Extracellular vesicles in the diagnosis and treatment of central nervous system diseases

Extracellular vesicles,including exosomes and microvesicles,play a fundamental role in the activity of the nervous system,participating in signal transmission between neurons and providing the interaction of central nervous system with all body systems.In many neurodegenerative diseases,neurons pack toxic substances into vesicles and release them into the extracellular space,which leads to the spread of misfolded neurotoxic proteins.The contents of neuron-derived extracellular vesicles may indicate pathological changes in the central nervous system,and the analysis of extracellular vesicle molecular content contributes to the development of non-invasive methods for the diagnosis of many central nervous system diseases.Extracellular vesicles of neuronal origin can be isolated from various biological fluids due to their ability to cross the blood-brain barrier.Today,the diagnostic potential of almost all toxic proteins involved in nervous system disease pathogenesis,specificallyα-synuclein,tau protein,superoxide dismutase 1,FUS,leucine-rich repeat kinase 2,as well as some synaptic proteins,has been well evidenced.Special attention is paid to extracellular RNAs mostly associated with extracellular vesicles,which are important in the onset and development of many neurodegenerative diseases.Depending on parental cell type,extracellular vesicles may have different therapeutic properties,including neuroprotective,regenerative,and anti-inflammatory.Due to nano size,biosafety,ability to cross the blood-brain barrier,possibility of targeted delivery and the lack of an immune response,extracellular vesicles are a promising vehicle for the delivery of therapeutic substances for the treatment of neurodegenerative diseases and drug delivery to the brain.This review describes modern approaches of diagnosis and treatment of central nervous system diseases using extracellular vesicles.     

The dual role of necrostatin-1 in Parkinson’s disease models

Neuronal cell death is the main hallmark of Parkinson’s disease(PD).It is an irreversible process promoted by neurotoxins and/or genetic mutations.Different types of cell death have been associated with PD.The mechanisms by which neurons decide to specific type of cell death remain elusive.However,it is well known that cell death can be either programmed or not.Apoptosis is a programmed cell death that involves the release of cytochrome c from damaged mitochondria to cytosol and the activation of caspases leading to nuclear condensation.Necrosis is a caspase-independent cell death characterized by a gain in cell volume,rupture of plasma membrane and leak of cell contents,inflammation,and affects neighbouring cells.It was classified as a nonprogrammed cell death,but there are types of necrotic death triggered by a protein activation cascade,including necroptosis.     

Exosomes in Parkinson's Disease

Exosomes, nano-sized extracellular vesicles secreted by most cell types, are found in all kinds of biological fluids and tissues, including the central nervous system(CNS). The proposed functions of these vesicles include roles in cell–cell signaling, removal of cellular debris, and transfer of pathogens between cells. Many studies have revealed that exosomes derived from the CNS occur in the cerebrospinal fluid and peripheral body fluids,and their contents are altered during disease, making them an appealing target for biomarker development in Parkinson's disease(PD). Exosomes have been shown to spread toxic a-synuclein(asyn) between cells and induce apoptosis, which suggests a key mechanism underlying the spread of asyn aggregates in the brain and the acceleration of pathology in PD. However, potential neuroprotective roles of exosomes in PD have also been reported. On the treatment side, as drug delivery vehicles, exosomes have been used to deliver small interfering RNAs and catalase to the brain, and have shown clear therapeutic effects in a mouse model of PD. These features of exosomes in PD make them extremely interesting from the point of view of developing novel diagnostic and therapeutic approaches.     

Role of platelet-derived extracellular vesicles in traumatic brain injury-induced coagulopathy and inflammation

Extracellular vesicles are composed of fragments of exfoliated plasma membrane, organelles or nuclei and are released after cell activation, apoptosis or destruction. Platelet-derived extracellular vesicles are the most abundant type of extracellular vesicle in the blood of patients with traumatic brain injury. Accumulated laboratory and clinical evidence shows that platelet-derived extracellular vesicles play an important role in coagulopathy and inflammation after traumatic brain injury. This ...     

Exosomes as mediators of neuron-glia communication in neuroinflammation

In recent years,a type of extracellular vesicles named exosomes has emerged that play an important role in intercellular communication under physiological and pathological conditions.These nanovesicles (30–150 nm) contain proteins,RNAs and lipids,and their internalization by bystander cells could alter their normal functions.This review focuses on recent knowledge about exosomes as messengers of neuron-glia communication and their participation in the physiological and pathological functions in the central nervous system.Special emphasis is placed on the role of exosomes under toxic or pathological stimuli within the brain,in which the glial exosomes containing inflammatory molecules are able to communicate with neurons and contribute to the pathogenesis of neuroinflammation and neurodegenerative disorders.Given the small size and characteristics of exosomes,they can cross the blood-brain barrier and be used as biomarkers and diagnosis for brain disorders and neuropathologies.Finally,although the application potential of exosome is still limited,current studies indicate that exosomes represent a promising strategy to gain pathogenic information to identify therapeutically targets and biomarkers for neurological disorders and neuroinflammation.     

Environmental factors in the development and progression of late-onset Alzheimer＇s disease

Late-onset Alzheimer＇s disease （LOAD） is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.     

Growth differentiation factor 5:a neurotrophic factor with neuroprotective potential in Parkinson’s disease

Parkinson’s disease is the most common movement disorder worldwide,affecting over 6 million people.It is an age-related disease,occurring in 1%of people over the age of 60,and 3%of the population over 80 years.The disease is characterized by the progressive loss of midbrain dopaminergic neurons from the substantia nigra,and their axons,which innervate the striatum,resulting in the characteristic motor and non-motor symptoms of Parkinson’s disease.This is paralleled by the intracellular accumulation ofα-synuclein in several regions of the nervous system.Current therapies are solely symptomatic and do not stop or slow disease progression.One promising disease-modifying strategy to arrest the loss of dopaminergic neurons is the targeted delivery of neurotrophic factors to the substantia nigra or striatum,to protect the remaining dopaminergic neurons of the nigrostriatal pathway.However,clinical trials of two well-established neurotrophic factors,glial cell line-derived neurotrophic factor and neurturin,have failed to meet their primary end-points.This failure is thought to be at least partly due to the downregulation byα-synuclein of Ret,the common co-receptor of glial cell line-derived neurorophic factor and neurturin.Growth/differentiation factor 5 is a member of the bone morphogenetic protein family of neurotrophic factors,that signals through the Ret-independent canonical Smad signaling pathway.Here,we review the evidence for the neurotrophic potential of growth/differentiation factor 5 in in vitro and in vivo models of Parkinson’s disease.We discuss new work on growth/differentiation factor 5’s mechanisms of action,as well as data showing that viral delivery of growth/differentiation factor 5 to the substantia nigra is neuroprotective in theα-synuclein rat model of Parkinson’s disease.These data highlight the potential for growth/differentiation factor 5 as a disease-modifying therapy for Parkinson’s disease.     

Targeting the noradrenergic system for anti-inflammatory and neuroprotective effects: implications for Parkinson's disease

Degeneration of the locus coeruleus noradrenergic system is thought to play a key role in the pathogenesis of Parkinson's disease(PD), whereas pharmacological approaches to increase noradrenaline bioavailability may provide neuroprotection. Noradrenaline inhibits microglial activation and suppresses pro-inflammatory mediator production(e.g., tumor necrosis factor-α, interleukin-1β inducible nitric oxide synthase activity), thus limiting the cytotoxicity of midbrain dopaminergic neurons in response to an inflammatory stimulus. Neighbouring astrocyte populations promote a neurotrophic environment in response to β_2-adrenoceptor(β_2-AR) stimulation via the production of growth factors(e.g., brain derived neurotrophic factor, cerebral dopamine neurotrophic factor glial cell derived neurotrophic factor which have shown promising neuroprotective and neuro-restorative effects in the nigrostriatal dopaminergic system. More recent findings have demonstrated a role for the β_2-AR in down-regulating expression levels of the human α-synuclein gene SNCA and relative α-synuclein protein abundance. Given that α-synuclein is a major protein constituent of Lewy body pathology, a hallmark neuropathological feature in Parkinson's disease, these findings could open up new avenues for pharmacological intervention strategies aimed at alleviating the burden of α-synucleinopathies in the Parkinsonian brain. In essence, the literature reviewed herein supports our hypothesis of a tripartite neuroprotective role for noradrenaline in combating PD-related neuropathology and motor dysfunction via(1) inhibiting nigral microglial activation pro-inflammatory mediator production,(2) promoting the synthesis of neurotrophic factors from midbrain astrocytes and(3) downregulating α-synuclein gene expression and protein abundance in a β_2-AR-dependent manner. Thus, taken together, either pharmacologically enhancing extra-synaptic noradrenaline bioavailability or targeting glial β_2-ARs directly makes itself as a promising treatment option aimed at slowing/halting PD progression.     

Alpha-synuclein and Parkinson disease

OBJECTIVE:To review the recent progresses on the studies ofα-synuclein in the pathogenesis of Parkinson disease(PD)and look into the perspective ofα-synuclein as a new therapy target.DATA SOURCES:To search the literatures on the progresses of PD studies,especially on the structure,gene expression of α-synuclein and the pathogenesis of PD in Medline from January 1998 to February 2007.Search terms were “Parkinson's disease,α-synuclein” in English.STUDY SELECTION:Initial check the data and choose the original and review articles directly linked to the role of α-synuclein in PD pathogenesis and screening out indirectly discussing articles.Collect the full text and trace the quoting articles and the quoted articles.Only the latest reviews were chosen in Chinese articles.DATA EXTRACTION:There were 424 articles on α-synuclein and its role in the pathogenesis of PD and 43 articles directly related withα-synuclein were chosen among which 12 were reviews.DATA SYNTHESIS:α-synuclein is a kind of soluble protein expressed in pre-synapse in central nervous system encoded by gene in homologous chromosome 4q21.It has physiological function in modulating the stability of membrane and neural plasticity.There is a close relationship between gene mutation inα-synuclein and the pathogenesis of PD.Environmental and genetic factors can induce the misfolding ofα-synuclein,and secondary structural change can result in oligomer formation which induces a series of cascade reaction to damage dopaminergic system subsequently.Cell and animal transgenic and non-transgenic models are established recently and the important role of α-synuclein in the pathogenesis both of familial and sporadic PD is confirmed.Studies reveal that inhibiting the aggregation of α-synuclein can prevent its neurotoxicity;gene parkin can intercept the cell death pathway triggered by the aggregation ofα-synuclein in cytoplasm.CONCLUSION:Gene mutation ofα-synuclein and the impairment in its structure and function are importan in the pathogenesis of PD.Intervention of the gene mutations and abnormal protein aggregation ofα-synuclein may be a new strategy for preventing and treating PD.     

Cross-correlations between pairs of neurons in cerebellar cortex in vivo

In the present paper we apply a new neurophysiological technique to make single-electrode, dual loose-patch recordings from pairs of neuronal elements in the cerebellar cortex in vivo. The analyzed cell pairs consisted of an inhibitory molecular layer interneuron and a Purkinje cell (PC) or a Golgi cell and a granule cell, respectively. To detect the magnitude of the unitary inhibitory synaptic inputs we used histograms of the spike activity of the target cell, triggered by the spikes of the inhibitory cell. Using this analysis, we found that single interneurons had no detectable effect on PC firing, which could be explained by an expected very low synaptic weight of individual interneuron–PC connections. However, interneurons did have a weak delaying effect on the overall series of interspike intervals of PCs. Due to the very high number of inhibitory synapses on each PC, a concerted activation of the interneurons could still achieve potent PC inhibition as previously shown. In contrast, in the histograms of the Golgi cell–granule cell pairs, we found a weak inhibitory effect on the granule cell but only at the time period defined as the temporal domain of the slow IPSP previously described for this connection. Surprisingly, the average granule cell firing frequency sampled at one second was strongly modulated with a negative correlation to the overall firing level of the Golgi cell when the latter was modified through current injection via the patch pipette. These findings are compatible with that tonic inhibition is the dominant form of Golgi cell–granule cell inhibition in the adult cerebellum in vivo.     

Changes in neuron number in the cerebellar cortex of the ageing mouse

The cerebellar cortex of mice aged 6, 15, 22, 25, 28 and 31 months was examined in parasagittal sections using quantitative histological techniques. The number Purkinje cells per mm declined from 13.0 +/- 0.2 at 6 months to 9.1 +/- 0.8 at 31 months. Granule cell density remained constant (2.63 x 10(6) per mm3) between 6 and 31 months of age. The granule cell to Purkinje cell ratio increased from 132.4 +/- 4.2 at 6 months of age to 184.2 +/- 6.9 at 31 months of age. The stellate and basket cell density declined from 83.8 +/- 8.1 per 10(6) mm at 6 months to 58.4 +/- 5.7 at 31 months. The stellate and basket cell to Purkinje cell ratio remained constant at 5.0 +/- 0.1 from 6 to 31 months of age whilst the granule cell to stellate and basket cell ratio increased from 25.5 +/- 0.6 at 6 months to 35.1 +/- 0.6 at 31 months of age. Since granule cell number does not increase in the adult the increase in the granule cell to Purkinje cell and granule cell to stellate and basket cell ratios must be due to loss of Purkinje cells and stellate and basket cells with increasing age. The correlation between Purkinje cell and stellate and basket cell loss is statistically significant (P less than 0.01).     

The role of the sympathetic nervous system in moxibustion-induced immunomodulation in rats

The effects of chemical sympathectomy on moxibustion-induced changes in splenic natural killer (NK) cell cytotoxicity, T and B cell proliferation were studied. Direct moxibustion was applied to the unilateral Zusanli region. NK cell cytotoxicity was suppressed by moxibustion in both vehicle-treated rats and sympathectomized rats. T cell proliferation was not affected by moxibustion. B cell proliferation showed no significant change in vehicle-treated rats, but an increase was seen in sympathectomized rats treated with moxibustion. Sympathectomy alone induced an augmentation of NK cell cytotoxicity and a suppression of T cell proliferation. These results suggest that the sympathetic nervous system (SNS) has no significant role in the mechanism of moxibustion-induced immunomodulation.     

Identification of modulators of hair cell regeneration in the zebrafish lateral line

The external location of the zebrafish lateral line makes it a powerful model for studying mechanosensory hair cell regeneration. We have developed a chemical screen to identify FDA-approved drugs and biologically active compounds that modulate hair cell regeneration in zebrafish. Of the 1680 compounds evaluated, we identified two enhancers and six inhibitors of regeneration. The two enhancers, dexamethasone and prednisolone, are synthetic glucocorticoids that potentiated hair cell numbers during regeneration and also induced hair cell addition in the absence of damage. BrdU analysis confirmed that the extra hair cells arose from mitotic activity. We found that dexamethasone and prednisolone, like other glucocorticoids, suppress zebrafish caudal fin regeneration, indicating that hair cell regeneration occurs by a distinctly different process. Further analyses of the regeneration inhibitors revealed that two of the six, flubendazole and topotecan, significantly suppress hair cell regeneration by preventing proliferation of hair cell precursors. Flubendazole halted support cell division in M-phase, possibly by interfering with normal microtubule activity. Topotecan, a topoisomerase inhibitor, killed both hair cells and proliferating hair cell precursors. A third inhibitor, fulvestrant, moderately delayed hair cell regeneration by reducing support cell proliferation. Our observation that hair cells do not regenerate when support cell proliferation is impeded confirms previous observations that cell division is the primary route for hair cell regeneration after neomycin treatment in zebrafish.     

Quantification of Normal Cell Death in the Rat Retina: Implications for Clone Composition in Cell Lineage Analysis

Naturally occurring cell death complicates the analysis of cell lineage studies by making the surviving members of a clone appear more closely related than they actually are. Here we ask how much normal cell death occurs during rat retinal development, and whether that amount of death is sufficient to confuse the analysis of cell lineage relationships. We measure total cell death in the retina by combining relative counts of dead cells with absolute measurements of total cell loss. For most cell types, but not rods, we find that half of the cells generated die during normal retinal development. We use a computer model to quantify the effects of different amounts of cell death in a simulated lineage study. The simulation indicates that 50% cell death means that clonal variability analysed after the cell death period is not necessarily a good indicator of how much variability actually occurs in the underlying lineage.     

Genetic basis of kainate-induced excitotoxicity in mice: phenotypic modulation of seizure-induced cell death

Excitotoxicity, a process in which excessive excitation of glutamate receptors results in cell death, has been implicated in a number of neurological disorders. However, the genetic characteristics and molecular mechanisms that can modulate the extent of cell death are unclear. Previously, we had reported that the extent of excitotoxic cell death is conferred by differences in the genetic background of several mouse strains. As a first step in the identification of loci that can modulate the extent of excitotoxin-induced cell death, we tested C57BL/6 and FVB/N mice, their F1 hybrids and backcross progeny for differences in apparent excitotoxic cell death induced by kainic acid (KA). While no strain dependent differences in seizure duration were observed, phenotypic analysis of cell death indicated that C57BL/6 mice showed no seizure-induced cell death, while FVB/N mice exhibited extensive cell death. Studies of seizure-induced cell death in hybrid and backcross progeny revealed an association between seizure-induced cell death and genotype. Mice from the F1 cross exhibited little to no seizure-induced cell death, indicative that the extent of cell death is conferred as a dominant genetic trait. Phenotypic assessment of cell death in backcross progeny suggests that differences in apparent cell death are conferred by a single gene locus. These findings implicate genetic factors in individual differences in excitotoxin-induced cell death.     

胶质瘤细胞端粒酶活性依赖细胞周期的调控

目的 研究胶质瘤细胞的端粒酶表达是否与细胞周期相关。方法 利用血清饥饿法和特异的细胞周期抑制剂，使细胞同步化于Go、G1、S、G2、M期，采用PCR—ELISA试剂盒对处于不同周期时相的胶质瘤细胞端粒酶活性进行检测。结果 不同周期时相胶质瘤细胞的端粒酶活性不同，去除血清不能影响活性，G0期细胞与非同步化细胞的活性相同；G1期较非同步化细胞的活性为高；S期活性最高；G2期活性比S期明显降低；M期则几乎无活性。结论 端粒酶活性与细胞周期明显相关，胶质瘤形成可能是由于细胞周期的异常调节使端粒酶活性增高，细胞获得不死性所致。     

Age-related quantitative changes in mitochondria of satellite cell sheaths enveloping spinal ganglion neurons in the rabbit

We studied mitochondria in the satellite cell sheaths which envelope the spinal ganglion neurons of rabbits aged 12, 42, and 79 months. While the mean cytoplasmic volume of satellite cell sheaths did not change significantly with age, the mean percentage of cytoplasmic volume occupied by mitochondria decreased with age. This decrease is mainly due to a reduction in the total mitochondrial mass and only in minor part is a consequence of lipofuscin accumulation. Mitochondrial structure did not change, while mitochondrial size increased with age. Comparison between mitochondria in nerve cell bodies and those in satellite cell sheaths showed that: (1) the mean percentage of cytoplasmic volume occupied by mitochondria was greater in nerve cell bodies than satellite cell sheaths and the ratio between these two percentages remained constant with advancing age; (2) the total mitochondrial mass was much greater in nerve cell bodies than satellite cell sheaths and the ratio between these two values increased with age; (3) the extent of increase of mitochondrial size with age was similar in nerve cell bodies and satellite cell sheaths. The results of the present study suggest that: (1) the ability of satellite cell sheaths to produce energy decreases with age; (2) the decreased ability of sensory neurons in old animals to meet high energy demands may be partly due to the diminished contribution of their associated satellite cell sheaths.     

Projection of the cochlea to cochlear nuclei in Merriam's kangaroo rat

Projection of the cochlea onto the cochlear nuclei was studied by lesioning the spiral ganglion and tracing degenerating fibers and terminals by the Nauta-Gygax and Fink-Heimer methods. Basal turn lesions result in degeneration in the medial portion of anterior ventral cochlear nucleus (AVCN) including globular cell area, small spherical cell area, and a small portion of the large spherical cell area; in dorsomedial portion of posterior ventral cochlear nucleus (PVCN) including globular cell area and octopus cell area; and in the medial extreme of the central region of dorsal cochlear nucleus (DCN). Apical turn lesions result in degeneration in the lateral portion of AVCN including globular cell area, small spherical cell area, and a substantial amount of the large spherical cell area; in the ventrolateral portion of PVCN including globular cell area and multipolar cell area; and in the lateral extreme of the central region of DCN. Lesions in intermediate turns of the cochlea result in degeneration in correspondingly intermediate regions of the cochlear nuclear complex. The basal portion of the cochlea (high frequency) projects most strongly to the small spherical cell area and the octopus cell area. The apical turns (low frequency) project most strongly to the large spherical cell area and the multipolar cell area.     

Identification of neuronal cell death in a model of degeneration in the hippocampus

Neuronal cell death and microglial changes are both hallmarks of neurodegenerative disorders. Therefore, analysis of degenerating neurons related to microglial changes are addressed in many studies of neurosciences. Here we compared different lesion models and two markers for neurodegeneration (Fluoro-Jade and propidium iodide) in an in vivo as well as an in vitro approach. Fluoro-Jade is a specific and selective marker to identify neurons undergoing degeneration. We also tested this marker to analyze neurodegeneration in organotypic hippocampal slice cultures. We could show that activation of microglia is followed by neuronal cell death. Most degeneration markers, such as propidium iodide, only stain the neuronal cell body excluding the axonal and dendritic processes. Fluoro-Jade is able to stain the distal portion as well as the proximal portion of the dissected axon including the axotomized neuron, as so called anterograde and retrograde degeneration after axotomy. To analyze the specificity of Fluoro-Jade, we used primary microglial and BV-2 cells, a well-described murine microglial cell line. Treatment of microglial and BV-2 cells with an excess of L-glutamate induces cell death which could be detected by propidium iodide staining, but not by Fluoro-Jade, demonstrating its specificity to monitor neuronal cell death.