Neurodegeneration as a consequence of failed mitochondrial maintenance

Maintaining the functional integrity of mitochondria is pivotal for cellular survival. It appears that neuronal homeostasis depends on high-fidelity mitochondria, in particular. Consequently, mitochondrial dysfunction is a fundamental problem associated with a significant number of neurological diseases, including Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and various peripheral neuropathies, as well as the normal aging process. To ensure optimal mitochondrial function, diverse, evolutionarily conserved mitochondrial quality control mechanisms are in place, including the scavenging of toxic reactive oxygen species (ROS) and degradation of damaged mitochondrial proteins, but also turnover of whole organelles. In this review we will discuss various mitochondria-associated conditions, focusing on the role of protein turnover in mitochondrial maintenance with special emphasis on neurodegenerative disorder

Primary cell culture of meningothelial cells—a new model to study the arachnoid in glaucomatous optic neuropathy

Background: In a previous report, we found that the occurrence and amount of meningothelial cell nests in the subarachnoid space are significantly increased in glaucomatous optic nerves compared to normals. In order to allow research into the role of meningothelial cells during diseases of the optic nerve, an in vitro model is necessary. For this purpose, we developed a culture method for porcine meningothelial cells from the arachnoid layer covering the optic nerve. Methods: Meningothelial cells were scraped from the arachnoid layer of porcine optic nerves and cultured for 2-3weeks until the cells formed a monolayer. To eliminate contaminating fibroblasts from the culture, cells were negatively selected using magnetic anti-fibroblast beads after the first passage. Cells were detached using 0.05% Trypsin-EDTA, incubated with anti-fibroblast beads, separated using a magnetic column and the flow-through was collected. The purified primary meningothelial cells were characterized by electron microscopy and immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) and anti-keratan sulfate antibodies. Results: Primary cells grew out after dissection and formed a monolayer within 2-3weeks, which was composed of two morphologically different cell types, flattened cells with round nuclei and fibroblast-like cells with long processes. The fibroblast-like cells in the culture could be labelled and selected using anti-fibroblast microbeads. The second cell type did not bind to the anti-fibroblast beads, and upon immunocytochemistry showed a marked expression of both GFAP and keratan sulphate. In addition, examination of these cells by electron microscopy revealed morphological characteristics of meningothelial cells, including hemidesmosomes and cytoplasmatic filaments. Conclusions: The technique described in this paper for the primary culture of meningothelial cells from the subarachnoid space of the optic nerve and using magnetic beads for the removal of fibroblasts is effective in obtaining a highly enriched meningothelial cell cultur

L-PGDS (Betatrace Protein) Inhibits Astrocyte Proliferation and Mitochondrial ATP Production in Vitro

L-PGDS is the most abundant protein present in the cerebrospinal fluid (CSF). Although CSF was believed to be homogenous in content, a previous study has showed that a marked concentration gradient of L-PGDS exists between the spinal CSF and the CSF in the subarachnoid space of patients with optic nerve disease (papilledema and normal-tension glaucoma). Astrocytes play a critical role in maintaining the integrity of axon function in the central nervous system and specifically in the optic nerve, and we therefore investigated the biochemical effects of L-PGDS on the proliferation of astrocytes and on the production of adenosine triphosphate (ATP) by astrocyte mitochondria. We found an inhibitory effect of L-PGDS on both proliferation of astrocytes and production of astrocyte ATP. The concentrations that inhibited astrocyte proliferation and ATP production were in the range measured in patients with idiopathic intracranial hypertension and in patients with normal-tension glaucoma. As the CSF is in contact with axons and mitochondria of the optic nerve (Bristow et al. Archives of Ophthalmology, 120, 791-796, 2002), we postulate that a change in the concentration of CSF protein such as L-PGDS could exercise a harmful effect on these structure

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant–expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch

Large-scale morphometry of the subarachnoid space of the optic nerve.

BACKGROUND The meninges, formed by dura, arachnoid and pia mater, cover the central nervous system and provide important barrier functions. Located between arachnoid and pia mater, the cerebrospinal fluid (CSF)-filled subarachnoid space (SAS) features a variety of trabeculae, septae and pillars. Like the arachnoid and the pia mater, these structures are covered with leptomeningeal or meningothelial cells (MECs) that form a barrier between CSF and the parenchyma of the optic nerve (ON). MECs contribute to the CSF proteome through extensive protein secretion. In vitro, they were shown to phagocytose potentially toxic proteins, such as α-synuclein and amyloid beta, as well as apoptotic cell bodies. They therefore may contribute to CSF homeostasis in the SAS as a functional exchange surface. Determining the total area of the SAS covered by these cells that are in direct contact with CSF is thus important for estimating their potential contribution to CSF homeostasis. METHODS Using synchrotron radiation-based micro-computed tomography (SRµCT), two 0.75 mm-thick sections of a human optic nerve were acquired at a resolution of 0.325 µm/pixel, producing images of multiple terabytes capturing the geometrical details of the CSF space. Special-purpose supercomputing techniques were employed to obtain a pixel-accurate morphometric description of the trabeculae and estimate internal volume and surface area of the ON SAS. RESULTS In the bulbar segment, the ON SAS microstructure is shown to amplify the MECs surface area up to 4.85-fold compared to an "empty" ON SAS, while just occupying 35% of the volume. In the intraorbital segment, the microstructure occupies 35% of the volume and amplifies the ON SAS area 3.24-fold. CONCLUSIONS We provided for the first time an estimation of the interface area between CSF and MECs. This area is of importance for estimating a potential contribution of MECs on CSF homeostasis

PGC-1α Determines Light Damage Susceptibility of the Murine Retina

The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases

Neuronal Mitochondrial Dysfunction Activates the Integrated Stress Response to Induce Fibroblast Growth Factor 21.

Stress adaptation is essential for neuronal health. While the fundamental role of mitochondria in neuronal development has been demonstrated, it is still not clear how adult neurons respond to alterations in mitochondrial function and how neurons sense, signal, and respond to dysfunction of mitochondria and their interacting organelles. Here, we show that neuron-specific, inducible in vivo ablation of the mitochondrial fission protein Drp1 causes ER stress, resulting in activation of the integrated stress response to culminate in neuronal expression of the cytokine Fgf21. Neuron-derived Fgf21 induction occurs also in murine models of tauopathy and prion disease, highlighting the potential of this cytokine as an early biomarker for latent neurodegenerative conditions

Meningothelial Cells React to Elevated Pressure and Oxidative Stress

BACKGROUND: Meningothelial cells (MECs) are the cellular components of the meninges enveloping the brain. Although MECs are not fully understood, several functions of these cells have been described. The presence of desmosomes and tight junctions between MECs hints towards a barrier function protecting the brain. In addition, MECs perform endocytosis and, by the secretion of cytokines, are involved in immunological processes in the brain. However, little is known about the influence of pathological conditions on MEC function; e.g., during diseases associated with elevated intracranial pressure, hypoxia or increased oxidative stress. METHODS: We studied the effect of elevated pressure, hypoxia, and oxidative stress on immortalized human as well as primary porcine MECs. We used MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) bioreduction assays to assess the proliferation of MECs in response to treatment and compared to untreated control cells. To assess endocytotic activity, the uptake of fluorescently labeled latex beads was analyzed by fluorescence microscopy. RESULTS: We found that exposure of MECs to elevated pressure caused significant cellular proliferation and a dramatic decrease in endocytotic activity. In addition, mild oxidative stress severely inhibited endocytosis. CONCLUSION: Elevated pressure and oxidative stress impact MEC physiology and might therefore influence the microenvironment of the subarachnoid space and thus the cerebrospinal fluid within this compartment with potential negative impact on neuronal function