Loss of Cholinergic and Monoaminergic Afferents in APPswe/PS1ΔE9 Transgenic Mouse Model of Cerebral Amyloidosis Preferentially Occurs Near Amyloid Plaques.

Alzheimer's disease (AD) is characterized by a loss of neurons in the cortex and subcortical regions. Previously, we showed that the progressive degeneration of subcortical monoaminergic (MAergic) neurons seen in human AD is recapitulated in the APPswe/PS1ΔE9 (APP/PS) transgenic mouse model. Because degeneration of cholinergic (Ach) neurons is also a prominent feature of AD, we examined the integrity of the Ach system in the APP/PS model. The overall density of Ach fibers is reduced in APP/PS1 mice at 12 and 18 months of age but not at 4 months of age. Analysis of basal forebrain Ach neurons shows no loss of Ach neurons in the APP/PS model. Thus, since MAergic systems show overt cell loss at 18 months of age, the Ach system is less vulnerable to neurodegeneration in the APP/PS1 model. We also examined whether the proximity to Aß deposition affected the degeneration of Ach and 5-HT afferents. We found that the areas closer to the edges of compact Aß deposits exhibit a more severe loss of afferents than the areas that are more distal to Aß deposits. Collectively, the results indicate that the APP/PS model recapitulates the degeneration of multiple subcortical neurotransmitter systems, including the Ach system. In addition, the results indicate that Aß deposits cause global as well as local toxicity to subcortical afferents.

Dysregulation of astrocytic Ca2+ signaling and gliotransmitter release in mouse models of α-synucleinopathies.

α-Synuclein is a major component of Lewy bodies (LB) and Lewy neurites (LN) appearing in the postmortem brain of Parkinson's disease (PD) and other α-synucleinopathies. While most studies of α-synucleinopathies have focused on neuronal and synaptic alterations as well as dysfunctions of the astrocytic homeostatic roles, whether the bidirectional astrocyte-neuronal communication is affected in these diseases remains unknown. We have investigated whether the astrocyte Ca2+ excitability and the glutamatergic gliotransmission underlying astrocyte-neuronal signaling are altered in several transgenic mouse models related to α-synucleinopathies, i.e., mice expressing high and low levels of the human A53T mutant α-synuclein (G2-3 and H5 mice, respectively) globally or selectively in neurons (iSyn mice), mice expressing human wildtype α-synuclein (I2-2 mice), and mice expressing A30P mutant α-synuclein (O2 mice). Combining astrocytic Ca2+ imaging and neuronal electrophysiological recordings in hippocampal slices of these mice, we have found that compared to non-transgenic mice, astrocytes in G2-3 mice at different ages (1-6 months) displayed a Ca2+ hyperexcitability that was independent of neurotransmitter receptor activation, suggesting that the expression of α-synuclein mutant A53T altered the intrinsic properties of astrocytes. Similar dysregulation of the astrocyte Ca2+ signal was present in H5 mice, but not in I2-2 and O2 mice, indicating α-synuclein mutant-specific effects. Moreover, astrocyte Ca2+ hyperexcitability was absent in mice expressing the α-synuclein mutant A53T selectively in neurons, indicating that the effects on astrocytes were cell-autonomous. Consistent with these effects, glutamatergic gliotransmission was enhanced in G2-3 and H5 mice, but was unaffected in I2-2, O2 and iSyn mice. These results indicate a cell-autonomous effect of pathogenic A53T expression in astrocytes that may contribute to the altered neuronal and synaptic function observed in α-synucleinopathies.

The UPR Maintains Proteostasis and the Viability and Function of Hippocampal Neurons in Adult Mice.

The unfolded protein response (UPR), which comprises three branches: PERK, ATF6α, and IRE1, is a major mechanism for maintaining cellular proteostasis. Many studies show that the UPR is a major player in regulating neuron viability and function in various neurodegenerative diseases; however, its role in neurodegeneration is highly controversial. Moreover, while evidence suggests activation of the UPR in neurons under normal conditions, deficiency of individual branches of the UPR has no major effect on brain neurons in animals. It remains unclear whether or how the UPR participates in regulating neuronal proteostasis under normal and disease conditions. To determine the physiological role of the UPR in neurons, we generated mice with double deletion of PERK and ATF6α in neurons. We found that inactivation of PERK and ATF6α in neurons caused lysosomal dysfunction (as evidenced by decreased expression of the V0a1 subunit of v-ATPase and decreased activation of cathepsin D), impairment of autophagic flux (as evidenced by increased ratio of LC3-II/LC3-I and increased p62 level), and accumulation of p-tau and Aß42 in the hippocampus, and led to impairment of spatial memory, impairment of hippocampal LTP, and hippocampal degeneration in adult mice. These results suggest that the UPR is required for maintaining neuronal proteostasis (particularly tau and Aß homeostasis) and the viability and function of neurons in the hippocampus of adult mice.

Astrocyte-neuronal network interplay is disrupted in Alzheimer's disease mice.

Alzheimer's disease (AD) is associated with senile plaques of beta-amyloid (Aß) that affect the function of neurons and astrocytes. Brain activity results from the coordinated function of neurons and astrocytes in astroglial-neuronal networks. However, the effects of Aß on astroglial and neuronal network function remains unknown. Simultaneously monitoring astrocyte calcium and electric neuronal activities, we quantified the impact of Aß on sensory-evoked cortical activity in a mouse model of AD. At rest, cortical astrocytes displayed spontaneous hyperactivity that was related to Aß density. Sensory-evoked astrocyte responsiveness was diminished in AD mice, depending on the density and distance of Aß, and the responses showed altered calcium dynamics. Hence, astrocytes were spontaneously hyperactive but hypo-responsive to sensory stimulation. Finally, AD mice showed sensory-evoked electrical cortical hyperresponsiveness associated with altered astrocyte-neuronal network interplay. Our findings suggest dysfunction of astrocyte networks in AD mice may dysregulate cortical electrical activity and contribute to cognitive decline.

Short-term dual antiplatelet therapy in diabetic patients admitted for acute coronary syndrome treated with a new-generation drug-eluting stent.

BACKGROUND: The optimal duration of dual antiplatelet therapy (DAPT) in patients with diabetes mellitus (DM) admitted with acute coronary syndrome (ACS) and treated with a drug-eluting stent (DES) remains unclear. This is a prespecified sub-study from the Randomised Evaluation of short-term DUal antiplatelet therapy in patients with acute Coronary syndromE treated with a new generation DES (REDUCE) trial that was designed to determine the efficacy and safety of short-term versus standard 12 months DAPT in diabetic patients with ACS undergoing percutaneous coronary intervention (PCI) using the COMBO stent. METHODS: In this study we included ACS diabetic patients enroled in the REDUCE trial treated with the COMBO stent and randomly assigned to either 3 or 12 months of DAPT. The primary study endpoint was the composite of all-cause mortality, myocardial infarction (MI), stent thrombosis (ST), stroke, target vessel revascularisation (TVR), and bleeding complications at 12 and 24 months follow-up. RESULTS: A total of 307 diabetic patients were included, of which 162 (52.8%) in the 3 months DAPT group and 145 (47.2%) in the 12 months DAPT group. Patient characteristics, PCI success, and number of stents used were similar in the 3 and 12 months DAPT groups. Occurrence of the primary study endpoint at 12 and 24 months follow-up was comparable between the two groups (3.1 vs. 3.5%, p = 0.865, and 15.8 vs. 14.9%, p = 0.824, respectively). Moreover, the prevalence of the specific clinical outcome parameters (all-cause mortality), MI, ST, stroke, TVR, and bleeding was similar in both study groups. CONCLUSIONS: This sub-analysis shows similar clinical outcomes following 3 months DAPT as compared to 12 months DAPT in diabetic patients undergoing PCI for ACS using the COMBO stent. These results suggest that, even in this particular subset of patients, short duration of DAPT might be considered safe. Future larger studies are warranted to provide more precise estimations in terms of safety and efficacy of short term DAPT in these high-risk patients.

BIOSOLVE-IV-registry: Safety and performance of the Magmaris scaffold: 12-month outcomes of the first cohort of 1,075 patients.

OBJECTIVES: We aimed to assess the safety and performance of the Magmaris sirolimus-eluting bioresorbable magnesium scaffold in a large patient population. BACKGROUND: Magmaris has shown good outcomes in small-sized controlled trials, but further data are needed to confirm its usability, safety, and performance. METHODS: BIOSOLVE-IV is an international, single arm, multicenter registry including patients with a maximum of two single de novo lesions. Follow-up is scheduled up to 5 years; the primary outcome is target lesion failure (TLF) at 12 months. RESULTS: A total of 1,075 patients with 1,121 lesions were enrolled. Mean patient age was 61.3 ± 10.5 years and 19.2% (n = 206) presented with non-ST-elevation myocardial infarction (NSTEMI). Lesions were 3.2 ± 0.3 mm in diameter and 14.9 ± 4.2 mm long; 5.1% (n = 57) were bifurcation lesions. Device success was 97.3% (n = 1,129) and procedure success 98.9% (n = 1,063). The Kaplan-Meier estimate of TLF at 12 months was 4.3% [95% confidence interval, CI: 3.2, 5.7] consisting of 3.9% target lesion revascularizations, 0.2% cardiac death, and 1.1% target-vessel myocardial infarction. Definite/probable scaffold thrombosis occurred in five patients (0.5% [95% CI: 0.2, 1.1]), thereof four after early discontinuation of antiplatelet/anticoagulation therapy. CONCLUSION: BIOSOLVE-IV confirms the safety and performance of the Magmaris scaffold in a large population with excellent device and procedure success and a very good safety profile up to 12 months in a low-risk population.

Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits.

Mounting evidence indicates that soluble oligomeric forms of amyloid proteins linked to neurodegenerative disorders, such as amyloid-ß (Aß), tau, or α-synuclein (αSyn) might be the major deleterious species for neuronal function in these diseases. Here, we found an abnormal accumulation of oligomeric αSyn species in AD brains by custom ELISA, size-exclusion chromatography, and nondenaturing/denaturing immunoblotting techniques. Importantly, the abundance of αSyn oligomers in human brain tissue correlated with cognitive impairment and reductions in synapsin expression. By overexpressing WT human αSyn in an AD mouse model, we artificially enhanced αSyn oligomerization. These bigenic mice displayed exacerbated Aß-induced cognitive deficits and a selective decrease in synapsins. Following isolation of various soluble αSyn assemblies from transgenic mice, we found that in vitro delivery of exogenous oligomeric αSyn but not monomeric αSyn was causing a lowering in synapsin-I/II protein abundance. For a particular αSyn oligomer, these changes were either dependent or independent on endogenous αSyn expression. Finally, at a molecular level, the expression of synapsin genes SYN1 and SYN2 was down-regulated in vivo and in vitro by αSyn oligomers, which decreased two transcription factors, cAMP response element binding and Nurr1, controlling synapsin gene promoter activity. Overall, our results demonstrate that endogenous αSyn oligomers can impair memory by selectively lowering synapsin expression.

A53T Mutant Alpha-Synuclein Induces Tau-Dependent Postsynaptic Impairment Independently of Neurodegenerative Changes.

Abnormalities in α-synuclein are implicated in the pathogenesis of Parkinson's disease (PD). Because α-synuclein is highly concentrated within presynaptic terminals, presynaptic dysfunction has been proposed as a potential pathogenic mechanism. Here, we report novel, tau-dependent, postsynaptic deficits caused by A53T mutant α-synuclein, which is linked to familial PD. We analyzed synaptic activity in hippocampal slices and cultured hippocampal neurons from transgenic mice of either sex expressing human WT, A53T, and A30P α-synuclein. Increased α-synuclein expression leads to decreased spontaneous synaptic vesicle release regardless of genotype. However, only those neurons expressing A53T α-synuclein exhibit postsynaptic dysfunction, including decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. We also found that long-term potentiation and spatial learning were impaired by A53T α-synuclein expression. Mechanistically, postsynaptic dysfunction requires glycogen synthase kinase 3ß-mediated tau phosphorylation, tau mislocalization to dendritic spines, and calcineurin-dependent AMPA receptor internalization. Previous studies reveal that human A53T α-synuclein has a high aggregation potential, which may explain the mutation's unique capacity to induce postsynaptic deficits. However, patients with sporadic PD with severe tau pathology are also more likely to have early onset cognitive decline. Our results here show a novel, functional role for tau: mediating the effects of α-synuclein on postsynaptic signaling. Therefore, the unraveled tau-mediated signaling cascade may contribute to the pathogenesis of dementia in A53T α-synuclein-linked familial PD cases, as well as some subgroups of PD cases with extensive tau pathology.SIGNIFICANCE STATEMENT Here, we report mutation-specific postsynaptic deficits that are caused by A53T mutant α-synuclein, which is linked to familial Parkinson's disease (PD). The overexpression of WT, A53T, or A30P human α-synuclein leads to decreased spontaneous synaptic vesicle release. However, only those neurons expressing A53T α-synuclein exhibit tau phosphorylation-dependent postsynaptic dysfunction, which is characterized by decreased miniature postsynaptic current amplitude and decreased AMPA to NMDA receptor current ratio. The mutation-specific postsynaptic effects caused by human A53T α-synuclein will help us better understand the neurobiological basis of this specific form of familial PD. The differential effects of exogenous human WT, A53T, A30P, and E46K α-synuclein on glutamatergic synaptic responses will help to explain the clinical heterogeneity of sporadic and familial PD.

Tau is required for progressive synaptic and memory deficits in a transgenic mouse model of α-synucleinopathy.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically and neuropathologically highly related α-synucleinopathies that collectively constitute the second leading cause of neurodegenerative dementias. Genetic and neuropathological studies directly implicate α-synuclein (αS) abnormalities in PDD and DLB pathogenesis. However, it is currently unknown how αS abnormalities contribute to memory loss, particularly since forebrain neuronal loss in PDD and DLB is less severe than in Alzheimer's disease. Previously, we found that familial Parkinson's disease-linked human mutant A53T αS causes aberrant localization of the microtubule-associated protein tau to postsynaptic spines in neurons, leading to postsynaptic deficits. Thus, we directly tested if the synaptic and memory deficits in a mouse model of α-synucleinopathy (TgA53T) are mediated by tau. TgA53T mice exhibit progressive memory deficits associated with postsynaptic deficits in the absence of obvious neuropathological and neurodegenerative changes in the hippocampus. Significantly, removal of endogenous mouse tau expression in TgA53T mice (TgA53T/mTau-/-), achieved by mating TgA53T mice to mouse tau-knockout mice, completely ameliorates cognitive dysfunction and concurrent synaptic deficits without affecting αS expression or accumulation of selected toxic αS oligomers. Among the known tau-dependent effects, memory deficits in TgA53T mice were associated with hippocampal circuit remodeling linked to chronic network hyperexcitability. This remodeling was absent in TgA53T/mTau-/- mice, indicating that postsynaptic deficits, aberrant network hyperactivity, and memory deficits are mechanistically linked. Our results directly implicate tau as a mediator of specific human mutant A53T αS-mediated abnormalities related to deficits in hippocampal neurotransmission and suggest a mechanism for memory impairment that occurs as a consequence of synaptic dysfunction rather than synaptic or neuronal loss. We hypothesize that these initial synaptic deficits contribute to network hyperexcitability which, in turn, exacerbate cognitive dysfunction. Our results indicate that these synaptic changes present potential therapeutic targets for amelioration of memory deficits in α-synucleinopathies.

Conditional Deletion of Prnp Rescues Behavioral and Synaptic Deficits after Disease Onset in Transgenic Alzheimer's Disease.

Biochemical and genetic evidence implicate soluble oligomeric amyloid-ß (Aßo) in triggering Alzheimer's disease (AD) pathophysiology. Moreover, constitutive deletion of the Aßo-binding cellular prion protein (PrPC) prevents development of memory deficits in APPswe/PS1ΔE9 mice, a model of familial AD. Here, we define the role of PrPC to rescue or halt established AD endophenotypes in a therapeutic disease-modifying time window after symptom onset. Deletion of Prnp at either 12 or 16 months of age fully reverses hippocampal synapse loss and completely rescues preexisting behavioral deficits by 17 months. In contrast, but consistent with a neuronal function for Aßo/PrPC signaling, plaque density, microgliosis, and astrocytosis are not altered. Degeneration of catecholaminergic neurons remains unchanged by PrPC reduction after disease onset. These results define the potential of targeting PrPC as a disease-modifying therapy for certain AD-related phenotypes after disease onset.SIGNIFICANCE STATEMENT The study presented here further elucidates our understanding of the soluble oligomeric amyloid-ß-Aßo-binding cellular prion protein (PrPC) signaling pathway in a familial form of Alzheimer's disease (AD) by implicating PrPC as a potential therapeutic target for AD. In particular, genetic deletion of Prnp rescued several familial AD (FAD)-associated phenotypes after disease onset in a mouse model of FAD. This study underscores the therapeutic potential of PrPC deletion given that patients already present symptoms at the time of diagnosis.

Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of αS toxic features.

Bidirectional modulation of Alzheimer phenotype by alpha-synuclein in mice and primary neurons.

α-Synuclein (αSyn) histopathology defines several neurodegenerative disorders, including Parkinson's disease, Lewy body dementia, and Alzheimer's disease (AD). However, the functional link between soluble αSyn and disease etiology remains elusive, especially in AD. We, therefore, genetically targeted αSyn in APP transgenic mice modeling AD and mouse primary neurons. Our results demonstrate bidirectional modulation of behavioral deficits and pathophysiology by αSyn. Overexpression of human wild-type αSyn in APP animals markedly reduced amyloid deposition but, counter-intuitively, exacerbated deficits in spatial memory. It also increased extracellular amyloid-ß oligomers (AßOs), αSyn oligomers, exacerbated tau conformational and phosphorylation variants associated with AD, and enhanced neuronal cell cycle re-entry (CCR), a frequent prelude to neuron death in AD. Conversely, ablation of the SNCA gene encoding for αSyn in APP mice improved memory retention in spite of increased plaque burden. Reminiscent of the effect of MAPT ablation in APP mice, SNCA deletion prevented premature mortality. Moreover, the absence of αSyn decreased extracellular AßOs, ameliorated CCR, and rescued postsynaptic marker deficits. In summary, this complementary, bidirectional genetic approach implicates αSyn as an essential mediator of key phenotypes in AD and offers new functional insight into αSyn pathophysiology.

Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone.

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. Above: Iron (Fe) normally moves from capillaries to glia to neurons. It is exported from the glia by ferroportin (Fpn) with ferroxidases ceruloplasmin (Cp) and/or Hephaestin (Heph). Below: In mice with mutation of Cp and Heph, iron accumulates in glia, while neurons have low iron levels. Both neurons and glia degenerate and mice become ataxic unless given an iron chelator.

Neurodegenerative phenotypes in an A53T α-synuclein transgenic mouse model are independent of LRRK2.

Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrain.

Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

Internalized α-synuclein fibrils become truncated and resist degradation in neurons while glial cells rapidly degrade α-synuclein fibrils.

Parkinson's disease (PD) and other α-synucleinopathies are characterized by the accumulation of α-synuclein (αS) pathology that can spread via the cell-to-cell transmission of αS aggregates. To better understand how various brain cells contribute to the spreading of αS pathology, we examined the metabolism of αS aggreges or pre-formed fibrils (PFFs) in neuronal and glial cells (microglia, astrocytes, and oligodendrocytes). In neurons, while the full-length αS rapidly disappeared following αS PFF uptake, truncated αS accumulated with a half-life of days rather than hours. Epitope mapping and fractionation studies indicate that αS PFF was truncated at the C-terminal region following uptake and remained insoluble/aggregated. In contrast, microglia and astrocytes rapidly metabolized αS PFF as the half-lives of αS PFF in these glial cells were <6 hours. Differential processing of αS by neurons was recapitulated in cell lines as differentiated CLU neuronal cell lines stably accumulate truncated αS while undifferentiated cells rapidly metabolize αS. Immunolocalization and subcellular fractionation studies show that internalized αS PFF is initially localized to endosomes followed by lysosomes. The lysosome is largely responsible for the degradation of internalized αS PFF as the inhibition of lysosomal function leads to the stabilization of αS in all cell types. Significantly, αS PFF causes lysosomal dysfunction in neurons. In summary, we show that neurons are inefficient in metabolizing internalized αS aggregates, partially because αS aggregates cause lysosomal dysfunction, potentially generating aggregation-prone truncated αS. In contrast, glial cells may protect neurons from αS aggregates by rapidly clearing αS aggregates.

Deletion of Glyoxalase 1 Exacerbates Acetaminophen-Induced Hepatotoxicity in Mice.

Acetaminophen (APAP) overdose triggers a cascade of intracellular oxidative stress events, culminating in acute liver injury. The clinically used antidote, N-acetylcysteine (NAC), has a narrow therapeutic window, and early treatment is essential for a satisfactory therapeutic outcome. For more versatile therapies that can be effective even at late presentation, the intricacies of APAP-induced hepatotoxicity must be better understood. Accumulation of advanced glycation end products (AGEs) and the consequent activation of the receptor for AGEs (RAGE) are considered one of the key mechanistic features of APAP toxicity. Glyoxalase 1 (Glo-1) regulates AGE formation by limiting the levels of methylglyoxal (MEG). In this study, we studied the relevance of Glo-1 in the APAP-mediated activation of RAGE and downstream cell death cascades. Constitutive Glo-1-knockout mice (GKO) and a cofactor of Glo-1, ψ-GSH, were used as tools. Our findings showed elevated oxidative stress resulting from the activation of RAGE and hepatocyte necrosis through steatosis in GKO mice treated with high-dose APAP compared to wild-type controls. A unique feature of the hepatic necrosis in GKO mice was the appearance of microvesicular steatosis as a result of centrilobular necrosis, rather than the inflammation seen in the wild type. The GSH surrogate and general antioxidant ψ-GSH alleviated APAP toxicity irrespective of the Glo-1 status, suggesting that oxidative stress is the primary driver of APAP toxicity. Overall, the exacerbation of APAP hepatotoxicity in GKO mice suggests the importance of this enzyme system in antioxidant defense against the initial stages of APAP overdose.

Accumulation of toxic α-synuclein oligomer within endoplasmic reticulum occurs in α-synucleinopathy in vivo.

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed using in vitro systems are not generally seen to be associated with α-synucleinopathy in vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.

Endoplasmic reticulum stress is important for the manifestations of α-synucleinopathy in vivo.

Accumulation of misfolded α-synuclein (αS) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other α-synucleinopathies. However, how αS causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in α-synucleinopathy. Using the A53T mutant human αS transgenic (A53TαS Tg) mouse model of α-synucleinopathy, we show that disease onset in the αS Tg model is coincident with induction of ER chaperones in neurons exhibiting αS pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2α, indicating that α-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated αS monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M αS than the control cases. Moreover, αS interacts with ER chaperones and overexpression of αS sensitizes neuronal cells to ERS-induced toxicity, suggesting that αS may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both the A53TαS Tg mouse model and the adeno-associated virus-transduced rat model of A53TαS-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation αS within ER leads to chronic ER stress conditions that contribute to neurodegeneration in α-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other α-synucleinopathies.