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Beneficial effects of anti-inflammatory therapy in a mouse model ofNiemann-Pick disease type C1
David Smith, Kerri-Lee Wallom, Ian M. Williams, Mylvaganam Jeyakumar ⁎, Frances M. Platt ⁎Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK
Article history:Received 1 June 2009Revised 10 July 2009Accepted 16 July 2009Available online 24 July 2009
Keywords:Niemann-Pick disease type CCNS inflammationNon-steroidal anti-inflammatory drugsNSAIDsMiglustat
Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal disorder characterized bysphingolipid and cholesterol storage in the late endocytic system. In common with other neurodegenerativediseases, activation of the innate immune system occurs in the brain resulting in neuro-inflammation.Targeting inflammation in the brain therefore represents a potential clinical intervention strategy that aimsto slow the rate of disease progression and improve quality of life.We evaluated non-steroidal anti-inflammatory drugs (NSAIDs) and an anti-oxidant to determine whetherthese agents are disease modifying in an acute mouse model of NPC1. NSAIDs significantly prolonged thelifespan of NPC1 mice and slowed the onset of clinical signs. However, anti-oxidant therapy was of nosignificant benefit. Combining NSAID therapy with substrate reduction therapy (SRT) resulted in additivebenefit. These data suggest that anti-inflammatory therapy may be a useful adjunctive treatment in theclinical management of NPC1, alone or combined with SRT.
Central nervous system (CNS) inflammation is a common featureof neurodegenerative diseases (Perry et al., 2007), includinglysosomal storage disorders of the brain. The hallmark of theneuro-inflammatory process is atypical microglial activation, prior tothe development of clinical signs (Jeyakumar et al., 2005; Jeyakumaret al., 2003; Wada et al., 2000). CNS inflammation signals therecruitment of peripheral monocytes that amplify the inflammatoryprocess (Wu and Proia, 2004). A key question is whether braininflammation plays a significant enough role in pathogenesis to be aviable target for adjunctive therapy (Jeyakumar et al., 2005).
Targeting inflammation is a viable therapeutic approach in thelysosomal disorder mouse model, Sandhoff disease (hexb−/−). Micenull for both hexb and macrophage-inflammatory-protein-1-alpha(MIP1α−/−) lived 30% longer than standard hexb−/− mice indicat-ing monocyte/macrophage-induced CNS inflammation contributesto the disease process (Wu and Proia, 2004). Treatment of hexb−/−
mice with non-steroidal anti-inflammatory drugs (NSAIDs) andanti-oxidants also increased life expectancy, despite therapy beinginitiated after macrophage infiltration of the CNS (Jeyakumar et al.,2004]. Monotherapy treatment with NSAIDS and anti-oxidants ledto a 12–23% increase in life expectancy and improved/protected
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neuromuscular function (Jeyakumar et al., 2004 #2561), while thecombination of NSAIDs with substrate reduction therapy (SRT),using the GSL biosynthesis inhibitor, miglustat (Platt et al., 1994),resulted in a synergistic improvement. Therefore, targeting morethan one aspect of disease pathology resulted in a better therapeuticoutcome (Jeyakumar et al., 2005).
Niemann-Pick disease type C1 (NPC) is a complex lysosomal dis-order resulting from defects in the late endosomal/lysosomal proteinNPC1 (Sturley et al., 2004). When this protein is defective a broadrange of lipids are stored including sphingolipids and cholesterol(Liscum, 2000). In NPC there is a unique defect in acidic store calciumas a result of sphingosine storage (Lloyd-Evans et al., 2008). Thefailure to release adequate levels of calcium from acidic stores causesa malfunction of late endosome/lysosome fusion and the secondarystorage of glycosphingolipids, sphingomyelin and cholesterol (Lloyd-Evans et al., 2008). Brain inflammation occurs in NPC, with microglialactivation and proliferation pre-dating the onset of clinical signs inthe NPC1 mouse (Baudry et al., 2003), suggesting that activation ofmicroglia/macrophages in the brain may contribute to NPC patho-genesis, as demonstrated in Sandhoff disease (Jeyakumar et al.,2004). Apoptosis in NPC1 mice correlates with up-regulation of theTNF-α death pathway, including caspase-8, FAD, TNFRp55, TRADDand RIP, suggesting apoptosis results from the pro-inflammatoryresponse to lysosomal storage in the brain (Wu et al., 2005). Anti-oxidant therapy showed a modest improvement in the NPC1 mouse(Bascunan-Castillo et al., 2004) suggesting that microglia-mediatedoxidative damage could be a factor in NPC pathology. The mechanismof action of neurosteroid therapy in NPC has been linked to anti-
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oxidant action in in vitro studies (Zampieri et al., 2008). Based onthese findings, we hypothesised that brain inflammation maycontribute to the pathology of NPC disease and may therefore beresponsive to anti-inflammatory therapy. In this study, the effects ofibuprofen, vitamin C and ibuprofen combined with miglustat havebeen evaluated.
Materials and methods
Mice
BALBc/NPCnih mice were bred as heterozygotes to generateNpc1−/− mice and control genotypes (Pentchev et al., 1980). Allmice were bred and housed under non-sterile conditions, withfood and water available ad lib. All animal studies were conductedusing protocols approved by the UK Home Office for the conduct ofregulated procedures under licence (Animal scientific ProceduresAct, 1986).
Fig. 1. Evaluation of GSL storage and inflammatory marker expression in the CNS of the Npc1PAS to demonstrate the glycosphingolipid storage in NPC1 brain. Note the lack of PAS stainedof PAS-positive cells in the NPC homozygous knockout (−/−) mouse brain indicates storage(C–D) demonstrates the lack of significant macrophage/B-cell infiltration in Npc1−/− miccerebellum (F). Blue staining: haematoxylin counterstain in all panels. Red staining in pane(developed with DAB).
Drug treatments
Aspirin (Sigma, 200 mg/kg/day) and ibuprofen (Sigma, 100 mg/kg/day) were supplemented as dry admix to powdered RM1 mousechow (SDS, UK) (starting at 6 weeks of age). Miglustat (600 mg/kg/day, Oxford GlycoSciences/Celltech, UK) was administered as dryadmix as above (starting at 3weeks of age). The anti-oxidant L-ascorbicacid (vitamin C, 2.5% in powdered chow) was administered from6 weeks of age. The untreated mice were fed on powdered chow.
Tissue processing
Mice were killed by CO2 asphyxiation, perfused through the leftcardiac ventricle with heparinised phosphate-buffered saline (PBS)and brain and spinal cord tissues were freeze-embedded in OCT(BDH) for sectioning. Coronal sections (10 μm) at 1 in 10 intervalswere collected onto glass microscope slides, air dried for 4–6 h andstored at −70 °C for histological examination.
−/− mice. 10-micron frozen brain sections of 8-week old cerebellumwere stained withcell bodies in NPC heterozygous (+/−) control mouse brain (A), while the high number. The arrows pinpoint examples of storage cells/regions. MHC class II (TIB-120) staininge, but CD68 staining (E–F) indicates massive CD68+ microglial activation in Npc1−/−
ls A–B: PAS. Brown staining: anti-MHC class II in panels C–D, anti-CD68 in panels E–F
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Periodic acid/Schiff reagent (PAS) staining
Frozen sections were warmed to room temperature (RT), fixed informalin–ethanol fixative (5 min, 3.7% formaldehyde in 95% ethanol),stained with periodic acid-Schiff (PAS) according to the manufac-turer's instructions (Sigma, Poole, Dorset, UK), counterstained withhaematoxylin (Gill No. 3, Vector Lab) and mounted in DePeX (BDH).
Immunohistochemistry (IHC)
Cryostat sections were fixed in 4% paraformaldehyde, andprocessed as described previously (Jeyakumar et al., 2003). Briefly,the sections were incubated at room temperature for 1 to 2 h withone of the following primary antibodies; MHC class II (1:10, MAbM5/114, rat Ig; ATCC TIB120), Rat anti-CD68 (1:100, Serotec) orRabbit anti-nitrotyrosine (1:500, Chemicon). The primary antibodieswere detected using one of the following species-specific secondaryantibodies: biotinylated rabbit anti-rat IgG (Vector Lab), biotinylatedmouse anti-rabbit IgG (Sigma). The secondary (biotinylated)antibodies were detected using ABC kit (Vector Lab) and developed
Fig. 2. Evaluation of CNS inflammation in Npc1−/− mice. Images of CD68 expression in cerebCD68+ signal is observed in control (Npc1+/−) brain tissue, while in Npc1−/− mice CD68+
with 3.3′-diaminobenzidine (DAB), and counterstained with hae-matoxylin counterstain (Gill's formula, Vector Lab). For thefluorescent CD68 labelling, brain slices were incubated overnightwith a Rat anti-mouse CD68 primary antibody (1:100, Serotec).A 2-hour incubation at room temperature with fluorescein-conju-gated Anti-Rat IgG (1:200, made in horse, Vector Labs) was used forCD68 detection, and counterstained using DAPI (Fluka).
Image analysis
To measure the levels of CD68+ microglial activation in the cere-bellum of these mice, a series of images were taken using a Zeiss AXIOImager A1 fluorescence microscope connected to a Zeiss AxioCamHRcdigital camera. Images were taken from aminimum of 13 fields selectedat random from several cerebellar sections per animal, includingexamples from both vermal and hemispheric regions of cerebellartissue. For each field, images of the FITC channel and the DAPI channelwere superimposed in Adobe Photoshop, then analysed using ImageJ(NIH). As the cell density and CD68 expressionwere not uniform acrossthe cerebellum, each field was split into different zones, the deep
ral cortex (A–B), caudate putamen (C–D) and spinal cord (E–F). No above-backgroundcells are clearly present throughout the CNS. CD68 labeled with FITC (green).
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cerebellar nuclei and surroundingparenchyma (DCN), the lobularwhitematter and internal granular layer (WM and IGL), and finally themolecular layer (ML). The area of these zones was calculated in mm2,and the number of DAPI-labeled nuclei surrounded with a CD68+ stainwas counted in each area. For every animal, the areas and cell countswere pooled together and the total number of CD68+ cells per mm2 ofDCN,WMand IGL orMLwas calculated. The data set for each treatmentconsisted of three separate animals (n=3).
In order to assess the effect of drug treatments on Purkinje cellsurvival, other sections from the same animals were stained with anantibody against calbindin, which in the cerebellum is a specificmarker for Purkinje cells. As two of the cerebella in each data set werecut as transverse sections, (the other was cut parasagittaly) the crus1zone of the ansiform lobule was chosen as a common area present inthe remaining sections in which to count the surviving Purkinje cells.As this region is not in lobule IX or X it will therefore allow anunbiased representation of NPC-Purkinje cell loss. For each individualanimal the number of Purkinje cells present in the crus1 zone wascounted in multiple sections and the mean calculated. This value wasused to portray the level of Purkinje cell survival in each treatmentdata set (n=2).
Mouse behavioral analysis
Open field
The spontaneous activity of each mouse was recorded individually.After 5 min acclimatization in the room, the mouse was placed in the‘open field’ (a plastic box measuring 45×25×12 cm). Rearing wasrecorded manually for 5 min recording the number of times the mousereared on its hind legs (either without support or against the cagewall).
Fig. 3.Nitrotyrosine staining in the thalamus (stria terminalis) to show oxidative stress/damthe thalamus of NPC1−/− mice stain heavily for nitrotyrosine (B). Brown staining: anti-nitr
Tremor
Tremor was measured with a commercial tremor monitor (SanDiego Instruments) according to the manufacturer's instructions. Thetremor monitor was housed on an anti-vibration table and the micewere monitored weekly for 256 s, after 30 s acclimatization time. Thetremor monitor was connected to a computer via a NationalInstruments PCI card and the output (amplitude/time) was analysed(fast Fourier transform) using LabView software, to give a measure-ment of power at each frequency (0–64 Hz).
Statistics
Survival curves were created by the method of Kaplan and Meier.Quantitative data were statistically evaluated with one-way analysisof variance with either a Dunnett's post hoc test or Tukey's post hoctest where statistical analysis was required for comparisons betweenall data sets in the experiment. The statistical software used wasGraphPad Prism version 4.0c (GraphPad Software, San Diego,California, USA) and values were considered statistically significantlydifferent when pb0.05.
Results
CD68 expression in NPC disease
Inflammatory marker expression was investigated in the brain ofan 8-week old NPC1 mouse in relation to storage defined by PASstaining (Figs. 1 and 2). In Npc1+/− mouse, PAS staining showed nosignificant glycosphingolipid storage (Fig. 1A). The opposite was truein Npc1−/− mice, with PAS-labeled cells clearly visible (Fig. 1B). In
age. No nitrotyrosine staining (brown) is observed in control (NPC+/−) tissue (A), whileotyrosine developed with DAB. Blue staining: haematoxylin counterstain.
Fig. 4. Effect of test drugs on survival of Npc1−/− mice. (A) Kaplan–Meier survival curve(%) and (B) average survival ages (±SD). All treated had significantly longer survivalfrom untreated NPC−/− except vitamin C and aspirin (pb0.05 for ibuprofen, pb0.001for miglustat and miglustat/ibuprofen combination).
Fig. 5. Effect of test drugs on weight of Npc1−/− mice. Average weight over time (A), at11 weeks (B) and at endstage (C) (all±SEM). All treated mice were significantlydifferent from WT (pb0.01) but not from untreated Npc1−/− mice (pN0.05) at both11 weeks and individual endstages.
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the brain of Npc1−/− mice MHC class II was not up-regulated(Figs. 1C–D) where as the macrophage/microglial activation markerCD68 (macrosialin) was highly up-regulated and broadly expressedby activated microglia/recruited macrophages throughout the brainand spinal cord (Figs. 1F and 2B, D, F). No CD68 was detected in braintissue from heterozygous mice (NPC+/−, Figs. 1E and 2A, C, E).Significantly, while Npc1+/− controls were negative for nitrotyrosinestaining (Fig. 3A), increased nitrotyrosine levels in Npc1−/− mousebrain indicated that the CD68+ activated microglia were responsiblefor increased oxidative stress (Fig. 3B).
Effects of NSAIDS, vitamin C and miglustat on survival of NPC1 mice
To test the potential efficacy of anti-inflammatory drugs and ananti-oxidant Npc1 mice were treated with aspirin, ibuprofen orvitamin C. An untreated control group was monitored in parallel tothe treatment groups. In addition, ibuprofen was combined withmiglustat therapy. The timing of initiation of treatment was pre-symptomatic (3–5 weeks), early symptomatic (6–8 weeks) or latesymptomatic (8–10 weeks).
Treatment ofmicewithNSAIDS or vitamin C prior to symptom onset(3–5 weeks) or when initiated during advanced disease (N8 weeks)was of no clinical benefit in terms of survival (data not shown).However, mice treated from 6weeks of age (the agewhen overt clinicalsigns were first present) showed a statistically significant improvementin survival with ibuprofen (survival increased by an average of1.67 weeks, pb0.05) (Figs. 4A–B). Aspirin and vitamin C showed nobenefit (survival on average increased by 1.38 weeks and 3 daysrespectively, pN0.05). Miglustat monotherapy resulted in a 4.97 weekincrease in survival (pb0.001) and the combination of miglustat withibuprofen resulted in a 6.77 week increase in survival (pb0.001). Thecombination treatment significantly increased survival in an additivefashion when compared to miglustat monotherapy (pb0.05).
Effects of NSAIDS, vitamin C and miglustat on body weight of NPC1 mice
None of the therapeutic interventions prevented loss of bodyweight as the disease progressed. However, with therapies that
prolonged survival no further body weight loss occurred i.e. bodyweights remained stable as animals lived beyond their naturalendpoint (Fig. 5A). At 11 weeks of age (just prior to the naturalend point of untreated Npc1−/− mice when a humane endpoint isapplied) all treatment groups had significantly lower body weightsthan wild type NPC1 mice (Npc1+/+) (pb0.05) but were notsignificantly different from each other (Fig. 5B) (pN0.05). All micehad similar body weights at their respective end points despitedifferent ages when the humane endpoint was applied, reflectingthe differential effects of the therapies tested (Fig. 5C).
Effects of NSAIDS, vitamin C and miglustat on tremor of NPC1 mice
A major clinical sign in Npc1−/− mice is tremor that occurs atlow (5–30 Hz) and high frequencies (30–50 Hz). In contrast toother clinical signs tremor can be measured to a high degree ofnumerical precision using a commercial tremor monitor. For thisreason tremor monitoring was used as a functional measure of theeffects of the different treatments on the CNS function of Npc1−/−
mice. Wild type mice (profiles are averages of n=2–10 mice pertimepoint) gave a profile over 0–55 Hz with very low amplitudethat showed no statistically significant changes with increasing age(Fig. 6A) (pN0.05). In contrast, the Npc1−/− mice show an agedependent progressive tremor (pb0.001) that was statisticallydifferent from wild type mice after 7 weeks of age (Fig. 6B). Todetermine the effects of treatment on tremor the amplitude of thesignal was compared at an arbitrary frequency of 43 Hz (i.e. themidpoint of the high frequency tremor). Wild type mice wereclustered as a tight group and exhibited minimal variation (Figs. 6Cand D). However, untreated Npc1−/− mice at 7–9 weeks showed abroad range of amplitudes of tremor that were characteristicallyhighly variable ranging from wild type levels through amplitudesthree times greater than wild type mice (Fig. 6C). A similar profilewas observed with ibuprofen treatment at 7–9 weeks of age.
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Interestingly, miglustat therapy, either alone or in combination withibuprofen, showed a tremor profile indistinguishable from WTcontrols (pN0.05) (Fig. 6C). By late stage disease (10–12 weeks) themagnitude of the tremor in untreated NPC mice had increased andwas still highly variable, as was the tremor in ibuprofen treatedmice and the lack of tremor in the miglustat treated groups wasmaintained i.e. they were indistinguishable from untreated wildtype mice (pN0.05) (Fig. 6D).
Motor function and coordination
A sensitive measure of functional decline in NPC mice is theability of mice to rear on their hind legs, either unaided or againstthe cage wall. This parameter was measured by observationalcounting to avoid episodes of hyperactivity. Hyperactivity (includingjumping) is commonly observed in late stage disease and canerroneously be scored as rearing events in an automated system.Rearing activity in Npc1−/− mice at 7–9 weeks of age showed asignificant reduction in rearing activity (85.3% of wild type control)(Fig. 7A). Ibuprofen showed no protective effects, in terms ofrearing, as the ibuprofen group was indistinguishable fromuntreated Npc1−/− mice. However, miglustat therapy resulted inretention of 56.5% rearing activity relative to untreated controls(pb0.01) as did the miglustat/ibuprofen co-treated group (53.8%,pb0.01). By 10–12 weeks of age (Fig. 7B) the untreated group hadlost rearing function completely, where as this activity was still
Fig. 6. Effect of test drugs on tremor of Npc1−/− mice. Tremor amplitude was monitored betwsymptomatic (7–9weeks) (D) late symptomatic (10–12weeks) timepoints. 43 Hz is an arbitrmade between treatment groups. The low frequency tremor range overlaps at the low endvariable. In both early and late symptomatic mice treated with miglustat, or a combination(pb0.05, pb0.01 respectively).
present (45.1% of wild type) in the miglustat treated group. Therewas also detectable rearing in the ibuprofen group (20.2% of wildtype) but neither was statistically significant relative to untreatedcontrols. However, the miglustat and ibuprofen combination treatedmice had significantly higher rearing activity than untreated mice(pb0.01, 92.5% of wild type), indicating that the mice treated withboth miglustat and ibuprofen retained their motor function longerthan those treated with either monotherapy.
Effects of NSAIDS and miglustat on brain inflammation
To test whether the treatments had any impact on braininflammation, mice were treated with miglustat, ibuprofen, miglu-stat and ibuprofen or fed standard chow as a control (n=3 for eachtreatment group). The animals were sacrificed at 7.4 weeks of ageand the cerebella analysed for CD68 expression as a measure ofmicroglial activation. All the treated Npc1−/− animals presentedwith significantly less CD68+ cells in all areas of the cerebellumcompared to untreated Npc1−/− animals (Fig. 8, compare theuntreated mice (panels A–C) to ibuprofen treated mice (panels D–F,pb0.01), miglustat treated mice (panels G–I, pb0.001) or miglustat/ibuprofen co-treated mice (panels J–L, pb0.001). Fig. 8M depicts thedata in a graphical format). However, there were distinct differencesin the efficacy of the various treatments (Fig. 8M). A significantreduction in the number of CD68+ cells in ibuprofen treated micewas observed when compared to untreated Npc1−/− mice, in the
een 0 and 55 Hz in (A) Npc1+/+ and (B) Npc1−/−, and quantified at 43 Hz in (C) earlyary point selected in the high frequency tremor range to allowa simple comparison to bewith grooming activity frequencies and so was not analysed to avoid this as a potentialof miglustat and ibuprofen, tremor was significantly lower than in untreated Npc1−/−
Fig. 7. Effectof test drugs on rearingofNpc1−/−miceat (A) early symptomatic (7–9weeks)and (B) late symptomatic (10–12 weeks) timepoints. In early symptomatic mice treatedwith miglustat alone, or in combination with ibuprofen, rearing was significantly higherthan in untreatedNpc1−/− (pb0.01), whereas in late symptomaticmice only combinationtreatment resulted in significantly higher rearing activity (pb0.01).
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deep cerebellar nuclei (DCN; ibuprofen 390.6 cells/mm2; NPC−/−
504.20 cells/mm2; pb0.001), the white matter and internal granularlayer (WM and IGL; ibuprofen 237.72 cells/mm2; NPC−/−
328.06 cells/mm2; pb0.01) and the molecular layer (ML; ibuprofen200.59 cells/mm2; NPC−/− 314.58 cells/mm2). However, miglustattreatment reduced the levels of CD68+ cells by a significant margin,not only relative to untreated Npc1−/− mice but also whencompared to the ibuprofen treated animals (DCN; ibuprofen390.95 cells/mm2 vs miglustat 310.95 cells/mm2, pb0.05. WMand IGL; ibuprofen 237.72 cells/mm2 vs miglustat 155.34 cells/mm2, pb0.05. ML; ibuprofen 200.59 cells/mm2 vs miglustat98.94 cells/mm2, pb0.01). There was no significant difference inthe number of CD68+ cells in WM and IGL or ML of miglustattreated mice compared to miglustat/ibuprofen co-treated mice,but a difference was observed in the DCN of these treatmentgroups (miglustat 310.95 cells/mm2 vs miglustat/ibuprofen216.43 cells/mm2, pb0.01, compare panels G and J), thusindicating a modest but significant cooperative effect of miglustatand ibuprofen on NPC1 brain inflammation.
Effects of NSAIDS and miglustat on Purkinje cell survival
The effects of the treatments on Purkinje cell survival wereassessed (Fig. 8N). In keeping with previously published data(Zervas et al., 2001) miglustat protected against Purkinje cell loss.However, ibuprofen therapy had no effect on Purkinje cell survival
with comparable numbers present as in the untreated NPC1−/−
mice. Combination therapy (miglustat and ibuprofen) resulted in nosignificant difference than miglustat alone.
The third brain from each treatment set that had been cutparasagittaly (n=1) and therefore could not be directly comparedwith the transverse sectioned material analysed above, exhibited thesame pattern of Purkinje cell survival when comparing the meannumber of Purkinje cells in lobule V (data not shown).
Discussion
Inflammation in the brain is a hallmark of many neurodegenerativediseases, irrespective of the underlying cause (Jeyakumar et al., 2005).Immune activationmaywell be a two edged sword, playing a reparativerole initially but rapidly progressing to the creation of a poorlycontrolled chronic inflammatory environment that promotes oxidativedamage and further neuronal dysfunction. As the majority ofneurodegenerative diseases are currently without treatment, targetingsecondary disease components, such as inflammation, is an attractiveapproach. This would employ existing approved drugs with typicallygood safety profiles that are available for rapid translation into theclinic. The application of anti-inflammatory drugswould not be curativebut may improve quality of life and slow disease progression. Theycould also be combined with other therapies, with a view to achievingadditive or synergistic benefit (Jeyakumar et al., 2004).
The mechanism(s) that causes macrophage/microglial activationin NPC and other neurodegenerative diseases remain ill defined.Activated macrophages/microglia commonly up-regulate MHC classII expression, as has been described in Sandhoff disease and GM1gangliosidosis. However, in NPC this is not the case. These datasuggest that in NPC1 the microglia are in a different activation statethan in the gangliosidoses, hence the relative absence of class IIexpression but up-regulation of CD68. CD68 is a member of theLAMP macrosialin family of hematopoietic mucin-like molecules andis present primarily in late endosomes/lysosomes with relativelylittle expressed at the cell surface (Holness et al., 1993).
CD68 is also up-regulated in prion disorders (Betmouni et al.,1996). Why there is differential activationmarker expression amongstneurodegenerative diseases remains unknown. However, the acti-vated microglial cells andmacrophages in the NPC1 mouse brainwereresponsible for oxidative stress as detected by nitrotyrosine staining,in keeping with the gangliosidosis mouse models.
Collectively, these data suggest that anti-inflammatory interven-tion may be of potential benefit in NPC disease, either alone or morelikely (in the acute mouse model) in conjunction with othertherapeutic modalities.
Anti-inflammatory drugs have also been evaluated in the idio-pathic neurodegenerative conditions. To date, a protective effect ofNSAIDs on the development of Alzheimer's disease has beenreported in an observational study (Etminan et al., 2003). The roleof the immune system in contributing to the neurodegenerativeprocess has also been highlighted in epidemiological studies thatshowed that patients with dementia deteriorated when they hadepisodes of peripheral infection (Perry et al., 2003). Once theimmune system is activated in the CNS, pro-inflammatory events inthe periphery can therefore exacerbate the inflammatory process inthe brain (Perry et al., 2003).
Our findings in the NPC1 mouse in this study illustrate that anti-inflammatory therapy slows the disease course suggesting it may bea useful adjunctive therapy in patients. However, careful clinicalstudies will be required to determine whether these findings extra-polate to the human disease. One limitation of the NPC1 mouse isits acute clinical course with death at 10–12 weeks of age. It is moreextreme than most patients and so is not an ideal system in whichto model adjunctive therapies that will have subtle disease modi-fying effects. Therefore the fact that NSAIDs, such as ibuprofen, do
Fig. 8. Effect of test drugs on NPC−/− cerebellar microglial activation and Purkinje cell survival. (A–L) Cerebellar sections (20 μm thick) were immunostained with anti-CD68antibody and revealed with a FITC-conjugated secondary to label activated miroglia (green). The tissue was counterstained with DAPI (blue) to outline cerebellar structure. Layers ofdense blue staining correspond to the internal granular layer, while the arrows indicate the lobular white matter tracts. The cerebellum of untreated Npc1−/− mice (A–C) wascompared to ibuprofen treated (D–F), miglustat treated (G–I) and miglustat/ibuprofen co-treated (J–L) animals, n=3 for each treatment group. The images illustrate the microglialactivation in the deep cerebellar nucleus and surrounding parenchyma (first column, dark green), the lobular white matter and internal granular layer (second column, mid-green),and the molecular layer (third column, light green), note the clear reduction in the number of green CD68-labeled cells in treated cases compared to the untreated Npc1−/− animalsin all areas of the cerebellum. The number of CD68+ cells was calculated per mm2 of each cerebellar zone. The data is presented in (M), with each cerebellar zone colour-coded asbefore (all bars±SEM). Note the cooperative effect of co-treatment in the deep cerebellar nuclei when compared to miglustat treatment alone (pb0.01) (see graph M, comparepanels G and J). In other sections, the number of calbindin-labeled Purkinje cells in the crus1 zone was counted to assess the impact of drug treatment on Purkinje cell survival. Theresults depicted in graph (N) indicate that ibuprofen did not have a protective effect on Purkinje cell survival, while miglustat treated animals had significantly more Purkinje cellsthan the untreated NPC−/− controls. ⁎⁎⁎pb0.001. ⁎⁎pb0.01. ⁎pb0.05. ns = not significant. Scale bar=200 mm.
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250 D. Smith et al. / Neurobiology of Disease 36 (2009) 242–251
show a significant effect in this mouse suggest that it may be ofgreater benefit in a more slowly progressing model or in a clinicalsetting. The dose of ibuprofen used in this study was 100 mg/kg/day, higher than the typical human dose range of 400–1000 mg/day(equivalent to 5–20 mg/kg/day in the mouse (Lim et al., 2000)).Further studies will be required to investigate whether lower dosetherapy in less acute animal models or in the clinic will also achievebenefit. The benefits of aspirin treatment in the NPC1 mouse did notreach statistical significance based on survival. This suggests thathigher dosing may be required in this very severe disease modeland/or testing on a much larger cohort of mice to determinewhether it can be beneficial.
What is more difficult to predict is whether anti-oxidants haveany potential role to play in clinical management. In two studies inthe NPC1 mouse, anti-oxidants have failed to show much benefit(this study and Bascunan-Castillo et al., 2004). However, somecaution is required in interpreting these results. In the Sandhoffmouse that lives 16 weeks, anti-oxidant therapy with vitamin C didprovide a modest but significant clinical improvement as a mono-therapy. It could be argued that the very acute nature of the NPC1mouse model may mask the potential benefits of anti-oxidanttherapy that could be useful in a clinical setting in patients with lesssevere forms of the disease relative to themouse NPC1model used inthis study. Future studies will be needed in less severe models ofNPC1 and/or using more powerful anti-oxidant strategies withcompounds that more effectively cross the blood-brain barrier (Aguset al., 1997).
An interesting observation we made in this study is that NPC micetolerate liver metabolized drugs poorly relative to other diseasemodels or standard inbred mouse strains (lower dosing is required). Itis possible that liver disease in this acute NPC1 mouse affects livermetabolism of drugs and leads to toxic side effects. This likely explainsthe negative effects of very early NSAID therapy that had negativeeffects on survival. At later timepoints, the positive effects of reducingestablished inflammation outweigh the toxic effects of the drug, hencebenefit is achieved. This potential problem in metabolizing drugs inthe liver in the NPC1 mouse requires detailed investigation. However,this hypothesis is supported by the fact that the renally excreted non-metabolized drugmiglustat, can be dosed in NPC1mice comparably tohealthy inbred strains.
Several therapies have shown efficacy in the feline and mousemodels of NPC including miglustat (Zervas et al., 2001), anti-oxidants (Bascunan-Castillo et al., 2004; Zampieri et al., 2008),pregnane X receptor (PXR) activators (Langmade et al., 2006),cyclodextrin (Camargo et al., 2001; Liu et al., 2009) and theneurosteroid allopregnanolone either alone (Ahmad et al., 2005;Griffin et al., 2004) or in combinations with a drug that activates PXR(Langmade et al., 2006). Until very recently, the clinical managementof NPC disease has been confined to symptomatic management andpalliative care. However, very recently substrate reduction therapyusing miglustat has been approved in Europe for treating NPC.Miglustat has shown disease modifying effects in clinical trials(Patterson et al., 2007) and in off-label use (Galanaud et al., 2009;Paciorkowski et al., 2008; Santos et al., 2008). In addition todemonstrating efficacy, the clinical trial identified clinical endpointsthat can now be applied to future clinical trials. What is now requiredis the combined use of miglustat with other approved drugs thattarget different aspects of the pathogenic cascade, with a view tomore effective management of NPC patients. Based on our findings,NSAIDs may be good candidates to test in the clinic as adjunctivetherapeutics in NPC disease.
Acknowledgments
DS was funded by a grant from the National Niemann-Pick DiseaseFoundation (NNPDF). IW was funded by the Niemann-Pick Disease
Group (UK). MJ and KLW were supported by the Medical ResearchCouncil, UK.
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