Small But Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration Part 3

Microglia-Derived Exosomes: Microglial cells are the macrophages of the brain and spinal cord. They account for approximately 10% of glia. 

Microglia are a type of non-neuronal cell in the brain and are the most abundant cells in the nervous system. In the past, microglia were thought to be neuron-supporting cells that mainly provided oxygen and nutrients.

However, recent studies have found that microglia play an important role in human health and cognitive function. Studies have shown that microglia play a vital role in the learning and memory process.

Microglia mainly promote the growth and development of neurons by releasing a substance called neuronal growth factor. This growth factor not only helps neurons connect and transmit information but also stimulates the generation of new neurons. These effects are critical for maintaining healthy learning and memory.

On the other hand, microglia can also remove excess neurons and synapses in the brain and promote the healthy development of the nervous system. This removal process can help reduce interfering signals in the brain and improve cognitive function.

In addition, microglia can protect neurons from oxidative damage by promoting the arrival of oxygen and nutrients in the brain. These cells also play a very important role in resisting cognitive degeneration and neurodegeneration.

In short, the importance of microglia cannot be underestimated. They can both protect neurons and help the learning and memory process. We should protect and improve the number and function of microglia as much as possible to ensure the health and vitality of our brains. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

Click know ways to improve brain function

Although their number is comparatively low, they have important functions of maintaining homeostasis by monitoring the presence of tissue infection and damage in the CNS. 

Under homeostatic conditions, microglia cells are maintained in a quiescent state, but they are constantly scanning their environment. When activated, they exert their phagocytic activity and can release inflammatory molecules such as cytokines, and chemokines. 

Microglia can acquire two phenotypes, the M1 (inflammatory type) and M2 subtypes (pro-regenerative type), depending upon the stimuli and can switch between these two opposing phenotypes [140], exemplifying their remarkable plasticity. 

Similar to neurons and other glial cells, microglia release exosomes to communicate with neighboring cells and cells at a distance. The bioactive cargo of microglial exosomes depends upon the phenotype of the releasing microglia, either pro-inflammatory or pro-regenerative. Microglial exosomes contain all enzymes essential for anaerobic glycolysis and lactate production. 

Therefore, it is proposed that lactate packaged in exosomes could function as a supplementary energy source for neurons during synaptic activity [141]. Microglial exosomes regulate synaptic transmission by promoting ceramide and sphingolipid production in neurons. 

Enhanced sphingolipid metabolism positively affects excitatory neurotransmission presenting a novel way by which microglia influence synaptic activity [142]. Faced with infection and/or injury, microglial cells quickly engage in a complex inflammatory response and acquire an M1 phenotype. 

Microglia now with a pro-inflammatory phenotype (M1 phenotype) release exosomes that help with the development of neuroinflammation. Evidence for microglial exosome involvement in neuroinflammation comes from a study conducted with lipopolysaccharide (LPS). 

Exposure of microglia to LPS, a major component of Gram-negative bacteria, increases the release of exosomes enriched with IL-1β, a pro-inflammatory cytokine, and microRNAs such as miR-155 and miR-375. MicroRNA 155 is an important regulatory microRNA in the immune system and its increased levels are detected in inflammatory diseases [143]. 

In another study, microglial cells were treated with LPS that increased expression of the N-myc downregulated gene 2 (NDRG2) protein levels. Increased NDRG2 protein in turn stimulated microglia to release miR-375 enriched exosomes. Internalization of miR-375 enriched exosomes reduced the cell viability of N2A neurons indicating the neurotoxic nature of these exosomes [144]. 

It appears that exposure to LPS alters microRNAs and proteins packaged in exosomes. The confirmatory evidence came from a study involving BV2 cells. The BV2 cells of C57 Black mice are immortalized microglial cells. When BV2 cells were exposed to LPS, they released exosomes rich in pro-inflammatory cytokines IL-6 and TNFα and proteins related to translation and transcription. 

The proteomic profile of exosomes analyzed by mass spectrometry identified 49 unique proteins present in exosomes from LPS-treated BV2 cells as compared to control BV2 cells. 

It is worth noting here that exosomes from LPS-activated microglia had 58 proteins while exosomes from control BV2 cells had 37 proteins [145]. The foregoing discussion demonstrates that microglial exosomes are important in facilitating neuroprotective and neuroinflammatory functions of microglial in the CNS.

4. Role of Exosomes in Neurodegenerative Diseases

Many neurodegenerative diseases are associated with the accumulation of abnormal, misfolded proteins leading to progressive neural and glial dysfunction. Generally, neurodegenerative diseases begin with dysfunction in a discrete brain region. 

Upon release into the extracellular space, misfolded proteins are transferred to healthy cells and begin to induce endogenous counterpart proteins to misfold like a domino effect [160]. 

This "infective" process leads to the amplification of pathology and the spread of disease to wider areas of the brain. Intercellular communication is important in the transmission and progression of neurodegenerative diseases. Exosomes released by all cells in the brain have become an integral player in neuro–glia communications. 

The ability of exosomes to transport and transfer bioactive cargo such as lipids, RNAs, and proteins from one cell to another makes them an attractive candidate as mediators of neurodegeneration. Below, we discuss the impact of exosomes in selective neurodegenerative diseases. 

Alzheimer's Disease: Alzheimer's disease is the most common form of neurodegenerative dementia characterized by a progressive loss of memory and cognitive abilities. 

Central to the pathology of Alzheimer's disease is the formation of extracellular aggregates of β-amyloid (Aβ) known as amyloid plaques combined with neurofibrillary tangles of tau. The (Aβ) peptides are derived from sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. 

As APP is an intracellular protein, a hypothesis was formulated that the pathological lesions of neurodegenerative disease involve the physical spread of the misfolded protein from neuron to neuron [161]. 

However, the mechanism(s) for the transmission of misfolded proteins remained an intriguing question. One of the earliest reports that started to shed light on the possible mechanisms of how Aβ is shed into the extracellular space came from the study conducted by Rajendran and Colleagues [162]. 

While investigating the location of APP cleavage, they observed that β-secretase cleavage of APP occurs in a subset of early endosomes with subsequent trafficking of Aβ peptide to multivesicular bodies. 

A small fraction of Aβ peptide associated with the exosome membrane was secreted into the extracellular space. Exosomes containing amyloid plaques had exosomal marker proteins, flotillin-1, and Alix. Thus, exosome membrane-associated Aβ peptide may represent a novel mechanism that contributes to amyloid plaque formation in the extracellular space [162]. 

Since this initial observation, full-length APP and many of its metabolites and several members of the secretase family of proteases involved in APP processing were detected in exosomes [163]. 

Aβ can exist in different conformational states that have different properties and intermediate products of fibril formation. Of these, low-molecular-weight Aβ and protofibrils have been suggested to be particularly neurotoxic and act as seeds for protein aggregation [164,165]. Exosomes isolated from postmortem brains of Alzheimer's disease patients were shown to have increased levels of Aβ oligomers. These exosomes were internalized when incubated with cultured neurons. 

Most importantly, they were able to spread Aβ oligomers to other neurons, causing cytotoxicity [166]. The initial observations were confirmed by incubating exosomes containing APP with primary cultures of normal neurons in vitro [167] and in vivo [168]. 

The question is why are Aβ or Aβ oligomers packaged into exosomes? Do neurons sense the toxic nature of Aβ or Aβ oligomers and hence try to clear toxic proteins from intracellularly present Aβ or Aβ oligomers similar to transferrin receptors in reticulocytes [7,8]? Monoubiquitination is required for sorting into MVB/exosomes. That raises a second question as to whether Aβ undergoes ubiquitination to be sorted into MVB/exosomes. The amyloid precursor protein (APP) has five lysine residues (Lys-724, Lys-725, Lys-726, Lys-751, and Lys-763) at its C-terminal end [169]. 

These residues have been mutated individually or in combination to examine the effect on APP processing to form Aβ peptide. Ubiquitination of APP at Lys-726 mediated by the F-box and leucine-rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase, reduced Aβ generation [170]. On the other hand, APP ubiquitination at Lys-763 sequestered APP in the Golgi complex and prevented APP maturation [171]. 

Inhibition of ubiquitination by substitution of all five lysine residues to arginine in the C-terminal fragment of APP (C99) prevented efficient degradation of APP and accumulation of protein in structures with Golgi-like appearance. This was attributed to a deficiency in endoplasmic reticulum-associated degradation. The C99 undergoes cleavage by γ-secretase to produce Aβ [172]. 

Mutation of three lysine residues (Lys-724, Lys-725, and Lys-726) simultaneously caused the protein to be retained in the limiting membrane of endosomes instead of becoming internalized into intraluminal vesicles of MVBs [173]. When all five lysine residues were mutated to prevent ubiquitination, the protein did not efficiently sort to MVB/exosomes, and a selective increase in Aβ40 was observed [174]. 

This finding is comparable to the presence of Aβ40 in amyloid deposits in cerebral amyloid angiopathy [175]. While it is evident that ubiquitination of APP may direct the protein to MVB/exosomes, direct evidence using neuronal cultures or in vivo models is required to prove that APP, Aβ, or Aβ oligomers are monoubiquitinated for their targeting to MVB/exosomes and that they are not ubiquitinated to be targeted for endoplasmic reticulum-associated degradation (ERAD). 

Another pathological hallmark of Alzheimer's disease is abnormally phosphorylated tau protein in neurofibrillary tangles (NFT). Tau is a cytoplasmic protein known to stabilize microtubules. Increasing evidence suggests that the pathological tau protein can spread between cells, recruiting native tau to form aggregates. More recent data implicated exosomes as a carrier of tau protein [176,177]. 

Cell scrutiny suggested that exosomes involved in tau pathology originated from microglial cells. Exosomes from microglia transferred tau protein to neurons. To obtain experimental evidence, studies were carried out in which either exosome biogenesis was inhibited or microglial cells were depleted of tau. Results from these studies showed attenuation of tau deposition in normal neurons [178]. 

The authors suggested that microglia phagocytose tau-containing cytopathic neurons recycle tau through exosomes thus incriminating exosomes in the propagation of Alzheimer's disease. In healthy brains, several protein kinases and phosphatases are responsible for phosphorylation and de-phosphorylation of tau, respectively. 

Dysregulation of these important enzymes may lead to abnormal phosphorylation patterns of tau in AD. A recent study compared exosomal protein cargo by Nano-LC–MS/MS. Exosomes were purified from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) and normal human iPSC neurons. 

A total of 1117 proteins were identified in both exosome groups and 733 proteins were common to both populations of exosomes. Among differentially associated proteins with mPS1 were phosphatases and protein kinases and their protein levels associated with mPS1 exosomes were significantly lower as compared to control neurons [179]. 

Furthermore, these exosomes contained distinct proteins absent in control exosomes. Specifically, the distinct proteins were those involved in extracellular matrix structure and function suggesting another mechanism for the propagation of tau pathology in AD [179]. The major emphasis of research in Alzheimer's disease has been on neurons. 

However, studies revealed that atrophy of the astroglia occurs at early stages of the neurodegenerative process. The lack of neuronal support from atrophied astroglia results in disruptions of synaptic connectivity, loss of synapses, and, imbalance of neurotransmitter homeostasis. At a later stage of Alzheimer's disease, astrocytes and microglia become activated and release inflammatory molecules and neurotoxic substances. Neurotoxic chemicals result in neuroinflammation and neuronal death leading to atrophy of the brain [180]. 

In the early stages of Alzheimer's disease, microglial cells activated through Toll-like receptor 4 acquire a neuroprotective role and clear Aβ [181]. Phagocytosis and degradation of purified polymorphous beta-amyloid protein deposits and Aβ associated with exosomes were confirmed using cultured microglial cells [182,183]. Curiously, astrocytes appear to relieve microglial cells of their neuroprotective function. Media and colleagues noticed that incubation with Aβ peptide activated glial cells and subsequently resulted in inflammation [184]. 

Co-culturing with astrocytes or culturing in astrocyte-conditioned medium-inhibited phagocytic action of microglia. Inhibition of microglial phagocytosis was highly specific as the conditioned medium from fibroblasts did not affect microglial phagocytic activity. From these studies, it is evident that astrocytes released signals in the form of soluble factors that interfered with the phagocytic activity of microglia [182]. Roles of astrocytes and microglia can be reversed following chronic stimulation of microglia. 

Several independent studies lead to the conclusion that exosomes released by neurons, astrocytes, and microglia act as scavengers and soak up seed-free soluble Aβ to promote Aβ aggregation that is internalized by microglia for degradation [185–187]. This observation is not surprising given the closely monitored intercellular communication among brain cells (briefly discussed in Section 3). 

As mentioned earlier, the Aβ aggregates interact with glycosphingolipids, ceramide, and/or the GPI-anchored protein PrPc (cellular prion protein) present on the surface of exosomes and on neurons [185,187]. Neuronal exosomes bind Aβ aggregates more efficiently as compared to astrocytic or microglial exosomes due to abundantly present ganglioside GM1 and sialylated glycosphingolipids especially trisialoganglioside GT1 on their surface [188–190]. Astrocytic exosomes are enriched with the sphingolipid ceramide [187]. 

The Aβ aggregates bound to astrocytic exosomes are internalized by neurons and are directed to mitochondria, causing mitochondrial clustering and simultaneously increasing the fission protein Drp-1 levels. At the outer membrane of mitochondria, exosomal Aβ forms a complex with the ADP/ATP transporter, voltage-dependent anion channel 1, and activates caspases. 

Active caspases induce neurite fragmentation and eventually neuronal cell death [191]. To better understand possible dynamic interactions between proteins, lipids, and RNA in Alzheimer's disease, high-throughput techniques have recently been applied. Cohn and colleagues took an 'omics' integrative approach to analyze microglial exosomes [192]. 

In this study, the authors isolated microglial exosomes from the parietal cortex of late-stage Alzheimer's disease patients. They performed an integrative analysis combining proteomic, transcriptomic, and lipidomic analyses. They used shotgun proteomics, which refers to a bottom-up protein analysis where proteins are characterized by analysis of peptides released from the protein through proteolysis [193], targeted lipidomics, and NanoString nCounter technology a multiplex nucleic acid hybridization technology [194]. 

Using this combinatorial approach, the research group showed a significant reduction in homeostatic microglia markers P2RY12 and TMEM119 and increased levels of disease-associated microglia markers FTH1 and TREM2. In addition, tau protein levels in AD brain-derived microglial exosomes were significantly higher suggesting that microglia-derived exosomes appear to be important in the spread of tau pathology. 

Synaptic and neuron-specific proteins were also differentially enriched in AD brain-derived microglial exosomes. The authors hypothesized, however, that the synaptic and myelin-specific proteins have been phagocytosed before entering microglial exosomes. The lipidomic analyses revealed a proinflammatory phenotype and a potential defect in acyl-chain remodeling. 

Lastly, miRNAs associated with immune and cellular senescence signaling pathways were increased in AD brain-derived microglial exosomes [192]. These data suggest that a significant change in the molecular composition of exosomes reflects changes in microglia consistent with a diseased state. We have amassed a large amount of data that implicates exosomes in Alzheimer's disease. 

An important question is to what extent do exosomes promote or prevent the clearance of misfolded proteins? Additionally, elucidating the contributions made by exosomes released from different brain cells will further promote our understanding of disease transmission.

Parkinson's Disease: Parkinson's disease is one of the most common age-related brain disorders-it is primarily considered a movement disorder, with typical symptoms of resting tremor, rigidity, bradykinesia, and motor instability [195]. 

Additionally associated with this disease are cognitive decline, depression, and psychosis [196]. Pathologically, it is characterized by the degeneration of nigrostriatal dopaminergic neurons and the presence of Lewy bodies which contain misfolded α-synuclein protein in surviving neurons. Alpha-synuclein is detected in many body fluids including cerebrospinal fluid and plasma [197,198]. 

Alpha-synuclein is found in a culture medium when cells expressing α-synuclein are cultured in vitro [199]. Considering that α-synuclein has been detected extracellularly in the absence of an extracellular sorting signal, how does this protein reach the extracellular space? Several investigators have examined the mechanism of α-synuclein secretion including the role of exosomes in α-synuclein secretion and pathology. 

The first indication that exosomes could indeed be involved in the pathogenesis came from an in vitro study employing SH-SY5Y cells expressing α-synuclein. The authors demonstrated extracellular secretion of α-synuclein via exosomes in a calcium-dependent manner and suggested their involvement in the spread of Parkinson's disease pathology [200]. 

Following this study, α-synuclein-containing exosomes have been identified from different cells, cerebrospinal fluid, and plasma of Parkinson's disease patients [201–203]. However, there is a lot of variability reported in different studies [204]. Interestingly, the amount of α-synuclein in exosomes is relatively low as compared to free α-synuclein detected in cerebrospinal fluid or conditioned media. 

A close examination revealed that α-synuclein expressing neuroglioma cells use two pathways to release α-synuclein oligomers, one through exosomes and a second, direct release of free α-synuclein as oligomers [205]. 

Although exosomes have low levels of α-synuclein, exosomes are more effective in exerting their toxic effects than the free α-synuclein. Alpha-synuclein oligomers present in exosomes were internalized more efficiently by human H4 neuroglioma cells as compared to free α-synuclein oligomers [205]. 

As a follow-up of this study, this group of researchers reported similar results using exosomes purified from the cerebrospinal fluid of Parkinson's disease patients. Efficient uptake of exosomal oligomerized α-synuclein was observed in human H4 neuroglioma cell cultures as compared to free α-synuclein oligomers [206]. 

Evidence for secretion of free α-synuclein to the extracellular space or fluid came from a study examining the role of vacuolar protein sorting 4 (VPS4) in loading α-synuclein to multivesicular bodies. Normally, the VPS4 regulates the sorting of proteins to multivesicular bodies. In Parkinson's disease, VPS4 directs α-synuclein to lysosomes for its degradation and to recycling endosomes for extracellular secretion of α-synuclein [207]. 

When lysosomal function was inhibited, the release of α-synuclein packaged in exosomes was seen to increase in cultured SH-SY5Y cells [208]. The endosomal fraction of α-synuclein escapes degradation in conditions of lysosomal impairment [209].

For more information:1950477648nn@gmail.com