Modest proteasomal inhibition by aberrant ubiquitin exacerbates aggregate formation in a Huntington disease mouse model

UBB⁺¹, a mutant form of ubiquitin, is both a substrate and an inhibitor of the proteasome which accumulates in the neuropathological hallmarks of Huntington disease (HD). In vitro, expression of UBB⁺¹ and mutant huntingtin synergistically increase aggregate formation and polyglutamine induced cell death. We generated a UBB⁺¹ transgenic mouse line expressing UBB⁺¹ within the neurons of the striatum. In these mice lentiviral driven expression of expanded huntingtin constructs in the striatum results in a significant increase in neuronal inclusion formation. Although UBB⁺¹ transgenic mice show neither a decreased lifespan nor apparent neuronal loss, they appear to be more vulnerable to toxic insults like expanded polyglutamine proteins due to a modest proteasome inhibition. These findings underscore the relevance of an efficient ubiquitin–proteasome system in HD.     

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B⁺¹ (UBB⁺¹), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB⁺¹-expressing transgenic mice display widespread labeling for UBB⁺¹ in brain and exhibit behavioral deficits. Here, we show that UBB⁺¹ is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB⁺¹-expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.     

Review: The ubiquitin‐proteasome system: contributions to cell death or survival in neurodegeneration

N. Rogers, S. Paine, L. Bedford and R. Layfield (2010) Neuropathology and Applied Neurobiology 36, 113–124
The ubiquitin‐proteasome system: contributions to cell death or survival in neurodegeneration The significance of the accumulation of ubiquitin‐positive intraneuronal inclusions in the brains of those affected with different neurodegenerative diseases is currently unclear. While one interpretation is that the disease mechanism(s) involves dysfunction of an ubiquitin‐mediated process, such as the ubiquitin‐proteasome system, the inclusions are also found in surviving neurones, suggesting a possible neuroprotective role. Here we review recent evidence in support of these seemingly opposing notions gleaned from cell and animal models as well as investigations of patient samples, with particular emphasis on studies relevant to Parkinson's disease.     

Parkin reverses intracellular β‐amyloid accumulation and its negative effects on proteasome function

The significance of intracellular β‐amyloid (Aβ₄₂) accumulation is increasingly recognized in Alzheimer's disease (AD) pathogenesis. Aβ removal mechanisms that have attracted attention include IDE/neprilysin degradation and antibody‐mediated uptake by immune cells. However, the role of the ubiquitin‐proteasome system (UPS) in the disposal of cellular Aβ has not been fully explored. The E3 ubiquitin ligase Parkin targets several proteins for UPS degradation, and Parkin mutations are the major cause of autosomal recessive Parkinson's disease. We tested whether Parkin has cross‐function to target misfolded proteins in AD for proteasome‐dependent clearance in SH‐SY5Y and primary neuronal cells. Wild‐type Parkin greatly decreased steady‐state levels of intracellular Aβ₄₂, an action abrogated by proteasome inhibitors. Intracellular Aβ₄₂ accumulation decreased cell viability and proteasome activity. Accordingly, Parkin reversed both effects. Changes in mitochondrial ATP production from Aβ or Parkin did not account for their effects on the proteasome. Parkin knock‐down led to accumulation of Aβ. In AD brain, Parkin was found to interact with Aβ and its levels were reduced. Thus, Parkin is cytoprotective, partially by increasing the removal of cellular Aβ through a proteasome‐dependent pathway. © 2009 Wiley‐Liss, Inc.     

Aβ induces endoplasmic reticulum stress causing possible proteasome impairment via the endoplasmic reticulum–associated degradation pathway

Background: Accumulation of β‐amyloid is a major pathology of Alzheimer’s disease (AD). As in other neurodegenerative diseases, it is also reported that proteasome activity is deteriorated in post‐mortem brains of AD patients. However, the mechanism of proteasomal dysfunction in AD remains unexplained. There is, however, increasing reported evidence that the unfolded protein response (UPR) is involved in AD pathology. Here we show that Aβ causes not only the UPR leading to endoplasmic reticulum (ER) stress mediated cell death, but also proteasomal dysfunction in cultured cells. Methods: Mouse primary cultured neurons and other cultured cells such as HEK 293T or SH‐SY5Y were treated with Aβ or other reagents, such as thapsigargin and lactacystin, to study UPR or proteasome activity. The UPR was investigated using proteins or mRNA expression. To ascertain proteasome activity, we also recruited SH‐SY5Y cells stably transfected with GFP^u. Results: In vitro study showed that UPR, phosphorylation of eIF‐2α and BiP degradation preceded proteasome dysfunction. It is known that the UPR of ER occurs with the assistance of proteasome as ER‐associated protein degradation (ERAD). Conclusion: This evidence, taken together, suggests that Aβ may induce proteasome dysfunction by preceding the UPR through ER‐associated protein degradation.     

Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3)

K. Seidel, M. Meister, G. J. Dugbartey, M. P. Zijlstra, J. Vinet, E. R. P. Brunt, F. W. van Leeuwen, U. Rüb, H. H. Kampinga and W. F. A. den Dunnen (2012) Neuropathology and Applied Neurobiology 38, 548–558 Cellular protein quality control and the evolution of aggregates in spinocerebellar ataxia type 3 (SCA3) Aims: A characteristic of polyglutamine diseases is the increased propensity of disease proteins to aggregate, which is thought to be a major contributing factor to the underlying neurodegeneration. Healthy cells contain mechanisms for handling protein damage, the protein quality control, which must be impaired or inefficient to permit proteotoxicity under pathological conditions. Methods: We used a quantitative analysis of immunohistochemistry of the pons of eight patients with the polyglutamine disorder spinocerebellar ataxia type 3. We employed the anti‐polyglutamine antibody 1C2, antibodies against p62 that is involved in delivering ubiquitinated protein aggregates to autophagosomes, antibodies against the chaperones HSPA1A and DNAJB1 and the proteasomal stress marker UBB⁺¹. Results: The 1C2 antibody stained neuronal nuclear inclusions (NNIs), diffuse nuclear staining (DNS), granular cytoplasmic staining (GCS) and combinations, with reproducible distribution. P62 always co‐localized with 1C2 in NNI. DNS and GCS co‐stained with a lower frequency. UBB⁺¹ was present in a subset of neurones with NNI. A subset of UBB⁺¹‐containing neurones displayed increased levels of HSPA1A, while DNAJB1 was sequestered into the NNI. Conclusion: Based on our results, we propose a model for the aggregation‐associated pathology of spinocerebellar ataxia type 3: GCS and DNS aggregation likely represents early stages of pathology, which progresses towards formation of p62‐positive NNI. A fraction of NNI exhibits UBB⁺¹ staining, implying proteasomal overload at a later stage. Subsequently, the stress‐inducible HSPA1A is elevated while DNAJB1 is recruited into NNIs. This indicates that the stress response is only induced late when all endogenous protein quality control systems have failed.     

The pattern of neuronal loss and survival may reflect differential expression of proteasome activators in Parkinson's disease

The basis of neuronal vulnerability, degeneration, and sparing in PD are unknown, but there is increasing evidence to suggest that the ubiquitin‐proteasome system (UPS) plays an important role in the pathogenesis of the disorder. In this study, we employed an immunocytochemical approach to determine if the differential expression of key UPS components in various brain regions and cells might underlie the pattern of neuronal degeneration and survival seen in PD. We showed that the ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and 26/20S proteasome α‐ and β‐subunits, are abundantly expressed in the substantia nigra pars compacta (SNc) and in cultured dopaminergic neurons. Although the proteasome activator PA700 is expressed in the medial SNc, levels are low in the lateral region, and expression of the other proteasome activator, PA28, is near absent in the entire SNc. PA28 (but not PA700) was found to be poorly expressed in noradrenergic neurons in the locus coeruleus (LC) compared with adjacent cells in the mesencephalic nucleus. PA700 and PA28 are also poorly expressed in dopaminergic neurons compared with other cell types in culture. Inhibition of proteasomal function, generation of misfolded proteins, induction of oxidative stress or impairment of mitochondrial complex I activity, caused a compensatory upregulation in PA700 and PA28 in a variety of cells but not in dopaminergic neurons in culture. These findings are consistent with the demonstration that, in sporadic PD, proteasomal activity and levels of PA700/PA28 are reduced in the SNc butare markedly upregulated in regions/cells that are spared from the neurodegenerative process. Thus, the differential distribution and activity of proteasome activations could play a significant role in the pathogenesis of PD. Synapse 64:241–250, 2010. © 2009 Wiley‐Liss, Inc.     

Reductions of the components of the calreticulin/calnexin quality‐control system by proteasome inhibitors and their relevance in a rodent model of Parkinson's disease

Evidence indicates that the ubiquitin‐proteasome system and the endoplasmic retculum (ER) quality‐control system work in concert to ensure that proteins are correctly folded in the ER and that misfolded proteins are retrotransported to the cytosol for degradation by proteasomes. Dysfunction of either system results in developmental abnormalities and even death in animals. This study investigates whether and how proteasome inhibition impacts the components of the calreticulin (CRT)/calnexin (CNX) glycoprotein folding machinery, a typical ER protein quality‐control system, in the context of early neuronal injury. Here we report that proteasome inhibitor treatments, at nonlethal levels, reduced protein levels of CRT and ERp57 but not of CNX. These treatments increased protein levels of CRT in culture media, an effect blocked by brefeldin A, an inhibitor of protein trafficking; by contrast, ERp57 was not detected in culture media. Knockdown of CRT levels alone increased the vulnerability of SH‐SY5Y, a neuronal cell line, to 6‐hydroxydopamine (6‐OHDA) toxicity. In a rat model of Parkinson's disease, intrastriatal 6‐OHDA lesions resulted in decreased levels of CRT and ERp57 in the midbrain. These findings suggest that reduction of the components of CRT/CNX glycoprotein quality‐control system may play a role in neuronal injury in Parkinson's disease and other neurodegenerative disorders associated with dysfunction of the ubiquitin‐proteasome system. © 2014 Wiley Periodicals, Inc.     

Ischemic tolerance in an in vivo model of glutamate preconditioning

Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. This study focuses on glutamate‐mediated neuronal tolerance to ischemia–reperfusion. We employed an animal model of lifelong excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo glutamate preconditioning. Nine‐ and twenty‐two‐month‐old Tg and wild‐type (wt) mice were subjected to 90 min of middle cerebral artery occlusion, followed by 24 hr of reperfusion. The Tg mice suffered significantly reduced infarction and edema volume compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and microtubule‐associated protein‐2A (MAP2A) expression to understand the mechanism of neuroprotection observed in the Tg mice. We found that, in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes, whereas there was no significant difference in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising, significant increase was observed in MAP2A expression in neurons of the Tg hippocampus following ischemia–reperfusion compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia–reperfusion. © 2014 Wiley Periodicals, Inc.     

Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn²⁺). The present studies were performed to identify the processes responsible for this effect. One of the well‐established mechanisms for protein‐level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4‐2 (neural precursor cells expressed developmentally downregulated 4‐2), and that Mn²⁺ increases both Nedd4‐2 mRNA and protein levels. Additionally, we have found that Mn²⁺ exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn²⁺ effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid‐inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4‐2. Additional findings establish that Mn²⁺ increases astrocytic caspase‐like proteolytic proteasome activity and that the Mn²⁺‐dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N‐acetyl‐leu‐leu‐norleucinal and lactacystin. Combined, these results demonstrate that Mn²⁺‐induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin‐mediated proteolytic system. © 2010 Wiley‐Liss, Inc.     

Mutant ubiquitin-mediated ��-secretase stability via activation of caspase-3 is related to ��-amyloid accumulation in ischemic striatum in rats

Previous studies have demonstrated that ischemic stroke increases β-amyloid (Aβ) production by increasing β-secretase (BACE1) through activation of caspase-3, and stimulates generation of mutant ubiquitin (UBB⁺¹) in rat brains. In this study, we examined whether caspase-3 activation participates in the regulation of UBB⁺¹ generation and UBB⁺¹-mediated BACE1 stability in ischemic injured brains. The results showed that UBB⁺¹ and activated caspase-3-immunopositive-stained cells were time dependently increased in the ipsilateral striatum of rat brains after middle cerebral artery occlusion. UBB⁺¹-immunopositive cells could be co-stained with caspase-3, Aβ (UBB⁺¹–Aβ), and BACE1 (UBB⁺¹–BACE1). BACE1 protein could also be pulled down by immunoprecipitation with UBB⁺¹ antibody. Z-DEVD-FMK (DEVD), a caspase-3 inhibitor, significantly decreased the level of UBB⁺¹ protein and the number of UBB⁺¹–Aβ and UBB⁺¹–BACE1 double-stained cells in the ischemic striatum, as well as the level of UBB⁺¹/BACE1 protein complex. We conclude that activation of caspase-3 might be upstream of UBB⁺¹ formation and that excessive UBB⁺¹ could bind to BACE1 and increase the stability of BACE1, thereby increasing Aβ in ischemic injured brains. These results suggest new biological and pathological effects of caspases and regulation of the ubiquitin–proteasome system in the brain. Our results provide new therapeutic targets to prevent further neurodegeneration in patients after stroke.     

MPTP treatment of common marmosets impairs proteasomal enzyme activity and decreases expression of structural and regulatory elements of the 26S proteasome

Dysfunction of the ubiquitin‐proteasome system occurs in the substantia nigra (SN) in Parkinson's disease (PD). However, it is unknown whether this is a primary cause or a secondary consequence of other components of the pathogenic process. We have investigated in nonhuman primates whether initiating cell death through mitochondrial complex I inhibition using 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine hydrochloride (MPTP) altered proteasomal activity or the proteasomal components in the SN. Chymotrypsin‐like, trypsin‐like and peptidylglutamyl‐peptide hydrolase (PGPH) activating of 20S proteasome were decreased in SN homogenates of MPTP‐treated marmosets compared to naïve animals. Western blotting revealed a marked decrease in the expression of 20S‐α subunits, but no change in 20S‐β subunits in the SN of MPTP‐treated marmoset compared to naïve animals. There was a marked decrease in the expression of the proteasome activator 700 (PA700) and proteasome activator 28 (PA28) regulatory complexes. The 20S‐α4 subunit immunoreactivity was decreased in the nucleus of colocalized tyrosine hydroxylase (TH)‐positive cells of MPTP‐treated animals compared to naïve animals but no difference in the intensity of 20S‐β1i subunit staining. Immunoreactivity for PA700‐Rpt5 and PA28‐α subunits within surviving TH‐positive cells of MPTP‐treated marmoset was reduced compared to naïve controls. Overall, the changes in proteasomal function and structure occurring follow MPTP‐induced destruction of the SN in common marmosets were very similar to those found in PD. This suggests that altered proteasomal function in PD could be a consequence of other pathogenic processes occurring in SN as opposed to initiating cell death as previously suggested.     

Amyloid-β peptide is a substrate of the human 20S proteasome

Intraneuronal accumulation of ubiquitin conjugates is a pathological feature of neurodegenerative disorders such as Alzheimer's disease (AD). Previous reports propose that accumulation of ubiquitinated species in AD is a result of inhibition of proteasomal activity by amyloid-β (Aβ) peptides, which leads to blocking of ubiquitin-dependent protein degradation by the proteasome. Here, we provide additional insight into proteasomal dysfunction by Aβ peptides by revealing that aggregated forms of Aβ(1-42) peptides (especially small oligomers) are, in fact, competitive substrates for the chymotrypsin-like activity of the human 20S (h20S) proteasome. In addition to examining the kinetics of the h20S proteasome activity in the presence or absence of Aβ peptides, we use gel electrophoresis, LC-MS, and TOF-MS/MS analyses to examine the degradation of Aβ(1-42) by the h20S proteasome. The observed peptide fragments resulting from proteolytic cleavage of Aβ were consistent with predicted cleavage sites from proteasome degradation. These results support that the interaction of Aβ peptides with the proteasome may play a mechanistic role in proteasomal dysfunction in AD pathology. These results may also reveal a previously unknown natural pathway for clearance of Aβ in normal or diseased cells.     

Immunohistochemical analysis of Marinesco bodies,using antibodies against proteins implicated in the ubiquitin‐proteasome system,autophagy and aggresome formation

Marinesco bodies (MBs) are spherical eosinophilic intranuclear inclusions in pigmented neurons in the substantia nigra and locus ceruleus. Previous immunohistochemical studies have shown that MBs are positive for ubiquitin, p62 and SUMO‐1, suggesting the involvement of ubiquitination and related proteins in the formation or disaggregation of MBs. However, the involvement is not thoroughly understood. Therefore, we immunohistochemically examined the midbrain from five control subjects ranged from 53 to 84 years old. MBs were positive for various proteins implicated in the ubiquitin‐proteasome system (ubiquitin, p62, EDD1, NEDD8, NUB1, SUMO‐1 and SUMO‐2), aggresome formation (HDAC6) and autophagy (ubiquitin, p62, LC3, GABARAP and GATE‐16). These findings suggest that proteins related to ubiquitination, proteasomal degradation and autophagy are involved in the formation or disaggregation of MBs.     

Quercetin improves the activity of the ubiquitin‐proteasomal system in 150Q mutated huntingtin‐expressing cells but exerts detrimental effects on neuronal survivability

Quercetin, a strong free radical scavenger, is investigated for neuroprotective effects in a Neuro 2a cell line conditionally transfected with 16Q huntingtin (Htt) and 150Q Htt, which express the protein upon stimulation. Cells were protected from death by a 20‐µM dose of quercetin on the second day of Htt induction, but 30–100‐µM doses of the drug caused further toxicity in both 16Q and 150Q cells, as indicated by MTT assay and by significant reductions in the number of cells bearing neurites on the second day. A significant decrease in the number of cells containing aggregate was seen in induced 150Q cells treated with 20 µM but not for those treated with 40 or 50 µM quercetin up to 4 days of induction. Mutated Htt (mHtt)‐induced reduction in proteasomal activity of the ubiquitin‐proteasomal system (UPS) was significantly attenuated by 20 µM quercetin. However, neither mitochondrial membrane potential loss nor colocalization of 20S proteasome with mHtt aggregate was corrected by quercetin treatment. Our results imply that the neuroprotective effect of quercetin arises out of the upregulation of UPS activity, which causes a decrease in the number of mHtt aggregate‐harboring cells. The increased neurotoxicity could result from the continued association of mHtt with 20S proteasome and the failure of quercetin to correct mitochondrial membrane potential loss. These results suggest that, although quercetin at a low dose protects against mHtt‐mediated cell death, higher doses are toxic to the cells, clearly demarcating a narrow therapeutic window for this dietary flavonoid. © 2015 Wiley Periodicals, Inc.     

Effect of ubiquitin expression on neuropathogenesis in a mouse model of familial amyotrophic lateral sclerosis

The ubiquitin-proteasome system (UPS) is a central component in the cellular defence against potentially toxic protein aggregates. UPS dysfunction is linked to the pathogenesis of both sporadic and inherited neurodegenerative diseases, including dominantly inherited familial amyotrophic lateral sclerosis (fALS). To investigate the role of the UPS in fALS pathogenesis, transgenic mice expressing mutant G9 3A Cu,Zn superoxide dismutase (SOD1) were crossed with transgenic mice expressing epitope tagged, wild-type or dominant-negative mutant ubiquitin (Ub(K48R)). RNase protection assays were used to confirm expression of the Ub transgenes in spinal cord and ubiquitin transgene levels were estimated to account for 9-12% of total ubiquitin. Mice expressing the G9 3A transgene exhibited neurological symptoms and histopathological changes typical of this model irrespective of ubiquitin transgene status. Impaired rotarod performance was observed in all G9 3A transgenics by 7 weeks of age irrespective of ubiquitin genotype. The presence of wild-type or mutant ubiquitin transgenes resulted in a small but significant delay in the onset of clinical symptoms and mild acceleration of disease progression, without influencing overall survival. These data suggest that relatively small changes in ubiquitin expression can influence the development of neurodegenerative disease and are consistent with a neuroprotective role for the UPS.     

Limbic structures are prone to age-related impairments in proteasome activity and neuronal ubiquitinated inclusions in SAMP10 mouse: a model of cerebral degeneration

AIMS: Neurodegenerative diseases are characterized by ubiquitinated inclusions in selective brain regions. Here we investigated whether the dysfunction of the ubiquitin proteasome system might be involved in the pathogenesis and regional selectivity of neuronal ubiquitinated inclusions using the SAMP10 strain of mouse, an inbred model of age-related cerebral degeneration. METHODS: By comparing SAMP10 mice at various ages with SAMR1 and C57BL mice as normal brain ageing controls, we studied morphological features and distribution of inclusions. We measured tissue proteasome activity in different brain regions of mice at various ages by fluorogenic substrate assays. We induced inclusions in cultured neurones by inhibiting the proteasome and analysed changes in the dendritic morphology. RESULTS: Inclusions were formed in association with lipofuscin in neuronal perikarya and occurred most frequently in the limbic-related forebrain structures. There were sparse inclusion-bearing neurones in the non-limbic forebrain. In aged SAMR1 and C57BL, there were far fewer inclusions in the limbic-related forebrain than in aged SAMP10. The proteasome activity in the limbic-related forebrain decreased much more rapidly and remarkably upon ageing (26% activity was detected in 17-month-old compared with 3-month-old mice) in SAMP10 than in SAMR1. The proteasome activity in the non-limbic forebrain did not change significantly with advancing age in either SAMP10 or SAMR1. Proteasomal inhibition enhanced the formation of ubiquitinated inclusions in cultured neurones. Neurones bearing inclusions had shortened neurites. CONCLUSIONS: We propose that the regional selectivity of proteasomal impairment is causally related to the selectivity of inclusion formation and associated dendritic degeneration in neurones of ageing SAMP10 mice.     

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.