**6. Extracellular vesicles' perspective use in brain pathology**

The discovery of the EV involvement in several biological processes gave hope that some questions regarding neurodegenerative diseases will be answered. First of all, it is important to clarify which are the cellular mechanisms involved in the progression of the disease and if exosomes play any role. Based on the spatio-temporal spreading of the pathological proteins in the brain, an appealing theory is the prionlike propagation theory [113]. It is presumed that exosomes play an important role by facilitating the interneuronal transport of the proteins [114]. As well, there is a critical need in finding accessible biomarkers that can diagnose a neurodegenerative disease in the asymptomatic stage [115]. Dosing certain free proteins in biofluids can be an option, but several problems are experienced because of their low concentrations [115]. Therefore, a new approach is being attempted consisting in finding the proteins encapsulated in extracellular vesicles [116]. Micro-RNAs and different proteins carried by exosomes are attractive options for finding new biomarkers in several diseases as well as in neurodegenerative diseases [117].

Recently, it was discovered that an important player in the field of neural diseases is EV derived from stem cells [118], mainly in the case of stroke [118–120]. In stroke pathophysiology, inflammation plays a significant role, circulating EV-activating immune cells. Neurons quickly depolarize and die, being next phagocytosed by infiltrating circulating macrophages and microglia [84, 121].

Because of their β1 and α2b integrin-enriched content, human neural stem cell-derived EV administration recovers both tissue and sensorimotor function and may protect the BBB integrity, in the preclinical mouse thromboembolic model of stroke [122, 123]. Similarly, multipotent mesenchymal stromal cells (MSCs), through their capacity to secrete soluble factors, play an important role in brain repair. It was demonstrated that MSC cargos modulate cell signaling in ischemic stroke by PI3K/Akt pathway activation [123, 124] and EVs facilitated secretion of miRs sustaining MSC neuroprotective effects in ischemic stroke as well. Previous research demonstrated that intravenous administration of bone marrow-MSCs containing exosomes transferred miR-133b to astrocytes and neurons into the ischemic boundary zone [120, 123], and MSCs cultured in the presence of extracts from rat ischemic brain induced increased expression of exosomal miR133b [123, 125]. Also, EVs released by human MSCs seem to have an anti-inflammatory effect on mast cells, by increased prostaglandin E2 (PGE2) synthesis and up-regulation of EP4 receptor which might prevent the rupture of intracranial aneurysms [126]. All these data suggest that EVs from various sources may contribute to the neurogenesis and angiogenesis during brain repair processes in cerebral diseases.

*Extracellular Vesicles and Their Importance in Human Health*

occurs especially in the cerebral cortex [92].

neurofibrillary tangles [99].

valuable research theme.

biomarkers for AD.

attempt to clean the intracellular space [107].

accumulation of the proteins induces an apoptotic response with neuronal loss and

It has been observed that the pathological findings in AD have a typical spatial

The evolution of AD is insidious with an asymptomatic stage that lasts several years [100]. Although asymptomatic, the pathological changes in the brain are present in this stage [93]. These findings suggest the value of discovering biomarkers that can anticipate the onset of the clinical symptoms or can facilitate a window for the possibility of a future therapy that could stop the progression. Amyloid beta and hyperphosphorylated tau proteins are of great value as biomarkers when dosed from the cerebrospinal fluid [101]. Nevertheless, performing lumbar puncture in a wide population is almost impossible. Thereby, the discovery of new biomarkers is a

Several types of miRs isolated from cerebrospinal fluid are differentially expressed in AD, such as miR-100, miR-146, miR-505, and miR-1274a [102]. The presence of several types of exosomal miRs isolated from serum (miR-361-5p, miR-93-5p, miR-335-5p, and miR-30e-5p) correlates with the neuropsychological evaluation and brain imaging [103]. It is to be mentioned that exosome-containing proteins like tau, apolipoprotein E, cystatin E, and HSP-90 alpha were isolated in the cerebrospinal fluid and were present in patients with AD [104]. This evidence proposes both miRs and protein-containing exosomes as a promising source of

**Parkinson's disease** (PD) is a neurodegenerative disease that consists in the loss of dopaminergic neurons localized in the substantia nigra [105]. The pathology of the disease implies the deposition of Lewy bodies in the neurons which are mostly made of misfolded and aggregated alpha-synuclein protein [105]. The Braak staging explains the spatio-temporal dissemination of Lewy bodies into the neurons from caudal to rostral, starting in the medulla oblongata and spreading to the level of the cerebral cortex, damaging various structures on this way [106]. The starting point is thought to be either the enteric nervous system or the olfactory bulb [106]. Thereby, it is suggested that PD has a prion-like propagation [94]. Are exosomes responsible for carrying alpha-synuclein from neuron to neuron? The mechanism could be similar to the one described in AD, but there are fewer studies to draw certain conclusions regarding the involvement of exosomes in the pathogenesis of PD. Excessive intracellular alpha-synuclein is thought to be transported out of the cell via multivesicular body containing exosomes in a similar manner as in AD in an

distribution suggesting a neuron-to-neuron spread of the amyloid and hyperphosphorylated tau proteins, which promote aggregation, acting as "seeds" [93]. Therefore AD is considered to have a prion-like model of propagation [94]. The immediate question that rises is: what is the mechanism of the propagation? Amyloid beta is mainly formed extracellularly from the cleavage of APP by beta and gamma secretases, which are found at the level of the plasma membrane [95]. But in some degree, the secretases are present in endoplasmic reticulum and Golgi apparatus, and there is a certain intracellular production of amyloid beta [96]. It is removed from the cell via exosomes embedded in a multivesicular body as an alternative pathway to lysosomal degradation [97]. A new study suggests that exosomes containing amyloid beta are present in higher concentrations in the AD brain compared to the healthy brain [98]. Moreover, the study shows that exosomes are the carriers of the toxic amyloid beta from one neuron to another [98]. Also, it is thought that exosomes mediate the intercellular transfer of hyperphosphorylated tau protein, and the exosome-mediated tau protein induces the formation of

**144**
