fuse with lysosomes for degradation of α-synuclein or result in the formation of multivesicular bodies to release α-synuclein associated with exosomes. 

Evidence suggesting that the endosomal pathway may promote the incorporation of α-synuclein into exosomes comes from ubiquitination studies. Davies and colleagues found that α-synuclein is ubiquitinated by the E3 ligase Nedd4 and ubiquitinated α-synuclein is targeted to endosomes [211]. This process is negatively regulated by USP8 [212]. 

SUMOylation appears to be another mechanism for sorting of α-synuclein to exosomes. SUMO protein modification is a ubiquitin-independent ESCRT mechanism that appears to regulate α-synuclein release via exosomes [213]. 

Neutral sphingomyelinase-2 hydrolyses sphingomyelins to ceramide and phosphocholine. Inhibiting neutral sphingomyelinase-2 with cannabinol (DDL-112) for five weeks reduced α-synuclein aggregates and exosome biogenesis and improved motor function in the PD mouse model [214]. 

A fundamental question to understand the pathogenesis of Parkinson's disease is how exosomes relay the toxic effects of α-synuclein. Exosomes may aid Parkinson's disease pathogenesis by promoting aggregation of α-synuclein due to their lipid and/or protein composition thus facilitating uptake of α-synuclein by cells. 

Several studies pointed out that exosomes contain α-synuclein as oligomers. Several cellular processes and enrichment of certain molecules within cells cause α-synuclein oligomerization and often in combination with other proteins. Exosomal ganglioside lipids GM1 or GM3 accelerate α-synuclein aggregation [215]. 

A combination of ceramides and neurodegeneration-linked proteins including α-synuclein and tau in exosomes is capable of inducing aggregation of wildtype α-synuclein [216]. Oxidation of two adjacent amino acids, methionine [Met(38)] and tyrosine [Tyr(39)], results in aggregation of γ-synuclein and seed aggregation of α-synuclein. 

Neuronal exosomes containing γ-synuclein upon internalization can cause aggregation of intracellular proteins in astrocytes, resulting in synucleinopathies [217]. Levels of Golgi complex localized the gamma adaptin ear-containing, ARF-binding protein 3 (GGA3) were downregulated in postmortem substantia nigra of PD patients as compared to controls. 

GGA3 induces oligomerization of α-synuclein in endosomes, resulting in secretion of α-synuclein oligomers [218]. In another study, researchers reported an interaction between α-synuclein and the autophagy protein, LC3B that resulted in the formation of detergent-insoluble oligomeric aggregates. 

Alpha-synuclein oligomers are deposited on the surface of late endosomes and are eventually secreted out of human pluripotent stem cells through exosomes [219]. Another intriguing question is how toxic α-synuclein is transferred between cells. Several studies have addressed the transfer of pathological α-synuclein from neuron to neuron and neuron to microglia and vice versa. 

The first evidence for transmission of α-synuclein in Parkinson's disease pathogenesis came from two independent research groups. In one study, neurons from the substantia nigra were transplanted into the striatum of an individual with Parkinson's disease. 

When examined fourteen years later, transplanted neurons were positive for α-synuclein aggregates similar to host dopamine neurons in the substantia nigra of the subject [220]. Simultaneously, Li and colleagues reported similar findings using two human subjects with Parkinson's disease [221]. These results demonstrate that cell-to-cell transmission is a continuous, insidious process. 

Once exosomes were established as an important entity in intercellular communication, exosomes packaged with α-synuclein were identified. Neuron-to-neuron transmission occurs through the internalization of α-synuclein-containing exosomes in Parkinson's disease pathology [208]. 

Normal embryonic dopaminergic neurons transplanted into the striatum of rat brain overexpressing human α-synuclein quickly endocytosed α-synuclein and was found in early endosomes. Results of this study lent strong support to data obtained in 2008 which showed transmission of disease in vivo [220–222]. Lipid peroxidation is associated with the late onset of Parkinson's disease. 

Lipid peroxidation results in the formation of 4-hydroxynonenal. Exposure of primary neurons to this product increases aggregation of endogenous α-synuclein. Extracellular vesicles released by primary neurons contained cytotoxic oligomeric α-synuclein. Endocytosis of these extracellular vesicles caused degeneration of healthy neurons in vitro. 

Injection of neuronal extracellular vesicles containing cytotoxic oligomeric α-synuclein to the striatum of normal healthy mice resulted in the transmission of α-synuclein pathology not only in the striatum but surrounding brain regions as well [223]. 

Implantation of exosomes from bone marrow mesenchymal stem cells rescued the pathogenic features of Parkinson's disease by altering the inflammatory microenvironment in the substantia nigra and repairing the injury to dopaminergic neuron nerves These exosomes were enriched with Wnt5a [224]. The long-term rescue efforts of exosomes are not clear from the study. 

Microglia are a double-edged sword in the CNS as they can be either neuroprotective or neurotoxic. Incubation of microglial cell line BV2 with α-synuclein released an increased number of exosomes enriched with MHC class II molecules and membrane TNFα. Internalization of these exosomes by neurons was neurotoxic suggesting a role for microglia in α-synuclein-induced neurodegeneration [225]. 

The question is how α-synuclein is internalized by microglia. Microglial cells selectively express Toll-like receptor 2 (TLR2) which acts as a ligand of α-synuclein. The binding of α-synuclein to TLR2 activates microglia. Since α-synuclein is present on the surface of exosomes, they are internalized by microglia. The excessive exosome uptake by microglia causes an inflammatory response [226], inhibition of autophagy, and reduced scavenger activity. 

Reduced phagocytosis of α-synuclein containing exosomes was seen in mouse microglia and human monocytes from aged donors. This observation suggests an age-dependent predisposition to the incidence of misfolded proteins in Parkinson's disease [227], which is in line with the onset of Parkinson's disease occurring predominantly after 60 years of age.

Surprisingly, only limited studies have addressed the role of astrocytes in the pathogenesis of Parkinson's even though these cells are known to play important roles in the CNS. As mentioned earlier in this section, astrocytes internalize neuronal exosomes containing γ-synuclein. 

In this case, γ-synuclein induced aggregation of intracellular proteins of astrocytes [217], thereby interfering with astrocyte function. The involvement of astrocytes in Parkinson's disease came from another study involving mutations in the leucine-rich repeat kinase 2 (LRRK2) protein. The LRRK2 protein plays a role in vesicle trafficking, possibly via phosphorylation of Rab GTPase substrates [228]. 

Mutations in the LRRK2 gene are associated with late-onset Parkinson's disease. In particular, the G2019S mutation resulted in increased kinase activity of LRRK2 by auto-phosphorylating Ser residue at 1292 [229]. Ser(P)-1292 LRRK2 protein was detected in exosomes from the urine of Parkinson's disease patients [229,230]. 

Astrocytes generated from LRRK2 G2019S patient-derived-induced pluripotent stem cells (iPSC) released exosomes. These exosomes had an abnormal shape and were enriched with LRRK2 and phospho-S129 α-synuclein. Dopaminergic neurons internalized LRRK2 G2019S astrocytic exosomes. Interestingly, internalized exosomes accumulated to a greater extent in neurites as compared to soma suggesting that neurite-localized astrocytic exosomes either interfered with neuronal function or were processed for recycling through neurites [231]. 

The neurotoxic effects of α-synuclein are caused by an increase in Ca2+ I through the activation of voltage-operated Ca2+ channels and a significant increase in mitochondrial Ca2+ sequestration [232]. Several studies examined microRNAs packaged in exosomes from cerebrospinal fluid and plasma of Parkinson's disease patients, cultured cells, and rat models of Parkinson's disease [233–236]. 

Several microRNAs were differentially associated with exosomes from the diseased samples as compared to controls. Pathway analyses of differentially present microRNAs in exosomes suggested their involvement in the following pathways: ubiquitin-mediated proteolysis, long-term potentiation, axon guidance, cholinergic synapse, gap junction, dopaminergic synapse, and glutamatergic synapse. 

One microRNA, miR23b-3p, was strikingly reduced in Parkinson's disease exosomes. MicroRNA-23b-3p binds to 30 -untranslated region of α-synuclein. Reduction of miR-23b-3p in exosomes leads to upregulation of α-synuclein mRNA [235], thereby increasing the expression of the α-synuclein protein in Parkinson's disease. 

The underlying cause of Parkinson's disease is not only an increase in α-synuclein but a combination of several dysregulated pathways that lead to the etiology of the disease. Continuous dysregulation of these pathways is perhaps important to the progression of the disease. Can we reset the dysregulation of affected pathways? 

At least one study examined this possibility. Exosomes from adipose-derived stem cells (ADSC) are enriched with miR-188-3p. This microRNA targets NAcht leucine-rich repeat protein 3 (NLRP3) and cell division protein kinase 5 (CDK5), and both targets are involved in autophagy. Internalization of ADSC exosomes reduced autophagy in MN9D cells [237]. 

In summary, there has been substantial interest in exosome research in the context of Parkinson's disease. From the foregoing discussion, it is apparent that exosomes are important mediators of α-synuclein transmission among brain cells. In addition, the ability of exosomes to transfer proteins and miRNAs contributes to pathogenesis. 

Amyotrophic Lateral Sclerosis: Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal neurodegenerative disease with a median survival of only 2–5 years-it affects upper motor neurons which project from the cortex to the brain stem and spinal cord, as well as lower motor neurons that project from the spinal cord to muscles. 

Patients develop progressive muscle paralysis and death usually occurs due to respiratory failure. Most cases are sporadic but some are familial cases. ALS is characterized by misfolding of Cu/Zn dismutase (SOD-1) [238] and TAR DNA-binding protein 43 (TDP-43) [239]. 

SOD 1 is a cytosolic mitochondrial enzyme involved in the clearance of superoxide molecules, while TDP-43 is a highly conserved nuclear RNA/DNA-binding protein involved in RNA processing. Posttranslational modifications such as cleavage, hyper-phosphorylation, and ubiquitination of TDP-43 can lead to cytoplasmic accumulation and aggregation of TDP-43. Both SOD-1 and TDP-43 are packaged in exosomes [240,241]. 

By overexpressing both wild-type and mutated SOD-1 in NSC-34 motor neuron-like cells, Grad and colleagues observed that misfolded SOD-1 protein was transferred from cell to cell via exosomes in addition to direct uptake of SOD-1 protein aggregates by micropinocytosis [241]. Studies have suggested that astrocytes may play a role in the pathogenesis of ALS. 

Exosomes released by primary astrocyte cultures expressing mutant SOD-1 efficiently transferred mutant SOD-1 protein to spinal neurons, causing selective motor neuron death [242]. 

A study utilizing a SOD-1 transgenic mouse model demonstrated that mutant SOD-1 was enriched in exosomes derived from both neurons and astrocytes, suggesting that these two cell types may contribute to the spread of pathology in ALS [153]. TDP-43, another protein involved in the pathogenesis of ALS, was detected in exosomes purified from the cerebrospinal fluid of ALS patients [243], supporting the idea that exosomes contribute to disease propagation. Indeed, cerebrospinal fluid enriched with TDP-43-containing exosomes was able to promote the accumulation of toxic TDP-43 in human glioma U251 cells [244]. Furthermore, TDP-43 oligomers present in exosomes were transmitted intercellularly [245]. Interestingly, levels of exosomal TDP-43 (full-length protein and C-terminal fragments) are upregulated in the brains of ALS patients. 

When Neuro2a cells were exposed to exosomes from ALS brains, TDP-43 was redistributed in the cytoplasm of Neuro2a cells [246]. Compared to other neurodegenerative diseases, research into the pathogenesis of this devastating fatal disease is much more limited. 

Much of the research has been performed in vitro. With the refinement of exosome isolation techniques from brain tissue, it is hoped that we will have a clearer picture of the role played by exosomes in the spread of ALS. From the foregoing discussion on neurodegenerative diseases, it is clear that exosomes provide a vehicle for the transmission of misfolded proteins (or toxic proteins) thus playing a role in the propagation of disease. 

Certainly, transmission of toxic proteins through exosomes is not the only mode of transmission. However, an important aspect to consider is that exosomes may provide a suitable environment for proteins to aggregate and stay in an aggregated form. 

At this time, we do not understand whether exosomes lie at the core of the pathology of neurodegenerative diseases or whether they are released as a consequence of the disease process. A better understanding of how toxic proteins are packaged into exosomes and how they are transferred to naive cells will provide important insight into the pathogenesis of devastating diseases involving misfolded proteins. 

This information will provide opportunities for improved therapeutic strategies and hopefully personalized treatments. Exosomes and the blood-brain barrier: The blood-brain barrier is a physical barrier between the brain and the peripheral circulation, controlling a strict influx and efflux of molecules to maintain homeostasis. 

Accumulating evidence suggests that exosomes have the remarkable ability to cross the blood-brain barrier from both directions. Exosomes carry cargos of membrane and cytosolic proteins and genetic material such as mRNAs, non-coding RNAs including miRNAs that otherwise generally do not cross the plasma membrane. 

Exosomes released from cancer cells have been shown to destroy the blood-brain barrier through the action of microRNA-181c, leading to actin mislocalization and perhaps, resulting in the breakdown of blood-brain barrier integrity. Such leakiness of the blood-brain barrier is also seen in cases of neurodegeneration often as a result of neuroinflammation. 

Furthermore, glioblastoma-specific mRNAs have been detected in exosomes in the peripheral circulation [247]. Experimental evidence suggests that exosomes can cross the blood-brain barrier from the periphery and localize in the brain. Analyses of fluorescent or luciferase-labeled exosomes demonstrated that they can accumulate in the brain from the periphery [248,249]. 

Exosomes loaded with siRNA were able to deliver their cargo to neurons, microglia, and oligodendrocytes in the brain when administered intravenously [208]. Exosomes derived from hematopoietic cells can be transferred to Purkinje cells in the brain and importantly were able to modulate gene expression in these cells. This observation suggests that the transfer of exosomes via the blood-brain barrier can have functional implications. 

The ability of exosomes to cross the blood-brain barrier presents a great potential for exosomes as a drug delivery system. Equally important is that uptake of the exosomal cargo by recipient cells can have profound functional impacts on the CNS. 

Thus, understanding how exosomes traverse the blood-brain barrier bidirectionally can have great therapeutic potential and diagnostic utility. Concluding remarks: As the exosome field is witnessing exponential growth, it is perhaps an understatement to say that there is a requirement for more uniformity in exosome isolation and characterization methods. Refinement of exosome isolation in an in vivo setting will enable the discovery of novel biological functions of exosomes. 

Many exosome studies have been performed using cells cultured in vitro. Future studies involving animal and clinical research will be a key to unlocking the potential of exosome biology. Particularly, a better understanding of the role played by exosomes in the pathogenesis of neurodegeneration will pave the way for new therapeutic avenues. 

This is specifically significant as the aging population increases and with it a growing incidence of neurodegenerative diseases. The biological content of exosomes can be harnessed for biomarker discovery aiding in diagnosis and prognostic follow-up studies. This is of particular importance as exosomes are present in most biological fluids and the biological cargo is stable and protected within the boundaries of exosome membranes. 

Future perspective of exosomes in neurodegeneration: Slow and progressive deterioration of the quality of life of patients suffering from neurodegenerative diseases has a devastating effect not only on patients but also on family members and medical professionals. Researchers worldwide are engaged in efforts to identify biomarkers that will unequivocally detect early signs of these crippling diseases. Olfactory dysfunction resulting in loss (anosmia) or reduction (hyposmia) of smell is considered an early sign of neurodegenerative diseases [250–252]. 

Unfortunately, impairment of smell is not unique to neurodegenerative diseases alone as exposure to drugs of abuse such as alcohol, viral infections such as COVID-19, trauma, or simple sinusitis or polyposis nasi also interfere with olfactory abilities [253–255]. 

Identification of gene-specific mutations; post-translationally modified and/or misfolded protein levels in CSF; and PET imaging have made significant contributions to our understanding of disease progression. More recently, exosomes have shown great promise in helping us understand the pathogenesis of disease spread and in identifying exosome-associated unique protein(s), non-coding RNA(s), lipid(s), or metabolite(s) as a biomarker(s) for a specific neurodegenerative disease. 

Biomarker discovery for neurodegenerative diseases is particularly critical because these diseases progress silently sometimes for decades before obvious clinical manifestations. Substantial neuronal death has already occurred in the late stages of the disease when the diagnosis is made. Hence, current treatments are only palliative once the disease is diagnosed in the late stages of the disease. 

Identification of changes that take place before the appearance of visible signs of disease is therefore crucial to our ability to identify biomarkers of disease. Identification of biomarkers in the neurodegenerative disease field is currently impeded due to a lack of systematic analysis of exosomes from the beginning of the disease. 

Since biological fluids are enriched with exosomes, analysis of exosomes regularly from members of families with known mutations for neurodegenerative diseases and disease models may be one opportunity to identify biomarkers. However, such studies require a commitment from funding agencies, family members, and researchers since these are long-term studies and come with a substantial price tag.

Author Contributions: Both authors contributed equally to writing this review article. All authors have read and agreed to the published version of the manuscript.

Funding: This research received intramural funding from the Department of Anatomy and Physiology, College of Veterinary Medicine at Kansas State University.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Acknowledgments: We apologize to our colleagues whose work we were unable to include in this review due to space limitations. The authors are thankful to Lekchnov, Konoshenko, and KrämerAlbers for allowing us to reuse figures from their respective manuscripts as well as providing us with original high-quality images of the figures. The authors are indebted to John Wiley & Sons, Inc., Hindawi Publishers, and Rockefeller University Press for permitting us to reuse images from manuscripts originally published in one of their journals. We are also grateful to Rockefeller University Press for waiving the fee to reuse three images from a manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

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