Review: transactive response DNA-binding protein 43 (TDP-43): mechanisms of neurodegeneration.

Since the identification of phosphorylated and truncated transactive response DNA-binding protein 43 (TDP-43) as a primary component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions, and the discovery that mutations in the TDP-43 gene cause ALS, much effort has been directed towards establishing how TDP-43 contributes to the development of neurodegeneration. Although few in vivo models are presently available, findings thus far strongly support the involvement of abnormally modified TDP-43 in promoting TDP-43 aggregation and cellular mislocalization. Therefore, TDP-43-mediated neurotoxicity is likely to result from a combination of toxic gains of function conferred by TDP-43 inclusions as well as from the loss of normal TDP-43 function. Nonetheless, the exact neurotoxic TDP-43 species remain unclear, as do the mechanism(s) by which they cause neuronal death. Moreover, little is currently known about the roles of TDP-43, both in the nucleus and the cytoplasm, making it difficult to truly appreciate the detrimental consequences of aberrant TDP-43 function. This review will summarize what is currently understood regarding normal TDP-43 function and the involvement of TDP-43 in neurodegeneration, and will also highlight some of the many remaining questions in need of further investigation.

Single dose of palonosetron plus dexamethasone to control nausea, vomiting and to warrant an adequate food intake in patients treated with highly emetogenic chemotherapy (HEC). Preliminary results.

INTRODUCTION: The aim of our study was to evaluate the efficacy of a single-dose palonosetron plus dexamethasone to control emesis in patients (pts) receiving HEC. Moreover, we evaluated the amount of their food intake (FI) in the week following therapy, in order to measure any reduction of calories consumption related to Chemotherapy-induced nausea and vomiting (CINV). METHODS: Patients affected with advanced cancer were treated with palonosetron 250 mcg plus dexamethasone 20 mg before HEC. Nausea, vomiting, and FI were monitored by a 7-day diary. Complete Response (CR: no vomiting and no rescue therapy) was the primary endpoint, Complete Control (CC: CR and no more than mild nausea) and the evaluation of FI were the secondary endpoints. The endpoints were evaluated during the acute (0-24 h), the delayed (25-168 h) and overall (0-168 h) phases. RESULTS: Thirty-five patients were enrolled; 85.7% and 82.9% of patients achieved CR and CC respectively, during the acute phase; 82.9% and 77.1% of patients achieved CR and CC, during the delayed phase; 80% and 77.1% of patients achieved CR and CC, during the overall phase. During the acute phase, patients with a CC without nausea had a median daily FI of 1,575 kcal, whereas patients with CC and presence of mild nausea had a median daily FI of 1,040 kcal (-535 kcal; p < 0.0001). CONCLUSIONS: Our preliminary results confirm the efficacy of a single-dose palonosetron plus dexamethasone to prevent both acute and delayed nausea and vomiting. Moreover, the efficacy of palonosetron in nausea and vomiting control seems to warrant adequate caloric intake in these patients.

alpha-Synuclein shares physical and functional homology with 14-3-3 proteins.

alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease. Mutations in alpha-synuclein cause some cases of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). In addition, many neurodegenerative diseases show accumulation of alpha-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et al., 1998). Here, we show that alpha-synuclein shares physical and functional homology with 14-3-3 proteins, which are a family of ubiquitous cytoplasmic chaperones. Regions of alpha-synuclein and 14-3-3 proteins share over 40% homology. In addition, alpha-synuclein binds to 14-3-3 proteins, as well as some proteins known to associate with 14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of alpha-synuclein inhibits protein kinase C activity. The association of alpha-synuclein with BAD and inhibition of protein kinase C suggests that increased expression of alpha-synuclein could be harmful. Consistent with this hypothesis, we observed that overexpression of wild-type alpha-synuclein is toxic, and overexpression of alpha-synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity and binding profile of alpha-synuclein suggests that it might act as a protein chaperone and that accumulation of alpha-synuclein could contribute to cell death in neurodegenerative diseases.

Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells.

The discovery of mutations in the gene for alpha-synuclein in familial Parkinson's disease (PD) has led to an increased interest in this pre-synaptic protein. Synphilin-1, a potential synuclein-binding protein, was cloned using yeast two-hybrid assays. The function of synphilin-1 is currently unknown, although it has been reported to be present along with alpha-synuclein in Lewy bodies in PD. In the present study, we monitored synphilin-1 aggregation directly using fusion proteins of synphilin-1 and green fluorescent protein (EGFP). Transfection of synphilin-EGFP fusion proteins formed cytoplasmic inclusions in HEK293 cells. Although these inclusions overlapped with the distribution of alpha-synuclein, they were unlike Lewy bodies in that they were not eosinophilic, and instead were membrane-bound, lipid-rich cytoplasmic inclusions.

SEPT5_v2 is a parkin-binding protein.

Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.

Co-association of parkin and alpha-synuclein.

Parkin and alpha-synuclein are two proteins that are associated with the pathophysiology of Parkinson's disease (PD). Parkin is present in Lewy bodies and axonal spheroids in brains affected by PD, and mutations in parkin cause hereditary forms of Parkinsonism. Alpha-synuclein is a major component of Lewy bodies and is associated with rare cases of PD. We now show that parkin binds to alpha-synuclein, including conditions associated with alpha-synuclein aggregation. Parkin and alpha-synuclein complexes were observed in BE-M17 cells under basal conditions, in BE- M17 cells under oxidative conditions and in brains from control or PD donors. Double staining of PD brains shows parkin and alpha-synuclein co-localize to the same pathological structures (both Lewy bodies and axonal spheroids). These results suggest that parkin interacts with alpha-synuclein and could contribute to the pathophysiology of PD more generally than was previously considered.

Presence of D-alanine in proteins of normal and Alzheimer human brain.

This report constitutes the first demonstration of the presence of D-alanine in the proteins of the human nervous system. Proteins of the frontal lobe white and gray matter of human brains, both normal and Alzheimer subjects, contain D-alanine at concentrations between 0.50 and 1.28 mumol/g of wet tissue, 50-70-times lower than the concentration of L-alanine. Both white and gray matter of Alzheimer brains contain D-alanine 1.4-times higher than the respective regions of normal brains. The gray matter proteins of Alzheimer brains show a highly significant 8% decrease in total alanine content, when compared with normal brain gray matter proteins. Since Alzheimer's disease is exhibited by deterioration of the gray matter, the occurrence of elevated D-alanine levels in the gray matter of Alzheimer brains is a significant discovery and raises the question whether this enantiomer causes the degeneration of the gray matter proteins in Alzheimer's disease, or whether it is an effect of the disease.

Quantification of D-aspartate in normal and Alzheimer brains.

Using a new procedure to hydrolyze proteins without provoking racemization of the amino acids and using enzymatic methods to determine D- and L-aspartate (Asp), we have quantified the content of protein-bound D-aspartate (both D-aspartic acid and D-asparagine) of human brain white and gray matter proteins from normal and Alzheimer subjects. The D-enantiomer is present in brain proteins at mean concentrations between 0.48 and 0.90 mumol/g of wet tissue, corresponding to concentrations 34-82 times lower than that of L-aspartate. The highest levels of D-aspartate were found in Alzheimer gray matter (0.60-0.90, mean 0.69 mumol/g of wet tissue). When expressed as the percentage of total (i.e. D- plus L-) aspartate, %D = [D/(D + L)] x 100, the Alzheimer brains show a significantly higher content of D-aspartate in both gray matter (2.08%) and white matter (1.80%) than in the corresponding tissues of normal brains (1.65% in gray, 1.58% in white).

Regional decreases of free D-aspartate levels in Alzheimer's disease.

N-methyl-D-aspartate (NMDA) receptors have been shown to be involved in learning and memory processes. In Alzheimer's disease, there is a reduction of NMDA receptors. Since D-aspartate is an endogenous agonist for the NMDA receptor, we hypothesised that if there are reduced levels of this amino acid in the Alzheimer's brain, this could raise the reduction of NMDA receptor signal transduction system and contribute to the marked memory deficits seen in these patients. Therefore, using a chromatographic HPLC method, the regional distribution of free D-aspartate levels in post-mortem human brain samples from patients with Alzheimer's disease (AD) (n = 5) and age-matched controls (n = 5) were determined. We found that the levels of D-aspartate are significantly lower in Alzheimer's patients compared to controls (range: from -35 to -47%; P < 0.01). However, no differences were found in the cerebellum, a region spared from the neuropathological changes of AD. These data suggest that decreased levels of D-aspartate could contribute to a lower NMDA receptor function and consequently contribute to the memory deficits seen in AD.

Involvement of D-aspartic acid in the synthesis of testosterone in rat testes.

D-Aspartic acid (D-Asp) is an endogenous amino acid which occurs in many marine and terrestrial animals. In fetal and young rats, this amino acid occurs prevalently in nervous tissue, whereas at sexual maturity it occurs in endocrine glands and above all in pituitary and testes. Here, we have studied if a relationship exists between the presence of D-Asp and the hormonal activity. The following results were obtained: 1) Both D-Asp and testosterone are synthesized in rat testes in two periods of the animal's life: before birth, about the 17th day after fertilization and, after birth, at sexual maturity. 2) Immunocytochemical studies have demonstrated that this enantiomer is localized in Leydig and Sertoli cells. 3) In vivo experiments, consisting of i.p. injection of D-Asp to adult male rats, demonstrated that this amino acid accumulates in pituitary and testis (after 5 h, the accumulation was of 12 and 4-fold over basal values, respectively); simultaneously, luteinizing hormone, testosterone and progesterone significantly increased in the blood (1.6-fold, p < 0.05; 3.0-fold, p < 0.01 and 2.9-fold, p < 0.01, respectively). 4) Finally, in vitro experiments, consisting of the incubation of D-Asp with isolated testes also demonstrated that this amino acid induces the synthesis of testosterone. These results suggest that free D-Asp is involved in the steroidogenesis.

Non-pegylated liposome-encapsulated doxorubicin citrate plus cyclophosphamide or vinorelbine in metastatic breast cancer not previously treated with chemotherapy:a multicenter phase III study.

We conducted a phase III multicenter randomized trial to compare the efficacy of the combination of liposome encapsulated doxorubicin (Myocet(©)) plus either cyclophosphamide (MC) or vinorelbine (MV). Since July 2006, 233 patients affected with metastatic breast cancer were randomized to receive the combination of Myocet (M) 60 mg/m(2) i.v. plus cyclophosphamide (C) 600 mg/m2 on Day 1 of a 21­day cycle (Arm A) or Myocet (M) at 50 mg/m2 plus vinorelbine (V) 25 mg/m2 i.v. on Day 1 and V 60 mg/m2 orally on Day 8 on a 21­day cycle (Arm B). The primary endpoints of the study was time to progression (TTP); secondary endpoints were RR, toxicity and OS. Response was observed in 53/116 (45.7%) evaluable patients of Arm A vs. 51/112 (45.5%) of Arm B, respectively (P=NS). Median TTP was 41 weeks (95% CI, 32­51) and 34 weeks (95% CI, 26­39), for M/C and M/V, respectively (P=0.0234). The difference in median OS was not statistically significant (131 vs. 122 weeks; P=0.107). With regard to toxicity, patients treated with MV showed a slight increase of neutropenia and constipation, as compared to those treated with MC. No clinical signs of cardiotoxicity were observed. The MC combination remains as an unbeaten 'standard' in first line treatment of MBC.

Targeting heat shock proteins in tauopathies.

Heat shock proteins are members of a large family that function normally in nascent protein folding and the removal of damaged proteins and are able to respond to cellular stresses such as thermal insult to prevent catastrophic protein aggregation. A number of the most common neurodegenerative disorders such as Alzheimer's and Parkinson's diseases are characterized by such abnormal protein folding and aggregation, and the induction of the heat shock response is observed in these cases through their increased expression and often localization within the inclusions. Tau proteins form the major structural component of the neurofibrillary protein aggregates that correlate with cognitive decline in Alzheimer's disease, and appropriately this abnormal tau is targeted for corrective action by the heat shock proteins that recognize sequence motifs that are normally masked though microtubule binding. This specific heat shock response to the formation of abnormal tau can also be targeted pharmacologically to inhibit the refolding pathways and drive the degradation of tau species that are thought to be pathogenic. This review discusses the recent advances of the roles of heat shock proteins in this process.

D-aspartate and D-glutamate in microwaved versus conventionally heated milk.

OBJECTIVE: It has been reported that microwave heating of infant formulae can isomerize and racemize amino acids in the milk proteins, causing toxicity or affecting the nutritional value of the milk formulae. Therefore, we investigated whether microwave heating vs conventional heating would produce any D- enantiomers of aspartic acid (Asp) and glutamic acid (Glu) in milk. METHODS: Whole and skim milk samples were heated for 10 minutes in either a microwave oven at medium power or on a hot water bath at 80 degrees C. D-Asp and D-Glu were determined by high performance liquid chromatography. RESULTS: Unheated (control) samples were found to contain 0.40-0.45% D-Asp and D-Glu, inherent from the original pasteurizing process. Both conventional heating and microwave heating induce < 0.25% more racemization when compared to the control samples. CONCLUSION: Within experimental error, there is no significant difference in the levels of these D-amino acids between the conventionally heated and microwave heated milks, thus having no significant effect on the nutritional value of the milk proteins.

Improved method for hydrolyzing proteins and peptides without inducing racemization and for determining their true D-amino acid content.

A new method of hydrolyzing proteins and peptides without racemizing the amino acids has been developed. This method consists of performing a brief partial chemical hydrolysis for 15 min in 6 M HCl at 80-90 degrees C, followed by an enzymatic hydrolysis with pronase for 12-16 h at 50 degrees C, and finally an enzymatic hydrolysis with leucine aminopeptidase and peptidyl-D-amino acid hydrolase for 24 h. Using this new method the time required for complete hydrolysis of proteins is less than 3 days. The total hydrolysis averages 97-100%, and the amount of racemization of the amino acids is less than 0.002%. This method may then be used as a tool to easily determine the intrinsic D-amino acid content of peptides or proteins from animal or vegetable tissues.

Further study on the specificity of D-amino acid oxidase and D-aspartate oxidase and time course for complete oxidation of D-amino acids.

1. D-Amino acid oxidase (D-AAO) oxidizes: D-Met, D-Pro, D-Phe, D-Tyr, D-Ile, D-Leu, D-Ala and D-Val. D-Ser, D-Arg, D-His, D-norleucine and D-Trp are oxidized at a low rate. D-Ornithine, cis-4-hydroxy-D-proline, D-Thr, D-Trp-methyl ester, N-acetyl-D-Ala and D-Lys are oxidized at a very low rate. 2. D-Asp, D-Glu and their derivatives, Gly and all the L-amino acids are not oxidized (or are at a rate which is undetectable). 3. Among all D-amino acids, D-Met is the most highly oxidized compound. The Km value is 1.7 mM. 4. D-Aspartate oxidase (D-Aspo) either purified from Octopus vulgaris or from beef kidney oxidizes only D-Asp, D-Glu and their following derivatives: D-Asn, D-Gln, D-Asp-dimethyl-ester and N-methyl-D-Asp. 5. However, D-Pro, D-Leu, D-Ala and D-Met, are also oxidized by this enzyme, but at a very low rate (between 0.2 and 0.6% of D-Asp). 6. All other D-amino acids, glycine and all the L-amino acids are not oxidized. 7. Under experimental conditions, 1 U of D-AAO is able to totally oxidize 0.2 micromol of the following amino acids: D-Met, D-Pro, D-Phe, D-Thy, D-Ile, D-Leu, D-Ala, D-Val, D-Ser and D-Arg. 8. Similarly, 1 U of D-AspO in 1 hr of incubation totally oxidizes 0.1 micromol of D-Asp, D-Glu, D-Asn and D-Gln.

The A53T alpha-synuclein mutation increases iron-dependent aggregation and toxicity.

Parkinson's disease (PD) is the most common motor disorder affecting the elderly. PD is characterized by the formation of Lewy bodies and death of dopaminergic neurons. The mechanisms underlying PD are unknown, but the discoveries that mutations in alpha-synuclein can cause familial PD and that alpha-synuclein accumulates in Lewy bodies suggest that alpha-synuclein participates in the pathophysiology of PD. Using human BE-M17 neuroblastoma cells overexpressing wild-type, A53T, or A30P alpha-synuclein, we now show that iron and free radical generators, such as dopamine or hydrogen peroxide, stimulate the production of intracellular aggregates that contain alpha-synuclein and ubiquitin. The aggregates can be identified by immunocytochemistry, electron microscopy, or the histochemical stain thioflavine S. The amount of aggregation occurring in the cells is dependent on the amount of alpha-synuclein expressed and the type of alpha-synuclein expressed, with the amount of alpha-synuclein aggregation following a rank order of A53T > A30P > wild-type > untransfected. In addition to stimulating aggregate formation, alpha-synuclein also appears to induce toxicity. BE-M17 neuroblastoma cells overexpressing alpha-synuclein show up to a fourfold increase in vulnerability to toxicity induced by iron. The vulnerability follows the same rank order as for aggregation. These data raise the possibility that alpha-synuclein acts in concert with iron and dopamine to induce formation of Lewy body pathology in PD and cell death in PD.

Identification and characterization of the human parkin gene promoter.

Compound mutations and homozygous loss of function of the parkin gene causes juvenile and early onset, autosomal recessive parkinsonism. Pathologically, the disease is associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, usually without Lewy body pathology. Hemizygous families have been described that may harbor mutations outside of the open reading frame. The parkin gene promoter has yet to be characterized, and therein, mutations in hemizygous families may plausibly be identified. To identify the promoter of the parkin gene, the transcription start site was defined by a combination of primer extension and 5' RACE. Five kilobases of DNA 5' to the parkin start codon were directly sequenced from a BAC containing parkin exon 1 and evaluated for promoter motifs. The parkin promoter lacks TATA or CAAT boxes and appears to share homology to the alpha-synuclein promoter. Deletion constructs demonstrated core promoter activity and tissue specific enhancing regions in HEK-293T and SH-SY5Y cells.