Experimental Characterization Of Alzheimer Disease Type Neuropathological Comorbidity In Temporal Lobe Epilepsy

Part I Expression of Amyloid and Tau Pathogenic Proteins in Epileptic CD1Mouse ForebrainRationale and Goal:Early studies suggest that human temporal lobe epilepsy (TLE) is associated with Alzheimer’s disease (AD)-type amyloid and tau pathology. Rodent pilocarpine rodent model of epilepsy recapitulates aspects of behavioral, electrophysiological and neuropathological features of human TLE, although the majority of studies use rats as model. We used CD1mice to establish the mouse model of TLE and explore potential alterations in the expression of proteins involved in AD neuropathology. The experimental model was evaluated by behavioral monitoring, neuronal activity recording and histological confirmation of aberrant mossy fiber sprouting.Materials and Methods:Adult CD1mice aged at2months were treated with pilocarpine or saline as control intraperitoneally. Animals survived the initial convulsive seizures were allowed to survive for24-48hours and7,14,30and60days, and perfused transcardially with paraformaldehyde. All animals were behaviorally monitored for recurrent convulsive epilepsy, with in vivo and hippocampal slice recording carried out in animals surviving7days. Nissl, Timm and neuropeptide Y (NPY) stainings were used to evaluate cell death and aberrant mossy fiber sprouting. Sections were processed immunohistochemically using antibodies to β-amyloid (Aβ) and its obligatory enzyme β-secretase-1(BACE1), phosphorylated human tau and relevant neuronal markers.Results:Pilocarpine-treated animals with recurrent convulsive epilepsy showed increased epileptic neuronal firing in field potential recording in the hippocampal formation. Cell death was detected clearly in hippocampal CA1and CAS sectors. Timm stain and NPY labeling revealled characteristic mossy fiber sprouting in the inner molecular layer in epileptic mice. BACE1elevation occurred in parallel with mossy fiber sprouting and axonal sprouting in subareas of CA1, the amygdala, and the piriform/entorhinal cortex. The regionally elevated BACE1expression in epileptic CD1mice colocalized locally with increased reactivity for the growth associated protein43, but reduced reactivity for the microtubule associated protein2.Summary and Conclusion:Pilocarpine epileptic CD1mice exhibit behavioral and pathological features of TLE. BACE1is upregulated in a time and region-specific manner in the limbic substructures in these mice in partnership with aberrant limbic axonal sprouting. In the absence of pre-existing high brain levels of soluble AP, TLE is associated with an intrinsic cellular modulation towards amyloidogenesis, although this does not lead AD-like plaque and tau pathology in adult wildtype mice.Part II Development of Alzheimer-like Neuropathology in3X Tg-AD Transgenic MiceRationale and Goal:The3X Tg-AD mice develop brain amyloid plaque and tau pathology with age. This study was to characterize these neuropathologies using in-house bred animals to facilitate further studies in Part Ⅲ. Also, it remains unknown whether BACE1alteration exists in this transgenic line and involves in plaque formation. Therefore, we carried out a thorough comparative study on BACE1expression relative to the age-related amyloid plaque pathogenesis.Materials and Methods:Brains from3X Tg-AD and non-transgenic mice aged1-26months were examined. Adjacent sets of sections were stained for BACE1, Aβ and phosphorylated tau (p-tau), and by double immunofluorescence for (1) BACE1with Aβ or p-tau;(2) Antibodies to the N-terminal, mid-region and C-terminal residues of Aβ domain;(3) BACE1with neuronal/axonal markers. Levels of BACE1and APP P-carboxyl terminal fragments (β-CTF), and BACE1activity, were measured in brain extracts from aged-matched transgenics and controls.Results:(1) Brain Aβ deposition, appearing mainly in diffuse form, developed in the transgenics, beginning at the subiculum by12-14months and spreading with age into the hippocampal and cortical areas. BACE1labeled dystrophic neurites developed in the forebrain spatiotemporally in parallel with the above plaque pathogenesis.(2) Antibodies targeting the Aβ N-terminal and mid-region labeled hippocampal and cortical principal neurons before plaque onset, as early as2months of age. With strong formic acid treatment, these antibodies also detected early swollen axonal terminals in the cortex and hippocampus, which exhibited colabeling for BACE1and APP P-CTF.(3) End-specific Aβ C-terminal antibodies only labeled extracellular amyloid plaques.(4) Hyperphosphorylation of tau occurred in the cortical and hippocampal principal neurons, detectable by2months of age and tended to increase with age.(5) BACE1/β-CTF protein levels and BACE1activity became elevated in the brain in3X Tg-AD relative to non-transgenic mice around mid-age, statistically significant by18-22months of age.Summary and Conclusion:The3X Tg-AD mice develop brain amyloid and tau pathology age-dependently; although there is no correlation between these two AD-like changes. Plauqes appear as compact and diffuse forms, both involve early BACE1elevation associated with axonal pathology. Axonal Aβ antibody reactivity might relate to a cross-reactivity of putative APP β-carboxyl terminal fragments. BACE1upregulation may play a crucial role in cerebral plaque pathogenesis in3×Tg-AD mice.Part III Development of Alzheimer-like Neuropathology in Epileptic3X Tg-AD MiceRationale and Goal:As BACE1elevation is associated with epilepsy-induced aberrant limbic axonal sprouting in CD1mice (Part I), and with neuritic dystrophy during plaque development in3X Tg-AD mice (Part Ⅱ), in this part of experiment we aimed to determine if the age-related amyloid and tau pathogenesis is altered in epileptic3X Tg-AD mice. In particular, we sort to test if experimentally induced aberrant axonal sprouting would lead to site-specific plaque formation, and to understand potential upstream factor(s) that might promote the amyloid and tau pathogenesis in the transgenic AD model animals.Materials and Methods:3×Tg-AD mice aged6-8months were treated with pilocarpine (i.p.) to induce acute convulsant seizures, with age-matched controls treated with saline. Animals with recurrent convulsant epilepsy were examined at9,11and14months of age (n=4/age group). BACE1, Aβ and phosphorylated tau (p-tau) immunoreactivity, and laminar (Nissl stain) and neuronal (immunoreactivity for the neuron-specific antigen, i.e., NeuN) integrity were comparatively assessed in the epileptics relative to age-matched controls.Results:(1) In contrast to controls, mossy fiber sprouting was present in all epileptic transgenics, characterized by a new BACE1immunoreactive band in the inner molecular layer.(2) BACE1immunoreactive neuritic clusters occurred in the hippocampal formation, piriform cortex and amygdala in9and11month-old epileptic, but not control, mice. These profiles were more numerous in the14month-old epileptics relative to controls.(3) The BACE1-labeled axonal dystrophic neurites colocalized with local extracellular Aβ deposition.(4) Densitometry carried over the subiculum and CA1confirmed increases in the amount of dystrophic neurites and amyloid plaques in the epileptic groups relative to controls.(5) Increased p-tau immunoreactivity occurred in dentate granule cells, but reduced in CA1pyramidal neurons in the epileptic mice.(6) Cell loss and neuronal death occurred in CA1, CA3and local areas of the piriform cortex in the epileptic animals.Summary and Conclusion:Pilocarpine-induced chronic epilepsy was associated with accelerated and enhanced neuritic plaque formation and altered intraneuronal p-tau expression in temporal lobe structures in3×Tg-AD mice relative to aged-match controls, with these pathologies occurred in regions showing neuronal death and axonal dystrophy. The excessive amyloid plaque formation in the epileptic transgenics may be a consequence of enhanced BACE1expression intrinsic to axonal pathology and neuritic dystrophy in affected limbic brain regions.