Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc boxdependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein.Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.proteomics | liquid chromatography-tandem mass spectrometry | U1A | RNA-seq | premature cleavage and polyadenylation

Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C- terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.

The precise localization of Dl and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against Dl and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. Dl and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing Dl were rare whereas DS-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 p.m sections, Dl was localized to 53% of neurons, and D2 to 48% of neurons, while mixing Dl and D2 antibodies labeled 78%. By electron microscopy, Dl was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining Dl and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for Dl and D2, colocalization was not observed. lpsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with Dl than DS-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with Dl and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of Dl and D2 receptorsin striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.[

A family of muscarinic ACh receptor genes are expressed in hippocampus, but little is known about the localization of the encoded proteins and their regulation by cholinergic innervation. Subtype-specific antibodies were used to localize m1-m4 proteins in the hippocampal formation by immunocytochemistry and to determine the alterations in the subtypes following deafferentation. Each of the receptors is differentially localized in Ammon's horn and dentate gyrus, with highly complementary distributions. m1 is widely expressed in somata and dendrites of pyramidal neurons and granule cells in dentate gyrus. m2 immunoreactivity is expressed mostly in nonpyramidal neurons, and in several discrete bands of fibers and puncta surrounding pyramidal neurons and other layers. m3 is enriched in pyramidal neurons, the neuropil in stratum lacunosum-moleculare and the outer third of the molecular layer of dentate gyrus. m4 is enriched in nonpyramidal neurons, in fiber pathways (alveus, fimbria, and hippocampal commissure), and in the inner third of the molecular layer. Fimbria- fornix lesions decreased ipsilateral m2- and m4-immunoreactive axons in the fimbria, with no apparent changes in the distribution of any of the receptors in hippocampus. 192-IgG immunotoxin lesions of the cholinergic septohippocampal projections, which spare noncholinergic projections, produced a small decrease in m2-immunoreactive fibers in the fimbria with no other major changes in the distribution of subtypes. Immunoprecipitation studies at 3–28 d following fimbria- fornix lesions revealed a 25% loss of m2 at 3 d in hippocampus, and upregulation of both m1 (20–29% at 7–14 d) and m4 (44% at 28 d). Thus, the vast majority of muscarinic receptor subtypes are intrinsic to the hippocampal formation and/or nonseptal hippocampal afferents. A subset of m2 and m4 are presynaptically localized, with m2 in cholinergic axons and m2 and m4 possibly in noncholinergic axons that comprise the septohippocampal pathway. The unique laminar and regional distributions of m1-m4 in the hippocampus reflect differential cellular and subcellular distributions of the subtypes and/or selective association of receptor subtypes with certain afferent and intrinsic connections. These results indicate that each subtype likely has a different role in cholinergic modulation of excitatory and inhibitory hippocampal circuits.