ResearchPad - neuronal-death Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[BAHD1 haploinsufficiency results in anxiety-like phenotypes in male mice]]> BAHD1 is a heterochomatinization factor recently described as a component of a multiprotein complex associated with histone deacetylases HDAC1/2. The physiological and patho-physiological functions of BAHD1 are not yet well characterized. Here, we examined the consequences of BAHD1 deficiency in the brains of male mice. While Bahd1 knockout mice had no detectable defects in brain anatomy, RNA sequencing profiling revealed about 2500 deregulated genes in Bahd1-/- brains compared to Bahd1+/+ brains. A majority of these genes were involved in nervous system development and function, behavior, metabolism and immunity. Exploration of the Allen Brain Atlas and Dropviz databases, assessing gene expression in the brain, revealed that expression of the Bahd1 gene was limited to a few territories and cell subtypes, particularly in the hippocampal formation, the isocortex and the olfactory regions. The effect of partial BAHD1 deficiency on behavior was then evaluated on Bahd1 heterozygous male mice, which have no lethal or metabolic phenotypes. Bahd1+/- mice showed anxiety-like behavior and reduced prepulse inhibition (PPI) of the startle response. Altogether, these results suggest that BAHD1 plays a role in chromatin-dependent gene regulation in a subset of brain cells and support recent evidence linking genetic alteration of BAHD1 to psychiatric disorders in a human patient.

<![CDATA[Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity]]>


Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.

Principle Findings

Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.


Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

<![CDATA[Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration]]>

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.

<![CDATA[ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease]]>

Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

<![CDATA[Neutralization of Schwann Cell-Secreted VEGF Is Protective to In Vitro and In Vivo Experimental Diabetic Neuropathy]]>

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy.

<![CDATA[Neuronal Cell Fate Specification by the Convergence of Different Spatiotemporal Cues on a Common Terminal Selector Cascade]]>

Specification of the myriad of unique neuronal subtypes found in the nervous system depends upon spatiotemporal cues and terminal selector gene cascades, often acting in sequential combinatorial codes to determine final cell fate. However, a specific neuronal cell subtype can often be generated in different parts of the nervous system and at different stages, indicating that different spatiotemporal cues can converge on the same terminal selectors to thereby generate a similar cell fate. However, the regulatory mechanisms underlying such convergence are poorly understood. The Nplp1 neuropeptide neurons in the Drosophila ventral nerve cord can be subdivided into the thoracic-ventral Tv1 neurons and the dorsal-medial dAp neurons. The activation of Nplp1 in Tv1 and dAp neurons depends upon the same terminal selector cascade: col>ap/eya>dimm>Nplp1. However, Tv1 and dAp neurons are generated by different neural progenitors (neuroblasts) with different spatiotemporal appearance. Here, we find that the same terminal selector cascade is triggered by Kr/pdm>grn in dAp neurons, but by Antp/hth/exd/lbe/cas in Tv1 neurons. Hence, two different spatiotemporal combinations can funnel into a common downstream terminal selector cascade to determine a highly related cell fate.

<![CDATA[Anti-oxidative effects of 4-hydroxybenzyl alcohol in astrocytes confer protective effects in autocrine and paracrine manners]]>

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata Blume (GEB), a traditional herbal medicine used in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in the brain, and in particular, its anti-inflammatory, anti-oxidative, and anti-zinc-toxic effects have been implicated in the postischemic brain. Here, the authors investigated the anti-oxidative effect of 4-HBA on astrocytes and sought to identify the underlying molecular mechanisms involved. 4-HBA dose-dependently suppressed H2O2-induced astrocyte cell death. More specifically, pre-incubation of C6 cells (an astrocyte cell line) with 100 μM 4-HBA for 6 hrs increased survival when cells were treated with H2O2 (100 μM, 1 hr) from 54.2±0.7% to 85.9±1.5%. In addition, 4-HBA was found to up-regulate and activate Nrf2, and subsequently, to induce the expressions of several anti-oxidative genes, such as, HO-1, NQO1, and GCLM. Notably, HO-1 was induced by 3.4-fold in 4-HBA-treated C6 cells, and siRNA-mediated HO-1 knockdown demonstrated that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of 4-HBA. ERK and Akt signaling pathways were activated by 4-HBA in C6 cells, suggesting their involvements in protective effect of 4-HBA. In addition, 4-HBA-conditioned astrocyte culture medium was found to have neuroprotective effects on primary neuronal cultures or fresh C6 cells exposed to oxidative stress, and these effects seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), which both accumulated in 4-HBA-treated astrocyte culture media. Thus, the 4-HBA-mediated activation of Nrf2 and induction of HO-1 in astrocytes were found to act via autocrine and paracrine mechanisms to confer protective effects. Furthermore, given the pleiotropic effects of 4-HBA with respect to its targeting of various brain cell types and functions, it would appear that 4-HBA has therapeutic potential for the prevention and amelioration of various brain diseases.

<![CDATA[Autophagy Constitutes a Protective Mechanism against Ethanol Toxicity in Mouse Astrocytes and Neurons]]>

Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.

<![CDATA[Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging]]>

TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro.

<![CDATA[Oroxylin A promotes retinal ganglion cell survival in a rat optic nerve crush model]]>


To investigate the effect of oroxylin A on the survival of retinal ganglion cells (RGC) and the activation of microglial cells in a rat optic nerve (ON) crush model.


Oroxylin A (15mg/Kg in 0.2ml phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush once by subcutaneous injection. Rats were euthanized at 2 weeks after the crush injury. The density of RGC was counted by retrograde labeling with FluoroGold and immunostaining of retina flat mounts for Brn3a. Electrophysiological visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, immunoblotting analysis of glial fibrillary acidic protein (GFAP), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the retinas, and immunohistochemistry of GFAP in the retinas and ED1 in the ON were evaluated.


Two weeks after the insult, the oroxylin A-treated group had significantly higher FG labeled cells and Brn3a+ cells suggesting preserved RGC density in the central and mid-peripheral retinas compared with those of the PBS-treated group. FVEP measurements showed a significantly better preserved latency of the P1 wave in the ON-crushed, oroxylin A-treated rats than the ON-crushed, PBS treated rats. TUNEL assays showed fewer TUNEL positive cells in the ON-crushed, oroxylin A-treated rats. The number of ED1 positive cells was reduced at the lesion site of the optic nerve in the ON-crushed, oroxylin A-treated group. Increased GFAP expression in the retina was reduced greatly in ON-crushed, oroxylin A-treated group. Furthermore, administration of oroxylin A significantly attenuated ON crush insult-induced iNOS and COX-2 expression in the retinas.


These results demonstrated that oroxylin A hasss neuroprotective effects on RGC survival with preserved visual function and a decrease in microglial infiltration in the ONs after ON crush injury.

<![CDATA[The Machado–Joseph Disease Deubiquitinase Ataxin-3 Regulates the Stability and Apoptotic Function of p53]]>

As a deubiquitinating enzyme (DUB), the physiological substrates of ataxin-3 (ATX-3) remain elusive, which limits our understanding of its normal cellular function and that of pathogenic mechanism of spinocerebellar ataxia type 3 (SCA3). Here, we identify p53 to be a novel substrate of ATX-3. ATX-3 binds to native and polyubiquitinated p53 and deubiquitinates and stabilizes p53 by repressing its degradation through the ubiquitin (Ub)-proteasome pathway. ATX-3 deletion destabilizes p53, resulting in deficiency of p53 activity and functions, whereas ectopic expression of ATX-3 induces selective transcription/expression of p53 target genes and promotes p53-dependent apoptosis in both mammalian cells and the central nervous system of zebrafish. Furthermore, the polyglutamine (polyQ)-expanded ATX-3 retains enhanced interaction and deubiquitination catalytic activity to p53 and causes more severe p53-dependent neurodegeneration in zebrafish brains and in the substantia nigra pars compacta (SNpc) or striatum of a transgenic SCA3 mouse model. Our findings identify a novel molecular link between ATX-3 and p53-mediated cell death and provide an explanation for the direct involvement of p53 in SCA3 disease pathogenesis.

<![CDATA[Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures]]>

Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.

<![CDATA[24S-hydroxycholesterol and 25-hydroxycholesterol differentially impact hippocampal neuronal survival following oxygen-glucose deprivation]]>

N-methyl-D-aspartate receptors (NMDARs), a major subtype of glutamate receptor mediating excitatory transmission throughout the CNS, participate in ischemia-induced neuronal death. Unfortunately, undesired side effects have limited the strategy of inhibiting/blocking NMDARs as therapy. Targeting endogenous positive allosteric modulators of NMDAR function may offer a strategy with fewer downsides. Here, we explored whether 24S-hydroxycholesterol (24S-HC), an endogenous positive NMDAR modulator characterized recently by our group, participates in NMDAR-mediated excitotoxicity following oxygen-glucose deprivation (OGD) in primary neuron cultures. 24S-HC is the major brain cholesterol metabolite produced exclusively in neurons near sites of glutamate transmission. By selectively potentiating NMDAR current, 24S-HC may participate in NMDAR-mediated excitotoxicity following energy failure, thus impacting recovery after stroke. In support of this hypothesis, our findings indicate that exogenous application of 24S-HC exacerbates NMDAR-dependent excitotoxicity in primary neuron culture following OGD, an ischemic-like challenge. Similarly, enhancement of endogenous 24S-HC synthesis reduced survival rate. On the other hand, reducing endogenous 24S-HC synthesis alleviated OGD-induced cell death. We found that 25-HC, another oxysterol that antagonizes 24S-HC potentiation, partially rescued OGD-mediated cell death in the presence or absence of exogenous 24S-HC application, and 25-HC exhibited NMDAR-dependent/24S-HC-dependent neuroprotection, as well as NMDAR-independent neuroprotection in rat tissue but not mouse tissue. Our findings suggest that both endogenous and exogenous 24S-HC exacerbate OGD-induced damage via NMDAR activation, while 25-HC exhibits species dependent neuroprotection through both NMDAR-dependent and independent mechanisms.

<![CDATA[Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats]]>

The underlying mechanisms for increased neurodegeneration and neurocognitive deficits in HIV-infected people are unclear. Therefore, this study was aimed to investigate the mechanisms of increased neurodegeneration in 5-month old male HIV-1 Transgenic (Tg) rats compared to the age- and gender-matched wild-type (WT) by evaluating histological changes and biochemical parameters of the key proteins involved in the cell death signaling and apoptosis. Histological and immunohistochemical analyses revealed decreased neuronal cells with elevated astrogliosis in HIV-1 Tg rats compared to WT. Mechanistic studies revealed that increased levels of nitroxidative stress marker proteins such as NADPH-oxidase, cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), the stress-activated mitogen-activated protein kinases such as JNK and p38K, activated cell-cycle dependent CDK5, hypoxia-inducible protein-1α, nitrated proteins, hyperphosphorylated tau, and amyloid plaques in HIV-Tg rats were consistently observed in HIV-1 Tg rats. Confocal microscopy and cell viability analyses showed that treatment with an antioxidant N-acetylcysteine or a specific inhibitor of iNOS 1400W significantly prevented the increased apoptosis of neuro-2A cells by HIV-1 Tat or gp120 protein, demonstrating the causal role of HIV-1 mediated nitroxidative stress and protein nitration in promoting neuronal cell death. Immunoprecipitation and immunoblot analysis confirmed nitration of Hsp90, evaluated as an example of nitrated proteins, suggesting possible involvement of nitrated proteins in neuronal damage. Further, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyperphosphorylation and tauopathy. These changes were accompanied with elevated levels of many apoptosis-related proteins Bax and cleaved (activated) caspase-3 as well as proinflammatory cytokines including TNF-α, IL-6 and MCP-1. Collectively, these results indicate that raised nitroxidative stress accompanied by elevated inflammation, cell death signaling pathway including activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyperphosphorylation are responsible for increased apoptosis of neuronal cells and neurodegeneration in 5-month old HIV-Tg rats.

<![CDATA[Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL/6 Mice]]>

The neurotoxicity of paraquat dichloride (PQ) was assessed in two inbred strains of 9- or 16-week old male C57BL/6 mice housed in two different laboratories and compared to the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). PQ was administered by intraperitoneal injections; either once (20 mg/kg) or twice (10 mg/kg) weekly for 3 weeks, while MPTP-HCl was injected 4 times on a single day (20 mg/kg/dose). Brains were collected 8, 16, 24, 48, 96 or 168 hours after the last PQ treatment, and 48 or 168 hours after MPTP treatment. Dopamine neurons in the substantia nigra pars compacta (SNpc) were identified by antibodies to tyrosine hydroxylase (TH+) and microglia were identified using Iba-1 immunoreactivity. The total number of TH+ neurons and the number of resting and activated microglia in the SNpc at 168 hours after the last dose were estimated using model- or design-based stereology, with investigators blinded to treatment. In a further analysis, a pathologist, also blinded to treatment, evaluated the SNpc and/or striatum for loss of TH+ neurons (SNpc) or terminals (striatum), cell death (as indicated by amino cupric silver uptake, TUNEL and/or caspase 3 staining) and neuroinflammation (as indicated by Iba-1 and/or GFAP staining). PQ, administered either once or twice weekly to 9- or 16-week old mice from two suppliers, had no effect on the number of TH+ neurons or microglia in the SNpc, as assessed by two groups, each blinded to treatment, using different stereological methods. PQ did not induce neuronal cell loss or degeneration in the SNpc or striatum. Additionally, there was no evidence of apoptosis, microgliosis or astrogliosis. In MPTP-treated mice, the number of TH+ neurons in the SNpc was significantly decreased and the number of activated microglia increased. Histopathological assessment found degenerating neurons/terminals in the SNpc and striatum but no evidence of apoptotic cell death. MPTP activated microglia in the SNpc and increased the number of astrocytes in the SNpc and striatum.

<![CDATA[Smart Soup, a Traditional Chinese Medicine Formula, Ameliorates Amyloid Pathology and Related Cognitive Deficits]]>

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that causes substantial public health care burdens. Intensive efforts have been made to find effective and safe disease-modifying treatment and symptomatic intervention alternatives against AD. Smart Soup (SS), a Chinese medicine formula composed of Rhizoma Acori Tatarinowii (AT), Poria cum Radix Pini (PRP) and Radix Polygalae (RP), is a typical prescription against memory deficits. Here, we assessed the efficacy of SS against AD. Oral administration of SS ameliorated the cognitive impairment of AD transgenic mice, with reduced Aβ levels, retarded Aβ amyloidosis and reduced Aβ-induced gliosis and neuronal loss in the brains of AD mice. Consistently, SS treatment reduced amyloid-related locomotor dysfunctions and premature death of AD transgenic Drosophila. Mechanistic studies showed that RP reduced Aβ generation, whereas AT and PRP exerted neuroprotective effects against Aβ. Taken together, our study indicates that SS could be effective against AD, providing a practical therapeutic strategy against the disease.

<![CDATA[The Proapoptotic Protein BNIP3 Interacts with VDAC to Induce Mitochondrial Release of Endonuclease G]]>

BNIP3 is a proapoptotic protein that induces cell death through a mitochondria-mediated pathway. We reported previously that mitochondrial localization of BNIP3 and translocation of EndoG from mitochondria to the nucleus are critical steps of the BNIP3 pathway. It is not clear, however, that how BNIP3 interacts with mitochondria. Here we show that expression of BNIP3 resulted in mitochondrial release and nuclear translocation of EndoG. Incubation of a recombinant GST-BNIP3 protein with freshly isolated mitochondria led to the integration of BNIP3 into mitochondria, reduction in the levels of EndoG in mitochondria and the presence of EndoG in the supernatant that was able to cleave chromatin DNA. Co-immunoprecipitation and mass spectrometry analysis reveals that BNIP3 interacted with the voltage-dependent anion channel (VDAC) to increase opening probabilities of mitochondrial permeability transition (PT) pores and induce mitochondrial release of EndoG. Blocking VDAC with a VDAC antibody largely abolished mitochondrial localization of BNIP3 and prevented EndoG release. Together, the data identify VDAC as an interacting partner of BNIP3 and support endonuclease G as a mediator of the BNIP3 pathway.

<![CDATA[CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature]]>

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature.

<![CDATA[Wnt Signaling Prevents the Aβ Oligomer-Induced Mitochondrial Permeability Transition Pore Opening Preserving Mitochondrial Structure in Hippocampal Neurons]]>

Alzheimer’s disease (AD) is a neurodegenerative disorder mainly known for synaptic impairment and neuronal cell loss, affecting memory processes. Beside these damages, mitochondria have been implicated in the pathogenesis of AD through the induction of the mitochondrial permeability transition pore (mPTP). The mPTP is a non-selective pore that is formed under apoptotic conditions, disturbing mitochondrial structure and thus, neuronal viability. In AD, Aβ oligomers (Aβos) favor the opening of the pore, activating mitochondria-dependent neuronal cell death cascades. The Wnt signaling activated through the ligand Wnt3a has been described as a neuroprotective signaling pathway against amyloid-β (Aβ) peptide toxicity in AD. However, the mechanisms by which Wnt signaling prevents Aβos-induced neuronal cell death are unclear. We proposed here to study whether Wnt signaling protects neurons earlier than the late damages in the progression of the disease, through the preservation of the mitochondrial structure by the mPTP inhibition. To study specific events related to mitochondrial permeabilization we performed live-cell imaging from primary rat hippocampal neurons, and electron microscopy to analyze the mitochondrial morphology and structure. We report here that Wnt3a prevents an Aβos-induced cascade of mitochondrial events that leads to neuronal cell death. This cascade involves (a) mPTP opening, (b) mitochondrial swelling, (c) mitochondrial membrane potential loss and (d) cytochrome c release, thus leading to neuronal cell death. Furthermore, our results suggest that the activation of the Wnt signaling prevents mPTP opening by two possible mechanisms, which involve the inhibition of mitochondrial GSK-3β and/or the modulation of mitochondrial hexokinase II levels and activity. This study suggests a possible new approach for the treatment of AD from a mitochondrial perspective, and will also open new lines of study in the field of Wnt signaling in neuroprotection.

<![CDATA[BBB-Permeable, Neuroprotective, and Neurotrophic Polysaccharide, Midi-GAGR]]>

An enormous amount of efforts have been poured to find an effective therapeutic agent for the treatment of neurodegenerative diseases including Alzheimer’s disease (AD). Among those, neurotrophic peptides that regenerate neuronal structures and increase neuron survival show a promise in slowing neurodegeneration. However, the short plasma half-life and poor blood-brain-barrier (BBB)-permeability of neurotrophic peptides limit their in vivo efficacy. Thus, an alternative neurotrophic agent that has longer plasma half-life and better BBB-permeability has been sought for. Based on the recent findings of neuroprotective polysaccharides, we searched for a BBB-permeable neuroprotective polysaccharide among natural polysaccharides that are approved for human use. Then, we discovered midi-GAGR, a BBB-permeable, long plasma half-life, strong neuroprotective and neurotrophic polysaccharide. Midi-GAGR is a 4.7kD cleavage product of low acyl gellan gum that is approved by FDA for human use. Midi-GAGR protected rodent cortical neurons not only from the pathological concentrations of co-/post-treated free reactive radicals and Aβ42 peptide but also from activated microglial cells. Moreover, midi-GAGR showed a good neurotrophic effect; it enhanced neurite outgrowth and increased phosphorylated cAMP-responsive element binding protein (pCREB) in the nuclei of primary cortical neurons. Furthermore, intra-nasally administered midi-GAGR penetrated the BBB and exerted its neurotrophic effect inside the brain for 24 h after one-time administration. Midi-GAGR appears to activate fibroblast growth factor receptor 1 (FGFR1) and its downstream neurotrophic signaling pathway for neuroprotection and CREB activation. Additionally, 14-day intranasal administration of midi-GAGR not only increased neuronal activity markers but also decreased hyperphosphorylated tau, a precursor of neurofibrillary tangle, in the brains of the AD mouse model, 3xTg-AD. Taken together, midi-GAGR with good BBB-permeability, long plasma half-life, and strong neuroprotective and neurotrophic effects has a great therapeutic potential for the treatment of neurodegenerative diseases, especially AD.