Reduction in Nicotinic Receptors in Anterior Cingulate of Postmortem Human Alzheimer’s Disease and Parkinson’s Disease Brain

Objectives: Anterior cingulate in the human brain is innervated with nicotinic acetylcholinergic receptors (nAChRs) α4β2[asterisk] in the human brain. This receptor system is involved in cognition, learning and memory and may be adversely affected in neurodegeneration. In our efforts to translate use of human [¹⁸F]Nifene PET in Alzheimer’s disease (AD) and Parkinson’s disease (PD) to examine potential receptor alterations in the anterior cingulate, we have carried out quantitative autoradiographic evaluation of nAChRs α4β2[asterisk] in postmortem brain tissue consisting of anterior cingulate and corpus callosum. Control, AD and PD brains were studied using [³H]cytisine, which is known to bind to nAChRs α4β2[asterisk].

Methods: Human post-mortem brain tissues (male and female) consisting of anterior cingulate and corpus callosum (AD, n=6, age 64-89, PD, n=6, and cognitively normal (CN) controls, n=6; age 73-93; with confirmed pathology) were obtained from Banner Health, Sun City, Arizona. Brain slices (10 μm thick) consisting of both anterior cingulate and corpus callosum of each subject were obtained on a Leica 1850 cryotome. Adjacent brain slices were incubated [³H]Cytisine (0.03 μCi/cc) in Tris/pH 7.4 buffer at 2 ^oC for 90 mins. Nonspecific binding was measured using 300 μM nicotine. Adjacent sections were tested for Aβ using [³H]PIB (0.03 μCi/cc) in 10% ethanol and 90% PBS at 37 ^oC for 1 hr. Using the Optiquant program (Packard Instruments Co), regions of interest were drawn and digital light units/ mm² (DLU/mm²) were used to quantify the percentage change in binding of the radiotracers.

Results: All CN subjects exhibited significant [³H]Cytisine binding in the anterior cingulate whereas corpus callosum had very low levels of [³H]Cytisine. This is consistent with our PET [¹⁸F]Nifene results in healthy human subjects with distribution volume ratios (DVR) greater than 1.30 in the anterior cingulate region of the frontal cortex (using corpus callosum as the reference region). In the case of the autoradiographic studies with [³H]Cytisine, the ratio for anterior cingulate to corpus callosum in CN subjects was found to be 3.71. This ratio went down to 2.96 for the AD subjects suggesting a 20% decrease in [³H]Cytisine binding. In the case of the PD subjects, the ratio was found to be 3.3, suggesting a 11% decrease compared to CN subjects. When only anterior cingulate binding was considered, a greater decrease in the binding of [³H]Cytisine was observed in both AD and PD subjects compared to CN subjects. These findings are consistent with our previous observation suggesting a decreased binding of [³H]Cytisine in the frontal cortex of AD.

Conclusions: Our results indicate that nAChRs α4β2[asterisk] are reduced significantly in anterior cingulate of the AD postmortem brains. They are also affected in PD, with the anterior cingulate to corpus callosum ratio showing a smaller effect than compared to AD. Autoradiographic studies on these brain specimens are now being performed using [¹⁸F]Nifene in order to assess the sensitivity of the PET radiotracer and confirm the reductions in these postmortem AD and PD brains. The extent of reduction (>20%) in AD using the ratio method is greater than the 5-10% test-retest error found in our human [¹⁸F]Nifene PET studies in CN subjects. This suggests that [¹⁸F]Nifene may be a useful PET probe for AD and possibly PD patients. Research Support:NIH/NIA R33 AG029479 (JM)