The Difference between Intraneuronal Aβ Accumulation in dup(15) Autism, Idiopathic Autism and Control Groups
In all subjects with dup15/autism spectrum disorder (ASD) and the majority of individuals with idiopathic ASD, intraneuronal Aβ immunoreactivity was observed in more neurons, and the amount of immunoreactive material was increased in comparison to the control subjects (). The morphology of the intracellular deposits of Aβ-positive material was cell type–specific. Cortical pyramidal neurons showed significant heterogeneity of intraneuronal deposits with a mixture of fine granular material and several times larger 4G8-positive granules. In Purkinje cells, fine granular deposits were accumulated in the cell body. In the dentate nucleus, large neurons accumulated fine granular material, whereas small neurons accumulated a few much larger Aβ-positive granules. Neurons in the reticulate nucleus in the thalamus contained a mixture of fine granular material and large 4G8-positive granules.
Enhanced intraneuronal accumulation of amino-terminally truncated Aβ in autism.
Immunocytochemistry with monoclonal antibodies (mAbs) 4G8 (17–24 aa of Aβ) and 6E10 (4–13 aa of Aβ) revealed that almost all intraneuronal Aβ is 4G8-positive, but only a very small proportion is labeled with 6E10.
Quantitative evaluation of 12 brain subregions/cell types (frontal, temporal and occipital cortex, Purkinje cells, amygdala, thalamus, lateral geniculate body (LGB), dentate gyrus, CA1 and CA4 sectors and dentate nucleus) revealed that in 11 subregions intraneuronal Aβ load was significantly greater in the dup(15) autism group than in the control and idiopathic autism cohorts (p<0.0001 for all comparisons). In eight regions (all three cortical subregions, Purkinje cells, amygdala, thalamus, LGB, and dentate gyrus), intraneuronal Aβ load differed significantly between the idiopathic autism and control groups (p<0.0001). In structures with almost all neurons positive for Aβ–the dentate nucleus and the inferior olive–the amyloid load was insignificantly higher in control subjects than in subjects with idiopathic autism.
Quantitative study revealed different patterns of immunoreactivity in brain subregions (, and Supporting Information, Fig. S1
). The characteristic feature distinguishing the amygdala, thalamus and Purkinje cells of subjects with dup(15) autism was the very high percentage of neurons with strong cytoplasmic Aβ immunoreactivity (46%, 46% and 35%, respectively); the percentage was significantly lower in the idiopathic autism group (32%, 38% and 19%, respectively), and very low in control subjects (6%, 6% and 12%, respectively). However, in pyramidal neurons in the frontal, temporal and occipital cortex, the percentage of neurons with strong Aβ immunoreactivity was low (3–10%), whereas the total percentage of Aβ-positive neurons was significantly higher in the dup(15) group (81–83%) than in the idiopathic autism group (56–71%) and in control subjects (45–51%).
Two major patterns of alterations in intraneuronal Aβ accumulation.
The percentage of Aβ-positive neurons and neuronal amyloid load was smaller in the hippocampal formation, especially in the CA1 sector and dentate gyrus of control subjects. The amyloid load was significantly higher in the dup(15) autism group than in control subjects, but the difference in amyloid load between the idiopathic autism and control groups was insignificant (Fig. S1
The feature distinguishing the LGB, inferior olive and dentate nucleus from other brain structures is the childhood onset of lipofuscin accumulation. In LGB, strong Aβ immunoreactivity was observed in 73% of neurons in dup(15) autism and in 62% in idiopathic autism but only 16% of LGB neurons were strongly Aβ-positive in control subjects. In the dentate nucleus, the percentage of strongly positive neurons was comparable in all three groups (41%, 35% and 41%, respectively), but overall amyloid load was statistically higher in dup(15) autism. The percentage of strongly Aβ-positive neurons in the inferior olive was the same in the idiopathic autism and in the dup(15) (32%) group, and there was no difference in overall amyloid load between autistic and control subjects (Fig. S1
Aβ in Glial Cells
Astrocytes and microglia in the control brains were usually Aβ-negative or contained only traces of Aβ immunoreactivity. Enhanced neuronal Aβ accumulation in the brains of individuals with autism was associated with Aβ accumulation in the astrocytes’ cytoplasm and in some microglial cells (). Two patterns of Aβ immunoreactivity were observed in astroglia. The most common form was a condensed aggregate of Aβ in one pole of the astrocyte soma typical for CA4 sector, some cortical areas but without clear anatomical predilection, and the cerebellar cortex border zone between granule and molecular layers. The less common form was deposition of Aβ-immunoreactive granular material in the entire astrocyte body and in a proximal portion of processes radiating from the cell body (frequent in the molecular layer of the cerebral cortex). The increase in the amount of cytoplasmic Aβ was often paralleled by (a) a several-fold increase in the number of astrocytes, all of which were Aβ-positive (), (b) clustering of astrocytes in groups of 3–10 cells (), (c) numerous mitoses as a sign of astrocyte proliferation () and (d) astrocyte death resulting in deposition of extracellular remnants of Aβ aggregates () similar to those seen in astrocyte cytoplasm. Extracellular Aβ deposits were found in neuropil, but larger aggregates (more than 10) were more often in the perivascular space. Confocal microscopy confirmed Aβ accumulation in GFAP-positive astrocytes (, lower panel).
Enhanced accumulation of amino-terminally truncated Aβ in autistic subjects astrocytes.
Intracellular Distribution of Amino-terminally Truncated Aβ in Neurons
Intraneuronal Aβ deposits revealed striking neuron type–specific differences in amount, morphology and cytoplasmic distribution; however, they had the same immunoproperties. They revealed no reaction or traces of reaction with mAb 6E10 () or 6F3D (not shown). The morphological diversity of Aβ deposits suggested that Aβ was present in different compartments of the endosomal-lysosomal pathway and in lipofuscin in neuron type–specific amounts. The number and size of Lamp1– () lysosomes was from 2 to 3 times more than the number of Aβ-positive deposits; however, only about 10% of Aβ was detected in rab5-positive endosomal vesicles and in LC3B-positive autophagic vacuoles. Colocalization of Aβ with COXIV-positive mitochondria was observed in only a very few mitochondria.
Aβ in endocytic vesicles, autophagic vacuoles, lysosomes and mitochondria.
Immunoreaction for Aβ detected with mAb 4G8 was present in some intracellular autofluorescent granules; however, the 4G8-immunoreactive deposits were detected also in neurons with scanty lipofuscin () and in neurons with abundant autofluorescent granules. On the other hand, some neurons with scanty immunoreaction for Aβ contained numerous autofluorescent granules. The autofluorescent granules were not immunostained with mAb 6E10. Immunoreaction with polyclonal antibody (pAb) R226, specific for the C-terminus of Aβ42, showed only a fraction of labeling colocalized with autofluorescent granules. These results indicate that the detected intraneuronal immunostaining reflects accumulation of N-terminally truncated Aβ in several cellular compartments, including lipofuscin granules.
Specificity of Immunohistochemical Detection of Aβ with mAb 4G8 and 6E10
The epitopes of mAbs 6E10 and 4G8 (4–13 aa and 17–24 aa of the Aβ sequence, respectively) are present in full-length APP and APP C-terminal fragments. In brain tissue that has been fixed in formalin for several months, embedded in polyethylene glycol (PEG) and pretreated with 70% formic acid for 20 min, the immunostaining with mAb 4G8 () and with 6E10 and 7F3D (8–17 aa of Aβ; not shown) is consistent with the distribution and amount of Aβ, but different from the distribution and amount of neuronal APP. In control brains, antibody R57 detects abundant intraneuronal APP immunoreactivity, but mAb 4G8 reveals only a very limited reaction with Aβ. In numerous neuronal populations in autistic subjects, the immunoreactivity for Aβ increases very significantly, but most R57 immunoreactive material is 4G8-negative, and most 4G8-positive granules are negative for APP. These results indicate that in the examined material, mAbs 6E10, 4G8 and 7F3D detect Aβ but do not bind to neuronal APP detected with pAb R57.
Immunoreactivity of mAb 4G8 with Aβ.
Diffuse Plaque Distribution and Immunoproperties in the Brain of Autistic Subjects
Aβ-positive plaques were detected in one of the nine examined subjects diagnosed with dup15 (AN11931), and in two of the 11 subjects diagnosed with idiopathic autism (AN17254 and BB1376). All three subjects were the oldest in each group. In the dup(15) group, a 39-year-old female with autistic features and intractable epilepsy (onset at 9 years of age) and whose death was epilepsy-related had clusters of plaques in several neocortical regions, including the frontal, temporal and insular cortex (). Plaques were also found in the brains of two individuals diagnosed with idiopathic autism, including a 51-year-old subject who had had only one grand mal seizure (), and a 52-year-old individual whose records do not contain information about epilepsy or brain trauma. In both brains, the postmortem examination revealed numerous plaques within the entire cortical ribbon (Fig. S2
) and in the amygdala, thalamus and subiculum (not shown).
Full-length Aβ in diffuse plaques and amino-terminally truncated Aβ in astrocytes in autism/dup15.
Full-length Aβ in diffuse plaques, and truncated Aβ in astrocytes in idiopathic autism.
In all three cases, thioflavin S staining did not reveal fluorescence in the plaques (not shown), suggesting that the amyloid plaques detected in the examined subjects with autism/dup(15) and idiopathic autism were nonfibrillar. However, positive immunoreactivity with all six antibodies used, including 6E10, 6F3, 4G8, Rabm38, Rabm40 and Rabm42 ( and ) and 6F3D (not shown), revealed full-length Aβ1–40/42 peptides. In the plaque area, numerous glial cells, mainly with the morphology of astrocytes, and less numerous, glial cells with the morphology of microglial cells, contained Aβ-immunoreactive granular material. In contrast to the presence of full-length Aβ peptides in plaques, the Aβ peptides in both astrocytes and microglial cells in the plaque perimeter and surrounding tissue were mAb 6E10- and 6F3D-negative, indicating that they were the product of α-secretase. They were positive for the three other antibodies, Rabm38, Rabm40 and Rabm42, demonstrating that both astrocytes and microglia accumulate Aβ17–40/42.
The extracts from the areas of the cerebral cortex in which diffuse plaques were detected by immunohistochemistry contained Aβ, mainly Aβ1–42, revealed by immunoblotting as a 4-kD band reacting with pAb R226 and mAb 6E10. The levels of Aβ1–42 in the samples exceeded 1.5 fmol per 1 µg of extracted proteins, whereas the levels of extracted Aβ 1–40 were low, below 0.2 fmol per 1 µg of extracted proteins ().
Properties of Aβ in plaque-rich cortex characterized by Western blotting.
Immunoblotting of lysates from the cerebral cortex of autistic subjects without plaques and age-matched control subjects detected Aβ42 () and Aβ40 (not shown) as a 3- to 4-kD band reacting with the pAb R226 and pAb R162, respectively. The levels of Aβ42 in the samples were in the range below 0.5 fmol per 40 µg of total proteins.
Detection by Western blots of Aβ in plaque-free subjects.
A very few neurofibrillary tangles (NFTs) were found in the entorhinal cortex and amygdala in a 43-year-old control subject and in the entorhinal cortex and cornu Ammonis of a 47-year-old control subject. A few NFTs were found in the entorhinal cortex, CA1 and parasubiculum in a 51-year-old autistic subject and in the entorhinal and temporal cortex and the amygdala of a 52-year-old autistic subject. Neurofibrillary changes were not found in the dup(15) autism cohort with the oldest examined subject who died at the age of 39 years.