Impaired Glucose Tolerance and Reduced Plasma Insulin Precede Decreased AKT Phosphorylation and GLUT3 Translocation in the Hippocampus of Old 3xTg-AD Mice.

Several studies have demonstrated that mouse models of Alzheimer's disease (AD) can exhibit impaired peripheral glucose tolerance. Further, in the APP/PS1 mouse model, this is observed prior to the appearance of AD-related neuropathology (e.g., amyloid-ß plaques; Aß) or cognitive impairment. In the current study, we examined whether impaired glucose tolerance also preceded AD-like changes in the triple transgenic model of AD (3xTg-AD). Glucose tolerance testing (GTT), insulin ELISAs, and insulin tolerance testing (ITT) were performed at ages prior to (1-3 months and 6-8 months old) and post-pathology (16-18 months old). Additionally, we examined for altered insulin signaling in the hippocampus. Western blots were used to evaluate the two-primary insulin signaling pathways: PI3K/AKT and MAPK/ERK. Since the PI3K/AKT pathway affects several downstream targets associated with metabolism (e.g., GSK3, glucose transporters), western blots were used to examine possible alterations in the expression, translocation, or activation of these targets. We found that 3xTg-AD mice display impaired glucose tolerance as early as 1 month of age, concomitant with a decrease in plasma insulin levels well prior to the detection of plaques (∼14 months old), aggregates of hyperphosphorylated tau (∼18 months old), and cognitive decline (≥18 months old). These alterations in peripheral metabolism were seen at all time points examined. In comparison, PI3K/AKT, but not MAPK/ERK, signaling was altered in the hippocampus only in 18-20-month-old 3xTg-AD mice, a time point at which there was a reduction in GLUT3 translocation to the plasma membrane. Taken together, our results provide further evidence that disruptions in energy metabolism may represent a foundational step in the development of AD.

Lack of human-like extracellular sortilin neuropathology in transgenic Alzheimer's disease model mice and macaques.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder bearing multiple pathological hallmarks suggestive of complex cellular/molecular interplay during pathogenesis. Transgenic mice and nonhuman primates are used as disease models for mechanistic and translational research into AD; the extent to which these animal models recapitulate AD-type neuropathology is an issue of importance. Putative C-terminal fragments from sortilin, a member of the vacuolar protein sorting 10 protein (Vps10p) family, have recently been shown to deposit in the neuritic ß-amyloid (Aß) plaques in the human brain. METHODS: We set out to explore if extracellular sortilin neuropathology exists in AD-related transgenic mice and nonhuman primates. Brains from different transgenic strains and ages developed overt cerebral Aß deposition, including the ß-amyloid precursor protein and presenilin 1 double-transgenic (APP/PS1) mice at ~ 14 months of age, the five familial Alzheimer's disease mutations transgenic (5×FAD) mice at ~ 8 months, the triple-transgenic Alzheimer's disease (3×Tg-AD) mice at ~ 22 months, and aged monkeys (Macaca mulatta and Macaca fascicularis) were examined. Brain samples from young transgenic mice, middle-aged/aged monkeys, and AD humans were used as negative and positive pathological controls. RESULTS: The C-terminal sortilin antibody, which labeled senile plaques in the AD human cerebral sections, did not display extracellular immunolabeling in the transgenic mouse or aged monkey brain sections with Aß deposition. In Western blot analysis, sortilin fragments ~ 15 kDa were not detectable in transgenic mouse cortical lysates, but they occurred in control AD lysates. CONCLUSIONS: In reference to their human brain counterparts, neuritic plaques seen in transgenic AD model mouse brains represent an incomplete form of this AD pathological hallmark. The species difference in neuritic plaque constituents also indicates more complex secondary proteopathies in the human brain relative to rodents and nonhuman primates during aging and in AD.

Evidence for altered insulin receptor signaling in Alzheimer's disease.

Epidemiological data have shown that metabolic disease can increase the propensity for developing cognitive decline and dementia, particularly Alzheimer's disease (AD). While this interaction is not completely understood, clinical studies suggest that both hyper- and hypoinsulinemia are associated with an increased risk for developing AD. Indeed, insulin signaling is altered in post-mortem brain tissue from AD patients and treatments known to enhance insulin signaling can improve cognitive function. Further, clinical evidence has shown that AD patients and mouse models of AD often display alterations in peripheral metabolism. Since insulin is primarily derived from the periphery, it is likely that changes in peripheral insulin levels lead to alterations in central nervous system (CNS) insulin signaling and could contribute to cognitive decline and pathogenesis. Developing a better understanding of the relationship between alterations in peripheral metabolism and cognitive function might provide a foundation for the development of better treatment options for patients with AD. In this article we will begin to piece together the present data defining this relationship by briefly discussing insulin signaling in the periphery and CNS, its role in cognitive function, insulin's relationship to AD, peripheral metabolic alterations in mouse models of AD and how information from these models helps understand the mechanisms through which these changes potentially lead to impairments in insulin signaling in the CNS, and potential ways to target insulin signaling that could improve cognitive function in AD. This article is part of the Special Issue entitled 'Metabolic Impairment as Risk Factors for Neurodegenerative Disorders.'

Differential Fasting Plasma Glucose and Ketone Body Levels in GHRKO versus 3xTg-AD Mice: A Potential Contributor to Aging-Related Cognitive Status?

Cognitive function declines with age and appears to correlate with decreased cerebral metabolic rate (CMR). Caloric restriction, an antiaging manipulation that extends life-span and can preserve cognitive function, is associated with decreased glucose uptake, decreased lactate levels, and increased ketone body (KB) levels in the brain. Since the majority of brain nutrients come from the periphery, this study examined whether the capacity to regulate peripheral glucose levels and KB production differs in animals with successful cognitive aging (growth hormone receptor knockouts, GHRKOs) versus unsuccessful cognitive aging (the 3xTg-AD mouse model of Alzheimer's disease). Animals were fasted for 5 hours with their plasma glucose and KB levels subsequently measured. Intriguingly, in GHRKO mice, compared to those in controls, fasting plasma glucose levels were significantly decreased while their KB levels were significantly increased. Conversely, 3xTg-AD mice, compared to controls, exhibited significantly elevated plasma glucose levels and significantly reduced plasma KB levels. Taken together, these results suggest that the capacity to provide the brain with KBs versus glucose throughout an animal's life could somehow help preserve cognitive function with age, potentially through minimizing overall brain exposure to reactive oxygen species and advanced glycation end products and improving mitochondrial function.

Glucose tolerance and insulin sensitivity are impaired in APP/PS1 transgenic mice prior to amyloid plaque pathogenesis and cognitive decline.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aß) deposition, neurofibrillary tangles and cognitive decline. Clinical data suggests that both type 1 and type 2 diabetes are risk factors for AD-related dementia and several clinical studies have demonstrated that AD patients show alterations in peripheral glucose regulation characterized by insulin resistance (hyperinsulinemia) or hypoinsulinemia. Whether animal models of AD exhibit a pre-diabetic phenotype without additional dietary or experimental manipulation is unclear however, with contradictory data available. Further, most studies have not examined the time course of potential pre-diabetic changes relative to AD pathogenesis and cognitive decline. Thus, in this study we tested the hypothesis that a pre-diabetic phenotype (peripheral metabolic dysregulation) exists in the APP/PS1 transgenic model of AD under normal conditions and precedes AD-related pathology. Specifically, we examined glucose tolerance in male APP/PS1 mice on a C57BL/6J congenic background at 2, 4-6 and 8-9months of age by assessing fasting glucose levels, glucose tolerance, plasma insulin levels and insulin sensitivity as well as the development of pathological characteristics of AD and verified that our APP/PS1 mice develop cognitive impairment. Here we show that APP/PS1 mice, compared to wild-type controls, exhibit a significant impairment in glucose tolerance during an intraperitoneal glucose tolerance test (ipGTT) and a trend for increased fasting plasma insulin concentrations as early as 2months of age, while extracellular Aß1-42 deposition occurs later and cognitive decline exists at 8-9months of age. Moreover, APP/PS1 mice did not respond as well to exogenous insulin as the wild-type controls during an intraperitoneal insulin tolerance test (ipITT). Taken together, these data reveal that male APP/PS1 mice on a C57BL/6J congenic background exhibit a pre-diabetic phenotype prior to the development of AD-like pathology and that this metabolic deficit persists when they exhibit neuropathology and cognitive decline. This raises the question of whether altered glucose regulation and insulin production/secretion could contribute to AD pathogenesis.

Experimentally induced diabetes worsens neuropathology, but not learning and memory, in middle aged 3xTg mice.

Alzheimer's disease (AD) is the primary cause of dementia in the elderly. The cause of the disease is still unknown, but amyloid plaques and neurofibrillary tangles in the brain are thought to play a role. However, transgenic mouse models expressing these neuropathological features do not show severe or consistent cognitive impairments. There is accumulating evidence that diabetes increases the risk for developing AD. We tested the hypothesis that experimentally induced diabetes would exacerbate cognitive symptoms in a mouse model of AD. Diabetes was induced in 12-month old 3xTg mice using streptozotocin (STZ; 90mg/kg, i.p., on two successive days). Hyperglycemia was verified by sampling blood glucose levels. Three months after injection (at 15 months of age), the mice were behaviorally tested in the Morris water maze and contextual fear conditioning. Subsequently, the hippocampal region was examined using immunohistochemistry (6E10 antibody for amyloid) and immunoblotting (AT8 antibody for phosphorylated tau). No differences were found in learning or memory between the vehicle-treated control and STZ-treated groups. A significant increase in the number of amyloid-positive plaques was observed in the subiculum of STZ-treated mice; very few plaques were seen in other hippocampal regions in either group. No differences in AT8 load were observed. These results reinforce that amyloid plaques, per se, are not sufficient to cause memory impairments. Further, while diabetes can enhance this aspect of brain pathology, the combination of disrupted glucose metabolism and the transgenes is still not sufficient to cause the severe cognitive impairments associated with clinical AD.

Aberrant expression of the pore-forming KATP channel subunit Kir6.2 in hippocampal reactive astrocytes in the 3xTg-AD mouse model and human Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aß) deposition, neurofibrillary tangles and cognitive decline. Recent pharmacologic studies have found that ATP-sensitive potassium (KATP) channels may play a role in AD and could be a potential therapeutic target. Interestingly, these channels are found in both neurons and astrocytes. One of the hallmarks associated with AD is reactive gliosis and a change in astrocytic function has been identified in several neuropathological conditions including AD. Thus the goal of this study was to examine whether the pore-forming subunits of KATP channels, Kir6.1 and Kir6.2, are altered in the hippocampus in a cell type-specific manner of the 3xTg-AD mouse model of AD and in human AD tissue obtained from the Chinese brain bank. Specifically, in old 3xTg-AD mice, and age-matched controls, we examined glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), Kir6.1 and Kir6.2 in hippocampal region CA1 with a combination of immunoblotting and immunohistochemistry (IHC). A time point was selected when memory impairment and histopathological changes have been reported to occur in 3xTg-AD mice. In human AD and age-matched control tissue IHC experiments were performed using GFAP and Kir6.2. In the hippocampus of 3xTg-AD mice, compared to wild-type controls, Western blots showed a significant increase in GFAP indicating astrogliosis. Further, there was an increase in Kir6.2, but not Kir6.1 in the plasma membrane fraction. IHC examination of hippocampal region CA1 in 3xTg-AD sections revealed an increase in Kir6.2 immunoreactivity (IR) in astrocytes as identified by GFAP and GS. In human AD tissue similar data were obtained. There was an increase in GFAP-IR in the stratum oriens (SO) and alveus (ALV) of CA1 concomitant with an increase in Kir6.2-IR in cells with an astrocytic-like morphology. Dual immunofluorescence revealed a dramatic increase in co-localization of Kir6.2-IR and GFAP-IR. Taken together, these data demonstrate that increased Kir6.2 is seen in reactive astrocytes in old 3xTg-AD mice and human AD tissue. These changes could dramatically alter astrocytic function and subsequently contribute to AD phenotype in either a compensatory or pathophysiological manner.

Sp8 expression in putative neural progenitor cells in guinea pig and human cerebrum.

Neural stem/progenitor cells have been characterized at neurogenic sites in adult mammalian brain with various molecular markers. Here it has been demonstrated that Sp8, a transcription factor typically expressed among mature GABAergic interneurons, also labels putative neural precursors in adult guinea pig and human cerebrum. In guinea pigs, Sp8 immunoreactive (Sp8+) cells were localized largely in the superficial layers of the cortex including layer I, as well as the subventricular zone (SVZ) and subgranular zone (SGZ). Sp8+ cells at the SGZ showed little colocalization with mature and immature neuronal markers, but co-expressed neural stem cell markers including Sox2. Some layer I Sp8+ cells also co-expressed Sox2. The amount of Sp8+ cells in the dentate gyrus was maintained 2 weeks after X-ray irradiation, while that of doublecortin (DCX+) cells was greatly reduced. Mild ischemic insult caused a transient increase of Sp8+ cells in the SGZ and layer I, with the subgranular Sp8+ cells exhibited an increased colabeling for the mitotic marker Ki67 and pulse-chased bromodeoxyuridine (BrdU). Sp8+ cells in the dentate gyrus showed an age-related decline in guinea pigs, in parallel with the loss of DCX+ cells in the same region. In adult humans, Sp8+ cells exhibited comparable morphological features as seen in guinea pigs, with those at the SGZ and some in cortical layer I co-expressed Sox2. Together, these results suggested that Sp8 may label putative neural progenitors in guinea pig and human cerebrum, with the labeled cells in the SGZ appeared largely not mitotically active under normal conditions. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 939-955, 2016.

Prenatal genesis of layer II doublecortin expressing neurons in neonatal and young adult guinea pig cerebral cortex.

Cells expressing doublecortin (DCX+) occur at cortical layer II, predominantly over the paleocortex in mice/rats, but also across the neocortex among larger mammals. Here, we explored the time of origin of these cells in neonatal and 2-month-old guinea pigs following prenatal BrdU pulse-chasing. In the neocortex, BrdU+ cells birth-dated at embryonic day 21 (E21), E28, and E35 laminated over the cortical plate with an inside-out order. In the piriform cortex, cells generated at E21 and E28 occurred with a greater density in layer II than III. Many cells were generated at later time points until birth, occurring in the cortex without a laminar preference. DCX+ cells in the neocortex and piriform cortex partially co-colocalized with BrdU (up to 7.5%) in the newborns after pulse-chasing from E21 to E49 and in the 2 month-old animals after pulse-chasing from E28 to E60/61, with higher rates seen among the E21-E35 groups. Together, layer II DCX+ cells in neonatal and young adult guinea pigs may be produced over a wide prenatal time window, but mainly during the early phases of corticogenesis. Our data also show an earlier establishment of the basic lamination in the piriform relative to neocortical areas in guinea pigs.

Experimental microembolism induces localized neuritic pathology in guinea pig cerebrum.

Microbleeds are a common finding in aged human brains. In Alzheimer's disease (AD), neuritic plaques composed of ß-amyloid (Aß) deposits and dystrophic neurites occur frequently around cerebral vasculature, raising a compelling question as to whether, and if so, how, microvascular abnormality and amyloid/neuritic pathology might be causally related. Here we used a guinea pig model of cerebral microembolism to explore a potential inductive effect of vascular injury on neuritic and amyloid pathogenesis. Brains were examined 7-30 days after experimental microvascular embolization occupying ~0.5% of total cortical area. Compared to sham-operated controls, glial fibrillary acidic protein immunoreactivity was increased in the embolized cerebrum, evidently around intracortical vasculature. Swollen/sprouting neurites exhibiting increased reactivity of nicotinamide adenine dinucleotide phosphate diaphorase, parvalbumin, vesicular glutamate transporter 1 and choline acetyltransferase appeared locally in the embolized brains in proximity to intracortical vasculature. The embolization-induced swollen/sprouting neurites were also robustly immunoreactive for ß-amyloid precursor protein and ß-secretase-1, the substrate and initiating enzyme for Aß genesis. These experimental data suggest that microvascular injury can induce multisystem neuritic pathology associated with an enhanced amyloidogenic potential in wild-type mammalian brain.

Lipolysaccharide-Induced Neuroinflammation Is Associated with Alzheimer-Like Amyloidogenic Axonal Pathology and Dendritic Degeneration in Rats.

Chronic neuroinflammation is thought to play an etiological role in Alzheimer's disease (AD), which is characterized pathologically by amyloid and tau formation, as well as neuritic dystrophy and synaptic degeneration. The causal relationship between these pathological events is a topic of ongoing research and discussion. Recent data from transgenic AD models point to a tight spatiotemporal link between neuritic and amyloid pathology, with the obligatory enzyme for ß-amyloid (Aß) production, namely ß-secretase-1 (BACE1), is overexpressed in axon terminals undergoing dystrophic change. However, the axonal pathology inherent with BACE1 elevation seen in transgenic AD mice may be secondary to increased soluble Aß in these genetically modified animals. Here we explored the occurrence of the AD-like axonal and dendritic pathology in adult rat brain affected by LPS-induced chronic neuroinflammation. Unilateral intracerebral LPS injection induced prominent inflammatory response in glial cells in the ipsilateral cortex and hippocampal formation. BACE1 protein levels were elevated the ipsilateral hippocampal lysates in the LPS treated animals relative to controls. BACE1 immunoreactive dystrophic axons appeared in the LPS-treated ipsilateral cortex and hippocampal formation, colocalizing with increased ß-amyloid precursor protein and Aß antibody (4G8) immunolabeling. Quantitative Golgi studies revealed reduction of dendritic branching points and spine density on cortical layer III and hippocampal CA3 pyramidal neurons in the LPS-treated ipsilateral cerebrum. These findings suggest that Alzheimer-like amyloidogenic axonal pathology and dendritic degeneration occur in wildtype mammalian brain in partnership with neuroinflammation following LPS injection.

Altered network timing in the CA3-CA1 circuit of hippocampal slices from aged mice.

Network patterns are believed to provide unique temporal contexts for coordinating neuronal activity within and across different regions of the brain. Some of the characteristics of network patterns modeled in vitro are altered in the CA3 or CA1 subregions of hippocampal slices from aged mice. CA3-CA1 network interactions have not been examined previously. We used slices from aged and adult mice to model spontaneous sharp wave ripples and carbachol-induced gamma oscillations, and compared measures of CA3-CA1 network timing between age groups. Coherent sharp wave ripples and gamma oscillations were evident in the CA3-CA1 circuit in both age groups, but the relative timing of activity in CA1 stratum pyramidale was delayed in the aged. In another sample of aged slices, evoked Schaffer collateral responses were attenuated in CA3 (antidromic spike amplitude) and CA1 (orthodromic field EPSP slope). However, the amplitude and timing of spontaneous sharp waves recorded in CA1 stratum radiatum were similar to adults. In both age groups unit activity recorded juxtacellularly from unidentified neurons in CA1 stratum pyramidale and stratum oriens was temporally modulated by CA3 ripples. However, aged neurons exhibited reduced spike probability during the early cycles of the CA3 ripple oscillation. These findings suggest that aging disrupts the coordination of patterned activity in the CA3-CA1 circuit.

γ-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aß.

Deposition of ß -amyloid (Aß) peptides, cleavage products of ß-amyloid precursor protein (APP) by ß-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aß approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aß peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aß efficacy. The present study compared active γ-secretase binding sites with Aß deposition in aged and AD human cerebrum, and explored the possibility of Aß production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and ß-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aß40 and Aß42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aß, probably at reduced levels in AD.

Amyloid plaque pathogenesis in 5XFAD mouse spinal cord: retrograde transneuronal modulation after peripheral nerve injury.

The spinal cord is composed of distinct neuronal groups with well-defined anatomic connections. In some transgenic (Tg) models of Alzheimer's disease (AD), amyloid plaques develop in this structure, although the underlying cellular mechanism remains elusive. We attempted to explore the origin, evolution, and modulation of spinal ß-amyloid (Aß) deposition using Tg mice harboring five familiar AD-related mutations (5XFAD) as an experiential model. Dystrophic neuritic elements with enhanced ß-secretase-1 (BACE1) immunoreactivity (IR) appeared as early as 2 months of age, and increased with age up to 12 months examined in this study, mostly over the ventral horn (VH). Extracellular Aß IR emerged and developed during this same period, site-specifically co-existing with BACE1-labeled neurites often in the vicinity of large VH neurons that expressed the mutant human APP. The BACE1-labeled neurites almost invariably colocalized with ß-amyloid precursor protein (APP) and synaptophysin, and frequently with the vesicular glutamate transporter-1 (VGLUT). Reduced IR for the neuronal-specific nuclear antigen (NeuN) occurred in the VH by 12 months of age. In 8-month-old animals surviving 6 months after a unilateral sciatic nerve transection, there were significant increases of Aß, BACE1, and VGLUT IR in the VN of the ipsilateral relative to contralateral lumbar spinal segments. These results suggest that extracellular Aß deposition in 5XFAD mouse spinal cord relates to a progressive and amyloidogenic synaptic pathology largely involving presynaptic axon terminals from projection neurons in the brain. Spinal neuritic plaque formation is enhanced after peripheral axotomy, suggesting a retrograde transneuronal modulation on pathogenesis.

Chronic temporal lobe epilepsy is associated with enhanced Alzheimer-like neuropathology in 3×Tg-AD mice.

The comorbidity between epilepsy and Alzheimer's disease (AD) is a topic of growing interest. Senile plaques and tauopathy are found in epileptic human temporal lobe structures, and individuals with AD have an increased incidence of spontaneous seizures. However, why and how epilepsy is associated with enhanced AD-like pathology remains unknown. We have recently shown ß-secretase-1 (BACE1) elevation associated with aberrant limbic axonal sprouting in epileptic CD1 mice. Here we sought to explore whether BACE1 upregulation affected the development of Alzheimer-type neuropathology in mice expressing mutant human APP, presenilin and tau proteins, the triple transgenic model of AD (3×Tg-AD). 3×Tg-AD mice were treated with pilocarpine or saline (i.p.) at 6-8 months of age. Immunoreactivity (IR) for BACE1, ß-amyloid (Aß) and phosphorylated tau (p-tau) was subsequently examined at 9, 11 or 14 months of age. Recurrent convulsive seizures, as well as mossy fiber sprouting and neuronal death in the hippocampus and limbic cortex, were observed in all epileptic mice. Neuritic plaques composed of BACE1-labeled swollen/sprouting axons and extracellular AßIR were seen in the hippocampal formation, amygdala and piriform cortices of 9 month-old epileptic, but not control, 3×Tg-AD mice. Densities of plaque-associated BACE1 and AßIR were elevated in epileptic versus control mice at 11 and 14 months of age. p-Tau IR was increased in dentate granule cells and mossy fibers in epileptic mice relative to controls at all time points examined. Thus, pilocarpine-induced chronic epilepsy was associated with accelerated and enhanced neuritic plaque formation and altered intraneuronal p-tau expression in temporal lobe structures in 3×Tg-AD mice, with these pathologies occurring in regions showing neuronal death and axonal dystrophy.

Age-related intraneuronal elevation of αII-spectrin breakdown product SBDP120 in rodent forebrain accelerates in 3×Tg-AD mice.

Spectrins line the intracellular surface of plasmalemma and play a critical role in supporting cytoskeletal stability and flexibility. Spectrins can be proteolytically degraded by calpains and caspases, yielding breakdown products (SBDPs) of various molecular sizes, with SBDP120 being largely derived from caspase-3 cleavage. SBDPs are putative biomarkers for traumatic brain injury. The levels of SBDPs also elevate in the brain during aging and perhaps in Alzheimer's disease (AD), although the cellular basis for this change is currently unclear. Here we examined age-related SBDP120 alteration in forebrain neurons in rats and in the triple transgenic model of AD (3×Tg-AD) relative to non-transgenic controls. SBDP120 immunoreactivity (IR) was found in cortical neuronal somata in aged rats, and was prominent in the proximal dendrites of the olfactory bulb mitral cells. Western blot and densitometric analyses in wild-type mice revealed an age-related elevation of intraneuronal SBDP120 in the forebrain which was more robust in their 3×Tg-AD counterparts. The intraneuronal SBDP120 occurrence was not spatiotemporally correlated with transgenic amyloid precursor protein (APP) expression, ß-amyloid plaque development, or phosphorylated tau expression over various forebrain regions or lamina. No microscopically detectable in situ activated caspase-3 was found in the nuclei of SBDP120-containing neurons. The present study demonstrates the age-dependent intraneuronal presence of an αII-spectrin cleavage fragment in mammalian forebrain which is exacerbated in a transgenic model of AD. This novel neuronal alteration indicates that impairments in membrane protein metabolism, possibly due to neuronal calcium mishandling and/or enhancement of calcium sensitive proteolysis, occur during aging and in transgenic AD mice.

BACE1 elevation is associated with aberrant limbic axonal sprouting in epileptic CD1 mice.

The brain is capable of remarkable synaptic reorganization following stress and injury, often using the same molecular machinery that governs neurodevelopment. This form of plasticity is crucial for restoring and maintaining network function. However, neurodegeneration and subsequent reorganization can also play a role in disease pathogenesis, as is seen in temporal lobe epilepsy and Alzheimer's disease. ß-Secretase-1 (BACE1) is a protease known for cleaving ß-amyloid precursor protein into ß-amyloid (Aß), a major constituent in amyloid plaques. Emerging evidence suggests that BACE1 is also involved with synaptic plasticity and nerve regeneration. Here we examined whether BACE1 immunoreactivity (IR) was altered in pilocarpine-induced epileptic CD1 mice in a manner consistent with the synaptic reorganization seen during epileptogenesis. BACE1-IR increased in the CA3 mossy fiber field and dentate inner molecular layer in pilocarpine-induced epileptic mice, relative to controls (saline-treated mice and mice 24-48 h after pilocarpine-status), and paralleled aberrant expression of neuropeptide Y. Regionally increased BACE1-IR also occurred in neuropil in hippocampal area CA1 and in subregions of the amygdala and temporal cortex in epileptic mice, colocalizing with increased IR for growth associated protein 43 (GAP43) and polysialylated-neural cell adhesion molecule (PSA-NCAM), but reduced IR for microtubule-associated protein 2 (MAP2). These findings suggest that BACE1 is involved in aberrant limbic axonal sprouting in a model of temporal lobe epilepsy, warranting further investigation into the role of BACE1 in physiological vs. pathological neuronal plasticity.

BACE1 elevation is involved in amyloid plaque development in the triple transgenic model of Alzheimer's disease: differential Aß antibody labeling of early-onset axon terminal pathology.

ß-amyloid precursor protein (APP) and presenilins mutations cause early-onset familial Alzheimer's disease (FAD). Some FAD-based mouse models produce amyloid plaques, others do not. ß-Amyloid (Aß) deposition can manifest as compact and diffuse plaques; it is unclear why the same Aß molecules aggregate in different patterns. Is there a basic cellular process governing Aß plaque pathogenesis? We showed in some FAD mouse models that compact plaque formation is associated with a progressive axonal pathology inherent with increased expression of ß-secretase (BACE1), the enzyme initiating the amyloidogenic processing of APP. A monoclonal Aß antibody, 3D6, visualized distinct axon terminal labeling before plaque onset. The present study was set to understand BACE1 and axonal changes relative to diffuse plaque development and to further characterize the novel axonal Aß antibody immunoreactivity (IR), using triple transgenic AD (3xTg-AD) mice as experimental model. Diffuse-like plaques existed in the forebrain in aged transgenics and were regionally associated with increased BACE1 labeled swollen/sprouting axon terminals. Increased BACE1/3D6 IR at axon terminals occurred in young animals before plaque onset. These axonal elements were also co-labeled by other antibodies targeting the N-terminal and mid-region of Aß domain and the C-terminal of APP, but not co-labeled by antibodies against the Aß C-terminal and APP N-terminal. The results suggest that amyloidogenic axonal pathology precedes diffuse plaque formation in the 3xTg-AD mice, and that the early-onset axonal Aß antibody IR in transgenic models of AD might relate to a cross-reactivity of putative APP ß-carboxyl terminal fragments.

The effects of aging and genotype on NMDA receptor expression in growth hormone receptor knockout (GHRKO) mice.

Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [(3)H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.

Electrical and Pharmacological Stimuli Reveal a Greater Susceptibility for CA3 Network Excitability in Hippocampal Slices from Aged vs. Adult Fischer 344 Rats.

Clinical data and experimental studies in rats have shown that the aged CNS is more susceptible to the proconvulsive effects of the excitotoxic glutamate analogues kainate (KA) and domoate (DA), which bind high-affinity receptors localized at mossy fiber (MF) synapses in the CA3 subregion of the hippocampus. Although decreased renal clearance appears to play a role in the hypersensitivity of the aged hippocampus to systemically-administered DA, it is unclear if the excitability of the CA3 network is also altered with age. Therefore, this study monitored CA3 field potential activity in hippocampal slices from aged and adult male Fischer 344 rats in response to electrical and pharmacological network stimulation targeted to the MF-CA3 circuit. Network challenges with repetitive hilar stimulation or bath application of nanomolar concentrations of KA more readily elicited excitable network activity (e.g. population spike facilitation, multiple population spikes, and epileptiform bursts) in slices from aged vs. adult rats, although basal network excitability was comparable between age groups. Additionally, exposure to 200 nM KA often abolished epileptiform activity and revealed theta or gamma oscillations instead. However, slices from aged rats were less sensitive to the rhythmogenic effects of 200 nM KA. Taken together, these findings suggest that aging decreases the capacity of the CA3 network to constrain the spread of excitability during focal excitatory network challenges.