Neurotoxicity In Primary Neurons Research Articles

ABBV-0805, a novel antibody selective for soluble aggregated α-synuclein, prolongs lifespan and prevents buildup of α-synuclein pathology in mouse models of Parkinson's disease

A growing body of evidence suggests that aggregated α-synuclein, the major constituent of Lewy bodies, plays a key role in the pathogenesis of Parkinson's disease and related α-synucleinopathies. Immunotherapies, both active and passive, against α-synuclein have been developed and are promising novel treatment strategies for such disorders. Here, we report on the humanization and pharmacological characteristics of ABBV-0805, a monoclonal antibody that exhibits a high selectivity for human aggregated α-synuclein and very low affinity for monomers. ABBV-0805 binds to a broad spectrum of soluble aggregated α-synuclein, including small and large aggregates of different conformations.Binding of ABBV-0805 to pathological α-synuclein was demonstrated in Lewy body-positive post mortem brains of Parkinson's disease patients. The functional potency of ABBV-0805 was demonstrated in several cellular assays, including Fcγ-receptor mediated uptake of soluble aggregated α-synuclein in microglia and inhibition of neurotoxicity in primary neurons. In vivo, the murine version of ABBV-0805 (mAb47) displayed significant dose-dependent decrease of α-synuclein aggregates in brain in several mouse models, both in prophylactic and therapeutic settings. In addition, mAb47 treatment of α-synuclein transgenic mice resulted in a significantly prolonged survival.ABBV-0805 selectively targets soluble toxic α-synuclein aggregates with a picomolar affinity and demonstrates excellent in vivo efficacy. Based on the strong preclinical findings described herein, ABBV-0805 has been progressed into clinical development as a potential disease-modifying treatment for Parkinson's disease.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration

BackgroundMutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson’s disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo.MethodsIn vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35.ResultsOur screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo.ConclusionsHere we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

Melatonin protects against Aβ-induced neurotoxicity in primary neurons via miR-132/PTEN/AKT/FOXO3a pathway.

Alzheimer's disease (AD) is a kind of neurodegenerative disorder associated with age. Investigations suggest that amyliod-β (Aβ) is implicated in the pathogenesis of AD. The accumulation of Aβ in the brain causes oxidative stress and synaptic toxicity, leads to synaptic dysfunction and neuronal death. Previous investigations suggest that melatonin an endogenous hormone can counteract Aβ-induced neurotoxicity. However, the molecular mechanisms of Aβ-induced toxicity and melatonin treatment remain elusive. Studies indicate that microRNA-132 is crucial for neuronal survival and plays a key role in the pathological process of AD. Moreover, PTEN and FOXO3a two key targets of miR-132 are upregulated in the AD brain. Here, we exposed the primary cultured cortical neurons with Aβ25-35 and treated with melatonin. Our investigations demonstrated that Aβ25-35 exposure significantly decreased the expression of miR-132 and elevated the expression of PTEN and FOXO3a. Whereas, melatonin treatment could rescue the expression of miR-132 and downregulate the level of PTEN and FOXO3a. Moreover, melatonin blocked the nuclear translocation of FOXO3a and thereby suppressed its pro-apoptotic pathways. In addition, our investigations suggested that the over-expression of miR-132 could block Aβ-induced neurotoxicity. We also found that VO-OHpic (PTEN inhibitor) could counteract Aβ-induced neuronal damage, and LY294002 (AKT inhibitor) suppressed the protective effect of melatonin. Together, these results indicate that melatonin exerts its neuroprotective effect in Aβ-induced neurotoxicity via miR-132/PTEN/AKT/FOXO3a pathway. © 2018 BioFactors, 44(6):609-618, 2018.

Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctiumlappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4h OGD with/without 4h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups ofneurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups ofneurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

Montelukast rescues primary neurons against Aβ1–42-induced toxicity through inhibiting CysLT1R-mediated NF-κB signaling

Amyloid-β peptide (Aβ), which can invoke a cascade of inflammatory responses, is considered to play a causal role in the development and progress of Alzheimer’s disease (AD). Montelukast, known as a cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, is currently used for treatment of inflammatory diseases such as asthma. We have previously reported that CysLT1R activation is involved in Aβ generation. In this study, we investigated rescuing effect of CysLT1R antagonist montelukast on Aβ1–42-induced neurotoxicity in primary neurons. Our data showed that Aβ1–42 elicited a marked increase of CysLT1R expression in primary mouse neurons. This increment of CysLT1R expression was accompanied by increases of inflammatory factors such as NF-κB p65, tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) as well as pro-apoptotic protein Caspase-3 activation and anti-apoptosis protein Bcl-2 reduction. Aβ1–42-mediated increase of CysLT1R expression was associated with Aβ1–42-induced cytotoxicity as measured by MTT reduction assay and lactate dehydrogenase (LDH) release assay. This observation was confirmed with treatment of montelukast, a selective CysLT1R antagonist, which had significant effect on Aβ1–42-induced cytotoxicity. Moreover, blockade of CysLT1R with montelukast reversed Aβ1–42-mediated increase of CysLT1R expression, and concomitant changes of the pro-inflammatory factors and the apoptosis-related proteins. The results demonstrate that montelukast rescued neurons against Aβ1–42-induced neurotoxicity, neuroinflammation and apoptosis by down-regulating CysLT1R-mediated NF-κB signaling, suggesting that CysLT1R may be a potential target for AD, and its antagonist may have beneficial effects for treatment of AD.

P4-311: PAN-811 protects primary neurons from Aβ neurotoxicity: A potential drug for Alzheimer’s disease

PAN–811 is a small lipophilic compound with a molecular mass of 195 Dalton, belonging to the (N)–heterocyclic carboxaldehyde thiosemicarbazone class of molecules originally developed for cancer therapy. Our previous result has demonstrated highly effective function in protecting ischemic neurotoxicity through a dual mechanism of divalent metal chelation and free radical scavenging. It has proved to be non–toxic in human clinical trials and crosses brain / blood barrier. We have evidence that oxidative stress is the most likely mediator of Aβ neurotoxicity and our objective is to demonstrate that PAN–811 could be an effective neuroprotector. To prove this hypothesis, first we established that Aβ has neurotoxicity in primary neurons in culture. Using Aβ1–42 or Aβ25–35, the rate and intensity of neurotoxicity was increased by age in culture and oxidative stress. For 2–week old neurons, the neurotoxicity was observed in 12 to 16 days after the insult and required 0% antioxidant level (high oxidative stress) whereas neurotoxicity occurred after 1 day for 3–week old neurons under 90% antioxidant level(mild oxidative stress). There was no neurotoxicity in young neurons low oxidative stress when treated with less than 80μm Aβ. The neuronal cell death was by apoptotic process. Neurotoxicty of Aβ1–42 and Aβ25–35 is specific since a reversed sequence peptide Aβ35–25 was unable to induce any toxicity. We have clearly demonstrated that PAN–811 inhibits Aβ–induced neurotoxity. PAN–811 at doses as low as 2μM and as late as 3–hours post insult (Aβ1–42 or Aβ25–35 treatment) effectively protects neurons. We conclude that PAN–811 protects primary neurons from Aβ neurotoxicity and should be pursued as a viable candidate for therapeutic drug development for Alzheimer's disease.

PAN-811 (3-Aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by up-regulating Bcl-2 expression

Neurotoxicity in primary neurons was induced using hypoxia/hypoglycemia (H/H), veratridine (10μM), staurosporine (1μM) or glutamate (100μM), which resulted in 72%, 67%, 75% and 66% neuronal injury, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811; 10μM; Panacea Pharmaceuticals, Gaithersburg, MD) pretreatment for 24 h provided maximal neuroprotection of 89%, 42%, 47% and 89% against these toxicities, respectively. Glutamate or H/H treatment of cells increased cytosolic cytochrome c levels, which was blocked by pretreatment of cells with PAN-811. Pretreatment of neurons with PAN-811 produced a time-dependent increase in the protein level of Bcl-2, which was evident even after glutamate or H/H treatments. An up-regulation in the expression of the p53 and Bax genes was also observed following exposure to these neurotoxic insults; however, this increase was not suppressed by PAN-811 pretreatment. Functional inhibition of Bcl-2 by HA14-1 reduced the neuroprotective efficacy of PAN-811. PAN-811 treatment also abolished glutamate or H/H-mediated internucleosomal DNA fragmentation.

Neuronal Cell Apoptosis by a Receptor-Binding Domain Peptide of ApoE4, Not through Low-Density Lipoprotein Receptor-Related Protein

Since an apolipoprotein E4 (ApoE4) peptide composed of the low-density lipoprotein (LDL) receptor-related protein (LRP)-binding domain [ApoE4(141–149)2 or ApoE(141–155)2] exerts neurotoxicity in primary neurons and neuronal cell lines, it has been controversial whether these effects are mediated by LRP. Here, we examined whether ApoE4(141–149)2-induced toxicity is mediated by LRP in a neuronal cell system where ApoE4 toxicity is mediated by LRP: serum-deprived F11 neuronal cells. In these cells, where ApoE4 exerted toxicity by apoptosis in a manner sensitive to both caspase inhibitors and pertussis toxin (PTX), ApoE4(141–149)2 also caused cell death by apoptosis but in a caspase-inhibitor-resistant, PTX-resistant manner. ApoE4(141–149)2-induced death was not inhibited by antisense oligonucleotides to LRP. Therefore, we conclude that ApoE4(141–149)2 is able to exert neurotoxicity without involving LRP.