130th Annual MeetingTuesday, September 27, 2005Abstracts: Plenary Session

(8:00 AM–8:15 AM)1. Functional Electrical Stimulation EnhancesNeuronal Differentiation of Transplanted EmbryonicStem Cells in Chronic Spinal Cord InjuryDaniel Becker, Carlo O. Martinez, and John W. McDonald;Nashville, TN; St. Louis, MO; and Baltimore, MD

We examined the effects of functional electrical stimulation(FES)–induced stepping-like hindlimb movements on thedifferentiation of transplanted embryonic stem (ES) cell–de-rived neural progenitor cells in rats with chronic spinal cordinjury (SCI). Six weeks following complete T8/9 suction–ablation SCI in adult female rats (postinjury day [PID] 43),we implanted two-channel FES electrodes into the hindlimbs(peroneal nerves; n � 8) and transplanted 100,000 GFP-expressing (4�/4� staged) neural induced ES cells one seg-ment above and below the injury level. FES implants wereactivated in half the rats for 3 hours per day (PID45–60).Animals were sacrificed on PID60. In non–FES-stimulatedrats, significantly fewer transplanted ES cells differentiatedinto neuronal phenotypes below the injury level than above,and FES restored this to values above the injury level(%NeuN�/GFP� cells: 9 � 1% below vs 27 � 9% abovein non–FES-stimulated rats; 32 � 4% below in FES-stimulated rats). This report represents the first demonstra-tion that FES can increase the percentage of neuronal differ-entiated ES cells after transplantation in adult chronic SCIand offers potential clinical approaches to alter or maximizethe efficacy of neural transplantation.

(8:15 AM–8:30 AM)2. Quantitation of In Vivo Amyloid-Beta Synthesisand Clearance Rates in Humans Using Stable IsotopeLabeling and Mass SpectrometryRandall J. Bateman, Ling Munsell, Kevin Yarasheski,John C. Morris, and David M. Holtzman; St. Louis, MO

The amyloid hypothesis suggests that amyloid-beta (A�)plays a critical role in the pathogenesis of Alzheimer’s disease(AD). This hypothesis proposes that accumulation of A�, intoxic forms, leads to downstream events that culminate indementia. To evaluate the physiology of A� in humans, wedeveloped a technique to quantify the synthesis and clearancerates of A� in vivo in humans. In healthy volunteers, 13C6-leucine was administered intravenously to metabolically labelA� in vivo, while serial cerebrospinal fluid (CSF) and bloodsamples were collected hourly through catheters. A� was im-munoisolated from CSF, the amount of 13C6-leucine incor-porated into A� was quantified using mass spectrometry, andrates of synthesis and clearance were calculated. The frac-tional synthesis rate of A�, as measured in CSF, was approx-imately 6% per hour—one of the fastest measured produc-tion rates of a protein. This technique may be used to studythe pathophysiology of A� in AD patients compared withcontrol subjects. It may also provide a biomarker of A� me-tabolism that can be used to monitor AD progression andthe effects of novel therapeutic agents on A� synthesis orclearance. Study supported by NIH grants P50-AG05681,MO1 RR00036, Washington University Biomedical Mass

Spectrometry Research Laboratory RR-00954, WashingtonUniversity Clinical Nutrition Research Unit DK-56341,m266 antibody provided by Eli Lilly.

(8:30 AM–8:45 AM)3. Stiff Person Syndrome with Cerebellar Disease andEpitope-Specific Anti-GAD AntibodiesGoran Rakocevic, Cristina Semino-Mora, Raghavan Raju,and Marinos C. Dalakas; Bethesda, MD

Stiff person syndrome (SPS) is an autoimmune disordercharacterized by stiffness and episodic spasms. High-titeranti-GAD antibodies, although diagnostic for SPS, are alsoseen in rare patients with autoimmune cerebellar disease. Wedescribe four anti-GAD–positive patients with typical SPSwho developed severe cerebellar disease (SPS � Cer) charac-terized by prominent gait or focal limb ataxia, dysarthria,and oculomotor signs. Brain magnetic resonance imagingscan was normal. Serum and cerebrospinal fluid anti-GADantibodies and intrathecal production of GAD-specific im-munoglobulin G (IgG) were high, but the titers were notdifferent between SPS � Cer and SPS without cerebellarsymptoms. When coded sera were applied to rat cerebellum,the SPS � Cer patients had distinct immunoreactivity. TheIgG from three of four SPS � Cer patients stained exclu-sively the granular cell layer and colocalized with GAD; incontrast, IgG from the other patients gave a scattered spottypattern in the cortex and colocalized with GAD. We con-clude that: a) a subset of SPS patients have cerebellar diseasewith high anti-GAD antibodies, b) GAD antibodies fromSPS � Cer patients recognize GAD epitopes unique to thecerebellum, and c) in SPS � Cer patients, anti-GAD orother related antibodies selectively suppress GABA-mediatedneurotransmission that corresponds to a cerebellar pheno-type.

(8:45 AM–9:00 AM)4. Histone Modifications in Huntington’s DiseaseGhazaleh Sadri-Vakili, Prianka Chawla, Kelly E. Glajch,Ryan P. Overland, and Jang-Ho J. Cha; Charlestown, MA

Transcriptional dysregulation has been proposed to play amajor role in Huntington’s disease (HD), but the mecha-nisms that cause selective gene downregulation remain un-known. Histone proteins regulate chromatin structure andcontrol gene expression; recent studies have suggested a rolefor histone deacetylase inhibitors in the treatment of HD.However, little is known about histones in HD; for example,it is not known whether downregulated genes in HD haveabnormal histone modifications. We hypothesized that mu-tant huntingtin expression can alter histone modificationsand lead to the transcriptional changes. We analyzed histoneH3 and H4 acetylation in the R6/2 mouse using westernblotting and immunohistochemistry. Our results demon-strate hypoacetylation of histones at 8 and 12 weeks in theR6/2 striatum but not the cortex, hippocampus, or cerebel-lum. In addition, there is a decrease in histone acetyltrans-ferase activity in the R6/2 striatum and STHdh Q111/111cell lines. Finally, there is a progressive decrease in acetylatedhistone H3 associated with specific genes in the R6/2 stria-tum as measured by a combination of chromatin immuno-precipitation (ChIP) and quantitative real-time polymerasechain reaction analysis. Together, these results suggest thathistone hypoacetylation can possibly be an underlying mech-anism of transcriptional dysregulation in HD. Study sup-ported by NIH NS38106, NS 45242, Huntington’s Disease

© 2005 American Neurological Association S1Published by Wiley-Liss, Inc., through Wiley Subscription Services

Society of America Coalition for the Cure, Hereditary Dis-ease Foundation, HighQ Foundation, Glendorn Foundation.

(9:05 AM–9:35 AM)5. Neuroprotection in the Peripheral Nervous SystemAhmet Hoke; Baltimore, MD

Recent advances in the field suggest that mechanisms under-lying axonal and neuronal degeneration differ. Consideringthat most degenerative disorders affecting the peripheral ner-vous system (PNS) do not cause significant neuronal loss,but lead to distal axonal degeneration, neuroprotective strat-egies aimed at rescuing the “neuronal body” may not haverelevance to recovery or rescue of function in the PNS. Bet-ter understanding of the underlying mechanisms of axonaldegeneration will lead to more rational “axonoprotective”and axonal-regenerative therapies. Using a combination of invitro culture techniques including compartmented culturesystem to study distal axonal events, and animal models, onecan dissect out mechanisms specific for model diseases andtest potential therapeutic compounds. Combining these ap-proaches, I will give insights into disease-specific mechanismsincluding human immunodeficiency virus andchemotherapy-induced neuropathies and the role of erythro-poietin as an endogenous and exogenous neuroprotectivecompound in peripheral neuropathies. One can use such ra-tional approaches to identify novel roles for endogenous tro-phic/tropic factors, and I will give an example of the rolepleiotrophin plays in motor neuron survival and regenera-tion.

(10:00 AM–10:15 AM)6. Evaluating Survival in Nontransplanted Boys withCerebral Phenotype of X-Linked AdrenoleukodystrophyAsif Mahmood, Prachi Dubey, Gerald V. Raymond, andHugo W. Moser; Baltimore, MD

Hematopoietic cell transplant (HCT) in X-linked adrenoleu-kodystrophy (X-ALD) demonstrated beneficial effects in pa-tients with cerebral phenotype of X-ALD (CERALD). In thisretrospective study, we analyzed the survival function inCERALD patients who did not receive bone marrow trans-plant to assess the comparative benefit obtained from HCT.Kaplan–Meier survival analysis was performed in 246 CER-ALD patients from 196 kindreds. Mean follow-up durationwas 4.8 � 4.9 years. The mean age of onset of symptomswas 7 � 2 years. A total of 111 patients died at the meanage of 12 � 4 years. Median survival time was 7 years fromthe age of onset of symptom. The 5-year survival rate fromthe onset of symptoms in these patients was 59%. Therewere no significant differences in survival time in patientsyounger than 7 years at baseline (median cutoff) versus older(p � 0.6). Results demonstrate that the 5-year survival rateof patients with CERALD from the time of onset of firstsymptoms is lower than the reported 90% in patients whoreceived HCT and did not have severe neuropsychologicaland magnetic resonance imaging abnormalities at the time ofHCT. These findings support the beneficial effect of HCTas a treatment for CERALD patients, fulfilling the eligibilitycriteria for HCT.

(10:15 AM–10:30 AM)7. Transcriptional Analysis in Amyotrophic LateralSclerosis: Mechanism for Loss for GlutamateTransporter EAAT2 and Evidence forAstroglial DysfunctionChristine Haenggeli, Melissa R. Regan, Natalie Perez,Andrew M. Watkins, and Jeffrey D. Rothstein;Baltimore, MD

Multiple pathways contribute to dysfunction and death ofneurons in amyotrophic lateral sclerosis (ALS). Studies in fa-milial and sporadic ALS tissue along with transgenic animalmodels suggest that glutamate mediated toxicity is part ofthis process. The loss of the astroglial glutamate transporterEAAT2/GLT1 appears to occur in all models examined, andits loss likely contributes to neurodegeneration. The mecha-nism for the loss of the protein remains unknown. Studies ofastroglial response to injury indicate that downregulation ofthe protein may occur in chronic and acute insults. We usedGLT1-BAC transgenic mice, expressing the full-length geneand promoter for GLT1, with an eGFP reporter, to evaluatethe regulation of GLT1 in ALS. G93A SOD1 ALS trans-genic mice were crossed with GLT1-BAC-eGFP mice, andtissues were examined at multiple time points. This tech-nique allowed analysis of the gene regulation at the tissueand cellular level. Confocal and tissue analysis of eGFP ex-pression revealed downregulation of the GLT-1 promoter incells surrounding motor neurons as early as 100 days of age,consistent with the hypothesis that astroglial dysfunction isthe principal mechanism for loss of transporter protein.Study supported by NIH.

(10:30 AM–10:45 AM)8. Increased Capillaries in Mitochondrial Myopathy:Implications for Mitochondrial Regulation ofCapillary GrowthTanja Taivassalo, Christine Kunesh, and Ronald G. Haller;Dallas, TX

Muscle capillary volume normally parallels myofiber oxida-tive capacity, increasing with training via hypoxia-mediatedinduction of vascular endothelial growth factor (VEGF). Im-paired OxPhos in mitochondrial myopathy (MM) causeshigh muscle O2 levels in exercise, but capillary effects havenot been evaluated. We assessed oxidative capacity (cycle er-gometry) and muscle capillaries and VEGF (CD31 andVEGF immunostaining) in needle biopsies of vastus lateralisin four patients with heteroplasmic mtDNA mutations andfour control subjects. In patients, respiratory incompetent fi-bers were identified as ragged red (Gomori trichrome) orCOX-negative fibers. Peak oxidative capacity in patients(0.76 � 0.3) was one-third that of control subjects (2.23 �0.4 L/min) but overall capillary to muscle fiber area washigher in patients (MM: 2.6 � 0.5; controls: 1.7 � 0.2, p �0.05) In each patient, capillary-to-myofiber area was three-fold higher (p � 0.05) in respiratory incompetent versusnormal fibers: 1) 6.9 � 2.4 vs 2.0 � 0.6; 2) 4.0 � 0.7 vs1.2 � 0.1; 3) 3.0 � 0.4 vs 1.4 � 0.2; 4) 4.5 � 1.8 vs 1.2 �0.4%). Qualitative increases in VEGF staining paralleled in-creased capillaries in respiratory incompetent fibers. Our re-sults suggest that impaired oxidative metabolism in MM dueto mtDNA mutations activates capillary growth by an hyp-oxia independent mechanism. Study supported by MDA andVA Merit Review

S2 Annals of Neurology Vol 58 (suppl 9) 2005

(10:45 AM–11:00 AM)9. An Alternative Route for F-box Protein–MediatedUbiquitination Links the Co-Chaperone/UbiquitinLigase CHIP to Glycoprotein Quality ControlRick F. Nelson, Victor M. Miller, Hsiang Wen, andHenry L. Paulson; Iowa City, IA

Defining the enzymes that regulate ubiquitin-dependent pro-tein quality control is vital to understanding neurodegenera-tive diseases. Here we describe a novel pathway for ubiquiti-nation in neurons. In SCF (Skp1/Cullin/F-box protein)ubiquitin ligases, substrate specificity is conferred by a di-verse array of F-box proteins that interact with Skp1 throughtheir F-box domain. Only in fully assembled SCF complexes,it is believed, can substrates bound to F-box proteins becomeubiquitinated. We now define an alternative route by whichan F-box protein can mediate ubiquitination. We show thatFbx2, a brain-enriched F-box protein implicated in ubiquiti-

nation of glycoproteins discarded from the endoplasmic re-ticulum, preferentially binds the co-chaperone/ubiquitin li-gase CHIP rather than its expected partner, Skp1. CHIP isknown to handle numerous brain substrates, including keyproteins in Parkinson disease and dementias. In Fbx2, a pre-dicted PEST domain flanking the F-box, mediates bindingto CHIP, which then directs ubiquitination and degradationof Fbx2-bound glycoproteins independent of SCF formation.Our results demonstrate a noncanonical pathway for proteinturnover that links CHIP to glycoprotein quality control andexpands the repertoire of pathways by which F-box proteinscan regulate ubiquitination. Study supported by NIH Pre-doctoral Grant F30 NS047872 (to RFN), T32 GM08629(to VMM and UI Program in Genetics), and NIH R01NS47535 (to HLP)

DOI: 10.1002/ana.11299

Program and Abstracts, American Neurological Association S3