Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson's diseases.

Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging-related diseases, in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP-induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α-synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α-synuclein oligomerization and S-129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.

Neurodegeneration in drop-dead mutant drosophila melanogaster is associated with the respiratory system but not with Hypoxia.

Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.

α-Synuclein in CSF of patients with severe traumatic brain injury.

OBJECTIVE: The study aims to examine α-synuclein in the CSF of patients with severe traumatic brain injury (TBI) and its relationship with clinical characteristics and long-term outcomes. METHODS: This prospective case-control study enrolled patients with severe TBI (Glasgow Coma Score ≤ 8) who underwent ventriculostomy. CSF samples were taken from each TBI patient at admission and daily for up to 8 days after injury and successively assessed by ELISA. Control CSF was collected for analysis from subjects receiving lumbar puncture for other medical reasons. We used trajectory analysis to identify distinct temporal profiles of CSF α-synuclein that were compared with clinical outcomes. RESULTS: CSF α-synuclein was elevated in TBI patients after injury as compared to controls (p = 0.0008). Overall, patients who died had higher concentrations (area under the curve) over 8 days of observation compared to those who survived at 6 months postinjury (p = 0.002). Two distinct temporal α-synuclein profiles were recognized over time. Subjects who died had consistently elevated α-synuclein levels compared to those who survived with α-synuclein levels near controls. High-risk trajectory was a strong and accurate predictor of death with 100% specificity and a very high sensitivity (83%). CONCLUSIONS: Taken together, these data support the hypothesis that in severe TBI patients, substantial increase of CSF α-synuclein may indicate widespread neurodegeneration and reflect secondary neuropathologic events occurring after injury. The determination of CSF α-synuclein may be a valuable prognostic marker, adding to the clinical assessment and creating opportunities for medical intervention.

Late-life hemoglobin and the incidence of Parkinson's disease.

Brain iron promotes neurodegeneration in Parkinson's disease (PD). While hemoglobin (Hb) is the most abundant source of peripheral iron in humans, its relationship with PD is uncertain. This report examines the association between Hb in late life and PD incidence. From 1991 to 1993, Hb was measured in 3507 men in the Honolulu-Asia Aging Study. Men were aged 71-93 years and without PD. Participants were followed until 2001 for incident PD. Hb levels declined markedly with age. For men aged 71-75 years, 14.8% had levels < 14 g/dL versus 53.6% in those aged 86 and older (p < 0.001). During follow-up, 47 men developed PD (19.8/10,000 person-years). After age adjustment, PD incidence rose significantly from 10.3 to 34.9/10,000 person-years as Hb increased from < 14 to ≥ 16 g/dL (p = 0.024; relative hazard 3.2; 95% confidence interval, 1.2-8.9). Associations persisted after accounting for early mortality and adjustments for concomitant risk factors. While Hb declines with advancing age, evidence suggests that Hb that remains high in elderly men is associated with an increased risk of PD.

Calcineurin inhibition at the clinical phase of prion disease reduces neurodegeneration, improves behavioral alterations and increases animal survival.

Prion diseases are fatal neurodegenerative disorders characterized by a long pre-symptomatic phase followed by rapid and progressive clinical phase. Although rare in humans, the unconventional infectious nature of the disease raises the potential for an epidemic. Unfortunately, no treatment is currently available. The hallmark event in prion diseases is the accumulation of a misfolded and infectious form of the prion protein (PrP(Sc)). Previous reports have shown that PrP(Sc) induces endoplasmic reticulum stress and changes in calcium homeostasis in the brain of affected individuals. In this study we show that the calcium-dependent phosphatase Calcineurin (CaN) is hyperactivated both in vitro and in vivo as a result of PrP(Sc) formation. CaN activation mediates prion-induced neurodegeneration, suggesting that inhibition of this phosphatase could be a target for therapy. To test this hypothesis, prion infected wild type mice were treated intra-peritoneally with the CaN inhibitor FK506 at the clinical phase of the disease. Treated animals exhibited reduced severity of the clinical abnormalities and increased survival time compared to vehicle treated controls. Treatment also led to a significant increase in the brain levels of the CaN downstream targets pCREB and pBAD, which paralleled the decrease of CaN activity. Importantly, we observed a lower degree of neurodegeneration in animals treated with the drug as revealed by a higher number of neurons and a lower quantity of degenerating nerve cells. These changes were not dependent on PrP(Sc) formation, since the protein accumulated in the brain to the same levels as in the untreated mice. Our findings contribute to an understanding of the mechanism of neurodegeneration in prion diseases and more importantly may provide a novel strategy for therapy that is beneficial at the clinical phase of the disease.

Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice.

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.

Long-term dietary administration of valproic acid does not affect, while retinoic acid decreases, the lifespan of G93A mice, a model for amyotrophic lateral sclerosis.

Mice bearing the mutated gene for Cu/Zn superoxide dismutase (G93A) are a good model for human amyotrophic lateral sclerosis (ALS). They develop progressive limb paralysis paralleled by loss of motor neurons of the cervical and lumbar spinal cord, which starts at 3-3.5 months of age and ends with death at 4-5 months. Several treatments have been attempted to delay clinical symptoms and to extend lifespan, and some have had modest beneficial effects. One such treatment, based on long-term administration of valproic acid (VPA), resulted in controversial results. We report here that, while dietary supplementation with high VPA dosage slows down motor neuron death, as assessed by measurement of a specific marker for cholinergic neurons in the spinal cord, it has no significant effect on lifespan. Recently, the hypothesis has been put forward that a deficiency of retinoic acid (RA) and its signaling may have a role in ALS. We report that long-term dietary supplementation with RA has no effect on the decrease of the cholinergic marker in the spinal cord, but it significantly shortens lifespan of G93A mice.

Gamma-synucleinopathy: neurodegeneration associated with overexpression of the mouse protein.

The role of alpha-synuclein in pathogenesis of familial and idiopathic forms of Parkinson's disease, and other human disorders known as alpha-synucleinopathies, is well established. In contrast, the involvement of two other members of the synuclein family, beta-synuclein and gamma-synuclein, in the development and progression of neurodegeneration is poorly studied. However, there is a growing body of evidence that alpha-synuclein and beta-synuclein have opposite neuropathophysiological effects. Unlike alpha-synuclein, overexpressed beta-synuclein does not cause pathological changes in the nervous system of transgenic mice and even ameliorates the pathology caused by overexpressed alpha-synuclein. To assess the consequences of excess expression of the third family member, gamma-synuclein, on the nervous system we generated transgenic mice expressing high levels of mouse gamma-synuclein under control of Thy-1 promoter. These animals develop severe age- and transgene dose-dependent neuropathology, motor deficits and die prematurely. Histopathological changes include aggregation of gamma-synuclein, accumulation of various inclusions in neuronal cell bodies and processes, and astrogliosis. These changes are seen throughout the nervous system but are most prominent in the spinal cord where they lead to loss of spinal motor neurons. Our data suggest that down-regulation of small heat shock protein HSPB1 and disintegration of neurofilament network play a role in motor neurons dysfunction and death. These findings demonstrate that gamma-synuclein can be involved in neuropathophysiological changes and the death of susceptible neurons suggesting the necessity of further investigations of the potential role of this synuclein in disease.

Neuroprotective effect of ACTH (4-9) in degeneration of hippocampal nerve cells caused by dexamethasone: morphological, immunocytochemical and ultrastructural studies.

Sustained exposure to glucocorticosteroids (GCs), adrenal hormones secreted during stress, can cause neural degeneration. This is particularly so in the hippocampus, a principal neural target site for GCs. The purpose of this research was an assessment of the neuroprotective effect of ACTH (4-9) in degenerative changes of hippocampal neurons induced by synthetic GC-dexamethasone. Experiments were conducted on male Albino-Swiss mice. We studied the morphology of neurons in the dorsal hippocampus in slides stained with cresyl violet. Immunocytochemical analysis was carried out with the use of monoclonal antibody anti-MAP2 in order to detect alterations in the neuronal cytoskeleton. We also performed ultrastructural examinations of hippocampal neurons. Quantitative analysis of morphological changes was completed using a computer analyser of histological pictures. It was shown that dexamethasone administered in toxic doses evokes neuronal death in layer CA3 of the hippocampus. Results indicate that ACTH (4-9) shows protective effects in that model. Dexamethasone-induced damage to hippocampal pyramidal neurons (assessed by cell counts, immunocytochemical analysis of cytoskeletal alterations and ultrastructural studies) was significantly reduced in animals administered ACTH (4-9).

Contribution of T cells to mortality in neurovirulent Sindbis virus encephalomyelitis.

Intranasal inoculation of C57BL/6 mice with a neurovirulent strain of Sindbis virus (SV) results in fatal encephalomyelitis. Mice with selective immune deficiencies were studied to determine the role of the immune response in fatal outcome. Mortality was decreased in mice deficient in alphabeta, but not gammadelta, T cells demonstrating a contribution of alphabeta T cells. Mice lacking either CD4+ or CD8+ T cells also had reduced mortality and mice lacking interferon (IFN)-gamma were completely protected. Clearance of infectious virus was identical in mice without T cells or IFN-gamma, but clearance of viral RNA was delayed compared to normal mice. Mice unable to produce antibody, perforin, Fas, TNF-alpha receptor1, IL-6 or IL-12 were not protected. These data suggest that T cells contribute to fatal acute viral encephalomyelitis through the production of IFN-gamma.

Protective effect of the antioxidant 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) in models of cerebral neurodegeneration.

In a previous study Dorey et al. [Bio. Org. Chem. Lett., 10 (2000) 935] a series of novel dihydroquinoline compounds were developed, based on the potent antioxidant 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin), and permitted the selection of the analogue 6-ethoxy-2,2-pentamethylen-1,2-dihydroquinoline (S 33113) lacking the hypothermic effects associated with ethoxyquin at equivalent doses. Herein, an extensive investigation of the neuroprotective capacity of S 33113 in different in vitro and in vivo paradigms of oxidative stress-mediated cellular degeneration was undertaken. In vitro S 33113 was a potent inhibitor (IC(50) = 0.29 microM) of Fenton-reaction-induced lipid peroxidation in mouse cortical membranes. Administration of S 33113 either intraperitoneally (< or =150 mg/kg i.p.) or orally (< or =600 mg/kg p.o.) did not significantly modify body temperature in NMRI mice. Furthermore, S 33113 (150 mg/kg i.p. or 600 mg/kg p.o.) markedly reduced the lethality induced by an intracerebroventricular injection of t-butylhydroperoxide in NMRI (naval medical research institute) mice for up to 5 h. Oral administration of S 33113, significantly attenuated alloxan-mediated hyperglycaemia in NMRI mice at 400 and 600 mg/kg (60%; P < 0.001). Administration of S 33113 (150 mg/kg i.p.) 30 min before transient global ischaemia significantly prevented delayed neuronal cell death in the CA1 region of the rat hippocampal formation, 7 days post-ischaemia (33% cell loss vs. 88% in ischaemia controls; P < 0.001). Similarly, a single pre-administration of S 33113 (150 mg/kg i.p.) prevented kainic acid-induced cell death in the CA3 hippocampal region at 7 days post-exposure (17% cell loss vs. 52% in kainate-treated controls; P < 0.01). Furthermore, D-methamphetamine-mediated dopamine depletion in the striatum of C57BL/6 mice (39-46%) was significantly prevented with S 33113 administered at either (2 x 150mg/kg i.p.) (11%; P < 0.01) or (2x150 mg/kg p.o.) (17%; P < 0.001). In conclusion, S 33113 represents a novel dihydroquinoline compound with potential for the treatment of cerebral pathologies implicating chronic neurodegeneration.

Intrathecal cyclosporin prolongs survival of late-stage ALS mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood-brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.