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Neural Progenitor Cells in the<br />
Huntington's Disease Hurnan Brain<br />
Maurice A. Curtis<br />
A thesis submitted in fulfilment of the requirements for the Degree of<br />
Doctor of Philosophy in Anatomy and Pharm<strong>ac</strong>ology at The University of<br />
Auckland, May, 2004<br />
THE UNIVERSITY OF AUCKLAND
" ... tkefunafional spec-ializaiton ef the brain imposes anthe neurones two great l<strong>ac</strong>unoc;<br />
p'rolifetation inqbility and irreversabitity af intrapratoplasmie fficrentiation It is for<br />
this reaaan tkat, iQn:ee the development was ended, the faants af grwth and regeneration<br />
af wcoas and derdriNes dried up irreuocab,Iy. In adult eenters tke nente paths are<br />
somahingfi'Ned, ended, imrnutabrc-. Everything may dle, nothing may he regenerated. lt'h<br />
for the science of thefutwre ta e,hange, if possible, this harsh d,ecree.,,<br />
Santiago,Rarnon Y. Cajal (lgl3)<br />
Nobel prize in Medicine IgA6<br />
(Ramon y Cajal, 1991)
The recent demonstration of endogenous progenitor cells in the adult mammalian brain<br />
raises the excitirrg possibility that these unclifferentiated cells may be able to generare<br />
new neurons fbr cell repl<strong>ac</strong>ement in diseases such as Huntington's disease (HD).<br />
hevious studies have shown that neural stem cells in the rodent brain subependymal<br />
layer (SEL), adj<strong>ac</strong>ent to the caudate nucleus. proliferate and differentiate into neurons<br />
and glial cells but no previous study has char<strong>ac</strong>terised the human SEL or shown<br />
neurogenesis in the diseased human brain. In this study. histochemical and<br />
immunohistochemical techniques were used to demonstrate the regional anatomy and<br />
staining char<strong>ac</strong>teristics of the normal and HD brain SEL using light and laser scanning<br />
confocal microscopy. The results demonstrated that the normal and HD SEL contained<br />
migrating neuroblasts, glial cells and precursor cells but there were more of e<strong>ac</strong>h cell type<br />
preseltt in the HD brain, and that the increase in cell numbers correlated with HD<br />
neuropathological grade. The nonnal and HD SEL was stained with a proliferative<br />
marker, proliferating cell nuclear antigen (PCNA), to label divicling cells. The results<br />
showed a significant increase in the number of dividing cells in the HD brain that<br />
correlated with HD grade and with CAG repeat length. Furthermore, the results showed<br />
that neurogenesis had occurred in the SEL as evidenced by co-localisation of pCNA and<br />
the neuronal marker Blll-tubulin. Also. gliogenesis had occumecl in the SEL as evidenced<br />
by the co-localisation of PCNA with the glial marker GFAP. These studies also revea.led<br />
a 2'6 fold increase in the number of new neurons in the HD SEL. PCNA positive cells<br />
were distributed throughout the SEL overlying the caudate nucleus but most notably the<br />
ventral and central regions of the SEL adj<strong>ac</strong>ent to the cauclate nucleus contained the<br />
highest number of prolif'erating cells. I examined the SEL fbr mature cell markers and<br />
demonstrated many of the same cell types that are present in the normal striaturn. With<br />
the exception of neuropepticle Y (NPY) neurons, there was a reduction in the number of<br />
mature neurons in the HD SEL. The NPY neurons were more abundant in the HD SEL<br />
suggesting they play a role in progenitor cell proliferation. The results in this thesis<br />
provide evidence of increased progenitor cell proliferation and neurogenesis in the<br />
diseased adult human brain and indicate the regenerative potential of the human brain.<br />
These findings may be of rnajor relevance to the development of therapeutic appro<strong>ac</strong>hes<br />
in the treatment of neurodegenerative diseases.<br />
llt
ACKNOWLEDGEMENTS<br />
I wish to specifically thank the members of the Anatomy department and a number of<br />
other people who have directly ancl indirectly influenced this phD work throughout the<br />
past 3 years.<br />
First and foremost I would like to thank rny principal supervisor hofessor Richard Faull.<br />
My undergraduate degree was not the prerequisite required to do a Masters degree in<br />
Richard's subject area but Richard successfully took my case before the appropriate<br />
committees and took me on as a Masters student and subsequently as a phD student.<br />
Richard has always been enthusiastic and positive throughout the highs and the lows and<br />
has inspired and mentorecl me in every aspect of this thesis. I will be forever grateful for<br />
the chance you took on me which has changed ancl improved my rife.<br />
I would like to thank Professor Mike Dragunow who, as my co-supervisor. has provided<br />
me with new ideas and fresh ways of looking at olcl problems. Mike has such an<br />
infectious enthusiasm for science that it is virtually impossible to leave Mike's office<br />
without being so fired up that you want to cancel your holidays and spend the time doing<br />
exciting experiments. Thank you for ail your ideas and enthusiasm.<br />
Also, I would like to thank Dr Bronwen Connor (co-supervisor). Bronwen has always<br />
been prompt with her fee
epresented in chapter 5 and has weathered some of my fairly inclement questions<br />
regarding statistics. Thank you for all your assistance. Thanks also to Jocelyn Bullock for<br />
your work cutting sections to obtain Weslern blotting tissues, discussion about interesting<br />
destinations and for lending/giving me quality literature of the adventure variety.<br />
While working toward this thesis I have had the privilige of being surrounded by some<br />
exceptionally bright PhD students who have provided me with scientific ideas and<br />
assistance, social inter<strong>ac</strong>tions and friendship. I would like to thank the PhD students in<br />
the Depertments of Anatomy and Pharrn<strong>ac</strong>ology, namely R<strong>ac</strong>hel Cameron, Palingu<br />
Dhanapala, Carissa Fonseca. Hannah Gibbons. Monica Kam, Andree Pearson, Suzanne<br />
Reid and Doris Thu. Best wishes in your studies and for the future. A special thanks to<br />
R<strong>ac</strong>hel for the much needed. sanity-inducing hot chocolate breaks, proofreading. hearing<br />
my crazy stories and for the listening ear.<br />
To John Klistiansen, Daniel McGowan and Glenn Pennycook, thanks for all the climbing<br />
adventures and CCMC trips to talk about it. You are top friends and have always offered<br />
light relief, hard case conversation and friendship regardless of the situation. I have been<br />
very fortunate to have some supportive church friends and in particular Andrew<br />
Schwamm, Tim Matsis, Jeremy Dixon and Leah Howard. Thank you for your church<br />
outre<strong>ac</strong>h, philosophical conversations and for coming sailing with me in gale force winds.<br />
These words poorly articulate the importance your friendship is to me.<br />
The two most supportive people in rny life have always been Ken and Lorraine Curtis<br />
(my parents). They have provided me with unerring and immeasurable love and support.<br />
They have always listened for hours on the telephone while I raved on about my<br />
disasters, my successes and my plans for the future. Thank you both for all you have done<br />
for me.<br />
Finally, I wish to <strong>ac</strong>knowledge and thank the Neurological Foundation of New Zealand<br />
for their very generous financial support of me as a Miller scholar. Also, the Health<br />
Research Council of New Zealand for their support in terms of working expenses and<br />
equipment. I am very grateful for this support.
1. B. connor, w. van Roon-Mom. M.A. curtis, M. Dragunow,<br />
R.L.M Faull (2001)<br />
Stem Cells and Neurodegenerative Diseases<br />
New Zealand Medical Journal. l14:477_479<br />
2. M.A. Curtis, E.B. penney, A.G. pearson, W.M.C. van Roon-<br />
Mom, N.J. Butterworth, M. Dragunow, B. connor, R.L.M. Faull<br />
(2003)<br />
Increased Cell Proliferation and Neurogenesis in the Adult Human<br />
Huntington's Disease Brain<br />
Proceedings of the National Academy of sciences of the united<br />
States of America 100: gO23-gOZ7<br />
3. M.A. Curtis, B. Connor, R.L.M. Faull (2003)<br />
Neurogenesis in the Diseased Aclult Human Brain: New Therapeutic<br />
Strategies for Neurodegenerati ve Diseases<br />
Cell Cycle 2: 4ZB-430<br />
vi
1r1l
2-'4.2Huntingtorr'sd.i'seascgadingseale..........|.r.'.!:r.]rt..ti.i<br />
2'5 The striaturn: cell types and relatisnship to the subependy,mal trayer.-...,.-.,.-.....,.......33<br />
2.6 Therap-e.utic appro<strong>ac</strong>hes to Huntington' s disease,.,....<br />
2.6.1 Neural transplaimtion..,........... r,..!...d......r,. ...-,_...........-..37<br />
2.6.2 hogenitor eell rep1<strong>ac</strong>ement.,,.............. .........40<br />
Chapten 3<br />
ANATOMY OF TI{E SI]BEPENDYMAL LAER<br />
IN TIffi NORIvIAL AND HL]-NTINGTOIN'S<br />
DISEASE HUMAN BF|AIN... .....r...................:42<br />
3.2ltIrtteriaIsandmethodS...'......,...".'.'.rn....i..it.i...i,..t.'l.!...r...<br />
3.2.1 Flur,nan tissue co11ec6on".......... .....r......rr.....:..i...i..i.i:.:.;.i .,.-..,43<br />
3,2.2 rissue proce$sins.......,........ ....--:....:.,::.."".":... ........4$5<br />
3.2.3 Heaurotoxylin & essin arrd luxol fast lue:staininE....... ....,._,......,...4S<br />
3.2,4 knage digitisarion<br />
3.2.5 trnage anal ysi s...., ; d. i.... ii.!..,.,.<br />
3.3.1 The anarom), of the SEL in the normal and<br />
Huntinglon's disease brain..,.,,.... ._r....r.........,. .........,.,....4j<br />
3.3.I.1Histological classification of the cells in the<br />
adult human subependymal lay_e.r......<br />
3,.3.l.?Iocalisation of the differenr cell tSpes in the<br />
subepcnd;ymal layer........ r..{ !...... r. ;,.<br />
vilt
3,3-2 comparison of the thickness of the subependymal layer in<br />
uorural and Huntington's disease brains.._...,..,i,.bj.........!.,. ..................52<br />
3.3.3 Quantification of cells in ttre subependyrnal layer in norural<br />
and, Huntington's tlisease brains.............r,.:.....,.-.. ............i,,...i.,......,.5g<br />
3.3,4 compnrison of the dif-ferent cell types present in the adulr<br />
subependymal layer in normal and Huntington's disease brains...........,,............61<br />
3.4.1 The general anatomy of the subependymal layer-,,,...<br />
342 The Huntington's disease subependy,mal layeris thicler<br />
than,that of the norrnal brain subependymat layer.............,......q...ii...._,,,.........65<br />
3,4.3 The Huntington's disea$e bmin subependynlal layrer contains<br />
mor€:cells than that of the normal subependyr,nal layer".,...,..,........:r....!.............66<br />
3.4.4.ThEre are rnore type A, B and C eells in the<br />
Huntin'gton's disease brain subependymal layer<br />
compared with the normal brain subependymallayer..........................ri .......,,..67<br />
3.6 Conclusion...........<br />
CIrapter 4<br />
ANATOMY OF THE SUBEPENDYMAL LAYER<br />
IN THE NORMAL AND TITINTINGTON'S<br />
D[sEAsEHI.'MANBRAIN....iil.};.'....j.;..i.......:..tt.....'.<br />
4.2 Materials and rne_thods.....--....... ........_..72<br />
4.2.1 Human tissue collection,. ......u........,......r1i...n..r.!- "...........72<br />
lx
4.2 :2 Imrnanohistoaherni sty.... -.......<br />
4.2.2.I Section preparation ...................,.;...........r... ......73<br />
4.2.2Z ptirary antibodies,........_...i.,.r...,.;....,...;.i.r..r .i..._.!.....r.. .,.oT4<br />
4.2,2,3 Secondary antibodiesr. .......r..,....r.r..................J4<br />
4.2.2ATertiaryantibodies..j].......r....i;j.....i....t,i.....r...<br />
4.2.3 Imrnunofluorgscence:i,..!-.............,r.,r..,r..;-..........i.,.i......-. ...................75<br />
4.2.3.1Section Fr€Faratiom..............r..!,......ri.,...--. ......J..!..............25<br />
4.2.3,2himury antibodies.. ..r.ij.,..i............75<br />
4.2.3.3 Fluorochromqs..........,.<br />
4.2.3.AIrnageanalysisandcellcounting.........'......<br />
4.2.4 IVestern blotting..,......i.,....!-.rr..r........,!,...,..t..i..,....i..,r.,. ...................,7V<br />
4.2'4..IProparationofgels..'.......'......'..l..i.<br />
4.2,42 preparation of riunples. ..i!.r....i......!. ..........-".....7g<br />
42,4.3lunrrunob-Iotting..............,..,r.,......j.i!.,.i...,!..,.......... .,.,............g0<br />
4.3.il hogenitor cell proliferation in the hurnan norrnal and<br />
Huntington's disease s_ubependymal 1ayer......,.,. ............g1<br />
4.3.2 comparison,of the numberof proriferating ceils in the<br />
normal and Huntingtoj's disease subependymal layer........,.j-.....*.-...r:.,r;r.ri;;.g2<br />
4.3.3, Comparison of the pathologieal grade of Huntington's<br />
disease with thenumber of proliferating cells in the<br />
subependymal layer......<br />
4.3 4 conrparissn of the number of cAG repeats in the rrr 5<br />
gene in Huntington's direase with the number of Broliferating<br />
cells in the subepe.ndymal |ayer........,.r..-..qi,.,....i;.;!..1............ .........g4<br />
4.3.5 ,{nalysis qf the specificity of the PCNA antibody for<br />
detecting cell proliferatiqn.................--r;.,......,.,;........i,..i ......-.........,..E5<br />
4.3.6 Western blot analysis of the subependymal laye.r...... ,.!.....ij..i..i.!....-.g5
4.5<br />
4.3.7'The lsealisation of proliferating, neuronal and glial<br />
cell rnarkers in the subependymal |ayer......r.......r...:.;n..o.r..!:.......'.i.......',,............86<br />
4.3.8 Neuroggnesis arid gliogenesis in the human subependymal layer......................90<br />
4.3.9 The nrumber of PCNA/Bill=tuhulin double-labe-lled cells in the<br />
normal and Huntington's disease subependym,al 1ayer.......... ".........93<br />
4,4.I lncreased numbers of progenitor cells in the HD subependymal layer.....'..,.....97<br />
4.4.2 Neurogenesis and gLi'ogenesis in the Huntington's disease<br />
subependyrnal layer...... .i...i!....;..'r......,.........98<br />
4.4,3 lncreased neurogenesis in the Hunti4gton's<br />
diseases,ubependyrnalIa er.......... .......;..-...j.;....i;..,;...,.,......,.....'......101<br />
xl
5.24 &ll counting,......:........,..r..........-...i.,...,.... j,...iij.i..!.. .,r.r..r:i.......,,......113<br />
5.2.5Stuisticalanalysis................;!............'ji.i<br />
5'3 Results ............ilT<br />
5.3.1 Cornparison;of pCNA positive cell'umbers in:the<br />
normal and Huntington's disease sub,ependymal layer......<br />
5.3.2The numbEr of pCNA.positive cells in the subependyanal<br />
layer in dorsal, nriddre and ventral areas of normar and<br />
Huntington's disease braing.....-. q..j...ri.?i!...,....,....,.........1 I E<br />
5.3.3 The number sf pcNA positive cells in the zubependral<br />
layer in nostal, cenhar and caudar regions. of the nort'al and<br />
Huntington' s disease brains........<br />
5.3,4 The nurnber of FCNA positive ceils in the subependymal<br />
layer in different roc.'atio''s in the nqrmar and Huntington,s<br />
diileaee brains..,...." ...i..;....i.i...r.._..,.-...,......121<br />
5.4.1 There is a variation in theregional distibution of pCNA<br />
poniti]vecellsinfienorrrra|brainsubepend,yma|Iayetr.,.......'..<br />
5.4.2 Ttnere is a vari.ation in the regional distribution sf pcNA<br />
positive eells in the Huntington's disease brain<br />
su'beperdfmalLlayer......... .,......It:s<br />
5-4.3 There is a variation in rhe disfiburion of pcNApositive<br />
cefls in different locati n of the subependymal layer...... ...........,.,126<br />
xu
Chapten 6<br />
CI,MMICAL ANATOVTY Or<br />
THE SUBEPENDYI\{AL LAYER.....!....IIr r,. t...<br />
6.1 Introduc<br />
6"2 Mabrial<br />
6,2-l Human bgste collection.. ...;i.a,q....i,.!i.!....!:....?.t... ....,,....Lg'z<br />
6,?2Immunohistocherrristry.............---.b.r,.i..r...i..;i!,..,..._.....r<br />
.........".........133<br />
6.2.2.1 Section preparation<br />
6.2.22 himary anribodies. .i..r........r.i.. ....,135<br />
5,2.2,3 Secondary antibodies...i.,...;.rr.!..!... ...........!..i.j.r..",.,..-..........13d<br />
6.2.2.4 Tefli4ry,arrtibodies..i. r.q.t,... r.i...<br />
i5.2.3luu4e digitisation .,,.136<br />
6.3.1 Calbinditr illrnunore<strong>ac</strong>tivit5r in the subependyraal layer......!...........ii.ri.-.........13?<br />
6.3.|.tN:grnral....'.!....-.'l.]...'r;..bi.r'.i.i.t'....'...t.<br />
6.3. N .2 Elunfi ngton' s disease.,.<br />
6'3.2 Enkephalin ininnunore<strong>ac</strong>tivity in the subependyr,nar layer<br />
6.3.2.L Ngnnal.,..,.tri...!.....r.......i..r...i..r.......,....{...rr-..,<br />
-........-......139<br />
6.3.2,,2 Huntington'g dieease... .r,....i.-.......ir...,.i....".,.,..,r,...,.........,."I39<br />
63'3 substane-e P imhunoreastivity in the subepindymal layer<br />
6.3.3.1 Nt0rmal,",,.. ...i,.!,.....,..i....!i!..ir!.....,... ........".....,.. t3g<br />
6.3 .3.2 Huntingtoa' s disease,..<br />
6.3.4 Parvalburnin irmunore<strong>ac</strong>tivity in the subependynral layer<br />
63,4.INormel.'.".'.l........J.a..-.|...iJrri.i|i;.|r..|.l<br />
6 "3.4.2 Hun tington' s drsease...<br />
xiii
6.3.5 eahetinin irnrnunore<strong>ac</strong>tivity in the subeper'rdy-mal layer<br />
6.3.5.1 Norural...... ........"........142<br />
6.3.5.2Huntiugton's disease",...................,.......,.....r.....i..d..i...!ii.i..,.-. ..............L42<br />
6.3.6 Choline-<strong>ac</strong>etyl transferase immunore<strong>ac</strong>tivity in the subependymal layer<br />
6.3,6.lNormal......,:.i.i.i.t.',,........!.!r,.r.j..i.....r.<br />
6.3.6,2 Huntington''s disease... .,....,.....i."...,...............,.143,<br />
6.3.7 fl|.{ourqpsptide Y immur,aore<strong>ac</strong>tivity in the subependymal layer<br />
6.3,7,L Norrn'al........i........'......,.<br />
6.3.7.2.Huntington's disease... .....r.r-1ii.i....,,.."..........".144<br />
6,3.E Glial frbri'llary asidic piotein irnmunoreastivity in the<br />
subependymal layer<br />
6.3.8.1 Norma]..,........-..,......r.. .,....r.......q!r.,.,..............145<br />
6,.3-8.2,[Iuntington's disease-.. .....-..........,145<br />
6.3.9 Vimentin .ir[rnunoreaotivity, in the subependymal layer<br />
6.3.9.1Nonbal'..........!.t...ir!..'i!.!...'.........<br />
6.3-92 Huntington's disease... ......rii!.i........,,......,......147<br />
6.3.10 Ferrifin irii[runoreaetivity in the subependymal layer<br />
6.3.10;1 Nonrral..........i!.!.........r,..:................ ...............148<br />
6.3.10"2 Huntingtoll's disease...... ...,r!.....r.i.....,......,.,..148<br />
6.3.I I,GABAA u,l subunitirnmunoreaetivity in the subependymal layer<br />
6.3.11.1 Normal"..... .,............,149<br />
6.3. 1 1,2 Huntingbn's disease...,..<br />
6.3..12 GABA.A p2, 3 subunit imrnunore<strong>ac</strong>tivity in the subependynal layer<br />
5.3.121 Normal...... r...i.....,..!,............,.....151<br />
6,3.|2.2Huntington'sdisease.............'..'..i........'..|...'..;..|.<br />
xlv
6.3.13 GABAAy2 subunit irinmunsre<strong>ac</strong>tivity in the sr,rbependymal laya<br />
6.3.13,1 Nsrlnal,.....!.....'.,...i.!...1 ......,..-.....151<br />
6"3.132 Huntington's disease... ,.,..i..i.....;............,...d,...i..."..,,."...."..152<br />
6.4 Diseussion..............<br />
6.4.1 The adult no'rraral and Huntington"s disease subependymal<br />
layer contain sorne projection neurons ,.."..154<br />
6.4.2T\e adtdt nornial and Huntington's disease subependymal<br />
Iayor contain some interneurons................ .-..,..,..........156<br />
64.3 The adult normal arrd fluntinglon's disease s.ubependymal<br />
laye! contain some glial cells.. .........,....."..158<br />
6:4.4The adult normal and Huntington's disease subependymal<br />
layer contain GABA6 reoeptor subunits..,.-.-.,...?......,r.. ...............160
Chapter 2<br />
LIST OF TABLES AND FIGURES<br />
Table 2.1<br />
Table of antibodies and the cell or cellular component detected by the antibody.............19<br />
Figure 2.1<br />
Stem and progenitor cells during development and in the adult brain. .............5<br />
Figure 2.2<br />
hogenitor cells during in vitro and in vivo expansion, and<br />
during symmetric or asymmetric division .........,...........6<br />
Figure 2.3<br />
The subependymal layer and granule cell layer in the rodent brain.......... ........9<br />
Figure 2.4<br />
Diagrammatic representation of the mouse subependymal layer<br />
(as described by Doetsch et al (1997))...... ...................21<br />
Figure 2.5<br />
Diagrammatic representation of the 3 types of lateral ventricle wall<br />
cytoarchitecture (as described by Rodriguez-Perez. et al (2003)).... ................................24<br />
Figure 2.6<br />
Cell types in the striatum and the relationship of the striatuln to<br />
the subependymal 1ayer.......... ....................36<br />
Chanter 3<br />
Table 3.1<br />
Table of normal and Huntington's disease cases.......... .................44<br />
Figure 3.1<br />
Anatomy of the ependymal and subependymal region in the normal brain......................48<br />
Figure 3.2<br />
Cell types in the subependymal layer of the normal adult human brain............................50<br />
Figure 3.3<br />
Comparison of the thickness of the subependymal layer in normal and<br />
Huntington's disease brains........ ...............54
Figure 3.4<br />
Thickness of the subependymal layer in normal<br />
and Huntington's disease brains........ .......55<br />
Figure 3.5<br />
Thickness of the subependymal layer in normal<br />
and Huntington's disease (grade l-3) brains. ..............56<br />
Figure 3.6<br />
The subependymal layer in normal and Huntington's<br />
disease (grades I and 3) brains........ ..........57<br />
Figure 3.7<br />
Number of cells/mm in the subependyrnal layer of normal<br />
and Huntington's disease brains........ ........59<br />
Figure 3.8<br />
Number of cells/mm in the subependymal layer of normal and Huntington's<br />
disease (grades l-3) brains. ......60<br />
Figure 3.9<br />
Cell types in the subependymal layer and ependymal layer of the<br />
normal and Huntington's disease brain.......... ..............62<br />
Figure 3.10<br />
Comparison of the numbers of the different cell types present in the<br />
normal and Huntington's disease (grades l-3) subependymal Iayer.................................63<br />
Chapter 4<br />
Table 4.1<br />
Normal and huntington's disease cases used for Western blotting analysis......................79<br />
Figure 4.1<br />
The number of proliferating cells in the Huntington's disease subependymal<br />
layer increase as the grade and CAG repeat length increase..... .....................83<br />
Figure 4.2<br />
Western blot analysis of normal and Huntington's disease<br />
subependymal layer homogenates................. ..............87<br />
Figure 4.3<br />
Localisation of proliferating, neuronal and glial cells in the<br />
Huntington's disease subependymal layer...... .............89<br />
xvll
Figure 4.4<br />
Neurogenesis and gliogenesis occur in the subependyrnal<br />
layer in Huntington's disease... ...,.,..:......... ..................91<br />
Figure 4.5<br />
A. The number of fluorescently labelled pcNA positive cells in<br />
the normal and Huntington's disease subependyirar rayer<br />
B. The number of pcNA/plll-tubulin positive cells in the normal<br />
and Huntington's disease subependyrnal layer...... ......g4<br />
Figure 4.6<br />
Neurogenesis in the normal brain subependymal 1ayer.......... .......95<br />
Figure 4.7<br />
Neurogenesis in the Huntington's disease subependymal layer...... ................96<br />
Chapter 5<br />
Table 5.1<br />
Table of normal and Huntington's clisease cases examined for the<br />
distribution of PCNA positive ceils in the subependymal |ayer.......... ..........1 l0<br />
Table 5.2<br />
ANOVA table for fixed effects................ ..................117<br />
Figure 5.1<br />
Diagram of a coronal section of the hurnan brain demonstrating<br />
'Areas' within the subependymal |ayer.......... ..........:....... ...........114<br />
Figure 5.2<br />
Diagrrun of a lateral section of the human brain clemonstrating .Areas,,<br />
'Regions' and 'Locations' within the subependyrnal 1ayer.......... .................1l5<br />
Figure 5.3<br />
Comparison of the number of pCNA positive cells in the<br />
normal and Huntington's disease subepenclymal layer..-... ..........1l9<br />
Figure 5.4<br />
The number of PCNA positive celrs in e<strong>ac</strong>h area (clorsal, middle, ventral)<br />
of the subependymal layer in normal and Huntingron.s disease brains...........................120<br />
Figure 5.5<br />
The number of PCNA positive cells in e<strong>ac</strong>h region (rostral, central caudal)<br />
of the subependymal layer in normal and Huntington's disease brains........ ...................122<br />
xvill
Figure 5.6<br />
The number of PCNA positive cells in different locations of the<br />
subependymal layer in normal and Huntington's disease brains........ ...........123<br />
Chanter 6<br />
Table 6.1<br />
Normal and Huntington's disease cases examined for the presence<br />
of mature cell markers in the subependymal layer...... ................L34<br />
Table 6.2<br />
Table summarising the staining demonstrated in the ependyma, subependyma<br />
and caudate nucleus in normal and Huntington's disease brains........ ...........153<br />
Figure 6.1<br />
hojection neuron markers in the subependyrnal layer<br />
of normal and Huntinston's disease brains........ ........138<br />
Figure 6.2<br />
Interneuron markers in the subependymal layer of normal<br />
and Huntington's disease brains........ ......141<br />
Figure 6.3<br />
Glial markers in the subependymal layer of normal<br />
and Huntinglon's disease brains........ ......146<br />
Figure 6.4<br />
GABAA receptor subunits in the subependymal layer of<br />
normal and Huntington's disease brains........ ............150<br />
Chapter 7<br />
Figure 7.1<br />
Possible method of targeting the subependymal layer for the delivery substances<br />
that f<strong>ac</strong>ilitate cell proliferation and differentiation.... ..................175<br />
Figure 7.2<br />
Diagram of the anatomy of the subependymal region in<br />
the Huntington's disease brain showing the possible path of<br />
migration for repl<strong>ac</strong>ement cells moving fi'om the subependymal<br />
layer to the caudate nucleus...... ...............I77<br />
xix
APS<br />
AraiC<br />
bctr-z<br />
BDhIF<br />
BLBP<br />
BrdU<br />
Calr<br />
ChAT<br />
,{<br />
CREB<br />
CT<br />
DAB<br />
DNA<br />
EGF<br />
ENK<br />
EPI<br />
FGF.2<br />
GABA.<br />
GAD<br />
GFAP<br />
HD<br />
IP,<br />
IGF.1<br />
u<br />
LV<br />
MEAO<br />
Iv{nn<br />
NCAM<br />
NeuN<br />
an'rmonium porsulfate<br />
cytosi ne-p-D-arabj nofu rancsi de<br />
B-ccll lymphoma protein-Z<br />
brain derived neurofrophic f<strong>ac</strong>tor<br />
brain lipid binding protein<br />
2-5 bromodeoxyiiridine<br />
sahetinin<br />
choline-<strong>ac</strong>etyl transferase<br />
caudate nuclzus<br />
cyclio adenosine monophosphate response element binding protein<br />
computed tomogr4phy<br />
3, 3 diamino be<strong>nz</strong>idine<br />
deoxy-ribose nueleic <strong>ac</strong>id<br />
epidermal growth f,<strong>ac</strong>tor<br />
onkephalin<br />
egendymal layer<br />
fi broblast growth f<strong>ac</strong>tor-2<br />
y-arrino butyric <strong>ac</strong>id<br />
glutfiric aeid decarboxyl ase<br />
gl-iat fibrillay <strong>ac</strong>idic protein<br />
Huntington's disease<br />
in ,apefltoneal<br />
insulin-like growth f<strong>ac</strong>ton- I<br />
labelling index<br />
lateratr venfiicle<br />
middlc cerebral artery occlusion<br />
rnagnetic resonance imaging<br />
nelral cell adhesion molecule<br />
neuronal nuclei<br />
xx
NGF nerve griwth f<strong>ac</strong>tor<br />
NMDA N-methyl D-aspartate<br />
NPY neuropepridq y<br />
NSE nouron*pecific enolase<br />
PErv parv:albumin<br />
PBS phosphate brdfered saline<br />
PCNA proliferatingcell nuolear andgen<br />
PS.A,-NCAM polysiarylated-neurdoefladhesionmorecule<br />
PVDF polyvinylidinerdi-fluoride<br />
r,pm revolutions per minute<br />
SEt eubeBendyrnal layer<br />
SP substancep<br />
STR s,g'iatr'ri<br />
t time.<br />
T6 total cell,eycietime<br />
Ts ttrre sp-ent in the S-phase sf the cell cyc-le<br />
TUI{EL transf,erase merrliated t4deoxyuridine triphosphate nick end labelling<br />
UV ultra violet<br />
VIM vfunentin<br />
xxr