Differential Regional Responses in Soluble Monomeric Alpha Synuclein Abundance Following Traumatic Brain Injury.

Alpha synuclein (α-synuclein) is a neuronal protein found predominately in presynaptic terminals. While the pathological effect of α-synuclein aggregates has been a topic of intense study in several neurodegenerative conditions, less attention has been placed on changes in monomeric α-synuclein and related physiological consequences on neuronal function. A growing body of evidence supports an important physiological role of α-synuclein in neurotransmission. In the context of traumatic brain injury (TBI), we hypothesized that the regional abundance of soluble monomeric α-synuclein is altered over a chronic time period post-injury. To this end, we evaluated α-synuclein in the cortex, hippocampus, and striatum of adult rats at 6 h, 1 day, 1, 2, 4, and 8 weeks after controlled cortical impact (CCI) injury. Western blot analysis demonstrated decreased levels of monomer α-synuclein protein in the ipsilateral hippocampus at 6 h, 1 day, 1, 2, and 8 weeks, as well as in the ipsilateral cortex at 1 and 2 weeks and in the ipsilateral striatum at 6 h after CCI compared with sham animals. Immunohistochemical analysis revealed lower α-synuclein and a modest reduction in synaptophysin staining in the ipsilateral hippocampus at 1 week after CCI compared with sham animals, with no evidence of intracellular or extracellular α-synuclein aggregates. Collectively, these findings demonstrate that monomeric α-synuclein protein abundance in the hippocampus is reduced over an extensive (acute-to-chronic) post-injury interval. This deficit may contribute to the chronically impaired neurotransmission known to occur after TBI.

Hippocampal plasticity during the progression of Alzheimer's disease.

Neuroplasticity involves molecular and structural changes in central nervous system (CNS) throughout life. The concept of neural organization allows for remodeling as a compensatory mechanism to the early pathobiology of Alzheimer's disease (AD) in an attempt to maintain brain function and cognition during the onset of dementia. The hippocampus, a crucial component of the medial temporal lobe memory circuit, is affected early in AD and displays synaptic and intraneuronal molecular remodeling against a pathological background of extracellular amyloid-beta (Aß) deposition and intracellular neurofibrillary tangle (NFT) formation in the early stages of AD. Here we discuss human clinical pathological findings supporting the concept that the hippocampus is capable of neural plasticity during mild cognitive impairment (MCI), a prodromal stage of AD and early stage AD.

Precuneus amyloid burden is associated with reduced cholinergic activity in Alzheimer disease.

OBJECTIVE: This study examined the relationship between postmortem precuneus cholinergic enzyme activity, Pittsburgh compound B (PiB) binding, and soluble amyloid-ß concentration in mild cognitive impairment (MCI) and Alzheimer disease (AD). METHODS: Choline acetyltransferase (ChAT) activity, [(3)H]PiB binding, and soluble amyloid-ß(1-42) (Aß42) concentration were quantified in precuneus tissue samples harvested postmortem from subjects with no cognitive impairment (NCI), MCI, and mild AD and correlated with their last antemortem Mini-Mental State Examination (MMSE) score and postmortem pathologic evaluation according to the National Institute on Aging-Reagan criteria, recommendations of the Consortium to Establish a Registry for Alzheimer's Disease, and Braak stage. RESULTS: Precuneus ChAT activity was lower in AD than in NCI and was comparable between MCI and NCI. Precuneus [(3)H]PiB binding and soluble Aß42 levels were elevated in MCI and significantly higher in AD than in NCI. Across all case subjects, reduced ChAT activity was associated with increased [(3)H]PiB binding, increased soluble Aß42, lower MMSE score, presence of the APOE*4 allele, and more advanced AD pathology. CONCLUSIONS: Despite accumulating amyloid burden, cholinergic enzyme activity is stable in the precuneus during prodromal AD. A decline in precuneus ChAT activity occurs only in clinical AD, when PiB binding and soluble Aß42 levels are substantially elevated compared with those in MCI. Anti-amyloid interventions in MCI case subjects with a positive PiB PET scan may aid in reducing cholinergic deficits and cognitive decline later in the disease process.

Anti-Amyloid Effects of Small Molecule Aß-Binding Agents in PS1/APP Mice.

AIMS: One promising approach for treatment of Alzheimer's disease (AD) is use of anti-amyloid therapies, based on the hypothesis that increases in amyloid-beta (Aß) deposits in brain are a major cause of AD. Several groups have focused on Aß immunotherapy with some success. Small molecules derivatives of Congo red have been shown to inhibit Aß aggregation and protect against Aß neurotoxicity in vitro. The agents described here are all small molecule Aß-binding agents (SMAßBA's) derivatives of Congo red. MAIN METHODS: Here, we have explored the anti-amyloid properties of these SMAßBA's in mice doubly transgenic for human prensenilin-1 (PS1) and APP gene mutations that cause early-onset AD. Mice were treated with either methoxy-X04, X:EE:B34 and X:034-3-OMe1. After treatment, brains were examined for Aß-deposition, using histochemistry, and soluble and insoluble Aß levels were determined using ELISA. KEY FINDINGS: A range of anti-amyloid activity was observed with these three compounds. PS1/APP mice treated with methoxy-X04 and X:EE:B34 showed decrease in total Aß load, a decrease in Aß fibril load, and a decrease in average plaque size. Treatment with methoxy-X04 also resulted in a decrease in insoluble Aß levels. The structurally similar compound, X:034:3-OMe1, showed no significant effect on any of these measures. The effectiveness of the SMAßBA's may be related to a combination of binding affinity for Aß and entry into brain, but other factors appear to apply as well. SIGNIFICANCE: These data suggest that SMAßBA's may significantly decrease amyloid burden in brain during the pathogenesis of AD and could be useful therapeutics alone, or in combination with immunotherapy.

Biochemical analysis of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of patients with Alzheimer's disease neuropathology.

Alzheimer's disease (AD) is characterized by selective vulnerability of specific neuronal populations within particular brain regions. For example, hippocampal glutamatergic cell populations within the CA1/subicular pyramidal cell fields have been found to be particularly vulnerable early in AD progression. In contrast, hippocampal GABA-ergic neurons and receptors appear resistant to neurodegeneration. Despite relative sparing of GABA(A) receptors in AD, it is possible that the specific subunit composition of these receptors may undergo alterations with disease progression. In order to address this issue, we employed quantitative Western blot analysis to examine protein levels of GABA(A) receptor subunits alpha 1, alpha 5, beta 1, beta 2 in the hippocampus of subjects displaying increasing severity of AD neuropathology. Subjects were categorized into three groups based upon Braak staging pathologic criteria: pathologically mild (stages I/II, n=9); moderate (stages III/IV, n=8); and severe (stages V/VI, n=7). Across all subject groups, levels of subunit protein were heterogeneously distributed throughout the five hippocampal subregions analyzed (subiculum, CA1-3, dentate gyrus). Statistical analyses revealed differential preservation of GABA(A) receptor subunits in AD. In particular, alpha 1, beta 1, and beta 2 displayed little difference in protein levels among pathologically mild, moderate, and severe subject groups. In contrast, although relatively modest, protein levels of the alpha 5 subunit were significantly reduced between subjects with severe neuropathology compared with pathologically mild subjects (13.5% reduction). Collectively, our data provide evidence for heterogeneous distribution and relative sparing of GABA(A) receptor subunits in the hippocampus of AD patients.

Age-related loss of the AMPA receptor subunits GluR2/3 in the human nucleus basalis of Meynert.

Magnocellular cholinergic neurons in the basal forebrain have long been recognized as vulnerable to the pathology of Alzheimer's disease. Despite numerous anatomical, pharmacological, behavioral, and physiological investigations of these neurons the cellular mechanism that underlines their selective vulnerability remains unclear. As part of an ongoing investigation into the molecular mechanism(s) underlying neuronal vulnerability in Alzheimer's disease and normal aging, we employed immunocytochemical techniques and examined the cellular localization of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) glutamate receptor subunits GluR1 and GluR2/3 in the basal forebrain of eight nondemented elderly human subjects (66-102 years). For each case we observed GluR1-positive magnocellular cells darkly labeled within all main divisions of the basal forebrain (Ch1-Ch4). Double-labeling immunohistochemical techniques confirmed that the overwhelming majority (94%) of these neurons were also positive for the p75NGFr antibody, thus substantiating the cholinergic nature of these neurons. In contrast, GluR2/3 immunolabeling upon magnocellular neurons was relatively faint or nonexistent. The latter observations were most apparent in cases of advanced age and in the posterior part of the nucleus basalis of Meynert (NBM) (i.e., Ch4). In contrast, in adjacent structures (e.g., globus pallidus), a number of robustly labeled GluR2/3-positive cells were observed. In addition to the eight elderly subjects, we examined GluR1 and GluR2/3 immunostaining in the NBM of five younger cases, 5, 33, 36, 47, and 48 years of age. Although practical considerations limited our observations to the Ch4 region, we observed both GluR1 and GluR2/3 labeling upon NBM neurons in this latter region. On average, the distribution of labeled cells and intensity of immunoreaction were comparable between GluR1 and GluR2/3. The presence of GluR2/3- and GluR1-labeled neurons in the Ch4 region of younger cases but primarily GluR1 in cases of advanced age suggests an age-related decrease in GluR2/3. Functionally, the loss of GluR2 from the AMPA receptor complex results in ion channels highly permeable to Ca(2+). These alterations in cation permeability of the AMPA receptor together with the occurrence of a number of other intrinsic and extrinsic events (i.e., decrease Ca(2+)-binding protein) likely contribute to the vulnerability of these neurons in aging and in AD.

Distribution of glutamate receptor subunit NMDAR1 in the hippocampus of normal elderly and patients with Alzheimer's disease.

Immunocytochemical techniques were employed to study the distribution and cytological features of NMDAR1-immunoreactive elements in the human hippocampal formation. Subjects with Alzheimer's disease (AD), presenting with a wide range of neuropathology and classified into six Braak stage (I-VI), and nondemented age-matched controls were examined. In control cases, the most intense NMDAR1 immunoreactivity was observed within the soma and dendrites of granule cells in the dentate gyrus and pyramidal neurons in Ammon's horn. Whereas small variations in the pattern of immunoreactivity were noted in control cases, AD subjects were characterized with intersubject variability which in most instances correlated with neuropathologic severity. For example, AD cases, particularly those with mild/modest pathology (Braak I-III), were indistinguishable from controls in the overall pattern of immunolabeling. In contrast, in those more severe AD cases (Braak IV-VI) the intensity of immunolabeling within the CA fields was greater than observed in controls and those with mild AD pathology. In addition, in pathologically severe cases numerous NMDAR1-positive pyramidal neurons were characterized by unique morphologic features including long and often tortuous apical dendrites. These latter findings were most prevalent in the CA1 region and subiculum. In contrast to the marked increase in immunolabeling in the CA fields, in the dentate gyrus we observed a reduction in NMDAR1 labeling particularly within the outer molecular layer (i.e., termination zone of the perforant pathway). This latter region was also the site of a number of NMDAR1-labeled plaques. Notably, the overall pattern of NMDAR1 immunoreactivity is distinct from that observed with antibodies against AMPA receptor subunits and suggests a differential role of various inotropic glutamate receptors in hippocampal plasticity in AD.

Immunohistochemical study of GABAA receptor alpha1 subunit in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes.

Immunocytochemical techniques were employed to examine the distribution of the gamma-aminobutyric acid (GABA)A receptor alpha1 subunit within the hippocampus of 19 elderly subjects with Alzheimer-related neuropathologic changes. In mild cases (i.e., Braak stages I and II), the most intense neuropil immunolabeling was observed in the molecular layer of the dentate gyrus, the stratum pyramidale of the CA1 subregion and subiculum, while the weakest labeling was observed in the CA3 subfield. In CA4 region, the proximal dendrites and cell bodies of mossy cells were intensely alpha1 positive. Throughout the hippocampus, we observed a number of alpha1 labeled interneurons. These cells consisted of both large and small multipolar cells as well as small bipolar neurons. In moderate cases (i.e., Braak stages III and IV), the pattern and intensity of alpha1 immunolabeling appeared indistinguishable from mild cases. In severe cases (i.e., Braak stages V and VI), we observed a marked decrease in neuropil immunolabeling within the CA2, CA1 subregions and prosubiculum, while the labeling of the molecular layer of the dentate gyrus, subiculum proper and presubiculum was indistinguishable from mild and moderate cases. These data together with our previous immunocytochemical study in which we demonstrated a marked preservation of the GABAA receptor subunit beta2/3 suggest that responses of selected GABAA receptor subunits to AD pathology are variable with the alpha1 subunit displaying a high degree of vulnerability.

GABAA receptor beta 2 and beta 3 subunits mRNA in the hippocampal formation of aged human brain with Alzheimer-related neuropathology.

Our work on the role of glutamate in Alzheimer's disease (AD)-related neuronal vulnerability and death provided significant insight into the potential contribution of the gamma-aminobutyric acid (GABA) neurotransmitter system as it participates in countering the neurotoxic effects of excessive glutamate receptor stimulation. Our previous studies demonstrate that beta2/3 GABAA receptor subunit immunoreactivity is relatively well preserved in hippocampi with AD pathology. To further elucidate the molecular basis for this observation, we employed in situ hybridization histochemistry to examine the levels of beta2 and beta3 receptor subunit mRNAs in the hippocampus of 19 elderly subjects presenting with a broad range of pathologic severity (i.e., Braak stage I-VI). Semi-quantitative analysis with film autoradiograms revealed that beta2 mRNA signal was highest in the granule cell layer, CA2 and CA1 subfields, while beta3 mRNA hybridization was highest in the granule cell layer, followed by CA2>/=CA3>/=CA1 regions. No significant difference in beta2 mRNA expression was detected among the pathologically mild, moderate or severe groups. In contrast, levels of beta3 mRNA in the pathologically severe group was significantly decreased compared to the mild group within all subregions examined except CA4. Our data suggest that alterations in the expression of GABAA receptor subunits in the AD hippocampus differ between specific receptor subunits with the amount of beta2 mRNA being relatively well-preserved, while beta3 mRNA levels were decreased.

Alterations of AMPA-selected glutamate subtype immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in immunolabeling of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the dentate gyrus 1, 3, 7, 14, 30, and 90 days after a unilateral perforant pathway lesion in the rat brain. Completeness of the lesion was confirmed following examination of Nissl-stained tissue sections at all times post-lesion and acetylcholinesterase (AChE)-stained sections 14, 30 and 90 days post-lesion, the latter providing evidence of compensatory sprouting of cholinergic fibers in the outer molecular layer of the dentate gyrus. Compared to the non-lesioned hippocampus there was no difference in the staining pattern of AMPA receptor subunits in the dentate gyrus of the deafferented hippocampus 1, 3, 7 and 14 days following lesioning of the perforant pathway. In contrast, 30 and 90 days post-lesion, GluR1 immunolabeling was increased in the outer molecular layer of the dentate gyrus (i.e., deafferented zone) ipsilateral to lesion. Likewise, GluR2/3 immunolabeling was increased within the same region although the intensity of the response was less than that which was observed for GluR1. These data suggest that the loss of the perforant pathway fibers results in a compensatory increase in GluR1 and to a lesser extent GluR2/3 immunolabeling of the outer molecular layer at 30 and 90 days post-lesion and further suggest that AMPA receptor subunits play a role in perforant pathway signal transduction.

Alterations of GABA(A)beta2/3 immunoreactivity in the dentate gyrus after perforant pathway lesion.

Immunocytochemical techniques were employed to examine the changes in the GABA receptor subunits beta2/3 within the dentate gyrus of the rat brain 1, 3, 7, 14, 30 and 90 days after a unilateral perforant pathway lesion. Three days post-lesion we observed a decrease in beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus followed by a comparable decrease in the outer molecular layer 7 days post-lesion. These decreases were transient; 30 and 90 days post-lesion, beta2/3 immunolabeling appeared similar to controls in the inner portion of the molecular layer, while in the outer region the labeling was increased. In this latter region we also observed a dense band of AChE fibers. Following survival times of 3 days we observed a diffuse staining of the neuropil in the hilar region, and a dense amorphous accumulation of peroxidase reaction product in the polymorphic region. These responses were transient and by 14 days the hilar/polymorphic region appeared indistinguishable from controls. These data suggest a unique pattern of immunoabeling in the molecular and polymorphic region in response to perforant pathway lesion. A putative explanation for this response is discussed.

Immunohistochemical study of GABA(A) receptor beta2/3 subunits in the hippocampal formation of aged brains with Alzheimer-related neuropathologic changes.

In AD, it is hypothesized that one factor contributing to the vulnerability of neurons is a delicate balance of excitatory and inhibitory inputs. To examine this hypothesis we have initiated a number of studies examining the role of the excitatory neurotransmitter glutamate and the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the neurodegeneration of AD. As an initial investigation into the GABAergic system in AD, we employed immunocytochemical techniques and examined the distribution and density of the GABAA receptor subunits beta2/3 within the hippocampus of 13 subjects with a clinical diagnosis of AD and 6 nondemented elderly subjects. Collectively, these 19 subjects presented with a broad range of pathologic severity (i.e., Braak stages I-VI). Density measurements of nine hippocampal regions demonstrated highest levels of beta2/3 immunolabeling in the inner molecular layer of the dentate gyrus > CA1 > CA2, while the lowest levels were found in the granular layer of the dentate gyrus < or = CA4 < CA3 field. Despite these regional variations no significant difference in the mean density of beta2/3 immunolabeling was observed when comparing the pathologically mild (stages I and II), moderate (stages III and IV), and severe (stages V and VI) groups. These data suggest that in the hippocampus receptor subunits associated with GABAergic neurotransmission are relatively maintained even until the terminal stages of the disease.

The loss of GluR2(3) immunoreactivity precedes neurofibrillary tangle formation in the entorhinal cortex and hippocampus of Alzheimer brains.

Double-immunolabeling techniques were employed to examine the distribution of GluR2(3) subunits and markers of early cytoskeletal changes (mab MC1) within the entorhinal cortex (EC) and hippocampus of cases with varying degrees of Alzheimer disease (AD) pathology (stages I-VI by Braak and Braak). In addition near-adjacent tissue sections were double-immunolabeled using antibodies against GluR2(3) and a marker of normal neuronal cytoskeleton (MAP2). In those cases classified as stages I-II, most layer II neurons of the EC and pyramidal neurons in the CA1/subiculum were double-labeled with GluR2(3) and MAP2. An occasional MC1-labeled cell was observed, yet in no instance were these neurons double-labeled with GluR2(3). In cases with moderate AD pathology (stages III-IV), layer II of the EC and CA1/subiculum were characterized by a substantial loss of GluR2(3)-labeled neurons, while many were still immunoreactive to MAP2. Notably, the loss of GluR2(3) immunolabeling was accompanied by an increasing number of MC1-positive neurons. In no instance were GluR2(3) and MC1 co-localized within the same neuron. In cases with severe AD pathology (stages V-VI), the EC and CA1/subiculum were almost completely devoid of GluR2(3)-positive neurons. MAP2-labeled neurons also were reduced in number. In contrast, both regions contained an abundance of MC1-positive cells. That GluR2(3) and MC1 are not observed in the same neuron, together with the observation that the number of GluR2(3)-labeled neurons decreases as the number of MC1-positive cells increases, suggest that a loss of GluR2(3) immunolabeling precedes the appearance of MC1 immunolabeling.

GABA and NMDA in the prevention of apoptotic-like cell death in vitro.

We have shown recently that cerebellar granule neurons die in the absence of depolarizing concentrations of KCl through an apoptosis-like process. To study the contributions of inhibitory (gamma-aminobutyric acid; GABA) and excitatory (glutamate) neurotransmitters in the prevention of apoptotic-like cell death in cultures grown in the presence of reduced concentrations of KCl (12.5 mM), we treated these cultures either acutely or chronically with GABA, bicuculline methiodide, a GABAA receptor antagonist, N-methyl-D-aspartate (NMDA) and/or the NMDA receptor antagonist, MK-801. Cell viability was measured with fluorescein diacetate/propidium iodide (FDA/PI) and trypan blue exclusion tests. In addition, DNA fragmentation was assessed quantitatively using an in situ terminal deoxynucleotidyl transferase assay. Our results demonstrate that treatment of cerebellar granule cell cultures maintained in 12.5 mM KCl with the glutamate receptor agonist NMDA and/or bicuculline protects against cell death and reduces DNA fragmentation. In contrast, GABA potentiated cerebellar granule cell apoptosis mediated by KCl deprivation. These data indicate that signal transduction pathways activated following NMDA receptor stimulation mimic the anti-apoptotic action of high potassium in primary cultures of cerebellar granule neurons. Also, our data support an inhibitory (hyperpolarizing) role for GABA in these cultures. Collectively, the results suggest that the neurotrophic actions of NMDA on granule cells maintained in low KCl and GABA on granule cells cultured in high KCl are due to the necessity for maintaining appropriate intraneuronal calcium concentrations.

AMPA-selective glutamate receptor subtype immunoreactivity in the hippocampal dentate gyrus of patients with Alzheimer disease. Evidence for hippocampal plasticity.

Immunocytochemical techniques were employed in order to examine the distribution and relative intensity of immunolabeling of the alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionate (AMPA) receptor subunits GluR1 and GluR2/3 within the hippocampal formation of patients with Alzheimer disease (AD). Within sectors of the hippocampus that are particularly vulnerable to AD pathology (i.e., CA1, subiculum), we observed a variable loss of GluR1 and GluR2/3 immunolabeling correlating with the extent of cell loss and neurofibrillary pathology. In contrast, in less vulnerable sectors of the hippocampus (i.e., CA2/3, dentate gyrus), the intensity of immunolabeling was markedly increased in AD cases, particularly in the molecular and polymorphic layers of the dentate gyrus. Importantly, these latter regions correspond to termination zones of glutamatergic perforant pathway axons and mossy fiber collaterals, respectively. The increase in immunolabeling within these projection fields is hypothesized to occur in response to the deafferentation of selected glutamatergic pathways, and suggests a critical role for AMPA receptor subunits in hippocampal plasticity.

Distribution of AMPA receptor subunits in the nucleus basalis of Meynert in aged humans: implications for selective neuronal degeneration.

Immunocytochemical techniques using polyclonal antibodies directed against GluR1 and GluR2/3 subunits of the AMPA-selective receptor complex were used to examine the distribution of these receptor subunits within the nucleus basalis of Meynert (NBM) of non-demented elderly humans. Both somata and processes of magnocellular neurons within the NBM were intensely immunoreactive to GluR1 antibodies. In contrast, within the same region GluR2/3 immunolabeling was largely absent, although GluR2/3-positive neurons were abundantly distributed within adjacent brain regions (i.e., amygdala, entorhinal cortex and hippocampus). These data suggest that NBM neurons may be unique compared to those of other brain regions, in their response to glutamatergic excitation as mediated via non-NMDA receptors and be particularly vulnerable to glutamate excitotoxicity via a mechanism involving the destabilization of intracellular calcium.

Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists.

Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr after serum removal) we found significant impairment of mitochondrial functioning [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] and an increase in the percentage of neurons showing signs of DNA fragmentation (insitu terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants [1mM: 1,4-diazobicyclo(2.2.2)octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01-1 microM ascorbic acid]. We also measured neuronal oxidation utilizing the oxidation-sensitive fluorescent dye 2', 7'-dichloro- fluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min after serum deprivation. The blockade of glutamate receptors by (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK-801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress-induced apoptosis.

Atomic force microscopy of paired helical filaments isolated from the autopsied brains of patients with Alzheimer's disease and immunolabeled against microtubule-associated protein tau.

Atomic force microscopy was employed to study the structural features of paired helical filaments isolated from autopsied brains of Alzheimer's disease patients. The identity of paired helical filaments was confirmed following a specific immunogold labeling using antibodies directed against the microtubule-associated protein tau, which is the main constituent of paired helical filaments. Computer-assisted analysis of high resolution, three-dimensional images allowed us to study the longitudinal and cross-sectional profiles of individual filaments. Vertical dimensions of filaments were assessed along these sectional profiles. The smallest vertical diameter (6.66 +/- 0.78 nm) was obtained at the level of the greatest lateral profile (ie, "loop"), while the greatest vertical diameter was two times larger (13.68 +/- 1.46 nm) and was obtained at the level of the smallest lateral profile (ie, "crossover") of the filament. Based on the shapes of these sectional profiles and their vertical dimensions, paired helical filaments appeared to be composed of two identical integral subunits, each of a circular cross-sectional profile of approximately 7 nm in diameter, wound around one another in a left helical manner, with a 7-nm center-to-center separation. Half-period of this helix was estimated at 81.4 +/- 2.1 nm. Serial cross-sectional profiles of paired helical filaments were further utilized to construct a theoretical model of their internal organization. This model suggests that each structural subunit of the paired helical filament incorporates at least four identical protofilaments.

AMPA-selective glutamate receptor subtype immunoreactivity in the aged human hippocampal formation.

It has been hypothesized that, in Alzheimer's disease, glutamate-mediated excitotoxicity contributes to the degeneration of selected populations of neurons. In the present study, immunocytochemical techniques were used to determine the distribution and anatomical features of GluR1- and GluR2/3-immunolabeled cell bodies and processes within the hippocampal formation of normal (i.e., no pathology) elderly humans. The results of this study provide an essential baseline with which to compare the expression and distribution of glutamate receptor subunits within the brains of patients with Alzheimer's disease. With respect to GluR1 immunoreactivity, the molecular layer of the dentate gyrus displays the most intense immunolabeling of any hippocampal structure. Contributing to this intense labeling are apical dendrites that arise from neurons within the adjacent granule cell layer. Interestingly, GluR1-labeled neurons account for a relatively small percentage of the total number of neurons as revealed by Nissl staining in the granule cell layer. In contrast, GluR2/3-labeled neurons are densely distributed throughout the granule cell layer, yet they provide relatively few processes to the adjacent molecular layer compared to GluR1-positive processes. GluR1 labeling is also prominent within the CA fields of Ammon's horn, with CA2 > CA3 > CA1 > or = CA4. Most prominent within the CA fields are the labeled dendrites of pyramidal neurons. In many instances, apical dendrites can be traced into the adjacent stratum radiatum, where they impart a deep striated appearance to this region of the hippocampus. Robust GluR2/3 labeling is also observed within the pyramidal layer of Ammon's horn, with an order of staining intensity similar to that observed for GluR1. However, unlike GluR1 labeling, which is localized predominantly along dendrites, GluR2/3 labeling is observed primarily in association with cell bodies. Collectively, these data suggest that the molecular composition of the AMPA receptor complex may differ between the dendrite and soma of granule and pyramidal neurons within the hippocampal formation, so functionally we may predict that these two regions of the neuron would respond differently following glutamate receptor stimulation.

Neuronal apoptosis in an in vitro model of photochemically induced oxidative stress.

In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.