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Changes of brain structure and functions in people with autism may result from altered neuronal development, however, no adequate cellular or animal models are available to study neurogenesis in autism. Neuronal development can be modeled in culture of neuronal progenitor cells (NPCs) stimulated with serum to differentiate into neurons. Because sera from people with autism and age-matched controls contain different levels of numerous biologically active factors, we hypothesized that development of human NPCs induced to differentiate into neurons with sera from children with autism reflects the altered early neuronal development that leads to autism. The control and autistic sera were collected from siblings aged below 6 years that lived in the same environment. The effect of sera on differentiation of NPC neurospheres into neuronal colonies was tested in 72-h-long cultures by morphometry, immunocytochemistry and immunoblotting. We found that sera from children with autism significantly reduced NPCs' proliferation, but stimulated cell migration, development of small neurons with processes, length of processes and synaptogenesis. These results suggest that development of network of processes and synaptogenesis--the specific events in the brain during postnatal ontogenesis--are altered in autism. Further studies in this cell culture model may explain some of the cellular alterations described in autistic patients.

Transplantation of human neuronal progenitor cells (HNPC) is being considered for neuroreplacement therapy in beta-amyloidosis associated with neuronal loss in Down's syndrome and Alzheimer's disease. However, the influence of amyloid-beta-containing brain environment on the development of HNPCs is unknown. Recently, we demonstrated that amyloid-beta peptide (Abeta) impaired differentiation of HNPCs in culture through oxidative stress. Now we studied the effect of neprilysin, an Abeta-degrading enzyme, on development of neuronal colonies from neurospheres of HNPCs in the presence of Abeta1-40. Neprilysin increased the number of neurospheres that formed colonies of neuron-like cells. This effect of neprilysin was associated with reduced amounts of the monomeric and dimeric Abeta that remained in culture supernatants as well as the Abeta uptaken by differentiating HNPCs. Phosphoramidon, a neprilysin inhibitor, attenuated these effects of neprilysin. In control cultures of HNPCs that grew without exogenous Abeta1-40, the treatment with neprilysin reduced the number of developing colonies. This effect might result from degradation by neprilysin of endogenous Abeta produced and secreted by HNPCs or other peptides that are involved in neuronal development. The results demonstrate that even a partial reduction of extracellular Abeta levels by neprilysin may facilitate development of HNPCs into neurons in an environment overloaded with Abeta. This finding suggests that neprilysin could facilitate neuroreplacement therapy with HNPCs in treatment of neurodegenerative diseases.

Recently, we found that brain vascular smooth muscle cells from Tg2576 mice over-expressed the APP transgene in culture, secreted amyloid-beta peptide (Abeta) and accumulated Abeta intracellularly. Now we detected this intracellular Abeta inside lysosomes, which were also rich in C-terminal domain of APP, but not in endoplasmic reticulum, Golgi apparatus, or trans-Golgi network. Treatment of cultures with ferrous ions (50-150 microM) increased the proportion of muscle cells with Abeta immunoreactive granules and the amounts of intracellular Abeta1-40 and Abeta1-42 in a dose-dependent manner. This increase of intracellular Abeta1-40 by iron was inhibited by alpha-tocopherol, but not by a water-soluble antioxidant melatonin. The increase of intracellular Abeta1-42 by iron was not inhibited by alpha-tocopherol or melatonin. Cell treatment with iron did not alter the lysosomal localization of Abeta immunoreactivity. Cell treatment with iron (II and III), copper (II), zinc (II) and aluminum (III) increased cellular levels of carbonyls. However, the effect of zinc on Abeta accumulation in cultures was weak, and there were no effects of copper and aluminum. The data suggest that iron may be the factor that triggers vascular amyloidosis. Lysosomal accumulation of APP and Abeta initiates deposition of amyloid in blood vessels in Tg2576 mice.

Apolipoprotein E (apoE) is a multifunctional molecule that is active during brain development, maintenance, and injury. Allele epsilon 4 of apoE is recognized as a risk factor for beta-amyloidosis, but the responsible mechanisms are not clear. Recently, we showed that vascular smooth muscle cells (SMCs) from epsilon 4/ epsilon 4 carriers are the most susceptible to oxidative protein damage that was associated with the appearance of apoE-Abeta-immunoreactive granules in cells. Here, we demonstrate that apoE4 is more readily accumulated in SMCs treated with ferrous ions than is apoE3. ApoE accumulated in lysosomes in the form of monomers, dimers, apoE-containing complexes, and apoE fragments. ApoE4 and apoE4-containing complexes persisted in SMCs longer than apoE3 and its complexes. Both isoforms of apoE stimulated formation of apoE-Abeta deposits and increased immobilization of iron in cultures treated with ferrous ions. The accumulation of apoE-Abeta deposits in lysosomes was associated with the appearance of lipid peroxidation products such as malondialdehyde and 4-hydroxynonenal-2-nonenal. The higher cellular accumulation of apoE4 than apoE3 in SMCs exposed to oxidative stress may facilitate development of beta-amyloid angiopathy that is more frequent in epsilon 4/ epsilon 4 carriers.

L-3-hydroxyacyl-coenzyme A dehydrogenase type II (HADH) was described as an endoplasmic reticulum amyloid beta-peptide-binding protein (ERAB), which enhances Abeta toxicity, and accumulates in neurons in Alzheimer's disease (AD). Hence, HADH/ERAB was suggested to mediate the amyloid-induced neurodegeneration. We estimated the in vivo interactions of HADH and Abeta in an immunocytochemical study of ten Alzheimer's disease and seven normal brains using five monoclonal HADH-specific antibodies. We found no HADH in amyloid plaques or vascular amyloid. The neuronal expression of HADH was not correlated with the severity of amyloid load in neuropil. HADH was expressed in vascular smooth muscle cells in young and old controls and in amyloid-free blood vessels in AD cases, but little or no HADH was in smooth muscle cells in arteries with amyloid deposits. The putative intracellular interaction between HADH and Abeta in amyloid-producing cells was further studied in vascular smooth muscle cells isolated from brain blood vessels with amyloid-beta angiopathy - the cells that were shown previously to accumulate Abeta intracellularly ['Research advances in Alzheimer's disease and related disorders' (1995) 747; Brain Res. 676 (1995) 225; Neurosci. Lett. 183 (1995) 120]. HADH had a mitochondrial localization and did not co-localize with an endoplasmic reticulum marker. Cells that accumulated Abeta were those with low expression of HADH and the proteins did not co-localize. Explanation of the association between low levels of HADH and deposition of Abeta by brain smooth muscle cells requires further studies.

Smooth muscle cells (SMCs) isolated from amyloid-angiopathy affected brain vessels accumulate intracellularly amyloid-beta peptide (A beta). Now we demonstrate that accumulation of A beta in SMCs can be reduced by factors secreted by macrophages - IL-1alpha, IL-6, TNF-alpha, TGF-beta1 or PGE2 - probably by stimulating the non-amyloidogenic processing of A beta precursor protein (PP). It is suggested that brain macrophages may regulate A betaPP/A beta metabolism under physiological conditions and prevent beta-amyloidosis. The disturbance of this regulatory function of brain macrophages may result in excessive production and accumulation of A beta.

Cultured brain vascular smooth muscle cells (SMCs) accumulate beta-peptide in intracytoplasmic granules [9,10,33]. We show here that apoE3 and E4 induces the intracytoplasmic beta-peptide accumulation in cultured human and canine SMCs. The induction is dose-dependent and the accumulated granules also contain apoE and some were thioflavine S-positive. The deposits induced with apoE3 were more abundant though less stable than with apoE4. Transthyretin at physiological concentrations blocked the effects of apoE3/E4. Thus, accumulation of beta-peptide appears to be regulated by beta-peptide carrier proteins.

Cultured smooth muscle cells isolated from beta-amyloid-affected blood vessels from old dogs accumulate beta-protein at early passages [5,24]. Now, we show that smooth muscle cells derived from amyloid-free brain blood vessels and peripheral arteries from old and young animals are induced by culture conditions to deposit intracellularly fibrillar and non-fibrillar beta-protein. Accumulation of beta-protein is associated with a higher secretion of beta-protein, but not with a higher secretion of beta-amyloid precursor protein (beta APP) or higher cellular content of beta APP. Gradual cessation of proliferative activity was observed in cultures that accumulate beta-protein.