ABSTRACT
Alzheimer's disease (AD) is characterized by the accumulation of intraneuronal tau and extracellular amyloid-ß (Aß) peptide. A triple transgenic (Tg) mouse (3xTg-AD) was reported to develop Aß plaques and tau inclusions as well as remarkable accumulations of intracellular Aß that were suggested to be the initiators of AD pathogenesis. However, it was unclear whether the anti-Aß antibodies were able to distinguish Aß peptide from the same Aß epitopes within the amyloid precursor protein (APP). To further elucidate the identity of the immunoreactive intraneuronal material in 3xTg-AD mice, we conducted immunohistochemical, biochemical, and ultrastructural studies using a well characterized panel of antibodies that distinguish Aß within APP from cleaved Aß peptides. We found that the intraneuronal material shared epitopes with full-length APP but not free Aß. To demonstrate unequivocally that this intraneuronal material was not free Aß peptide, we generated 3xTg-AD mice deficient for ß-secretase (BACE), the protease required for Aß generation from APP. In the absence of Aß production, robust intraneuronal APP immunostaining was detected in the 3xTg-AD/BACE(-/-) mice. Finally, we found that the formation of tau lesions was not different between 3xTg-AD versus 3xTg-AD/BACE(-/-) mice, thereby demonstrating that tau pathology forms independently from Aß peptide generation in this mouse model. Although we cannot corroborate the presence of intraneuronal Aß peptide in 3xTg-AD mice, our findings warrant further study as to the role of aberrant APP accumulation in this unique model of AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/physiology , Amyloid beta-Protein Precursor/physiology , Disease Models, Animal , Nerve Degeneration/metabolism , Neurons/metabolism , tau Proteins/physiology , Alzheimer Disease/pathology , Animals , Mice , Mice, Transgenic , Nerve Degeneration/pathology , Neurons/pathologyABSTRACT
We used cross-linking and immunoprecipitation coupled with high-throughput sequencing to identify binding sites in 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein that, when mutated, causes amyotrophic lateral sclerosis. Massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs were changed (including Fus (Tls), progranulin and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events were detected (including in sortilin, the receptor for progranulin) following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels were most depleted by reduction in TDP-43 were derived from genes with very long introns and that encode proteins involved in synaptic activity. Lastly, we found that TDP-43 autoregulates its synthesis, in part by directly binding and enhancing splicing of an intron in the 3' untranslated region of its own transcript, thereby triggering nonsense-mediated RNA degradation.
Subject(s)
Alternative Splicing/genetics , Amyotrophic Lateral Sclerosis/genetics , DNA-Binding Proteins/genetics , Nerve Degeneration/genetics , Neurons/pathology , RNA Precursors/genetics , RNA, Messenger/genetics , 3' Untranslated Regions/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/deficiency , Female , Homeostasis/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology , Neurons/metabolism , Oligonucleotides, Antisense/genetics , RNA Precursors/antagonists & inhibitors , RNA, Messenger/antagonists & inhibitorsABSTRACT
Heat shock proteins (Hsps) facilitate refolding of denatured polypeptides, but there is limited understanding about their roles in neurodegenerative diseases characterized by misfolded proteins. Because Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy are alpha-synucleinopathies characterized by filamentous alpha-synuclein (alpha-syn) inclusions, we assessed which Hsps might be implicated in these disorders by examining human brain samples, transgenic mouse models, and cell culture systems. Light and electron microscopic multiple-label immunohistochemistry showed Hsp90 was the predominant Hsp examined that co-localized with alpha-syn in Lewy bodies, Lewy neurites, and glial cell inclusions and that Hsp90 co-localized with alpha-syn filaments of Lewy bodies in PD. Hsp90 levels were most predominantly increased in PD brains, which correlated with increased levels of insoluble alpha-syn. These alterations in Hsp90 were recapitulated in a transgenic mouse model of PD-like alpha-syn pathologies. Cell culture studies also revealed that alpha-syn co-immunoprecipitated preferentially with Hsp90 and Hsc70 relative to other Hsps, and exposure of cells to proteasome inhibitors resulted in increased levels of Hsp90. These data implicate predominantly Hsp90 in the formation of alpha-syn inclusions in PD and related alpha-synucleinopathies.