Microtubule affinity-regulating kinase 2 (MARK2) turns on phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) at Thr-313, a mutation site in Parkinson disease: effects on mitochondrial transport

The kinase MARK2/Par-1 plays key roles in several cell processes, including neurodegeneration such as Alzheimer disease by phosphorylating tau and detaching it from microtubules. In search of interaction partners of MARK2, we identified phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which is important for the survival of neurons and whose mutations are linked to familial Parkinson disease (PD). MARK2 phosphorylated and activated the cleaved form of PINK1 (ΔN-PINK1; amino acids 156-581). Thr-313 was the primary phosphorylation site, a residue mutated to a non-phosphorylatable form (T313M) in a frequent variant of PD. Mutation of Thr-313 to Met or Glu in PINK1 showed toxic effects with abnormal mitochondrial distribution in neurons. MARK2 and PINK1 were found to colocalize with mitochondria and regulate their transport. ΔN-PINK1 promoted anterograde transport and increased the fraction of stationary mitochondria, whereas full-length PINK1 promoted retrograde transport. In both cases, MARK2 enhanced the effects. The results identify MARK2 as an upstream regulator of PINK1 and ΔN-PINK1 and provide insights into the regulation of mitochondrial trafficking in neurons and neurodegeneration in PD.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 controls homeostasis of the colon to prevent spontaneous development of murine inflammatory bowel disease

Fine-tuning of host cell responses to commensal bacteria plays a crucial role in maintaining homeostasis of the gut. Here, we show that tumor necrosis factor receptor-associated factor (Traf)2(-/-) mice spontaneously developed severe colitis and succumbed within 3 weeks after birth. Histological analysis revealed that apoptosis of colonic epithelial cells was enhanced, and B cells diffusely infiltrated into the submucosal layer of the colon of Traf2(-/-) mice. Expression of proinflammatory cytokines, including Tnfa, Il17a, and Ifng, was up-regulated, whereas expression of antimicrobial peptides was down-regulated in the colon of Traf2(-/-) mice. Moreover, a number of IL-17-producing helper T cells were increased in the colonic lamina propria of Traf2(-/-) mice. These cellular alterations resulted in drastic changes in the colonic microbiota of Traf2(-/-) mice compared with Traf2(+/+) mice. Treatment of Traf2(-/-) mice with antibiotics ameliorated colitis along with down-regulation of proinflammatory cytokines and prolonged survival, suggesting that the altered colonic microbiota might contribute to exacerbation of colitis. Finally, deletion of Tnfr1, but not Il17a, dramatically ameliorated colitis in Traf2(-/-) mice by preventing apoptosis of colonic epithelial cells, down-regulation of proinflammatory cytokines, and restoration of wild-type commensal bacteria. Together, TRAF2 plays a crucial role in controlling homeostasis of the colon.     

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.     

Activity-dependent neurotrophic factor peptide (ADNF9) protects neurons against oxidative stress-induced death

Activity-dependent neurotrophic factor (ADNF) and a 14-amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid beta-peptide, N-methyl-D-aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine-amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.     

A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein

Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.     

Sirtuin 2 (SIRT2) Enhances 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Nigrostriatal Damage via Deacetylating Forkhead Box O3a (Foxo3a) and Activating Bim Protein

Sirtuins are NAD-dependent protein deacetylases that were shown to have beneficial effects against age-related diseases. SIRT2 is a strong deacetylase that is highly expressed in brain. It has been associated with neurodegenerative diseases. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a dopaminergic neurotoxin that replicates most of the clinical features of Parkinson disease (PD) and produces a reliable and reproducible lesion of the nigrostriatal dopaminergic pathway and neurodegeneration after its systemic administration. Chronic administration of MPTP induces lesion via apoptosis. We show here that SIRT2 deacetylates Foxo3a, increases RNA and protein levels of Bim, and as a result, enhances apoptosis in the MPTP model of PD. We also show that neurodegeneration induced by chronic MPTP regimen is prevented by genetic deletion of SIRT2 in mouse. Deletion of SIRT2 leads to the reduction of apoptosis due to an increase in acetylation of Foxo3a and a decrease in Bim levels. We demonstrate that SIRT2 deacetylates Foxo3a, activates Bim, and induces apoptosis only in 1-methyl-4-phenylpyridinium-treated cells. Therefore, designing SIRT2 inhibitors might be helpful to develop effective treatments for PD.     

Loss of lysosomal ion channel transient receptor potential channel mucolipin-1 (TRPML1) leads to cathepsin B-dependent apoptosis

Mucolipidosis type IV (MLIV) is a lysosomal storage disease caused by mutations in the gene MCOLN1, which codes for the transient receptor potential family ion channel TRPML1. MLIV has an early onset and is characterized by developmental delays, motor and cognitive deficiencies, gastric abnormalities, retinal degeneration, and corneal cloudiness. The degenerative aspects of MLIV have been attributed to cell death, whose mechanisms remain to be delineated in MLIV and in most other storage diseases. Here we report that an acute siRNA-mediated loss of TRPML1 specifically causes a leak of lysosomal protease cathepsin B (CatB) into the cytoplasm. CatB leak is associated with apoptosis, which can be prevented by CatB inhibition. Inhibition of the proapoptotic protein Bax prevents TRPML1 KD-mediated apoptosis but does not prevent cytosolic release of CatB. This is the first evidence of a mechanistic link between acute TRPML1 loss and cell death.     

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: Comparison with the effects of the neurotrophins

We compared the effects of glial cell line-derived neurotrophic factor (GDNF) on dorsal root ganglion (DRG) sensory neurons to that of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3). All of these factors were retrogradely transported to sub-populations of sensory neuron cell bodies in the L4/L5 DRG of neonatal rats. The size distribution of ¹²⁵I-GDNF-labeled neurons was variable and consisted of both small and large DRG neurons (mean of 506.60 μm²). ¹²⁵I-NGF was preferentially taken up by small neurons with a mean cross-sectional area of 383.03 μm². Iodinated BDNF and NT-3 were transported by medium to large neurons with mean sizes of 501.48 and 529.27 μm², respectively. A neonatal, sciatic nerve axotomy-induced cell death model was used to determine whether any of these factors could influence DRG neuron survival in vivo. GDNF and NGF rescued nearly 100% of the sensory neurons. BDNF and NT-3 did not promote any detectable level of neuronal survival despite the fact that they underwent retrograde transport. We examined the in vitro survival-promoting ability of these factors on neonatal DRG neuronal cultures derived from neonatal rats. GDNF, NGF, and NT-3 were effective in vitro, while BDNF was not. The range of effects seen in the models described here underscores the importance of testing neuronal responsiveness in more than one model. The biological responsiveness of DRG neurons to GDNF in multiple models suggests that this factor may play a role in the development and maintenance of sensory neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 22–32, 1997.     

Blocking c-Jun N-terminal Kinase (JNK) Translocation to the Mitochondria Prevents 6-Hydroxydopamine-induced Toxicity in Vitro and in Vivo

Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-Sab_KIM1 protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-Sab_KIM1 via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-Sab_KIM1 decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-Sab_KIM1 at day 7 were 750 nm, which was ∼3.4-fold higher than the IC₅₀ for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.     

Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes

The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.     

Insulin-like Growth Factor I (IGF-I)-induced Chronic Gliosis and Retinal Stress Lead to Neurodegeneration in a Mouse Model of Retinopathy

Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factor''s extensively described neuroprotective actions, transgenic mice with increased intraocular levels of IGF-I showed progressive impairment of electroretinographic amplitudes up to complete loss of response, with loss of photoreceptors and bipolar, ganglion, and amacrine neurons. Neurodegeneration was preceded by the overexpression of genes related to retinal stress, acute-phase response, and gliosis, suggesting that IGF-I altered normal retinal homeostasis. Indeed, gliosis and microgliosis were present from an early age in transgenic mice, before other alterations occurred, and were accompanied by signs of oxidative stress and impaired glutamate recycling. Older mice also showed overproduction of pro-inflammatory cytokines. Our results suggest that, when chronically increased, intraocular IGF-I is responsible for the induction of deleterious cellular processes that can lead to neurodegeneration, and they highlight the importance that this growth factor may have in the pathogenesis of conditions such as ischemic or diabetic retinopathy.     

Inactivation of Pink1 gene in vivo sensitizes dopamine-producing neurons to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and can be rescued by autosomal recessive Parkinson disease genes, Parkin or DJ-1

Mutations in the mitochondrial PTEN-induced kinase 1 (Pink1) gene have been linked to Parkinson disease (PD). Recent reports including our own indicated that ectopic Pink1 expression is protective against toxic insult in vitro, suggesting a potential role for endogenous Pink1 in mediating survival. However, the role of endogenous Pink1 in survival, particularly in vivo, is unclear. To address this critical question, we examined whether down-regulation of Pink1 affects dopaminergic neuron loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the adult mouse. Two model systems were utilized: virally delivered shRNA-mediated knockdown of Pink1 and germ line-deficient mice. In both instances, loss of Pink1 generated significant sensitivity to damage induced by systemic MPTP treatment. This sensitivity was associated with greater loss of dopaminergic neurons in the Substantia Nigra pars compacta and terminal dopamine fiber density in the striatum region. Importantly, we also show that viral mediated expression of two other recessive PD-linked familial genes, DJ-1 and Parkin, can protect dopaminergic neurons even in the absence of Pink1. This evidence not only provides strong evidence for the role of endogenous Pink1 in neuronal survival, but also supports a role of DJ-1 and Parkin acting parallel or downstream of endogenous Pink1 to mediate survival in a mammalian in vivo context.     

Ras-related proteins (Rab) are key proteins related to male fertility following a unique activation mechanism

Ras-related protein Rab (Rab) proteins, member of Ras superfamily of monomeric G proteins, are well known key regulators of intracellular vesicular transport. Recently, it has been reported that Rab 2A and 3A are related to acrosomal exocytosis in spermatozoa and Rab 2A can be used to fertility-related biomarker in male. However, the role and mechanism of Rab proteins in spermatozoa has not been fully understood yet. Therefore, the study to analyze the expression and location of various Rab proteins in spermatozoa is required to understand the role and mechanism of Rab proteins in spermatozoa. In present study, to analyze the expression level and location of various Rab proteins (Rab 2A, Rab3A, Rab4, Rab5, Rab8A, Rab9, Rab11, Rab14, Rab25, Rab27A, and Rab34) and Rab protein regulators (RabGAP, RabGDI, RabGEF) in spermatozoa following capacitation, immunofluorescence and western blot analysis were performed. All of 11 Rab proteins were expressed in acrosomal region and tail of spermatozoa. Furthermore, all Rab proteins and Rab protein regulators, except RabGAP, have decreased expression patterns after capacitation. Taken together, Rab proteins were located in sperm head and tail. In addition, expression patterns of Rab proteins in spermatozoa were altered following capacitation. Therefore, our results suggested that Rab proteins may be key proteins related with capacitation as well as playing important role with uniquely activation pathway for male fertility.     

Paraoxonase 2 Serves a Proapopotic Function in Mouse and Human Cells in Response to the Pseudomonas aeruginosa Quorum-sensing Molecule N-(3-Oxododecanoyl)-homoserine Lactone

Pseudomonas aeruginosa use quorum-sensing molecules, including N-(3-oxododecanoyl)-homoserine lactone (C12), for intercellular communication. C12 activated apoptosis in mouse embryo fibroblasts (MEF) from both wild type (WT) and Bax/Bak double knock-out mice (WT MEF and DKO MEF that were responsive to C12, DKO_R MEF): nuclei fragmented; mitochondrial membrane potential (Δψ_mito) depolarized; Ca²⁺ was released from the endoplasmic reticulum (ER), increasing cytosolic [Ca²⁺] (Ca_cyto); and caspase 3/7 was activated. DKO_R MEF had been isolated from a nonclonal pool of DKO MEF that were non-responsive to C12 (DKO_NR MEF). RNAseq analysis, quantitative PCR, and Western blots showed that WT and DKO_R MEF both expressed genes associated with cancer, including paraoxonase 2 (PON2), whereas DKO_NR MEF expressed little PON2. Adenovirus-mediated expression of human PON2 in DKO_NR MEF rendered them responsive to C12: Δψ_mito depolarized, Ca_cyto increased, and caspase 3/7 activated. Human embryonic kidney 293T (HEK293T) cells expressed low levels of endogenous PON2, and these cells were also less responsive to C12. Overexpression of PON2, but not PON2-H114Q (no lactonase activity) in HEK293T cells caused them to become sensitive to C12. Because [C12] may reach high levels in biofilms in lungs of cystic fibrosis (CF) patients, PON2 lactonase activity may control Δψ_mito, Ca²⁺ release from the ER, and apoptosis in CF airway epithelia. Coupled with previous data, these results also indicate that PON2 uses its lactonase activity to prevent Bax- and Bak-dependent apoptosis in response to common proapoptotic drugs like doxorubicin and staurosporine, but activates Bax- and Bak-independent apoptosis in response to C12.     

A Competitive binding mechanism between Skp1 and exportin 1 (CRM1) controls the localization of a subset of F-box proteins

SCF-type E3 ubiquitin ligases are crucial regulators of cell cycle progression. As the F-box protein is the substrate-specifying subunit of this family of ligases, their availability dictates the timing and the location of the ubiquitination of substrates. We report here our investigation into the regulation of the localization of F-box proteins, in particular Fbxo7, whose mislocalization is associated with human disease. We identified a motif in Fbxo7 that we have characterized as a functional leucine-rich nuclear export sequence (NES), and which allowed binding to the nuclear export protein, exportin 1 (CRM1). Unusually, the NES was embedded within the F-box domain, which is bound by Skp1 and enables the F-box protein to form part of an E3 ubiquitin ligase. The NES of Fbxo7 controlled its localization and was conserved in Fbxo7 homologues in other species. Skp1 binding prevented Fbxo7 from contacting CRM1. We propose that this competitive binding allowed Fbxo7 to accumulate within the nucleus starting at the G1/S transition. More than ten other F-box proteins also contain an NES at the same location in their F-box domains, indicating that this competitive binding mechanism may contribute to the regulation of a sixth of the known F-box proteins.