Cathepsin D Deficiency - Molecular and Cellular Mechanisms of Neurodegeneration

Cathepsin D (CTSD) is a lysosomal protease, the deficiency of which is fatal and associated with neurodegeneration. CTSD knock-out mice, which die at the age of four weeks, show intestinal necrosis, loss of lymphoid cells and moderate pathological changes in the brain. An active-site mutation in the CTSD gene underlies a neurodegenerative disease in newborn sheep, characterized by brain atrophy without any changes to visceral tissues. The CTSD deficiences belong to the group of neuronal ceroid-lipofuscinoses (NCLs), severe neurodegenerative lysosomal storage disorders. The aim of this thesis was to examine the molecular and cellular mechanisms behind neurodegeneration in CTSD deficiency. We found the developmental expression pattern of CTSD to resemble that of synaptophysin and the increasing expression of CTSD to coincide with the active period of myelination in the rat brain, suggesting a role for CTSD in early rat brain development. An active-site mutation underlying the congenital ovine NCL not only affected enzymatic activity, but also changed the stability, processing and transport of the mutant protein, possibly contributing to the disease pathogenesis. We also provide CTSD deficiency as a first molecular explanation for human congenital NCL, a lysosomal storage disorder, characterized by neuronal loss and demyelination in the central nervous system. Finally, we show the first evidence for synaptic abnormalities and thalamocortical changes in CTSD-deficient mice at the molecular and ultrastructural levels. Keywords: cathepsin D, congenital, cortex, lysosomal storage disorder, lysosome, mutation, neurodegeneration, neuronal ceroid-lipofuscinosis, overexpression, synapse, thalamus

New evidences on Tau-DNA interactions and relevance to neurodegeneration

Tau is mainly distributed in cytoplasm and also found to be localized in the nucleus. There is limited data on DNA binding potential of Tau.We provide novel evidence on nicking of DNA by Tau. Tau nicks the supercoiled DNA leading to open circular and linear forms. The metal ion magnesium (a co-factor for endonuclease) enhanced the Tau DNA nicking ability, while an endonuclease specific inhibitor,aurinetricarboxylic acid (ATA) inhibited the Tau DNA nicking ability Further, we also evidenced that Tau induces B-C-A mixed conformational transition in DNA and also changes DNA stability. Tau-scDNA complex is more sensitive to DNAse I digestion indicating stability changes in DNA caused by Tau. These findings indicate that Tau alters DNA helicity and integrity and also nicks the DNA. The relevance of these novel intriguing findings regarding the role Tau in neuronal dysfunction is discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Putative neuroprotective compounds in in vitro models of dopaminergic neurodegeneration

Parkinson´s disease (PD) is a debilitating age-related neurological disorder that affects various motor skills and can lead to a loss of cognitive functions. The motor symptoms are the result of the progressive degeneration of dopaminergic neurons within the substantia nigra. The factors that influence the pathogenesis and the progression of the neurodegeneration remain mostly unclear. This study investigated the role of various programmed cell death (PCD) pathways, oxidative stress, and glial cells both in dopaminergic neurodegeneration and in the protective action of various drugs. To this end, we exposed dopaminergic neuroblastoma cells (SH-SY5Y cells) to 6-OHDA, which produces oxidative stress and activates various PCD modalities that result in neuronal degeneration. Additionally, to explore the role of glia, we prepared rat midbrain primary mixed-cell cultures containing both neurons and glial cell types such as microglia and astroglia and then exposed the cultures to either MPP plus or lipopolysaccharide. Our results revealed that 6-OHDA activated several PCD pathways including apoptosis, autophagic stress, lysosomal membrane permeabilization, and perhaps paraptosis in SH-SY5Y cells. Furthermore, we found that minocycline protected SH-SY5Y cells from 6-OHDA by inhibiting both apoptotic and non-apoptotic PCD modalities. We also observed an inconsistent neuroprotective effect of various dietary anti-oxidant compounds against 6-OHDA toxicity in vitro in SH-SY5Y cells. Specifically, quercetin and curcumin exerted neuroprotection only within a narrow concentration range and a limited time frame, whereas resveratrol and epigallocatechin 3-gallate provided no protection whatsoever. Lastly, we found that molecules such as amantadine may delay or even halt the neurodegeneration in primary cell cultures by inhibiting the release of neurotoxic factors from overactivated microglia and by enhancing the pro-survival actions of astroglia. Together these data suggest that the strategy of dampening oxidative species with anti-oxidants is less effective than preventing the production of toxic factors such as oxidative and pro-inflammatory molecules by pathologically activated microglia. This would subsequently prevent the activation of various PCD modalities that cause neuronal degeneration.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.

Transgenic Drosophila model to study apolipoprotein E4-induced neurodegeneration

The epsilon 4 isoform of apolipoprotein E (ApoE4) that is involved in neuron-glial lipid metabolism has been demonstrated as the main genetic risk factor in late-onset of Alzheimer's disease. However, the mechanism underlying ApoE4-mediated neurodegeneration remains unclear. We created a transgenic model of neurodegenerative disorder by expressing epsilon 3 and epsilon 4 isoforms of human ApoE in the Drosophila melanogaster. The genetic models exhibited progressive neurodegeneration, shortened lifespan and memory impairment. Genetic interaction studies between amyloid precursor protein and ApoE in axon pathology of the disease revealed that over expression of hApoE in Appl-expressing neurons of Drosophila brain causes neurodegeneration. Moreover, acute oxidative damage in the hApoE transgenic flies triggered a neuroprotective response of hApoE3 while chronic induction of oxidative damage accelerated the rate of neurodegeneration. This Drosophila model may facilitate analysis of the molecular and cellular events implicated in hApoE4 neurotoxicity. (C) 2015 Elsevier B.V. All rights reserved.

Expression of C5a receptor in mouse brain: Role in signal transduction and neurodegeneration

In this study we explored the potential role of the complement derived anaphylatoxin C5a and the expression of its receptor in mouse brain. Using in situ hybridization, we found that C5a receptor messenger RNA is expressed in mouse brain. In response to intraventricular kainic acid injection, there was marked increase in the C5a receptor messenger RNA expression, particularly in hippocampal formation and cerebral cortex. C5a ligand-binding autoradiography confirmed the functional expression and elevation of the C5a receptor post-lesioning. The expression of Cia receptor messenger RNA in brain was confirmed by northern blot hybridization of total RNA from neuronal and glial cells in vitiro. Based on these findings we explored the role of C5a in mechanisms of signal transduction in brain cells. Treatment of primary cultures of mouse astrocytes with human recombinant C5a resulted in the activation of mitogen-activated extracellular signal-regulated protein kinase. This response appeared to be mediated by the C5a receptor since astrocyte cultures derived from C5a receptor knockout mice were not responsive to the treatment. Understanding the regulation of C5a receptor in brain and mechanisms by which pro-inflammatory C5a modulates specific signal transduction pathways in brain cells is crucial to studies of inflammatory mechanisms in neurodegeneration. (C) 1998 IBRO. Published by Elsevier Science Ltd.

Sustained intraocular VEGF neutralization results in retinal neurodegeneration in the Ins2Akita diabetic mouse

Current therapies that target vascular endothelial growth factor (VEGF) have become a mainstream therapy for the management of diabetic macular oedema. The treatment involves monthly repeated intravitreal injections of VEGF inhibitors. VEGF is an important growth factor for many retinal cells, including different types of neurons. In this study, we investigated the adverse effect of multiple intravitreal anti-VEGF injections (200 ng/μl/eye anti-mouse VEGF164, once every 2 weeks totalling 5-6 injections) to retinal neurons in Ins2(Akita) diabetic mice. Funduscopic examination revealed the development of cotton wool spot-like lesions in anti-VEGF treated Ins2(Akita) mice after 5 injections. Histological investigation showed focal swellings of retinal nerve fibres with neurofilament disruption. Furthermore, anti-VEGF-treated Ins2(Akita) mice exhibited impaired electroretinographic responses, characterized by reduced scotopic a- and b-wave and oscillatory potentials. Immunofluorescent staining revealed impairment of photoreceptors, disruptions of synaptic structures and loss of amacrine and retinal ganglion cells in anti-VEGF treated Ins2(Akita) mice. Anti-VEGF-treated WT mice also presented mild amacrine and ganglion cell death, but no overt abnormalities in photoreceptors and synaptic structures. At the vascular level, exacerbated albumin leakage was observed in anti-VEGF injected diabetic mice. Our results suggest that sustained intraocular VEGF neutralization induces retinal neurodegeneration and vascular damage in the diabetic eye.

Aggregating Brain Cell Cultures as a Model to Study Ischaemia-induced Neurodegeneration.

Rotation-mediated aggregating brain cell cultures at two different maturational stages (DIV 11 and DIV 20) were subjected for 1 or 2 hours to ischaemic conditions by transient immobilization (arrest of media circulation). During recovery, cell damage was evaluated by measuring changes in cell type-specific enzyme activities and total protein content. It was found that in immature cultures (DIV 11), immobilization for 1 or 2 hours did not affect the parameters measured. By contrast, at DIV 20, ischaemic conditions for 1 hour caused a pronounced decrease in the activities of glutamic acid decarboxylase and choline acetyltransferase. A significant decrease in these neuron-specific enzyme activities was found at post-ischaemic days 1-14, indicating immediate and irreversible neuronal damage. The activity of the astrocyte-specific enzyme, glutamine synthetase, was significantly increased at 4 days post-treatment; equal to control values at 6 days; and significantly decreased at 14 days after the ischaemic insult. Immobilization of DIV 20 cultures for 2 hours caused a drastic reduction in all the parameters measured at post-ischaemic day 6. Generally, the ischaemic conditions appeared to be more detrimental to neurons than to astrocytes, and GABAergic neurons were more affected than cholinergic neurons.

Oligodendroglia metabolically support axons and contribute to neurodegeneration.

Oligodendroglia support axon survival and function through mechanisms independent of myelination, and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been proposed. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and use. Here we show that the most abundant lactate transporter in the central nervous system, monocarboxylate transporter 1 (MCT1, also known as SLC16A1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and in mouse models of, amyotrophic lateral sclerosis, suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.

Histological and functional aspects of Alpha-synuclein in neurodegeneration

Alpha-synuclein has been implicated in the cellular mechanisms that control auditory sensitivity. In other systems it can also confer protection against cellular injury. Auditory brainstem response thresholds and immunohistochemistry were used to assess the ability of alpha-synuclein to protect against oxidative damage to the cochlea.

Tumour necrosis factor alpha induces rapid reduction in AMPA receptor-mediated calcium entry in motor neurones by increasing cell surface expression of the GluR2 subunit: relevance to neurodegeneration

The AMPA receptor (AMPAR) subunit GluR2, which regulates excitotoxicity and the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) have both been implicated in motor neurone vulnerability in Amyotrophic Lateral Sclerosis/Motor Neurone Disease. TNF alpha has been reported to increase cell surface expression of AMPAR subunits to increase synaptic strength and enhance excitotoxicity, but whether this mechanism occurs in motor neurones is unknown. We used primary cultures of mouse motor neurones and cortical neurones to examine the interaction between TNF alpha receptor activation, GluR2 availability, AMPAR-mediated calcium entry and susceptibility to excitotoxicity. Short exposure to a physiologically relevant concentration of TNFalpha (10 ng/ml, 15 min) caused a marked redistribution of both GluR1 and GluR2 to the cell surface as determined by cell surface biotinylation and immunofluorescence. Using Fura-2 AM microfluorimetry we showed that exposure to TNFalpha caused a rapid reduction in the peak amplitude of AMPA-mediated calcium entry in a PI3-kinase and p38 kinase-dependent manner, consistent with increased insertion of GluR2-containing AMPAR into the plasma membrane. This resulted in a protection of motor neurones against kainate-induced cell death. Our data therefore, suggests that TNF alpha acts primarily as a physiological regulator of synaptic activity in motor neurones rather than a pathological drive in ALS

New insights on regulation of LMTK2, a membrane kinase integrating pathways central to neurodegeneration

In a short communication in this issue (Manser et al. 2012), Christopher Miller’s group at the Institute of Psychiatry, King’s College London present an elegant and convincing set of experiments using molecular techniques to show that a brain-enriched membrane-associated protein kinase, lemur tyrosine kinase-2 (LMTK2), is directly phosphorylated by the cyclin-dependent kinase-5/p35 and this event is sufficient for LMTK2 to phosphorylate an abundant protein phosphatase, PP1C. LMTK2 has been little studied to date and, despite its name, is a kinase which phosphorylates serine or threonine residues of protein substrates. The paper adds to the evidence that this enzyme is a potentially important mediator positioned to integrate a number of intracellular signalling pathways relevant to neurodegeneration.

Hypoxia and neurodegeneration

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimer's disease (AD). AD is characterized in part by the increased production of amyloid beta peptide (Abeta), which forms the extracellular plaques by which the disease can be identified post mortem. Numerous studies have now shown that hypoxia, even in vitro, can increase production of Abeta in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Abeta precursor, APP, and also the expression of the secretase enzymes, which cleave Abeta from APP. Other studies implicate reduced Abeta degradation as a possible means by which hypoxia increases Abeta levels. Such variability may be attributable to cell-specific responses to hypoxia. Further evidence indicates that some, but not all of the cellular adaptations to chronic hypoxia (including alteration of Ca(2+) homeostasis) require Abeta formation. However, other aspects of hypoxic remodeling of cell function appear to occur independently of this process. The molecular and cellular responses to hypoxia contribute to our understanding of the clinical association of hypoxia and increased incidence of AD. However, it remains to be determined whether inhibition of one or more of the effects of hypoxia may be of benefit in arresting the development of this neurodegenerative disease.