14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

Synergistic effects of environmental risk factors and gene mutations in Parkinson's disease accelerate age-related neurodegeneration

As Parkinson's disease appears to be a multifactoral disorder, the use of animal models to investigate combined effects of genetic and environmental risk factors are of great importance especially in the context of aging which is the single major risk factor for the disorder. Here, we assessed the combined effects of neonatal iron feeding and environmental paraquat exposure on age-related nigrostriatal degeneration in transgenic mice expressing the A53T familial mutant form of human α-synuclein within these neurons. We report here that A53T α-synuclein mice exhibit greater susceptibility to paraquat. Increased oral intake of iron in the neonatal period leads to a progressive age-related enhancement of dopaminergic neurodegeneration associated with paraquat neurotoxicity. Furthermore, neurodegeneration associated with these combined genetic and environmental risk factors could be attenuated by systemic treatment with the bioavailable antioxidant compound EUK-189. These data suggest that environmental factors previously identified as contributors to neurodegeneration associated with sporadic Parkinson's disease may also be candidates for observed variations in symptoms and disease progression in monogenic forms and that this may mechanistically involve increased levels of oxidatively-induced post-translational nitration of α-synuclein.     

Intrastriatal Grafts of Fetal Mesencephalic Cell Suspensions in MPP+-Lesioned Rats: A Microdialysis Study In Vivo

The striatum of rats was lesioned by unilateral administration of MPP⁺. Two weeks later, a suspension of fetal mesencephalic cells (FMC), obtained from 14-day rat embryos, was injected into the lesioned striatum. Two weeks after grafting, the success of implantation and recovery of dopamine function were assessed by tyrosine hydroxylase immunocytochemistry (TH) and the measurement of striatal dopamine content. In addition, the extracellular concentrations of dopamine and dopamine metabolites were studied by microdialysis in vivo before and after perfusion of MPP⁺ to induce dopamine release from vesicular stores. TH+ cell bodies were seen in the lesioned grafted striata, indicating that fetal cells survived in these striata. In addition, there was a marked increase in TH-immunoreactivity in the neuronal fibers and terminals in the area surrounding the cell implant, suggesting a compensatory response of the host tissue which may involve fiber sprouting. Grafting induced a recovery in indices of dopamine function, including recovery in dopamine content, and basal and MPP⁺-induced dopamine release. Thus, grafts of FMC may provide a significant recovery of dopamine function in MPP⁺-lesioned striata.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

Suppressive effects of 4-phenylbutyrate on the aggregation of Pael receptors and endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress is defined as an accumulation of unfolded proteins in the endoplasmic reticulum. 4-phenylbutyrate (4-PBA) has been demonstrated to promote the normal trafficking of the DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutant from the ER to the plasma membrane and to restore activity. We have reported that 4-PBA protected against cerebral ischemic injury and ER stress-induced neuronal cell death. In this study, we revealed that 4-PBA possesses chemical chaperone activity in vitro, which prevents the aggregation of denatured alpha-lactalbumin and bovine serum albumin (BSA). Furthermore, we investigated the effects of 4-PBA on the accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R) pathologically relevant to the loss of dopaminergic neurons in autosomal recessive juvenile parkinsonism (AR-JP). Interestingly, 4-PBA restored the normal expression of Pael-R protein and suppressed ER stress induced by the overexpression of Pael-R. In addition, we showed that 4-PBA attenuated the activation of ER stress-induced signal transduction pathways and subsequent neuronal cell death. Moreover, 4-PBA restored the viability of yeasts that fail to induce an ER stress response under ER stress conditions. These results suggest that 4-PBA suppresses ER stress by directly reducing the amount of misfolded protein, including Pael-R accumulated in the ER.     

A single intramuscular injection of rAAV‐mediated mutant erythropoietin protects against MPTP‐induced parkinsonism

Erythropoietin (Epo) is neuroprotective in a number of preparations, but can lead to unacceptably high and even lethal hematocrit levels. Recent reports show that modified Epo variants confer neuroprotection in models of glaucoma and retinal degeneration without raising hematocrit. In this study, neuroprotective effects of two Epo variants (EpoR76E and EpoS71E) were assessed in a model of Parkinson's disease. The constructs were packaged in recombinant adeno‐associated viral (rAAV) vectors and injected intramuscularly. After 3 weeks, mice received five daily injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and were killed 5 weeks later. The MPTP‐lesioned mice pretreated with rAAV.eGFP (negative control) exhibited a 7‐ to 9‐Hz tremor and slower latencies to move on a grid test (akinesia). Both of these symptomatic features were absent in mice pretreated with either modified Epo construct. The rAAV.eGFP‐treated mice lesioned with MPTP exhibited a 41% reduction in tyrosine hydroxylase (TH)‐positive neurons in the substantia nigra. The rAAV.EpoS71E construct did not protect nigral neurons, but neuronal loss in mice pretreated with rAAV.EpoR76E was only half that of rAAV.eGFP controls. Although dopamine levels were normal in all groups, 3,4‐dihydroxyphenylacetic acid (DOPAC) was significantly reduced only in MPTP‐lesioned mice pretreated with rAAV.eGFP, indicating reduced dopamine turnover. Analysis of TH‐positive fibers in the striatum showed normalized density in MPTP‐lesioned mice pretreated with rAAV.EpoS71E, suggesting that enhanced sprouting induced by EpoS71E may have been responsible for normal behavior and dopaminergic tone in these mice. These results show that systemically administered rAAV‐generated non‐erythropoietic Epo may protect against MPTP‐induced parkinsonism by a combination of neuroprotection and enhanced axonal sprouting.     

Mechanistic contributions of FBXO7 to Parkinson disease

Parkinson disease (PD) is, without doubt, a burden on modern society as the prevalence increases significantly with age. Owing to this growing number of PD cases, it is more critical than ever to understand the pathogenic mechanisms underlying PD to identify therapeutic targets. The discovery of genetic mutations associated with PD and parkinsonism paves the way toward this goal. Even though, familial forms of the disease represent the minority of PD cases and some forms are so rare that there are only a few affected families, the research on the associated genes is invaluable. Recent additions to PARK mutations are those in PARK15 that encodes the F‐box protein O‐type 7 (FBXO7). In this review, we highlight the recent research on FBXO7, which advances our knowledge of the etiopathological pathways and fills unexpected gaps therein, justifying the dedicated study of rare variants of PD.

     

Parkin Transcript Variants in Rat and Human Brain

Alternative splicing has an important role in expanding protein diversity. We have identified complementary DNA species from adult rat and fetal human brain encoding seven new splice variants of parkin, a gene mutated in autosomal recessive juvenile parkinsonism (ARJP). Alternative splicing affects almost all previously characterized exons, plus 3 new exons of 72, 156, and 180 nucleotides. This creates the potential to express hundreds of different isoforms. The encoded parkin isoforms have different amino acid composition, post-translational modifications, and, most important, molecular architectures. They diverge for the presence or absence of the ubiquitin-like domain, one or two C3HC4 ring fingers, the in-between ring fingers (IBR) domain, and a thiol proteases active site, which has not been previously characterized. Distinct expression patterns occur in primary cultures of neuronal and glial cells. Extensive splicing of parkin produces regional and structural diversity and may have important implications for the pathogenetic mechanisms underlying ARJP.     

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini-gene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing.