Check GAD antibody positivity with the Diamyd anti-GAD RIA plate*

References1. Chattopadhyay, S., et al,An autoantibody inhibitory to glutamic acid decarboxylasein the neurodegeneartive disorder Batten disease.Hum. Mol. Genet. 11, 1421-1431, 2002.2. International Batten Disease Consortium. Isolation ofa novel gene underlying Batten disease.Cell 82, 949-957, 1995.3. Mitchison, H. M., et al,Targeted disruption of the Cln3 gene provides a mousemodel for Batten disease.Neurobiol. Dis. 6, 321-334, 1999.4. Chattopadhyay, S., et al,An autoantibody to GAD65 in sera of patients withjuvenile neuronal ceroid lipofuscinoses.Neurology. 59, 1816-1817, 2002.David Pearce is Assistant Professor ofBiochemistry and Biophysics in the Centerfor Aging and Developmental Biology at theUniversity of Rochester School of Medicineand Dentistry, Rochester, NY. Pearce is researchingthe molecular basis of juvenileneuronal ceroid lipofuscinosis (JNCL), orBatten disease. Pearce’s group discovered thepresence of autoantibodies to GAD65 inindividuals with Batten disease as well as ina mouse model for the human disease.GAD and Batten’s DiseaseTDavid Pearcehe discovery that individualswith juvenile neuronal ceroidlipofuscinosis, or Batten diseasehave circulating autoantibodiesto GAD65 is perhapsthe most recent chapter onGAD65 autoantibodies and disease, and certainlyone that requires further exploration. We firstpublished that individuals with Batten disease, anda mouse model for the disease had circulatingautoantibodies to GAD65 in Human MolecularGenetics in 2002 (1). The circumstances that ledto this report do not necessarily follow conventionalscientific reasoning, as much as to intuition.Batten disease is a pediatric onset devastatingfatal neurodegenerative disease. Individuals withBatten disease have inherited mutations in bothcopies of a still to be functionally characterizedgene product, designated CLN3 (2). We were studyinga mouse model that lacks a CLN3-gene productconstructed by others (3). Gene expressionstudies indicated that in the brain of cln3-knockoutmice, a shift in the expression of enzymesassociated to the synthesis and utilization of theneurotransmitter glutamate was altered. We nextconfirmed that endogenous levels ofglutamate/glutamic acid were elevated in thebrains of cln3-knockout mice. There was no reasonto suspect, and still isn’t, that the CLN3-gene productis directly involved in glutamate metabolism.I convinced Subrata Chattopadhyay the postdoctoralfellow working with me on this to use serumdrawn from the cln3-knockout mice as the primaryantibody in a western blot against a brain proteinextract. The basis for this was that a block inGAD activity, which converts glutamate to GABAcould be mediated by the presence of an autoantibodyto GAD, thus causing the elevation in glutamatethat we observed. It turned out that thecln3-knockout mice sera had circulating autoantibodiesreactive to a number of brain proteins.Most notably, there was a predominant protein ofaround 65kD. We knew that this would beGAD65, we just had to prove it. Thanks to thevast amount of research on GAD65 in diabetesand stiff persons syndrome we were able to benefitfrom reagents available and a plethora ofpublished techniques. Within the space of a fewmonths Subrata confirmed that the cln3-knockoutmice in addition to having elevated glutamate inthe brain, also had a decrease in enzymatic activityof GAD, most likely due to a demonstrated associationof autoantibody in the brain. His in vitrostudies showed that serum drawn from the cln3-knockout mice also contained an element thatcould inhibit the activity of GAD, and that wecould block the reactivity of the autoantibody toGAD65 by pre-incubation with recombinantGAD. At this point I felt we had biochemicallymet the criteria of showing that the autoantibodycould block an enzyme, GAD, resulting in accumulationof it’s substrate, glutamate, and that associationto the brain resulted in a decrease in theactivity of the enzyme. The final piece of this initialstudy was to show a possible link between theautoantibody and the disease. I might add that thisin many ways is an ongoing task.Nevertheless, this is where Jim Powers, a neuropathologistcame to the rescue. With the aid of hispostdoctoral fellow, Masumi Ito, he confirmed adecrease in GAD65 positive neurons in Batten diseasepost-mortem brain. I might add that Jim did ahuge amount of other work that aided our understandingof this phenomenon, and most importantly,he believed what initially was a strange idea,namely that GAD65 autoantibodies may be presentin the disease. Of course we have gone on toshow that individuals with Batten disease haveautoantibodies to GAD65, in fact to date everychild we have tested has come up positive.Furthermore, we have shown that other pediatricneurodegenerative diseases that fall in a similarcategory to Batten disease do not have circulatingautoantibodies to GAD65 (4). Our focus right nowis to deduce whether GAD65 autoantibodies arereally pathological, or simply epi-phenomenal inBatten disease. This of course is a mammoth biologicaltask, however, thanks again to the resourcesalready put forth in researching GAD65 autoantibodiesin other diseases, and some excellent collaborationswith researchers in this field, I believe wewill again benefit in expediting answering thisquestion. Importantly, establishing the potentialcontribution of the autoantibody to Batten disease,will reveal whether or not this is an element of thedisease process that should be targeted.page 40 dmccad june 2003