Material examined, cause of death, and the prevalence of epilepsy.

<p>Sudden unexpected and unexplained death of subject with known epilepsy (SUDEP), Intractable epilepsy (IE), Epilepsy (E), Years (y), Months (m).</p

Properties of Aβ in plaque-rich cortex characterized by Western blotting.

<p>Panels A1 and A2 show Aβ40 and Aβ42 detected with pAbs R162 and R226, respectively, in blots of extracts (3 µg of total proteins per line) from cerebral cortex containing diffuse plaques of a 39-year-old subject with dup(15) (lane 1), of 51- and 52-year-old individuals with idiopathic autism (lanes 2 and 3), and of 48- and 47-year-old controls (lane 4 and 5). Blots reveal full-length Aβ, mainly Aβ42, in samples from plaque-positive subjects but not in controls. As standards, 1, 2 and 4 fmols of synthetic Aβ1–40, 17–40 (panel A1) and Aβ1–42, 17–42 (panel A2) were used. Panel B shows Aβ detected with mAb 6E10 specific for the N-terminal portion of Aβ in extract from the cortex of the 52-year-old subject (lane 3; 6 µg of protein per lane) and 4 fmol of synthetic Aβ1–40 (st). Panels A1, A2 and B demonstrate that in the extracts from diffuse plaque–positive cortical samples of autistic subjects, the levels of Aβ1–40 and 1–42 exceeded 1.5 fmol per 1 µg of extracted protein.</p

Antibodies used for immunocytochemistry, immunofluorescence and western blotting.

<p>Mouse monoclonal (M-m), Rabbit monoclonal (R-m) or polyclonal (R-p), Goat polyclonal (G-p). Immunocytochemistry (ICH), Confocal microscopy (CM), Western blots (WB).</p

Two major patterns of alterations in intraneuronal Aβ accumulation.

<p>Graphs show a high percentage of neurons with strong cytoplasmic immunoreactivity (mAb 4G8) in the amygdala, thalamus and Purkinje cells in subjects diagnosed with dup(15) autism (D15), a lower percentage in idiopathic autism (IA) subjects, and a low percentage in control subjects. In contrast, the characteristic feature of pyramidal neurons in the frontal, temporal and occipital cortex is a low percentage of neurons with strong Aβ immunoreactivity, whereas the total percentage of Aβ-positive neurons is significantly higher in the dup(15) group than in the idiopathic autism group or in control subjects. Differences in Aβ immunoreactivity in the dup(15) autism vs. control cohort, the idiopathic autism vs. control group, and the dup(15) autism vs. idiopathic autism are significant (p<0.0001).</p

Detection by Western blots of Aβ in plaque-free subjects.

<p>Aβ42 detected with pAb R266 in lysates from cerebral cortex of control individuals 31, 32 and 51 years old (lanes 1–3, respectively), and individuals with idiopathic autism 8, 22, 29, and 29 years old (lane 4, 6, respectively). 40 µg of total lysate proteins were loaded per lane. Synthetic Aβ1–42 was used as a standard.</p

Immunoreactivity of mAb 4G8 with Aβ.

<p>mAb4G8 detects Aβ but does not detect APP in immunohistochemical staining in formalin-fixed and PEG-embedded samples of the frontal cortex of an 8-year-old control subject and a 10-year-old subject diagnosed with dup(15) and autism. Neurons in the control brain contain numerous granules that are immunoreactive with C-terminal APP–specific pAb R57 and are 4G8 negative. In the neurons of an autistic subject, only a few very numerous 4G8-positive deposits are R57-positive, whereas the majority of very numerous APP-immunoreactive granules are 4G8-negative.</p

Full-length Aβ in diffuse plaques, and truncated Aβ in astrocytes in idiopathic autism.

<p>Diffuse plaques in the frontal cortex of a 51-year-old subject (AN17254) diagnosed with idiopathic autism, who had had only one grand mal seizure and died because of cardiac arrest, are immunopositive when stained with all five antibodies (6E10, 4G8, Rabm38, Rabm 40 and Rabm 42), but granular material in the cytoplasm of glial cells is immunopositive for all antibodies used except 6E10.</p

Enhanced accumulation of amino-terminally truncated Aβ in autistic subjects astrocytes.

<p>Clusters of 4G8-positive astrocytes, especially numerous in the molecular layer (a, b); very frequent mitotic divisions (c, d); and extracellular 4G8-positive Aβ deposits, with morphology of astrocytes’ cytoplasmic aggregates (e) may reflect the enhanced proliferation, degeneration and death of Aβ-positive astrocytes in the brain of autistic subjects. Confocal microscopy confirmed the presence of Aβ (green; arrows) in the cytoplasm of GFAP-positive astrocytes (red). Cell nuclei were stained with TO-PRO-3-iodide (blue).</p

Enhanced intraneuronal accumulation of amino-terminally truncated Aβ in autism.

<p>Mapping of Aβ_17–24 in the brain AN09402 reveals brain region– and cell type–specific patterns of abnormal Aβ accumulation in the cytoplasm of neurons and glial cells of a male diagnosed with dup(15), autism and intractable epilepsy, whose sudden unexpected death at the age of 11 years was seizure-related. Almost all neurons in the frontal (FC) and temporal cortex (TC) are 4G8-positive, but the reaction intensity varies from weak to strong. Strong immunoreactivity is observed in many neurons in the lateral geniculate body (LGB), thalamus (Th), amygdala (Amy), Purkinje neurons and basket and stellate neurons in the molecular layer in the cerebellar (Crb) cortex, in many neurons and astrocytes in the CA4, and large and small neurons in the dentate nucleus (DN). Some types of neurons (in the reticular nucleus in the thalamus and small neurons in the dentate nucleus) have different types of deposits: fine-granular and 2- to 3-µm in diameter 4G8-positive deposits. No reaction or only traces of a reaction detected with mAb 6E10 in the frontal cortex, thalamus, cerebellum and dentate nucleus indicate that in intraneuronal Aβ the amino-terminal portion is missing, and the prevalent form of Aβ is α-secretase product. Immunoreactivity with mAb 4G8 is present in the brain of the control subject (14 years of age), but fewer neurons are positive, and immunoreactivity in the frontal cortex, thalamus, cerebellum and dentate nucleus is weaker than in the affected subject. In the control subject, glial cells are usually 4G8-immunonegative.</p

Full-length Aβ in diffuse plaques and amino-terminally truncated Aβ in astrocytes in autism/dup15.

<p>Diffuse plaques in the frontal cortex of a 39-year-old female (AN11931) diagnosed with dup(15), autistic features, and intractable seizures (age of onset 9 years) and whose death was epilepsy-related, are 6E10-, 4G8-, Rabm38-, Rabm40- and Rabm42-positive. Reaction with Rabm38 and Rabm42 was weaker than with other antibodies. Almost all glial cells with the morphology of astrocytes detected in the plaque perimeter had a large cluster of granular material located usually at one cell pole and positive with all antibodies detecting Aβ, except 6E10.</p