Autophagy and its neuroprotection in neurodegenerative diseases

It has been suggested that protein misfolding and aggregation contribute significantly to the development of neurodegenerative diseases.Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and by both Micro and Macro autophagy lysosomal pathway (ALP).Autophagosomal dysfunction has been implicated in an increasing number of diseases including neurodegenerative diseases.Autophagy is a cellular self-eating process that plays an important role in neuroprotection as well as neuronal injury and death.While a decrease in autophagic activity interferes with protein degradation and possibly organelle turnover,increased autophagy has been shown to facilitate the clearance of aggregation-prone proteins and promote neuronal survival in a number of disease models.On the other hand,too much autophagic activity can be detrimental,suggesting the regulation of autophagy is critical in dictating cell fate.In this review paper,we will discuss various aspects of ALP biology and its dual functions in neuronal cell death and survival.We will also evaluate the role of autophagy in neurodegenerative diseases including Alzheimer’s disease,Parkinson’s disease,Huntington’s disease,amyotrophic lateral sclerosis.Finally,we will explore the therapeutic potential of autophagy modifiers in several neurodegenerative diseases.     

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expressionin vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase (ROCK) pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.     

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible fac-tor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increas-ing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hy-poxia-inducible factor-1α and cytoglobin during hypoxic injury.     

State and perspectives on flavonoid neuroprotection against aminochrome-induced neurotoxicity

正Parkinson's disease(PD) motor symptoms are induced by the loss of dopaminergic neurons containing neuromelanin in the nigrostriatal system. The exact mechanism that triggers the degeneration of the nigrostriatal neurons is still unknown but there is general consensus in the scientific community that mitochondrial dysfunction, alpha-synuclein aggregation to neurotoxic oligomers, protein degradation dysfunction of both lysosomal and proteasomal systems, endoplasmic reticulum stress,     

Oxidative stress, induced by 6-hydroxydopamine, reduces proteasome activities in PC12 cells

Mutations in familial Parkinson’s disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10–100 μM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.     

Expression of Mammalian BM88/CEND1 in Drosophila Affects Nervous System Development by Interfering with Precursor Cell Formation

We used Drosophila melanogaster as an experimental model to express mouse and pig BM88/CEND1(cell cycle exit and neuronal differentiation 1) in order to investigate its potential functional effects on Drosophila neurogenesis. BM88/CEND1 is a neuron-specific protein whose function is implicated in triggering cells to exit from the cell cycle and differentiate towards a neuronal phenotype. Transgenic flies expressing either mouse or pig BM88/CEND1 in the nervous system had severe neuronal phenotypes with variable expressivity at various stages of embryonic development. In early embryonic stage 10,BM88/CEND1 expression led to an increase in the neuralspecific antigenicity of neuroectoderm at the expense of precursor cells [neuroblasts(Nbs) and ganglion mother cells(GMCs)] including the defective formation and differentiation of the MP2 precursors, whereas at later stages(12–15), protein accumulation induced gross morphological defects primarily in the CNS accompanied by a reduction of Nb and GMC markers. Furthermore, the neuronal precursor cells of embryos expressing BM88/CEND1 failed to carry out proper cell-cycle progression as revealed by the disorganized expression patterns of specific cell-cycle markers. BM88/CEND1 accumulation in the Drosophila eye affected normal eye disc development by disrupting the ommatidia. Finally, we demonstrated that expression of BM88/CEND1 modified/reduced the levels of activated MAP kinase indicating a functional effect of BM88/CEND1 on the MAPK signaling pathway. Our findings suggest that the expression of mammalian BM88/CEND1 in Drosophila exerts specific functional effects associated with neuronal precursor cell formation during embryonic neurogenesis and proper eye disc development.This study also validates the use of Drosophila as a powerful model system in which to investigate gene function and the underlying molecular mechanisms.     

Heat shock protein 72 confers protection in retinal ganglion cells and lateral geniculate nucleus neurons via blockade of the SAPK/JNK pathway in a chronic ocular-hypertensive rat model

Optic nerve transection increased the expression of heat shock protein 72 （HSP72） in the lateral geniculate body, indicating that this protein is involved in the prevention of neuronal injury. Zinc sulfate and quercetin induced and inhibited the expression of HSP72, respectively. Intraperitoneal injections of zinc sulfate, SP600125 （c-Jun N-terminal kinase inhibitor）, or quercetin were performed on retinal ganglion cells in a Wistar rat model of chronic ocular hypertension. Our results showed that compared with the control group, the expression of HSP72 in retinal ganglion cells and the lateral geniculate body was increased after the injection of zinc sulfate, but was decreased after the injection of quercetin. The expression of phosphorylated c-Jun N-terminal kinases and phosphorylated c-Jun were visible 3 days after injection in the control group, and reached apeak at 7 days. Zinc sulfate and SP600125 significantly decreased the expression of p-c-Jun, whereas quercetin significantly enhanced the expression of this protein. These results suggest that HSP72 protects retinal ganglion cells and lateral geniculate body in a rat model of chronic ocular hypertension from injury by blocking the activation of the stress-activated kinase/c-Jun N-terminal kinase apoptotic pathway.     

Neural stem cells over-expressing brain-derived neurotrophic factor promote neuronal survival and cytoskeletal protein expression in traumatic brain injury sites

Cytoskeletal proteins are involved in neuronal survival.Brain-derived neurotrophic factor can increase expression of cytoskeletal proteins during regeneration after axonal injury.However,the effect of neural stem cells genetically modified by brain-derived neurotrophic factor transplantation on neuronal survival in the injury site still remains unclear.To examine this,we established a rat model of traumatic brain injury by controlled cortical impact.At 72 hours after injury,2 × 10~7 cells/m L neural stem cells overexpressing brain-derived neurotrophic factor or naive neural stem cells(3 m L) were injected into the injured cortex.At 1–3 weeks after transplantation,expression of neurofilament 200,microtubule-associated protein 2,actin,calmodulin,and beta-catenin were remarkably increased in the injury sites.These findings confirm that brain-derived neurotrophic factor-transfected neural stem cells contribute to neuronal survival,growth,and differentiation in the injury sites.The underlying mechanisms may be associated with increased expression of cytoskeletal proteins and the Wnt/β-catenin signaling pathway.     

20S proteasome and glyoxalase 1 activities decrease in erythrocytes derived from Alzheimer’s disease patients

As a result of accumulating methylglyoxal and advanced glycation end products in the brains of patients with Alzheimer’s disease,it is considered a protein precipitation disease.The ubiquitin proteasome system is one of the most important mechanisms for cells to degrade proteins,and thus is very important for maintaining normal physiological function of the nervous system.This study recruited 48 individuals with Alzheimer’s disease(20 males and 28 females aged 75±6 years)and 50 healthy volunteers(21 males and 29 females aged 72±7 years)from the Affiliated Hospital of Youjiang Medical University for Nationalities(Baise,China)between 2014 and 2017.Plasma levels of malondialdehyde and H2O2 were measured by colorimetry,while glyoxalase 1 activity was detected by spectrophotometry.In addition,20S proteasome activity in erythrocytes was measured with a fluorescent substrate method.Ubiquitin and glyoxalase 1 protein expression in erythrocyte membranes was detected by western blot assay.The results demonstrated that compared with the control group,patients with Alzheimer’s disease exhibited increased plasma malondialdehyde and H2O2 levels,and decreased glyoxalase 1 activity;however,expression level of glyoxalase 1 protein remained unchanged.Moreover,activity of the 20S proteasome was decreased and expression of ubiquitin protein was increased in erythrocytes.These findings indicate that proteasomal and glyoxalase activities may be involved in the occurrence of Alzheimer’s disease,and erythrocytes may be a suitable tissue for Alzheimer’s disease studies.This study was approved by the Ethics Committee of Youjiang Medical University for Nationalities(approval No.YJ12017013)on May 3,2017.     

SIRT1 facilitates amyloid beta peptide degradation by upregulating lysosome number in primary astrocytes

Previous studies have shown that sirtuin 1(SIRT1) reduces the production of neuronal amyloid beta(Aβ) and inhibits the inflammatory response of glial cells, thereby generating a neuroprotective effect against Aβ neurotoxicity in animal models of Alzheimer's disease. However, the protective effect of SIRT1 on astrocytes is still under investigation. This study established a time point model for the clearance of Aβ in primary astrocytes. Results showed that 12 hours of culture was sufficient for endocytosis of oligomeric Aβ, and 36 hours sufficient for effective degradation. Immunofluorescence demonstrated that Aβ degradation in primary astrocytes relies on lysosome function. Enzymatic agonists or SIRT1 inhibitors were used to stimulate cells over a concentration gradient. Aβ was co-cultured for 36 hours in medium. Western blot assay results under different conditions revealed that SIRT1 relies on its deacetylase activity to promote intracellular Aβ degradation. The experiment further screened SIRT1 using quantitative proteomics to investigate downstream, differentially expressed proteins in the Aβ degradation pathway and selected the ones related to enzyme activity of SIRT1. Most of the differentially expressed proteins detected are close to the primary astrocyte lysosomal pathway. Immunofluorescence staining demonstrated that SIRT1 relies on its deacetylase activity to upregulate lysosome number in primary astrocytes. Taken together, these findings confirm that SIRT1 relies on its deacetylase activity to upregulate lysosome number, thereby facilitating oligomeric Aβ degradation in primary astrocytes.     

溶酶体自噬途径降解α—synuclein蛋白作用研究

目的探讨α—synuclein蛋白细胞内溶酶体途径降解机制。方法用神经生长因子NGF诱导分化PCI2细胞作为研究多巴胺能神经元的细胞载体,应用鱼藤酮处理PCI2细胞建立α-synuclein蛋白细胞模型。使用溶酶体途径降解抑制剂E64处理神经元样分化的PCI2细胞,应用免疫荧光双标方法观察PCI2细胞内硫黄素S、α—synuclein蛋白阳性聚集包涵体形成情况,比较各组的差异。结果用E64处理鱼藤酮预处理过的PCI2细胞后α—synuclein蛋白聚集且较多包涵体形成（15．36±0．85）％,与对照组相比差异有统计学意义（P〈0．05）。结论溶酶体自噬途径可能在α—synuclein蛋白降解、聚集和多巴胺神经元死亡过程中发挥重要作用。     

Coordinated and widespread expression of gamma-secretase in vivo: evidence for size and molecular heterogeneity

Gamma-secretase is a high molecular weight protein complex composed of four subunits, namely, presenilin (PS; 1 or 2), nicastrin, anterior pharynx defective-1 (Aph-1; A or B), and presenilin enhancer-2 (Pen-2), and is responsible for the cleavage of a number of type-1 transmembrane proteins. A fundamental question is whether different gamma-secretase complexes exist in vivo. We demonstrate here by in situ hybridization and by Northern and Western blotting that the gamma-secretase components are widely distributed in all tissues investigated. The expression of the different subunits seems tightly coregulated. However, some variation in the expression of the Aph-1 proteins is observed, Aph-1A being more general and abundantly distributed than Aph-1B. The previously uncharacterized rodent-specific Aph-1C mRNA is highly expressed in the kidney and testis but not in brain or other tissues, indicating some tissue specificity for the Aph-1 component of the gamma-secretase complex. Blue-native electrophoresis revealed size heterogeneity of the mature gamma-secretase complex in various tissues. Using co-immunoprecipitations and blue-native electrophoresis at endogenous protein levels, we find evidence that several independent gamma-secretase complexes can coexist in the same cell type. In conclusion, our results suggest that gamma-secretase is a heterogeneous family of protein complexes widely expressed in the adult organism.     

蛋白酶体在信号转导和神经退行性疾病中的作用

哺乳动物细胞内有两条蛋白降解途径：泛素-蛋白酶体通路和自噬-溶酶体通路。蛋白酶体由多种蛋白亚单位组成,在降解细胞短寿命蛋白中有重要作用。近来的研究发现蛋白酶体降解系统在细胞信号转导和细胞功能中有重要作用。蛋白酶体功能障碍或失调可能参与某些神经退行性疾病的致病机制。本文综述蛋白酶体在细胞信号转导中的生物学功能和在神经退行性疾病中的可能作用。     

Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin–proteasome system in PC12D cells

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson’s disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin–proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-⁴⁰Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin–proteasome system in PC12D cells.     

c-Jun N-terminal kinase pathway mediates Lactacystin-induced cell death in a neuronal differentiated Neuro2a cell line

The ubiquitin-proteasome pathway is an intracellular protein degradation pathway responsible for degradation of many regulatory proteins that must be rapidly eliminated normally. Some recent studies reported that a proteasome dysfunction was involved in the pathogenesis of neurodegenerative diseases. Thus, there is now considerable interest in the possible role of proteasome in this regard. Here we show that inhibition of proteasomal function by Lactacystin-induced cell death in a neuronal differentiated Neuro2a (nN2a) cell line but not in an undifferentiated Neuro2a (N2a) cell line. Cell death was accompanied by both the activation of c-Jun N-terminal kinase, p38 and caspase-3. A pan-caspase inhibitor, Z-VAD-FMK, or SB203580, a p38 inhibitor could not inhibit cell death induced by Lactacystin, whereas nN2a cell lines with stable expression of the dominant negative mutant of c-Jun N-terminal kinase showed a remarkable suppression of cell death. Lactacystin-induced cell death is mediated through the c-Jun N-terminal kinase pathway but not the caspase-dependent pathway in a nN2a cell line. Our results shed light on the association among the proteasomal dysfunction, JNK pathway and neuronal cell death, leading to the elucidation of its possible role in the pathogenesis of neurodegenerative diseases.     

Expression patterns of synaptic vesicle protein 2A in focal cortical dysplasia and TSC‐cortical tubers

Purpose: Synaptic vesicle protein 2A (SV2A), the binding site for the antiepileptic drug (AED) levetiracetam, has been shown to be involved in the control of neuronal excitability. The aim of the study was to define the expression and cell‐specific distribution of SV2A in developmental focal lesions associated with medically intractable epilepsy. Methods: SV2A immunocytochemistry and Western blotting was performed in focal cortical dysplasia (FCD type IIB) and cortical tubers from patients with tuberous sclerosis complex (TSC). Results: Autopsy and surgical control neocortical specimens were characterized by strong SV2A immunoreactivity throughout all cortical layers, with punctate labeling around the somata and dendrites of neurons. In FCD and cortical tuber specimens less intense, SV2A immunoreactivity was observed in the neuropil. The reduction in expression was confirmed by Western blot analysis. In both FCD and tuber specimens, clusters of punctate labeling were detected along cell borders and processes (perisomatic synapses) of dysplastic neuronal cells localized in both gray and white matter. The large majority of balloon cells in FCD, or giant cells in tubers, did not show punctate labeling around their somata. SV2A immunoreactivity was observed occasionally within the neuronal perikarya. Conclusions: The pattern of SV2A immunoreactivity with reduced neuropil expression and altered cellular and subcellular distribution suggests a possible contribution of SV2A to the epileptogenicity of these malformations of cortical development. Knowledge of the expression pattern of SV2A in epilepsy‐associated pathologies may be valuable for the evaluation of the effectiveness of AEDs targeting this protein.     

Low levels of mutant ubiquitin are degraded by the proteasome in vivo

The ubiquitin‐proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin‐ containing proteinaceous deposits. UBB⁺¹, a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB⁺¹ properties shift from a proteasomal ubiquitin‐fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB⁺¹. In these mice, UBB⁺¹ protein is scarcely detectable in the neuronal cell population. Accumulation of UBB⁺¹ commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB⁺¹ protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB⁺¹ is continuously degraded and disease‐related UBB⁺¹ accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB⁺¹ and its role in neurodegenerative disease. © 2010 Wiley‐Liss, Inc.     

Brain‐derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells,possibly by triggering the Wnt/β‐catenin signaling pathway

Brain‐derived neurotrophic factor (BDNF) has critical functions in promoting survival, expansion, and differentiation of neural stem cells (NSCs), but its downstream regulation mechanism is still not fully understood. The role of BDNF in proliferation and differentiation of NSCs through Wnt/β‐catenin signaling was studied via cell culture of cortical NSCs, Western blotting, immunocytochemistry, and TOPgal (Wnt reporter) analysis in mice. First, BDNF stimulated NSC proliferation dose dependently in cultured neurospheres that exhibited BrdU incorporation and neuronal and glial differentiation abilities. Second, BDNF effectively enhanced cell commitment to neuronal and oligodendrocytic fates, as indicated by increased differentiation marker Tuj‐1 (neuronal marker), CNPase (oligodendrocyte marker), and neuronal process extension. Third, BDNF upregulated expression of Wnt/β‐catenin signaling (Wnt1 and free β‐catenin) molecules. Moreover, these promoting effects were significantly inhibited by application of IWR1, a Wnt signaling‐specific blocker in culture. The TOPgal mouse experiment further confirmed BDNF‐triggered Wnt signaling activation by β‐gal labeling. Finally, an MEK inhibition experiment showed a mediating role of the microtubule‐associated protein kinase pathway in BDNF‐triggered Wnt/β‐catenin signaling cascades. This study overall has revealed that BDNF might contribute to proliferation and neuronal and oligodendrocytic differentiation of NSCs in vitro, most possibly by triggering the Wnt/β‐catenin signaling pathway. Nevertheless, determining the exact cross‐talk points at which BDNF might stimulate Wnt/β‐catenin signaling pathway in NSC activity requires further investigation. © 2012 Wiley Periodicals, Inc.     

大鼠局灶性脑缺血再灌注后LPA1的表达对神经元凋亡的影响

目的 探讨大鼠局灶性脑缺血再灌注后溶血磷脂酸受体1(LPA1)的表达对神经元凋亡的影响及其可能机制。方法 将24只SPF级SD雄性大鼠随机分为4组,每组各6只,分别为假手术组(A组)、大脑中动脉栓塞(MCAO)组(B组)、MCAO+溶剂组(C组)、MCAO+LPA1拮抗剂(Ki16425)组(D组); 4组均于手术后48 h取标本; 利用HE染色观察大鼠脑组织细胞形态的变化; 四氮唑红(TTC)染色观察大鼠脑梗死面积; 免疫荧光技术检测LPA1在大鼠皮层半暗带神经元表达水平; 免疫印迹、免疫组化技术检测大鼠脑组织中Caspase-3蛋白及p-Akt蛋白表达水平。结果 与A组比较,B组有明显的缺血再灌注损伤,表现为细胞肿胀,细胞溶解坏死,大鼠皮层半暗带神经元的LPA1表达水平较高; 与C组比较,D组大鼠脑梗死面积显著增大(P<0.05),缺血半暗带细胞肿胀更加明显,胞浆空泡区增大,细胞核固缩更加严重,细胞间隙增宽更明显; D组较C组缺血半暗带Caspase-3蛋白表达水平明显升高(P<0.05),而p-Akt蛋白表达水平明显降低(P<0.05)。结论 抑制大鼠局灶性脑缺血再灌后LPA1的表达可使大鼠脑梗死面积增大,细胞凋亡增加,同时p-Akt蛋白表达减少,这说明在大鼠局灶性缺血再灌注过程中LPA1对神经元具有保护作用,其机制可能是通过Akt途径来发挥保护作用的。