Publications by authors named "David N Palmer"

34 Publications

Intravitreal gene therapy protects against retinal dysfunction and degeneration in sheep with CLN5 Batten disease.

Exp Eye Res 2021 Jun 28;207:108600. Epub 2021 Apr 28.

Faculty of Agricultural and Life Sciences, Lincoln University, Lincoln, 7647, New Zealand; Department of Radiology, University of Otago, Christchurch, 8140, New Zealand. Electronic address:

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases primarily affecting children. A common feature across most NCLs is the progressive loss of vision. We performed intravitreal injections of self-complementary AAV9 vectors packaged with either ovine CLN5 or CLN6 into one eye of 3-month-old CLN5 or CLN6 animals, respectively. Electroretinography (ERG) was performed every month following treatment, and retinal histology was assessed post-mortem in the treated compared to untreated eye. In CLN5 animals, ERG amplitudes were normalised in the treated eye whilst the untreated eye declined in a similar manner to CLN5 affected controls. In CLN6 animals, ERG amplitudes in both eyes declined over time although the treated eye showed a slower decline. Post-mortem examination revealed significant attenuation of retinal atrophy and lysosomal storage body accumulation in the treated eye compared with the untreated eye in CLN5 animals. This proof-of-concept study provides the first observation of efficacious intravitreal gene therapy in a large animal model of NCL. In particular, the single administration of AAV9-mediated intravitreal gene therapy can successfully ameliorate retinal deficits in CLN5 sheep. Combining ocular gene therapy with brain-directed therapy presents a promising treatment strategy to be used in future sheep trials aiming to halt neurological and retinal disease in CLN5 Batten disease.
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http://dx.doi.org/10.1016/j.exer.2021.108600DOI Listing
June 2021

Urine proteomics analysis of patients with neuronal ceroid lipofuscinoses.

iScience 2021 Feb 31;24(2):102020. Epub 2020 Dec 31.

Inborn Errors of Metabolism Section, Genetics & Genomic Medicine Unit, Great Ormond Street Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

The neuronal ceroid lipofuscinoses (NCL) are a group of 13 rare neurodegenerative disorders characterized by accumulation of cellular storage bodies. There are few therapeutic options, and existing tests do not monitor disease progression and treatment response. However, urine biomarkers could address this need. Proteomic analysis of CLN2 patient urine revealed activation of immune response pathways and pathways associated with the unfolded protein response. Analysis of CLN5 and CLN6 sheep model urine showed subtle changes. To confirm and investigate the relevance of candidate biomarkers a targeted LC-MS/MS proteomic assay was created. We applied this assay to additional CLN2 samples as well as other patients with NCL (CLN1, CLN3, CLN5, CLN6, and CLN7) and demonstrated that hexosaminidase-A, aspartate aminotransferase-1, and LAMP1 are increased in NCL samples and betaine-homocysteine S-methyltransferase-1 was specifically increased in patients with CLN2. These proteins could be used to monitor the effectiveness of future therapies aimed at treating systemic NCL disease.
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http://dx.doi.org/10.1016/j.isci.2020.102020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822952PMC
February 2021

Computed tomography provides enhanced techniques for longitudinal monitoring of progressive intracranial volume loss associated with regional neurodegeneration in ovine neuronal ceroid lipofuscinoses.

Brain Behav 2018 09 23;8(9):e01096. Epub 2018 Aug 23.

Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, New Zealand.

Introduction: The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well-established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral-mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans.

Methods: Computed tomography (CT)-based three-dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL.

Results: ICVs of NCL-affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5 sheep and 11-15 months in CLN6 sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (-12; 23).

Conclusions: Computed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.
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http://dx.doi.org/10.1002/brb3.1096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160654PMC
September 2018

Longitudinal In Vivo Monitoring of the CNS Demonstrates the Efficacy of Gene Therapy in a Sheep Model of CLN5 Batten Disease.

Mol Ther 2018 10 17;26(10):2366-2378. Epub 2018 Jul 17.

Department of Molecular Biosciences, Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand; Department of Radiology, University of Otago, Christchurch 8140, New Zealand. Electronic address:

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.
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http://dx.doi.org/10.1016/j.ymthe.2018.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171082PMC
October 2018

Practical implications of motion correction with motion insensitive radial k-space acquisitions in MRI.

Br J Radiol 2018 Jul 23;91(1087):20170593. Epub 2018 Mar 23.

1 New Zealand Brain Research Institute , Christchurch , New Zealand.

Objective: To highlight specific instances when radial k-space acquisitions in MRI result in image artifacts and how to ameliorate such artifacts.

Methods: We acquired axial T weighted MR images on (1) the American College of Radiology (ACR) phantom and (2) a sedated sheep with rectilinear and multiblade radial k-space filling acquisitions. Images were acquired on four (2 × 1.5T and 2 × 3T) different MRI scanners. For the radial k-space acquisitions, we acquired images with and without motion correction. All images were visually inspected for the presence of artifact.

Results: Images collected via the conventional rectilinear method were of diagnostic quality and free of artifact. Both ACR and sheep images acquired with radial k-space acquisitions and motion correction suffered significant artifact at different slice locations, scan sessions and across all the four scanners. Severity of the artifact was associated with echo train length. However, the artifact was eliminated when motion correction was not employed.

Conclusion: When little to no motion is present, the use of motion correction with radial k-space acquisitions can compromise image quality. However, image quality is quickly improved, and the artifact eliminated, by repeating the scan without motion correction or by using a conventional rectilinear alternative. Advances in Knowledge: By improving awareness and understanding of this artifact, MRI users will be able to adjust MRI protocols, resulting in more successful scanning sessions, better image quality, fewer call backs and increased diagnostic confidence.
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http://dx.doi.org/10.1259/bjr.20170593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221774PMC
July 2018

An EEG Investigation of Sleep Homeostasis in Healthy and CLN5 Batten Disease Affected Sheep.

J Neurosci 2016 08;36(31):8238-49

Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, United Kingdom,

Unlabelled: Sheep have large brains with human-like anatomy, making them a useful species for studying brain function. Sleep homeostasis has not been studied in sheep. Here, we establish correlates of sleep homeostasis in sheep through a sleep deprivation experiment. We then use these correlates to elucidate the nature of sleep deficits in a naturally occurring ovine model of neuronal ceroid lipofuscinosis (NCL, Batten disease) caused by a mutation in CLN5 In humans, mutations in this gene lead to cortical atrophy and blindness, as well as sleep abnormalities. We recorded electroencephalograms (EEGs) from unaffected and early stage CLN5(-/-) (homozygous, affected) sheep over 3 consecutive days, the second day being the sleep deprivation day. In unaffected sheep, sleep deprivation led to increased EEG delta (0.5-4 Hz) power during non-rapid eye movement (NREM) sleep, increased time spent in the NREM sleep state, and increased NREM sleep bout length. CLN5(-/-) sheep showed comparable increases in time spent in NREM sleep and NREM sleep bout duration, verifying the presence of increased sleep pressure in both groups. Importantly, CLN5(-/-) sheep did not show the increase in NREM sleep delta power seen in unaffected sheep. This divergent delta power response is consistent with the known cortical degeneration in CLN5(-/-) sheep. We conclude that, whereas sleep homeostasis is present in CLN5(-/-) sheep, underlying CLN5(-/-) disease processes prevent its full expression, even at early stages. Such deficits may contribute to early abnormalities seen in sheep and patients and warrant further study.

Significance Statement: Sleep abnormalities pervade most neurological diseases, including the neuronal ceroid lipofuscinoses (NCLs). Here, we show that, in an ovine model of a variant late-infantile NCL, there is abnormal expression of sleep homeostasis. Whereas some sleep pressure correlates respond to sleep deprivation, the strongest electroencephalogram (EEG) correlate of sleep pressure, non-REM delta power, failed to increase. This highlights the relevance of sleep deficits in this disease, in which the drive for sleep exists but the underlying disease prevents its full expression. Sleep abnormalities could contribute to early disease symptoms such as behavioral disorder and cognitive decline. Our study also shows sleep homeostatic EEG correlates in sheep, opening up new opportunities for studying sleep in a large social mammal with complex human-like brain neuroanatomy.
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http://dx.doi.org/10.1523/JNEUROSCI.4295-15.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601950PMC
August 2016

Molecular neuropathology of the synapse in sheep with CLN5 Batten disease.

Brain Behav 2015 Nov 9;5(11):e00401. Epub 2015 Oct 9.

Centre for Integrative Physiology University of Edinburgh Hugh Robson Building Edinburgh UK ; Euan MacDonald Centre for Motor Neurone Disease Research University of Edinburgh Hugh Robson Building Edinburgh UK.

Aims: Synapses represent a major pathological target across a broad range of neurodegenerative conditions. Recent studies addressing molecular mechanisms regulating synaptic vulnerability and degeneration have relied heavily on invertebrate and mouse models. Whether similar molecular neuropathological changes underpin synaptic breakdown in large animal models and in human patients with neurodegenerative disease remains unclear. We therefore investigated whether molecular regulators of synaptic pathophysiology, previously identified in Drosophila and mouse models, are similarly present and modified in the brain of sheep with CLN5 Batten disease.

Methods: Gross neuropathological analysis of CLN5 Batten disease sheep and controls was used alongside postmortem MRI imaging to identify affected brain regions. Synaptosome preparations were then generated and quantitative fluorescent Western blotting used to determine and compare levels of synaptic proteins.

Results: The cortex was particularly affected by regional neurodegeneration and synaptic loss in CLN5 sheep, whilst the cerebellum was relatively spared. Quantitative assessment of the protein content of synaptosome preparations revealed significant changes in levels of seven out of eight synaptic neurodegeneration proteins investigated in the motor cortex, but not cerebellum, of CLN5 sheep (α-synuclein, CSP-α, neurofascin, ROCK2, calretinin, SIRT2, and UBR4).

Conclusions: Synaptic pathology is a robust correlate of region-specific neurodegeneration in the brain of CLN5 sheep, driven by molecular pathways similar to those reported in Drosophila and rodent models. Thus, large animal models, such as sheep, represent ideal translational systems to develop and test therapeutics aimed at delaying or halting synaptic pathology for a range of human neurodegenerative conditions.
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http://dx.doi.org/10.1002/brb3.401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667763PMC
November 2015

Rapid and Progressive Regional Brain Atrophy in CLN6 Batten Disease Affected Sheep Measured with Longitudinal Magnetic Resonance Imaging.

PLoS One 2015 10;10(7):e0132331. Epub 2015 Jul 10.

Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, UK.

Variant late-infantile Batten disease is a neuronal ceroid lipofuscinosis caused by mutations in CLN6. It is a recessive genetic lysosomal storage disease characterised by progressive neurodegeneration. It starts insidiously and leads to blindness, epilepsy and dementia in affected children. Sheep that are homozygous for a natural mutation in CLN6 have an ovine form of Batten disease Here, we used in vivo magnetic resonance imaging to track brain changes in 4 unaffected carriers and 6 affected Batten disease sheep. We scanned each sheep 4 times, between 17 and 22 months of age. Cortical atrophy in all sheep was pronounced at the baseline scan in all affected Batten disease sheep. Significant atrophy was also present in other brain regions (caudate, putamen and amygdala). Atrophy continued measurably in all of these regions during the study. Longitudinal MRI in sheep was sensitive enough to measure significant volume changes over the relatively short study period, even in the cortex, where nearly 40% of volume was already lost at the start of the study. Thus longitudinal MRI could be used to study the dynamics of progression of neurodegenerative changes in sheep models of Batten disease, as well as to assess therapeutic efficacy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132331PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498759PMC
April 2016

The relevance of the storage of subunit c of ATP synthase in different forms and models of Batten disease (NCLs).

Authors:
David N Palmer

Biochim Biophys Acta 2015 Oct 17;1852(10 Pt B):2287-91. Epub 2015 Jun 17.

Molecular Biosciences, Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 85084, Lincoln 7647, New Zealand; BARN (www.BARN.org.nz). Electronic address:

The discoveries of specific protein storage in the NCLs, particularly of subunit c of ATP synthase in most, and the sphingolipid activator proteins, SAPs or saposins A and D in CLN1, CLN10 and an unassigned form are reviewed. The subunit c stored in the relevant NCLs is the complete mature molecule including an unusual modification found only in animal species, trimethylation of its lysine-43. Because of its strongly hydrophobic and lipid-like properties subunit c is easily overlooked or incorrectly described. This is becoming more of a problem as subunit c is not detected in standard proteomic investigations. Methods are reviewed that allow its unequivocal characterisation. Subunit c storage and cellular storage body accumulation do not cause the neuropathology characteristic of these diseases. The function of the trimethyl group on lysine-43 of subunit c is considered, along with some indications of where its normal turnover may be disrupted in the NCLs.
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http://dx.doi.org/10.1016/j.bbadis.2015.06.014DOI Listing
October 2015

Recent studies of ovine neuronal ceroid lipofuscinoses from BARN, the Batten Animal Research Network.

Biochim Biophys Acta 2015 Oct 12;1852(10 Pt B):2279-86. Epub 2015 Jun 12.

Molecular Biosciences, Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand; Batten Animal Research Network (BARN) (www.BARN.org.nz).

Studies on naturally occurring New Zealand and Australian ovine models of the neuronal ceroid-lipofuscinoses (Batten disease, NCLs) have greatly aided our understanding of these diseases. Close collaborations between the New Zealand groups at Lincoln University and the University of Otago, Dunedin, and a group at the University of Sydney, Australia, led to the formation of BARN, the Batten Animal Research Network. This review focusses on presentations at the 14th International Conference on Neuronal Ceroid Lipofuscinoses (Batten Disease), recent relevant background work, and previews of work in preparation for publication. Themes include CLN5 and CLN6 neuronal cell culture studies, studies on tissues from affected and control animals and whole animal in vivo studies. Topics include the effect of a CLN6 mutation on endoplasmic reticulum proteins, lysosomal function and the interactions of CLN6 with other lysosomal activities and trafficking, scoping gene-based therapies, a molecular dissection of neuroinflammation, identification of differentially expressed genes in brain tissue, an attempted therapy with an anti-inflammatory drug in vivo and work towards gene therapy in ovine models of the NCLs. This article is part of a Special Issue entitled: "Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease)".
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http://dx.doi.org/10.1016/j.bbadis.2015.06.013DOI Listing
October 2015

Translational neurophysiology in sheep: measuring sleep and neurological dysfunction in CLN5 Batten disease affected sheep.

Brain 2015 Apr 26;138(Pt 4):862-74. Epub 2015 Feb 26.

1 Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK

Creating valid mouse models of slowly progressing human neurological diseases is challenging, not least because the short lifespan of rodents confounds realistic modelling of disease time course. With their large brains and long lives, sheep offer significant advantages for translational studies of human disease. Here we used normal and CLN5 Batten disease affected sheep to demonstrate the use of the species for studying neurological function in a model of human disease. We show that electroencephalography can be used in sheep, and that longitudinal recordings spanning many months are possible. This is the first time such an electroencephalography study has been performed in sheep. We characterized sleep in sheep, quantifying characteristic vigilance states and neurophysiological hallmarks such as sleep spindles. Mild sleep abnormalities and abnormal epileptiform waveforms were found in the electroencephalographies of Batten disease affected sheep. These abnormalities resemble the epileptiform activity seen in children with Batten disease and demonstrate the translational relevance of both the technique and the model. Given that both spontaneous and engineered sheep models of human neurodegenerative diseases already exist, sheep constitute a powerful species in which longitudinal in vivo studies can be conducted. This will advance our understanding of normal brain function and improve our capacity for translational research into neurological disorders.
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http://dx.doi.org/10.1093/brain/awv026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014075PMC
April 2015

Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan adenosine triphosphate (ATP) synthases.

Mol Cell Proteomics 2015 Apr 21;14(4):828-40. Epub 2015 Jan 21.

From the ‡Mitochondrial Biology Unit, Medical Research Council, Hills Road, Cambridge, CB2 0XY, United Kingdom and

The rotors of ATP synthases turn about 100 times every second. One essential component of the rotor is a ring of hydrophobic c-subunits in the membrane domain of the enzyme. The rotation of these c-rings is driven by a transmembrane proton-motive force, and they turn against a surface provided by another membrane protein, known as subunit a. Together, the rotating c-ring and the static subunit a provide a pathway for protons through the membrane in which the c-ring and subunit a are embedded. Vertebrate and invertebrate c-subunits are well conserved. In the structure of the bovine F1-ATPase-c-ring subcomplex, the 75 amino acid c-subunit is folded into two transmembrane α-helices linked by a short loop. Each bovine rotor-ring consists of eight c-subunits with the N- and C-terminal α-helices forming concentric inner and outer rings, with the loop regions exposed to the phospholipid head-group region on the matrix side of the inner membrane. Lysine-43 is in the loop region and its ε-amino group is completely trimethylated. The role of this modification is unknown. If the trimethylated lysine-43 plays some important role in the functioning, assembly or degradation of the c-ring, it would be expected to persist throughout vertebrates and possibly invertebrates also. Therefore, we have carried out a proteomic analysis of c-subunits across representative species from different classes of vertebrates and from invertebrate phyla. In the twenty-nine metazoan species that have been examined, the complete methylation of lysine-43 is conserved, and it is likely to be conserved throughout the more than two million extant metazoan species. In unicellular eukaryotes and prokaryotes, when the lysine is conserved it is unmethylated, and the stoichiometries of c-subunits vary from 9-15. One possible role for the trimethylated residue is to provide a site for the specific binding of cardiolipin, an essential component of ATP synthases in mitochondria.
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http://dx.doi.org/10.1074/mcp.M114.047456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4390263PMC
April 2015

Inhibition of storage pathology in prenatal CLN5-deficient sheep neural cultures by lentiviral gene therapy.

Neurobiol Dis 2014 Feb 19;62:543-50. Epub 2013 Nov 19.

Faculty of Agriculture and Life Sciences, PO Box 85084, Lincoln University, Lincoln 7647, New Zealand. Electronic address:

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are inherited neurodegenerative lysosomal storage diseases caused by mutations in several different genes. Mutations in CLN5 cause a variant late-infantile human disease and some cases of juvenile and adult clinical disease. NCLs also occur in animals, and a flock of New Zealand Borderdale sheep with a CLN5 splice-site mutation has been developed for model studies. Dissociated mixed neural cells from CLN5-deficient foetal sheep brains contained no obvious storage bodies at plating but these accumulated rapidly in culture, mainly in microglial cells and also in neurons and astrocytes. Accumulation was very obvious after a week, as monitored by fluorescent microscopy and immunostaining for subunit c of mitochondrial ATP synthase. Photography at intervals revealed the dynamic nature of the cultures and a flow of storage bodies between cells, specifically the phagocytosis of storage-body containing cells by microglia and incorporation of the storage bodies into the host cells. No storage was observed in cultured control cells. Transduction of cell cultures with a lentiviral vector expressing a C-terminal Myc tagged CLN5 resulted in secretion of post-translationally glycosylated and processed CLN5. Transduction of CLN5-deficient cultures with this construct rapidly reversed storage body accumulation, to less than half in only six days. These results show that storage body accumulation is reversible with enzyme correction and support the use of these cultures for testing of therapeutics prior to whole animal studies.
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http://dx.doi.org/10.1016/j.nbd.2013.11.011DOI Listing
February 2014

Chronic oral administration of minocycline to sheep with ovine CLN6 neuronal ceroid lipofuscinosis maintains pharmacological concentrations in the brain but does not suppress neuroinflammation or disease progression.

J Neuroinflammation 2013 Jul 30;10:97. Epub 2013 Jul 30.

Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 85084, Lincoln 7647, New Zealand.

Background: The neuronal ceroid lipofuscinoses (NCLs; or Batten disease) are fatal inherited human neurodegenerative diseases affecting an estimated 1:12,500 live births worldwide. They are caused by mutations in at least 11 different genes. Currently, there are no effective treatments. Progress into understanding pathogenesis and possible therapies depends on studying animal models. The most studied animals are the CLN6 South Hampshire sheep, in which the course of neuropathology closely follows that in affected children. Neurodegeneration, a hallmark of the disease, has been linked to neuroinflammation and is consequent to it. Activation of astrocytes and microglia begins prenatally, starting from specific foci associated with the later development of progressive cortical atrophy and the development of clinical symptoms, including the occipital cortex and blindness. Both neurodegeneration and neuroinflammation generalize and become more severe with increasing age and increasing clinical severity. The purpose of this study was to determine if chronic administration of an anti-inflammatory drug, minocycline, from an early age would halt or reverse the development of disease.

Method: Minocycline, a tetracycline family antibiotic with activity against neuroinflammation, was tested by chronic oral administration of 25 mg minocycline/kg/day to presymptomatic lambs affected with CLN6 NCL at 3 months of age to 14 months of age, when clinical symptoms are obvious, to determine if this would suppress neuroinflammation or disease progression.

Results: Minocycline was absorbed without significant rumen biotransformation to maintain pharmacological concentrations of 1 μM in plasma and 400 nM in cerebrospinal fluid, but these did not result in inhibition of microglial activation or astrocytosis and did not change the neuronal loss or clinical course of the disease.

Conclusion: Oral administration is an effective route for drug delivery to the central nervous system in large animals, and model studies in these animals should precede highly speculative procedures in humans. Minocycline does not inhibit a critical step in the neuroinflammatory cascade in this form of Batten disease. Identification of the critical steps in the neuroinflammatory cascade in neurodegenerative diseases, and targeting of specific drugs to them, will greatly increase the likelihood of success.
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http://dx.doi.org/10.1186/1742-2094-10-97DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733893PMC
July 2013

NCL disease mechanisms.

Biochim Biophys Acta 2013 Nov 23;1832(11):1882-93. Epub 2013 May 23.

Department of Wine, Food and Molecular Biosciences, Faculty of Agricultural and Life Sciences, Lincoln University, PO Box 85084, Lincoln 7647, Christchurch, New Zealand. Electronic address:

Despite the identification of a large number of disease-causing genes in recent years, it is still unclear what disease mechanisms operate in the neuronal ceroid lipofuscinoses (NCLs, Batten disease). As a group they are defined by the specific accumulation of protein, either subunit c of mitochondrial ATP synthase or SAPs A and D in lysosome-derived organelles, and regionally specific neurodegeneration. Evidence from biochemical and cell biology studies indicates related lesions in intracellular vesicle trafficking and lysosomal function. There is also extensive immunohistological evidence of a causative role of disease associated neuroinflammation. However the nature of these lesions is not clear nor is it clear why they lead to the defining pathology. Several different theories have proposed a range of potential mechanisms, but it remains to be determined which are central to pathogenesis, and whether there is a mechanism consistent across the group, or if it differs between disease forms. This review summarises the evidence that is currently available and the progress that has been made in understanding these profoundly disabling disorders. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.
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http://dx.doi.org/10.1016/j.bbadis.2013.05.014DOI Listing
November 2013

Increased zinc and manganese in parallel with neurodegeneration, synaptic protein changes and activation of Akt/GSK3 signaling in ovine CLN6 neuronal ceroid lipofuscinosis.

PLoS One 2013 14;8(3):e58644. Epub 2013 Mar 14.

Department of Pathology, The University of Melbourne, Victoria, Australia.

Mutations in the CLN6 gene cause a variant late infantile form of neuronal ceroid lipofuscinosis (NCL; Batten disease). CLN6 loss leads to disease clinically characterized by vision impairment, motor and cognitive dysfunction, and seizures. Accumulating evidence suggests that alterations in metal homeostasis and cellular signaling pathways are implicated in several neurodegenerative and developmental disorders, yet little is known about their role in the NCLs. To explore the disease mechanisms of CLN6 NCL, metal concentrations and expression of proteins implicated in cellular signaling pathways were assessed in brain tissue from South Hampshire and Merino CLN6 sheep. Analyses revealed increased zinc and manganese concentrations in affected sheep brain in those regions where neuroinflammation and neurodegeneration first occur. Synaptic proteins, the metal-binding protein metallothionein, and the Akt/GSK3 and ERK/MAPK cellular signaling pathways were also altered. These results demonstrate that altered metal concentrations, synaptic protein changes, and aberrant modulation of cellular signaling pathways are characteristic features in the CLN6 ovine form of NCL.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058644PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597713PMC
September 2013

The locus for an inherited cataract in sheep maps to ovine chromosome 6.

Mol Vis 2012 31;18:1384-94. Epub 2012 May 31.

Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln, New Zealand.

Purpose: Cataracts are an important cause of blindness in humans but there are few large animal models available. One of these animal models is Ovine Heritable Cataract, a bilateral cortical cataract which develops after birth. This cataract has been used as a model for human cataracts in drug trials, but the gene responsible for the cataract trait is unknown. A genetic test for cataract would improve the efficiency of the model by predicting which animals would develop cataracts. Identifying the genetic basis of the cataract would indicate its relevance to human cataract.

Methods: A genome scan was performed on 20 sheep chromosomes, representing 86% of the genome, to determine the position of the cataract locus. Additional microsatellite markers were tested on chromosome 6 using a larger pedigree. Fine mapping was performed using a breakpoint panel of 36 animals and novel microsatellite markers taken from the bovine genome assembly. All exons of the candidate gene nudix (nucleoside diphosphate linked moiety X)-type motif 9 (NUDT9) were sequenced in normal and affected sheep.

Results: Significant linkage was found between cataract status and markers on chromosome 6. Linkage analysis on the larger pedigree showed the most likely position of the cataract locus was between 112.3 and 132.9 cM from the centromere. During fine mapping, NUDT9 was considered as a positional candidate for the cataract gene because it was located within the linked interval and is expressed in the lens. The gene was ruled out as the cataract gene after extensive genotype analysis, but a single nucleotide polymorphism (SNP) inside it provided a useful restriction fragment length polymorphism (RFLP) marker for further fine mapping. Twelve new markers were found and used to map the cataract locus to between 131.1 and 131.8 cM from the centromere.

Conclusions: A region of ovine chromosome 6 strongly linked to cataract has been identified, and a genetic test for cataract based on a SNP within this region has been developed. The best candidate gene within this region is AF4/FMR2 family, member 1 (AFF1), the mouse equivalent of which is associated with an inherited cataract.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370893PMC
November 2012

Lentiviral-mediated gene transfer to the sheep brain: implications for gene therapy in Batten disease.

Hum Gene Ther 2011 Aug 19;22(8):1011-20. Epub 2011 May 19.

Department of Biochemistry, School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand.

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are inherited neurodegenerative lysosomal storage diseases with common clinical features of blindness and seizures culminating in premature death. Gene-therapy strategies for these diseases depend on whether the missing activity is a secreted lysosomal protein taken up by neighboring cells, or an intramembrane protein that requires careful targeting. Therapies are best developed in animal models with large complex human-like brains. Lentiviral-mediated gene delivery to neural cell cultures from normal sheep and sheep affected with an NCL resulted in green fluorescent protein (GFP) expression in neurons and neuroblasts, more efficiently than in astrocytes. Similar transgene expression was obtained from two constitutive promoters, the viral MND promoter and the human EF1α promoter. In vivo studies showed stable and persistent GFP expression throughout the cell bodies, axons, and dendrites from intracortical injections and indicated ependymal and subependymal transduction. The sheep showed no ill effects from the injections. These data support continuing gene-therapy trials in the sheep models of Batten disease.
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http://dx.doi.org/10.1089/hum.2011.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159522PMC
August 2011

The specific loss of GnRH-positive neurons from the hypothalamus of sheep with CLN6 neuronal ceroid lipofuscinosis occurs without glial activation and has only minor effects on reproduction.

Neurobiol Dis 2011 Mar 24;41(3):614-23. Epub 2010 Nov 24.

Faculty of Agriculture and Life Sciences, PO Box 84, Lincoln University, Lincoln 7647, New Zealand.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are characterized by progressive neurodegeneration resulting in widespread brain atrophy. Each form is assumed to be the consequence of some universal intracellular event; however, time course studies on the cerebral cortex of a sheep model of the CLN6 form revealed distinct regional neurodegeneration preceded by regional glial activation, spreading from quite localized foci. Previous neurological investigations have concentrated on obviously affected cortical functions. This study investigated the impact of ovine CLN6 NCL on a subcortical structure and function, the discrete gonadotrophin-releasing hormone (GnRH) secreting neurons of the hypothalamus, and the effect of changes in the neuroendocrine system on reproductive efficiency and embryonic development. The number of immunopositive GnRH neurons in the hypothalamus and median eminence of affected sheep was reduced by 80%, but the rest of the hypothalamus showed no changes or atrophy. This specific loss of neuron type was not accompanied by either microglial or astrocyte activation, which was absent from the hypothalamus and was not associated with cell-type-specific storage body accumulation. Ovarian responsiveness to follicle stimulating hormone, ovulation rates, sperm production, fertilization rates, embryonic development, and reproductive efficiency were sub-par but reproduction was still functional. This remains when the sheep are profoundly blind. We conclude that physiological functionality and connectivity, not genotype, determine neuron fate in CLN6 NCL.
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http://dx.doi.org/10.1016/j.nbd.2010.11.008DOI Listing
March 2011

Neuropeptide changes and neuroactive amino acids in CSF from humans and sheep with neuronal ceroid lipofuscinoses (NCLs, Batten disease).

Neurochem Int 2009 Dec 5;55(8):783-8. Epub 2009 Aug 5.

Agriculture and Life Sciences Faculty, Lincoln University, Lincoln 7647, New Zealand.

Anomalies in neuropeptides and neuroactive amino acids have been postulated to play a role in neurodegeneration in a variety of diseases including the inherited neuronal ceroid lipofuscinoses (NCLs, Batten disease). These are often indicated by concentration changes in cerebrospinal fluid (CSF). Here we compare CSF neuropeptide concentrations in patients with the classical juvenile CLN3 form of NCL and the classical late infantile CLN2 form with neuropeptide and neuroactive amino acid concentrations in CSF from sheep with the late infantile variant CLN6 form. A marked disease related increase in CSF concentrations of neuron specific enolase and tau protein was noted in the juvenile CLN3 patients but this was not observed in an advanced CLN2 patient nor CLN6 affected sheep. No changes were noted in S-100b, GFAP or MBP in patients or of S-100b, GFAP or IGF-1 in affected sheep. There were no disease related changes in CSF concentrations of the neuroactive amino acids, aspartate, glutamate, serine, glutamine, glycine, taurine and GABA in these sheep. The changes observed in the CLN3 patients may be progressive markers of neurodegeneration, or of underlying metabolic changes perhaps associated with CLN3 specific changes in neuroactive amino acids, as have been postulated. The lack of changes in the CLN2 and CLN6 subjects indicate that these changes are not shared by the CLN2 or CLN6 forms and changes in CSF concentrations of these compounds are unreliable as biomarkers of neurodegeneration in the NCLs in general.
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http://dx.doi.org/10.1016/j.neuint.2009.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2764820PMC
December 2009

Location and connectivity determine GABAergic interneuron survival in the brains of South Hampshire sheep with CLN6 neuronal ceroid lipofuscinosis.

Neurobiol Dis 2008 Oct 25;32(1):50-65. Epub 2008 Jun 25.

Agriculture and Life Sciences Division, Lincoln University, Lincoln 7647, New Zealand.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are fatal inherited neurodegenerative diseases. Sheep affected with the CLN6 form provide a valuable model to investigate underlying disease mechanisms from preclinical stages. Excitatory neuron loss in these sheep is markedly regional, localized early reactive changes accurately predicting neuron loss and subsequent symptom development. This investigation of GABAergic interneuron loss revealed similar regional effects that correlate with symptoms. Loss of parvalbumin positive neurons from the affected cortex was apparent at four months and became profound by 19 months, as was somatostatin positive neuron loss to a lesser extent. Conversely calbindin and neuropeptide Y positive neurons were relatively preserved and calretinin staining temporarily increased. Staining of subcortical regions was more intense but subcortical architecture remained relatively intact. Discrete subcortical changes followed from cortical changes in interconnected regions. These data highlight cellular location and interconnectivity as the major determinants of neuron survival, rather than phenotype.
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http://dx.doi.org/10.1016/j.nbd.2008.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2647510PMC
October 2008

A new large animal model of CLN5 neuronal ceroid lipofuscinosis in Borderdale sheep is caused by a nucleotide substitution at a consensus splice site (c.571+1G>A) leading to excision of exon 3.

Neurobiol Dis 2008 Feb 29;29(2):306-15. Epub 2007 Sep 29.

Lincoln University, Agriculture and Life Sciences Division, Cell Biology Group, PO Box 84, Lincoln 7647, Canterbury, New Zealand.

Batten disease (neuronal ceroid lipofuscinoses, NCLs) are a group of inherited childhood diseases that result in severe brain atrophy, blindness and seizures, leading to premature death. To date, eight different genes have been identified, each associated with a different form. Linkage analysis indicated a CLN5 form in a colony of affected New Zealand Borderdale sheep. Sequencing studies established the disease-causing mutation to be a substitution at a consensus splice site (c.571+1G>A), leading to the excision of exon 3 and a truncated putative protein. A molecular diagnostic test has been developed based on the excision of exon 3. Sequence alignments support the gene product being a soluble lysosomal protein. Western blotting of isolated storage bodies indicates the specific storage of subunit c of mitochondrial ATP synthase. This flock is being expanded as a large animal model for mechanistic studies and trial therapies.
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http://dx.doi.org/10.1016/j.nbd.2007.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2249613PMC
February 2008

Metabolomic investigation of CLN6 neuronal ceroid lipofuscinosis in affected South Hampshire sheep.

J Neurosci Res 2007 Nov;85(15):3494-504

Department of Biochemistry, University of Cambridge, Cambridge, UK.

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal inherited neurodegenerative diseases in humans and animals distinguished by a common clinical pathology, characteristic storage body accumulation in cells, and gross brain atrophy. An (1)H NMR spectroscopy- and GC-MS-based metabolomic investigation of changes in the cerebellum, frontal and occipital lobes, and cerebrospinal fluid (CSF) of CLN6 NCL affected South Hampshire sheep charted changes from the preclinical state to advanced disease. Glutamine and succinate concentrations increased in all brain regions in affected sheep relative to controls, whereas concentrations of aspartate, acetate, glutamate, N-acetyl aspartate (NAA), and gamma-aminobutyric acid (GABA) decreased. Changes in the concentrations of inositols, NAA, and GABA were consistent with glial cell activation and neurodegeneration beginning in the frontal and occipital lobes, in agreement with previous histopathological data. Further metabolic deficits were defined in all regions at earlier time points, including the cerebellum, where very little neurological degeneration has been reported. Biochemical abnormalities in the CSF of affected sheep at 18-31 months include relative increases in lactate, acetate, tyrosine, and creatine/creatinine concentrations and decreases in myo- and scyllo-inositol and citrate concentrations. The changes detected in the CSF and brain tissue mirrored those previously apparent in NCL mouse models, suggesting that they are common to all NCLs. However, the changes in glutamate and glutamine concentrations in CSF occurred after clinical disease, indicating that any changes in glutamate/glutamine cycling occur as a consequence of the primary deficits associated with the NCLs.
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http://dx.doi.org/10.1002/jnr.21343DOI Listing
November 2007

A missense mutation (c.184C>T) in ovine CLN6 causes neuronal ceroid lipofuscinosis in Merino sheep whereas affected South Hampshire sheep have reduced levels of CLN6 mRNA.

Biochim Biophys Acta 2006 Oct 12;1762(10):898-905. Epub 2006 Sep 12.

Centre for Advanced Technologies in Animal Genetics and Reproduction (Reprogen), Faculty of Veterinary Science, The University of Sydney, PMB3, Camden, NSW, Australia.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal recessively inherited neurodegenerative diseases of humans and animals characterised by common clinical signs and pathology. These include blindness, ataxia, dementia, behavioural changes, seizures, brain and retinal atrophy and accumulation of fluorescent lysosome derived organelles in most cells. A number of different variants have been suggested and seven different causative genes identified in humans (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8 and CTSD). Animal models have played a central role in the investigation of this group of diseases and are extremely valuable for developing a better understanding of the disease mechanisms and possible therapeutic approaches. Ovine models include flocks of affected New Zealand South Hampshires and Borderdales and Australian Merinos. The ovine CLN6 gene has been sequenced in a representative selection of these sheep. These investigations unveiled the mutation responsible for the disease in Merino sheep (c.184C>T; p.Arg62Cys) and three common ovine allelic variants (c.56A>G, c.822G>A and c.933_934insCT). Linkage analysis established that CLN6 is the gene most likely to cause NCL in affected South Hampshire sheep, which do not have the c.184C>T mutation but show reduced expression of CLN6 mRNA in a range of tissues as determined by real-time PCR. Lack of linkage precludes CLN6 as a candidate for NCL in Borderdale sheep.
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http://dx.doi.org/10.1016/j.bbadis.2006.09.004DOI Listing
October 2006

Neuronal ceroid lipofuscinosis in Devon cattle is caused by a single base duplication (c.662dupG) in the bovine CLN5 gene.

Biochim Biophys Acta 2006 Oct 25;1762(10):890-7. Epub 2006 Jul 25.

Centre for Advanced Technologies in Animal Genetics and Reproduction (Reprogen), Faculty of Veterinary Science, The University of Sydney, PMB3, Camden NSW, Australia.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are recessively inherited neurodegenerative disorders that affect humans and other animals, characterised by brain atrophy and the accumulation of lysosome derived fluorescent storage bodies in neurons and most other cells. Common clinical signs include blindness, ataxia, dementia, seizures and premature death. The associated genes for six different human forms have been identified (CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8), and three other human forms suggested (CLNs 4, 7 and 9). A form of NCL in Australian Devon cattle is caused by a single base duplication (c.662dupG) in bovine CLN5. This mutation causes a frame-shift and premature termination (p.Arg221GlyfsX6) which is predicted to result in a severely truncated protein, analogous to disease causing mutations in human Finnish late infantile variant NCL (CLN5), and a simple genetic diagnostic test has been developed. The symptoms and disease course in cattle also matches CLN5. Only one initiation site was found in the bovine gene, equivalent to the third of four possible initiation sites in the human gene. As cattle are anatomically and physiologically similar to humans with a human-like central nervous system and easy to maintain and breed, they provide a valuable alternative model for CLN5 studies.
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http://dx.doi.org/10.1016/j.bbadis.2006.07.008DOI Listing
October 2006

Activation of non-neuronal cells within the prenatal developing brain of sheep with neuronal ceroid lipofuscinosis.

Brain Pathol 2006 Apr;16(2):110-6

Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealand.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are fatal inherited lysosomal storage diseases of children characterized by increasing blindness, seizures and profound neurodegeneration but the mechanisms leading to these pathological changes remain unclear. Sheep with a CLN6 form that have a human-like brain and disease progression are invaluable for studying pathogenesis. A study of preclinical pathology in these sheep revealed localized glial activation at only 12 days of age, particularly in cortical regions that subsequently degenerate. This has been extended by examining fetal tissue from 60 days of gestation onwards. A striking feature was the presence of reactive astrocytes and the hypertrophy and proliferation of perivascular cells noted within the developing white matter of the cerebral cortex 40 days before birth. Astrocytic activation was evident within the cortical gray matter 20 days before birth, and was confined to the superficial laminae 12 days after birth. Clusters of activated microglia were detected in upper neocortical gray matter laminae shortly after birth. Neuronal development in affected sheep was undisturbed at these early ages. This prenatal activation of non-neuronal cells within the affected brain indicates the onset of pathogenesis during brain development and that an ordered sequence of glial activation precedes neurodegeneration.
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http://dx.doi.org/10.1111/j.1750-3639.2006.00002.xDOI Listing
April 2006

The development and characterisation of complex ovine neuron cultures from fresh and frozen foetal neurons.

J Neurosci Methods 2006 Jul 17;155(1):98-108. Epub 2006 Feb 17.

Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealand.

Cultures of ovine cerebral and cerebellar neurons from mid-term sheep foetal brains, 9-15 weeks old, have been established for the first time. These foetal brains are relatively mature, being at similar stages of development as peri and post-natal rodent brains. Cultures were routinely maintained for 3-4 weeks, and longer. Nearly all the cells from the younger foetuses adhered as neurons. The proportion of glial cells increased with age, as did the risk of cultures being overtaken by glial cells. Cultured neurons were bipolar, tripolar and multipolar, similar to the morphologies of neurons in vivo. Older foetuses also yield more complex neurons, notably giant cells. Other properties of the cultured neurons also mimic in vivo observations, including neurite beading, complexity in neurotransmitter class (GABAergic and glutamatergic) and calcium binding protein (calbindin and calretinin) content. Single cell divisions of neurons were observed in younger cultures by time-lapse photography and the occurrence of telophase nuclei. The advantage of the high yield of genetically identical cells obtained from a single sheep foetus, 150 million, was extended by cryopreservation of neurons after snap freezing, and later culture. These cultures showed the same characteristics as cultures from the freshly plated cells.
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http://dx.doi.org/10.1016/j.jneumeth.2006.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1525139PMC
July 2006

Glial activation spreads from specific cerebral foci and precedes neurodegeneration in presymptomatic ovine neuronal ceroid lipofuscinosis (CLN6).

Neurobiol Dis 2005 Oct;20(1):49-63

Agriculture and Life Sciences Division, Lincoln University, PO Box 84, Canterbury, New Zealand.

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are fatal inherited neurodegenerative diseases characterized by gross brain atrophy, blindness, and intracellular accumulation of lysosome-derived storage bodies. A CLN6 form in sheep is studied as a large animal model of the human diseases. This study describes neuropathological changes in brains from presymptomatic affected sheep. Activated astrocytes and focal clusters of activated microglia were present in outer layers of occipital and somatosensory cortical regions as early as 12 days of age, together with activated perivascular macrophages. Astrocytic activation and progressive transformation of microglia to brain macrophages preceded neurodegeneration and spread to different cortical areas, most prominently in regions associated with clinical symptoms. In contrast, storage body accumulation was much more evenly spread across regions. These data support suggestions that neurodegeneration and storage body accumulation may be independent manifestations of CLN6 mutation and indicate that glial cell activation may be an important mediator in pathogenesis.
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http://dx.doi.org/10.1016/j.nbd.2005.01.025DOI Listing
October 2005

Lysine 43 is trimethylated in subunit C from bovine mitochondrial ATP synthase and in storage bodies associated with batten disease.

J Biol Chem 2004 May 9;279(21):21883-7. Epub 2004 Mar 9.

Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge, CB2 2XY, United Kingdom.

The hydrophobic membrane protein, subunit c, has been isolated from ATP synthase purified from bovine heart mitochondria. It has also been obtained from lysosomal storage bodies associated with ceroid lipofuscinosis from ovine liver and from human brain tissue of a victim of Batten disease. It is likely that the lysosomal protein has originated from the mitochondrion. These samples have been characterized by mass spectrometric methods. Irrespective of its source, subunit c has an intact molecular mass of 7650 Da, 42 Da greater than the value calculated from the amino acid sequence, and the protein has been modified post-translationally. In all three samples, the modification is associated with lysine 43, which lies in a polar loop region linking the two transmembrane alpha-helices of the protein. This residue is conserved throughout vertebrate sequences. The additional mass arises from trimethylation and not acetylation at the epsilon-N-position of the residue. These experiments show that the post-translational modification of subunit c is not, as has been suggested, an abnormal phenomenon associated with the etiology of Batten disease and ceroid lipofucinoses. Evidently, it occurs either before or during import of the protein into mitochondria or at a mitochondrial location after completion of the import process. The function of the trimethyllysine residue in the assembled ATP synthase complex is obscure. The residue and the modification are not conserved in all ATP synthases, and their role in the assembly and (or) functioning of the enzyme appear to be confined to higher organisms.
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http://dx.doi.org/10.1074/jbc.M402074200DOI Listing
May 2004