Intraneuronal Aβ immunoreactivity is not a predictor of brain amyloidosis-β or neurofibrillary degeneration

Amyloid β (Aβ) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer’s disease to determine if intraneuronal Aβ immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Aβ immunoreactivity in neurons in infants and stable neuron-type specific Aβ immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4–13 aa and 8–17 aa of Aβ in neurons indicated that intraneuronal Aβ was mainly a product of α- and γ-secretases (Aβ(17–40/42)). The presence of N-terminally truncated Aβ(17–40) and Aβ(17–42) in the control brains was confirmed by Western blotting and the identity of Aβ(17–40) was confirmed by mass spectrometry. The prevalence of products of α- and γ -secretases in neurons and β- and γ-secretases in plaques argues against major contribution of Aβ-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Aβ(17–42) immunoreactivity was observed in structures with low susceptibility to fibrillar Aβ deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Aβ immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Aβ immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Aβ represents a product of normal neuronal metabolism

Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

<div><h3>Background</h3><p>It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount.</p> <h3>Methodology/Principal Findings</h3><p>In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ_17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ_1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.</p> <h3>Conclusions/Significance</h3><p>The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.</p> </div

Intraneuronal Aβ immunoreactivity is not a predictor of brain amyloidosis-β or neurofibrillary degeneration

Amyloid β (Aβ) immunoreactivity in neurons was examined in brains of 32 control subjects, 31 people with Down syndrome, and 36 patients with sporadic Alzheimer’s disease to determine if intraneuronal Aβ immunoreactivity is an early manifestation of Alzheimer-type pathology leading to fibrillar plaque formation and/or neurofibrillary degeneration. The appearance of Aβ immunoreactivity in neurons in infants and stable neuron-type specific Aβ immunoreactivity in a majority of brain structures during late childhood, adulthood, and normal aging does not support this hypothesis. The absence or detection of only traces of reaction with antibodies against 4–13 aa and 8–17 aa of Aβ in neurons indicated that intraneuronal Aβ was mainly a product of α- and γ-secretases (Aβ17–40/42). The presence of N-terminally truncated Aβ17–40 and Aβ17–42 in the control brains was confirmed by Western blotting and the identity of Aβ17–40 was confirmed by mass spectrometry. The prevalence of products of α- and γ -secretases in neurons and β- and γ-secretases in plaques argues against major contribution of Aβ-immunopositive material detected in neuronal soma to amyloid deposit in plaques. The strongest intraneuronal Aβ17–42 immunoreactivity was observed in structures with low susceptibility to fibrillar Aβ deposition, neurofibrillary degeneration, and neuronal loss compared to areas more vulnerable to Alzheimer-type pathology. These observations indicate that the intraneuronal Aβ immunoreactivity detected in this study is not a predictor of brain amyloidosis or neurofibrillary degeneration. The constant level of Aβ immunoreactivity in structures free from neuronal pathology during essentially the entire life span suggests that intraneuronal amino-terminally truncated Aβ represents a product of normal neuronal metabolism

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

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

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